Harry B Greenberg

All Publications

• Rhesus rotavirus NSP1 mediates extra-intestinal infection and is a contributing factor for biliary obstruction. PLoS pathogens Li, E., Feng, N., Zeng, Q., Sanchez-Tacuba, L., Kawagishi, T., Branham, G., Hou, G., Wang, Z., Greenberg, H. B., Ding, S. 2024; 20 (9): e1012609

  Abstract

  We previously demonstrated that in Ifnar1-/-Ifngr1-/- or Stat1-/- suckling mice lacking intact type I and type II interferon (IFN) signaling, rhesus rotavirus (RRV) infection causes a lethal disease with clinical manifestations similar to biliary atresia, including acholic stools, oily fur, growth retardation, and excess mortality. Elevated levels of viral RNA are detected in the bile ducts and liver of diseased pups together with severe inflammatory responses in these tissues. However, the viral determinants and the molecular mechanisms driving this process remain incompletely understood. Using an optimized rotavirus (RV) reverse genetics system, we generated a panel of recombinant RVs that encode non-structural protein 1 (NSP1) derived from different RV strains. We found that compared to the parental simian SA11 strain that is less biliary pathogenic, SA11 containing an RRV-derived NSP1 resulted in severe biliary obstructive disease comparable to that associated with RRV infection, reflected by high levels of viral RNA and inflammation in the biliary tract, liver, and pancreas. In contrast, RRV containing an SA11-originated NSP1 showed only mild biliary obstruction comparable to what was observed during SA11 infection. Infection with a monoreassortant RRV virus carrying NSP1 from the bovine RV UK strain also showed substantially reduced viral replication in extra-intestinal organs and did not develop clinical biliary diseases. Mechanistically, RRV NSP1 seemed to promote active viral replication in hepatocytes and this expanded tropism led to enhanced infiltration of CD4 and CD8 T cells, causing immunopathology and damage in the hepatobiliary system. These results highlight an unexpectedly important role of RV NSP1 in viral replication and disease progression in extra-intestinal tissues.

  View details for DOI 10.1371/journal.ppat.1012609

  View details for PubMedID 39348381

• Safety and immunogenicity of the intranasal H3N2 M2-deficient single-replication influenza vaccine alone or coadministered with an inactivated influenza vaccine (Fluzone High-Dose Quadrivalent) in adults aged 65-85 years in the USA: a multicentre, randomised, double-blind, double-dummy, phase 1b trial. The Lancet. Infectious diseases Eiden, J., Fierro, C., White, A., Davis, M., Rhee, M., Turner, M., Murray, B., Herber, R., Aitchison, R., Marshall, D., Moser, M. J., Belshe, R., Greenberg, H., Coelingh, K., Kawaoka, Y., Neumann, G., Bilsel, P. 2024

  Abstract

  BACKGROUND: Older adults (aged ≥65 years) show increased susceptibility to severe disease with influenza virus infection, accounting for 70-85% of annual influenza-related fatalities in the USA. Stimulating mucosal antibodies and T cells might enhance the low vaccine effectiveness seen in older adults for currently licensed inactivated influenza vaccines, which induce mainly serum antibodies. We aimed to evaluate the safety and immunogenicity of the intranasal H3N2 M2-deficient single-replication (M2SR) vaccine, alone or coadministered with a licensed inactivated influenza vaccine (Fluzone High-Dose Quadrivalent; hereafter referred to as Fluzone HD), in older adults.METHODS: In this multicentre, randomised, double-blind, double-dummy, phase 1b trial, individuals aged 65-85 years who were considered healthy or with stable chronic conditions with no recent (<6 months) influenza vaccinations were recruited from five clinical trial sites in the USA and randomly assigned (3:3:3:1) using a permuted block design to receive the H3N2 M2SR vaccine and Fluzone HD, the H3N2 M2SR vaccine and placebo, Fluzone HD and placebo, or placebo alone. All participants received a single intranasal spray and a single intramuscular injection, whether active or placebo, to maintain masking. The primary outcome was to assess the safety of H3N2 M2SR, administered alone or with Fluzone HD, in the safety analysis set, which included all participants who were randomly assigned and received treatment. Serum and mucosal antibodies were assessed as a secondary endpoint, and cell-mediated immunity as an exploratory endpoint, in participants in the per-protocol population, which included individuals in the safety analysis set without major protocol deviations. This trial is registered with ClinicalTrials.gov, NCT05163847.FINDINGS: Between June 14 and Sept 15, 2022, 305 participants were enrolled and randomly assigned to receive the H3N2 M2SR vaccine plus placebo (n=89), H3N2 M2SR vaccine plus Fluzone HD (n=94), Fluzone HD plus placebo (n=92), or placebo alone (n=30). All randomly assigned participants were included in the safety analysis set. The most frequently reported local symptoms up to day 8 in groups that received M2SR were rhinorrhoea (43% [38 of 89] in the H3N2 M2SR plus placebo group and 38% [36 of 94] in the H3N2 M2SR plus Fluzone HD group), nasal congestion (51% [45 of 89] and 35% [33 of 94]), and injection-site pain (8% [seven of 89] and 49% [46 of 94]), and the most frequently reported solicited systemic symptoms were sore throat (28% [25 of 89]) for M2SR and decreased activity (26% [24 of 94]) for the M2SR plus Fluzone HD group. In the Fluzone HD plus placebo group, the most frequently reported local symptom was injection-site pain (48% [44 of 92]) and systemic symptom was muscle aches (22% [20 of 92]). The frequency of participants with any treatment-emergent adverse event related to vaccination was low across all groups (2-5%). One serious adverse event was reported, in a participant in the Fluzone HD plus placebo group. M2SR with Fluzone HD induced seroconversion (≥four-fold increase in haemagglutination inhibition antibodies from baseline to day 29) in 44 (48%) of 91 participants, compared with 28 (31%) of 90 participants who seroconverted in the Fluzone HD plus placebo group (p=0·023). M2SR with Fluzone HD also induced mucosal and cellular immune responses.INTERPRETATION: The H3N2 M2SR vaccine coadministered with Fluzone HD in older adults was well tolerated and provided enhanced immunogenicity compared with Fluzone HD administered alone, suggesting potential for improved efficacy of influenza vaccination in this age group. Additional studies are planned to assess efficacy.FUNDING: US Department of Defense.

  View details for DOI 10.1016/S1473-3099(24)00351-7

  View details for PubMedID 39004096

• Reverse Genetics of Murine Rotavirus: A Comparative Analysis of the Wild-Type and Cell-Culture-Adapted Murine Rotavirus VP4 in Replication and Virulence in Neonatal Mice. Viruses Kawagishi, T., Sánchez-Tacuba, L., Feng, N., Greenberg, H. B., Ding, S. 2024; 16 (5)

  Abstract

  Small-animal models and reverse genetics systems are powerful tools for investigating the molecular mechanisms underlying viral replication, virulence, and interaction with the host immune response in vivo. Rotavirus (RV) causes acute gastroenteritis in many young animals and infants worldwide. Murine RV replicates efficiently in the intestines of inoculated suckling pups, causing diarrhea, and spreads efficiently to uninoculated littermates. Because RVs derived from human and other non-mouse animal species do not replicate efficiently in mice, murine RVs are uniquely useful in probing the viral and host determinants of efficient replication and pathogenesis in a species-matched mouse model. Previously, we established an optimized reverse genetics protocol for RV and successfully generated a murine-like RV rD6/2-2g strain that replicates well in both cultured cell lines and in the intestines of inoculated pups. However, rD6/2-2g possesses three out of eleven gene segments derived from simian RV strains, and these three heterologous segments may attenuate viral pathogenicity in vivo. Here, we rescued the first recombinant RV with all 11 gene segments of murine RV origin. Using this virus as a genetic background, we generated a panel of recombinant murine RVs with either N-terminal VP8* or C-terminal VP5* regions chimerized between a cell-culture-adapted murine ETD strain and a non-tissue-culture-adapted murine EW strain and compared the diarrhea rate and fecal RV shedding in pups. The recombinant viruses with VP5* domains derived from the murine EW strain showed slightly more fecal shedding than those with VP5* domains from the ETD strain. The newly characterized full-genome murine RV will be a useful tool for dissecting virus-host interactions and for studying the mechanism of pathogenesis in neonatal mice.

  View details for DOI 10.3390/v16050767

  View details for PubMedID 38793648

• MYADM binds human parechovirus 1 and is essential for viral entry. Nature communications Qiao, W., Richards, C. M., Kim, Y., Zengel, J. R., Ding, S., Greenberg, H. B., Carette, J. E. 2024; 15 (1): 3469

  Abstract

  Human parechoviruses (PeV-A) are increasingly being recognized as a cause of infection in neonates and young infants, leading to a spectrum of clinical manifestations ranging from mild gastrointestinal and respiratory illnesses to severe sepsis and meningitis. However, the host factors required for parechovirus entry and infection remain poorly characterized. Here, using genome-wide CRISPR/Cas9 loss-of-function screens, we identify myeloid-associated differentiation marker (MYADM) as a host factor essential for the entry of several human parechovirus genotypes including PeV-A1, PeV-A2 and PeV-A3. Genetic knockout of MYADM confers resistance to PeV-A infection in cell lines and in human gastrointestinal epithelial organoids. Using immunoprecipitation, we show that MYADM binds to PeV-A1 particles via its fourth extracellular loop, and we identify critical amino acid residues within the loop that mediate binding and infection. The demonstrated interaction between MYADM and PeV-A1, and its importance specifically for viral entry, suggest that MYADM is a virus receptor. Knockout of MYADM does not reduce PeV-A1 attachment to cells pointing to a role at the post-attachment stage. Our study suggests that MYADM is a multi-genotype receptor for human parechoviruses with potential as an antiviral target to combat disease associated with emerging parechoviruses.

  View details for DOI 10.1038/s41467-024-47825-0

  View details for PubMedID 38658526

  View details for PubMedCentralID 6893760

• A Viral Protein 4-Based Trivalent Nanoparticle Vaccine Elicited High and Broad Immune Responses and Protective Immunity against the Predominant Rotaviruses. ACS nano Xia, M., Huang, P., Vago, F., Kawagishi, T., Ding, S., Greenberg, H. B., Jiang, W., Tan, M. 2024

  Abstract

  The current live rotavirus (RV) vaccines show reduced effectiveness in developing countries, calling for vaccine strategies with improved efficacy and safety. We generated pseudovirus nanoparticles (PVNPs) that display multiple ectodomains of RV viral protein 4 (VP4), named S-VP4e, as a nonreplicating RV vaccine candidate. The RV spike protein VP4s that bind host receptors and facilitate viral entry are excellent targets for vaccination. In this study, we developed scalable methods to produce three S-VP4e PVNPs, each displaying the VP4e antigens from one of the three predominant P[8], P[4], and P[6] human RVs (HRVs). These PVNPs were recognized by selected neutralizing VP4-specific monoclonal antibodies, bound glycan receptors, attached to permissive HT-29 cells, and underwent cleavage by trypsin between VP8* and VP5*. 3D PVNP models were constructed to understand their structural features. A trivalent PVNP vaccine containing the three S-VP4e PVNPs elicited high and well-balanced VP4e-specific antibody titers in mice directed against the three predominant HRV P types. The resulting antisera neutralized the three HRV prototypes at high titers; greater than 4-fold higher than the neutralizing responses induced by a trivalent vaccine consisting of the S60-VP8* PVNPs. Finally, the trivalent S-VP4e PVNP vaccine provided 90-100% protection against diarrhea caused by HRV challenge. Our data supports the trivalent S-VP4e PVNPs as a promising nonreplicating HRV vaccine candidate for parenteral delivery to circumvent the suboptimal immunization issues of all present live HRV vaccines. The established PVNP-permissive cell and PVNP-glycan binding assays will be instrumental for further investigating HRV-host cell interactions and neutralizing effects of VP4-specific antibodies and antivirals.

  View details for DOI 10.1021/acsnano.4c00544

  View details for PubMedID 38353701

• Lactobacillus acidophilus Expressing Murine Rotavirus VP8 and Mucosal Adjuvants Induce Virus-Specific Immune Responses. Vaccines Gilfillan, D., Vilander, A. C., Pan, M., Goh, Y. J., O'Flaherty, S., Feng, N., Fox, B. E., Lang, C., Greenberg, H. B., Abdo, Z., Barrangou, R., Dean, G. A. 2023; 11 (12)

  Abstract

  Rotavirus diarrhea-associated illness remains a major cause of global death in children under five, attributable in part to discrepancies in vaccine performance between high- and low-middle-income countries. Next-generation probiotic vaccines could help bridge this efficacy gap. We developed a novel recombinant Lactobacillus acidophilus (rLA) vaccine expressing rotavirus antigens of the VP8* domain from the rotavirus EDIM VP4 capsid protein along with the adjuvants FimH and FliC. The upp-based counterselective gene-replacement system was used to chromosomally integrate FimH, VP8Pep (10 amino acid epitope), and VP8-1 (206 amino acid protein) into the L. acidophilus genome, with FliC expressed from a plasmid. VP8 antigen and adjuvant expression were confirmed by flow cytometry and Western blot. Rotavirus naïve adult BALB/cJ mice were orally immunized followed by murine rotavirus strain ECWT viral challenge. Antirotavirus serum IgG and antigen-specific antibody-secreting cell responses were detected in rLA-vaccinated mice. A day after the oral rotavirus challenge, fecal antigen shedding was significantly decreased in the rLA group. These results indicate that novel rLA constructs expressing VP8 can be successfully constructed and used to generate modest homotypic protection from rotavirus challenge in an adult murine model, indicating the potential for a probiotic next-generation vaccine construct against human rotavirus.

  View details for DOI 10.3390/vaccines11121774

  View details for PubMedID 38140179

  View details for PubMedCentralID PMC10747613

• Corrected and republished from: "VP4 Is a Determinant of Alpha-Defensin Modulation of Rotaviral Infection". Journal of virology Hu, C. T., Diaz, K., Yang, L. C., Sharma, A., Greenberg, H. B., Smith, J. G. 2023: e0096223

  Abstract

  Fecal-oral pathogens encounter constitutively expressed enteric alpha-defensins in the intestine during replication and transmission. Alpha-defensins can be potently antiviral and antibacterial; however, their primary sequences, the number of isoforms, and their activity against specific microorganisms often vary greatly between species, reflecting adaptation to species-specific pathogens. Therefore, alpha-defensins might influence not only microbial evolution and tissue tropism within a host but also species tropism and zoonotic potential. To investigate these concepts, we generated a panel of enteric and myeloid alpha-defensins from humans, rhesus macaques, and mice and tested their activity against group A rotaviruses, an important enteric viral pathogen of humans and animals. Rotaviral adaptation to the rhesus macaque correlated with resistance to rhesus enteric but not myeloid alpha-defensins and sensitivity to human alpha-defensins. Infection by mouse and human rotaviruses was either resistant to or increased by host enteric alpha-defensins, although the effects of cross-species alpha-defensins did not follow an obvious pattern. Because infection by all rotaviruses tested was resistant to or enhanced by enteric alpha-defensins from their hosts, exposure to alpha-defensins may have shaped their evolution. We then used a genetic approach to identify the viral attachment and penetration protein, VP4, as a determinant of alpha-defensin sensitivity. Our results provide a foundation for future studies of the VP4-dependent mechanism of defensin neutralization, highlight the species-specific activities of alpha-defensins, and focus future efforts on a broader range of rotaviruses that differ in VP4 to uncover the potential for enteric alpha-defensins to influence species tropism. IMPORTANCE Rotavirus is a leading cause of severe diarrhea in young children. Like other fecal-oral pathogens, rotaviruses encounter abundant, constitutively expressed defensins in the small intestine. These peptides are a vital part of the vertebrate innate immune system. By investigating the impact that defensins from multiple species have on the infectivity of different strains of rotavirus, we show that some rotaviral infections can be inhibited by defensins. We also found that rotaviruses may have evolved resistance to defensins in the intestine of their host species, and some even appropriate defensins to increase their infectivity. Because rotaviruses infect a broad range of animals and rotaviral infections are highly prevalent in children, identifying immune defenses against infection and how they vary across species and among viral genotypes is important for our understanding of the evolution, transmission, and zoonotic potential of these viruses as well as the improvement of vaccines.

  View details for DOI 10.1128/jvi.00962-23

  View details for PubMedID 37787534

• Mucosal and systemic neutralizing antibodies to norovirus induced in infant mice orally inoculated with recombinant rotaviruses. Proceedings of the National Academy of Sciences of the United States of America Kawagishi, T., Sanchez-Tacuba, L., Feng, N., Costantini, V. P., Tan, M., Jiang, X., Green, K. Y., Vinje, J., Ding, S., Greenberg, H. B. 2023; 120 (9): e2214421120

  Abstract

  Rotaviruses (RVs) preferentially replicate in the small intestine and frequently cause severe diarrheal disease, and the following enteric infection generally induces variable levels of protective systemic and mucosal immune responses in humans and other animals. Rhesus rotavirus (RRV) is a simian RV that was previously used as a human RV vaccine and has been extensively studied in mice. Although RRV replicates poorly in the suckling mouse intestine, infection induces a robust and protective antibody response. The recent availability of plasmid only-based RV reverse genetics systems has enabled the generation of recombinant RVs expressing foreign proteins. However, recombinant RVs have not yet been experimentally tested as potential vaccine vectors to immunize against other gastrointestinal pathogens invivo. This is a newly available opportunity because several live-attenuated RV vaccines are already widely administered to infants and young children worldwide. To explore the feasibility of using RV as a dual vaccine vector, we rescued replication-competent recombinant RRVs harboring bicistronic gene segment 7 that encodes the native RV nonstructuralprotein 3 (NSP3) protein and a human norovirus (HuNoV) VP1 protein or P domain from the predominant genotype GII.4. The rescued viruses expressed HuNoV VP1 or P protein in infected cells invitro and elicited systemic and local antibody responses to HuNoV and RRV following oral infection of suckling mice. Serum IgG and fecal IgA from infected suckling mice bound to and neutralized both RRV and HuNoV. These findings have encouraging practical implications for the design of RV-based next-generation multivalent enteric vaccines to target HuNoV and other human enteric pathogens.

  View details for DOI 10.1073/pnas.2214421120

  View details for PubMedID 36821582

• Intranasal M2SR (M2-deficient Single Replication) H3N2 Influenza Vaccine Provides Enhanced Mucosal and Serum Antibodies In Adults. The Journal of infectious diseases Eiden, J., Fierro, C., Schwartz, H., Adams, M., Ellis, K. J., Aitchison, R., Herber, R., Hatta, Y., Marshall, D., Moser, M. J., Belshe, R., Greenberg, H., Coelingh, K., Kawaoka, Y., Neumann, G., Bilsel, P. 2022

  Abstract

  BACKGROUND: We previously demonstrated that an intranasal dose of 108 TCID50 M2SR (M2-deficient single replication) influenza vaccine protected against highly drifted H3N2 influenza challenge in a subset of subjects who demonstrated ≥2-fold increase in microneutralization (MN) antibodies to Belgium2015 (the challenge strain) after vaccination. Here, we describe a phase 1b, observer-blinded, dose-escalation study demonstrating an increased proportion of responders with this signal of immune protection.METHODS: Sero-susceptible subjects ages 18-49 years were randomized to receive two doses (108-109TCID50) of M2SR or placebo administered 28 days apart. Clinical specimens were collected before and after each dose. The primary objective was to demonstrate safety of M2SR vaccines (ClinicalTrials.gov number NCT03999554).RESULTS: The vaccine was well-tolerated at all dose levels. Against Belgium2015, ≥2-fold increases in MN antibodies were noted among 40% (95% CI 24.9-56.7) of subjects following a single 108 TCID50 M2SR dose and among 80.6% (95% CI 61.4-92.3) after 109 dose (P<0.001). A single 109 TCID50 dose of M2SR generated ≥4-fold HAI seroconversion against the vaccine strain in 71% (95% CI 52.0-85.8) of recipients. Mucosal and cellular immune responses were also induced.CONCLUSIONS: These results indicate that M2SR may provide substantial protection against infection with highly drifted strains of H3N2 influenza.

  View details for DOI 10.1093/infdis/jiac433

  View details for PubMedID 36350017

• Rotavirus Infection Elicits Host Responses via P2Y1 Purinergic Signaling Engevik, K., Kawagishi, T., Gebert, J., Scribano, F., Perry, J., Greenberg, H., Hyser, J. ELSEVIER SCIENCE INC. 2022: S5

  View details for Web of Science ID 000873068000015

• Rotavirus VP4 Epitope of a Broadly Neutralizing Human Antibody Defined by Its Structure Bound with an Attenuated-Strain Virion. Journal of virology Jenni, S., Li, Z., Wang, Y., Bessey, T., Salgado, E. N., Schmidt, A. G., Greenberg, H. B., Jiang, B., Harrison, S. C. 2022: e0062722

  Abstract

  Rotavirus live-attenuated vaccines, both mono- and pentavalent, generate broadly heterotypic protection. B-cells isolated from adults encode neutralizing antibodies, some with affinity for VP5*, that afford broad protection in mice. We have mapped the epitope of one such antibody by determining the high-resolution cryo-EM structure of its antigen-binding fragment (Fab) bound to the virion of a candidate vaccine strain, CDC-9. The Fab contacts both the distal end of a VP5* β-barrel domain and the two VP8* lectin-like domains at the tip of a projecting spike. Its interactions with VP8* do not impinge on the likely receptor-binding site, suggesting that the mechanism of neutralization is at a step subsequent to initial attachment. We also examined structures of CDC-9 virions from two different stages of serial passaging. Nearly all the VP4 (cleaved to VP8*/VP5*) spikes on particles from the earlier passage (wild-type isolate) had transitioned from the "upright" conformation present on fully infectious virions to the "reversed" conformation that is probably the end state of membrane insertion, unable to mediate penetration, consistent with the very low in vitro infectivity of the wild-type isolate. About half the VP4 spikes were upright on particles from the later passage, which had recovered substantial in vitro infectivity but had acquired an attenuated phenotype in neonatal rats. A mutation in VP4 that occurred during passaging appears to stabilize the interface at the apex of the spike and could account for the greater stability of the upright spikes on the late-passage, attenuated isolate. IMPORTANCE Rotavirus live-attenuated vaccines generate broadly heterotypic protection, and B-cells isolated from adults encode antibodies that are broadly protective in mice. Determining the structural and mechanistic basis of broad protection can contribute to understanding the current limitations of vaccine efficacy in developing countries. The structure of an attenuated human rotavirus isolate (CDC-9) bound with the Fab fragment of a broadly heterotypic protective antibody shows that protection is probably due to inhibition of the conformational transition in the viral spike protein (VP4) critical for viral penetration, rather than to inhibition of receptor binding. A comparison of structures of CDC-9 virus particles at two stages of serial passaging supports a proposed mechanism for initial steps in rotavirus membrane penetration.

  View details for DOI 10.1128/jvi.00627-22

  View details for PubMedID 35924923

• The Dengue Virus Nonstructural Protein 1 (NS1) Interacts with the Putative Epigenetic Regulator DIDO1 to Promote Flavivirus Replication in Mosquito Cells. Journal of virology Caraballo, G. I., Rosales, R., Viettri, M., Castillo, J. M., Cruz, R., Ding, S., Greenberg, H. B., Ludert, J. E. 2022: e0070422

  Abstract

  Dengue virus (DENV) NS1 is a multifunctional protein essential for viral replication. To gain insights into NS1 functions in mosquito cells, the protein interactome of DENV NS1 in C6/36 cells was investigated using a proximity biotinylation system and mass spectrometry. A total of 817 mosquito targets were identified as protein-protein interacting with DENV NS1. Approximately 14% of them coincide with interactomes previously obtained in vertebrate cells, including the oligosaccharide transferase complex, the chaperonin containing TCP-1, vesicle localization, and ribosomal proteins. Notably, other protein pathways not previously reported in vertebrate cells, such as epigenetic regulation and RNA silencing, were also found in the NS1 interactome in mosquito cells. Due to the novel and strong interactions observed for NS1 and the epigenetic regulator DIDO1 (Death-Inducer Obliterator 1), the role of DIDO1 in viral replication was further explored. Interactions between NS1 and DIDO1 were corroborated in infected mosquito cells, by colocalization and proximity ligation assays. Silencing DIDO1 expression results in a significant reduction in DENV and ZIKV replication and progeny production. Comparison of transcription analysis of mock or DENV infected cells silenced for DIDO1 revealed variations in multiple gene expression pathways, including pathways associated with DENV infection such as RNA surveillance, IMD, and Toll. These results suggest that DIDO1 is a host factor involved in the negative modulation of the antiviral response necessary for flavivirus replication in mosquito cells. Our findings uncover novel mechanisms of NS1 to promote DENV and ZIKV replication, and add to the understanding of NS1 as a multifunctional protein. IMPORTANCE Dengue is the most important mosquito-borne viral disease to humans. Dengue virus NS1 is a multifunctional protein essential for replication and modulation of innate immunity. To gain insights into NS1 functions, the protein interactome of dengue virus NS1 in Aedes albopictus cells was investigated using a proximity biotinylation system and mass spectrometry. Several protein pathways, not previously observed in vertebrate cells, such as transcription and epigenetic regulation, were found as part of the NS1 interactome in mosquito cells. Among those, DIDO1 was found to be a necessary host factor for dengue and Zika virus replication in mosquito cells. Transcription analysis of infected mosquito cells silenced for DIDO1 revealed alterations of the IMD and Toll pathways, part of the antiviral response in mosquitoes. The results suggest that DIDO1 is a host factor involved in modulation of the antiviral response and necessary for flavivirus replication.

  View details for DOI 10.1128/jvi.00704-22

  View details for PubMedID 35652656

• VP4 Is a Determinant of Alpha-Defensin Modulation of Rotaviral Infection. Journal of virology Hu, C. T., Diaz, K., Yang, L. C., Sharma, A., Greenberg, H. B., Smith, J. G. 2022: e0205321

  Abstract

  Fecal-oral pathogens encounter constitutively expressed enteric alpha-defensins in the intestine during replication and transmission. Alpha-defensins can be potently antiviral and antibacterial; however, their primary sequences, the number of isoforms, and their activity against specific microorganisms often vary greatly between species, reflecting adaptation to species-specific pathogens. Therefore, alpha-defensins might influence not only microbial evolution and tissue tropism within a host but also species tropism and zoonotic potential. To investigate these concepts, we generated a panel of enteric and myeloid alpha-defensins from humans, rhesus macaques, and mice and tested their activity against group A rotaviruses, an important enteric viral pathogen of humans and animals. Rotaviral adaptation to the rhesus macaque correlated with resistance to rhesus enteric, but not myeloid, alpha-defensins and sensitivity to human alpha-defensins. While mouse rotaviral infection was increased in the presence of mouse enteric alpha-defensins, two prominent genotypes of human rotaviruses were differentially sensitive to human enteric alpha-defensins. Furthermore, the effects of cross-species alpha-defensins on human and mouse rotaviruses did not follow an obvious pattern. Thus, exposure to alpha-defensins may have shaped the evolution of some, but not all, rotaviruses. We then used a genetic approach to identify the viral attachment and penetration protein, VP4, as a determinant of alpha-defensin sensitivity. Our results provide a foundation for future studies of the VP4-dependent mechanism of defensin neutralization, highlight the species-specific activities of alpha-defensins, and focus future efforts on a broader range of rotaviruses that differ in VP4 to uncover the potential for enteric alpha-defensins to influence species tropism. IMPORTANCE Rotavirus is a leading cause of severe diarrhea in young children. Like other fecal-oral pathogens, rotaviruses encounter abundant, constitutively expressed defensins in the small intestine. These peptides are a vital part of the vertebrate innate immune system. By investigating the impact that defensins from multiple species have on the infectivity of different strains of rotavirus, we show that some rotaviral infections can be inhibited by defensins. We also found that some, but not all, rotaviruses may have evolved resistance to defensins in the intestine of their host species, and some even appropriate defensins to increase their infectivity. Because rotaviruses infect a broad range of animals and rotaviral infections are highly prevalent in children, identifying immune defenses against infection and how they vary across species and among viral genotypes is important for our understanding of the evolution, transmission, and zoonotic potential of these viruses as well as the improvement of vaccines.

  View details for DOI 10.1128/jvi.02053-21

  View details for PubMedID 35285683

• The Role of the VP4 Attachment Protein in Rotavirus Host Range Restriction in an In Vivo Suckling Mouse Model Journal of Virology Sánchez-Tacuba, L., Kawagishi, T., Feng, N., Jiang, B., Ding, S., Greenberg, H. 2022

  View details for DOI 10.1128/jvi.00550-22

• A recombinant murine-like rotavirus with Nano-Luciferase expression reveals tissue tropism, replication dynamics, and virus transmission. Frontiers in immunology Zhu, Y., Sanchez-Tacuba, L., Hou, G., Kawagishi, T., Feng, N., Greenberg, H. B., Ding, S. 2022; 13: 911024

  Abstract

  Rotaviruses (RVs) are one of the main causes of severe gastroenteritis, diarrhea, and death in children and young animals. While suckling mice prove to be highly useful small animal models of RV infection and pathogenesis, direct visualization tools are lacking to track the temporal dynamics of RV replication and transmissibility in vivo. Here, we report the generation of the first recombinant murine-like RV that encodes a Nano-Luciferase reporter (NLuc) using a newly optimized RV reverse genetics system. The NLuc-expressing RV was replication-competent in cell culture and both infectious and virulent in neonatal mice in vivo. Strong luciferase signals were detected in the proximal and distal small intestines, colon, and mesenteric lymph nodes. We showed, via a noninvasive in vivo imaging system, that RV intestinal replication peaked at days 2 to 5 post infection. Moreover, we successfully tracked RV transmission to uninoculated littermates as early as 3 days post infection, 1 day prior to clinically apparent diarrhea and 3 days prior to detectable fecal RV shedding in the uninoculated littermates. We also observed significantly increased viral replication in Stat1 knockout mice that lack the host interferon signaling. Our results suggest that the NLuc murine-like RV represents a non-lethal powerful tool for the studies of tissue tropism and host and viral factors that regulate RV replication and spread, as well as provides a new tool to facilitate the testing of prophylactic and therapeutic interventions in the future.

  View details for DOI 10.3389/fimmu.2022.911024

  View details for PubMedID 35967392

• Rotavirus NSP1 Contributes to Intestinal Viral Replication, Pathogenesis, and Transmission. mBio Hou, G., Zeng, Q., Matthijnssens, J., Greenberg, H. B., Ding, S. 1800: e0320821

  Abstract

  Rotavirus (RV)-encoded nonstructural protein 1 (NSP1), the product of gene segment 5, effectively antagonizes host interferon (IFN) signaling via multiple mechanisms. Recent studies with the newly established RV reverse genetics system indicate that NSP1 is not essential for the replication of the simian RV SA11 strain in cell culture. However, the role of NSP1 in RV infection in vivo remains poorly characterized due to the limited replication of heterologous simian RVs in the suckling mouse model. Here, we used an optimized reverse genetics system and successfully recovered recombinant murine RVs with or without NSP1 expression. While the NSP1-null virus replicated comparably with the parental murine RV in IFN-deficient and IFN-competent cell lines in vitro, it was highly attenuated in 5-day-old wild-type suckling pups in both the 129sv and C57BL/6 backgrounds. In the absence of NSP1 expression, murine RV had significantly reduced replication in the ileum, systemic spread to mesenteric lymph nodes, fecal shedding, diarrhea occurrence, and transmission to uninoculated littermates. The defective replication of the NSP1-null RV in small intestinal tissues occurred as early as 1day postinfection. Of interest, the replication and pathogenesis defects of NSP1-null RV were only minimally rescued in Stat1 knockout pups, suggesting that NSP1 facilitates RV replication in an IFN-independent manner. Our findings highlight a pivotal function of NSP1 during homologous RV infections in vivo and identify NSP1 as an ideal viral protein for targeted attenuation for future vaccine development. IMPORTANCE Rotavirus remains one of the most important causes of severe diarrhea and dehydration in young children worldwide. Although NSP1 is dispensable for rotavirus replication in cell culture, its exact role in virus infection in vivo remains unclear. In this study, we demonstrate, for the first time in a pathologically valid homologous small animal model, that in the context of a fully replication-competent, pathogenic, and transmissible murine rotavirus, loss of NSP1 expression substantially attenuated virus replication in the gastrointestinal tract, diarrheal disease, and virus transmission. Notably, the NSP1-deficient murine rotavirus also replicated poorly in mice lacking host interferon or inflammasome signaling. Our data provide the first piece of evidence that NSP1 is essential for murine rotavirus replication in vivo, making it an attractive target for developing improved next-generation rotavirus vaccines better suited for socioeconomically disadvantaged and immunocompromised individuals.

  View details for DOI 10.1128/mBio.03208-21

  View details for PubMedID 34903043

• Safety and Immunogenicity of M2-Deficient, Single Replication, Live Influenza Vaccine (M2SR) in Adults. Vaccines Eiden, J., Gordon, G., Fierro, C., Herber, R., Aitchison, R., Belshe, R., Greenberg, H., Hoft, D., Hatta, Y., Moser, M. J., Tary-Lehmann, M., Kawaoka, Y., Neumann, G., Radspinner, P., Bilsel, P. 1800; 9 (12)

  Abstract

  M2SR (M2-deficient single replication) is an investigational live intranasal vaccine that protects against multiple influenza A subtypes in influenza-naive and previously infected ferrets. We conducted a phase 1, first-in-human, randomized, dose-escalation, placebo-controlled study of M2SR safety and immunogenicity. Adult subjects received a single intranasal administration with either placebo or one of three M2SR dose levels (106, 107 or 108 tissue culture infectious dose (TCID50)) expressing hemagglutinin and neuraminidase from A/Brisbane/10/2007 (H3N2) (24 subjects per group). Subjects were evaluated for virus replication, local and systemic reactions, adverse events (AE), and immune responses post-vaccination. Infectious virus was not detected in nasal swabs from vaccinated subjects. At least one AE (most commonly mild nasal rhinorrhea/congestion) was reported among 29%, 58%, and 83% of M2SR subjects administered a low, medium or high dose, respectively, and among 46% of placebo subjects. No subject had fever or a severe reaction to the vaccine. Influenza-specific serum and mucosal antibody responses and B- and T-cell responses were significantly more frequent among vaccinated subjects vs. placebo recipients. The M2SR vaccine was safe and well tolerated and generated dose-dependent durable serum antibody responses against diverse H3N2 influenza strains. M2SR demonstrated a multi-faceted immune response in seronegative and seropositive subjects.

  View details for DOI 10.3390/vaccines9121388

  View details for PubMedID 34960134

• Rotavirus NSP1 Contributes to Intestinal Viral Replication, Pathogenesis, and Transmission MBIO Hou, G., Zeng, Q., Matthijnssens, J., Greenberg, H. B., Ding, S. 2021; 12 (6)

  View details for Web of Science ID 000736928500006

• Single Replication M2SR Influenza Vaccine Induced Immune Responses Associated with Protection Against Human Challenge with Highly Drifted H3N2 Influenza Strain. The Journal of infectious diseases Eiden, J., Volckaert, B., Rudenko, O., Aitchison, R., Herber, R., Belshe, R., Greenberg, H., Coelingh, K., Marshall, D., Kawaoka, Y., Neumann, G., Bilsel, P. 2021

  Abstract

  BACKGROUND: Current influenza vaccines are strain-specific and demonstrate low vaccine efficacy against H3N2 influenza disease, especially when vaccine is mis-matched to circulating virus. The novel influenza vaccine candidate, M2SR (M2-deficient Single Replication), induces a broad, multi-effector immune response.METHODS: A phase 2 challenge study was conducted to assess efficacy of M2SR vaccine expressing HA and NA from A/Brisbane/10/2007 (H3N2, clade 1). Four weeks after vaccination subjects were challenged with antigenically distinct H3N2 virus (A/Belgium/4217/2015, clade 3C.3b), and assessed for infection and clinical symptoms.RESULTS: Adverse events following vaccination were mild and similar in frequency between placebo and M2SR recipients. A single dose of Bris2007 M2SR induced neutralizing antibody to the vaccine (48% of recipients) and challenge strain (27% of recipients). Overall, 54% of M2SR subjects were infected after challenge, compared to 71% of placebo subjects. The subset of M2SR subjects with a vaccine-induced microneutralization response against the challenge virus had reduced rates of infection after challenge (38% vs. 71% of placebo subjects, P=0.0505) and reduced illness.CONCLUSIONS: Subjects with vaccine-induced neutralizing antibodies were protected against infection and illness following challenge with an antigenically distinct virus. This is the first demonstration of vaccine-induced protection against a highly drifted H3N2 challenge virus.

  View details for DOI 10.1093/infdis/jiab374

  View details for PubMedID 34323977

• Perspectives for the optimization and utility of the rotavirus reverse genetics system. Virus research Ding, S., Greenberg, H. B. 2021: 198500

  Abstract

  Following Kobayashi and colleagues' seminal paper in 20171, in the past four years the rotavirus (RV) field has witnessed a burst in research and publications based on the use of a fully plasmid-based RV reverse genetics systems and subsequent modifications2,3. However, in most cases, the rotaviral strain under interrogation has been the prototypic simian RV SA11-L2 strain (G3P [2]). Of note, a variety of other weakly-to-modestly replication-competent animal and human RV strains, bioluminescent and fluorescent reporter viruses, and clinical isolates of human RVs have proved hard or impossible to rescue using the original reverse genetics system2,4, highlighting a critical need to further enhance the recovery efficiency and expand the rescue tool kit. A number of further modifications of the initial reverse genetics system have enabled the rescue of other RV strains such as the human RV KU and CDC-9 strains, and a murine RV D6/2-like strain4,5. Here, we discuss future possible modifications of existing RV reverse genetics systems to further increase efficiency based on past experience with the improvement of influenza A virus recovery. The development of RV to accommodate the insertion and expression of heterologous sequences have substantial potential in the design of next-generation RV vaccine candidates and enteric viral vectors.

  View details for DOI 10.1016/j.virusres.2021.198500

  View details for PubMedID 34331991

• A CD22-Shp1 phosphatase axis controls integrin beta 7 display and B cell function in mucosal immunity Ballet, R., Brennan, M., Brandl, C., Feng, N., Berri, J., Cheng, J., Ocon, B., Sheikh, A., Marki, A., Bi, Y., Abram, C., Lowell, C. WILEY. 2021

  View details for DOI 10.1096/fasebj.2021.35.S1.02188

  View details for Web of Science ID 000669893101305

• A CD22-Shp1 phosphatase axis controls integrin beta7 display and B cell function in mucosal immunity. Nature immunology Ballet, R., Brennan, M., Brandl, C., Feng, N., Berri, J., Cheng, J., Ocon, B., Alborzian Deh Sheikh, A., Marki, A., Bi, Y., Abram, C. L., Lowell, C. A., Tsubata, T., Greenberg, H. B., Macauley, M. S., Ley, K., Nitschke, L., Butcher, E. C. 2021

  Abstract

  The integrin alpha4beta7 selectively regulates lymphocyte trafficking and adhesion in the gut and gut-associated lymphoid tissue (GALT). Here, we describe unexpected involvement of the tyrosine phosphatase Shp1 and the B cell lectin CD22 (Siglec-2) in the regulation of alpha4beta7 surface expression and gut immunity. Shp1 selectively inhibited beta7 endocytosis, enhancing surface alpha4beta7 display and lymphocyte homing to GALT. In B cells, CD22 associated in a sialic acid-dependent manner with integrin beta7 on the cell surface to target intracellular Shp1 to beta7. Shp1 restrained plasma membrane beta7 phosphorylation and inhibited beta7 endocytosis without affecting beta1 integrin. B cells with reduced Shp1 activity, lacking CD22 or expressing CD22 with mutated Shp1-binding or carbohydrate-binding domains displayed parallel reductions in surface alpha4beta7 and in homing to GALT. Consistent with the specialized role of alpha4beta7 in intestinal immunity, CD22 deficiency selectively inhibited intestinal antibody and pathogen responses.

  View details for DOI 10.1038/s41590-021-00862-z

  View details for PubMedID 33589816

• Elucidating the Role of Host Purinergic Signaling During Rotavirus Infection Engevik, K. A., Gebert, J. T., Perry, J. L., Scribano, F. J., Kawagishi, T., Greenberg, H., Hyser, J. M. ELSEVIER SCIENCE INC. 2021: S4-S5

  View details for Web of Science ID 000704491400014

• Inhibitor of growth protein 3 epigenetically silences endogenous retroviral elements and prevents innate immune activation. Nucleic acids research Song, Y., Hou, G., Diep, J., Ooi, Y. S., Akopyants, N. S., Beverley, S. M., Carette, J. E., Greenberg, H. B., Ding, S. 2021

  Abstract

  Endogenous retroviruses (ERVs) are subject to transcriptional repression in adult tissues, in part to prevent autoimmune responses. However, little is known about the epigenetic silencing of ERV expression. Here, we describe a new role for inhibitor of growth family member 3 (ING3), to add to an emerging group of ERV transcriptional regulators. Our results show that ING3 binds to several ERV promoters (for instance MER21C) and establishes an EZH2-mediated H3K27 trimethylation modification. Loss of ING3 leads to decreases of H3K27 trimethylation enrichment at ERVs, induction of MDA5-MAVS-interferon signaling, and functional inhibition of several virus infections. These data demonstrate an important new function of ING3 in ERV silencing and contributing to innate immune regulation in somatic cells.

  View details for DOI 10.1093/nar/gkab1070

  View details for PubMedID 34791430

• An optimized reverse genetics system suitable for efficient recovery of simian, human and murine-like rotaviruses. Journal of virology Sanchez-Tacuba, L., Feng, N., Meade, N. J., Mellits, K. H., Jais, P. H., Yasukawa, L. L., Resch, T. K., Jiang, B., Lopez, S., Ding, S., Greenberg, H. B. 2020

  Abstract

  An entirely plasmid-based reverse genetics (RG) system was recently developed for rotavirus (RV), opening new avenues for in-depth molecular dissection of RV biology, immunology, and pathogenesis. Several improvements to further optimize the RG efficiency have now been described. However, only a small number of individual RV strains have been recovered to date. None of the current methods have supported the recovery of murine RV, impeding the study of RV replication and pathogenesis in an in vivo suckling mouse model. Here, we describe useful modifications to the RG system that significantly improve rescue efficiency of multiple RV strains. In addition to the 11 RVA segment-specific (+)ssRNAs, a chimeric plasmid was transfected, from which the capping enzyme NP868R of African swine fever virus (ASFV) and the T7 RNA polymerase were expressed. Secondly, a genetically modified MA104 cell line was used in which several compounds of the innate immune were degraded. Using this RG system, we successfully recovered the simian RV RRV strain, the human RV CDC-9 strain, a reassortant between murine RV D6/2 and simian RV SA11 strains, and several reassortants and reporter RVs. All these recombinant RVs were rescued at a high efficiency (≥80% success rate) and could not be reliably rescued using several recently published RG strategies (<20%). This improved system represents an important tool and great potential for the rescue of other hard-to-recover RV strains such as low replicating attenuated vaccine candidates or low cell culture passage clinical isolates from humans or animals.IMPORTANCE Group A rotavirus (RV) remains as the single most important cause of severe acute gastroenteritis among infants and young children worldwide. An entirely plasmid-based reverse genetics (RG) system was recently developed opening new ways for in-depth molecular study of RV. Despite several improvements to further optimize the RG efficiency, it has been reported that current strategies do not enable the rescue of all cultivatable RV strains. Here, we described helpful modification to the current strategies and established a tractable RG system for the rescue of the simian RRV strain, the human CDC-9 strain and a murine-like RV strain, which is suitable for both in vitro and in vivo studies. This improved RV reverse genetics system will facilitate study of RV biology in both in vitro and in vivo systems that will facilitate the improved design of RV vaccines, better antiviral therapies and expression vectors.

  View details for DOI 10.1128/JVI.01294-20

  View details for PubMedID 32759316

• TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes. Science immunology Zang, R., Gomez Castro, M. F., McCune, B. T., Zeng, Q., Rothlauf, P. W., Sonnek, N. M., Liu, Z., Brulois, K. F., Wang, X., Greenberg, H. B., Diamond, M. S., Ciorba, M. A., Whelan, S. P., Ding, S. 2020; 5 (47)

  Abstract

  Gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA are frequently observed in COVID-19 patients. However, it is unclear whether SARS-CoV-2 replicates in the human intestine and contributes to possible fecal-oral transmission. Here, we report productive infection of SARS-CoV-2 in ACE2+ mature enterocytes in human small intestinal enteroids. Expression of two mucosa-specific serine proteases, TMPRSS2 and TMPRSS4, facilitated SARS-CoV-2 spike fusogenic activity and promoted virus entry into host cells. We also demonstrate that viruses released into the intestinal lumen were inactivated by simulated human colonic fluid, and infectious virus was not recovered from the stool specimens of COVID-19 patients. Our results highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression.

  View details for DOI 10.1126/sciimmunol.abc3582

  View details for PubMedID 32404436

• alphavbeta8 integrin-expression by BATF3-dependent dendritic cells facilitates early IgA responses to Rotavirus. Mucosal immunology Nakawesi, J., This, S., Hutter, J., Boucard-Jourdin, M., Barateau, V., Muleta, K. G., Gooday, L. J., Thomsen, K. F., Lopez, A. G., Ulmert, I., Poncet, D., Malissen, B., Greenberg, H., Thaunat, O., Defrance, T., Paidassi, H., Lahl, K. 2020

  Abstract

  Secretory intestinal IgA can protect from re-infection with rotavirus (RV), but very little is known about the mechanisms that induce IgA production during intestinal virus infections. Classical dendritic cells (cDCs) in the intestine can facilitate both T cell-dependent and -independent secretory IgA. Here, we show that BATF3-dependent cDC1, but not cDC2, are critical for the optimal induction of RV-specific IgA responses in the mesenteric lymph nodes. This depends on the selective expression of the TGFbeta-activating integrin alphavbeta8 by cDC1. In contrast, alphavbeta8 on cDC1 is dispensible for steady state immune homeostasis. Given that cDC2 are crucial in driving IgA during steady state but are dispensable for RV-specific IgA responses, we propose that the capacity of DC subsets to induce intestinal IgA responses reflects the context, as opposed to an intrinsic property of individual DC subsets.

  View details for DOI 10.1038/s41385-020-0276-8

  View details for PubMedID 32161355

• Reverse genetics reveals a role of the rotavirus VP3 phosphodiesterase activity in inhibiting RNase L signaling and contributing to intestinal viral replication in vivo. Journal of virology Song, Y., Feng, N., Sanchez-Tacuba, L., Yasukawa, L. L., Ren, L., Silverman, R. H., Ding, S., Greenberg, H. B. 2020

  Abstract

  Our understanding of how rotavirus (RV) subverts host innate immune signaling has greatly increased over the past decade. However, the relative contribution of each virus-encoded innate immune antagonist has not been fully studied in the context of RV infection in vivo Here, we present both in vitro and in vivo evidence that the host IFN-inducible 2'-5'-oligoadenylate synthetase (OAS) and ribonuclease L (RNase L) pathway effectively suppresses the replication of heterologous RV strains. VP3 from homologous RVs relies on its 2'-5'-phosphodiesterase (PDE) domain to counteract RNase L mediated antiviral signaling. Using a RV reverse genetics system, we show that compared to the parental strain, VP3 PDE mutant RVs replicated at a lower level in the small intestine and shed less in the feces of wild-type mice and such defects were rescued in Rnasel -/- suckling mice. Collectively, these findings highlight an important role of VP3 in promoting viral replication and pathogenesis in vivo in addition to its well characterized function as the viral RNA capping enzyme.ImportanceRotaviruses are significant human pathogens that result in diarrhea, dehydration, and deaths in many children around the world. Rotavirus vaccines have suboptimal efficacy in low to middle income countries, where the burden of the diseases is the most severe. With the ultimate goal to improve current vaccines, we aim to better understand how rotavirus interacts with the host innate immune system in the small intestine. Here, we demonstrate that the interferon-activated RNase L signaling blocks rotavirus replication in a strain-specific manner. In addition, virus encoded VP3 antagonizes RNase L activity both in vitro and in vivo These studies highlight an ever-evolving arms race between antiviral factors and viral pathogens and provide a new means of targeted attenuation for the next-generation rotavirus vaccine design.

  View details for DOI 10.1128/JVI.01952-19

  View details for PubMedID 32051268

• Rotavirus re-programs multiple IFN receptors and restricts their intestinal antiviral and inflammatory functions. Journal of virology Sen, A. n., Namsa, N. D., Feng, N. n., Greenberg, H. B. 2020

  Abstract

  Rotaviruses (RV) cause acute severe diarrhea in the absence of substantial intestinal inflammation. They are also highly infectious in their homologous host species. The replication capacity of RV in the small bowel is substantially due to its ability to inhibit different types of interferons (IFNs). Here, we find that during RV infection in vitro, both virus-infected and uninfected bystander cells resist STAT1 phosphorylation and IRF7 induction in response to exogenous IFN. Functionally, cellular transcription in response to stimulation with IFN, but not intracellular dsRNA, was inhibited by RV. Further, IFNAR1 stimulation during RV infection significantly repressed a set of virus-induced transcripts. Regulation of IFN signaling in vivo was studied in suckling mice using the highly infectious murine EW RV strain. Kinetic studies indicated that sustained EW RV replication and IFN induction in the small intestine is accompanied by significant decreases in IFN-stimulated transcripts. LPS-mediated intestinal damage, driven by STAT1-induced inflammation, was also prevented in EW RV-infected mice. Remarkably, by ectopically stimulating either IFNAR1 or IFNGR1 in EW RV-infected mice we could eliminate several intestinal antiviral and inflammatory transcriptional responses to RV. In contrast to homologous RV, infection with a STAT1-sensitive heterologous RV strain induced IFN-stimulated transcripts, inflammatory cytokines, and intestinal expression of STAT1-pY701. Finally, RV strain-specific STAT1 regulation also likely determines the intestinal activation of multiple caspases. The simian RRV strain, but not murine EW RV, uniquely triggers the cleavage of both extrinsic and intrinsic caspases (-8, -9, and -3) in a STAT1-mediated manner. Collectively, our findings reveal efficient re-programing of multiple IFN receptors towards a negative feedback mode of signaling, accompanied by suppression of IFN-mediated antiviral, apoptotic, and inflammatory functions, during natural RV intestinal infection.IMPORTANCE Rotavirus is a highly infectious pathogen that causes severe diarrhea. Replication of RV in the small intestine is restricted to homologous host species, and host range restriction is substantially determined by the interferon response. In this study, we demonstrate that during infection, RV bystander cells resist exogenous IFN-mediated STAT1 signaling and transcription. In a suckling mouse model, ectopically stimulating different intestinal interferon receptors during RV infection eliminates several innate and inflammatory antiviral responses. Different intestinal inflammatory cytokines were also suppressed by homologous RV, as was intestinal damage in response to endotoxin. The ability of RV to suppress IFN-mediated receptors likely impacts intestinal cell homeostasis as the cleavage of multiple intestinal caspases during RV infection is mediated by the IFN-STAT1 signaling pathway. Together, our results provide a mechanism underlying both the remarkable interferon resistance of homologous RV, and its ability to prevent substantial inflammatory damage to the small bowel.

  View details for DOI 10.1128/JVI.01775-19

  View details for PubMedID 31896593

• Retinoic Acid and Lymphotoxin Signaling Promote Differentiation of Human Intestinal M Cells. Gastroenterology Ding, S. n., Song, Y. n., Brulois, K. F., Pan, J. n., Co, J. Y., Ren, L. n., Feng, N. n., Yasukawa, L. L., Sánchez-Tacuba, L. n., Wosen, J. E., Mellins, E. D., Monack, D. M., Amieva, M. R., Kuo, C. J., Butcher, E. C., Greenberg, H. B. 2020

  Abstract

  Intestinal microfold (M) cells are a unique subset of intestinal epithelial cells in the Peyer's patches that regulate mucosal immunity, serving as portals for sampling and uptake of luminal antigens. The inability to efficiently develop human M cells in cell culture has impeded studies of the intestinal immune system. We aimed to identify signaling pathways required for differentiation of human M cells and establish a robust culture system using human ileum enteroids.We analyzed transcriptome data from mouse Peyer's Patches to identify cell populations in close proximity to M cells. We used the human enteroid system to determine which cytokines were required to induce M cell differentiation. We performed transcriptome, immunofluorescence, scanning electron microscope, and transcytosis experiments to validate the development of phenotypic and functional human M cells.A combination of retinoic acid and lymphotoxin induced differentiation of glycoprotein 2-positive human M cells, which lack apical microvilli structure. Upregulated expression of innate immune-related genes within M cells correlated with a lack of viral antigens after rotavirus infection. Human M cells, developed in the enteroid system, internalized and transported enteric viruses, such as rotavirus and reovirus, across the intestinal epithelium barrier in the enteroids.We identified signaling pathways required for differentiation of intestinal M cells, and used this information to create a robust culture method to develop human M cells with capacity for internalization and transport of viruses. Studies of this model might increase our understanding of antigen presentation and the systemic entry of enteric pathogens in the human intestine.

  View details for DOI 10.1053/j.gastro.2020.03.053

  View details for PubMedID 32247021

• Our New President-M. Bishr Omary, MD, PhD, AGAF. Gastroenterology Greenberg, H. B., Owyang, C. n., Williams, J. A., Landis, E. n., Rustgi, A. K. 2020; 158 (6): 1811–21

  View details for DOI 10.1053/j.gastro.2020.03.061

  View details for PubMedID 32364115

• The Role of Innate Immunity in Regulating Rotavirus Replication, Pathogenesis, and Host Range Restriction and the Implications for Live Rotaviral Vaccine Development MUCOSAL VACCINES greenberg, h. Elsevier. 2020; 2
• The Role of Innate Immunity in Regulating Rotavirus Replication, Pathogenesis, and Host Range Restriction and the Implications for Live Rotaviral Vaccine Development MUCOSAL VACCINES: INNOVATION FOR PREVENTING INFECTIOUS DISEASES, 2ND EDITION Sen, A., Ding, S., Greenberg, H. B., Kiyono, H., Pascual, D. W. 2020: 683–97

  View details for DOI 10.1016/B978-0-12-811924-2.00041-9

  View details for Web of Science ID 000538933600041

• Diminished B-Cell Response After Repeat Influenza Vaccination JOURNAL OF INFECTIOUS DISEASES Sanyal, M., Holmes, T. H., Maecker, H. T., Albrecht, R. A., Dekker, C. L., He, X., Greenberg, H. B. 2019; 219 (10): 1586–95

  View details for DOI 10.1093/infdis/jiy685

  View details for Web of Science ID 000473776700010

• Profiling of rotavirus 3UTR-binding proteins reveals the ATP synthase subunit ATP5B as a host factor that supports late-stage virus replication JOURNAL OF BIOLOGICAL CHEMISTRY Ren, L., Ding, S., Song, Y., Li, B., Ramanathan, M., Co, J., Amieva, M. R., Khavari, P. A., Greenberg, H. B. 2019; 294 (15): 5993–6006

  View details for DOI 10.1074/jbc.RA118.006004

  View details for Web of Science ID 000465077500030

• Influenza Virus Vaccination Elicits Poorly Adapted B Cell Responses in Elderly Individuals. Cell host & microbe Henry, C., Zheng, N., Huang, M., Cabanov, A., Rojas, K. T., Kaur, K., Andrews, S. F., Palm, A. E., Chen, Y., Li, Y., Hoskova, K., Utset, H. A., Vieira, M. C., Wrammert, J., Ahmed, R., Holden-Wiltse, J., Topham, D. J., Treanor, J. J., Ertl, H. C., Schmader, K. E., Cobey, S., Krammer, F., Hensley, S. E., Greenberg, H., He, X., Wilson, P. C. 2019

  Abstract

  Influenza is a leading cause of death in the elderly, and the vaccine protects only a fraction of this population. A key aspect of antibody-mediated anti-influenza virus immunity is adaptation to antigenically distinct epitopes on emerging strains. We examined factors contributing to reduced influenza vaccine efficacy in the elderly and uncovered a dramatic reduction in the accumulation of de novo immunoglobulin gene somatic mutations upon vaccination. This reduction is associated with a significant decrease in the capacity of antibodies to target the viral glycoprotein, hemagglutinin (HA), and critical protective epitopes surrounding the HA receptor-binding domain. Immune escape by antigenic drift, in which viruses generate mutations in key antigenic epitopes, becomes highly exaggerated. Because of this reduced adaptability, most Bcells activated in the elderly cohort target highly conserved but less potent epitopes. Given these findings, vaccines driving immunoglobulin gene somatic hypermutation should be a priority to protect elderly individuals.

  View details for PubMedID 30795982

• Human VP8* mAbs neutralize rotavirus selectively in human intestinal epithelial cells. The Journal of clinical investigation Feng, N., Hu, L., Ding, S., Sanyal, M., Zhao, B., Sankaran, B., Ramani, S., McNeal, M., Yasukawa, L. L., Song, Y., Prasad, B. V., Greenberg, H. B. 2019; 130

  Abstract

  We previously generated 32 rotavirus-specific (RV-specific) recombinant monoclonal antibodies (mAbs) derived from B cells isolated from human intestinal resections. Twenty-four of these mAbs were specific for the VP8* fragment of RV VP4, and most (20 of 24) were non-neutralizing when tested in the conventional MA104 cell-based assay. We reexamined the ability of these mAbs to neutralize RVs in human intestinal epithelial cells including ileal enteroids and HT-29 cells. Most (18 of 20) of the "non-neutralizing" VP8* mAbs efficiently neutralized human RV in HT-29 cells or enteroids. Serum RV neutralization titers in adults and infants were significantly higher in HT-29 than MA104 cells and adsorption of these sera with recombinant VP8* lowered the neutralization titers in HT-29 but not MA104 cells. VP8* mAbs also protected suckling mice from diarrhea in an in vivo challenge model. X-ray crystallographic analysis of one VP8* mAb (mAb9) in complex with human RV VP8* revealed that the mAb interaction site was distinct from the human histo-blood group antigen binding site. Since MA104 cells are the most commonly used cell line to detect anti-RV neutralization activity, these findings suggest that prior vaccine and other studies of human RV neutralization responses may have underestimated the contribution of VP8* antibodies to the overall neutralization titer.

  View details for DOI 10.1172/JCI128382

  View details for PubMedID 31403468

• Enterovirus pathogenesis requires the host methyltransferase SETD3. Nature microbiology Diep, J. n., Ooi, Y. S., Wilkinson, A. W., Peters, C. E., Foy, E. n., Johnson, J. R., Zengel, J. n., Ding, S. n., Weng, K. F., Laufman, O. n., Jang, G. n., Xu, J. n., Young, T. n., Verschueren, E. n., Kobluk, K. J., Elias, J. E., Sarnow, P. n., Greenberg, H. B., Hüttenhain, R. n., Nagamine, C. M., Andino, R. n., Krogan, N. J., Gozani, O. n., Carette, J. E. 2019

  Abstract

  Enteroviruses (EVs) comprise a large genus of positive-sense, single-stranded RNA viruses whose members cause a number of important and widespread human diseases, including poliomyelitis, myocarditis, acute flaccid myelitis and the common cold. How EVs co-opt cellular functions to promote replication and spread is incompletely understood. Here, using genome-scale CRISPR screens, we identify the actin histidine methyltransferase SET domain containing 3 (SETD3) as critically important for viral infection by a broad panel of EVs, including rhinoviruses and non-polio EVs increasingly linked to severe neurological disease such as acute flaccid myelitis (EV-D68) and viral encephalitis (EV-A71). We show that cytosolic SETD3, independent of its methylation activity, is required for the RNA replication step in the viral life cycle. Using quantitative affinity purification-mass spectrometry, we show that SETD3 specifically interacts with the viral 2A protease of multiple enteroviral species, and we map the residues in 2A that mediate this interaction. 2A mutants that retain protease activity but are unable to interact with SETD3 are severely compromised in RNA replication. These data suggest a role of the viral 2A protein in RNA replication beyond facilitating proteolytic cleavage. Finally, we show that SETD3 is essential for in vivo replication and pathogenesis in multiple mouse models for EV infection, including CV-A10, EV-A71 and EV-D68. Our results reveal a crucial role of a host protein in viral pathogenesis, and suggest targeting SETD3 as a potential mechanism for controlling viral infections.

  View details for DOI 10.1038/s41564-019-0551-1

  View details for PubMedID 31527793

• Diminished B-cell response after repeat influenza vaccination. The Journal of infectious diseases Sanyal, M., Holmes, T. H., Maecker, H., Albrecht, R. A., Dekker, C. L., He, X., Greenberg, H. B. 2018

  Abstract

  Annual vaccination with influenza vaccines is recommended for protection against influenza in the United States. Past clinical studies and meta-analysis, however, have reported conflicting results on the benefits of annual vaccination. B-cell responses elicited following repeat influenza vaccinations over multiple seasons have not been examined in detail. We analyzed the B-cell and antibody responses in volunteers vaccinated yearly with seasonal trivalent inactivated influenza vaccines (TIV) from 2010 or 2011 to 2014. Statistical analyses were designed to help correct for possible bias due to reduced sample size in the later years of the study. We show that after the second annual vaccination the frequency of vaccine-specific plasmablasts and the binding reactivity of plasmablast-derived polyclonal antibodies (PPAb) are reduced and do not increase in subsequent years. Similar trends are observed with the serum hemagglutination inhibition antibody response after each annual vaccination, as well as the binding reactivity of PPAb for the hemagglutinin of influenza A vaccine components, even with changes in the seasonal vaccine components during the study. Our findings indicate a diminished B-cell response to annually repeated TIV vaccination. These results emphasize the need of developing improved strategies to enhance the immunogenicity and efficacy of annual influenza vaccination.

  View details for PubMedID 30496437

• Rotavirus VP3 targets MAVS for degradation to inhibit type III interferon expression in intestinal epithelial cells. eLife Ding, S., Zhu, S., Ren, L., Feng, N., Song, Y., Ge, X., Li, B., Flavell, R. A., Greenberg, H. B. 2018; 7

  Abstract

  Rotaviruses (RVs), a leading cause of severe diarrhea in young children1 and many mammalian species, have evolved multiple strategies to counteract the host innate immunity, specifically interferon (IFN) signaling through RV non-structural protein 1 (NSP1)2. However, whether RV structural components also subvert antiviral response remains under-studied. Here, we found that MAVS, critical for the host RNA sensing pathway upstream of IFN induction3, is degraded by the RV RNA methyl- and guanylyl-transferase (VP3) in a host-range-restricted manner. Mechanistically, VP3 localizes to the mitochondria and mediates the phosphorylation of a previously unidentified SPLTSS motif within the MAVS proline-rich region, leading to its proteasomal degradation and blockade of IFN-lambda production in RV-infected intestinal epithelial cells. Importantly, VP3 inhibition of MAVS activity contributes to enhanced RV replication and to viral pathogenesis in vivo. Collectively, our findings establish RV VP3 as a viral antagonist of MAVS function in mammals and uncover a novel pathogen-mediated inhibitory mechanism of MAVS signaling.

  View details for PubMedID 30460894

• New mitochondrial DNA synthesis enables NLRP3 inflammasome activation NATURE Zhong, Z., Liang, S., Sanchez-Lopez, E., He, F., Shalapour, S., Lin, X., Wong, J., Ding, S., Seki, E., Schnabl, B., Hevener, A. L., Greenberg, H. B., Kisseleva, T., Karin, M. 2018; 560 (7717): 198-+

  Abstract

  Dysregulated NLRP3 inflammasome activity results in uncontrolled inflammation, which underlies many chronic diseases. Although mitochondrial damage is needed for the assembly and activation of the NLRP3 inflammasome, it is unclear how macrophages are able to respond to structurally diverse inflammasome-activating stimuli. Here we show that the synthesis of mitochondrial DNA (mtDNA), induced after the engagement of Toll-like receptors, is crucial for NLRP3 signalling. Toll-like receptors signal via the MyD88 and TRIF adaptors to trigger IRF1-dependent transcription of CMPK2, a rate-limiting enzyme that supplies deoxyribonucleotides for mtDNA synthesis. CMPK2-dependent mtDNA synthesis is necessary for the production of oxidized mtDNA fragments after exposure to NLRP3 activators. Cytosolic oxidized mtDNA associates with the NLRP3 inflammasome complex and is required for its activation. The dependence on CMPK2 catalytic activity provides opportunities for more effective control of NLRP3 inflammasome-associated diseases.

  View details for PubMedID 30046112

• Identification and Characterization at the Single-Cell Level of Cytokine-Producing Circulating Cells in Children With Dengue JOURNAL OF INFECTIOUS DISEASES Perdomo-Celis, F., Romero, F., Salgado, D. M., Vega, R., Rodriguez, J., Angel, J., Franco, M. A., Greenberg, H. B., Narvaez, C. F. 2018; 217 (9): 1472–80

  Abstract

  In this study, we identified, at the single-cell level, naturally induced cytokine-producing circulating cells (CPCCs) in children with dengue virus (DENV) infection ranging clinically from mild to severe disease. Tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) CPCCs were detected in children with primary or secondary acute dengue virus (DENV) infection, and the pattern of these cytokines was similar to that seen in the supernatant of cultured peripheral blood mononuclear cells and partially comparable to that found in plasma. Monocytes, B cells, and myeloid dendritic cells (mDCs) were the primary CPCCs detected, and the frequency of mDCs was significantly higher in severe disease. B cells isolated from children with dengue spontaneously secreted TNF-α, IL-6, and interleukin 10, and supernatants from cultures of purified B cells induced activation of allogeneic T cells, supporting an antibody-independent function of these cells during DENV infection. Thus, CPCCs could be a new immune parameter with potential use to evaluate pathogenesis in this infection.

  View details for PubMedID 29390091

• STAG2 deficiency induces interferon responses via cGAS-STING pathway and restricts virus infection. Nature communications Ding, S., Diep, J., Feng, N., Ren, L., Li, B., Ooi, Y. S., Wang, X., Brulois, K. F., Yasukawa, L. L., Li, X., Kuo, C. J., Solomon, D. A., Carette, J. E., Greenberg, H. B. 2018; 9 (1): 1485

  Abstract

  Cohesin is a multi-subunit nuclear protein complex that coordinates sister chromatid separation during cell division. Highly frequent somatic mutations in genes encoding core cohesin subunits have been reported in multiple cancer types. Here, using a genome-wide CRISPR-Cas9 screening approach to identify host dependency factors and novel innate immune regulators of rotavirus (RV) infection, we demonstrate that the loss of STAG2, an important component of the cohesin complex, confers resistance to RV replication in cell culture and human intestinal enteroids. Mechanistically, STAG2 deficiency results in spontaneous genomic DNA damage and robust interferon (IFN) expression via the cGAS-STING cytosolic DNA-sensing pathway. The resultant activation of JAK-STAT signaling and IFN-stimulated gene (ISG) expression broadly protects against virus infections, including RVs. Our work highlights a previously undocumented role of the cohesin complex in regulating IFN homeostasis and identifies new therapeutic avenues for manipulating the innate immunity.

  View details for PubMedID 29662124

• Editorial overview: Viral pathogenesis: New technologies to advance research in human viral pathogenesis CURRENT OPINION IN VIROLOGY Greenberg, H. B., Griffin, D. E. 2018; 29: IV-VI

  View details for PubMedID 29735182

• Rotavirus Degrades Multiple Interferon (IFN) Type Receptors To Inhibit IFN Signaling and Protects against Mortality from Endotoxin in Suckling Mice JOURNAL OF VIROLOGY Sen, A., Sharma, A., Greenberg, H. B. 2018; 92 (1)

  View details for DOI 10.1128/JVI.01394-17

  View details for Web of Science ID 000417922700018

• Rotavirus infection NATURE REVIEWS DISEASE PRIMERS Crawford, S. E., Ramani, S., Tate, J. E., Parashar, U. D., Svensson, L., Hagbom, M., Franco, M. A., Greenberg, H. B., O'Ryan, M., Kang, G., Desselberger, U., Estes, M. K. 2017; 3: 17083

  Abstract

  Rotavirus infections are a leading cause of severe, dehydrating gastroenteritis in children <5 years of age. Despite the global introduction of vaccinations for rotavirus over a decade ago, rotavirus infections still result in >200,000 deaths annually, mostly in low-income countries. Rotavirus primarily infects enterocytes and induces diarrhoea through the destruction of absorptive enterocytes (leading to malabsorption), intestinal secretion stimulated by rotavirus non-structural protein 4 and activation of the enteric nervous system. In addition, rotavirus infections can lead to antigenaemia (which is associated with more severe manifestations of acute gastroenteritis) and viraemia, and rotavirus can replicate in systemic sites, although this is limited. Reinfections with rotavirus are common throughout life, although the disease severity is reduced with repeat infections. The immune correlates of protection against rotavirus reinfection and recovery from infection are poorly understood, although rotavirus-specific immunoglobulin A has a role in both aspects. The management of rotavirus infection focuses on the prevention and treatment of dehydration, although the use of antiviral and anti-emetic drugs can be indicated in some cases.

  View details for PubMedID 29119972

• Rotavirus degrades multiple type interferon receptors to inhibit IFN signaling and protects against mortality from endotoxin in suckling mice. Journal of virology Sen, A., Sharma, A., Greenberg, H. B. 2017

  Abstract

  STAT1 phosphorylation in response to exogenous IFN administration can be inhibited by rotaviral replication both in vitro and in vivo. In addition many rotavirus strains are resistant to the actions of different IFN types . The regulation of multiple IFN type-mediated antiviral pathways by RVs is not well understood. In this study, we find that during infection in vitro and in vivo RV significantly depletes IFN-type I, II, and III receptors (IFNRs). Regulation of IFNRs occurred exclusively within RV-infected cells and could be abrogated by inhibiting the lysosomal-endosomal degradation pathway. In vitro, IFNR degradation was conserved across multiple RV strains that differ in their modes of regulating IFN induction. In suckling mice, exogenously administered types I, II, or III IFN each induced phosphorylation of STAT1-Y701 within intestinal epithelial cells (IECs) of suckling mice. Murine EW strain RV infection transiently activated intestinal STAT1 at 1dpi, but not subsequently at 2-3dpi. In response to injection of purified IFN-α/β or -λ, IECs in EW-infected mice exhibited impaired STAT1-Y701 phosphorylation, correlating with depletion of different intestinal IFNRs and impaired IFN-mediated transcription. The ability of EW murine RV to inhibit multiple IFN types led us to test protection of suckling mice from endotoxin-mediated shock, an outcome that is dependent on the host IFN response. Compared to controls, mice infected with EW murine RV were substantially protected against mortality following parenteral endotoxin administration. These studies identify a novel mechanism of IFN subversion by RV and reveal an unexpected protective effect of RV infection on endotoxin-mediated shock in suckling mice.IMPORTANCE Antiviral functions of types 1, 2, and 3 IFN are mediated by receptor-dependent activation of STAT1. Here we find that RV degrades the types 1, 2, and 3 IFN receptors (IFNR) in vitro In a suckling mouse model, RV effectively blocked STAT1 activation and transcription following injection of different purified IFNs. This correlated with significantly decreased protein expression of intestinal types 1 and 2 IFNRs. Recent studies demonstrate that in mice, LPS-induced lethality is prevented by genetic ablation of IFN signaling genes such as IFNAR1 and STAT1. When suckling mice were infected with RV they were substantially protected from lethal exposure to endotoxin. These findings provide novel insights into the mechanisms underlying rotavirus regulation of different interferons and are likely to stimulate new research into both rotavirus pathogenesis and endotoxemia.

  View details for DOI 10.1128/JVI.01394-17

  View details for PubMedID 29070687

  View details for PubMedCentralID PMC5730789

• Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells NATURE Zhu, S., Ding, S., Wang, P., Wei, Z., Pan, W., Palm, N. W., Yang, Y., Yu, H., Li, H., Wang, G., Lei, X., de Zoete, M. R., Zhao, J., Zheng, Y., Chen, H., Zhao, Y., Jurado, K. A., Feng, N., Shan, L., Kluger, Y., Lu, J., Abraham, C., Fikrig, E., Greenberg, H. B., Flavell, R. A. 2017; 546 (7660): 667-+

  Abstract

  Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens.

  View details for PubMedID 28636595

  View details for PubMedCentralID PMC5787375

• VP4-and VP7-specific antibodies mediate heterotypic immunity to rotavirus in humans SCIENCE TRANSLATIONAL MEDICINE Nair, N., Feng, N., Blum, L. K., Sanyal, M., Ding, S., Jiang, B., Sen, A., Morton, J. M., He, X., Robinson, W. H., Greenberg, H. B. 2017; 9 (395)

  Abstract

  Human rotaviruses (RVs) are the leading cause of severe diarrhea in young children worldwide. The molecular mechanisms underlying the rapid induction of heterotypic protective immunity to RV, which provides the basis for the efficacy of licensed monovalent RV vaccines, have remained unknown for more than 30 years. We used RV-specific single cell-sorted intestinal B cells from human adults, barcode-based deep sequencing of antibody repertoires, monoclonal antibody expression, and serologic and functional characterization to demonstrate that infection-induced heterotypic immunoglobulins (Igs) primarily directed to VP5*, the stalk region of the RV attachment protein, VP4, are able to mediate heterotypic protective immunity. Heterotypic protective Igs against VP7, the capsid glycoprotein, and VP8*, the cell-binding region of VP4, are also generated after infection; however, our data suggest that homotypic anti-VP7 and non-neutralizing VP8* responses occur more commonly in people. These results indicate that humans can circumvent the extensive serotypic diversity of circulating RV strains by generating frequent heterotypic neutralizing antibody responses to VP7, VP8*, and most often, to VP5* after natural infection. These findings further suggest that recombinant VP5* may represent a useful target for the development of an improved, third-generation, broadly effective RV vaccine and warrants more direct examination.

  View details for PubMedID 28637924

• Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Li, B., Ding, S., Feng, N., Mooney, N., Ooi, Y. S., Ren, L., Diep, J., Kelly, M. R., Yasukawa, L. L., Patton, J. T., Yamazaki, H., Shirao, T., Jackson, P. K., Greenberg, H. B. 2017; 114 (18): E3642-E3651

  Abstract

  Despite the wide administration of several effective vaccines, rotavirus (RV) remains the single most important etiological agent of severe diarrhea in infants and young children worldwide, with an annual mortality of over 200,000 people. RV attachment and internalization into target cells is mediated by its outer capsid protein VP4. To better understand the molecular details of RV entry, we performed tandem affinity purification coupled with high-resolution mass spectrometry to map the host proteins that interact with VP4. We identified an actin-binding protein, drebrin (DBN1), that coprecipitates and colocalizes with VP4 during RV infection. Importantly, blocking DBN1 function by siRNA silencing, CRISPR knockout (KO), or chemical inhibition significantly increased host cell susceptibility to RV infection. Dbn1 KO mice exhibited higher incidence of diarrhea and more viral antigen shedding in their stool samples compared with the wild-type littermates. In addition, we found that uptake of other dynamin-dependent cargos, including transferrin, cholera toxin, and multiple viruses, was also enhanced in DBN1-deficient cells. Inhibition of cortactin or dynamin-2 abrogated the increased virus entry observed in DBN1-deficient cells, suggesting that DBN1 suppresses dynamin-mediated endocytosis via interaction with cortactin. Our study unveiled an unexpected role of DBN1 in restricting the entry of RV and other viruses into host cells and more broadly to function as a crucial negative regulator of diverse dynamin-dependent endocytic pathways.

  View details for DOI 10.1073/pnas.1619266114

  View details for Web of Science ID 000400358000009

  View details for PubMedID 28416666

  View details for PubMedCentralID PMC5422808

• Trafficking receptor signatures define blood plasmablasts responding to tissue-specific immune challenge. JCI insight Seong, Y., Lazarus, N. H., Sutherland, L., Habtezion, A., Abramson, T., He, X., Greenberg, H. B., Butcher, E. C. 2017; 2 (6)

  Abstract

  Antibody-secreting cells are generated in regional lymphoid tissues and traffic as plasmablasts (PBs) via lymph and blood to target sites for local immunity. We used multiparameter flow cytometry to define PB trafficking programs (TPs, combinations of adhesion molecules and chemoattractant receptors) and their imprinting in patients in response to localized infection or immune insults. TPs enriched after infection or autoimmune inflammation of mucosae correlate with sites of immune response or symptoms, with different TPs imprinted during small intestinal, colon, throat, and upper respiratory immune challenge. PBs induced after intramuscular or intradermal influenza vaccination, including flu-specific antibody-secreting cells, display TPs characterized by the lack of mucosal homing receptors. PBs of healthy donors display diverse mucosa-associated TPs, consistent with homeostatic immune activity. Identification of TP signatures of PBs may facilitate noninvasive monitoring of organ-specific immune responses.

  View details for DOI 10.1172/jci.insight.90233

  View details for PubMedID 28352656

• DDX6 Represses Aberrant Activation of Interferon-Stimulated Genes. Cell reports Lumb, J. H., Li, Q. n., Popov, L. M., Ding, S. n., Keith, M. T., Merrill, B. D., Greenberg, H. B., Li, J. B., Carette, J. E. 2017; 20 (4): 819–31

  Abstract

  The innate immune system tightly regulates activation of interferon-stimulated genes (ISGs) to avoid inappropriate expression. Pathological ISG activation resulting from aberrant nucleic acid metabolism has been implicated in autoimmune disease; however, the mechanisms governing ISG suppression are unknown. Through a genome-wide genetic screen, we identified DEAD-box helicase 6 (DDX6) as a suppressor of ISGs. Genetic ablation of DDX6 induced global upregulation of ISGs and other immune genes. ISG upregulation proved cell intrinsic, imposing an antiviral state and making cells refractory to divergent families of RNA viruses. Epistatic analysis revealed that ISG activation could not be overcome by deletion of canonical RNA sensors. However, DDX6 deficiency was suppressed by disrupting LSM1, a core component of mRNA degradation machinery, suggesting that dysregulation of RNA processing underlies ISG activation in the DDX6 mutant. DDX6 is distinct among DExD/H helicases that regulate the antiviral response in its singular ability to negatively regulate immunity.

  View details for PubMedID 28746868

  View details for PubMedCentralID PMC5551412

• Comparative Proteomics Reveals Strain-Specific ß-TrCP Degradation via Rotavirus NSP1 Hijacking a Host Cullin-3-Rbx1 Complex. PLoS pathogens Ding, S., Mooney, N., Li, B., Kelly, M. R., Feng, N., Loktev, A. V., Sen, A., Patton, J. T., Jackson, P. K., Greenberg, H. B. 2016; 12 (10)

  Abstract

  Rotaviruses (RVs) are the leading cause of severe gastroenteritis in young children, accounting for half a million deaths annually worldwide. RV encodes non-structural protein 1 (NSP1), a well-characterized interferon (IFN) antagonist, which facilitates virus replication by mediating the degradation of host antiviral factors including IRF3 and β-TrCP. Here, we utilized six human and animal RV NSP1s as baits and performed tandem-affinity purification coupled with high-resolution mass spectrometry to comprehensively characterize NSP1-host protein interaction network. Multiple Cullin-RING ubiquitin ligase (CRL) complexes were identified. Importantly, inhibition of cullin-3 (Cul3) or RING-box protein 1 (Rbx1), by siRNA silencing or chemical perturbation, significantly impairs strain-specific NSP1-mediated β-TrCP degradation. Mechanistically, we demonstrate that NSP1 localizes to the Golgi with the host Cul3-Rbx1 CRL complex, which targets β-TrCP and NSP1 for co-destruction at the proteasome. Our study uncovers a novel mechanism that RV employs to promote β-TrCP turnover and provides molecular insights into virus-mediated innate immunity inhibition.

  View details for DOI 10.1371/journal.ppat.1005929

  View details for PubMedID 27706223

  View details for PubMedCentralID PMC5051689

• Total and Envelope Protein-Specific Antibody-Secreting Cell Response in Pediatric Dengue Is Highly Modulated by Age and Subsequent Infections PLOS ONE Toro, J. F., Salgado, D. M., Vega, R., Rodriguez, J. A., Rodriguez, L., Angel, J., Franco, M. A., Greenberg, H. B., Naravez, C. F. 2016; 11 (8)

  View details for DOI 10.1371/journal.pone.0161795

  View details for Web of Science ID 000382258600102

  View details for PubMedID 27560782

• Distinct Roles of Type I and Type III Interferons in Intestinal Immunity to Homologous and Heterologous Rotavirus Infections PLOS PATHOGENS Lin, J., Feng, N., Sen, A., Balan, M., Tseng, H., McElrath, C., Smirnov, S. V., Peng, J., Yasukawa, L. L., Durbin, R. K., Durbin, J. E., Greenberg, H. B., Kotenko, S. V. 2016; 12 (4)

  Abstract

  Type I (IFN-α/β) and type III (IFN-λ) interferons (IFNs) exert shared antiviral activities through distinct receptors. However, their relative importance for antiviral protection of different organ systems against specific viruses remains to be fully explored. We used mouse strains deficient in type-specific IFN signaling, STAT1 and Rag2 to dissect distinct and overlapping contributions of type I and type III IFNs to protection against homologous murine (EW-RV strain) and heterologous (non-murine) simian (RRV strain) rotavirus infections in suckling mice. Experiments demonstrated that murine EW-RV is insensitive to the action of both types of IFNs, and that timely viral clearance depends upon adaptive immune responses. In contrast, both type I and type III IFNs can control replication of the heterologous simian RRV in the gastrointestinal (GI) tract, and they cooperate to limit extra-intestinal simian RRV replication. Surprisingly, intestinal epithelial cells were sensitive to both IFN types in neonatal mice, although their responsiveness to type I, but not type III IFNs, diminished in adult mice, revealing an unexpected age-dependent change in specific contribution of type I versus type III IFNs to antiviral defenses in the GI tract. Transcriptional analysis revealed that intestinal antiviral responses to RV are triggered through either type of IFN receptor, and are greatly diminished when receptors for both IFN types are lacking. These results also demonstrate a murine host-specific resistance to IFN-mediated antiviral effects by murine EW-RV, but the retention of host efficacy through the cooperative action by type I and type III IFNs in restricting heterologous simian RRV growth and systemic replication in suckling mice. Collectively, our findings revealed a well-orchestrated spatial and temporal tuning of innate antiviral responses in the intestinal tract where two types of IFNs through distinct patterns of their expression and distinct but overlapping sets of target cells coordinately regulate antiviral defenses against heterologous or homologous rotaviruses with substantially different effectiveness.

  View details for DOI 10.1371/journal.ppat.1005600

  View details for Web of Science ID 000378156900066

  View details for PubMedID 27128797

  View details for PubMedCentralID PMC4851417

• High-dimensional immune profiling of total and rotavirus VP6-specific intestinal and circulating B cells by mass cytometry MUCOSAL IMMUNOLOGY Nair, N., Newell, E. W., Vollmers, C., Quake, S. R., Morton, J. M., DAVIS, M. M., He, X. S., Greenberg, H. B. 2016; 9 (1): 68-82

  Abstract

  In-depth phenotyping of human intestinal antibody secreting cells (ASCs) and their precursors is important for developing improved mucosal vaccines. We used single-cell mass cytometry to simultaneously analyze 34 differentiation and trafficking markers on intestinal and circulating B cells. In addition, we labeled rotavirus (RV) double-layered particles with a metal isotope and characterized B cells specific to the RV VP6 major structural protein. We describe the heterogeneity of the intestinal B-cell compartment, dominated by ASCs with some phenotypic and transcriptional characteristics of long-lived plasma cells. Using principal component analysis, we visualized the phenotypic relationships between major B-cell subsets in the intestine and blood, and revealed that IgM(+) memory B cells (MBCs) and naive B cells were phenotypically related as were CD27(-) MBCs and switched MBCs. ASCs in the intestine and blood were highly clonally related, but associated with distinct trajectories of phenotypic development. VP6-specific B cells were present among diverse B-cell subsets in immune donors, including naive B cells, with phenotypes representative of the overall B-cell pool. These data provide a high dimensional view of intestinal B cells and the determinants regulating humoral memory to a ubiquitous, mucosal pathogen at steady-state.Mucosal Immunology advance online publication, 22 April 2015; doi:10.1038/mi.2015.36.

  View details for DOI 10.1038/mi.2015.36

  View details for Web of Science ID 000367653800006

• Total and Envelope Protein-Specific Antibody-Secreting Cell Response in Pediatric Dengue Is Highly Modulated by Age and Subsequent Infections. PloS one Toro, J. F., Salgado, D. M., Vega, R., Rodríguez, J. A., Rodríguez, L., Angel, J., Franco, M. A., Greenberg, H. B., Narváez, C. F. 2016; 11 (8)

  Abstract

  The response of antibody-secreting cells (ASC) induced by dengue has only recently started to be characterized. We propose that young age and previous infections could be simple factors that affect this response. Here, we evaluated the primary and secondary responses of circulating ASC in infants (6-12 months old) and children (1-14 years old) infected with dengue showing different degrees of clinical severity. The ASC response was delayed and of lower magnitude in infants, compared with older children. In primary infection (PI), the total and envelope (E) protein-specific IgM ASC were dominant in infants but not in children, and a negative correlation was found between age and the number of IgM ASC (rho = -0.59, P = 0.03). However, infants with plasma dengue-specific IgG detectable in the acute phase developed an intense ASC response largely dominated by IgG and comparable to that of children with secondary infection (SI). IgM and IgG produced by ASC circulating in PI or SI were highly cross-reactive among the four serotypes. Dengue infection caused the disturbance of B cell subsets, particularly a decrease in the relative frequency of naïve B cells. Higher frequencies of total and E protein-specific IgM ASC in the infants and IgG in the children were associated with clinically severe forms of infection. Therefore, the ASC response induced by dengue is highly influenced by the age at which infection occurs and previous immune status, and its magnitude is a relevant element in the clinical outcome. These results are important in the search for correlates of protection and for determining the ideal age for vaccinating against dengue.

  View details for DOI 10.1371/journal.pone.0161795

  View details for PubMedID 27560782

  View details for PubMedCentralID PMC4999220

• High-dimensional immune profiling of total and rotavirus VP6-specific intestinal and circulating B cells by mass cytometry. Mucosal immunology Nair, N., Newell, E. W., Vollmers, C., Quake, S. R., Morton, J. M., Davis, M. M., He, X. S., Greenberg, H. B. 2015

  Abstract

  In-depth phenotyping of human intestinal antibody secreting cells (ASCs) and their precursors is important for developing improved mucosal vaccines. We used single-cell mass cytometry to simultaneously analyze 34 differentiation and trafficking markers on intestinal and circulating B cells. In addition, we labeled rotavirus (RV) double-layered particles with a metal isotope and characterized B cells specific to the RV VP6 major structural protein. We describe the heterogeneity of the intestinal B-cell compartment, dominated by ASCs with some phenotypic and transcriptional characteristics of long-lived plasma cells. Using principal component analysis, we visualized the phenotypic relationships between major B-cell subsets in the intestine and blood, and revealed that IgM(+) memory B cells (MBCs) and naive B cells were phenotypically related as were CD27(-) MBCs and switched MBCs. ASCs in the intestine and blood were highly clonally related, but associated with distinct trajectories of phenotypic development. VP6-specific B cells were present among diverse B-cell subsets in immune donors, including naive B cells, with phenotypes representative of the overall B-cell pool. These data provide a high dimensional view of intestinal B cells and the determinants regulating humoral memory to a ubiquitous, mucosal pathogen at steady-state.Mucosal Immunology advance online publication, 22 April 2015; doi:10.1038/mi.2015.36.

  View details for DOI 10.1038/mi.2015.36

  View details for PubMedID 25899688

• Distinct patterns of B-cell activation and priming by natural influenza virus infection versus inactivated influenza vaccination. journal of infectious diseases He, X., Holmes, T. H., Sanyal, M., Albrecht, R. A., García-Sastre, A., Dekker, C. L., Davis, M. M., Greenberg, H. B. 2015; 211 (7): 1051-1059

  Abstract

  The human B-cell response to natural influenza virus infection has not been extensively investigated at the polyclonal level.The overall B-cell response of patients acutely infected with the 2009 pandemic influenza A(H1N1)pdm09 virus (A[H1N1]pdm09) was analyzed by determining the reactivity of plasmablast-derived polyclonal antibodies (PPAbs) to influenza proteins. Recipients of inactivated influenza vaccine containing the same A(H1N1)pdm09 strain were studied for comparison.During acute infection, robust plasmablast responses to the infecting virus were detected, characterized by a greater PPAb reactivity to the conserved influenza virus nuclear protein and to heterovariant and heterosubtypic hemagglutinins, in comparison to responses to the inactivated A(H1N1)pdm09 vaccine. In A(H1N1)pdm09 vaccinees, the presence of baseline serum neutralizing antibodies against A(H1N1)pdm09, suggesting previous exposure to natural A(H1N1)pdm09 infection, did not affect the plasmablast response to vaccination, whereas repeated immunization with inactivated A(H1N1)pdm09 vaccine resulted in significantly reduced vaccine-specific and cross-reactive PPAb responses.Natural A(H1N1)pdm09 infection and inactivated A(H1N1)pdm09 vaccination result in very distinct patterns of B-cell activation and priming. These differences are likely to be associated with differences in protective immunity, especially cross-protection against heterovariant and heterosubtypic influenza virus strains.

  View details for DOI 10.1093/infdis/jiu580

  View details for PubMedID 25336731

  View details for PubMedCentralID PMC4366605

• Distinct Cross-reactive B-Cell Responses to Live Attenuated and Inactivated Influenza Vaccines. journal of infectious diseases Sasaki, S., Holmes, T. H., Albrecht, R. A., García-Sastre, A., Dekker, C. L., He, X., Greenberg, H. B. 2014; 210 (6): 865-874

  Abstract

  Background. The immunological bases for the efficacies of the two currently licensed influenza vaccines, the live attenuated influenza vaccine (LAIV) and inactivated influenza vaccine (IIV), are not fully understood. The goal of this study was to identify specific B-cell responses correlated with the known efficacies of these two vaccines.Methods. We compared the B-cell and antibody responses after immunization with 2010/2011 IIV versus LAIV in young adults, focusing on peripheral plasmablasts at days 6-8 post-vaccination.Results. The quantities of vaccine-specific plasmablasts and plasmablast-derived polyclonal antibodies (PPAb) were significantly higher in IIV recipients than in LAIV recipients. No significant difference was detected in the avidity of vaccine-specific PPAb between the two vaccine groups. Proportionally, LAIV induced a greater vaccine-specific IgA plasmablast response as well as a greater plasmablast response to the conserved influenza nuclear protein than did IIV. The cross-reactive plasmablast response to heterovariant strains, as indicated by the relative levels of cross-reactive plasmablasts and the cross-reactive PPAb binding reactivity, was also greater in the LAIV group.Conclusions. Distinct quantitative and qualitative patterns of plasmablast responses were induced by LAIV and IIV in young adults; a proportionally greater cross-reactive response was induced by LAIV.

  View details for DOI 10.1093/infdis/jiu190

  View details for PubMedID 24676204

• Efficacy of a monovalent human-bovine (116E) rotavirus vaccine in Indian children in the second year of life. Vaccine Bhandari, N., Rongsen-Chandola, T., Bavdekar, A., John, J., Antony, K., Taneja, S., Goyal, N., Kawade, A., Kang, G., Rathore, S. S., Juvekar, S., Muliyil, J., Arya, A., Shaikh, H., Abraham, V., Vrati, S., Proschan, M., Kohberger, R., Thiry, G., Glass, R., Greenberg, H. B., Curlin, G., Mohan, K., Harshavardhan, G. V., Prasad, S., Rao, T. S., Boslego, J., Bhan, M. K. 2014; 32: A110-6

  Abstract

  Rotavirus gastroenteritis is one of the leading causes of diarrhea in Indian children less than 2 years of age. The 116E rotavirus strain was developed as part of the Indo-US Vaccine Action Program and has undergone efficacy trials. This paper reports the efficacy and additional safety data in children up to 2 years of age. In a double-blind placebo controlled multicenter trial, 6799 infants aged 6-7 weeks were randomized to receive three doses of an oral human-bovine natural reassortant vaccine (116E) or placebo at ages 6, 10, and 14 weeks. The primary outcome was severe (≥11 on the Vesikari scale) rotavirus gastroenteritis. Efficacy outcomes and adverse events were ascertained through active surveillance. We randomly assigned 4532 and 2267 subjects to receive vaccine and placebo, respectively, with over 96% subjects receiving all three doses of the vaccine or placebo. The per protocol analyses included 4354 subjects in the vaccine and 2187 subjects in the placebo group. The overall incidence of severe RVGE per 100 person years was 1.3 in the vaccine group and 2.9 in the placebo recipients. Vaccine efficacy against severe rotavirus gastroenteritis in children up to 2 years of age was 55.1% (95% CI 39.9 to 66.4; p<0.0001); vaccine efficacy in the second year of life of 48.9% (95% CI 17.4 to 68.4; p=0.0056) was only marginally less than in the first year of life [56.3% (95% CI 36.7 to 69.9; p<0.0001)]. The number of infants needed to be immunized to prevent one episode of severe RVGE in the first 2 years of life was 40 (95% CI 28.0 to 63.0) and for RVGE of any severity, it was 21 (95% CI 16.0 to 32.0). Serious adverse events were observed at the same rates in the two groups. None of the eight intussusception events occurred within 30 days of a vaccine dose and all were reported only after the third dose. The sustained efficacy of the 116E in the second year of life is reassuring.The trial is registered with Clinical Trial Registry-India (# CTRI/2010/091/000102) and Clinicaltrials.gov (# NCT01305109).

  View details for DOI 10.1016/j.vaccine.2014.04.079

  View details for PubMedID 25091663

• Efficacy of a monovalent human-bovine (116E) rotavirus vaccine in Indian children in the second year of life. Vaccine Bhandari, N., Rongsen-Chandola, T., Bavdekar, A., John, J., Antony, K., Taneja, S., Goyal, N., Kawade, A., Kang, G., Rathore, S. S., Juvekar, S., Muliyil, J., Arya, A., Shaikh, H., Abraham, V., Vrati, S., Proschan, M., Kohberger, R., Thiry, G., Glass, R., Greenberg, H. B., Curlin, G., Mohan, K., Harshavardhan, G. V., Prasad, S., Rao, T. S., Boslego, J., Bhan, M. K. 2014; 32: A110-6

  View details for DOI 10.1016/j.vaccine.2014.04.079

  View details for PubMedID 25091663

• Efficacy of a monovalent human-bovine (116E) rotavirus vaccine in Indian infants: a randomised, double-blind, placebo-controlled trial. Lancet Bhandari, N., Rongsen-Chandola, T., Bavdekar, A., John, J., Antony, K., Taneja, S., Goyal, N., Kawade, A., Kang, G., Rathore, S. S., Juvekar, S., Muliyil, J., Arya, A., Shaikh, H., Abraham, V., Vrati, S., Proschan, M., Kohberger, R., Thiry, G., Glass, R., Greenberg, H. B., Curlin, G., Mohan, K., Harshavardhan, G. V., Prasad, S., Rao, T. S., Boslego, J., Bhan, M. K. 2014; 383 (9935): 2136-2143

  Abstract

  Rotavirus is the most common cause of severe dehydrating gastroenteritis in developing countries. Safe, effective, and affordable rotavirus vaccines are needed in these countries. We aimed to assess the efficacy and tolerability of a monovalent human-bovine rotavirus vaccine for severe rotavirus gastroenteritis in low-resource urban and rural settings in India.We did a randomised double-blind, placebo-controlled, multicentre trial at three sites in Delhi (urban), Pune (rural), and Vellore (urban and rural) between March 11, 2011, and Nov 5, 2012. Infants aged 6-7 weeks were randomly assigned (2:1), via a central interactive voice or web response system with a block size of 12, to receive either three doses of oral human-bovine natural reassortant vaccine (116E) or placebo at ages 6-7 weeks, 10 weeks, and 14 weeks. Infants' families, study investigators, paediatricians in referral hospitals, laboratory staff, and committee members were all masked to treatment allocation. The primary outcome was incidence of severe rotavirus gastroenteritis (≥11 on the Vesikari scale). Efficacy outcomes and adverse events were ascertained through active surveillance. Analysis was by intention to treat and per protocol. The trial is registered with Clinical Trial Registry-India (CTRI/2010/091/000102) and ClinicalTrials.gov (NCT01305109).4532 infants were assigned to receive the 116E vaccine and 2267 to receive placebo, of whom 4354 (96%) and 2187 (96%) infants, respectively, were included in the primary per-protocol efficacy analysis. 71 events of severe rotavirus gastroenteritis were reported in 4752 person-years in infants in the vaccine group compared with 76 events in 2360 person-years in those in the placebo group; vaccine efficacy against severe rotavirus gastroenteritis was 53·6% (95% CI 35·0-66·9; p=0·0013) and 56·4% (36·6-70·1; p<0·0001) in the first year of life. The number of infants needed to be immunised to prevent one severe rotavirus gastroenteritis episode was 55 (95% CI 37-97). The incidence of severe rotavirus gastroenteritis per 100 person-years was 1·5 in the vaccine group and 3·2 in the placebo group, with an incidence rate ratio of 0·46 (95% CI 0·33-0·65). Prevalence of immediate, solicited, and serious adverse events was similar in both groups. One case of urticaria in the vaccine group and one each of acute gastroenteritis and suspected sepsis in the placebo group were regarded as related to the study product. We recorded six cases of intussusception in the vaccine group and two in the placebo group, all of which happened after the third dose. 25 (<1%) infants in the vaccine group and 17 (<1%) in the placebo group died; no death was regarded as related to the study product.Monovalent human-bovine (116E) rotavirus vaccine is effective and well tolerated in Indian infants.Department of Biotechnology and the Biotechnology Industry Research Assistance Council, Government of India; Bill & Melinda Gates Foundation to PATH, USA; Research Council of Norway; UK Department for International Development; National Institutes of Health, Bethesda, USA; and Bharat Biotech International, Hyderabad, India.

  View details for DOI 10.1016/S0140-6736(13)62630-6

  View details for PubMedID 24629994

• Team science and the creation of a novel rotavirus vaccine in India: a new framework for vaccine development. Lancet Bhan, M. K., Glass, R. I., Ella, K. M., Bhandari, N., Boslego, J., Greenberg, H. B., Mohan, K., Curlin, G., Rao, T. S. 2014; 383 (9935): 2180-2183

  View details for DOI 10.1016/S0140-6736(14)60191-4

  View details for PubMedID 24629993

• Efficacy of a monovalent human-bovine (116E) rotavirus vaccine in Indian infants: a randomised, double-blind, placebo-controlled trial LANCET Bhandari, N., Rongsen-Chandola, T., Bavdekar, A., John, J., Antony, K., Taneja, S., Goyal, N., Kawade, A., Kang, G., Rathore, S. S., Juvekar, S., Muliyil, J., Arya, A., Shaikh, H., Abraham, V., Vrati, S., Proschan, M., Kohberger, R., Thiry, G., Glass, R., Greenberg, H. B., Curlin, G., Mohan, K., Harshavardhan, G. V., Prasad, S., Rao, T. S., Boslego, J., Bhan, M. K. 2014; 383 (9935): 2136-2143

  Abstract

  Rotavirus is the most common cause of severe dehydrating gastroenteritis in developing countries. Safe, effective, and affordable rotavirus vaccines are needed in these countries. We aimed to assess the efficacy and tolerability of a monovalent human-bovine rotavirus vaccine for severe rotavirus gastroenteritis in low-resource urban and rural settings in India.We did a randomised double-blind, placebo-controlled, multicentre trial at three sites in Delhi (urban), Pune (rural), and Vellore (urban and rural) between March 11, 2011, and Nov 5, 2012. Infants aged 6-7 weeks were randomly assigned (2:1), via a central interactive voice or web response system with a block size of 12, to receive either three doses of oral human-bovine natural reassortant vaccine (116E) or placebo at ages 6-7 weeks, 10 weeks, and 14 weeks. Infants' families, study investigators, paediatricians in referral hospitals, laboratory staff, and committee members were all masked to treatment allocation. The primary outcome was incidence of severe rotavirus gastroenteritis (≥11 on the Vesikari scale). Efficacy outcomes and adverse events were ascertained through active surveillance. Analysis was by intention to treat and per protocol. The trial is registered with Clinical Trial Registry-India (CTRI/2010/091/000102) and ClinicalTrials.gov (NCT01305109).4532 infants were assigned to receive the 116E vaccine and 2267 to receive placebo, of whom 4354 (96%) and 2187 (96%) infants, respectively, were included in the primary per-protocol efficacy analysis. 71 events of severe rotavirus gastroenteritis were reported in 4752 person-years in infants in the vaccine group compared with 76 events in 2360 person-years in those in the placebo group; vaccine efficacy against severe rotavirus gastroenteritis was 53·6% (95% CI 35·0-66·9; p=0·0013) and 56·4% (36·6-70·1; p<0·0001) in the first year of life. The number of infants needed to be immunised to prevent one severe rotavirus gastroenteritis episode was 55 (95% CI 37-97). The incidence of severe rotavirus gastroenteritis per 100 person-years was 1·5 in the vaccine group and 3·2 in the placebo group, with an incidence rate ratio of 0·46 (95% CI 0·33-0·65). Prevalence of immediate, solicited, and serious adverse events was similar in both groups. One case of urticaria in the vaccine group and one each of acute gastroenteritis and suspected sepsis in the placebo group were regarded as related to the study product. We recorded six cases of intussusception in the vaccine group and two in the placebo group, all of which happened after the third dose. 25 (<1%) infants in the vaccine group and 17 (<1%) in the placebo group died; no death was regarded as related to the study product.Monovalent human-bovine (116E) rotavirus vaccine is effective and well tolerated in Indian infants.Department of Biotechnology and the Biotechnology Industry Research Assistance Council, Government of India; Bill & Melinda Gates Foundation to PATH, USA; Research Council of Norway; UK Department for International Development; National Institutes of Health, Bethesda, USA; and Bharat Biotech International, Hyderabad, India.

  View details for DOI 10.1016/S0140-6736(13)62630-6

  View details for Web of Science ID 000337906600028

• Rotavirus NSP1 Protein Inhibits Interferon-Mediated STAT1 Activation JOURNAL OF VIROLOGY Sen, A., Rott, L., Nguyen Phan, N., Mukherjee, G., Greenberg, H. B. 2014; 88 (1): 41-53

  Abstract

  Rotavirus (RV) replicates efficiently in intestinal epithelial cells (IECs) in vivo despite the activation of a local host interferon (IFN) response. Previously, we demonstrated that homologous RV efficiently inhibits IFN induction in single infected and bystander villous IECs in vivo. Paradoxically, RV also induces significant type I IFN expression in the intestinal hematopoietic cell compartment in a relatively replication-independent manner. This suggests that RV replication and spread in IECs must occur despite exogenous stimulation of the STAT1-mediated IFN signaling pathway. Here we report that RV inhibits IFN-mediated STAT1 tyrosine 701 phosphorylation in human IECs in vitro and identify RV NSP1 as a direct inhibitor of the pathway. Infection of human HT29 IECs with simian (RRV) or porcine (SB1A or OSU) RV strains, which inhibit IFN induction by targeting either IFN regulatory factor 3 (IRF3) or NF-κB, respectively, resulted in similar regulation of IFN secretion. By flow cytometric analysis at early times during infection, neither RRV nor SB1A effectively inhibited the activation of Y701-STAT1 in response to exogenously added IFN. However, at later times during infection, both RV strains efficiently inhibited IFN-mediated STAT1 activation within virus-infected cells, indicating that RV encodes inhibitors of IFN signaling targeting STAT1 phosphorylation. Expression of RV NSP1 in the absence of other viral proteins resulted in blockage of exogenous IFN-mediated STAT1 phosphorylation, and this function was conserved in NSP1 from simian, bovine, and murine RV strains. Analysis of NSP1 determinants responsible for the inhibition of IFN induction and signaling pathways revealed that these determinants are encoded on discrete domains of NSP1. Finally, we observed that at later times during infection with SB1A, there was almost complete inhibition of IFN-mediated Y701-STAT1 in bystander cells staining negative for viral antigen. This property segregated with the NSP1 gene and was observed in a simian SA11 monoreassortant that encoded porcine OSU NSP1 but not in wild-type SA11 or a reassortant encoding simian RRV NSP1.

  View details for DOI 10.1128/JVI.01501-13

  View details for Web of Science ID 000329194600004

  View details for PubMedID 24131713

  View details for PubMedCentralID PMC3911692

• Rotavirus. Microbiology spectrum Franco, M. A., Greenberg, H. B. 2013; 1 (2)

  Abstract

  Rotaviruses (RV) are ubiquitous, highly infectious, segmented double-stranded RNA genome viruses of importance in public health because of the severe acute gastroenteritis they cause in young children and many animal species. They are very well adapted to their host, with symptomatic and asymptomatic reinfections being virtually universal during the first 3 years of life. Antibodies are the major arm of the immune system responsible for protecting infants from RV reinfection. The relationship between the virus and the B cells (Bc) that produce these antibodies is complex and incompletely understood: most blood-circulating Bc that express RV-specific immunoglobulin (Ig) on their surface (RV-Ig) are naive Bc and recognize the intermediate capsid viral protein VP6 with low affinity. When compared to non-antigen-specific Bc, RV-Bc are enriched in CD27+ memory Bc (mBc) that express IgM. The Ig genes used by naive RV-Bc are different than those expressed by RV-mBc, suggesting that the latter do not primarily develop from the former. Although RV predominantly infects mature villus enterocytes, an acute systemic viremia also occurs and RV-Bc can be thought of as belonging to either the intestinal or systemic immune compartments. Serotype-specific or heterotypic RV antibodies appear to mediate protection by multiple mechanisms, including intracellular and extracellular homotypic and heterotypic neutralization. Passive administration of RV-Ig can be used either prophylactically or therapeutically. A better understanding of the Bc response generated against RV will improve our capacity to identify improved correlates of protection for RV vaccines.

  View details for DOI 10.1128/microbiolspec.AID-0011-2013

  View details for PubMedID 26184961

• Permissive Replication of Homologous Murine Rotavirus in the Mouse Intestine Is Primarily Regulated by VP4 and NSP1 JOURNAL OF VIROLOGY Feng, N., Yasukawa, L. L., Sen, A., Greenberg, H. B. 2013; 87 (15): 8307-8316

  Abstract

  Homologous rotaviruses (RV) are, in general, more virulent and replicate more efficiently than heterologous RV in the intestine of the homologous host. The genetic basis for RV host range restriction is not fully understood and is likely to be multigenic. In previous studies, RV genes encoding VP3, VP4, VP7, NSP1, and NSP4 have all been implicated in strain- and host species-specific infection. These studies used different RV strains, variable measurements of host range, and different animal hosts and no clear consensus on the host range restriction determinants emerged. We used a murine model to demonstrate that enteric replication of murine RV EW is 1,000 to 10,000 fold greater than a simian rotavirus (RRV) in suckling mice. Intestinal replication of a series of EW × RRV reassortants was used to identify several RV genes that influenced RV replication in the intestine. The role of VP4 (encoded by gene 4) in enteric infection was strain specific. RRV VP4 only slightly reduced murine RV infectivity; however, a reassortant expressing VP4 from a bovine RV strain (UK) severely restricted intestinal replication in the suckling mice. The homologous murine EW NSP1 (encoded by gene 5) was necessary but not sufficient for promoting efficient enteric growth. Efficient enteric replication required a constellation of murine genes encoding VP3, NSP2, and NSP3 along with NSP1.

  View details for DOI 10.1128/JVI.00619-13

  View details for Web of Science ID 000321590200005

  View details for PubMedID 23698306

• Combinatorial tetramer staining and mass cytometry analysis facilitate T-cell epitope mapping and characterization NATURE BIOTECHNOLOGY Newell, E. W., Sigal, N., Nair, N., Kidd, B. A., Greenberg, H. B., Davis, M. M. 2013; 31 (7): 623-U81

  Abstract

  It is currently not possible to predict which epitopes will be recognized by T cells in different individuals. This is a barrier to the thorough analysis and understanding of T-cell responses after vaccination or infection. Here, by combining mass cytometry with combinatorial peptide-MHC tetramer staining, we have developed a method allowing the rapid and simultaneous identification and characterization of T cells specific for many epitopes. We use this to screen up to 109 different peptide-MHC tetramers in a single human blood sample, while still retaining at least 23 labels to analyze other markers of T-cell phenotype and function. Among 77 candidate rotavirus epitopes, we identified six T-cell epitopes restricted to human leukocyte antigen (HLA)-A*0201 in the blood of healthy individuals. T cells specific for epitopes in the rotavirus VP3 protein displayed a distinct phenotype and were present at high frequencies in intestinal epithelium. This approach should be useful for the comprehensive analysis of T-cell responses to infectious diseases or vaccines.

  View details for DOI 10.1038/nbt.2593

  View details for Web of Science ID 000321579700019

  View details for PubMedID 23748502

• Plasmacytoid dendritic cells promote rotavirus-induced human and murine B cell responses. journal of clinical investigation Deal, E. M., Lahl, K., Narváez, C. F., Butcher, E. C., Greenberg, H. B. 2013; 123 (6): 2464-2474

  Abstract

  B cell-dependent immunity to rotavirus, an important intestinal pathogen, plays a significant role in viral clearance and protects against reinfection. Human in vitro and murine in vivo models of rotavirus infection were used to delineate the role of primary plasmacytoid DCs (pDCs) in initiating B cell responses. Human pDCs were necessary and sufficient for B cell activation induced by rotavirus. Type I IFN recognition by B cells was essential for rotavirus-mediated B cell activation in vitro and murine pDCs and IFN-α/β-mediated B cell activation after in vivo intestinal rotavirus infection. Furthermore, rotavirus-specific serum and mucosal antibody responses were defective in mice lacking functional pDCs at the time of infection. These data demonstrate that optimal B cell activation and virus-specific antibody secretion following mucosal infection were a direct result of pDC-derived type I IFN. Importantly, viral shedding significantly increased in pDC-deficient mice, suggesting that pDC-dependent antibody production influences viral clearance. Thus, mucosal pDCs critically influence the course of rotavirus infection through rotavirus recognition and subsequent IFN production and display powerful adjuvant properties to initiate and enhance humoral immunity.

  View details for DOI 10.1172/JCI60945

  View details for PubMedID 23635775

  View details for PubMedCentralID PMC3668833

• Lineage structure of the human antibody repertoire in response to influenza vaccination. Science translational medicine Jiang, N., He, J., Weinstein, J. A., Penland, L., Sasaki, S., He, X., Dekker, C. L., Zheng, N., Huang, M., Sullivan, M., Wilson, P. C., Greenberg, H. B., Davis, M. M., Fisher, D. S., Quake, S. R. 2013; 5 (171): 171ra19-?

  Abstract

  The human antibody repertoire is one of the most important defenses against infectious disease, and the development of vaccines has enabled the conferral of targeted protection to specific pathogens. However, there are many challenges to measuring and analyzing the immunoglobulin sequence repertoire, including that each B cell's genome encodes a distinct antibody sequence, that the antibody repertoire changes over time, and the high similarity between antibody sequences. We have addressed these challenges by using high-throughput long read sequencing to perform immunogenomic characterization of expressed human antibody repertoires in the context of influenza vaccination. Informatic analysis of 5 million antibody heavy chain sequences from healthy individuals allowed us to perform global characterizations of isotype distributions, determine the lineage structure of the repertoire, and measure age- and antigen-related mutational activity. Our analysis of the clonal structure and mutational distribution of individuals' repertoires shows that elderly subjects have a decreased number of lineages but an increased prevaccination mutation load in their repertoire and that some of these subjects have an oligoclonal character to their repertoire in which the diversity of the lineages is greatly reduced relative to younger subjects. We have thus shown that global analysis of the immune system's clonal structure provides direct insight into the effects of vaccination and provides a detailed molecular portrait of age-related effects.

  View details for DOI 10.1126/scitranslmed.3004794

  View details for PubMedID 23390249

• Lineage Structure of the Human Antibody Repertoire in Response to Influenza Vaccination SCIENCE TRANSLATIONAL MEDICINE Jiang, N., He, J., Weinstein, J. A., Penland, L., Sasaki, S., He, X., Dekker, C. L., Zheng, N., Huang, M., Sullivan, M., Wilson, P. C., Greenberg, H. B., Davis, M. M., Fisher, D. S., Quake, S. R. 2013; 5 (171)

  Abstract

  The human antibody repertoire is one of the most important defenses against infectious disease, and the development of vaccines has enabled the conferral of targeted protection to specific pathogens. However, there are many challenges to measuring and analyzing the immunoglobulin sequence repertoire, including that each B cell's genome encodes a distinct antibody sequence, that the antibody repertoire changes over time, and the high similarity between antibody sequences. We have addressed these challenges by using high-throughput long read sequencing to perform immunogenomic characterization of expressed human antibody repertoires in the context of influenza vaccination. Informatic analysis of 5 million antibody heavy chain sequences from healthy individuals allowed us to perform global characterizations of isotype distributions, determine the lineage structure of the repertoire, and measure age- and antigen-related mutational activity. Our analysis of the clonal structure and mutational distribution of individuals' repertoires shows that elderly subjects have a decreased number of lineages but an increased prevaccination mutation load in their repertoire and that some of these subjects have an oligoclonal character to their repertoire in which the diversity of the lineages is greatly reduced relative to younger subjects. We have thus shown that global analysis of the immune system's clonal structure provides direct insight into the effects of vaccination and provides a detailed molecular portrait of age-related effects.

  View details for Web of Science ID 000314810000008

  View details for PubMedID 23390249

• Heterovariant Cross-Reactive B-Cell Responses Induced by the 2009 Pandemic Influenza Virus A Subtype H1N1 Vaccine JOURNAL OF INFECTIOUS DISEASES He, X., Sasaki, S., Baer, J., Khurana, S., Golding, H., Treanor, J. J., Topham, D. J., Sangster, M. Y., Jin, H., Dekker, C. L., Subbarao, K., Greenberg, H. B. 2013; 207 (2): 288-296

  Abstract

  The generation of heterovariant immunity is a highly desirable feature of influenza vaccines. The goal of this study was to compare the heterovariant B-cell response induced by the monovalent inactivated 2009 pandemic influenza A virus subtype H1N1 (A[H1N1]pdm09) vaccine with that induced by the 2009 seasonal trivalent influenza vaccine (sTIV) containing a seasonal influenza A virus subtype H1N1 (A[H1N1]) component in young and elderly adults.Plasmablast-derived polyclonal antibodies (PPAb) from young and elderly recipients of A(H1N1)pdm09 vaccine or sTIV were tested for binding activity to various influenza antigens.In A(H1N1)pdm09 recipients, the PPAb titers against homotypic A(H1N1)pdm09 vaccine were similar to those against the heterovariant seasonal A(H1N1) vaccine and were similar between young and elderly subjects. The PPAb avidity was higher among elderly individuals, compared with young individuals. In contrast, the young sTIV recipients had 10-fold lower heterovariant PPAb titers against the A(H1N1)pdm09 vaccine than against the homotypic seasonal A(H1N1) vaccine. In binding assays with recombinant head and stalk domains of hemagglutinin, PPAb from the A(H1N1)pdm09 recipients but not PPAb from the sTIV recipients bound to the conserved stalk domain.The A(H1N1)pdm09 vaccine induced production of PPAb with heterovariant reactivity, including antibodies targeting the conserved hemagglutinin stalk domain.

  View details for DOI 10.1093/infdis/jis664

  View details for Web of Science ID 000312886400015

  View details for PubMedID 23107783

  View details for PubMedCentralID PMC3532823

• Characterization of Rotavirus RNAs That Activate Innate Immune Signaling through the RIG-I-Like Receptors. PloS one Uzri, D., Greenberg, H. B. 2013; 8 (7)

  Abstract

  In mammalian cells, the first line of defense against viral pathogens is the innate immune response, which is characterized by induction of type I interferons (IFN) and other pro-inflammatory cytokines that establish an antiviral milieu both in infected cells and in neighboring uninfected cells. Rotavirus, a double-stranded RNA virus of the Reoviridae family, is the primary etiological agent of severe diarrhea in young children worldwide. Previous studies demonstrated that rotavirus replication induces a MAVS-dependent type I IFN response that involves both RIG-I and MDA5, two cytoplasmic viral RNA sensors. This study reports the isolation and characterization of rotavirus RNAs that activate IFN signaling. Using an in vitro approach with purified rotavirus double-layer particles, nascent single-stranded RNA (ssRNA) transcripts (termed in vitro ssRNA) were found to be potent IFN inducers. In addition, large RNAs isolated from rotavirus-infected cells six hours post-infection (termed in vivo 6 hr large RNAs), also activated IFN signaling, whereas a comparable large RNA fraction isolated from cells infected for only one hour lacked this stimulatory activity. Experiments using knockout murine embryonic fibroblasts showed that RIG-I is required for and MDA5 partly contributes to innate immune signaling by both in vitro ssRNA and in vivo 6 hr large RNAs. Enzymatic studies demonstrated that in vitro ssRNA and in vivo 6 hr large RNA samples contain uncapped RNAs with exposed 5' phosphate groups. RNAs lacking 2'-O-methylated 5' cap structures were also detected in the in vivo 6 hr large RNA sample. Taken together, our data provide strong evidence that the rotavirus VP3 enzyme, which encodes both guanylyltransferase and methyltransferase activities, is not completely efficient at either 5' capping or 2'-O-methylation of the 5' cap structures of viral transcripts, and in this way produces RNA patterns that activate innate immune signaling through the RIG-I-like receptors.

  View details for DOI 10.1371/journal.pone.0069825

  View details for PubMedID 23894547

  View details for PubMedCentralID PMC3720929

• The Battle between Rotavirus and Its Host for Control of the Interferon Signaling Pathway PLOS PATHOGENS Arnold, M. M., Sen, A., Greenberg, H. B., Patton, J. T. 2013; 9 (1)

  Abstract

  Viral pathogens must overcome innate antiviral responses to replicate successfully in the host organism. Some of the mechanisms viruses use to interfere with antiviral responses in the infected cell include preventing detection of viral components, perturbing the function of transcription factors that initiate antiviral responses, and inhibiting downstream signal transduction. RNA viruses with small genomes and limited coding space often express multifunctional proteins that modulate several aspects of the normal host response to infection. One such virus, rotavirus, is an important pediatric pathogen that causes severe gastroenteritis, leading to ~450,000 deaths globally each year. In this review, we discuss the nature of the innate antiviral responses triggered by rotavirus infection and the viral mechanisms for inhibiting these responses.

  View details for DOI 10.1371/journal.ppat.1003064

  View details for Web of Science ID 000314464600004

  View details for PubMedID 23359266

  View details for PubMedCentralID PMC3554623

• Innate immune response to homologous rotavirus infection in the small intestinal villous epithelium at single-cell resolution PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sen, A., Rothenberg, M. E., Mukherjee, G., Feng, N., Kalisky, T., Nair, N., Johnstone, I. M., Clarke, M. F., Greenberg, H. B. 2012; 109 (50): 20667-20672

  Abstract

  "Bulk" measurements of antiviral innate immune responses from pooled cells yield averaged signals and do not reveal underlying signaling heterogeneity in infected and bystander single cells. We examined such heterogeneity in the small intestine during rotavirus (RV) infection. Murine RV EW robustly activated type I IFNs and several antiviral genes (IFN-stimulated genes) in the intestine by bulk analysis, the source of induced IFNs primarily being hematopoietic cells. Flow cytometry and microfluidics-based single-cell multiplex RT-PCR allowed dissection of IFN responses in single RV-infected and bystander intestinal epithelial cells (IECs). EW replicates in IEC subsets differing in their basal type I IFN transcription and induces IRF3-dependent and IRF3-augmented transcription, but not NF-κB-dependent or type I IFN transcripts. Bystander cells did not display enhanced type I IFN transcription but had elevated levels of certain IFN-stimulated genes, presumably in response to exogenous IFNs secreted from immune cells. Comparison of IRF3 and NF-κB induction in STAT1(-/-) mice revealed that murine but not simian RRV mediated accumulation of IkB-α protein and decreased transcription of NF-κB-dependent genes. RRV replication was significantly rescued in IFN types I and II, as well as STAT1 (IFN types I, II, and III) deficient mice in contrast to EW, which was only modestly sensitive to IFNs I and II. Resolution of "averaged" innate immune responses in single IECs thus revealed unexpected heterogeneity in both the induction and subversion of early host antiviral immunity, which modulated host range.

  View details for DOI 10.1073/pnas.1212188109

  View details for Web of Science ID 000312605600104

  View details for PubMedID 23188796

  View details for PubMedCentralID PMC3528539

• Cell-free production of trimeric influenza hemagglutinin head domain proteins as vaccine antigens BIOTECHNOLOGY AND BIOENGINEERING Welsh, J. P., Lu, Y., He, X., Greenberg, H. B., Swartz, J. R. 2012; 109 (12): 2962-2969

  Abstract

  In order to effectively combat pandemic influenza threats, there is a need for more rapid and robust vaccine production methods. In this article, we demonstrate E. coli-based cell-free protein synthesis (CFPS) as a method to rapidly produce domains from the protein hemagglutinin (HA), which is present on the surface of the influenza virus. The portion of the HA coding sequence for the "head" domain from the 2009 pandemic H1N1 strain was first optimized for E. coli expression. The protein domain was then produced in CFPS reactions and purified in soluble form first as a monomer and then as a trimer by a C-terminal addition of the T4 bacteriophage foldon domain. Production of soluble trimeric HA head domain was enhanced by introducing stabilizing amino acid mutations to the construct in order to avoid aggregation. Trimerization was verified using size exclusion HPLC, and the stabilized HA head domain trimer was more effectively recognized by antibodies from pandemic H1N1 influenza vaccine recipients than was the monomer and also bound to sialic acids more strongly, indicating that the trimers are correctly formed and could be potentially effective as vaccines.

  View details for DOI 10.1002/bit.24581

  View details for Web of Science ID 000310465300008

  View details for PubMedID 22729608

• Plasmacytoid Dendritic Cells and Anti-Inflammatory Intestinal IgA Production Crohn's-and-Colitis-Foundation's-National-Clinical-and-Research Conference on Advances in Inflammatory Bowel Diseases Lahl, K., Deal, E., Greenberg, H., Butcher, E. WILEY-BLACKWELL. 2012: S108–S108

  View details for Web of Science ID 000311172600275

• Human Rotavirus-Specific IgM Memory B Cells Have Differential Cloning Efficiencies and Switch Capacities and Play a Role in Antiviral Immunity In Vivo JOURNAL OF VIROLOGY Narvaez, C. F., Feng, N., Vasquez, C., Sen, A., Angel, J., Greenberg, H. B., Franco, M. A. 2012; 86 (19): 10829-10840

  Abstract

  Protective immunity to rotavirus (RV) is primarily mediated by antibodies produced by RV-specific memory B cells (RV-mBc). Of note, most of these cells express IgM, but the function of this subset is poorly understood. Here, using limiting dilution assays of highly sort-purified human IgM(+) mBc, we found that 62% and 21% of total (non-antigen-specific) IgM(+) and RV-IgM(+) mBc, respectively, switched in vitro to IgG production after polyclonal stimulation. Moreover, in these assays, the median cloning efficiencies of total IgM(+) (17%) and RV-IgM(+) (7%) mBc were lower than those of the corresponding switched (IgG(+) IgA(+)) total (34%) and RV-mBc (17%), leading to an underestimate of their actual frequency. In order to evaluate the in vivo role of IgM(+) RV-mBc in antiviral immunity, NOD/Shi-scid interleukin-2 receptor-deficient (IL-2Rγ(null)) immunodeficient mice were adoptively transferred highly purified human IgM(+) mBc and infected with virulent murine rotavirus. These mice developed high titers of serum human RV-IgM and IgG and had significantly lower levels than control mice of both antigenemia and viremia. Finally, we determined that human RV-IgM(+) mBc are phenotypically diverse and significantly enriched in the IgM(hi) IgD(low) subset. Thus, RV-IgM(+) mBc are heterogeneous, occur more frequently than estimated by traditional limiting dilution analysis, have the capacity to switch Ig class in vitro as well as in vivo, and can mediate systemic antiviral immunity.

  View details for DOI 10.1128/JVI.01466-12

  View details for Web of Science ID 000308740700057

  View details for PubMedID 22855480

  View details for PubMedCentralID PMC3457288

• Rotavirus immune responses and correlates of protection CURRENT OPINION IN VIROLOGY Angel, J., Franco, M. A., Greenberg, H. B. 2012; 2 (4): 419-425

  Abstract

  Selected topics in the field of rotavirus immunity are reviewed focusing on recent developments that may improve efficacy and safety of current and future vaccines. Rotaviruses (RVs) have developed multiple mechanisms to evade interferon (IFN)-mediated innate immunity. Compared to more developed regions of the world, protection induced by natural infection and vaccination is reduced in developing countries where, among other factors, high viral challenge loads are common and where infants are infected at an early age. Studies in developing countries indicate that rotavirus-specific serum IgA levels are not an optimal correlate of protection following vaccination, and better correlates need to be identified. Protection against rotavirus following vaccination is substantially heterotypic; nonetheless, a role for homotypic immunity in selection of circulating postvaccination strains needs further study.

  View details for DOI 10.1016/j.coviro.2012.05.003

  View details for Web of Science ID 000312112900007

  View details for PubMedID 22677178

• High Throughput Sequencing of the Human Antibody Repertoire in Response to Influenza Vaccination 99th Annual Meeting of the American-Association-of-Immunologists Jiang, N., He, J., Weinstein, J., Penland, L., Saaki, S., He, X., Dekker, C., Wilson, P., Greenberg, H., Davis, M., Fisher, D., Quake, S. AMER ASSOC IMMUNOLOGISTS. 2012

  View details for Web of Science ID 000304659700423

• Rhesus Rotavirus Trafficking during Entry into MA104 Cells Is Restricted to the Early Endosome Compartment JOURNAL OF VIROLOGY Wolf, M., Deal, E. M., Greenberg, H. B. 2012; 86 (7): 4009-4013

  Abstract

  Endocytosis has recently been implicated in rotavirus (RV) entry. We examined the role of Rabs, which regulate endosomal trafficking, during RV entry. Several structural proteins of neuraminidase-sensitive and -insensitive RVs colocalized with Rab5, an early endosome marker, but not Rab7, a late endosome marker. Dominant-negative and constitutively active mutants demonstrated that Rab5 but not Rab4 or Rab7 affects rhesus RV (RRV) infectivity. These data suggest that early RRV trafficking is confined to the early endosome compartment and requires Rab5.

  View details for DOI 10.1128/JVI.06667-11

  View details for Web of Science ID 000301371500056

  View details for PubMedID 22278225

  View details for PubMedCentralID PMC3302545

• Cross-Linking of Rotavirus Outer Capsid Protein VP7 by Antibodies or Disulfides Inhibits Viral Entry JOURNAL OF VIROLOGY Aoki, S. T., Trask, S. D., Coulson, B. S., Greenberg, H. B., Dormitzer, P. R., Harrison, S. C. 2011; 85 (20): 10509-10517

  Abstract

  Antibodies that neutralize rotavirus infection target outer coat proteins VP4 and VP7 and inhibit viral entry. The structure of a VP7-Fab complex (S. T. Aoki, et al., Science 324:1444-1447, 2009) led us to reclassify epitopes into two binding regions at inter- and intrasubunit boundaries of the calcium-dependent trimer. It further led us to show that antibodies binding at the intersubunit boundary inhibit uncoating of the virion outer layer. We have now tested representative antibodies for each of the defined structural epitope regions and find that antibodies recognizing epitopes in either binding region neutralize by cross-linking VP7 trimers. Antibodies that bind at the intersubunit junction neutralize as monovalent Fabs, while those that bind at the intrasubunit region require divalency. The VP7 structure has also allowed us to design a disulfide cross-linked VP7 mutant which recoats double-layered particles (DLPs) as efficiently as does wild-type VP7 but which yields particles defective in cell entry as determined both by lack of infectivity and by loss of α-sarcin toxicity in the presence of recoated particles. We conclude that dissociation of the VP7 trimer is an essential step in viral penetration into cells.

  View details for DOI 10.1128/JVI.00234-11

  View details for Web of Science ID 000296254100008

  View details for PubMedID 21849465

  View details for PubMedCentralID PMC3187514

• Limited efficacy of inactivated influenza vaccine in elderly individuals is associated with decreased production of vaccine-specific antibodies JOURNAL OF CLINICAL INVESTIGATION Sasaki, S., Sullivan, M., Narvaez, C. F., Holmes, T. H., Furman, D., Zheng, N., Nishtala, M., Wrammert, J., Smith, K., James, J. A., Dekker, C. L., Davis, M. M., Wilson, P. C., Greenberg, H. B., He, X. 2011; 121 (8): 3109-3119

  Abstract

  During seasonal influenza epidemics, disease burden is shouldered predominantly by the very young and the elderly. Elderly individuals are particularly affected, in part because vaccine efficacy wanes with age. This has been linked to a reduced ability to induce a robust serum antibody response. Here, we show that this is due to reduced quantities of vaccine-specific antibodies, rather than a lack of antibody avidity or affinity. We measured levels of vaccine-specific plasmablasts by ELISPOT 1 week after immunization of young and elderly adults with inactivated seasonal influenza vaccine. Plasmablast-derived polyclonal antibodies (PPAbs) were generated from bulk-cultured B cells, while recombinant monoclonal antibodies (re-mAbs) were produced from single plasmablasts. The frequency of vaccine-specific plasmablasts and the concentration of PPAbs were lower in the elderly than in young adults, whereas the yields of secreted IgG per plasmablast were not different. Differences were not detected in the overall vaccine-specific avidity or affinity of PPAbs and re-mAbs between the 2 age groups. In contrast, reactivity of the antibodies induced by the inactivated seasonal influenza vaccine toward the 2009 pandemic H1N1 virus, which was not present in the vaccine, was higher in the elderly than in the young. These results indicate that the inferior antibody response to influenza vaccination in the elderly is primarily due to reduced quantities of vaccine-specific antibodies. They also suggest that exposure history affects the cross-reactivity of vaccination-induced antibodies.

  View details for DOI 10.1172/JCI57834

  View details for Web of Science ID 000293495500022

  View details for PubMedID 21785218

  View details for PubMedCentralID PMC3148747

• Reconciliation of Rotavirus Temperature-Sensitive Mutant Collections and Assignment of Reassortment Groups D, J, and K to Genome Segments JOURNAL OF VIROLOGY Criglar, J., Greenberg, H. B., Estes, M. K., Ramig, R. F. 2011; 85 (10): 5048-5060

  Abstract

  Four rotavirus SA11 temperature-sensitive (ts) mutants and seven rotavirus RRV ts mutants, isolated at the National Institutes of Health (NIH) and not genetically characterized, were assigned to reassortment groups by pairwise crosses with the SA11 mutant group prototypes isolated and characterized at Baylor College of Medicine (BCM). Among the NIH mutants, three of the RRV mutants and all four SA11 mutants contained mutations in single reassortment groups, and four RRV mutants contained mutations in multiple groups. One NIH mutant [RRVtsK(2)] identified the previously undefined 11th reassortment group (K) expected for rotavirus. Three NIH single mutant RRV viruses, RRVtsD(7), RRVtsJ(5), and RRVtsK(2), were in reassortment groups not previously mapped to genome segments. These mutants were mapped using classical genetic methods, including backcrosses to demonstrate reversion or suppression in reassortants with incongruent genotype and temperature phenotype. Once located to specific genome segments by genetic means, the mutations responsible for the ts phenotype were identified by sequencing. The reassortment group K mutant RRVtsK(2) maps to genome segment 9 and has a Thr280Ileu mutation in the capsid surface glycoprotein VP7. The group D mutant RRVtsD(7) maps to segment 5 and has a Leu140Val mutation in the nonstructural interferon (IFN) antagonist protein NSP1. The group J mutant RRVtsJ(5) maps to segment 11 and has an Ala182Gly mutation affecting only the NSP5 open reading frame. Rotavirus ts mutation groups are now mapped to 9 of the 11 rotavirus genome segments. Possible segment locations of the two remaining unmapped ts mutant groups are discussed.

  View details for DOI 10.1128/JVI.02691-10

  View details for Web of Science ID 000289787300040

  View details for PubMedID 21367894

• The Early Interferon Response to Rotavirus Is Regulated by PKR and Depends on MAVS/IPS-1, RIG-I, MDA-5, and IRF3 JOURNAL OF VIROLOGY Sen, A., Pruijssers, A. J., Dermody, T. S., Garcia-Sastre, A., Greenberg, H. B. 2011; 85 (8): 3717-3732

  Abstract

  In mouse embryonic fibroblasts (MEFs), the bovine rotavirus (UK strain) but not the simian rhesus rotavirus (RRV) robustly triggers beta interferon (IFN-β) secretion, resulting in an IFN-dependent restriction of replication. We now find that both rotavirus strains trigger antiviral transcriptional responses early during infection and that both transcriptional responses and IFN-β secretion are completely abrogated in MAVS/IPS-1(-/-) MEFs. Replication of UK virus could be rescued in MAVS/IPS-1(-/-) MEFs, and synthesis of viral RNA significantly increased early during virus infection. UK virus induced IFN-β secretion and transcription of IFN-stimulated genes (ISGs) in both RIG-I(-/-) and MDA-5(-/-) MEFs, and neither receptor was essential by itself for the antiviral response to UK rotavirus. However, when receptors RIG-I and MDA-5 were depleted using RNA interference, we found that both contribute to the magnitude of the IFN response. IRF3 was found to be essential for MAVS/IPS-1-directed ISG transcription and IFN-β secretion during rotavirus infection. Interestingly, absence of the double-stranded RNA-dependent protein kinase PKR led to a profound defect in the capacity of host cells to secrete IFN-β in response to virus. Both PKR and IRF3 restricted the early replication of UK as indicated by significant increases in viral RNA in fibroblasts lacking either gene. Despite the loss in IFN-β secretion in PKR(-/-) MEFs, we did not observe decreased IRF3- or NF-κB-dependent early ISG transcription in these cells. Levels of transcripts encoding IFN-α4, IFN-α5, and IFN-β were high in infected PKR(-/-) MEFs, indicating that during rotavirus infection, PKR functions at a stage between IFN gene transcription and subsequent IFN-β secretion. These findings reveal that activation of the antiviral response by rotavirus is dependent on MAVS/IPS-1 and IRF3 and involves both RIG-I and MDA-5 and that IFN-β secretion during rotavirus infection is regulated by PKR.

  View details for DOI 10.1128/JVI.02634-10

  View details for Web of Science ID 000288536100003

  View details for PubMedID 21307186

  View details for PubMedCentralID PMC3126121

• Rhesus Rotavirus Entry into a Polarized Epithelium Is Endocytosis Dependent and Involves Sequential VP4 Conformational Changes JOURNAL OF VIROLOGY Wolf, M., Vo, P. T., Greenberg, H. B. 2011; 85 (6): 2492-2503

  Abstract

  Rotavirus (RV) cell entry is an incompletely understood process, involving VP4 and VP7, the viral proteins composing the outermost layer of the nonenveloped RV triple-layered icosahedral particle (TLP), encasing VP6. VP4 can exist in three conformational states: soluble, cleaved spike, and folded back. In order to better understand the events leading to RV entry, we established a detection system to image input virus by monitoring the rhesus RV (RRV) antigens VP4, VP6, and VP7 at very early times postinfection. We provide evidence that decapsidation occurs directly after cell membrane penetration. We also demonstrate that several VP4 and VP7 conformational changes take place during entry. In particular, we detected, for the first time, the generation of folded-back VP5 in the context of the initiation of infection. Folded-back VP5 appears to be limited to the entry step. We furthermore demonstrate that RRV enters the cell cytoplasm through an endocytosis pathway. The endocytosis hypothesis is supported by the colocalization of RRV antigens with the early endosome markers Rab4 and Rab5. Finally, we provide evidence that the entry process is likely dependent on the endocytic Ca(2+) concentration, as bafilomycin A1 treatment as well as an augmentation of the extracellular calcium reservoir using CaEGTA, which both lead to an elevated intraendosomal calcium concentration, resulted in the accumulation of intact virions in the actin network. Together, these findings suggest that internalization, decapsidation, and cell membrane penetration involve endocytosis, calcium-dependent uncoating, and VP4 conformational changes, including a fold-back.

  View details for DOI 10.1128/JVI.02082-10

  View details for Web of Science ID 000288386500002

  View details for PubMedID 21191022

  View details for PubMedCentralID PMC3067957

• Roles of VP4 and NSP1 in Determining the Distinctive Replication Capacities of Simian Rotavirus RRV and Bovine Rotavirus UK in the Mouse Biliary Tract JOURNAL OF VIROLOGY Feng, N., Sen, A., Wolf, M., Vo, P., Hoshino, Y., Greenberg, H. B. 2011; 85 (6): 2686-2694

  Abstract

  Rotavirus replication and virulence are strongly influenced by virus strain and host species. The rotavirus proteins VP3, VP4, VP7, NSP1, and NSP4 have all been implicated in strain and species restriction of replication; however, the mechanisms have not been fully determined. Simian (RRV) and bovine (UK) rotaviruses have distinctive replication capacities in mouse extraintestinal organs such as the biliary tract. Using reassortants between UK and RRV, we previously demonstrated that the differential replication of these viruses in mouse embryonic fibroblasts is determined by the respective NSP1 proteins, which differ substantially in their abilities to degrade interferon (IFN) regulatory factor 3 (IRF3) and suppress the type I IFN response. In this study, we used an in vivo model of rotavirus infection of mouse gallbladder with UK × RRV reassortants to study the genetic and mechanistic basis of systemic rotavirus replication. We found that the low-replication phenotype of UK in biliary tissues was conferred by UK VP4 and that the high-replication phenotype of RRV was conferred by RRV VP4 and NSP1. Viruses with RRV VP4 entered cultured mouse cholangiocytes more efficiently than did those with UK VP4. Reassortants with RRV VP4 and UK NSP1 genes induced high levels of expression of IRF3-dependent p54 in biliary tissues, and their replication was increased 3-fold in IFN-α/β and -γ receptor or STAT1 knockout (KO) mice compared to wild-type mice. Our data indicate that systemic rotavirus strain-specific replication in the murine biliary tract is determined by both viral entry mediated by VP4 and viral antagonism of the host innate immune response mediated by NSP1.

  View details for DOI 10.1128/JVI.02408-10

  View details for Web of Science ID 000288386500019

  View details for PubMedID 21191030

  View details for PubMedCentralID PMC3067955

• Plasmablast-derived polyclonal antibody response after influenza vaccination JOURNAL OF IMMUNOLOGICAL METHODS He, X., Sasaki, S., Narvaez, C. F., Zhang, C., Liu, H., Woo, J. C., Kemble, G. W., Dekker, C. L., Davis, M. M., Greenberg, H. B. 2011; 365 (1-2): 67-75

  Abstract

  Conventional measurement of antibody responses to vaccines largely relies on serum antibodies, which are primarily produced by bone marrow plasma cells and may not represent the entire vaccine-induced B cell repertoire, including important functional components such as those targeted to mucosal sites. After immunization or infection, activated B cells differentiate into plasmablasts in local lymphoid organs, then traffic through circulation to the target sites where they further develop into plasma cells. On day 7 after influenza vaccination, a burst of plasmablasts, highly enriched for vaccine-specific antibody secreting cells, appears in the peripheral blood. This provides a unique window to the overall B cell response to the vaccine, without interference of pre-existing cross-reactive serum antibody. In this study we isolated B cells from volunteers on day 7 after immunization with the inactivated influenza vaccine and cultured them ex vivo to collect plasmablast-derived polyclonal antibodies (PPAb). The PPAb contained secreted IgG and IgA, which was approximately 0.2ng per antibody secreting cell. Influenza-specific IgG and IgA binding activity was detected in PPAb at dilutions up to 10(5) by ELISA. The ratio of the titers of influenza-specific IgA to IgG by ELISA was 4-fold higher in PPAb than in day 28 post-vaccination sera, suggesting that vaccine-induced IgA is enriched in PPAb compared to sera. Functional activity was also detected in PPAb as determined by microneutralization and hemagglutination inhibition assays. In addition to bulk B cell cultures, we also cultured plasmablast subsets sorted by cell surface markers to generate PPAb. These results suggest that PPAb better reflects the mucosal IgA response than serum samples. Since PPAb are exclusively produced by recently activated B cells, it allows assessing vaccine-induced antibody response without interference from pre-existing cross-reactive serum antibodies and permits an assessment of antibody avidity based on antigen specific binding and antibody quantity. Therefore this assay is particularly useful for studying vaccine/infection-induced antibodies against antigens that might have previously circulated, such as antibody responses to rotavirus, dengue or influenza viruses in which cross-reactive antibodies against different virus serotypes/subtypes play a critical role in immunity and/or pathogenesis.

  View details for DOI 10.1016/j.jim.2010.12.008

  View details for Web of Science ID 000288620600007

  View details for PubMedID 21182843

  View details for PubMedCentralID PMC3039424

• Membrane Vesicles Released by Intestinal Epithelial Cells Infected with Rotavirus Inhibit T-Cell Function VIRAL IMMUNOLOGY Barreto, A., Rodriguez, L., Lucia Rojas, O., Wolf, M., Greenberg, H. B., Franco, M. A., Angel, J. 2010; 23 (6): 595-608

  Abstract

  Rotavirus (RV) predominantly replicates in intestinal epithelial cells (IEC), and "danger signals" released by these cells may modulate viral immunity. We have recently shown that human model IEC (Caco-2 cells) infected with rhesus-RV release a non-inflammatory group of immunomodulators that includes heat shock proteins (HSPs) and TGF-β1. Here we show that both proteins are released in part in association with membrane vesicles (MV) obtained from filtrated Caco-2 supernatants concentrated by ultracentrifugation. These MV express markers of exosomes (CD63 and others), but not of the endoplasmic reticulum (ER) or nuclei. Larger quantities of proteins associated with MV were released by RV-infected cells than by non-infected cells. VP6 co-immunoprecipitated with CD63 present in these MV, and VP6 co-localized with CD63 in RV-infected cells, suggesting that this viral protein is associated with the MV, and that this association occurs intracellularly. CD63 present in MV preparations from stool samples from 36 children with gastroenteritis due or not due to RV were analyzed. VP6 co-immunoprecipitated with CD63 in 3/8 stool samples from RV-infected children, suggesting that these MV are released by RV-infected cells in vivo. Moreover, fractions that contained MV from RV-infected cells induced death and inhibited proliferation of CD4(+) T cells to a greater extent than fractions from non-infected cells. These effects were in part due to TGF-β, because they were reversed by treatment of the T cells with the TGF-β-receptor inhibitor ALK5i. MV from RV-infected and non-infected cells were heterogeneous, with morphologies and typical flotation densities described for exosomes (between 1.10 and 1.18 g/mL), and denser vesicles (>1.24 g/mL). Both types of MV from RV-infected cells were more efficient at inhibiting T-cell function than were those from non-infected cells. We propose that RV infection of IEC releases MV that modulate viral immunity.

  View details for DOI 10.1089/vim.2009.0113

  View details for Web of Science ID 000285093300007

  View details for PubMedID 21142445

• Rotavirus Structural Proteins and dsRNA Are Required for the Human Primary Plasmacytoid Dendritic Cell IFN alpha Response PLOS PATHOGENS Deal, E. M., Jaimes, M. C., Crawford, S. E., Estes, M. K., Greenberg, H. B. 2010; 6 (6)

  Abstract

  Rotaviruses are the leading cause of severe dehydrating diarrhea in children worldwide. Rotavirus-induced immune responses, especially the T and B cell responses, have been extensively characterized; however, little is known about innate immune mechanisms involved in the control of rotavirus infection. Although increased levels of systemic type I interferon (IFNalpha and beta) correlate with accelerated resolution of rotavirus disease, multiple rotavirus strains, including rhesus rotavirus (RRV), have been demonstrated to antagonize type I IFN production in a variety of epithelial and fibroblast cell types through several mechanisms, including degradation of multiple interferon regulatory factors by a viral nonstructural protein. This report demonstrates that stimulation of highly purified primary human peripheral plasmacytoid dendritic cells (pDCs) with either live or inactivated RRV induces substantial IFNalpha production by a subset of pDCs in which RRV does not replicate. Characterization of pDC responses to viral stimulus by flow cytometry and Luminex revealed that RRV replicates in a small subset of human primary pDCs and, in this RRV-permissive small subset, IFNalpha production is diminished. pDC activation and maturation were observed independently of viral replication and were enhanced in cells in which virus replicates. Production of IFNalpha by pDCs following RRV exposure required viral dsRNA and surface proteins, but neither viral replication nor activation by trypsin cleavage of VP4. These results demonstrate that a minor subset of purified primary human peripheral pDCs are permissive to RRV infection, and that pDCs retain functionality following RRV stimulus. Additionally, this study demonstrates trypsin-independent infection of primary peripheral cells by rotavirus, which may allow for the establishment of extraintestinal viremia and antigenemia. Importantly, these data provide the first evidence of IFNalpha induction in primary human pDCs by a dsRNA virus, while simultaneously demonstrating impaired IFNalpha production in primary human cells in which RRV replicates. Rotavirus infection of primary human pDCs provides a powerful experimental system for the study of mechanisms underlying pDC-mediated innate immunity to viral infection and reveals a potentially novel dsRNA-dependent pathway of IFNalpha induction.

  View details for DOI 10.1371/journal.ppat.1000931

  View details for Web of Science ID 000279806300009

  View details for PubMedID 20532161

  View details for PubMedCentralID PMC2880586

• Differential Transcriptional Responses to Interferon-alpha and Interferon-gamma in Primary Human Hepatocytes JOURNAL OF INTERFERON AND CYTOKINE RESEARCH He, X., Nanda, S., Ji, X., Calderon-Rodriguez, G. M., Greenberg, H. B., Liang, T. J. 2010; 30 (5): 311-320

  Abstract

  Interferon (IFN) plays a central role in the innate and adaptive antiviral immune responses. While IFN-alpha is currently approved for treating chronic hepatitis B and hepatitis C, in limited studies, IFN-gamma has not been shown to be effective for chronic hepatitis B or C. To identify the potential mechanism underlying the differential antiviral effects of IFN-alpha and IFN-gamma, we used cDNA microarray to profile the global transcriptional response to IFN-alpha and IFN-gamma in primary human hepatocytes, the target cell population of hepatitis viruses. Our results reveal distinct patterns of gene expression induced by these 2 cytokines. Overall, IFN-alpha induces more genes than IFN-gamma at the transcriptional level. Distinct sets of genes were induced by IFN-alpha and IFN-gamma with limited overlaps. IFN-alpha induces gene transcription at an early time point (6 h) but not at a later time point (18 h), while the effects of IFN-gamma are more prominent at 18 h than at 6 h, suggesting a delayed transcriptional response to IFN-gamma in the hepatocytes. These findings indicate differential actions of IFN-alpha and IFN-gamma in the context of therapeutic intervention for chronic viral infections in the liver.

  View details for DOI 10.1089/jir.2009.0082

  View details for Web of Science ID 000277723600005

  View details for PubMedID 20038212

  View details for PubMedCentralID PMC2947462

• Rotavirus Differentially Infects and Polyclonally Stimulates Human B Cells Depending on Their Differentiation State and Tissue of Origin JOURNAL OF VIROLOGY Narvaez, C. F., Franco, M. A., Angel, J., Morton, J. M., Greenberg, H. B. 2010; 84 (9): 4543-4555

  Abstract

  We have shown previously that rotavirus (RV) can infect murine intestinal B220(+) cells in vivo (M. Fenaux, M. A. Cuadras, N. Feng, M. Jaimes, and H. B. Greenberg, J. Virol. 80:5219-5232, 2006) and human blood B cells in vitro (M. C. Mesa, L. S. Rodriguez, M. A. Franco, and J. Angel, Virology 366:174-184, 2007). However, the effect of RV on B cells, especially those present in the human intestine, the primary site of RV infection, is unknown. Here, we compared the effects of the in vitro RV infection of human circulating (CBC) and intestinal B cells (IBC). RV infected four times more IBC than CBC, and in both types of B cells the viral replication was highly restricted to the memory subset. RV induced cell death in 30 and 3% of infected CBC and IBC, respectively. Moreover, RV induced activation and differentiation into antibody-secreting cells (ASC) of CBC but not IBC when the B cells were present with other mononuclear cells. However, RV did not induce these effects in purified CBC or IBC, suggesting the participation of other cells in activating and differentiating CBC. RV infection was associated with enhanced interleukin-6 (IL-6) production by CBC independent of viral replication. The infection of the anti-B-cell receptor, lipopolysaccharide, or CpG-stimulated CBC reduced the secretion of IL-6 and IL-8 and decreased the number of ASC. These inhibitory effects were associated with an increase in viral replication and cell death and were observed in polyclonally stimulated CBC but not in IBC. Thus, RV differentially interacts with primary human B cells depending on their tissue of origin and differentiation stage, and it affects their capacity to modulate the local and systemic immune responses.

  View details for DOI 10.1128/JVI.02550-09

  View details for Web of Science ID 000276358000040

  View details for PubMedID 20164228

  View details for PubMedCentralID PMC2863723

• VP5*Rearranges when Rotavirus Uncoats JOURNAL OF VIROLOGY Yoder, J. D., Trask, S. D., Vo, P. T., Binka, M., Feng, N., Harrison, S. C., Greenberg, H. B., Dormitzer, P. R. 2009; 83 (21): 11372-11377

  Abstract

  Trypsin primes rotavirus for efficient infectivity by cleaving the spike protein, VP4, into VP8* and VP5*. A recombinant VP5* fragment has a trimeric, folded-back structure. Comparison of this structure with virion spikes suggests that a rearrangement, analogous to those of enveloped virus fusion proteins, may mediate membrane penetration by rotavirus during entry. To detect this inferred rearrangement of virion-associated authentic VP5*, we raised conformation-specific monoclonal antibodies against the recombinant VP5* fragment in its putative post-membrane penetration conformation. Using one of these antibodies, we demonstrate that rotavirus uncoating triggers a conformational change in the cleaved VP4 spike to yield rearranged VP5*.

  View details for DOI 10.1128/JVI.01228-09

  View details for Web of Science ID 000270602300049

  View details for PubMedID 19692464

  View details for PubMedCentralID PMC2772785

• IRF3 Inhibition by Rotavirus NSP1 Is Host Cell and Virus Strain Dependent but Independent of NSP1 Proteasomal Degradation JOURNAL OF VIROLOGY Sen, A., Feng, N., Ettayebi, K., Hardy, M. E., Greenberg, H. B. 2009; 83 (20): 10322-10335

  Abstract

  Rotavirus host range restriction forms a basis for strain attenuation although the underlying mechanisms are unclear. In mouse fibroblasts, the inability of rotavirus NSP1 to mediate interferon (IFN) regulatory factor 3 (IRF3) degradation correlates with IFN-dependent restricted replication of the bovine UK strain but not the mouse EW and simian RRV strains. We found that UK NSP1 is unable to degrade IRF3 when expressed in murine NIH 3T3 cells in contrast to the EW and RRV NSP1 proteins. Surprisingly, UK NSP1 expression led to IRF3 degradation in simian COS7 cells, indicating that IRF3 degradation by NSP1 is host cell dependent, a finding further supported using adenovirus-expressed NSP1 from NCDV bovine rotavirus. By expressing heterologous IRF3 proteins in complementary host cells, we found that IRF3 is the minimal host factor constraining NSP1 IRF3-degradative ability. NSP1-mediated IRF3 degradation was enhanced by transfection of double-stranded RNA (dsRNA) in a host cell-specific manner, and in IRF3-dependent positive regulatory domain III reporter assays, NSP1 inhibited IRF3 function in response to pathway activation by dsRNA, TBK-1, IRF3, or constitutively activated IRF3-5D. An interesting observation arising from these experiments is the ability of transiently expressed UK NSP1 to inhibit poly(I:C)-directed IRF3 activity in NIH 3T3 cells in the absence of detectable IRF3 degradation, an unexpected finding since UK virus infection was unable to block IFN secretion, and UK NSP1 expression did not result in suppression of IRF3-directed activation of the pathway. RRV and EW but not UK NSP1 was proteasomally degraded, requiring E1 ligase activity, although NSP1 degradation was not required for IRF3 degradation. Using a chimeric RRV NSP1 protein containing the carboxyl 100 residues derived from UK NSP1, we found that the RRV NSP1 carboxyl 100 residues are critical for its IRF3 inhibition in murine cells but are not essential for NSP1 degradation. Thus, NSP1's ability to degrade IRF3 is host cell dependent and is independent of NSP1 proteasomal degradation.

  View details for DOI 10.1128/JVI.01186-09

  View details for Web of Science ID 000270121600001

  View details for PubMedID 19656876

  View details for PubMedCentralID PMC2753142

• Variation in Antagonism of the Interferon Response to Rotavirus NSP1 Results in Differential Infectivity in Mouse Embryonic Fibroblasts JOURNAL OF VIROLOGY Feng, N., Sen, A., Nguyen, H., Vo, P., Hoshino, Y., Deal, E. M., Greenberg, H. B. 2009; 83 (14): 6987-6994

  Abstract

  Rotavirus NSP1 has been shown to function as an E3 ubiquitin ligase that mediates proteasome-dependent degradation of interferon (IFN) regulatory factors (IRF), including IRF3, -5, and -7, and suppresses the cellular type I IFN response. However, the effect of rotavirus NSP1 on viral replication is not well defined. Prior studies used genetic analysis of selected reassortants to link NSP1 with host range restriction in the mouse, suggesting that homologous and heterologous rotaviruses might use their different abilities to antagonize the IFN response as the basis of their host tropisms. Using a mouse embryonic fibroblast (MEF) model, we demonstrate that heterologous bovine (UK and NCDV) and porcine (OSU) rotaviruses fail to effectively degrade cellular IRF3, resulting in IRF3 activation and beta IFN (IFN-beta) secretion. As a consequence of this failure, replication of these viruses is severely restricted in IFN-competent wild-type, but not in IFN-deficient (IFN-alpha/beta/gamma receptor- or STAT1-deficient) MEFs. On the other hand, homologous murine rotaviruses (ETD or EHP) or the heterologous simian rotavirus (rhesus rotavirus [RRV]) efficiently degrade cellular IRF3, diminish IRF3 activation and IFN-beta secretion and are not replication restricted in wild-type MEFs. Genetic reassortant analysis between UK and RRV maps the distinctive phenotypes of IFN antagonism and growth restriction in wild-type MEFs to NSP1. Therefore, there is a direct relationship between the replication efficiencies of different rotavirus strains in MEFs and strain-related variations in NSP1-mediated antagonism of the type I IFN response.

  View details for DOI 10.1128/JVI.00585-09

  View details for Web of Science ID 000267354300003

  View details for PubMedID 19420080

  View details for PubMedCentralID PMC2704795

• Structure of Rotavirus Outer-Layer Protein VP7 Bound with a Neutralizing Fab SCIENCE Aoki, S. T., Settembre, E. C., Trask, S. D., Greenberg, H. B., Harrison, S. C., Dormitzer, P. R. 2009; 324 (5933): 1444-1447

  Abstract

  Rotavirus outer-layer protein VP7 is a principal target of protective antibodies. Removal of free calcium ions (Ca2+) dissociates VP7 trimers into monomers, releasing VP7 from the virion, and initiates penetration-inducing conformational changes in the other outer-layer protein, VP4. We report the crystal structure at 3.4 angstrom resolution of VP7 bound with the Fab fragment of a neutralizing monoclonal antibody. The Fab binds across the outer surface of the intersubunit contact, which contains two Ca2+ sites. Mutations that escape neutralization by other antibodies suggest that the same region bears the epitopes of most neutralizing antibodies. The monovalent Fab is sufficient to neutralize infectivity. We propose that neutralizing antibodies against VP7 act by stabilizing the trimer, thereby inhibiting the uncoating trigger for VP4 rearrangement. A disulfide-linked trimer is a potential subunit immunogen.

  View details for DOI 10.1126/science.1170481

  View details for Web of Science ID 000266878700046

  View details for PubMedID 19520960

  View details for PubMedCentralID PMC2995306

• Broadening the age restriction for initiating rotavirus vaccination in regions with high rotavirus mortality: Benefits of mortality reduction versus risk of fatal intussusception VACCINE Patel, M. M., Clark, A. D., Glass, R. I., Greenberg, H., Tate, J., Santosham, M., Sanderson, C. F., Steele, D., Cortese, M., Parashar, U. D. 2009; 27 (22): 2916-2922

  Abstract

  Recently developed rotavirus vaccines have the potential to reduce diarrhea mortality in children in developing countries. Available data to date do not indicate risk of intussusception with these new vaccines. To avoid a potential unanticipated risk post-licensure, it is recommended that rotavirus immunization be initiated before 12 weeks of age when background intussusception rates are low. This policy could exclude a substantial number of children from vaccination, especially in developing countries where delays in vaccination are common.We conducted a scenario analysis to assess the potential benefits of mortality reduction from rotavirus versus the risk of fatal intussusception when the first dose of the vaccine is strictly administered by 12 weeks of age compared with a free strategy with vaccine administered before 1 year of age using data on rotavirus disease, vaccine safety and efficacy, and current diphtheria-tetanus-pertussis vaccination rates, and by incorporating hypothetical risks of intussusception.In developing countries, assuming vaccine efficacy of 50% and 75% for doses 1 and 2, respectively, and a hypothetical sixfold and threefold increased relative risk of intussusception within 7 days of doses 1 and 2, respectively, initiating rotavirus immunization before 12 weeks of age would prevent 194,564 of the 517,959 annual rotavirus-associated deaths among children <5 years, while potentially resulting in 1106 fatal intussusception events. Administration of the first dose to infants up to 1 year of age would prevent an additional 54,087 rotavirus-associated deaths (total=248,651) while potentially resulting in an additional 1226 intussusception deaths (total=2332).In developing countries, the additional lives saved by broadening the age restrictions for initiation of rotavirus vaccination would far outnumber the hypothetical excess intussusception deaths that would accompany such an approach.

  View details for DOI 10.1016/j.vaccine.2009.03.016

  View details for Web of Science ID 000266120100004

  View details for PubMedID 19428901

• Rotaviruses: From Pathogenesis to Vaccination GASTROENTEROLOGY Greenberg, H. B., Estes, M. K. 2009; 136 (6): 1939-1951

  Abstract

  Rotaviruses cause life-threatening gastroenteritis in children worldwide; the enormous disease burden has focused efforts to develop vaccines and led to the discovery of novel mechanisms of gastrointestinal virus pathogenesis and host responses to infection. Two live-attenuated vaccines for gastroenteritis (Rotateq [Merck] and Rotarix) have been licensed in many countries. This review summarizes the latest data on these vaccines, their effectiveness, and challenges to global vaccination. Recent insights into rotavirus pathogenesis also are discussed, including information on extraintestinal infection, viral antagonists of the interferon response, and the first described viral enterotoxin. Rotavirus-induced diarrhea now is considered to be a disease that can be prevented through vaccination, although there are many challenges to achieving global effectiveness. Molecular biology studies of rotavirus replication and pathogenesis have identified unique viral targets that might be useful in developing therapies for immunocompromised children with chronic infections.

  View details for DOI 10.1053/j.gastro.2009.02.076

  View details for Web of Science ID 000266090900009

  View details for PubMedID 19457420

• Characterization of rotavirus specific B cells and their relation with serological memory VIROLOGY Lucia Rojas, O., Fernando Narvaez, C., Greenberg, H. B., Angel, J., Franco, M. A. 2008; 380 (2): 234-242

  Abstract

  We quantified circulating total, rotavirus (RV) and Tetanus toxin (TT) memory B cells (mBc) in healthy adults using a limiting dilution assay (LDA) and a flow cytometry assay (FCA) that permit evaluation of both CD27+ and CD27- mBc. RV mBc were enriched in the CD27-, IgG+ and in the CD27+, IgM+ subsets. The numbers of RV mBc were higher by FCA than by LDA and results of the two assays did not correlate. TT IgGmBc and RV IgA mBc determined by FCA and by LDA correlated with TT plasma IgG and RV plasma IgA, respectively. The mean ratio of specific mBc/mug/ml of the corresponding plasma immunoglobulin was lower for TT IgG than for RV IgA mBc. Our studies contribute to understand the relationship between circulating mBc and serological memory, and enhance our capacity to develop better correlates of protection against RV disease.

  View details for DOI 10.1016/j.virol.2008.08.004

  View details for Web of Science ID 000260148400009

  View details for PubMedID 18789807

• The influence of CD4(+) CD25(+) Foxp3(+) regulatory T cells on the immune response to rotavirus infection VACCINE Kim, B., Feng, N., Narvaez, C. F., He, X., Eo, S. K., Lim, C. W., Greenberg, H. B. 2008; 26 (44): 5601-5611

  Abstract

  Rotavirus (RV) infection of the intestine is the major cause of severe dehydrating diarrhea in infants around the world. Although protective immunity against RV, especially acquired B and T-cell responses, has been extensively studied, our understanding of RV immunity remains incomplete. In addition, the interaction between various protective immune mechanisms in the gut and specific enteric immune suppressor systems that normally exert a regulatory function on mucosal immunity has not been extensively investigated. Among the candidate suppressor systems, we hypothesized that CD4+ CD25+ Foxp3+ regulatory T (Treg) cells may play a role in modulating RV immunity since such cells are naturally present in large numbers in the intestine and function nonspecifically. Here we demonstrate that neonatal murine RV (EC) infection induces an expansion of the Treg cell population and the magnitude of the T cell mediated immune response is modulated by Treg cells. Accordingly, when natural Treg cells in neonatal mice were depleted before virus infection, both CD4+ and CD8+ T-cell responses to RV, such as proliferation and IFN-gamma secretion, were enhanced in mesenteric lymph nodes (MLNs) and the spleen. Interestingly, increased proliferation of CD19+ B cells from Treg cell depleted animals was also observed. Finally, we analyzed the in vivo effect of the Treg cell depletion on diarrheal disease, virus shedding and IgA RV-specific response. Treg cell depletion did not affect these functions. Our studies of immune modulatory Treg cells in the RV infection model may promote a better understanding of the basis for RV immunity as well as providing valuable clues for the development of more immunogenic RV vaccines.

  View details for DOI 10.1016/j.vaccine.2008.07.099

  View details for Web of Science ID 000260286200009

  View details for PubMedID 18725261

  View details for PubMedCentralID PMC2574936

• Influence of Prior Influenza Vaccination on Antibody and B-Cell Responses PLOS ONE Sasaki, S., He, X., Holmes, T. H., Dekker, C. L., Kemble, G. W., Arvin, A. M., Greenberg, H. B. 2008; 3 (8)

  Abstract

  Currently two vaccines, trivalent inactivated influenza vaccine (TIV) and live attenuated influenza vaccine (LAIV), are licensed in the USA. Despite previous studies on immune responses induced by these two vaccines, a comparative study of the influence of prior influenza vaccination on serum antibody and B-cell responses to new LAIV or TIV vaccination has not been reported. During the 2005/6 influenza season, we quantified the serum antibody and B-cell responses to LAIV or TIV in adults with differing influenza vaccination histories in the prior year: LAIV, TIV, or neither. Blood samples were collected on days 0, 7-9 and 21-35 after immunization and used for serum HAI assay and B-cell assays. Total and influenza-specific circulating IgG and IgA antibody secreting cells (ASC) in PBMC were detected by direct ELISPOT assay. Memory B cells were also tested by ELISPOT after polyclonal stimulation of PBMC in vitro. Serum antibody, effector, and memory B-cell responses were greater in TIV recipients than LAIV recipients. Prior year TIV recipients had significantly higher baseline HAI titers, but lower HAI response after vaccination with either TIV or LAIV, and lower IgA ASC response after vaccination with TIV than prior year LAIV or no vaccination recipients. Lower levels of baseline HAI titer were associated with a greater fold-increase of HAI titer and ASC number after vaccination, which also differed by type of vaccine. Our findings suggest that the type of vaccine received in the prior year affects the serum antibody and the B-cell responses to subsequent vaccination. In particular, prior year TIV vaccination is associated with sustained higher HAI titer one year later but lower antibody response to new LAIV or TIV vaccination, and a lower effector B-cell response to new TIV but not LAIV vaccination.

  View details for DOI 10.1371/journal.pone.0002975

  View details for Web of Science ID 000264420900002

  View details for PubMedID 18714352

  View details for PubMedCentralID PMC2500171

• Role of interferon in homologous and heterologous rotavirus infection in the intestines and extraintestinal organs of suckling mice JOURNAL OF VIROLOGY Feng, N., Kim, B., Fenaux, M., Nguyen, H., Vo, P., Omary, M. B., Greenberg, H. B. 2008; 82 (15): 7578-7590

  Abstract

  Recent studies demonstrated that viremia and extraintestinal rotavirus infection are common in acutely infected humans and animals, while systemic diseases appear to be rare. Intraperitoneal infection of newborn mice with rhesus rotavirus (RRV) results in biliary atresia (BA), and this condition is influenced by the host interferon response. We studied orally inoculated 5-day-old suckling mice that were deficient in interferon (IFN) signaling to evaluate the role of interferon on the outcome of local and systemic infection after enteric inoculation. We found that systemic replication of RRV, but not murine rotavirus strain EC, was greatly enhanced in IFN-alpha/beta and IFN-gamma receptor double-knockout (KO) or STAT1 KO mice but not in mice deficient in B- or T-cell immunity. The enhanced replication of RRV was associated with a lethal hepatitis, pancreatitis, and BA, while no systemic disease was observed in strain EC-infected interferon-deficient mice. In IFN-alpha/beta receptor KO mice the extraintestinal infection and systemic disease were only moderately increased, while RRV infection was not augmented and systemic disease was not present in IFN-gamma receptor KO mice. The increase of systemic infection in IFN-deficient mice was also observed during simian strain SA11 infection but not following bovine NCDV, porcine OSU, or murine strain EW infection. Our data indicate that the requirements for the interferon system to inhibit intestinal and extraintestinal viral replication in suckling mice vary among different heterologous and homologous rotavirus strains, and this variation is associated with lethal systemic disease.

  View details for DOI 10.1128/JVI.00391-08

  View details for Web of Science ID 000257770500032

  View details for PubMedID 18495762

  View details for PubMedCentralID PMC2493311

• Baseline Levels of Influenza-Specific CD4 Memory T-Cells Affect T-Cell Responses to Influenza Vaccines PLOS ONE He, X., Holmes, T. H., Sasaki, S., Jaimes, M. C., Kemble, G. W., Dekker, C. L., Arvin, A. M., Greenberg, H. B. 2008; 3 (7)

  Abstract

  Factors affecting immune responses to influenza vaccines have not been studied systematically. We hypothesized that T-cell and antibody responses to the vaccines are functions of pre-existing host immunity against influenza antigens.During the 2004 and 2005 influenza seasons, we have collected data on cellular and humoral immune reactivity to influenza virus in blood samples collected before and after immunization with inactivated or live attenuated influenza vaccines in healthy children and adults. We first used cross-validated lasso regression on the 2004 dataset to identify a group of candidate baseline correlates with T-cell and antibody responses to vaccines, defined as fold-increase in influenza-specific T-cells and serum HAI titer after vaccination. The following baseline parameters were examined: percentages of influenza-reactive IFN-gamma(+) cells in T and NK cell subsets, percentages of influenza-specific memory B-cells, HAI titer, age, and type of vaccine. The candidate baseline correlates were then tested with the independent 2005 dataset. Baseline percentage of influenza-specific IFN-gamma(+) CD4 T-cells was identified as a significant correlate of CD4 and CD8 T-cell responses, with lower baseline levels associated with larger T-cell responses. Baseline HAI titer and vaccine type were identified as significant correlates for HAI response, with lower baseline levels and the inactivated vaccine associated with larger HAI responses. Previously we reported that baseline levels of CD56(dim) NK reactivity against influenza virus inversely correlated with the immediate T-cell response to vaccination, and that NK reactivity induced by influenza virus depended on IL-2 produced by influenza-specific memory T-cells. Taken together these results suggest a novel mechanism for the homeostasis of virus-specific T-cells, which involves interaction between memory helper T-cells, CD56(dim) NK and DC.These results demonstrate that assessment of baseline biomarkers may predict immunologic outcome of influenza vaccination and may reveal some of the mechanisms responsible for variable immune responses following vaccination and natural infection.

  View details for DOI 10.1371/journal.pone.0002574

  View details for Web of Science ID 000263288200039

  View details for PubMedID 18596908

  View details for PubMedCentralID PMC2440350

• Qualitative and quantitative characteristics of rotavirus-specific CD8 T cells vary depending on the route of infection JOURNAL OF VIROLOGY Jiang, J. Q., He, X., Feng, N., Greenberg, H. B. 2008; 82 (14): 6812-6819

  Abstract

  CD8 T-cell response provides an important defense against rotavirus, which infects a variety of systemic locations in addition to the gut. Here we investigated the distribution, phenotype, and function of rotavirus-specific CD8 T cells in multiple organs after rotavirus infection initiated via the intranasal, oral, or intramuscular route. The highest level of virus-specific CD8 T cells was observed in the Peyer's patches of orally infected mice and in the lungs of intranasally infected animals. Very low levels of virus-specific CD8 T cells were detected in peripheral blood or spleen irrespective of the route of infection. Rotavirus-specific CD8 T cells from Peyer's patches of orally infected mice expressed high levels of CCR9, while CXCR6 and LFA-1 expression was associated with virus-specific CD8 T cells in lungs of intranasally infected mice. Oral infection induced the highest proportion of gamma interferon(-) CD107a/b(+) CD8 T cells in Peyer's patches. When equal numbers of rotavirus-specific CD8 T cells were transferred into Rag-1 knockout mice chronically infected with rotavirus, the donor cells derived from Peyer's patches of orally infected mice were more efficient than those derived from lungs of intranasally infected animals in clearing intestinal infection. These results suggest that different routes of infection induce virus-specific CD8 T cells with distinct homing phenotypes and effector functions as well as variable abilities to clear infection.

  View details for DOI 10.1128/JVI.00450-08

  View details for Web of Science ID 000257545300003

  View details for PubMedID 18480435

  View details for PubMedCentralID PMC2446946

• Phenotypic changes in influenza-specific CD8(+) T cells after immunization of children and adults with influenza vaccines 26th Annual Meeting of the American-Society-for-Virology He, X., Holmes, T. H., Mahmood, K., Kemble, G. W., Dekker, C. L., Arvin, A. M., Greenberg, H. B. UNIV CHICAGO PRESS. 2008: 803–11

  Abstract

  The effect of trivalent inactivated influenza vaccine (TIV) or live attenuated influenza vaccine (LAIV) on the phenotypes of circulating influenza-specific CD8+ T cells was analyzed by interferon (IFN)-gamma flow cytometry and tetramer staining. In adults, the expression of the T cell differentiation marker CD27 on virus-specific CD8+ T cells decreased after LAIV but increased after TIV. In children, expression of the cytotoxicity molecule perforin in influenza-specific CD8+ T cells increased after TIV but not after LAIV. Among children aged 6 months to 4 years who had not been vaccinated previously and who received 2 doses of TIV, CD27 expression decreased after each dose, whereas perforin expression increased after the second dose. These findings indicate that the phenotypic changes of influenza-specific CD8+ T cells differ depending on the type of vaccine and the age of the vaccinee. These differences are potentially affected by the different routes of vaccination and pathways of antigen presentation for TIV and LAIV.

  View details for DOI 10.1086/528804

  View details for Web of Science ID 000253773900005

  View details for PubMedID 18279048

• Reg-II is an exocrine pancreas injury-response product that is up-regulated by keratin absence or mutation MOLECULAR BIOLOGY OF THE CELL Zhong, B., Strnad, P., Toivola, D. M., Tao, G., Ji, X., Greenberg, H. B., Omary, M. B. 2007; 18 (12): 4969-4978

  Abstract

  The major keratins in the pancreas and liver are keratins 8 and 18 (K8/K18), but their function seemingly differs in that liver K8/K18 are essential cytoprotective proteins, whereas pancreatic K8/K18 are dispensable. This functional dichotomy raises the hypothesis that K8-null pancreata may undergo compensatory cytoprotective gene expression. We tested this hypothesis by comparing the gene expression profile in pancreata of wild-type and K8-null mice. Most prominent among the up-regulated genes in K8-null pancreas was mRNA for regenerating islet-derived (Reg)-II, which was confirmed by quantitative reverse transcription-polymerase chain reaction and by an anti-Reg-II peptide antibody we generated. Both K8-null and wild-type mice express Reg-II predominantly in acinar cells as determined by in situ hybridization and immunostaining. Analysis of Reg-II expression in various keratin-related transgenic mouse models showed that its induction also occurs in response to keratin cytoplasmic filament collapse, absence, or ablation of K18 Ser52 but not Ser33 phosphorylation via Ser-to-Ala mutation, which represent situations associated with predisposition to liver but not pancreatic injury. In wild-type mice, Reg-II is markedly up-regulated in two established pancreatitis models in response to injury and during the recovery phase. Thus, Reg-II is a likely mouse exocrine pancreas cytoprotective candidate protein whose expression is regulated by keratin filament organization and phosphorylation.

  View details for DOI 10.1091/mbc.E07-02-0180

  View details for Web of Science ID 000251660800026

  View details for PubMedID 17898082

• Rotavirus vaccines: recent developments and future considerations NATURE REVIEWS MICROBIOLOGY Angel, J., Franco, M. A., Greenberg, H. B. 2007; 5 (7): 529-U18

  Abstract

  Two new vaccines have recently been shown to be safe and effective in protecting young children against severe rotavirus gastroenteritis. Although both vaccines are now marketed worldwide, it is likely that improvements to these vaccines and/or the development of future generations of rotavirus vaccines will be desirable. This Review addresses recent advances in our knowledge of rotavirus, the host immune response to rotavirus infection and the efficacy and safety of the new vaccines that will be helpful for improving the existing rotavirus vaccines, or developing new rotavirus vaccines in the future.

  View details for DOI 10.1038/nrmicro1692

  View details for Web of Science ID 000247344600015

  View details for PubMedID 17571094

• Humoral and cellular immune responses in children given annual immunization with trivalent inactivated influenza vaccine PEDIATRIC INFECTIOUS DISEASE JOURNAL Zeman, A. M., Holmes, T. H., Stamatis, S., Tu, W., He, X., Bouvier, N., Kemble, G., Greenberg, H. B., Lewis, D. B., Arvin, A. M., Dekker, C. L. 2007; 26 (2): 107-115

  Abstract

  There have been no prior reports of the frequency of circulating influenza-specific, interferon gamma-producing memory CD4+ and CD8+ T-cells in healthy children who have received multiple influenza immunizations.We evaluated 21 previously immunized children, ages 3 to 9 years, before and 1 month after administration of trivalent inactivated influenza vaccine. Frequencies of influenza-specific CD4+ and CD8+ T-cells stimulated with trivalent inactivated influenza vaccine or A/Panama (H3N2) virus were determined by flow cytometry, and antibody responses to vaccine strains and a drifted H3N2 strain were measured by hemagglutination inhibition assay and neutralizing antibody assays.Mean change in CD4+ and in CD8+ T-cell frequencies after immunization was 0.01% (P > 0.39) with postimmunization CD4+ frequencies higher than CD8+ frequencies. Children with more previous vaccinations had a higher baseline frequency of CD4+ T-cells (P = 0.0002) but a smaller increase or even a decline from baseline after immunization (P = 0.003). An association between age and change in frequency was not detected. Baseline geometric mean titers (GMTs) and seroprotection rates were significantly higher in older children against A/Panama (neutralizing baseline GMT, P = 0.0488) and A/New Caledonia (hemagglutination inhibition baseline GMT and seroprotection, P < 0.0297). Baseline GMTs against B/Hong Kong were not associated with age or quantity of prior vaccinations.These findings suggest that children may plateau in CD4+ T-cell responses to influenza antigens with repeated exposures and that the number of exposures may play a large role in building a memory CD4+ T-cell response to influenza A, perhaps independently from age.

  View details for DOI 10.1097/01.inf.0000253251.03785.9b

  View details for Web of Science ID 000243985700002

  View details for PubMedID 17259871

• Comparison of the influenza virus-specific effector and memory C-cell responses to immunization of children and adults with live attenuated or inactivated influenza virus vaccines JOURNAL OF VIROLOGY Sasaki, S., Jaimes, M. C., Holmes, T. H., Dekker, C. L., Mahmood, K., Kemble, G. W., Arvin, A. M., Greenberg, H. B. 2007; 81 (1): 215-228

  Abstract

  Cellular immune responses to influenza virus infection and influenza virus vaccination have not been rigorously characterized. We quantified the effector and memory B-cell responses in children and adults after administration of either live attenuated (LAIV) or inactivated (TIV) influenza virus vaccines and compared these to antibody responses. Peripheral blood mononuclear cells were collected at days 0, 7 to 12, and 27 to 42 after immunization of younger children (6 months to 4 years old), older children (5 to 9 years old), and adults. Influenza virus-specific effector immunoglobulin A (IgA) and IgG circulating antibody-secreting cells (ASC) and stimulated memory B cells were detected using an enzyme-linked immunospot assay. Circulating influenza virus-specific IgG and IgA ASC were detected 7 to 12 days after TIV and after LAIV immunization. Seventy-nine percent or more of adults and older children had demonstrable IgG ASC responses, while IgA ASC responses were detected in 29 to 53% of the subjects. The IgG ASC response rate to LAIV immunization in adults was significantly higher than the response rate measured by standard serum antibody assays (26.3% and 15.8% by neutralization and hemagglutination inhibition assays, respectively). IgG ASC and serum antibody responses were relatively low in the younger children compared to older children and adults. TIV, but not LAIV, significantly increased the percentage of circulating influenza virus-specific memory B cells detected at 27 to 42 days after immunization in children and adults. In conclusion, although both influenza vaccines are effective, we found significant differences in the B-cell and antibody responses elicited after LAIV or TIV immunization in adults and older children and between young children and older age groups.

  View details for DOI 10.1128/JVI.01957-06

  View details for Web of Science ID 000242958600020

  View details for PubMedID 17050593

  View details for PubMedCentralID PMC1797237

• Cellular immune responses in children and adults receiving inactivated or live attenuated influenza vaccines JOURNAL OF VIROLOGY He, X., Holmes, T. H., Zhang, C., Mahmood, K., Kemble, G. W., Lewis, D. B., Dekker, C. L., Greenberg, H. B., Arvin, A. M. 2006; 80 (23): 11756-11766

  Abstract

  The patterns of cellular immune responses induced by live attenuated influenza vaccine (LAIV) versus those of the trivalent inactivated influenza vaccine (TIV) have not been studied extensively, especially in children. The goals of this study were to evaluate the effects of TIV and LAIV immunization on cellular immunity to live influenza A virus in children and adults and to explore factors associated with variations in responses to influenza vaccines among individuals. A gamma interferon (IFN-gamma) flow cytometry assay was used to measure IFN-gamma-producing (IFN-gamma+) NK and T cells in peripheral blood mononuclear cell cultures stimulated with a live influenza A virus strain before and after LAIV or TIV immunization of children and adults. The mean percentages of influenza A virus-specific IFN-gamma+ CD4 and CD8 T cells increased significantly after LAIV, but not TIV, immunization in children aged 5 to 9 years. No increases in the mean levels of influenza A virus-reactive IFN-gamma+ T cells and NK cells were observed in adults given LAIV or TIV. TIV induced a significant increase in influenza A virus-reactive T cells in 6-month- to 4-year-old children; LAIV was not evaluated in this age group. The postvaccination changes (n-fold) in the percentages of influenza A virus-reactive IFN-gamma+ T and NK cells in adults were highly variable and correlated inversely with the prevaccination percentages, in particular with that of the CD56(dim) NK cell subset. In conclusion, our findings identify age, type of vaccine, and prevaccination levels of immune reactivity to influenza A virus as factors significantly associated with the magnitude of cellular immune responses to influenza vaccines.

  View details for DOI 10.1128/JVI.01460-06

  View details for Web of Science ID 000242222200032

  View details for PubMedID 16971435

  View details for PubMedCentralID PMC1642596

• Rotavirus anti-VP6 secretory immunoglobulin a contributes to protection via intracellular neutralization but not via immune exclusion JOURNAL OF VIROLOGY Corthesy, B., Benureau, Y., Perrier, C., Fourgeux, C., Parez, N., Greenberg, H., Schwartz-Cornil, I. 2006; 80 (21): 10692-10699

  Abstract

  Immunoglobulin A (IgA) monoclonal antibodies (MAbs) directed at the conserved inner core protein VP6 of rotavirus, such as the IgA7D9 MAb, provide protective immunity in adult and suckling mice when delivered systemically. While these antibodies do not have traditional in vitro neutralizing activity, they could mediate their antiviral activity either by interfering with the viral replication cycle along the IgA secretory pathway or by acting at mucosal surfaces as secretory IgA and excluding virus from target enterocytes. We sought to determine the critical step at which antirotaviral activity was initiated by the IgA7D9 MAb. The IgA7D9 MAb appeared to directly interact with purified triple-layer viral particles, as shown by immunoprecipitation and immunoblotting. However, protection was not conferred by passively feeding mice with the secretory IgA7D9 MAb. This indicates that the secretory IgA7D9 MAb does not confer protection by supplying immune exclusion activity in vivo. We next evaluated the capacity of polymeric IgA7D9 MAb to neutralize rotavirus intracellularly during transcytosis. We found that when polymeric IgA7D9 MAb was applied to the basolateral pole of polarized Caco-2 intestinal cells, it significantly reduced viral replication and prevented the loss of barrier function induced by apical exposure of the cell monolayer to rotavirus, supporting the conclusion that the antibody carries out its antiviral activity intracellularly. These findings identify a mechanism whereby the well-conserved immunodominant VP6 protein can function as a target for heterotypic antibodies and protective immunity.

  View details for DOI 10.1128/JVI.00927-06

  View details for Web of Science ID 000241606100039

  View details for PubMedID 16956954

• Phenotypic and functional status of intrahepatic T cells in chronic hepatitis C 10th International Meeting on Hepatitis C Virus and Related Viruses Wang, J., Holmes, T. H., de Guevara, L. L., Cheung, R., Wright, T. L., He, X., Greenberg, H. B. OXFORD UNIV PRESS INC. 2006: 1068–77

  Abstract

  Polychromatic flow-cytometric assays were used to analyze paired intrahepatic and peripheral lymphocyte samples from 37 patients with chronic hepatitis C. Compared with peripheral cells, intrahepatic T cells were selectively enriched with CD45RO+ memory T cells but had a lower percentage of CD4+ T cells expressing the differentiation markers CD27 and CD28. The percentage of intrahepatic CD45RO+ and CD28+ T cells correlated with the degree of liver inflammation, which suggests that memory T cells at relatively early stages of differentiation are directly involved in liver inflammation. Despite their memory phenotype, intrahepatic T cells were defective in proliferation capability, produced less interferon- gamma in response to stimulation by T cell receptor, and contained less perforin but expressed higher levels of Fas and Fas ligand, compared with their counterparts in peripheral blood. The distinct characteristics of intrahepatic T cells suggest that they play an important role in the immunopathogenesis of chronic hepatitis C.

  View details for Web of Science ID 000240548500007

  View details for PubMedID 16991081

• Quantitative evaluation of rotaviral antigenemia in children with acute rotaviral diarrhea 13th International Congress of Virology Ray, P., Fenaux, M., Sharma, S., Malik, J., Subodh, S., Bhatnagar, S., Greenberg, H., Glass, R. I., Gentsch, J., Bhan, M. K. UNIV CHICAGO PRESS. 2006: 588–93

  Abstract

  Rotaviral antigen and RNA have recently been identified in the serum of patients with rotaviral gastroenteritis, but the roles they play in disease remains undetermined.Rotaviral antigen and RNA were quantified by enzyme-linked immunosorbant assay and by quantitative reverse-transcription polymerase chain reaction in stool and serum specimens from children with rotaviral diarrhea (n=102), children with nonrotaviral diarrhea (n=40), and nondiarrheal control children (n=30).Rotaviral antigenemia was detected in 64%, 3%, and 0% of the children with rotaviral diarrhea, the children with nonrotaviral diarrhea, and the nondiarrheal control children, respectively. The level of rotaviral antigen in serum was approximately 2x10(2) -fold lower than that in stool, and a moderate correlation was observed between the 2 levels. Rotaviral RNA was detected in 93% of the antigen-positive serum specimens. The median number of RNA copies in serum was approximately 1 x 10(5) -fold lower than that in stool, and no correlation was observed between the 2 levels. Serum levels of both antigen and RNA were inversely associated with baseline titers of rotaviral serum immunoglobulin G (P<.01). Antigenemia was also associated with G1 serotype.Rotaviral antigenemia and viremia were common in children with rotaviral diarrhea, but antigen and RNA levels in serum were substantially lower than those in stool. Antigenemia was associated with infection with G1 strains and with low baseline titers of rotaviral serum antibody.

  View details for Web of Science ID 000240092800009

  View details for PubMedID 16897656

• Global transcriptional response to interferon is a determinant of HCV treatment outcome and is modified by race HEPATOLOGY He, X., Ji, X., Hale, M. B., Cheung, R., Ahmed, A., Guo, Y., Nolan, G. P., Pfeffer, L. M., Wright, T. L., Risch, N., Tibshirani, R., Greenberg, H. B. 2006; 44 (2): 352-359

  Abstract

  Interferon (IFN)-alpha-based therapy for chronic hepatitis C is effective in fewer than 50% of all treated patients, with a substantially lower response rate in black patients. The goal of this study was to investigate the underlying host transcriptional response associated with interferon treatment outcomes. We collected peripheral blood mononuclear cells from chronic hepatitis C patients before initiation of IFN-alpha therapy and incubated the cells with or without IFN-alpha for 6 hours, followed by microarray assay to identify IFN-induced gene transcription. The microarray datasets were analyzed statistically according to the patients' race and virological responses to subsequent IFN-alpha treatment. The global induction of IFN-stimulated genes (ISGs) was significantly greater in sustained virological responders compared with nonresponders and in white patients compared with black patients. In addition, a significantly greater global induction of ISGs was observed in sustained virological responders compared with nonresponders within the group of white patients. The level of IFN-induced signal transducer and activator of transcription (STAT) 1 activation, a key component of the Janus kinase (JAK)-STAT signaling pathway, correlated with the global induction of ISGs and was significantly higher in white patients than in black patients. In conclusion, both treatment outcome and race are associated with different transcriptional responses to IFN-alpha. Because this difference is evident in the global induction of ISGs rather than a selective effect on a subset of such genes, key factors affecting the outcome of IFN-alpha therapy are likely to act at the JAK-STAT pathway that controls transcription of downstream ISGs.

  View details for DOI 10.1002/hep.21267

  View details for PubMedID 16871572

• Safety and immunogenicity of two live attenuated human rotavirus vaccine candidates, 116E and I321, in infants: Results of a randomised controlled trial VACCINE Bhandari, N., Sharma, P., Glass, R. I., Ray, P., Greenberg, H., Taneja, S., Saksena, M., Rao, C. D., Gentsch, J. R., Parashar, U., Maldonado, Y., Ward, R. L., Bhan, M. K. 2006; 24 (31-32): 5817-5823

  Abstract

  We evaluated safety and immunogenicity of two orally administered human rotavirus vaccine candidates 116E and I321. Ninety healthy infants aged 8 weeks received a single dose of 116E (10(5)FFu (florescence focus units)), I321 (10(5)FFu) or placebo. There were no significant differences in the number of adverse events. Fever was reported by 6/30, 1/30 and 5/30 in the 116E, I321 and placebo groups; the corresponding figures for diarrhoea were 5/30, 8/29 and 3/30. Serum IgA seroconversion rates were 73%, 39% and 20% in the 116E, I321 and placebo groups, respectively. Vaccine virus was shed on days 3, 7 or 28 in 11/30 infants of the 116E and none in the other two groups. The 116E strain is attenuated, clinically safe and highly immunogenic with a single dose.

  View details for DOI 10.1016/j.vaccine.2006.05.001

  View details for Web of Science ID 000239470000008

  View details for PubMedID 16735085

• Active viremia in rotavirus-infected mice JOURNAL OF VIROLOGY Blutt, S. E., Fenaux, M., Warfield, K. L., Greenberg, H. B., Conner, M. E. 2006; 80 (13): 6702-6705

  Abstract

  Rotavirus circulates extraintestinally in animals used as models for rotavirus infection and in children. Rotavirus infection in mice was used to define host or viral factors that affect rotavirus viremia. Antigenemia was observed with homologous and heterologous rotaviruses, and neither age nor mouse strain genetics altered the occurrence of rotavirus antigenemia or viremia. Rotavirus RNA and infectious virus were present in sera and associated with the plasma fraction of blood in all infected mice. These findings indicate that antigenemia/viremia occurs routinely in rotavirus infections and imply that infectious rotavirus has access to any extraintestinal cell within contact of blood.

  View details for DOI 10.1128/JVI.00329-06

  View details for Web of Science ID 000238380700053

  View details for PubMedID 16775359

• Extraintestinal spread and replication of a homologous EC rotavirus strain and a heterologous rhesus rotavirus in BALB/c mice JOURNAL OF VIROLOGY Fenaux, M., Cuadras, A. A., Feng, N., Jaimes, M., Greenberg, H. B. 2006; 80 (11): 5219-5232

  Abstract

  Although rotavirus infection has generally been felt to be restricted to the gastrointestinal tract, over the last two decades there have been sporadic reports of children with acute or fatal cases of rotavirus gastroenteritis testing positive for rotavirus antigen and/or nucleic acid in various extraintestinal locations such as serum, liver, kidney, bladder, testes, nasal secretions, cerebrospinal fluid, and the central nervous system. Recently, studies in animals and people have demonstrated that rotavirus antigenemia is a common event during natural infection. In this study, we extend these observations and compare the intestinal and extraintestinal spread of wild-type homologous murine rotavirus EC and a heterologous strain, rhesus rotavirus (RRV), in newborn mice. A strand-specific quantitative reverse transcription-PCR (ssQRT-PCR) assay was used to quantify the ability of different rotavirus strains to spread and replicate extraintestinally. Both strain EC and RRV were detected extraintestinally in the mesenteric lymph nodes (MLN), livers, lungs, blood, and kidneys. Extraintestinal replication, as measured by ssQRT-PCR, was most prominent in the MLN and occurred to a lesser degree in the livers, kidneys, and lungs. In the MLN, strain EC and RRV had similar (P < 0.05) RNA copy numbers, although EC was present at a 10,000-fold excess over RRV in the small intestine. Rotavirus nonstructural protein 4 (NSP4) and/or assembled triple-layered particles, indicated by immunostaining with the VP7 conformation-dependent monoclonal antibody 159, were detected in the MLN, lungs, and livers of EC- and RRV-inoculated mice, confirming the ssQRT-PCR findings. Infectious RRV was detected in the MLN in quantities exceeding the amount present in the small intestines or blood. The cells in the MLN that supported rotavirus replication included dendritic cells and potentially B cells and macrophages. These data indicate that extraintestinal spread and replication occurs commonly during homologous and some heterologous rotaviral infections; that the substantial host range restrictions for rhesus rotavirus, a heterologous strain present in the intestine, are not necessarily apparent at systemic sites; that the level and location of extraintestinal replication varies between strains; that replication can occur in several leukocytes subsets; and that extraintestinal replication is likely a part of the normal pathogenic sequence of homologous rotavirus infection.

  View details for DOI 10.1128/JVI.02664-05

  View details for Web of Science ID 000237753400012

  View details for PubMedID 16699002

  View details for PubMedCentralID PMC1472171

• Redundant role of chemokines CCL25/TECK and CCL28/MEC in IgA(+) plasmablast recruitment to the intestinal lamina propria after rotavirus infection JOURNAL OF IMMUNOLOGY Feng, N., Jaimes, M. C., Lazarus, N. H., Monak, D., Zhang, C., Butcher, E. C., Greenberg, H. B. 2006; 176 (10): 5749-5759

  Abstract

  Rotaviruses (RV) are the most important cause of severe childhood diarrheal disease. In suckling mice, infection with RV results in an increase in total and virus-specific IgA(+) plasmablasts in the small intestinal lamina propria (LP) soon after infection, providing a unique opportunity to study the mechanism of IgA(+) cell recruitment into the small intestine. In this study, we show that the increase in total and RV-specific IgA(+) plasmablasts in the LP after RV infection can be blocked by the combined administration of Abs against chemokines CCL25 and CCL28, but not by the administration of either Ab alone. RV infection in CCR9 knockout mice still induced a significant accumulation of IgA(+) plasmablasts in the LP, which was blocked by the addition of anti-CCL28 Ab, confirming the synergistic role of CCL25 and CCL28. The absence of IgA(+) plasmablast accumulation in LP following combined anti-chemokine treatment was not due to changes in proliferation or apoptosis in these cells. We also found that coadministration of anti-CCL25 and anti-CCL28 Abs with the addition of anti-alpha(4) Ab did not further inhibit IgA(+) cell accumulation in the LP and that the CCL25 receptor, CCR9, was coexpressed with the intestinal homing receptor alpha(4)beta(7) on IgA(+) plasmablasts. Finally, we showed that RV infection was associated with an increase in both CCL25 and CCL28 in the small intestine. Hence, our findings indicate that alpha(4)beta(7) along with either CCR9 or CCR10 are sufficient for mediating the intestinal migration of IgA(+) plasmablasts during RV infection.

  View details for Web of Science ID 000237705200010

  View details for PubMedID 16670280

• Immunity and correlates of protection for rotavirus vaccines VACCINE Franco, M. A., Angel, J., Greenberg, H. B. 2006; 24 (15): 2718-2731

  Abstract

  Rotaviruses are the most common cause of severe, dehydrating diarrhea in children worldwide. The tremendous global incidence of rotavirus gastroenteritis, especially in developing countries, emphasizes the need for vaccines to prevent associated morbidity and mortality. However, immunity to rotavirus is not completely understood. At this time, total serum RV IgA, measured shortly after infection, appears to be the best marker of protection against rotavirus. This review describes the current understanding of rotavirus immunity, including mechanisms of protection against rotavirus from selected animal models, and correlates of protection associated with natural infection or vaccination from humans.

  View details for DOI 10.1016/j.vaccine.2005.12.048

  View details for Web of Science ID 000236931700004

  View details for PubMedID 16446014

• Dissecting rotavirus particle-raft interaction with small interfering RNAs: Insights into rotavirus transit through the secretory pathway JOURNAL OF VIROLOGY Cuadras, M. A., Bordier, B. B., Zambrano, J. L., Ludert, J. E., Greenberg, H. B. 2006; 80 (8): 3935-3946

  Abstract

  Studies of rotavirus morphogenesis, transport, and release have shown that although these viruses are released from the apical surface of polarized intestinal cells before cellular lysis, they do not follow the classic exocytic pathway. Furthermore, increasing evidence suggests that lipid rafts actively participate in the exit of rotavirus from the infected cell. In this study, we silenced the expression of VP4, VP7, and NSP4 by using small interfering RNAs (siRNAs) and evaluated the effect of shutting down the expression of these proteins on rotavirus-raft interactions. Silencing of VP4 and NSP4 reduced the association of rotavirus particles with rafts; in contrast, inhibition of VP7 synthesis slightly affected the migration of virions into rafts. We found that inhibition of rotavirus migration into lipid rafts, by either siRNAs or tunicamycin, also specifically blocked the targeting of VP4 to rafts, suggesting that the association of VP4 with rafts is mostly mediated by the formation of viral particles in the endoplasmic reticulum (ER). We showed that two populations of VP4 exist, one small population that is independently targeted to rafts and a second large pool of VP4 whose association with rafts is mediated by particle formation in the ER. We also present evidence to support the hypothesis that assembly of VP4 into mature virions takes place in the late stages of transit through the ER. Finally, we analyzed the progression of rotavirus proteins in the exocytic pathway and found that VP4 and virion-assembled VP7 colocalized with ERGIC-53, suggesting that rotavirus particles transit through the intermediate compartment between the ER and the Golgi complex.

  View details for DOI 10.1128/JVI.80.8.3935-3946.2006

  View details for Web of Science ID 000236685600025

  View details for PubMedID 16571810

  View details for PubMedCentralID PMC1440455

• New viral vaccines VIROLOGY Arvin, A. M., Greenberg, H. B. 2006; 344 (1): 240-249

  Abstract

  Vaccination is the most effective medical intervention against diseases caused by human viral pathogens. Viral vaccines prevent or modify the severity of illness in the individual and interrupt or reduce the transmission of the pathogens to other susceptible people. Through these mechanisms, vaccines against smallpox, polio, measles and hepatitis B have had an enormous impact on world health over the last 50 years. Advances in basic virology and understanding of human immunity promise more progress in the control of human viral diseases as the 21st century begins. Some important targets, including human immunodeficiency virus, respiratory syncytial virus and hepatitis C virus present challenges that require more basic research. The purpose of this review is to highlight four new viral vaccines that have recently, or will soon demonstrate the effective translation of basic investigations into clinical benefits for disease control in healthy and high-risk populations. These vaccines include the live attenuated vaccines against the RNA viruses, rotavirus and influenza A and B, and vaccines against human papilloma virus and varicella-zoster virus, which are DNA viruses that cause morbidity and mortality through their capacity to establish persistent infection. Although only the influenza vaccine has been licensed in the United States, these other new tools for disease prevention are likely to be introduced within the next few years, with profound effects on the diseases that they cause. Hence, as Virology celebrates its 50th anniversary, it is appropriate to examine these examples of recent advances in viral vaccines.

  View details for DOI 10.1016/j.virol.2005.09.057

  View details for Web of Science ID 000234611000026

  View details for PubMedID 16364754

• Multiple gene segments control the temperature sensitivity and attenuation phenotypes of ca B/Ann Arbor/1/66 JOURNAL OF VIROLOGY Hoffmann, E., Mahmood, K., Chen, Z. Y., Yang, C. F., Spaete, J., Greenberg, H. B., Herlocher, M. L., Jin, H., Kemble, G. 2005; 79 (17): 11014-11021

  Abstract

  Cold-adapted (ca) B/Ann Arbor/1/66 is the influenza B virus strain master donor virus for FluMist, a live, attenuated, influenza virus vaccine licensed in 2003 in the United States. Each FluMist vaccine strain contains six gene segments of the master donor virus; these master donor gene segments control the vaccine's replication and attenuation. These gene segments also express characteristic biological traits in model systems. Unlike most virulent wild-type (wt) influenza B viruses, ca B/Ann Arbor/1/66 is temperature sensitive (ts) at 37 degrees C and attenuated (att) in the ferret model. In order to define the minimal genetic components of these phenotypes, the amino acid sequences of the internal genes of ca B/Ann Arbor/1/66 were aligned to those of other influenza B viruses. These analyses revealed eight unique amino acids in three proteins: two in the polymerase subunit PA, two in the M1 matrix protein, and four in the nucleoprotein (NP). Using reverse genetics, these eight wt amino acids were engineered into a plasmid-derived recombinant of ca B/Ann Arbor/1/66, and these changes reverted both the ts and the att phenotypes. A detailed mutational analysis revealed that a combination of two sites in NP (A114 and H410) and one in PA (M431) controlled expression of ts, whereas these same changes plus two additional residues in M1 (Q159 and V183) controlled the att phenotype. Transferring this genetic signature to the divergent wt B/Yamanashi/166/98 strain conferred both the ts and the att phenotypes on the recombinant, demonstrating that this small, complex, genetic signature encoded the essential elements for these traits.

  View details for DOI 10.1128/JVI.79.17.11014-11021.2005

  View details for Web of Science ID 000231303900016

  View details for PubMedID 16103152

• Development of candidate rotavirus vaccines derived from neonatal strains in India JOURNAL OF INFECTIOUS DISEASES Glass, R. I., Bhan, M. K., Ray, P., Bahl, R., Parashar, U. D., Greenberg, H., Rao, C. D., Bhandari, N., Maldonado, Y., Ward, R. L., BERNSTEIN, D. I., Gentsch, J. R. 2005; 192: S30-S35

  Abstract

  The need for a rotavirus vaccine in India is based on the enormous burden associated with the >100,000 deaths due to rotavirus diarrhea that occur annually among Indian children. Two rotavirus strains identified during nosocomial outbreaks of rotavirus infection in New Delhi and Bangalore, India, more than a decade ago are being developed as live oral vaccines. Infected newborns had no symptoms, shed virus for up to 2 weeks after infection, mounted a robust immune response, and demonstrated protection against severe rotavirus diarrhea after reinfection. The 2 strains are naturally occurring bovine-human reassortants. The New Delhi strain, 116E, is characterized as having a P[11],G9 genotype, and the Bangalore strain, I321, is characterized as having a P[11],G10 genotype. The strains have been prepared as pilot lots for clinical trials to be conducted in New Delhi. This unique project, which is developing a new rotavirus vaccine in India with the use of Indian strains, an Indian manufacturer, and an Indian clinical development program, aims to expedite introduction of rotavirus vaccines in India.

  View details for Web of Science ID 000231113500005

  View details for PubMedID 16088802

• Natural evolution of a human virus-specific antibody gene repertoire by somatic hypermutation requires both hotspot-directed and randomly-directed processes HUMAN IMMUNOLOGY Weitkamp, J. H., LaFleur, B. J., Greenberg, H. B., Crowe, J. E. 2005; 66 (6): 666-676

  Abstract

  Somatic hypermutation of antibody genes is mediated by activation-induced cytidine deaminase and targets primarily hotspot motifs. We tested the hypothesis that the antibody variable genes of virus-specific B cells from infants exhibit a decreased frequency of somatic mutations compared with adults. We also sought to determine whether virus-specific B cells exhibit predominantly hotspot or randomly directed processes. We analyzed somatic mutations in rotavirus (RV)-specific B cells from otherwise healthy but recently RV-infected infants or adults in comparison with B cells from healthy volunteers not recently infected. We compared these antibody variable gene sequences with those derived from RV-specific B cells from an adult patient with X-linked hyper-IgM syndrome (XHIM). We found that the overall mutational frequency within the antibody variable region was lowest in RV-specific B cells from RV-infected infants, followed by randomly selected B cells, followed by RV-specific B cells from the patient with XHIM. RV-specific memory B cells from healthy adults exhibited the highest frequency of mutations. Approximately half of mutations in random or RV-specific B cells from adults or infants occurred at the DGYW/WRCH or WA/TW hotspot motifs. These findings suggest that virus-specific antibodies require both hotspot and randomly-directed processes.

  View details for DOI 10.1016/j.humimm.2005.02.008

  View details for Web of Science ID 000230868800004

  View details for PubMedID 15993712

• Characterization of homologous and heterologous rotavirus-specific T-Cell responses in infant and adult mice JOURNAL OF VIROLOGY Jaimes, M. C., Feng, N. G., Greenberg, H. B. 2005; 79 (8): 4568-4579

  Abstract

  During primary rotavirus (RV) infection, CD8+ T cells play an important role in viral clearance as well as providing partial protection against reinfection. CD4+ T cells are essential for maximal development of RV-specific intestinal immunoglobulin A. In this study, we took advantage of the cytokine flow cytometry technique to obtain a detailed map of H-2b- and H-2d-restricted CD8+ and CD4+ T-cell epitopes from the RV proteins VP6 and VP7. Three new CD8+ T-cell epitopes (H-2d and H-2b restricted) and one new CD4+ T-cell epitope (H-2d and H-2b restricted) were identified. Using these newly identified targets, we characterized the development and specificity of cellular immune responses in C57BL/6 and BALB/c mice during acute infection of infants and adults. We found that both the CD4+ and CD8+ responses peaked on days 5 to 7 after infection and then declined rapidly. Interestingly, both the response kinetics and tissue distributions were different when epitopes on VP6 and VP7 were compared. VP6 elicited a response which predominated in the intestine, while the response to VP7 was more systemic. Additionally, the T-cell responses elicited after homologous versus heterologous infection differed substantially. We found that during homologous infection, there was a greater response toward VP6 than that toward VP7, especially in the intestine, while after heterologous infection, this was not the case. Finally, in suckling mice, we found two peaks in the CD8 response on days 7 and 14 postinfection, which differed from the single peak found in adults and likely mimics the biphasic pattern of rotavirus shedding in infant mice.

  View details for DOI 10.1128/JVI.79.8.4568-4579.2005

  View details for Web of Science ID 000228073700002

  View details for PubMedID 15795243

  View details for PubMedCentralID PMC1069584

• VH1-46 is the dominant immunoglobulin heavy chain gene segment in rotavirus-specific memory B cells expressing the intestinal homing receptor alpha(4)beta(7) JOURNAL OF IMMUNOLOGY Weitkamp, J. H., Kallewaard, N. L., Bowen, A. L., LaFleur, B. J., Greenberg, H. B., Crowe, J. E. 2005; 174 (6): 3454-3460

  Abstract

  Memory B cells expressing the intestinal homing marker alpha4beta7 are important for protective immunity against human rotavirus (RV). It is not known whether the B cell repertoire of intestinal homing B cells differs from B cells of the systemic compartment. In this study, we analyzed the RV-specific VH and VL repertoire in human IgD- B cells expressing the intestinal homing marker alpha4beta7. The mean frequency of RV-specific B cells in the systemic compartment of healthy adult subjects was 0.6% (range, 0.2-1.2). The mean frequency of IgD- B cells that were both RV specific and alpha4beta7 was 0.04% (range, 0.01-0.1), and a mean of 10% (range, 1-32) of RV-specific peripheral blood human B cells exhibited an intestinal homing phenotype. We previously demonstrated that VH1-46 is the dominant Ab H chain gene segment in RV-specific systemic B cells from adults and infants. RV-specific systemic IgD- or intestinal homing IgD-/alpha4beta7+ B cells in the current study also used the gene segment VH1-46 at a high frequency, while randomly selected B cells with those phenotypes did not. These data show that VH1-46 is the immunodominant gene segment in human RV-specific effector B cells in both the systemic compartment and in intestinal homing lymphocytes. The mean replacement/silent mutation ratio of systemic compartment IgD- B cells was >2, consistent with a memory phenotype and antigenic selection. Interestingly, RV-specific intestinal homing IgD-/alpha4beta7+ B cells using the VH1-46 gene segment were not mutated, in contrast to systemic RV-specific IgD- B cells.

  View details for Web of Science ID 000227510900042

  View details for PubMedID 15749880

• Keratin mutation primes mouse liver to oxidative injury HEPATOLOGY Zhou, Q., Ji, X. H., Chen, L. X., Greenberg, H. B., Lu, S. C., Omary, M. B. 2005; 41 (3): 517-525

  Abstract

  Mutation of the cytoskeletal intermediate filament proteins keratin 8 and keratin 18 (K8/K18) is associated with cirrhosis in humans, whereas transgenic mice that overexpress K18 Arg89-->Cys (R89C) have significant predisposition to liver injury. To study the mechanism of keratin-associated predisposition to liver injury, we used mouse microarrays to examine genetic changes associated with hepatocyte keratin mutation and assessed the consequences of such changes. Liver gene expression was compared in R89C versus nontransgenic or wild-type K18-overexpressing mice. Microarray-defined genetic changes were confirmed by quantitative polymerase chain reaction. Nineteen genes had a more than two-fold altered expression (nine downregulated, 10 upregulated). Upregulated genes in keratin-mutant hepatocytes included the oxidative metabolism genes cytochrome P450, S-adenosylhomocysteine (SAH) hydrolase, cysteine sulfinic acid decarboxylase, and oxidation-reduction pathway genes. Downregulated genes included fatty acid binding protein 5, cyclin D1, and some signaling molecules. Several methionine metabolism-related and glutathione synthetic pathway intermediates, including S-adenosylmethionine (SAMe) and SAH, were modulated in R89C versus control mice. R89C livers had higher lipid and protein oxidation by-products as reflected by increased malondialdehyde and oxidized albumin. In conclusion, K18 point mutation in transgenic mice modulates several hepatocyte oxidative stress-related genes and leads to lipid and protein oxidative by-products. Mutation-associated decreases in SAH and SAMe could compromise needed cysteine availability to generate glutathione during oxidative stress. Hence keratin mutations may prime hepatocytes to oxidative injury, which provides a new potential mechanism for how keratin mutations may predispose patients to cirrhosis.

  View details for DOI 10.1002/hep.20578

  View details for Web of Science ID 000227298200013

  View details for PubMedID 15726665

• T cell-dependent production of IFN-gamma by NK cells in response to influenza A virus JOURNAL OF CLINICAL INVESTIGATION He, X. S., Draghi, M., Mahmood, K., Holmes, T. H., Kemble, G. W., Dekker, C. L., Arvin, A. M., Parham, P., Greenberg, H. B. 2004; 114 (12): 1812-1819

  Abstract

  The role of human NK cells in viral infections is poorly understood. We used a cytokine flow-cytometry assay to simultaneously investigate the IFN-gamma response of NK and T lymphocytes to influenza A virus (fluA). When PBMCs from fluA-immune adult donors were incubated with fluA, IFN-gamma was produced by both CD56(dim) and CD56(bright) subsets of NK cells, as well as by fluA-specific T cells. Purified NK cells did not produce IFN-gamma in response to fluA, while depletion of T lymphocytes reduced to background levels the fluA-induced IFN-gamma production by NK cells, which indicates that T cells are required for the IFN-gamma response of NK cells. The fluA-induced IFN-gamma production of NK cells was suppressed by anti-IL-2 Ab, while recombinant IL-2 replaced the helper function of T cells for IFN-gamma production by NK cells. This indicates that IL-2 produced by fluA-specific T cells is involved in the T cell-dependent IFN-gamma response of NK cells to fluA. Taken together, these results suggest that at an early stage of recurrent viral infection, NK-mediated innate immunity to the virus is enhanced by preexisting virus-specific T cells.

  View details for DOI 10.1172/JCI200422797

  View details for Web of Science ID 000225695800016

  View details for PubMedID 15599406

  View details for PubMedCentralID PMC535070

• Immunization against viral respiratory disease: a review. Pediatric infectious disease journal Greenberg, H. B., Piedra, P. A. 2004; 23 (11): S254-61

  Abstract

  Healthy and at risk children are susceptible to the morbidity and mortality associated with viral-induced respiratory diseases, including respiratory syncytial virus (RSV) and influenza. The World Health Organization is attempting to develop and distribute effective vaccines to prevent/reduce key viral respiratory diseases.The goals of a vaccination program for viral respiratory infections include the prevention of lower respiratory tract infections and prevention of infection-associated morbidities, hospitalization and mortality. This article explores influenza and RSV vaccine developments.There are 2 influenza vaccines, trivalent inactivated and live, cold-adapted, attenuated. Trivalent inactivated vaccine is indicated for persons older than 6 months of age. Currently <10%, <30% and <30% of healthy children, healthy adults and high risk children, respectively, are vaccinated. Efficacy is from 70 to 90% in healthy adults younger than 65 years of age and 30-90% in children, with lower efficacy in younger children. Live, cold-adapted, attenuated vaccine is indicated for healthy persons 5-49 years of age and usually is 70-90% effective. Various RSV vaccine formulations are being investigated. The Advisory Committee on Immunization Practices (ACIP) recommends influenza vaccination for children 6-23 months old. Studies support immunization of all children, not only those at high risk. Current ACIP recommendations focus on high risk persons and do not include school age children. A universal immunization program for all children could benefit the entire community.Effective vaccines are available for some viral respiratory pathogens (eg, influenza virus), but not for most mucosally restricted respiratory viral pathogens. Research should continue into safe and effective vaccines for all childhood viral illnesses.

  View details for PubMedID 15577581

• Maturation and trafficking markers on rotavirus-specific B cells during acute infection and convalescence in children JOURNAL OF VIROLOGY Jaimes, M. C., Rojas, O. L., Kunkel, E. J., Lazarus, N. H., Soler, D., BUTCHER, E. C., Bass, D., Angel, J., Franco, M. A., Greenberg, H. B. 2004; 78 (20): 10967-10976

  Abstract

  We have previously studied B cells, from people and mice, that express rotavirus-specific surface immunoglobulin (RV-sIg) by flow cytometry with recombinant virus-like particles that contain green fluorescent protein. In the present study we characterized circulating B cells with RV-sIg in children with acute and convalescent infection. During acute infection, circulating RV-sIgD(-) B cells are predominantly large, CD38(high), CD27(high), CD138(+/-), CCR6(-), alpha4beta7(+), CCR9(+), CCR10(+), cutaneous lymphocyte antigen-negative (CLA(-)), L-selectin(int/-), and sIgM(+), sIgG(-), sIgA(+/-) lymphocytes. This phenotype likely corresponds to gut-targeted plasma cells and plasmablasts. During convalescence the phenotype switches to small and large lymphocytes, CD38(int/-), CD27(int/-), CCR6(+), alpha4beta7(+/-), CCR9(+/-) and CCR10(-), most likely representing RV-specific memory B cells with both gut and systemic trafficking profiles. Of note, during acute RV infection both total and RV-specific murine IgM and IgA antibody-secreting cells migrate efficiently to CCL28 (the CCR10 ligand) and to a lesser extent to CCL25 (the CCR9 ligand). Our results show that CCR10 and CCR9 can be expressed on IgM as well as IgA antibody-secreting cells in response to acute intestinal infection, likely helping target these cells to the gut. However, these intestinal infection-induced plasmablasts lack the CLA homing receptor for skin, consistent with mechanisms of differential CCR10 participation in skin T versus intestinal plasma cell homing. Interestingly, RV memory cells generally lack CCR9 and CCR10 and instead express CCR6, which may enable recruitment to diverse epithelial sites of inflammation.

  View details for DOI 10.1028/JVI.78.20.10967-10976.2004

  View details for Web of Science ID 000224229000014

  View details for PubMedID 15452217

  View details for PubMedCentralID PMC521846

• Expression of chemokine receptors on intrahepatic and peripheral lymphocytes in chronic hepatitis C infection: Its relationship to liver inflammation 10th International Meeting on Hepatitis C Virus and Related Viruses Wang, J. H., Holmes, T. H., Cheung, R., Greengberg, H. B., He, X. S. UNIV CHICAGO PRESS. 2004: 989–97

  Abstract

  Intrahepatic lymphocytes are believed to be directly involved in the immunopathogenesis of chronic liver diseases. Little is known about the trafficking of lymphocytes into the liver and their role in chronic hepatitis C infection.The expression of 4 chemokine receptors and an activation marker on multiple lymphocyte subsets in paired liver biopsy and peripheral blood specimens from 23 patients with chronic hepatitis C infection were analyzed by a 6-color flow-cytometric assay.CCR5, CXCR3, and CXCR6 were expressed on intrahepatic CD4+ and CD8+ T cells, natural killer (NK) T cells, NK cells, and B cells at significantly higher frequencies than on peripheral lymphocyte subsets. The expression of these receptors and the activation marker CD38 tended to increase with the severity of liver inflammation. This increase was significant for several intrahepatic lymphocytes subsets. Correlations in expression differed among pairs of these extralymphoid homing receptors on the intrahepatic T cells.The homing program for intrahepatic lymphocytes involves multiple extralymphoid chemokine receptors that are regulated by >1 pathway. The expression of homing receptors on intrahepatic lymphocytes is associated with the immunopathogenesis of chronic hepatitis C disease. These preliminary results indicate that confirmational studies with larger sample sizes are warranted.

  View details for Web of Science ID 000223114800018

  View details for PubMedID 15295707

• Possible role of neonatal infection with the asymptomatic reassortant rotavirus (RV) strain I321 in the decrease in hospital admissions for RV diarrhea, Bangalore, India, 1988-1999 Symposium on Trends in Malaria and Vaccine Research Vethanayagam, R. R., Babu, M. A., Nagalaxmi, K. S., Maiya, P. P., Venkatesh, H. A., Purohit, S., Behl, R., Bhan, M. K., Ward, R. L., Greenberg, H. B., Rao, C. D. UNIV CHICAGO PRESS. 2004: 2282–89

  Abstract

  We sought to determine the proportion of rotavirus (RV) infections among children with severe diarrhea in Bangalore, India, and to determine the role of neonatal infection with the asymptomatic RV strain I321 in protection against subsequent RV diarrhea. At 2 major hospitals, there was a >42% decrease in diarrhea-specific admissions during the study period. At 6 hospitals, asymptomatic infections were found in 25%-50% of neonates, when screening was performed randomly, and in >58% of neonates, when screening was performed daily, with the majority of infections occurring within the first 7 days of life. All the RVs found in asymptomatic neonates were strain I321. A 24-month follow-up of a cohort of 44 children who had been neonatally infected with strain I321 and 28 children who had not (control group) revealed comparable rates of RV detection but a marked decrease in the number of RV diarrhea episodes in the strain I321-infected group (2.3%), compared with the control group (39.3%) (P<.0001). This preliminary study suggests a possible association between neonatal infection with strain I321 and protection against subsequent RV illness.

  View details for Web of Science ID 000221699500017

  View details for PubMedID 15181576

• Antiviral CD8 T cells in the control of primary human cytomegalovirus infection in early childhood 40th Annual Meeting of the Infectious-Diseases-Society-of-America Chen, S. F., Tu, W. W., Sharp, M. A., Tongson, E. C., He, X. S., Greenberg, H. B., Holmes, T. H., Wang, Z. T., Kemble, G., Manganello, A. M., Adler, S. P., Dekker, C. L., Levvis, D. B., Arvin, A. M. UNIV CHICAGO PRESS. 2004: 1619–27

  Abstract

  Human cytomegalovirus (CMV) establishes persistent infection, with control of replication thought to be mediated by CMV-specific CD8 T cells. Primary CMV infection commonly affects young children and causes prolonged viral shedding in saliva and urine. We investigated whether this virus-host interaction pattern reflects a developmental deficiency of antiviral CD8 T cell-mediated immunity during childhood. CMV-specific CD8 T cell responses in asymptomatic children with active infection were not different from adults with recent or long-term infection in frequency and functional analyses. High urine CMV concentrations were detected, despite these CMV-specific CD8 T cell responses. We conclude that delayed resolution of primary CMV infection in young children is not caused by a deficient CMV-specific CD8 T cell response. Because these healthy children continue to have local CMV replication, we suggest that CD8 T cells may function primarily to prevent symptomatic, disseminated disease.

  View details for PubMedID 15116298

• The use of class-I HLA tetramers for the detection of hepatitis C virus NS3-specific CD8+ T cells in patients with chronic infection JOURNAL OF IMMUNOLOGICAL METHODS Lopez-Labrador, F. X., He, X. S., Berenguer, M., Cheung, R. C., Wright, T. L., Greenberg, H. B. 2004; 287 (1-2): 91-99

  Abstract

  New methods to detect virus-specific T-cell responses have recently been developed. Several human leukocyte antigen (HLA)-peptide tetramers for the detection of hepatitis C virus (HCV)-specific CD8(+) T cells are under evaluation.Evaluation of one HLA class I-tetramer (HCVNS3-2) for the detection of HCV NS3-specific CD8(+) T cells in a series of 38 HLA-A2(+) chronically infected patients.Almost half (42%) of the patients had detectable NS3-specific CD8(+) T cells. The frequencies of such cells ranged from 0.01% to 0.22% of total CD8(+) T cells. No significant differences in clinical features or mean viral load were detected between patients with or without tetramer + CD8(+) T cells.The tetramer HCVNS3-2 may be very useful for the study of the HCV-specific CD8(+) immune response. Combination of this reagent with other tetramers based on other HCV peptides may help in the understanding of the immune response to the virus. However, a panel of tetramers based on several parts of the HCV polyprotein may be a mandatory requirement to explore the whole breadth of the CD8(+) T-cell response against HCV and to detect that response in the majority of patients with chronic infection.

  View details for DOI 10.1016/j.jim.2004.01.023

  View details for Web of Science ID 000221148800008

  View details for PubMedID 15099758

• Genetic variability of hepatitis C virus non-structural protein 3 and virus-specific CD8+response in patients with chronic hepatitis C JOURNAL OF MEDICAL VIROLOGY Lopez-Labrador, F. X., He, X. S., Berenguer, M., Cheung, R. C., Gonzalez-Candelas, F., Wright, T. L., Greenberg, H. B. 2004; 72 (4): 575-585

  Abstract

  Hepatitis C virus (HCV) variation in specific T-cell epitopes may represent a mechanism of viral persistence in chronic infection. We examined the HCV non-structural protein 3 (NS3), including the immunologically relevant epitopes HCV NS3-2 KLVALGINAV (human leukocyte antigen [HLA]-A2-restricted) and HCV NS3-1391 LIFCHSKKK (HLA-A3-restricted), in 22 HLA-A2+ patients with chronic infection. Significant amino acid variation was found in HCV NS3-2 epitope sequences when compared to the HCV-1 prototype virus. Six of the nine different HCV NS3-2 peptide variants were identified in patients with HCV NS3-2-specific CD8+ cells, detected with an HLA-A2 tetramer made with the HCV-1 prototype peptide. Phylogenetic analysis, including HCV reference sequences other than HCV-1, suggested however that most of the variations in the HCV NS3-2 epitope could be related to genetic heterogeneity between HCV reference subtypes. Variation was less common when comparing HCV NS3-2 epitope sequences from the clinical isolates to the most-closely related HCV reference subtype in each case. Some subtype-independent variations were found in epitopic residues probably important for T-cell receptor interaction. In contrast, no significant variation was found in HLA primary anchor sites, flanking regions, or in the contiguous HLA A3-restricted CD8+ T-cell epitope. Ongoing variation was not evident in two selected patients with follow-up. In conclusion, (i) the HCV NS3-2 epitope is not conserved between different HCV strains/subtypes, and (ii) an HLA-A2 tetramer loaded with the HCV-1 prototype NS3-2 peptide may still detect NS3-specific CD8+ cells in some patients with variant viruses. These data may be useful to improve T-cell assays using HCV NS3 peptides, taking into account the genetic diversity of this virus.

  View details for DOI 10.1002/jmv.20036

  View details for Web of Science ID 000220046100010

  View details for PubMedID 14981760

• A central role for the chemokine MEC/CCL28 in regulating IgA mediated passive immunity of the neonate Experimental Biology 2004 Annual Meeting Wilson, E., Feng, N. G., Greenberg, H., Butcher, E. FEDERATION AMER SOC EXP BIOL. 2004: A818–A818

  View details for Web of Science ID 000220470700284

• Detection and characterization of virus-specific CD8+ T cells using the tetramer approach. Methods in molecular medicine He, X., Greenberg, H. B. 2004; 96: 89-96

  View details for PubMedID 14762262

• Infant and adult human B cell responses to rotavirus share common immunodominant variable gene repertoires JOURNAL OF IMMUNOLOGY Weitkamp, J. H., Kallewaard, N., Kusuhara, K., Bures, E., Williams, J. V., LaFleur, B., Greenberg, H. B., Crowe, J. E. 2003; 171 (9): 4680-4688

  Abstract

  Ab repertoires exhibit marked restrictions during fetal life characterized by biases of variable gene usage and lack of junctional diversity. We tested the hypothesis that Ab repertoire restriction contributes to the observed poor quality of specific Ab responses made by infants to viral infections. We analyzed the molecular determinants of B cell responses in humans to two Ags of rotavirus (RV), a common and clinically important infection of human infants. We sequenced Ab H and L chain V region genes (V(H) and V(L)) of clones expanded from single B cells responding to RV virus protein 6 or virus protein 7. We found that adults exhibited a distinct bias in use of gene segments in the V(H)1 and V(H)4 families, for example, V(H)1-46, V(H)4-31, and V(H)4-61. This gene segment bias differed markedly from the V(H)3 dominant bias seen in randomly selected adult B cells. Recombinant Abs incorporating any of those three immunodominant V(H) segments bound to RV-infected cells and also to purified RV particles. The RV-specific B cell repertoires of infants aged 2-11 mo and those of adults were highly related when compared by V(H), D, J(H), V(L), and J(L) segment selection, extent of junctional diversity, and mean H chain complementarity determining region 3 length. These data suggest that residual fetal bias of the B cell repertoire is not a limiting determinant of the quality of Ab responses to viruses of infants beyond the neonatal period.

  View details for Web of Science ID 000186136800025

  View details for PubMedID 14568943

• Human rotavirus specific T cells: quantification by ELISPOT and expression of homing receptors on CD4+ T cells VIROLOGY Rojas, O. L., Gonzalez, A. M., GONZALEZ, R., Perez-Schael, I., Greenberg, H. B., Franco, M. A., Angel, J. 2003; 314 (2): 671-679

  Abstract

  Using an intracellular cytokine assay, we recently showed that the frequencies of rotavirus (RV)-specific CD4(+) and CD8(+) T cells secreting INFgamma, circulating in RV infected and healthy adults, are very low compared to the frequencies of circulating cytomegalovirus (CMV) reactive T cells in comparable individuals. In children with acute RV infection, these T cells were barely or not detectable. In the present study, an ELISPOT assay enabled detection of circulating RV-specific INFgamma-secreting cells in children with RV diarrhea but not in children with non-RV diarrhea without evidence of a previous RV infection. Using microbead-enriched CD4(+) and CD8(+) T cell subsets, IFNgamma-secreting RV-specific CD8(+) but not CD4(+) T cells were detected in recently infected children. Using the same approach, both CD4(+) and CD8(+) RV-specific T cells were detected in healthy adults. Furthermore, stimulation of purified subsets of PBMC that express lymphocyte homing receptors demonstrated that RV-specific INFgamma-secreting CD4(+) T cells from adult volunteers preferentially express the intestinal homing receptor alpha4beta7, but not the peripheral lymph node homing receptor L-selectin. In contrast, CMV-specific INFgamma-secreting CD4(+) T cells preferentially express L-selectin but not alpha4beta7. These results suggest that the expression of homing receptors on virus-specific T cells depends on the organ where these cells were originally stimulated and that their capacity to secrete INFgamma is independent of the expression of these homing receptors.

  View details for DOI 10.1016/S0042-6822(03)00507-5

  View details for Web of Science ID 000186009100018

  View details for PubMedID 14554094

• II, 11. Human adaptive immunity to rotaviruses: A model of intestinal mucosal adaptive immunity. Perspectives in medical virology Gonzalez, A. M., Jaimes, M. C., Rojas, O. L., Angel, J., Greenberg, H. B., A Franco, M. 2003; 9: 307-316

  Abstract

  This chapter discusses the human adaptive immune response to rotaviruses (RVs), placing the immune response to RVs in the context of the immune response to other mucosal viruses. The chapter discusses the studies of both RV-specific T and B cells. As children with T and/or B immunodeficiencies can develop chronic RV infection, prolonged symptoms, and extraintestinal infection, it is clear that both T and B cells are important for immunity to RV. The various reasons proposed to explain the absence of complete immunity to mucosal viruses such as RV, following primary infection, include a short incubation period after viral exposure, difficulty in maintaining a high level of protective antibody at respiratory and gastrointestinal mucosal surfaces, and a short-lived protective humoral mucosal immune response.

  View details for DOI 10.1016/S0168-7069(03)09018-9

  View details for PubMedID 32287601

  View details for PubMedCentralID PMC7133893

• Rotavirus infectious particles use lipid rafts during replication for transport to the cell surface in vitro and in vivo VIROLOGY Cuadras, M. A., Greenberg, H. B. 2003; 313 (1): 308-321

  Abstract

  The pathway by which rotavirus is released from the cell is poorly understood but recent work has shown that, prior to cell lysis, rotavirus is released almost exclusively from the apical surface of the infected cell. By virtue of their unique biochemical and physical properties, viruses have exploited lipid rafts for host cell entry and/or assembly. Here we characterized the association of rhesus rotavirus (RRV) with lipid rafts during the rotavirus replication cycle. We found that newly synthesized infectious virus associates with rafts in vitro and in vivo. RRV proteins cosegregated with rafts on density gradients. Viral infectivity and genomic dsRNA also cosegregated with the raft fractions. Confocal microscopic analysis of raft and RRV virion proteins demonstrated colocalization within the cell. In addition, cholesterol depletion interfered with the association of RRV particles with rafts and reduced the release of infectious particles from the cell. Furthermore, murine rotavirus associates with lipid rafts in intestinal epithelial cells during a natural infection in vivo. Our results confirm the association of rotavirus infectious particles with rafts during replication in vitro and in vivo and strongly support the conclusion that this virus uses these microdomains for transport to the cell surface during replication.

  View details for DOI 10.1016/S0042-6822(03)00326-X

  View details for Web of Science ID 000185209200028

  View details for PubMedID 12951042

• Keratin 20 helps maintain intermediate filament organization in intestinal epithelia MOLECULAR BIOLOGY OF THE CELL Zhou, Q., Toivola, D. M., Feng, N. G., Greenberg, H. B., Franke, W. W., Omary, M. B. 2003; 14 (7): 2959-2971

  Abstract

  Of the >20 epithelial keratins, keratin 20 (K20) has an unusual distribution and is poorly studied. We began to address K20 function, by expressing human wild-type and Arg80-->His (R80H) genomic (18 kb) and cDNA K20 in cells and mice. Arg80 of K20 is conserved in most keratins, and its mutation in epidermal keratins causes several skin diseases. R80H but not wild-type K20 generates disrupted keratin filaments in transfected cells. Transgenic mice that overexpress K20 R80H have collapsed filaments in small intestinal villus regions, when expressed at moderate levels, whereas wild-type K20-overexpressing mice have normal keratin networks. Overexpressed K20 maintains its normal distribution in several tissues, but not in the pancreas and stomach, without causing any tissue abnormalities. Hence, K20 pancreatic and gastric expression is regulated outside the 18-kb region. Cross-breeding of wild-type or R80H K20 mice with mice that overexpress wild-type K18 or K18 that is mutated at the conserved K20 Arg80-equivalent residue show that K20 plays an additive and compensatory role with K18 in maintaining keratin filament organization in the intestine. Our data suggest the presence of unique regulatory domains for pancreatic and gastric K20 expression and support a significant role for K20 in maintaining keratin filaments in intestinal epithelia.

  View details for DOI 10.1091/mbc.E03-02-0059

  View details for Web of Science ID 000184148400028

  View details for PubMedID 12857878

• Amphipathic helix-dependent localization of NS5A mediates hepatitis C virus RNA replication JOURNAL OF VIROLOGY Elazar, M., Cheong, K. H., Liu, P., Greenberg, H. B., Rice, C. M., Glenn, J. S. 2003; 77 (10): 6055-6061

  Abstract

  We identified an N-terminal amphipathic helix (AH) in one of hepatitis C virus (HCV)'s nonstructural proteins, NS5A. This AH is necessary and sufficient for membrane localization and is conserved across isolates. Genetically disrupting the AH impairs HCV replication. Moreover, an AH peptide-mimic inhibits the membrane association of NS5A in a dose-dependent manner. These results have exciting implications for the HCV life cycle and novel antiviral strategies.

  View details for DOI 10.1128/JVI.77.10.6055-6061.2003

  View details for PubMedID 12719597

• Novel generations of influenza vaccines 56th World Health Assembly Meeting Kemble, G., Greenberg, H. ELSEVIER SCI LTD. 2003: 1789–95

  Abstract

  Several strategies are being pursued to increase the quality and quantity of influenza vaccines that are used on an annual basis including increasing the immunogenicity of currently licensed inactivated vaccines, delivery of inactive vaccines directly to the nasal mucosa, the use of cell lines for virus production and the use of live, attenuated vaccines. In addition, modern molecular biological techniques are being used to create and evaluate new vaccine approaches. This report will briefly review these different strategies and outline some of the potential advantages and challenges associated with them.

  View details for DOI 10.1016/S0264-410X(03)00074-4

  View details for Web of Science ID 000182575500011

  View details for PubMedID 12686096

• Generation of recombinant human monoclonal antibodies to rotavirus from single antigen-specific B cells selected with fluorescent virus-like particles JOURNAL OF IMMUNOLOGICAL METHODS Weitkamp, J. H., Kallewaard, N., Kusuhara, K., Feigelstock, D., Feng, N. G., Greenberg, H. B., Crowe, J. E. 2003; 275 (1-2): 223-237

  Abstract

  Technical difficulties have severely limited the yield of methods for the generation of human antiviral monoclonal antibodies (Mabs) in the past. We describe here a novel method for the efficient development of human Mabs against viruses. Rotavirus (RV) is a major cause of gastroenteritis in infants and adults worldwide. We generated fluorescent virus-like particles (VLPs) to identify and physically sort single RV-specific B cells from healthy adult blood donors, or RV-infected infants or adults. We expanded the sorted single B cells in culture, tested for RV-specific antibody secretion, and cloned and sequenced the antibody heavy and light chain variable region (VH and VL) genes. The percentage of wells that produced antibodies after sorting and expanding RV-specific adult B cell clones was high at 23%. The overall efficiency of RV-specific antibody gene recovery after the isolation, confirmation, and cloning of RV-specific VH segments was 1.3% of sorted cells in adults. RV-specific variable gene segments also were obtained from acutely infected infants, although infant B cells did not proliferate and differentiate in culture as well as adult B cells. We expressed recombinant Fabs incorporating the VH and VL genes from RV-specific B cell clones using a new modified bacterial Fab expression vector that we describe. Finally, we demonstrated binding of purified Fabs to RV proteins by immunofluorescence and ELISA. This method for the generation of recombinant human Mabs to RV from single antigen-specific B cell clones selected with fluorescent VLPs could be used to generate human Mabs to many other viruses whose proteins can self-assemble into VLPs.

  View details for DOI 10.1016/S002201759(03)00013-9

  View details for Web of Science ID 000182145600022

  View details for PubMedID 12667686

• Analysis of the frequencies and of the memory T cell phenotypes of human CD8(+) T cells specific for influenza A viruses JOURNAL OF INFECTIOUS DISEASES He, X. S., Mahmood, K., Maecker, H. T., Holmes, T. H., Kemble, G. W., Arvin, A. M., Greenberg, H. B. 2003; 187 (7): 1075-1084

  Abstract

  We characterized the human CD8+ T cell response against influenza A viruses by a flow cytometry-based assay. Peripheral blood mononuclear cells (PBMCs) were incubated with inactivated influenza virus preparation, for 17 h, and were stained for intracellular interferon-gamma. Major histocompatibility complex class I-restricted memory CD8+ T cells specific for influenza antigens were detected in PBMCs from all 19 adult donors, at an average frequency of 0.39%. On average, 83% of influenza virus-specific CD8+ T cells expressed the differentiation-associated marker CD27, a percentage that is significantly higher than that of CD8+ T cells specific for pp65 of human cytomegalovirus (53%). These observations indicate that class I-restricted immunity against influenza A viruses is characterized by the persistence, after clearance of infection, of circulating antigen-specific CD8+ T cells. The different patterns of CD27 expression in influenza virus- and cytomegalovirus-specific CD8+ T cells suggest that influenza virus-specific memory and effector CD8+ T cells can be differentiated by phenotypic analysis.

  View details for Web of Science ID 000181713000007

  View details for PubMedID 12660922

• Interferon alfa regulated gene expression in patients initiating interferon treatment for chronic hepatitis C HEPATOLOGY Ji, X. H., Cheung, R., Cooper, S., Li, Q. Q., Greenberg, H. B., He, X. S. 2003; 37 (3): 610-621

  Abstract

  Interferon alfa (IFN-alpha) is an approved therapeutic agent for chronic hepatitis C. To directly characterize the effects of IFN-alpha in humans, we used microarrays to profile gene expression in peripheral blood mononuclear cells (PBMCs) from hepatitis C patients treated with IFN-alpha. Seven patients were studied using two strategies: (1) in vivo: PBMCs were collected immediately before the first dose of IFN-alpha, and 3 and 6 hours after the dose; (2) ex vivo: PBMCs that were collected before the first IFN-alpha dose were incubated with IFN-alpha for 3 and 6 hours. The microarray datasets were analyzed with significance analysis of microarrays (SAM) to identify genes regulated by IFN-alpha. We identified 516 named genes up-regulated at least 2-fold, at a false discovery rate (FDR) of less than 1%. In vivo and ex vivo studies generated similar results. No genes were identified as regulated differently between these 2 experimental conditions. The up-regulated genes belonged to a broad range of functional pathways and included multiple genes thought to be involved in the direct antiviral effect of IFN-alpha. Of particular interest, 88 genes directly relating to functions of immune cells were up-regulated, including genes involved in antigen processing and presentation, T-cell activation, lymphocyte trafficking, and effector functions, suggesting that IFN-alpha up-regulates multiple genes involving different aspects of immune responses to enhance immunity against hepatitis C virus. In conclusion, IFN-alpha-inducible genes can be identified in human PBMCs in vivo as well as ex vivo. Signature changes associated with different treatment outcomes may be found among these genes.

  View details for DOI 10.1053/jhep.2003.50105

  View details for Web of Science ID 000181276800017

  View details for PubMedID 12601359

• Multiple amino acid residues confer temperature sensitivity to human influenza virus vaccine strains (FluMist) derived from cold-adapted A/Ann Arbor/6/60 VIROLOGY Jin, H., Lu, B., Zhou, H., Ma, C. H., Zhao, J., Yang, C. F., Kemble, G., Greenberg, H. 2003; 306 (1): 18-24

  Abstract

  FluMist influenza A vaccine strains contain the PB1, PB2, PA, NP, M, and NS gene segments of ca A/AA/6/60, the master donor virus-A strain. These gene segments impart the characteristic cold-adapted (ca), attenuated (att), and temperature-sensitive (ts) phenotypes to the vaccine strains. A plasmid-based reverse genetics system was used to create a series of recombinant hybrids between the isogenic non-ts wt A/Ann Arbor/6/60 and MDV-A strains to characterize the genetic basis of the ts phenotype, a critical, genetically stable, biological trait that contributes to the attenuation and safety of FluMist vaccines. PB1, PB2, and NP derived from MDV-A each expressed determinants of temperature sensitivity and the combination of all three gene segments was synergistic, resulting in expression of the characteristic MDV-A ts phenotype. Site-directed mutagenesis analysis mapped the MDV-A ts phenotype to the following four major loci: PB1(1195) (K391E), PB1(1766) (E581G), PB2(821) (N265S), and NP(146) (D34G). In addition, PB1(2005) (A661T) also contributed to the ts phenotype. The identification of multiple genetic loci that control the MDV-A ts phenotype provides a molecular basis for the observed genetic stability of FluMist vaccines.

  View details for DOI 10.1016/S0042-6822(02)00035-1

  View details for Web of Science ID 000181511100003

  View details for PubMedID 12620793

• Rotavirus-specific B cells induced by recent infection in adults and children predominantly express the intestinal homing receptor alpha 4 beta 7 VIROLOGY Gonzalez, A. M., Jaimes, M. C., Cajiao, I., Rojas, O. L., Cohen, J., Pothier, P., Kohli, E., BUTCHER, E. C., Greenberg, H. B., Angel, J., Franco, M. A. 2003; 305 (1): 93-105

  Abstract

  In vivo replication of rotaviruses is generally limited to enterocytes. Because of this restriction, most blood circulating rotavirus-specific B cells are hypothesized to originate in Peyer's patches and should express the intestinal homing receptor alpha4beta7. To test this hypothesis in humans, we used a flow cytometry assay that identifies antigen-activated (IgD-) B cells (CD19+) that express surface rotavirus-specific immunoglobulin. With this assay we could detect rotavirus-specific B cells in both children and adults with an acute rotavirus (RV) infection. Staining with an anti-alpha4beta7 monoclonal antibody, we could determine that B cells that express rotavirus-specific surface immunoglobulin predominantly express alpha4beta7. The response of rotavirus-specific antibody-secreting cells in the peripheral blood of children and adults with acute rotavirus infection was also studied by ELISPOT. The antibody-secreting cells of children were mainly of the IgM isotype, while the antibody-secreting cells of adults were predominantly of the IgA and IgG isotype. alpha4beta7+ and alpha4beta7- subsets of peripheral blood mononuclear cells were purified using paramagnetic beads and then tested in the ELISPOT assay. Rotavirus-specific antibody-secreting cells were predominantly present in the alpha4beta7+ subpopulation. The flow cytometry assay we have described will permit future studies to characterize the phenotype of virus-specific B cells and could be useful in the study of the immunogenicity and protective efficacy of RV vaccines and the identification of markers of protective immunity.

  View details for DOI 10.1006/viro.2002.1708

  View details for Web of Science ID 000180214200010

  View details for PubMedID 12504544

• Liver-infiltrating lymphocytes in end-stage hepatitis C virus: Subsets, activation status, and chemokine receptor phenotypes JOURNAL OF HEPATOLOGY Boisvert, J., Kunkel, E. J., Campbell, J. J., Keeffe, E. B., BUTCHER, E. C., Greenberg, H. B. 2003; 38 (1): 67-75

  Abstract

  Hepatitis C virus (HCV) is a leading cause of chronic liver disease, yet little is known about the intrahepatic immune response in end-stage patients. Chemokines and their receptors are important regulators of immunity, particularly in the migration and localization of circulating leukocytes within peripheral tissues.This report provides a comprehensive comparison of the chemokine receptor and activation phenotype of the major leukocyte subsets present in end-stage HCV-infected and non-HCV infected livers.Lymphocytes were purified from homogenized explant liver tissue and analyzed by flow cytometry.NK cells are the predominant cell type, followed by T cells, B cells and NK-T cells, independent of HCV status. T cells displayed a memory phenotype and low levels of activation markers. CCR5, CXCR3 and CXCR6 were expressed on a large fraction of activated cells, while moderate to low expression of CCR2, CCR6 and CX(3)CR1 was observed. Several other tissue-specific and inflammatory chemokine receptors were absent from infiltrating lymphocytes.These results identify the chemokine receptors present on infiltrating lymphocytes during end-stage liver disease and suggest that such infiltration is predominantly controlled by non-tissue-specific inflammatory chemokines, a situation that may be distinct from liver homing pathways under normal conditions.

  View details for PubMedID 12480562

• A blood-borne antigen induces rapid T-B cell contact: a potential mechanism for tolerance induction IMMUNOLOGY Gutgemann, I., Darling, J. M., Greenberg, H. B., DAVIS, M. M., Chien, Y. H. 2002; 107 (4): 420-425

  Abstract

  Understanding the difference between the development of a productive T-cell response and tolerance is central to discerning how the immune system functions. Intravenous injection of soluble protein is thought to mimic the presentation of self-serum and orally introduced antigens. It is generally toleragenic. The current view is that this outcome reflects the failure of 'immunogenic' dendritic cells to relocate to the T-cell zone of the secondary lymphoid tissues. Here, using a peptide/I-Ek tetramer and antibodies to stain splenic sections, we showed that antigen-specific T cells were activated in the spleen within hours of injection or feeding of protein. The activated T cells were found to be located at the T-B junction, the bridging zone and the B-cell area, interacting directly with B cells. In addition, B cells gain the ability to present antigen. Our results suggest a way for T cells to be stimulated by blood-borne antigen presented by naïve B cells, a potential mechanism of tolerance induction.

  View details for Web of Science ID 000179547900005

  View details for PubMedID 12460186

• A prenylation inhibitor prevents production of infectious hepatitis delta virus particles JOURNAL OF VIROLOGY Bordier, B. B., Marion, P. L., Ohashi, K., Kay, M. A., Greenberg, H. B., Casey, J. L., Glenn, J. S. 2002; 76 (20): 10465-10472

  Abstract

  Hepatitis delta virus (HDV) causes both acute and chronic liver disease throughout the world. Effective medical therapy is lacking. Previous work has shown that the assembly of HDV virus-like particles (VLPs) could be abolished by BZA-5B, a compound with farnesyltransferase inhibitory activity. Here we show that FTI-277, another farnesyltransferase inhibitor, prevented the production of complete, infectious HDV virions of two different genotypes. Thus, in spite of the added complexity and assembly determinants of infectious HDV virions compared to VLPs, the former are also sensitive to pharmacological prenylation inhibition. Moreover, production of HDV genotype III virions, which is associated with particularly severe clinical disease, was as sensitive to prenylation inhibition as was that of HDV genotype I virions. Farnesyltransferase inhibitors thus represent an attractive potential class of novel antiviral agents for use against HDV, including the genotypes associated with most severe disease.

  View details for DOI 10.1128/JVI.76.20.10465-10472.2002

  View details for PubMedID 12239323

• Analysis of CD8+ T cell receptor(TCR) diversity to an NS3 epitope in spontaneously cleared and chronic HCV infection: Plasticity of TCR may influence outcome of HCV infection. 53rd Annual Meeting of the Association-for-the-Study-of-Liver-Diseases (AASLD) Darling, J. M., Erickson, A. L., Kanistanon, D., He, X. S., Wright, T. L., Greenberg, H. B., Cooper, S. WILEY-BLACKWELL. 2002: 263A–263A

  View details for Web of Science ID 000178301700384

• Impaired effector function of hepatitis C virus-specific CD8(+) T cells in chronic hepatitis C virus infection JOURNAL OF IMMUNOLOGY Wedemeyer, H., He, X. S., Nascimbeni, M., Davis, A. R., Greenberg, H. B., Hoofnagle, J. H., Liang, T. J., Alter, H., Rehermann, B. 2002; 169 (6): 3447-3458

  Abstract

  The cellular immune response contributes to clearance of hepatitis C virus (HCV) and persists for decades after recovery from infection. The immunological basis for the inefficiency of the cellular immune response in chronically infected persons is not known. Here, we used four HLA-A2 tetramers, specific for two HCV core and two HCV NS3 epitopes, to investigate at the single-cell level effector function and phenotype of HCV-specific CD8+ T cells in 20 chronically infected and 12 long-term recovered patients. Overall, HCV-specific, tetramer+ T cells were more frequently found in PBMCs of chronically infected patients than in those of recovered patients. However, when compared with HCV-tetramer+ T cells of recovered patients, they displayed an impaired proliferative capacity. As a result of the impaired proliferative capacity, HCV-specific T cell lines derived from chronically infected patients displayed less peptide-specific cytotoxicity than those from recovered patients. In addition, proliferation and ex vivo IFN-gamma production of HCV-tetramer+ cells, but not influenza-virus-specific T cells, were defective in chronically infected patients and could not be restored by in vitro stimulation with peptide and IL-2. At least three distinct phenotypes of HCV-specific CD8+ T cells were identified and associated with certain functional characteristics. In addition, impairment of proliferative, cytokine, and cytotoxic effector functions of tetramer+ T cells in viremic patients was associated with weak ex vivo HCV-specific CD4+ T cell responses. Thus, the defective functions of HCV-specific CD8+ T cells might contribute to viral persistence in chronically infected patients, and knowledge on their reversibility may facilitate the development of immunotherapeutic vaccines.

  View details for Web of Science ID 000177958200080

  View details for PubMedID 12218168

• Rescue of influenza B virus from eight plasmids PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hoffmann, E., Mahmood, K., Yang, C. F., Webster, R. G., Greenberg, H. B., Kemble, G. 2002; 99 (17): 11411-11416

  Abstract

  Influenza B virus causes a significant amount of morbidity and mortality, yet the systems to produce high yield inactivated vaccines for these viruses have lagged behind the development of those for influenza A virus. We have established a plasmid-only reverse genetics system for the generation of recombinant influenza B virus that facilitates the generation of vaccine viruses without the need for time consuming coinfection and selection procedures currently required to produce reassortants. We cloned the eight viral cDNAs of influenza B/Yamanashi/166/98, which yields relatively high titers in embryonated chicken eggs, between RNA polymerase I and RNA polymerase II transcription units. Virus was detected as early as 3 days after transfection of cocultured COS7 and Madin-Darby canine kidney cells and achieved levels of 10(6)-10(7) plaque-forming units per ml of cell supernatant 6 days after transfection. The full-length sequence of the recombinant virus after passage into embryonated chicken eggs was identical to that of the input plasmids. To improve the utility of the eight-plasmid system for generating 6 + 2 reassortants from recently circulating influenza B strains, we optimized the reverse transcriptase-PCR for cloning of the hemagglutinin (HA) and neuraminidase (NA) segments. The six internal genes of B/Yamanashi/166/98 were used as the backbone to generate 6 + 2 reassortants including the HA and NA gene segments from B/Victoria/504/2000, B/Hong Kong/330/2001, and B/Hawaii/10/2001. Our results demonstrate that the eight-plasmid system can be used for the generation of high yields of influenza B virus vaccines expressing current HA and NA glycoproteins from either of the two lineages of influenza B virus.

  View details for DOI 10.1073/pnas.172393399

  View details for Web of Science ID 000177606900083

  View details for PubMedID 12172012

• Heterologous protection induced by the inner capsid proteins of rotavirus requires transcytosis of mucosal immunoglobulins JOURNAL OF VIROLOGY Schwartz-Cornil, I., Benureau, Y., Greenberg, H., Hendrickson, B. A., Cohen, J. 2002; 76 (16): 8110-8117

  Abstract

  Protective immunization against rotavirus (RV) can be achieved with heterologous RV, i.e., virus isolated from another species, and with heterologous inner core VP2 and VP6 proteins assembled as virus-like particles (VLP). Although the antigenically conserved VP6 protein does not induce in vitro-neutralizing antibodies, it may, however, elicit immunoglobulins (Ig) involved in heterologous protection, as some IgA against VP6 prevent RV infection in a backpack mouse model. The protective role of Ig directed to the RV inner core proteins VP2 and VP6 was investigated in J-chain-deficient mice (J chain(-/-)), which have a defect in the polymeric Ig receptor (pIgR)-mediated transcytosis of IgA and IgM. J chain(-/-) mice and wild-type (WT) mice were intranasally vaccinated with bovine RV-derived VLP2/6 and then challenged with highly infectious murine ECw RV. Whereas WT mice were totally protected, immunized J chain(-/-) mice shed RV for several days. In addition, naïve J chain(-/-) mice exhibited a 2-day delay in clearing RV compared with WT mice. The immunized J chain(-/-) mice displayed unaltered VLP2/6-specific B-cell numbers in spleen and in mesenteric nodes and similar levels of serum anti-VLP2/6 Ig, confirming that the adaptive B-cell response is preserved in J chain(-/-) mice. These results indicate that J-chain-mediated transcytosis of Ig participates in the clearance of RV and that epithelial pIgR-mediated transport of Ig is involved in the heterologous protection induced by VLP2/6.

  View details for DOI 10.1128/JVI.76.16.8110-8117.2002

  View details for Web of Science ID 000177049500021

  View details for PubMedID 12134016

• Principles, organization, and operation of a DNA bank for clinical trials: a Department of Veterans Affairs cooperative study CONTROLLED CLINICAL TRIALS Lavori, P. W., Krause-Steinrauf, H., Brophy, M., Buxbaum, J., Cockroft, J., Cox, D. R., Fiore, L., Greely, H. T., Greenberg, H., Holmes, E. W., Nelson, L. M., Sugarman, J. 2002; 23 (3): 222-239

  Abstract

  The mapping and sequencing of the human genome promises rapid growth in understanding the genetically influenced mechanisms that underlie human disease. To realize this promise fully, it is necessary to relate genetic information to clinical phenotypes. Genetic tissue banking in clinical studies provides opportunities to analyze the genetic contribution to variation in response to treatments. The challenges to progress are likely to come from the complex organizational, social, political, and ethical issues that must be resolved in order to put clinical and DNA bank information together. Concerns about subjects' rights, informed consent, privacy, and ownership of genetic material require attention in the development of DNA banks. In this paper we describe one approach to the solution of these problems that was adopted by one clinical trials group, the Department of Veterans Affairs Cooperative Studies Program.

  View details for Web of Science ID 000175963200001

  View details for PubMedID 12057876

• Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6-specific IgA mAb. journal of clinical investigation Feng, N., Lawton, J. A., Gilbert, J., Kuklin, N., Vo, P., Prasad, B. V., Greenberg, H. B. 2002; 109 (9): 1203-1213

  Abstract

  Rotaviruses are the leading cause of severe diarrheal disease in young children. Intestinal mucosal IgA responses play a critical role in protective immunity against rotavirus reinfection. Rotaviruses consist of three concentric capsid layers surrounding a genome of 11 segments of double-stranded RNA. The outer layer proteins, VP4 and VP7, which are responsible for viral attachment and entry, are targets for protective neutralizing antibodies. However, IgA mAb's directed against the intermediate capsid protein VP6, which do not neutralize the virus, have also been shown to protect mice from rotavirus infection and clear chronic infection in SCID mice. We investigated whether the anti-VP6 IgA (7D9) mAb could inhibit rotavirus replication inside epithelial cells and found that 7D9 acted at an early stage of infection to neutralize rotavirus following antibody lipofection. Using electron cryomicroscopy, we determined the three-dimensional structure of the virus-antibody complex. The attachment of 7D9 IgA to VP6 introduces a conformational change in the VP6 trimer, rendering the particle transcriptionally incompetent and preventing the elongation of initiated transcripts. Based on these observations, we suggest that anti-VP6 IgA antibodies confers protection in vivo by inhibiting viral transcription at the start of the intracellular phase of the viral replication cycle.

  View details for PubMedID 11994409

• Inhibition of rotavirus replication by a non-neutralizing rotaviras VP6-specific IgA mAb JOURNAL OF CLINICAL INVESTIGATION Feng, N. G., Lawton, J. A., Gilbert, J., Kuklin, N., Vo, P., Prasad, B. V., Greenberg, H. B. 2002; 109 (9): 1203-1213

  View details for DOI 10.1172/JCI200214397

  View details for Web of Science ID 000175488000011

• Frequencies of virus-specific CD4(+) and CD8(+) T lymphocytes secreting gamma interferon after acute natural rotavirus infection in children and adults JOURNAL OF VIROLOGY Jaimes, M. C., Rojas, O. L., Gonzalez, A. M., Cajiao, I., Charpilienne, A., Pothier, P., Kohli, E., Greenberg, H. B., Franco, M. A., Angel, J. 2002; 76 (10): 4741-4749

  Abstract

  Human rotavirus-specific CD4(+) and CD8(+) T-cell responses in peripheral blood lymphocytes were studied using a flow cytometric assay that detects the intracellular accumulation of cytokines after short-term in vitro antigen stimulation. The frequencies of virus-specific T cells that secrete gamma interferon and interleukin-13 (IL-13) were determined in adults and children during the acute or convalescent phase of rotavirus-induced diarrhea, in asymptomatically infected adults and laboratory workers who worked with human stool samples containing rotavirus, and in healthy adults. Significantly higher frequencies of rotavirus-specific interferon gamma-secreting CD8(+) and CD4(+) T cells, but not IL-13-secreting T cells, were detected in symptomatically infected adults and exposed laboratory workers than in healthy adults and children with acute rotavirus diarrhea. The levels of rotavirus-specific T cells returned to levels found in healthy adults by 32 days after the onset of rotavirus diarrhea in most adult subjects. Children with rotavirus diarrhea had undetectable or very low levels of CD4(+) and CD8(+) T cells that secrete gamma interferon. Adult cytomegalovirus-seropositive individuals had frequencies of cytomegalovirus-specific T cells that secrete gamma interferon that were approximately 20 times the level of rotavirus-specific T cells. This result suggests that rotavirus is a relatively poor inducer of circulating memory T cells that secrete gamma interferon. The frequencies of gamma interferon-secreting CD4(+) and CD8(+) T cells and the frequencies of IL-13-secreting CD4(+) T cells responding to the T-cell superantigen staphylococcal enterotoxin B (SEB) were lower in children than in adults. In both adults and children, the frequencies of CD4(+) cells secreting gamma interferon in response to SEB were higher than the frequencies of cells secreting IL-13.

  View details for DOI 10.1128/JVI.76.10.4741-4749.2002

  View details for Web of Science ID 000175256500007

  View details for PubMedID 11967291

• Gene expression pattern in Caco-2 cells following rotavirus infection JOURNAL OF VIROLOGY Cuadras, M. A., Feigelstock, D. A., An, S. W., Greenberg, H. B. 2002; 76 (9): 4467-4482

  Abstract

  Rotaviruses are recognized as the leading cause of severe dehydrating diarrhea in infants and young children worldwide. Preventive and therapeutic strategies are urgently needed to fight this pathogen. In tissue culture and in vivo, rotavirus induces structural and functional alterations in the host cell. In order to better understand the molecular mechanisms involved in the events after rotavirus infection, we identified host cellular genes whose mRNA levels changed after infection. For this analysis, we used microarrays containing more than 38,000 human cDNAs to study the transcriptional response of the human intestinal cell line Caco-2 to rotavirus infection. We found that 508 genes were differentially regulated >2-fold at 16 h after rotavirus infection, and only one gene was similarly regulated at 1 h postinfection. Of these transcriptional changes, 73% corresponded to the upregulation of genes, with the majority of them occurring late, at 12 or more hours postinfection. Some of the regulated genes were classified according to known biological function and included genes encoding integral membrane proteins, interferon-regulated genes, transcriptional and translational regulators, and calcium metabolism-related genes. A new picture of global transcriptional regulation in the infected cell is presented and families of genes which may be involved in viral pathogenesis are discussed.

  View details for DOI 10.1128/JVI.76.9.4467-4482.2002

  View details for Web of Science ID 000174966700034

  View details for PubMedID 11932413

  View details for PubMedCentralID PMC155077

• Correlation of tissue distribution, developmental phenotype, and intestinal homing receptor expression of antigen-specific B cells during the murine anti-rotavirus immune response JOURNAL OF IMMUNOLOGY Youngman, K. R., Franco, M. A., Kuklin, N. A., Rott, L. S., BUTCHER, E. C., Greenberg, H. B. 2002; 168 (5): 2173-2181

  Abstract

  The intestinal homing receptor, alpha(4)beta(7), helps target lymphocytes to Peyer's patches (PP) and intestinal lamina propria (ILP). We have previously shown that protective immunity to rotavirus (RV), an intestinal pathogen, resides in memory B cells expressing alpha(4)beta(7). In this study, using a novel FACS assay, we have directly studied the phenotype of B cells that express surface RV-specific Ig during the in vivo RV immune response. During primary infection, RV-specific B cells first appear as large IgD(-)B220(low)alpha(4)beta(7)(-)and alpha(4)beta(7)(+) cells (presumptive extrafollicular, Ab-secreting B cells), and then as large and small IgD(-)B220(high)alpha(4)beta(7)(-)cells (presumptive germinal center B cells). The appearance of B cells with the phenotype of large IgD(-)B220(low)alpha(4)beta(7)(+) cells in PP and most notably in mesenteric lymph nodes coincides with the emergence of RV-specific Ab-secreting cells (ASC) in the ILP. Thus, these B lymphocytes are good candidates for the migratory population giving rise to the RV-specific ASC in the ILP. RV-specific long-term memory B cells preferentially accumulate in PP and express alpha(4)beta(7). Nine months after infection most RV-specific IgA ASC are found in PP and ILP and at lower frequency in bone marrow and spleen. This study is the first to follow changes in tissue-specific homing receptor expression during Ag-specific B cell development in response to a natural host, tissue-specific pathogen. These results show that alpha(4)beta(7) is tightly regulated during the Ag-specific B cell response to RV and is expressed concurrently with the specific migration of memory and effector B cells to intestinal tissues.

  View details for Web of Science ID 000173990200013

  View details for PubMedID 11859103

• The intestinal chemokine thymus-expressed chemokine (CCL25) attracts IgA antibody-secreting cells JOURNAL OF EXPERIMENTAL MEDICINE Bowman, E. P., Kuklin, N. A., Youngman, K. R., Lazarus, N. H., Kunkel, E. J., Pan, J. L., Greenberg, H. B., Butcher, E. C. 2002; 195 (2): 269-275

  Abstract

  Immunoglobulin A (IgA) provides protection against pathogens at mucosal surfaces. Chemotactic responses have been hypothesized to target IgA plasma cells involved in mucosal immune responses. We show here that thymus-expressed chemokine (TECK, CCL25) is a potent and selective chemoattractant for IgA antibody-secreting cells (ASC), efficiently recruiting IgA-producing cells from spleen, Peyer's patches, and mesenteric lymph node. Cells secreting IgA antibody in response to rotavirus, an intestinal pathogen, also respond well. In contrast, IgG- and IgM-ASC respond poorly. Epithelial cells in the small intestines, a principal site of IgA-ASC localization and IgA production in the body, highly and selectively express TECK. The migration of IgA-ASC to the intestinal epithelial cell chemokine TECK may help target IgA-producing cells to the gut wall, thus helping define and segregate the intestinal immune response.

  View details for Web of Science ID 000173759300013

  View details for PubMedID 11805153

• Expression of the chemokine receptors CCR4, CCR5, and CXCR3 by human tissue-infiltrating lymphocytes AMERICAN JOURNAL OF PATHOLOGY Kunkel, E. J., Boisvert, J., Murphy, K., Vierra, M. A., Genovese, M. C., Wardlaw, A. J., Greenberg, H. B., Hodge, M. R., Wu, L. J., BUTCHER, E. C., Campbell, J. J. 2002; 160 (1): 347-355

  Abstract

  Differential expression of adhesion molecules and chemokine receptors has been useful for identification of peripheral blood memory lymphocyte subsets with distinct tissue and microenvironmental tropisms. Expression of CCR4 by circulating memory CD4(+) lymphocytes is associated with cutaneous and other systemic populations while expression of CCR9 is associated with a small intestine-homing subset. CCR5 and CXCR3 are also expressed by discrete memory CD4(+) populations in blood, as well as by tissue-infiltrating lymphocytes from a number of sites. To characterize the similarities and differences among tissue-infiltrating lymphocytes, and to shed light on the specialization of lymphocyte subsets that mediate inflammation and immune surveillance in particular tissues, we have examined the expression of CCR4, CXCR3, and CCR5 on CD4(+) lymphocytes directly isolated from a wide variety of normal and inflamed tissues. Extra-lymphoid tissues contained only memory lymphocytes, many of which were activated (CD69(+)). As predicted by classical studies, skin lymphocytes were enriched in CLA expression whereas intestinal lymphocytes were enriched in alpha(4)beta(7) expression. CCR4 was expressed at high levels by skin-infiltrating lymphocytes, at lower levels by lung and synovial fluid lymphocytes, but never by intestinal lymphocytes. Only the high CCR4 levels characteristic of skin lymphocytes were associated with robust chemotactic and adhesive responses to TARC, consistent with a selective role for CCR4 in skin lymphocyte homing. In contrast, CXCR3 and CCR5 were present on the majority of lymphocytes from each non-lymphoid tissue examined, suggesting that these receptors are unlikely to determine tissue specificity, but rather, may play a wider role in tissue inflammation.

  View details for Web of Science ID 000173231700038

  View details for PubMedID 11786428

• CD8(+) T-Cell response against hepatitis C virus VIRAL IMMUNOLOGY He, X. S., Greenberg, H. B. 2002; 15 (1): 121-131

  Abstract

  CD8+ T-cell response is thought to be important for the control of hepatitis C virus (HCV) as well as for the liver cell injury caused by HCV infection. Studies on antigen-specific CD8+ T cells had long been hampered by lack of suitable techniques. Recently developed single-cell based assays, including peptide major histocompatibility complex (MHC) tetramer staining and intracellular cytokine staining, have greatly enhanced the opportunities for directly studying HCV-specific CD8+ T cells. Thanks to these novel assays the quantitative and qualitative nature of HCV-specific CD8+ T cells, including their number, phenotype, and effector functions, are starting to be revealed. However, much important information remains missing, including the signals for differentiation and migration of HCV-specific CD8+ T cells and the precise functions of antigen-specific effector cells in the virus-infected liver. The urgent need for effective immunotherapy and vaccines can not be met without a better understanding of the CD8+ T-cell response in HCV infection, which calls for a comprehensive strategy to study such cells directly using sensitive and quantitative assays.

  View details for Web of Science ID 000175313800010

  View details for PubMedID 11952134

• Localization of membrane permeabilization and receptor binding sites on the VP4 hemagglutinin of rotavirus: Implications for cell entry JOURNAL OF MOLECULAR BIOLOGY Tihova, M., Dryden, K. A., Bellamy, A. R., Greenberg, H. B., Yeager, M. 2001; 314 (5): 985-992

  Abstract

  The surface of rotavirus is decorated with 60 spike-like projections, each composed of a dimer of VP4, the viral hemagglutinin. Trypsin cleavage of VP4 generates two fragments, VP8*, which binds sialic acid (SA), and VP5*, containing an integrin binding motif and a hydrophobic region that permeabilizes membranes and is homologous to fusion domains. Although the mechanism for cell entry by this non-enveloped virus is unclear, it is known that trypsin cleavage enhances viral infectivity and facilitates viral entry. We used electron cryo-microscopy and difference map analysis to localize the binding sites for two neutralizing monoclonal antibodies, 7A12 and 2G4, which are directed against the SA-binding site within VP8* and the membrane permeabilization domain within VP5*, respectively. Fab 7A12 binds at the tips of the dimeric heads of VP4, and 2G4 binds in the cleft between the two heads of the spike. When these binding results are combined with secondary structure analysis, we predict that the VP4 heads are composed primarily of beta-sheets in VP8* and that VP5* forms the body and base primarily in beta-structure and alpha-helical conformations, respectively. Based on these results and those of others, a model is proposed for cell entry in which VP8* and VP5* mediate receptor binding and membrane permeabilization, and uncoating occurs during transfer across the lipid bilayer, thereby generating the transcriptionally active particle.

  View details for DOI 10.1006/jmbi.2001.5238

  View details for Web of Science ID 000173365300004

  View details for PubMedID 11743716

• Quantitative analysis of hepatitis C virus in peripheral blood and liver: Replication detected only in liver 10th International Symposium on Viral Hepatitis and Liver Disease Boisvert, J., He, X. S., Cheung, R., Keeffe, E. B., Wright, T., Greenberg, H. B. UNIV CHICAGO PRESS. 2001: 827–35

  Abstract

  Prior studies seeking evidence of viral replication in peripheral lymphocytes of hepatitis C virus (HCV)-infected patients have yielded conflicting results. This study sought to quantitatively determine whether a permissive HCV cell interaction could be detected in leukocytes from infected patients. Peripheral leukocytes from chronically infected patients were purified and were tested for HCV RNA. The results show that virus load is highest in B cells. Other subsets of peripheral leukocytes consistently had very low levels of viral RNA or were negative. Negative-strand HCV was found only in hepatocytes. To determine whether HCV replication could be induced by activation, B cells from HCV-infected patients were stimulated in vitro. No HCV replicating in peripheral leukocytes was detected by a highly sensitive assay. If HCV replication occurs in the leukocyte subsets analyzed here, it is at extremely low levels or occurs under alternate physiological conditions.

  View details for Web of Science ID 000171228400003

  View details for PubMedID 11550124

• Proteolysis of monomeric recombinant rotavirus VP4 yields an oligomeric VP5*core JOURNAL OF VIROLOGY Dormitzer, P. R., Greenberg, H. B., HARRISON, S. C. 2001; 75 (16): 7339-7350

  Abstract

  Rotavirus particles are activated for cell entry by trypsin cleavage of the outer capsid spike protein, VP4, into a hemagglutinin, VP8*, and a membrane penetration protein, VP5*. We have purified rhesus rotavirus VP4, expressed in baculovirus-infected insect cells. Purified VP4 is a soluble, elongated monomer, as determined by analytical ultracentrifugation. Trypsin cleaves purified VP4 at a number of sites that are protected on the virion and yields a heterogeneous group of protease-resistant cores of VP5*. The most abundant tryptic VP5* core is trimmed past the N terminus associated with activation for virus entry into cells. Sequential digestion of purified VP4 with chymotrypsin and trypsin generates homogeneous VP8* and VP5* cores (VP8CT and VP5CT, respectively), which have the authentic trypsin cleavages in the activation region. VP8CT is a soluble monomer composed primarily of beta-sheets. VP5CT forms sodium dodecyl sulfate-resistant dimers. These results suggest that trypsinization of rotavirus particles triggers a rearrangement in the VP5* region of VP4 to yield the dimeric spikes observed in icosahedral image reconstructions from electron cryomicroscopy of trypsinized rotavirus virions. The solubility of VP5CT and of trypsinized rotavirus particles suggests that the trypsin-triggered conformational change primes VP4 for a subsequent rearrangement that accomplishes membrane penetration. The domains of VP4 defined by protease analysis contain all mapped neutralizing epitopes, sialic acid binding residues, the heptad repeat region, and the membrane permeabilization region. This biochemical analysis of VP4 provides sequence-specific structural information that complements electron cryomicroscopy data and defines targets and strategies for atomic-resolution structural studies.

  View details for Web of Science ID 000170093000012

  View details for PubMedID 11462006

  View details for PubMedCentralID PMC114969

• Immune responses and protection obtained with rotavirus VP6 DNA vaccines given by intramuscular injection VACCINE Yang, K. J., Wang, S. X., Chang, K. O., Lu, S., SAIF, L. J., Greenberg, H. B., Brinker, J. P., Herrmann, J. E. 2001; 19 (23-24): 3285-3291

  Abstract

  Intramuscular (i.m.) injection of murine VP6 DNA vaccines raised high titers of rotavirus-specific serum IgG and IgA antibodies in BALB/c mice. A Th1-like antibody response was generated based on the ratio of serum IgG2a to IgG1 antibodies. Rotavirus-specific serum IgA but not fecal IgA was detected in mice prior to rotavirus challenge. Partial protection against rotavirus challenge was achieved as measured by reduction of rotavirus antigen shedding in feces. A similar level of protection was found with a bovine rotavirus VP6 DNA vaccine against a murine rotavirus challenge, suggesting that heterologous protection can be obtained by immunizing with VP6 DNA vaccines. We did not directly test for cytotoxic T lymphocyte (CTL) activity, but in vivo depletion of CD8+ T cells in mice immunized with a murine VP6 DNA vaccine did not significantly change the duration of virus shedding or the pattern of protection obtained. This finding suggested that CD8+ CTL activity was not essential for the partial protection we obtained by i.m. immunization of mice with VP6 DNA vaccines.

  View details for Web of Science ID 000168579000032

  View details for PubMedID 11312027

• Challenges for rotavirus vaccines VIROLOGY Franco, M. A., Greenberg, H. B. 2001; 281 (2): 153-155

  View details for Web of Science ID 000167800000001

  View details for PubMedID 11277688

• Bonzo/CXCR6 expression defines type 1-polarized T-cell subsets with extralymphoid tissue homing potential JOURNAL OF CLINICAL INVESTIGATION Kim, C. H., Kunkel, E. J., Boisvert, J., Johnston, B., Campbell, J. J., Genovese, M. C., Greenberg, H. B., BUTCHER, E. C. 2001; 107 (5): 595-601

  Abstract

  Chemokine receptor expression is finely controlled during T-cell development. We show that newly identified chemokine receptor Bonzo/CXCR6 is expressed by subsets of Th1 or T-cytotoxic 1 (Tc1) cells, but not by Th2 or Tc2 cells, establishing Bonzo as a differential marker of polarized type 1 T cells in vitro and in vivo. Priming of naive T cells by dendritic cells induces expression of Bonzo on T cells. IL-12 enhances this dendritic cell-dependent upregulation, while IL-4 inhibits it. In blood, 35-56% of Bonzo+ CD4 T cells are Th1 cells, and 60-65% of Bonzo+ CD8 T cells are Tc1 cells, while few Bonzo+ cells are type 2 T cells. Almost all Bonzo+ Tc1 cells contain preformed granzyme A and display cytotoxic effector phenotype. Most Bonzo+ T cells lack L-selectin and/or CCR7, homing receptors for lymphoid tissues. Instead, Bonzo+ T cells are dramatically enriched among T cells in tissue sites of inflammation, such as rheumatoid joints and inflamed livers. Bonzo may be important in trafficking of effector T cells that mediate type 1 inflammation, making it a potential target for therapeutic modulation of inflammatory diseases.

  View details for Web of Science ID 000167337800009

  View details for PubMedID 11238560

  View details for PubMedCentralID PMC199429

• Protective intestinal anti-rotavirus B cell immunity is dependent on alpha(4)beta(7) integrin expression but does not require IgA antibody production JOURNAL OF IMMUNOLOGY Kuklin, N. A., Rott, L., Feng, N. G., Conner, M. E., Wagner, N., Muller, W., Greenberg, H. B. 2001; 166 (3): 1894-1902

  Abstract

  Rotavirus (RV) is the main cause of severe gastroenteritis in young children; protection has been correlated with intestinal Ab responses. Using a mouse model of RV infection and beta(7)-deficient (beta(7)(-/-)) mice, which do not express alpha(4)beta(7) integrin, we demonstrated the importance of alpha(4)beta(7) integrin in B cell-mediated anti-RV immunity. beta(7)(-/-) mice acutely infected with murine RV resolved infection and developed normal serum IgG Abs but had diminished intestinal IgA responses. alpha(4)beta(7)(-/-) immune B cells did not resolve RV infection when adoptively transferred into RV-infected Rag-2-deficient mice. Fewer RV-specific B cells were found in the intestine of Rag-2-deficient mice transferred with beta(7)(-/-) B cells compared with wild type. The absence of alpha(4)beta(7) expression and/or a lower frequency of IgA-producing cells among transferred beta(7)(-/-) B cells could have accounted for the inability of these cells to resolve RV infection following passive transfer. To distinguish between these possibilities, we studied the importance of IgA production in RV infection using IgA-deficient (IgA(-/-)) mice. IgA(-/-) mice depleted of CD8(+) T cells were able to clear primary RV infection. Similarly, adoptive transfer of immune IgA(-/-) B cells into chronically infected Rag-2-deficient mice resolved RV infection. We further demonstrated in both wild-type and IgA(-/-) mice that, following oral RV infection, protective B cells reside in the alpha(4)beta(7)(high) population. Our findings suggest that alpha(4)beta(7) integrin expression is necessary for B cell-mediated immunity to RV independent of the presence of IgA.

  View details for Web of Science ID 000166622700059

  View details for PubMedID 11160237

• CCR7 expression and memory T cell diversity in humans JOURNAL OF IMMUNOLOGY Campbell, J. J., Murphy, K. E., Kunkel, E. J., Brightling, C. E., Soler, D., Shen, Z. M., Boisvert, J., Greenberg, H. B., Vierra, M. A., Goodman, S. B., Genovese, M. C., Wardlaw, A. J., BUTCHER, E. C., Wu, L. J. 2001; 166 (2): 877-884

  Abstract

  CCR7, along with L-selectin and LFA-1, mediates homing of T cells to secondary lymphoid organs via high endothelial venules (HEV). CCR7 has also been implicated in microenvironmental positioning of lymphocytes within secondary lymphoid organs and in return of lymphocytes and dendritic cells to the lymph after passage through nonlymphoid tissues. We have generated mAbs to human CCR7, whose specificities correlate with functional migration of lymphocyte subsets to known CCR7 ligands. We find that CCR7 is expressed on the vast majority of peripheral blood T cells, including most cells that express adhesion molecules (cutaneous lymphocyte Ag alpha(4)beta(7) integrin) required for homing to nonlymphoid tissues. A subset of CD27(neg) memory CD4 T cells from human peripheral blood is greatly enriched in the CCR7(neg) population, as well as L-selectin(neg) cells, suggesting that these cells are incapable of homing to secondary lymphoid organs. Accordingly, CD27(neg) T cells are rare within tonsil, a representative secondary lymphoid organ. All resting T cells within secondary lymphoid organs express high levels of CCR7, but many activated cells lack CCR7. CCR7 loss in activated CD4 cells accompanies CXC chemokine receptor (CXCR)5 gain, suggesting that the reciprocal expression of these two receptors may contribute to differential positioning of resting vs activated cells within the organ. Lymphocytes isolated from nonlymphoid tissues (such as skin, lung, or intestine) contain many CD27(neg) cells lacking CCR7. The ratio of CD27(neg)/CCR7(neg) cells to CD27(pos)/CCR7(pos) cells varies from tissue to tissue, and may correlate with the number of cells actively engaged in Ag recognition within a given tissue.

  View details for Web of Science ID 000166259600023

  View details for PubMedID 11145663

• Protective intestinal antirotavirus B cell immunity is dependent on alph4beta7 integrin expression but does not require IgA antibody production J Immunol Greenberg HB., Kuklin NA, Rott L, Feng N, Conner ME, Wagner N, Muller W 2001
• Protective intestinal antirotavirus B cell immunity is dependent on alph4beta7 integrin expression but does not require IgA antibody production. J Immunol. Greenberg HB., Kuklin NA, Rott L, Feng N, Conner ME, Wagner N, Muller W 2001: 1894-192
• Rotavirus assembly - interaction of surface protein VP7 with middle layer protein VP6 ARCHIVES OF VIROLOGY Gilbert, J. M., Feng, N., Patton, J. T., Greenberg, H. B. 2001; 146 (6): 1155-1171

  Abstract

  The interaction between the rotavirus proteins viral protein 6 (VP6) and VP7 was examined in several exogenous protein expression systems. These proteins associated in the absence of other rotaviral proteins as demonstrated by a coimmunoprecipitation assay. Deletion analysis of VP7 indicated that truncations of either the mature amino or carboxyl terminus disrupted the proper folding of the protein and were not able to coimmunoprecipitate VP6. Truncation analysis of VP6 indicated that trimerization of VP6 was necessary, but not sufficient, for VP7 binding. MAb mapping and coimmunoprecipitation interference assays indicate that the VP6 amino acid residues between 271 and 342 are required for VP7 interaction. The interaction of VP6 and VP7 was also examined by the assembly of soluble VP7 onto baculovirus-expressed virus-like particles containing VP2 and VP6. Abrogation of this binding by preincubation of the particles with VP6 MAbs mapped to this same domain of VP6, validated our coimmunoprecipitation results. VP6 IgA MAbs that have been shown to be protective in vivo, but not a nonprotective IgA MAb, can interfere with VP7 binding to VP6. This suggests that these IgA MAbs may protect against rotavirus infection by blocking rotavirus assembly.

  View details for Web of Science ID 000169605500008

  View details for PubMedID 11504422

• Direct functional analysis of epitope-specific CD8+ T cells in peripheral blood VIRAL IMMUNOLOGY He, X. S., Rehermann, B., Boisvert, J., Mumm, J., Maecker, H. T., Roederer, M., Wright, T. L., Maino, V. C., DAVIS, M. M., Greenberg, H. B. 2001; 14 (1): 59-69

  Abstract

  The functional status of virus-specific CD8+ T cells is important for the outcome and the immunopathogenesis of viral infections. We have developed an assay for the direct functional analysis of antigen-specific CD8+ T cells, which does not require prolonged in vitro cultivation and amplification of T cells. Whole blood samples were incubated with peptide antigens for <5 h, followed by staining with peptide-MHC tetramers to identify epitope-specific T cells. The cells were also stained for the activation marker CD69 or for the production of cytokines such as interferon-gamma (IFNgamma) or tumor necrosis factor-alpha (TNFalpha). With the combined staining with tetramer and antibodies to CD69 or cytokines the number of antigen-specific CD8+ T cells as well as the functional response of each individual cell to the cognate antigen can be determined in a single experiment. Virus-specific CD8+ T cells that are nonfunctional, as well as those that are functional under the same stimulating conditions can be simultaneously detected with this assay, which is not possible by using other T-cell functional assays including cytotoxicity assay, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay.

  View details for Web of Science ID 000167575500005

  View details for PubMedID 11270597

• alpha(4)beta(7) independent pathway for CD8+ T cell-mediated intestinal immunity to rotavirus JOURNAL OF CLINICAL INVESTIGATION Kuklin, N. A., Rott, L., Darling, J., Campbell, J. J., Franco, M., Feng, N. G., Muller, W., Wagner, N., Altman, J., BUTCHER, E. C., Greenberg, H. B. 2000; 106 (12): 1541-1552

  Abstract

  Rotavirus (RV), which replicates exclusively in cells of the small intestine, is the most important cause of severe diarrhea in young children worldwide. Using a mouse model, we show that expression of the intestinal homing integrin alpha(4)ss(7) is not essential for CD8(+) T cells to migrate to the intestine or provide immunity to RV. Mice deficient in ss7 expression (ss7(-/-)) and unable to express alpha(4)ss(7) integrin were found to clear RV as quickly as wild-type (wt) animals. Depletion of CD8(+) T cells in ss7(-/-) animals prolonged viral shedding, and transfer of immune ss7(-/-) CD8(+) T cells into chronically infected Rag-2-deficient mice resolved RV infection as efficiently as wt CD8(+) T cells. Paradoxically, alpha(4)ss(7)(hi) memory CD8(+) T cells purified from wt mice that had been orally immunized cleared RV more efficiently than alpha(4)ss(7)(low) CD8(+) T cells. We explained this apparent contradiction by demonstrating that expression of alpha(4)ss(7) on effector CD8(+) T cells depends upon the site of initial antigen exposure: oral immunization generates RV-specific CD8(+) T cells primarily of an alpha(4)ss(7)(hi) phenotype, but subcutaneous immunization yields both alpha(4)ss(7)(hi) and alpha(4)ss(7)(low) immune CD8(+) T cells with anti-RV effector capabilities. Thus, alpha(4)ss(7) facilitates normal intestinal immune trafficking to the gut, but it is not required for effective CD8(+) T cell immunity.

  View details for PubMedID 11120761

• Purified recombinant rotavirus VP7 forms soluble, calcium-dependent trimers VIROLOGY Dormitzer, P. R., Greenberg, H. B., HARRISON, S. C. 2000; 277 (2): 420-428

  Abstract

  Rotavirus is a major cause of severe, dehydrating childhood diarrhea. VP7, the rotavirus outer capsid glycoprotein, is a target of protective antibodies and is responsible for the calcium-dependent uncoating of the virus during cell entry. We have purified, characterized, and crystallized recombinant rhesus rotavirus VP7, expressed in insect cells. A critical aspect of the purification is the elution of VP7 from a neutralizing monoclonal antibody column by EDTA. Gel filtration chromatography and equilibrium analytical ultracentrifugation demonstrate that, in the presence of calcium, purified VP7 trimerizes. Trimeric VP7 crystallizes into hexagonal plates. Preliminary X-ray analysis suggests that the crystal packing reproduces the hexagonal component of the icosahedral lattice of VP7 on triple-layered rotavirus particles. These data indicate that the rotavirus outer capsid assembles from calcium-dependent VP7 trimers and that dissociation of these trimers is the biochemical basis for EDTA-induced rotavirus uncoating and loss of VP7 neutralizing epitopes.

  View details for Web of Science ID 000165628300020

  View details for PubMedID 11080489

• Lymphocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression distinguish the small intestinal immune compartment: Epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity JOURNAL OF EXPERIMENTAL MEDICINE Kunkel, E. J., Campbell, J. J., Haraldsen, G., Pan, J. L., Boisvert, J., Roberts, A. I., Ebert, E. C., Vierra, M. A., Goodman, S. B., Genovese, M. C., Wardlaw, A. J., Greenberg, H. B., Parker, C. M., Butcher, E. C., Andrew, D. P., Agace, W. W. 2000; 192 (5): 761-767

  Abstract

  The immune system has evolved specialized cellular and molecular mechanisms for targeting and regulating immune responses at epithelial surfaces. Here we show that small intestinal intraepithelial lymphocytes and lamina propria lymphocytes migrate to thymus-expressed chemokine (TECK). This attraction is mediated by CC chemokine receptor (CCR)9, a chemoattractant receptor expressed at high levels by essentially all CD4(+) and CD8(+) T lymphocytes in the small intestine. Only a small subset of lymphocytes in the colon are CCR9(+), and lymphocytes from other tissues including tonsils, lung, inflamed liver, normal or inflamed skin, inflamed synovium and synovial fluid, breast milk, and seminal fluid are universally CCR9(-). TECK expression is also restricted to the small intestine: immunohistochemistry reveals that intense anti-TECK reactivity characterizes crypt epithelium in the jejunum and ileum, but not in other epithelia of the digestive tract (including stomach and colon), skin, lung, or salivary gland. These results imply a restricted role for lymphocyte CCR9 and its ligand TECK in the small intestine, and provide the first evidence for distinctive mechanisms of lymphocyte recruitment that may permit functional specialization of immune responses in different segments of the gastrointestinal tract. Selective expression of chemokines by differentiated epithelium may represent an important mechanism for targeting and specialization of immune responses.

  View details for PubMedID 10974041

• Host-microbe interactions: viruses - Lessons in defense: hepatitic C, a case study - Editorial overview CURRENT OPINION IN MICROBIOLOGY Cooper, S. S., Glenn, J., Greenberg, H. B. 2000; 3 (4): 363-365

  View details for Web of Science ID 000088817300007

• Hepatitis C virus and the host: An imbalance induced by immunosuppression? HEPATOLOGY Berenguer, M., Lopez-Labrador, F. X., Greenberg, H. B., Wright, T. L. 2000; 32 (2): 433-435

  View details for Web of Science ID 000089090700036

  View details for PubMedID 10915755

• Protective immunity to rotavirus shedding in the absence of interleukin-6: Th1 cells and immunoglobulin A develop normally JOURNAL OF VIROLOGY VanCott, J. L., Franco, M. A., Greenberg, H. B., Sabbaj, S., Tang, B. Z., Murray, R., MCGHEE, J. R. 2000; 74 (11): 5250-5256

  Abstract

  We investigated whether interleukin-6 (IL-6) was required for the development of immunoglobulin A (IgA)- and T-helper 1 (Th1)-associated protective immune responses to rotavirus by using adult IL-6-deficient mice [BALB/c and (C57BL/6 x O1a)F(2) backgrounds]. Naive IL-6(-) mice had normal frequencies of IgA plasma cells in the gastrointestinal tract. Consistent with this, total levels of IgA in fecal extracts, saliva, and sera were unaltered. In specific response to oral infection with rhesus rotavirus, IL-6(-) and IL-6(+) mice exhibited efficient Th1-type gamma interferon responses in Peyer's patches with high levels of serum IgG2a and intestinal IgA. Although there was an increase in Th2-type IL-4 in CD4(+) T cells from IL-6(-) mice following restimulation with rotavirus antigen in the presence of irradiated antigen-presenting cells, unfractionated Peyer's patch cells failed to produce a significant increase in IL-4. Moreover, virus-specific IgG1 in serum was not significantly increased in IL-6(-) mice in comparison with IL-6(+) mice. Following oral inoculation with murine rotavirus, IL-6(-) and IL-6(+) mice mediated clearance of rotavirus and mounted a strong IgA response. When IL-6(-) and IL-6(+) mice [(C57BL/6 x O1a)F(2) background] were orally inoculated with rhesus rotavirus and later challenged with murine rotavirus, all of the mice maintained high levels of IgA in feces and were protected against reinfection. Thus, IL-6 failed to provide unique functions in the development of IgA-secreting B cells and in the establishment of Th1-associated protective immunity against rotavirus infection in adult mice.

  View details for Web of Science ID 000086948000035

  View details for PubMedID 10799601

• Rotavirus VP6 expressed by PVX vectors in Nicotiana benthamiana coats PVX rods and also assembles into viruslike particles VIROLOGY O'Brien, G. J., Bryant, C. J., Voogd, C., Greenberg, H. B., Gardner, R. C., Bellamy, A. R. 2000; 270 (2): 444-453

  Abstract

  The rotavirus major inner capsid protein (VP6) has been expressed in Nicotiana benthamiana plants using vectors based on potato virus X (PVX). VP6 was expressed either as a fusion with the PVX coat protein or from an additional subgenomic promoter inserted to enable both VP6 and PVX coat protein to be expressed independently. Both approaches yielded VP6, which retained the ability to form trimers. VP6 expressed from the subgenomic promoter assembled into paracrystalline sheets and tubes. Expression as a fusion protein yielded PVX rods that presented an external "overcoat" of VP6, but unexpectedly, some rotavirus protein also assembled into icosahedral viruslike particles (VLPs). The assembly of viral protein into VLPs suggests that prior display of VP6 on the flexuous PVX rod facilitates the subsequent assembly of VP6 into stable icosahedral particles.

  View details for Web of Science ID 000087227100022

  View details for PubMedID 10793003

• CCR6 mediates dendritic cell localization, lymphocyte homeostasis, and immune responses in mucosal tissue IMMUNITY Cook, D. N., Prosser, D. M., Forster, R., Zhang, J., Kuklin, N. A., Abbondanzo, S. J., Niu, X. D., Chen, S. C., Manfra, D. J., Wiekowski, M. T., Sullivan, L. M., Smith, S. R., Greenberg, H. B., Narula, S. K., Lipp, M., Lira, S. A. 2000; 12 (5): 495-503

  Abstract

  Chemokine-directed migration of leukocyte subsets may contribute to the qualitative differences between systemic and mucosal immunity. Here, we demonstrate that in mice lacking the chemokine receptor CCR6, dendritic cells expressing CD11c and CD11b are absent from the subepithelial dome of Peyer's patches. These mice also have an impaired humoral immune response to orally administered antigen and to the enteropathic virus rotavirus. In addition, CCR6(-/-) mice have a 2-fold to 15-fold increase in cells of select T lymphocyte populations within the mucosa, including CD4+ and CD8+ alphabeta-TCR T cells. By contrast, systemic immune responses to subcutaneous antigens in CCR6(-/-) mice are normal. These findings demonstrate that CCR6 is a mucosa-specific regulator of humoral immunity and lymphocyte homeostasis in the intestinal mucosa.

  View details for Web of Science ID 000087356400005

  View details for PubMedID 10843382

• Immunity to calicivirus infection International Workshop on Human Caliciviruses Matsui, S. M., Greenberg, H. B. UNIV CHICAGO PRESS. 2000: S331–S335

  Abstract

  The evolution of our understanding of immunity to calicivirus infection, using Norwalk virus as the prototype, is discussed in three stages: (1) "ancient times (1972-1978), when human volunteer studies prevailed, (2) the "middle ages (1978-1990), which were characterized by the development and implementation of solid-phase immunoassays based on native viral antigens, and (3) "modern times (1990 to present), which began with the cloning of the genome of the noncultivatable 8FIIa strain of Norwalk virus and resulted in a readily available source of recombinant virus-like particles that have revolutionized the study of caliciviruses. Throughout these stages, it has been shown repeatedly that short-term immunity develops to homologous virus. However, the search for determinants of long-term immunity continues. These studies will likely be facilitated by the newest reagents-the noninfectious recombinant virus-like particles-used in the setting of human volunteer studies and large epidemiologic studies.

  View details for Web of Science ID 000087439400015

  View details for PubMedID 10804146

• Intestinal homing receptor expression during the B cell response to murine rotavirus infection. Youngman, K. R., Franco, M. A., Kuklin, N. A., Rott, L. S., Greenberg, H. B., BUTCHER, E. C. FEDERATION AMER SOC EXP BIOL. 2000: A973–A973

  View details for Web of Science ID 000086643100361

• alpha 4 beta 7 independent pathway for CD8 T cell mediated intestinal immunity to rotavirus Kuklin, N. A., Rott, L., Darling, J., Campbell, J. J., Franco, M., Feng, N., Muller, W., Wagner, N., Altman, J. D., BUTCHER, E. C., Greenberg, H. B. FEDERATION AMER SOC EXP BIOL. 2000: A971–A971

  View details for Web of Science ID 000086643100350

• Sustained survival of human hepatocytes in mice: A model for in vivo infection with human hepatitis B and hepatitis delta viruses NATURE MEDICINE Ohashi, K., Marion, P. L., Nakai, H., Meuse, L., Cullen, J. M., Bordier, B. B., Schwall, R., Greenberg, H. B., Glenn, J. S., Kay, M. A. 2000; 6 (3): 327-331

  Abstract

  Persistence of hepatocytes transplanted into the same or related species has been established. The long-term engraftment of human hepatocytes into rodents would be useful for the study of human viral hepatitis, where it might allow the species, technical and size limitations of the current animal models to be overcome. Although transgenic mice expressing the hepatitis B virus (HBV) genome produce infectious virus in their serum, the viral life cycle is not complete, in that the early stages of viral binding and entry into hepatocytes and production of an episomal transcriptional DNA template do not occur. As for hepatitis delta virus (HDV), another cause of liver disease, no effective therapy exists to eradicate infection, and it remains resistant even to recent regimens that have considerably changed the treatment of HBV (ref. 13). Here, we demonstrate long-term engraftment of primary human hepatocytes transplanted in a matrix under the kidney capsule of mice with administration of an agonistic antibody against c-Met. These mice were susceptible to HBV infection and completion of the viral life cycle. In addition, we demonstrate super-infection of the HBV-infected mice with HDV. Our results describe a new xenotransplant model that allows study of multiple aspects of human hepatitis viral infections, and may enhance studies of human liver diseases.

  View details for PubMedID 10700236

• Immunity to homologous rotavirus infection in adult mice TRENDS IN MICROBIOLOGY Franco, M. A., Greenberg, H. B. 2000; 8 (2): 50-52

  View details for Web of Science ID 000085252800004

  View details for PubMedID 10664594

• Rotavirus entry into tissue culture cells. Methods in molecular medicine Gilbert, J. M., Greenberg, H. B. 2000; 34: 67-77

  Abstract

  Rotavirus (RV) is a triple-protein-layered icosahedral virus, for which studies have established that the two outer-layer proteins, viral protein 4 (VP4) and viral protein 7 (VP7), are required for viral infectivity (1,2). VP7, a glycoprotein, is the major component of the outer-layer, but its role in viral entry is unclear. VP4 forms dimers extending out from the VP7-coated viral surface (3,4) and have been shown to be a determinant of host range and virulence, and is directly involved in cell attachment and RV entry into cells (5-8). Proteolytic cleavage of VP4 into two noncovalently associated subunits, VP8* and VP5* (2,9,10), significantly enhances viral infectivity (11-13).

  View details for DOI 10.1385/1-59259-078-0:67

  View details for PubMedID 21318855

• Visualisation of hepatitis C virus-specific cytotoxic T cells using peptide-MHC tetramers: Variations in the viral epitope and relationship with the viral load Lopex-Labrador, F. X., He, X. S., Berenguer, M., Lu, Y., Boisvert, J., Cheung, R., Rehermann, B., DAVIS, M. M., Wright, T., Greenberg, H. B. ELSEVIER SCIENCE BV. 2000: 172–172

  View details for Web of Science ID 000087104500555

• In vivo study of immunity to rotaviruses : selected methods in mice. Methods in molecular medicine Franco, M. A., Greenberg, H. B. 2000; 34: 133-145

  Abstract

  Rotaviruses (RVs) are important human pathogens. The murine model of RV infection has been very useful in clarifying the mechanisms that mediate clearance of primary RV infection, and the mechanisms that mediate immunity to reinfection. The use of immunodeficient strains of mice, immunodepletion studies with specific monoclonal antibodies (MAbs), and passive transfer of purified cells are three basic, complementary experimental approaches that have been used for this purpose, and are the subject of this chapter. These experimental approaches analyze the outcome of RV infection under artificial conditions; thus, the relevance of the results obtained, to the physiological immune response of immunocompetent mice or humans, is at times difficult to establish. For example, immunodeficient strains of mice frequently develop compensatory immune mechanisms that are potentially absent or nonfunctional in immunocompetent mice. Immunodepletion experiments introduce into the experimental animal high (nonphysiological) levels of antibodies (Abs) that potentially have other immunomodulatory effects different from the desired one, and, many times, depletion strategies fail to completely eliminate the target cell population. Passive cell transfer experiments analyze the antiviral capacity of a specific cell population (many times abnormal in number), independent of other cells with which it may normally interact, and in an environment to which it is at least partially alien. Because of these and other limitations of such experimental approaches, one should be very careful in selecting adequate controls, and cautious in the interpretation of the results, by taking into account results obtained with two or three of the approaches and analysis of the characteristics of the immune response in normal animals. The combination of two or three strategies (for example, passive cell transfer into immunodeficient hosts and immunodepletion of selected cell populations in immunocompetent hosts) have proven particularly useful in the study of immunity to RV.

  View details for DOI 10.1385/1-59259-078-0:133

  View details for PubMedID 21318859

• Hepatitis C virus-like particles synthesized in insect cells as a potential vaccine candidate 48th Annual Meeting of the American-Association-for-the-Study-of-Liver-Diseases Baumert, T. F., Vergalla, J., Satoi, J., Thomson, M., Lechmann, M., Herion, D., Greenberg, H. B., Ito, S., Liang, T. J. W B SAUNDERS CO-ELSEVIER INC. 1999: 1397–1407

  Abstract

  Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. Successful vaccine development is crucial in controlling global HCV infection. We have previously described the generation of HCV-like particles (HCV-LPs) in insect cells using a recombinant baculovirus containing the complementary DNA of the HCV structural proteins. These HCV-LPs had similar morphological and biophysical properties as the putative virions. In this study, we analyzed the structural features, antigenic composition, seroreactivity, and immunogenicity of purified HCV-LPs.HCV-LPs were analyzed by electron microscopy and antibody immunolabeling and precipitation. An enzyme-linked immunosorbent assay (ELISA) using HCV-LPs was developed. The humoral response to HCV-LPs in mice was studies by core and envelope ELISAs, Western immunoblotting, and immunofluorescence.Structural and antigenic compositions of HCV-LPs were shown to be similar to those of putative HCV virions. Using the HCV-LP ELISA, high-titer anti-HCV antibodies were detected in individuals infected with various HCV genotypes. In vivo, HCV-LPs elicited a humoral response broadly directed against HCV structural proteins.HCV-LPs resemble HCV virions and are capable of inducing a humoral response targeted against various regions of HCV structural proteins, suggesting that HCV-LPs may be promising as a potential vaccine candidate.

  View details for Web of Science ID 000083952500021

  View details for PubMedID 10579981

• The epithelial cell response to rotavirus infection JOURNAL OF IMMUNOLOGY Rollo, E. E., Kumar, K. P., Reich, N. C., Cohen, J., Angel, J., Greenberg, H. B., Sheth, R., Anderson, J., Oh, B., Hempson, S. J., MACKOW, E. R., Shaw, R. D. 1999; 163 (8): 4442-4452

  Abstract

  Rotavirus is the most important worldwide cause of severe gastroenteritis in infants and young children. Intestinal epithelial cells are the principal targets of rotavirus infection, but the response of enterocytes to rotavirus infection is largely unknown. We determined that rotavirus infection of HT-29 intestinal epithelial cells results in prompt activation of NF-kappaB (<2 h), STAT1, and ISG F3 (3 h). Genetically inactivated rotavirus and virus-like particles assembled from baculovirus-expressed viral proteins also activated NF-kappaB. Rotavirus infection of HT-29 cells induced mRNA for several C-C and C-X-C chemokines as well as IFNs and GM-CSF. Mice infected with simian rotavirus or murine rotavirus responded similarly with the enhanced expression of a profile of C-C and C-X-C chemokines. The rotavirus-stimulated increase in chemokine mRNA was undiminished in mice lacking mast cells or lymphocytes. Rotavirus induced chemokines only in mice <15 days of age despite documented infection in older mice. Macrophage inflammatory protein-1beta and IFN-stimulated protein 10 mRNA responses occurred, but were reduced in p50-/- mice. Macrophage inflammatory protein-1beta expression during rotavirus infection localized to the intestinal epithelial cell in murine intestine. These results show that the intestinal epithelial cell is an active component of the host response to rotavirus infection.

  View details for Web of Science ID 000082974800047

  View details for PubMedID 10510386

• Direct visualisation of hepatitis C virus-specific cytotoxic T cells using peptide-MHC tetramers and analysis of aminoacid variations within the viral epitope. Lopez-Labrador, F. X., He, X. S., Berenguer, M., Lu, Y., Boisvert, J., Cheung, R., Rehermann, B. R., DAVIS, M. M., Wright, T., Greenberg, H. WILEY-BLACKWELL. 1999: 422A–422A

  View details for Web of Science ID 000082794701045

• Lessons learned from a review of the development of selected vaccines PEDIATRICS Peter, G., des-Vignes-Kendrick, M., Eickhoff, T. C., Fine, A., Galvin, V., LEVINE, M. M., Maldonado, Y. A., Marcuse, E. K., Monath, T. P., Osborn, J. E., Macy, J., Plotkin, S., Poland, G. A., Quinlisk, M. P., Smith, D. R., Sokol, M., Soland, D. B., Whitley-Williams, P. N., Williamson, D. E., Breiman, R. F., Arvin, A., Baylor, N., Breiman, R., Clements-Mann, M. L., Couch, R., Douglas, G., Galambos, L., Gellin, B., Gershon, A., Glass, R., Gordon, L., Glode, M., Greenberg, H., Hardegree, C., Heilman, C., Hilleman, M., Jordan, W., LEVINE, M. M., MARCUSE, E., Paradiso, P., Parkman, P., Plotkin, S., Quinnan, G., Rabinovich, R., Read, L., Russell, P., Sewell, J., Shepherd, H. R., Siber, G., Soland, D., Ward, J., WHITE, J., Wright, P. 1999; 104 (4): 942-950

  View details for Web of Science ID 000082907300014

• Analysis of differential gene expression at the response of human liver cells to hepatitis C virus core protein expression An, S., Kawai, H., Berry, C., Feinstone, S. M., He, X. S., Brown, P. O., Greenberg, H. B. WILEY-BLACKWELL. 1999: 512A–512A

  View details for Web of Science ID 000082794701403

• Immunity obtained by gene-gun inoculation of a rotavirus DNA vaccine to the abdominal epidermis or anorectal epithelium VACCINE Chen, S. C., Fynan, E. F., Greenberg, H. B., Herrmann, J. E. 1999; 17 (23-24): 3171-3176

  Abstract

  We have previously shown that gene-gun delivery of murine rotavirus DNA vaccines to the epidermis induced protection against rotavirus challenge in mice. In this study, we used a rotavirus group antigen (VP6)-specific DNA vaccine to compare epidermal immunization with immunization to the anorectal epithelium for efficacy in inducing protective immunity. The vaccine was administered into cells of the abdominal epidermis or anorectal epithelium of adult BALB/c mice with an Accell gene-gun (PowderJect, Inc). Vaccines administered by either route elicited rotavirus-specific ELISA antibodies and analysis of the IgG subtypes indicated Th2-type responses were generated by both routes of administration, in contrast to Th1-type responses generated by live rotavirus. Protection against virus challenge was obtained in mice inoculated by either route, as shown by significant reduction of virus excreted in stools. The protection obtained by immunization of the anorectal epithelium was greater than that for epidermal immunization at the same vaccine dose. These results suggest that mucosal immunization of DNA vaccines may be an effective means to generate protective immunity against mucosal pathogens.

  View details for Web of Science ID 000081957300027

  View details for PubMedID 10462253

• Immune responses and protection obtained by oral immunization with rotavirus VP4 and VP7 DNA vaccines encapsulated in microparticles VIROLOGY Herrmann, J. E., Chen, S. C., Jones, D. H., Tinsley-Bown, A., Fynan, E. F., Greenberg, H. B., Farrar, G. H. 1999; 259 (1): 148-153

  Abstract

  Protective immune responses in mice were obtained after oral immunization with rotavirus DNA vaccines encapsulated in poly(lactide-co-glycolide) (PLG) microparticles. The DNA vaccines used encoded outer capsid proteins VP4 and VP7; proteins that are the basis for rotavirus serotyping and the generation of virus neutralizing antibodies. One dose of vaccine was given to BALB/c mice by oral gavage (75 microg DNA/mouse). Rotavirus-specific serum antibodies and intestinal IgA antibodies were detectable by 6 weeks postimmunization. After challenge with homologous murine rotavirus at 12 weeks postimmunization, fecal rotavirus antigen was reduced significantly in immunized mice compared with controls. Protective immunity also was generated by oral delivery of unencapsulated VP 7 DNA vaccine but to a lesser degree. These results demonstrate that the oral route is effective for generating protective immune responses with rotavirus DNA vaccines targeting neutralization antigens.

  View details for Web of Science ID 000081025400016

  View details for PubMedID 10364499

• Lack of a role for type I and type II interferons in the resolution of rotavirus-induced diarrhea and infection in mice JOURNAL OF INTERFERON AND CYTOKINE RESEARCH Angel, J., Franco, M. A., Greenberg, H. B., Bass, D. 1999; 19 (6): 655-659

  Abstract

  Rotavirus infects the intestinal epithelium of most mammalian species and causes diarrhea in infants. Previously, we have shown that both type I and II human interferons (IFNs) have potent and mechanistically discreet antiviral effects in vitro against rotavirus. We have also shown that adult IFN-gamma knockout (-/-) mice have no alteration in clearance of primary rotavirus infection. In the present studies, we wished to determine the importance of both IFN types in modulation of degree and duration of disease and infection in mice. Immunocompetent suckling mice were treated orally (5,000 IU) or parenterally (500 IU) with type I and II murine IFNs before and after challenge with virulent murine rotavirus. Treated animals developed diarrhea indistinguishable from that observed in untreated control mice. In other experiments, type I IFN receptor -/- suckling mice and IFN-gamma-/- suckling mice developed diarrhea of similar characteristics and duration and had comparable quantities of viral antigen in their intestines as did immunocompetent mice. Furthermore, type I IFN receptor -/- adult mice infected with rotavirus shed equivalent quantities of viral antigen and with similar kinetics as the control mice. Thus, IFNs do not seem to be major inhibitors of rotavirus diarrhea or replication in mice.

  View details for Web of Science ID 000081510000014

  View details for PubMedID 10433367

• Genomic analysis of hepatitis C virus infection using dna microarrays. Barry, C. T., He, X. S., Greenberg, H. B., Brown, P. O. LIPPINCOTT WILLIAMS & WILKINS. 1999: S581–S581

  View details for Web of Science ID 000080624100175

• Quantitative analysis of hepatitis C virus-specific CD8(+) T cells in peripheral blood and liver using peptide-MHC tetramers PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA He, X. S., Rehermann, B., Lopez-Labrador, F. X., Boisvert, J., Cheung, R., Mumm, J., Wedemeyer, H., Berenguer, M., Wright, T. L., DAVIS, M. M., Greenberg, H. B. 1999; 96 (10): 5692-5697

  Abstract

  It is believed that the hepatitis C virus (HCV)-specific CD8(+) cytotoxic T lymphocytes (CTLs) play a role in the development of liver cell injury and in the clearance of the virus. To develop a direct binding assay for HCV-specific CTLs, we generated two peptide-MHC tetramers by using the recombinant HLA A2.1 molecule and A2-restricted T cell epitopes of the HCV NS3 protein. With these reagents we are able to detect specific CD8(+) cells in the blood of 15 of 20 HLA-A2(+), HCV-infected patients, at a frequency ranging from 0.01% to 1.2% of peripheral CD8(+) T cells. Phenotypic analysis of these specific cells indicated that there is a significant variation in the expression of the CD45 isoforms and CD27 in different patients. A 6-hour incubation of one patient's blood with NS3 peptides resulted in the activation of the epitope-specific CD8(+) cells, as indicated by their expression of CD69 and IFN-gamma. We also detected NS3-specific CD8(+) T cells in the intrahepatic lymphocyte population isolated from liver biopsies of two HCV-infected patients. The frequency of these specific CD8(+) cells in the liver was 1-2%, at least 30-fold higher than in the peripheral blood. All of the intrahepatic NS3-specific CD8(+) T cells were CD69(+), suggesting that they were activated CTLs. Direct quantitation and characterization of HCV-specific CTLs should extend our understanding of the immunopathogenesis and the mechanism of clearance or persistence of HCV.

  View details for Web of Science ID 000080246500068

  View details for PubMedID 10318946

• Immunity to rotavirus infection in mice Meeting on HIV-1 Infection, Mucosal Immunity, and Pathogenesis Franco, M. A., Greenberg, H. B. UNIV CHICAGO PRESS. 1999: S466–S469

  Abstract

  Recent findings from our laboratory regarding the immune response of mice to rotavirus (a mucosal pathogen) show that although in most situations an acquired (T or B cell or both) response is necessary for elimination of primary rotavirus infection, unidentified innate mechanisms can also play a role in some mouse strains. Similar to what is seen with many other viruses, CD8+ T cells appear to provide the first but not the exclusive mechanism that mediates clearance of a primary rotavirus infection. Antibodies are the critical mediators of prevention against rotavirus reinfection. Nonneutralizing IgA monoclonal antibodies directed against VP6 (an internal structural rotavirus protein) can mediate immunity against rotaviruses in vivo. Rotavirus-specific CD8+ T cells can mediate their antiviral effect in the absence of perforin, fas, or interferon-gamma and are preferentially represented in the subset that expresses high levels of the enteric mucosal homing receptor alpha4beta7.

  View details for Web of Science ID 000079973300019

  View details for PubMedID 10099121

• Isolation of a human rotavirus strain with a super-short RNA pattern and a new P2 subtype JOURNAL OF CLINICAL MICROBIOLOGY Nakagomi, T., Horie, Y., Koshimura, Y., Greenberg, H. B., Nakagomi, O. 1999; 37 (4): 1213-1216

  Abstract

  Super-short rotavirus strains that have a rearranged gene segment 11 are rarely found in humans, and only five isolates, all from Southeast Asia, have been described in the literature. We report the first isolation in Japan from an infant with severe diarrhea of a rotavirus possessing a super-short RNA pattern. This strain, designated AU19, had a G1 VP7 and is also the first isolate in Japan that possesses a P2[6] VP4. Furthermore, the P2[6] VP4 carried by AU19 was divergent in the hypervariable region of the amino acid sequence from the P2A[6] VP4s carried by asymptomatic neonatal strains or from the P2B[6] VP4 carried by porcine rotavirus strain Gottfried. Thus, AU19 is likely to represent a new VP4 subtype, which we propose to call P2C. Given the recent emergence of the P2[6] VP4s in India, Brazil, and the United States and the role of VP4 in protective immunity, further scrutiny is justified to see whether the emergence of the previously underrepresented P2[6] VP4 serotype is related to this new P2 subtype.

  View details for Web of Science ID 000079105500062

  View details for PubMedID 10074557

• The immunology of rotavirus infection in the mouse ADVANCES IN VIRUS RESEARCH, VOL 51 Rose, J., Franco, M., Greenberg, H. 1999; 51: 203-235

  View details for Web of Science ID 000087256500005

• Use of a prenylation inhibitor as a novel antiviral agent JOURNAL OF VIROLOGY Glenn, J. S., Marsters, J. C., Greenberg, H. B. 1998; 72 (11): 9303-9306

  Abstract

  No specific therapy exists for hepatitis delta virus (HDV), which can cause severe liver disease. Molecular genetic studies have implicated the prenylation site of large delta antigen as a critical determinant of HDV particle assembly. We have established a cell culture model which produces HDV-like particles, and we show that delta antigen prenylation can be pharmacologically inhibited by the prenylation inhibitor BZA-5B. Furthermore, BZA-5B specifically abolishes particle production in a dose-dependent manner. These results demonstrate that the use of such a prenylation inhibitor-based antiviral therapy may be feasible and identify a novel class of potential antiviral agents.

  View details for PubMedID 9765479

• Detection and characterization of novel rotavirus strains in the United States JOURNAL OF CLINICAL MICROBIOLOGY Ramachandran, M., Gentsch, J. R., Parashar, U. D., Jin, S., Woods, P. A., Holmes, J. L., Kirkwood, C. D., BISHOP, R. F., Greenberg, H. B., Urasawa, S., Gerna, G., Coulson, B. S., Taniguchi, K., Bresee, J. S., Glass, R. I. 1998; 36 (11): 3223-3229

  Abstract

  We recently established a rotavirus strain surveillance system in the United States to monitor the prevalent G serotypes before and after the anticipated implementation of a vaccination program against rotavirus and to identify the emergence of uncommon strains. In this study, we examined 348 rotavirus strains obtained in 1996 to 1997 from children with diarrhea in 10 U.S. cities. Strains were characterized for P and G types, subgroups, and electropherotypes by using a combination of monoclonal antibody immunoassay, reverse transcription-PCR, and hybridization. The four strains most commonly found worldwide comprised 83% of the isolates (P[8]G1, 66.4%; P[4]G2, 8.3%; P[8]G3, 6.9%; P[8]G4, 1.4%), but 9.2% were unusual strains (P[6]G9, 5.5%; P[8]G9, 1.7%; P[6]G1, 1.4%; and P[4]G1 and P[8]G2, 0. 3% each). Strains not typeable for P or G type accounted for 5.5% of the total, while 2.3% of the strains had more than one G type (mixed infections). All P[6]G9 strains tested had short electropherotypes and subgroup I specificity and were detected in 4 of 10 cities, while P[8]G9 strains had long electropherotypes and subgroup II VP6 antigens. Both sequence analysis of the VP7 open reading frame (about 94 to 95% amino acid identity with the VP7 gene of G9 prototype strain WI61) and binding to a G9-specific monoclonal antibody strongly suggest that U.S. G9 strains belong to serotype G9. The high detection rates of unusual rotaviruses with G9 (7.2%) or P[6] (6.9%) specificity in multiple U.S. cities suggest the emergence of new strains or inadequate diagnosis in the past. The epidemiologic importance of these strains remains to be determined.

  View details for Web of Science ID 000076491200021

  View details for PubMedID 9774569

  View details for PubMedCentralID PMC105305

• The memory B cell subset responsible for the secretory IgA response and protective humoral immunity to rotavirus expresses the intestinal homing receptor, alpha(4)beta(7) JOURNAL OF IMMUNOLOGY Williams, M. B., Rose, J. R., Rott, L. S., Franco, M. A., Greenberg, H. B., BUTCHER, E. C. 1998; 161 (8): 4227-4235

  Abstract

  Infection of mice with murine rotaviruses induces life-long immunity, characterized by high levels of IgA in the intestine and large numbers of rotavirus (RV)-specific Ab-secreting cells in gut-associated lymphoid tissues. Lymphocyte trafficking into gut-associated lymphoid tissues is mediated by interaction of the alpha4beta7 integrin on lymphocytes with the vascular mucosal addressin cell adhesion molecule-1. To determine whether B cell memory for RV correlates with alpha4beta7 expression, we transferred sorted B220+ phenotypically defined memory (IgD- alpha4beta7(high) and IgD- alpha4beta7-) and naive (IgD+ alpha4beta7+) splenocytes into recombination-activating gene-2 knockout mice (B and T cell-deficient) that were chronically infected with RV. Only mice receiving alpha4beta7(high) memory (IgD-) B cells produced RV-specific IgA in the stool, cleared the virus, and were immune to reinfection. Alpha4beta7(high) (but not alpha4beta7-) memory B cells from donors boosted as much as 7 mo previously also cleared the virus, indicating that alpha4beta7(high) memory B cells maintain long term functional immunity to RV. Although only alpha4beta7(high) memory cells provided mucosal immunity, alpha4beta7- cells from recently boosted donor animals could generate RV-specific serum IgG, but, like naive (IgD+) B cells, were unable to induce viral clearance even 60 days after cell transfer. These data indicate that protective immunity for an intestinal pathogen, RV, resides in memory phenotype B cells expressing the intestinal homing receptor, alpha4beta7.

  View details for Web of Science ID 000076343300057

  View details for PubMedID 9780197

• Protective immunity induced by oral immunization with a rotavirus DNA vaccine encapsulated in microparticles JOURNAL OF VIROLOGY Chen, S. C., Jones, D. H., Fynan, E. F., Farrar, G. H., CLEGG, J. C., Greenberg, H. B., Herrmann, J. E. 1998; 72 (7): 5757-5761

  Abstract

  DNA vaccines are usually given by intramuscular injection or by gene gun delivery of DNA-coated particles into the epidermis. Induction of mucosal immunity by targeting DNA vaccines to mucosal surfaces may offer advantages, and an oral vaccine could be effective for controlling infections of the gut mucosa. In a murine model, we obtained protective immune responses after oral immunization with a rotavirus VP6 DNA vaccine encapsulated in poly(lactide-coglycolide) (PLG) microparticles. One dose of vaccine given to BALB/c mice elicited both rotavirus-specific serum antibodies and intestinal immunoglobulin A (IgA). After challenge at 12 weeks postimmunization with homologous rotavirus, fecal rotavirus antigen was significantly reduced compared with controls. Earlier and higher fecal rotavirus-specific IgA responses were noted during the peak period of viral shedding, suggesting that protection was due to specific mucosal immune responses. The results that we obtained with PLG-encapsulated rotavirus VP6 DNA are the first to demonstrate protection against an infectious agent elicited after oral administration of a DNA vaccine.

  View details for Web of Science ID 000074160500050

  View details for PubMedID 9621034

• Cleavage of rhesus rotavirus VP4 after arginine 247 is essential for rotavirus-like particle-induced fusion from without JOURNAL OF VIROLOGY Gilbert, J. M., Greenberg, H. B. 1998; 72 (6): 5323-5327

  Abstract

  We recently described our finding that recombinant baculovirus-produced virus-like particles (VLPs) can induce cell-cell fusion similar to that induced by intact rotavirus in our assay for viral entry into tissue culture cells (J. M. Gilbert and H. B. Greenberg, J. Virol. 71:4555-4563, 1997). The conditions required for syncytium formation are similar to those for viral penetration of the plasma membrane during the course of viral infection. This VLP-mediated fusion activity was dependent on the presence of the outer-layer proteins, viral protein 4 (VP4) and VP7, and on the trypsinization of VP4. Fusion activity occurred only with cells that are permissive for rotavirus infection. Here we begin to dissect the role of VP4 in rotavirus entry by examining the importance of the precise trypsin cleavage of VP4 and the activation of VP4 function related to viral entry. We present evidence that the elimination of the three trypsin-susceptible arginine residues of VP4 by specific site-directed mutagenesis prevents syncytium formation. Two of the three arginine residues in VP4 are dispensable for syncytium formation, and only the arginine residue at site 247 appears to be required for activation of VP4 functions and cell-cell fusion. Using the recombinant VLPs in our syncytium assay will aid in understanding the conformational changes that occur in VP4 involved in rotavirus penetration into host cells.

  View details for Web of Science ID 000073497600097

  View details for PubMedID 9573313

• Antigenic and genomic diversity of human rotavirus VP4 in two consecutive epidemic seasons in Mexico JOURNAL OF CLINICAL MICROBIOLOGY Padilla-Noriega, L., Mendez-Toss, M., Menchaca, G., Contreras, J. F., Romero-Guido, P., Puerto, F. I., Guiscafre, H., MOTA, F., Herrera, I., Cedillo, R., Munoz, O., Calva, J., Guerrero, M. D., Coulson, B. S., Greenberg, H. B., Lopez, S., Arias, C. F. 1998; 36 (6): 1688-1692

  Abstract

  In the present investigation we characterized the antigenic diversity of the VP4 and VP7 proteins in 309 and 261 human rotavirus strains isolated during two consecutive epidemic seasons, respectively, in three different regions of Mexico. G3 was found to be the prevalent VP7 serotype during the first year, being superseded by serotype G1 strains during the second season. To antigenically characterize the VP4 protein of the strains isolated, we used five neutralizing monoclonal antibodies (MAbs) which showed specificity for VP4 serotypes P1A, P1B, and P2 in earlier studies. Eight different patterns of reactivity with these MAbs were found, and the prevalence of three of these patterns varied from one season to the next. The P genotype of a subset of 52 samples was determined by PCR. Among the strains characterized as genotype P[4] and P[8] there were three and five different VP4 MAb reactivity patterns, respectively, indicating that the diversity of neutralization epitopes in VP4 is greater than that previously appreciated by the genomic typing methods.

  View details for Web of Science ID 000073641300038

  View details for PubMedID 9620401

  View details for PubMedCentralID PMC104901

• Identification of mutations in the rotavirus protein VP4 that alter sialic-acid-dependent infection JOURNAL OF GENERAL VIROLOGY Ludert, J. E., Mason, B. B., Angel, J., Tang, B. Z., Hoshino, Y., Feng, N. G., Vo, P. T., Mackow, E. M., Ruggeri, F. M., Greenberg, H. B. 1998; 79: 725-729

  Abstract

  To explore further the role of VP4 as the rotavirus cell attachment protein, VP7 monoreassortants derived from the sialic-acid-dependent simian strain RRV and from the sialic-acid-independent human strains D, DS-1 and ST-3 were tested for susceptibility of infectivity of neuraminidase-treated MA-104 cells. Infectivity of RRV x D VP7 and RRV x ST-3 VP7 monoreassortants decreased when sialic acid was removed from the cell surface. However, of three separate RRV x DS-1 VP7 monoreassortants tested, only one was sialic-acid-dependent. Sequence analysis showed that both sialic-acid-independent strains contained a single amino acid change, Lys to Arg, at position 187. In addition, sialic-acid-independent infectivity was seen in one of 14 RRV VP4 neutralization escape mutants tested, and this strain was found to have a Gly to Glu change at amino acid position 150. These results indicate that positions 150 and 187 of VP4 play an important role in early rotavirus-cell interactions.

  View details for Web of Science ID 000072889400011

  View details for PubMedID 9568967

• Determinants of rotavirus immunity in the mouse. Greenberg, H. B., Feng, N., Franco, M. FEDERATION AMER SOC EXP BIOL. 1998: A362–A362

  View details for Web of Science ID 000076006402100

• Studies of the role for NSP4 in the pathogenesis of homologous murine rotavirus diarrhea JOURNAL OF INFECTIOUS DISEASES Angel, J., Tang, B. Z., Feng, N. G., Greenberg, H. B., Bass, D. 1998; 177 (2): 455-458

  Abstract

  A rotavirus (RV) nonstructural protein, NSP4, has recently been proposed to function as an enterotoxin in the pathogenesis of RV diarrhea. The role of NSP4 in the pathogenesis of RV diarrhea was examined by infecting cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice with virulent murine RV and by comparing deduced amino acid sequences of RV gene 10 encoding NSP4 from three distinct sets of virulent and tissue culture-adapted avirulent variant RVs. Homozygous CFTR (CFTR-/-) mice, which do not respond to any known intestinal secretagogues, experienced diarrhea comparable to that in normal CFTR+/+ littermates after RV challenge. Comparison of amino acid sequences of NSP4 from virulent and attenuated pairs of RVs failed to show consistent or significant changes. Together, these data suggest that enterotoxigenic properties of RV NSP4 are not critical in the pathogenesis of murine RV diarrhea and that attenuation of murine RVs is not usually mediated by mutations in the gene encoding NSP4.

  View details for Web of Science ID 000071801500026

  View details for PubMedID 9466536

• Three forms of AU-1 like human rotaviruses differentiated by their overall genomic constellation and by the sequence of their VP8 ARCHIVES OF VIROLOGY Gollop, R., Nakagomi, O., Silberstein, I., Shulman, L. M., Greenberg, H. B., Mendelson, E., Shif, I. 1998; 143 (2): 263-277

  Abstract

  Insight into the origin of human rotaviruses carrying the AU-1 VP4 allele was gained by examining their genomic RNA constellation using RNA-RNA hybridization and by sequencing the VP8* portion (nucleotides 1-750) of their gene 4. AU-1 like viruses isolated in Israel from children attending outpatient clinics were classified into three sub-genogroups based on RNA-RNA hybridization analysis: Subgenogroup 1 consists of two strains (Ro-5829 and Ro-5960) which belong to the AU-1 genogroup, since all their 11 segments hybridized to AU-1 segments. Subgenogroup 2 consists of one reassortant virus (Ro-5193) of which seven RNA segments hybridized to AU-1 segments and the remaining four segments hybridized to NCDV (bovine rotavirus). Subgenogroup 3 consists of four reassortant viruses (Ro-6460, Ro-6584, Ro-6784 and Ro-7044) which had a common genome constellation: only four of their RNA segments hybridized to AU-1 and the other seven segments hybridized to NCDV segments. Sequence analysis of the VP8* gene also revealed a three level pattern of homology with the AU-1 prototype and the local AU-1 like strains which was consistent with the overall genomic (RNA-RNA) constellation: Subgenogroup 1 had 98-98.1% homology with the AU-1 prototype; Subgenogroup 2 had 96.8% homology with the AU-1 prototype and 95.6-96.7% homology with Subgenogroup 1; Subgenogroup 3 had 95.3-95.6% homology with the prototype AU-1 and 93.4-94.3% homology with Subgenogroup 1. Possible evolutionary pathways are discussed.

  View details for Web of Science ID 000072379500005

  View details for PubMedID 9541612

• The immunology of rotavirus infection in the mouse. Advances in virus research Rosé, J., Franco, M., Greenberg, H. 1998; 51: 203-235

  View details for PubMedID 9891588

• Expression of the mucosal homing receptor alpha(4)beta(7) correlates with the ability of CD8(+) memory T cells to clear rotavirus infection JOURNAL OF VIROLOGY Rose, J. R., Williams, M. B., Rott, L. S., BUTCHER, E. C., Greenberg, H. B. 1998; 72 (1): 726-730

  Abstract

  The integrin alpha4beta7 plays an important role in lymphocyte homing to mucosal lymphoid tissues and has been shown to define a subpopulation of memory T cells capable of homing to intestinal sites. Here we have used a well-characterized intestinal virus, murine rotavirus, to investigate whether memory/effector function for an intestinal pathogen is associated with alpha4beta7 expression. Alpha4beta7(hi) memory phenotype (CD44hi), alpha4beta7- memory phenotype, and presumptively naive (CD44(lo)) CD8+ T lymphocytes from rotavirus-infected mice were sorted and transferred into Rag-2 (T- and B-cell-deficient) recipients that were chronically infected with murine rotavirus. Alpha4beta7(hi) memory phenotype CD8+ cells were highly efficient at clearing rotavirus infection, alpha4beta7- memory cells were inefficient or ineffective, depending on the cell numbers transferred, and CD44(lo) cells were completely unable to clear chronic rotavirus infection. These data demonstrate that functional memory for rotavirus resides primarily in memory phenotype cells that display the mucosal homing receptor alpha4beta7.

  View details for Web of Science ID A1998YL01000086

  View details for PubMedID 9420279

• Immunity to rotavirus in T cell deficient VIROLOGY Franco, M. A., Greenberg, H. B. 1997; 238 (2): 169-179

  Abstract

  Rotavirus infection was studied in adult nude mice (BALB/c background), alpha beta or gamma delta and alpha beta/gamma delta T cell receptor (TCR) knockout (-/-) mice (C57BL/6 and C57BL/6 x 129 backgrounds), and SCID mice (C57BL/6 background). The gamma delta TCR -/- mice cleared infection just like control mice. All of the nude mice, alpha beta, and alpha beta/gamma delta TCR -/- mice cleared primary rotavirus infection, with a short delay, compared to immunocompetent control mice and developed a rotavirus-specific intestinal IgA measured by ELISA. Elispot analysis with spleen and lamina propia cells showed that the virus-specific intestinal IgA response in immunocompetent C57BL/6 mice was similar to the gamma delta TCR -/- mice and 7- to 60-fold higher than in the alpha beta TCR -/- and alpha beta/gamma delta TCR -/- mice. Likewise, the response of nude +/- mice was 20 times greater than that of nude -/- littermates. While the intestinal IgA antibodies of C57BL/6 mice, gamma delta TCR -/- mice, and nude +/- mice recognized insect cells infected with recombinant baculovirus expressing rotavirus VP6 and VP4 proteins, those of the alpha beta TCR -/-, alpha beta/gamma delta TCR -/-, and nude -/- mice recognized only VP6. Immunocompetent C57BL/6 mice depleted of CD4+ T cell developed similar levels of rotavirus-specific intestinal IgA as the alpha beta TCR -/- mice, suggesting that this T cell-independent IgA response is present in normal mice. In contrast to previously published results with BALB/c SCID and RAG 2 -/- (C57BL/6 x 129 background) mice, all of which become chronically infected with murine rotavirus, 40% of the C57BL/6 SCID mice cleared primary rotavirus infection. These results suggest that both a T cell-independent antibody response and innate mechanisms can contribute to immunity to murine rotavirus and show that gamma delta T cells are not necessary for efficient clearance of primary rotavirus infection in mice.

  View details for Web of Science ID A1997YK65600001

  View details for PubMedID 9400590

• Hepatitis G virus infection in hemodialysis patients and the effects of interferon treatment AMERICAN JOURNAL OF GASTROENTEROLOGY Umlauft, F., WONG, D. T., Underhill, P. A., Oefner, P. J., Jin, L., Urbank, M., Gruenewald, K., Greenberg, H. B. 1997; 92 (11): 1986-1991

  Abstract

  To characterize the nature of hepatitis G virus (HGV) infections in hemodialysis patients and to determine the responsiveness of HGV to antiviral therapy in these patients.HGV, a recently identified flavivirus, is associated with non-A-E viral hepatitis infections. We studied HGV infections in hepatitis C virus (HCV)-infected hemodialysis patients over a 1-yr period, using two independent PCR assays and nucleic acid sequencing. Thirty-four of 63 study patients were treated with interferon.We observed a 27% prevalence (17/63 patients) and a 4% annual incidence of HGV infections in the study population. HGV was not detected in any of the 10 HGV-infected patients immediately after interferon therapy. Although seven of these 10 patients developed HGV relapses, three had long-term responses. The interferon responsiveness of HGV and HCV appeared to be unrelated. In contrast, all seven untreated HGV-infected patients remained viremic. Sequence analyses of the different HGV isolates revealed only very limited genetic variability in the polymerase chain reaction-amplified regions of HGV during 1 yr of observation.Our data suggest that HCV-infected hemodialysis patients are at substantial risk of acquiring HGV infection and that HGV infections are prevalent in this population. In addition, HGV infections become chronic but are responsive to interferon treatment.

  View details for Web of Science ID A1997YE27000007

  View details for PubMedID 9362177

• Comparison of the rotavirus gene 6 from different species by sequence analysis and localization of subgroup-specific epitopes using site-directed mutagenesis VIROLOGY Tang, B. Z., Gilbert, J. M., Matsui, S. M., Greenberg, H. B. 1997; 237 (1): 89-96

  Abstract

  The nucleotide sequence of gene 6 encoding the rotavirus major capsid protein VP6 of EDIM strain (EW) was determined and compared to that of 20 previously reported strains with known subgroup specificities. Multiple alignments of amino acid sequences exhibited a high level of sequence conservation (87 to 99.2%). Site-specific mutagenesis experiments were undertaken to localize regions involved in subgroup specificity. Amino acid positions 305, 315, and a region 296-299 (or 301 for equine strain H-2) were identified as contributing to subgroup epitopes. A single amino acid mutation at position 305 or 315 was sufficient to change the subgroup specificity of EW VP6 protein from non I/II to subgroup I- or subgroup II-like, respectively. Mutation at these sites may be another important mechanism for subgroup variation, along with gene reassortment.

  View details for Web of Science ID A1997YD91800009

  View details for PubMedID 9344910

• Expression of mucosal homing receptor alpha 4 beta 7 by circulating CD4(+) cells with memory for intestinal rotavirus JOURNAL OF CLINICAL INVESTIGATION Rott, L. S., Rose, J. R., Bass, D., Williams, M. B., Greenberg, H. B., BUTCHER, E. C. 1997; 100 (5): 1204-1208

  Abstract

  The integrin alpha4beta7 mediates lymphocyte binding to mucosal addressin cell adhesion molecule-1, and its expression defines lymphocytes capable of trafficking through the intestines and the intestinal lymphoid tissues. We examined the ability of discrete alpha4beta7(hi) and alpha4beta7- subsets of circulating memory phenotype (CD45RA-) CD4+ T cells to proliferate in response to rotavirus, a ubiquitous intestinal pathogen. alpha4beta7(hi) memory (CD45RA-) CD4+ T cells displayed much greater reactivity to rotavirus than alpha4beta7- memory or naive (CD45RA+) CD4+ T cells. In contrast, alpha4beta7- memory cells were the predominant population responsive to mumps antigen after intramuscular vaccination. Our results are consistent with the conclusion that natural rotavirus infection, an enteric pathogen, results in a specific circulating memory CD4+ response that is largely limited to the gut-homing alpha4beta7+ subpopulation. This phenotype is not shared with memory cells elicited by intramuscular immunization (shown here) or by skin contact allergens. The results support the hypothesis that gut trafficking memory CD4+ T cells comprise cellular memory for intestinal antigens and suggest that regulated expression of alpha4beta7 helps target and segregate intestinal versus systemic immune response.

  View details for Web of Science ID A1997XV75300030

  View details for PubMedID 9276738

• Hepatitis G virus: Is it a hepatitis virus? WESTERN JOURNAL OF MEDICINE Cheung, R. C., Keeffe, E. B., Greenberg, H. B. 1997; 167 (1): 23-33

  Abstract

  Hepatitis G virus (HGV) and GB virus C (GBV-C) are two newly discovered viral agents, different isolates of a positive-sense RNA virus that represents a new genus of Flaviviridae. The purpose of this review is to analyze new data that have recently been published on the epidemiology and associations between HGV and liver diseases such as posttransfusion hepatitis, acute and chronic non-A-E hepatitis, fulminant hepatitis, cryptogenic cirrhosis, and hepatocellular carcinoma. The role of HGV in coinfection with other hepatitis viruses, the response to antiviral therapy, and the impact of HGV on liver transplantation are also discussed. HGV is a transmissible blood-borne viral agent that frequently occurs as a coinfection with other hepatitis viruses due to common modes of transmission. The prevalence of HGV ranges from 0.9 to 10% among blood donors throughout the world and is found in 1.7% of volunteer blood donors in the United States. The majority of patients infected with HGV by blood transfusion do not develop chronic hepatitis, but hepatitis G viremia frequently persists without biochemical evidence of hepatitis. Serum HGV RNA has been found in 0 to 50% of patients with fulminant hepatitis of unknown etiology and 14 to 36% of patients with cryptogenic cirrhosis. The association between HGV and chronic non-A-E hepatitis remains unclear. Although HGV appears to be a hepatotrophic virus, its role in independently causing acute and chronic liver diseases remains uncertain.

  View details for Web of Science ID A1997XQ01100004

  View details for PubMedID 9265860

• Immune responses to individual rotavirus proteins following heterologous and homologous rotavirus infection in mice JOURNAL OF INFECTIOUS DISEASES Ishida, S., Feng, N. G., Gilbert, J. M., Tang, B. Z., Greenberg, H. B. 1997; 175 (6): 1317-1323

  Abstract

  Serum and intestinal humoral immune responses to rotavirus proteins VP2, VP4, VP6, VP7, NSP2, and NSP4 were quantitatively compared in mice infected with a homologous murine rotavirus (EHPw) or a heterologous simian rotavirus (RRV). Viral protein-specific antibody responses were measured by an immunohistochemistry assay that uses recombinant baculovirus-expressed rotavirus proteins as antigens. In serum, IgG responses to VP6 were dominant and comparable in both RRV- and EHPw-infected groups, but responses to VP2, VP4, VP7, and NSP2 were higher in RRV infection. In feces, IgA responses to VP2, VP4, and VP6 were higher in EHPw-infected mice, but responses to VP7 and NSP2 were detected only in the RRV-infected group. These findings indicate that immune responses to homologous and heterologous rotavirus infection vary both quantitatively and qualitatively. Differences in humoral responses may play a role in the differences in protection induced following homologous or heterologous rotavirus infection.

  View details for Web of Science ID A1997XC32200005

  View details for PubMedID 9180169

• Protective immunity induced by rotavirus DNA vaccines International Meeting on Nucleic Acid Vaccines for the Prevention of Infectious Diseases Chen, S. C., Fynan, E. F., Robinson, H. L., Lu, S., Greenberg, H. B., Santoro, J. C., Herrmann, J. E. ELSEVIER SCI LTD. 1997: 899–902

  Abstract

  It is estimated that Group A rotavirus diarrhea causes as many as one million deaths per year in children worldwide, and effective vaccines will be essential for their control. Plasmid DNA vaccines encoding murine rotaviral proteins VP4, VP6, or VP7 were tested in adult BALB/c mice for their ability to induce immune responses and provide protection against rotavirus challenge. The vaccines were administered by inoculation into cells of the epidermis with an Accell gene gun. (Auragen, Inc., Middleton, WI, USA). Each vaccine elicited rotavirus-specific serum antibodies as measured by ELISA. Virus neutralizing antibodies were detected in mice receiving plasmid DNAs encoding for outer capsid proteins VP4 and VP7, but not for VP6, an inner capsid protein, and all of the vaccines generated virus-specific CTL responses. Each vaccine was effective in protecting mice against infection after homotypic rotavirus (100 ID50) challenge, showing reductions (P < 0.0002) in viral excretion measured over a 9 day period. Increased rotavirus-specific intestinal IgA antibodies were seen in vaccinated mice after rotavirus challenge, particularly in mice that received the VP6 DNA vaccine. This suggests that intracellular IgA-mediated neutralization may be involved in protective immunity induced by the VP6 DNA vaccine, and may represent a new mechanism for protection by DNA vaccines.

  View details for Web of Science ID A1997XJ33600032

  View details for PubMedID 9234543

• Virus-like particle-induced fusion from without in tissue culture cells: Role of outer-layer proteins VP4 and VP7 JOURNAL OF VIROLOGY Gilbert, J. M., Greenberg, H. B. 1997; 71 (6): 4555-4563

  Abstract

  We recently described an assay that measures fusion from without induced in tissue culture cells by rotavirus, a nonenveloped, triple-protein-layered member of the Reoviridae family (M. M. Falconer, J. M. Gilbert, A. M. Roper, H. B. Greenberg, and J. S. Gavora, J. Virol. 69:5582-5591, 1995). The conditions required for syncytium formation are similar to those for viral penetration of the plasma membrane during the course of viral infection of host cells, as the presence of the outer-layer proteins VP4 and VP7 and the cleavage of VP4 are required. Here we present evidence that virus-like particles (VLPs) produced in Spodoptera frugiperda Sf-9 cells from recombinant baculoviruses expressing the four structural proteins of rotavirus can induce cell-cell fusion to the same extent as native rotavirus. This VLP-mediated fusion activity was dependent on trypsinization of VP4, and the strain-specific phenotype of individual VP4 molecules was retained in the syncytium assay similar to what has been seen with reassortant rotaviruses. We show that intact rotavirus and VLPs induce syncytia with cells that are permissive to rotavirus infection whereas nonpermissive cells are refractory to syncytium formation. This finding further supports our hypothesis that the syncytium assay accurately reflects very early events involved in viral infection and specifically the events related to viral entry into the cell. Our results also demonstrate that neither viral replication nor rotavirus proteins other than VP2, VP6, VP4, and VP7 are required for fusion and that both VP4 and VP7 are essential. The combination of a cell-cell fusion assay and the availability of recombinant VLPs will permit us to dissect the mechanisms of rotavirus penetration into host cells.

  View details for Web of Science ID A1997WZ57100046

  View details for PubMedID 9151849

• CD8(+) T cells can mediate almost complete short-term and partial long-term immunity to rotavirus in mice JOURNAL OF VIROLOGY Franco, M. A., Tin, C., Greenberg, H. B. 1997; 71 (5): 4165-4170

  Abstract

  We have recently shown that CD8+ T cells mediate clearance of rotavirus infection in mice. B-cell-deficient J(H)D knockout (-/-) mice depleted of CD8+ T cells become chronically infected with murine rotavirus, and beta2 microglobulin -/- and other mice depleted of CD8+ T cells have a 1- to 4-day delay in clearance of primary rotavirus infection. A role for CD8+ T cells in protection from reinfection with rotavirus was suggested by these studies, because J(H)D -/- mice rechallenged 6 to 8 weeks after primary infection shed smaller quantities of viral antigen and for fewer days than naive mice. Here we show that 8, 11, 13, and 18 days after primary infection the J(H)D -/- mice are almost completely resistant to reinfection and that they are still partially protected from reinfection 6 weeks, 5 months, and 8 months after primary infection. Protection against reinfection was dependent on CD8+ T cells, since J(H)D -/- mice depleted of CD8+ T cells by administration of an anti-CD8 monoclonal antibody became chronically infected with rotavirus upon rechallenge 13 days, 18 days, 6 weeks, and 5 months after primary infection. Thus, CD8+ T cells can actively mediate almost complete short-term and partial long-term protection from reinfection.

  View details for Web of Science ID A1997WT18900098

  View details for PubMedID 9094702

• Hepatitis G virus infection in hemodialysis patients and the effects of interferon Umlauft, F., WONG, D. T., Underhill, P. A., Oefner, P. J., Jin, L., Urbanek, M., Gruenewald, K., Greenberg, H. B. W B SAUNDERS CO-ELSEVIER INC. 1997: A1406–A1406

  View details for Web of Science ID A1997WV41905603

• Heterotypic protection following oral immunization with live heterologous rotaviruses in a mouse model JOURNAL OF INFECTIOUS DISEASES Feng, N. G., Vo, P. T., Chung, D., Vo, T. V., Hoshino, Y., Greenberg, H. B. 1997; 175 (2): 330-341

  Abstract

  A Jennerian approach using live animal viruses to immunize humans is the current lead strategy for developing rotavirus vaccines. This strategy has been modified by incorporating human rotavirus VP7 genes into vaccine strains to induce serotype-specific neutralizing antibodies to human strains. However, the role of homotypic versus heterotypic immunity in protection is unclear. To investigate the importance of serotype-specific immunity in a mouse model, mice were immunized with rhesus rotavirus (RRV: G3, P5[3]), RRV-based modified Jennerian vaccine strains DxRRV (G1, P5[3]), DS1xRRV (G2, P5[3]), or ST3xRRV (G4, P5[3]), or bovine rotavirus NCDV (G6, P6[1]) and challenged with murine rotavirus ECw (G3, P[16]). Mice immunized with modified Jennerian vaccines exhibited complete to near-complete protection from challenge. NCDV-immunized mice also showed partial protection. The protection was correlated with fecal IgA levels to VP6, not serum IgG responses. Modified Jennerian vaccines induce both heterotypic and homotypic immunity in mice.

  View details for Web of Science ID A1997WF06300013

  View details for PubMedID 9203654

• Evidence for CD8(+) T-Cell immunity to murine rotavirus in the absence of perforin, fas, and gamma interferon JOURNAL OF VIROLOGY Franco, M. A., Tin, C., Rott, L. S., VanCott, J. L., MCGHEE, J. R., Greenberg, H. B. 1997; 71 (1): 479-486

  Abstract

  We recently showed that class I-restricted CD8+ T cells mediate clearance of primary rotavirus infection in mice: JHD knockout (JHD -/-) (B-cell-deficient) mice depleted of CD8+ T cells become chronically infected with murine rotavirus, and beta2 microglobulin knockout (beta2m -/-) mice have delayed but complete clearance of primary rotavirus infection. In the present work we have analyzed the mechanism used by CD8+ T cells to clear rotavirus infection. We first determined that perforin knockout (perforin -/-) mice and lpr (fas-deficient) mice clear rotavirus infection with the same kinetics as control mice. When perforin -/- or perforin +/+ mice were depleted of CD8+ T cells by administration of an anti-CD8 monoclonal antibody, they showed a delay of 1 to 2 days in the clearance of rotavirus infection compared to the clearance time for untreated control mice, indicating that CD8+ T cells in both groups of mice participate in the resolution of primary rotavirus infection. In addition, passively transferred CD8+ T cells from rotavirus-infected perforin +/+ and perforin -/- mice were able to mediate viral clearance in Rag 2 knockout (Rag 2 -/-) mice chronically infected with rotavirus with similar kinetics, suggesting that CD8+ T cells from perforin -/- mice are as efficient as CD8+ T cells from perforin +/+ mice in clearing a rotavirus infection. Gamma interferon (IFN-gamma) was also shown to be unnecessary for the antirotavirus effect of CD8+ T cells: IFN-gamma knockout (IFN-gamma -/-) mice and JHD -/-, perforin -/-, and perforin +/+ mice depleted of IFN-gamma by administration of an anti-IFN-gamma monoclonal antibody cleared rotavirus infection with the same kinetics as those for control mice. Hence, CD8+ T cells have an antirotaviral effect that is not mediated by perforin and appears to be independent of fas and the release of IFN-gamma.

  View details for Web of Science ID A1997VX29200059

  View details for PubMedID 8985374

• Murine model of rotavirus infection 1st International Rushmore Conference on Mechanisms in the Pathogenesis of Enteric Diseases Feng, N. G., Franco, M. A., Greenberg, H. B. PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1997: 233–240

  Abstract

  The murine model of homologous rotavirus infection has been used to study the determinants of protection. The local IgA immune response appears to be the critical factor in generating protective immunity after natural infection. A series of knockout mice were used to evaluate the contribution of T cells and B cells to immunity and resolution from primary infection. Both arms of immune system played a role in the resolution of primary infection but antibody was much more important for prevention of reinfection.

  View details for Web of Science ID A1997BH98Z00035

  View details for PubMedID 9192019

• Production and characterization of murine IgA monoclonal antibodies to the surface antigens of rhesus rotavirus VIROLOGY Giammarioli, A. M., MACKOW, E. R., Fiore, L., Greenberg, H. B., Ruggeri, F. M. 1996; 225 (1): 97-110

  Abstract

  Rotavirus is the single most important cause of severe diarrhea in humans and is diffuse in most animal species worldwide, and an understanding of the antigenic properties of the virus is essential to the design of rational vaccine strategies. To better understand the localization of viral epitopes involved in antibody-mediated neutralization of virus infectivity, we have orally immunized mice with live rhesus rotavirus (RRV) and generated a panel of hybridoma cell clones secreting IgA class monoclonal antibodies. A total of 12 neutralizing IgA MAbs to VP4 and VP7 proteins were studied for their epitope specificity and topographical relationships by hemagglutination-inhibition assays, neutralization assays, and competitive-binding assays with previously mapped MAbs. In addition, neutralization-escape virus mutants were selected and gene segments for each variant were cloned and sequenced. Two IgA MAbs were found to be directed to the antigenic region A of the VP7 protein at amino acid 94, and 10 MAbs were directed at the VP8 trypsin cleavage fragment of VP4. Five of the VP4-specific MAbs identified the same neutralization epitope on the RRV VP8 protein, not previously associated with RRV neutralization. All neutralization-escape variants selected by this antibody group contained mutations at amino acids 132- 135 of VP4. One IgA MAb selected for a mutation at amino acid 190 of VP4, and the corresponding viral mutant failed to agglutinate erythrocytes. This MAb mapped to an epitope recognized by 2 additional IgA MAbs. These results suggest that oral immunization of mice with RRV elicits an IgA immune response which is predominantly directed toward antigenic determinants on the VP8 portion of VP4. As a consequence, the route of immunization may alter immunodominant neutralization responses elicited to rotavirus.

  View details for Web of Science ID A1996VT70500010

  View details for PubMedID 8918537

• Hepatitis C virus detection by single-round PCR specific for the terminal 3' noncoding region JOURNAL OF CLINICAL MICROBIOLOGY Umlauft, F., WONG, D. T., Oefner, P. J., Underhill, P. A., Cheung, R. C., Wright, T. L., Kolykhalov, A. A., Gruenewald, K., Greenberg, H. B. 1996; 34 (10): 2552-2558

  Abstract

  A single-round PCR method with primers specific for the 3' noncoding region (NCR) of hepatitis C virus (HCV) has been developed. Using a double RNAzol-B extraction, a high-temperature reverse-transcription step with SuperScript II reverse transcriptase, and a 40-cycle two-temperature PCR with a TaqStart antibody hot-start procedure, we were able to detect a 92-nucleotide fragment of the recently discovered 98-nucleotide highly conserved sequence at the 3' terminus of the HCV genome. Direct sequencing of the PCR products confirmed the specificity of the PCR and demonstrated conservation in this region. Only one nucleotide change in 14 specimens was found. End point dilution titration of sera with known viral RNA titers showed the sensitivity of the single-round 3' NCR PCR to be comparable to those of the established nested 5' NCR assays (fewer than 25 HCV genome equivalents). To evaluate specificity and sensitivity, a panel of 116 serum samples characterized by nested 5'-end PCR, genotyping, and quantitative assays was tested. A high degree of concordance (96%) between the 3' NCR and 5' NCR PCR results was found. The sequence conservation at the 3' end of the HCV genome among common genotypes and the savings in time, labor, and reagents from a single-round PCR make this assay a useful addition to the detection systems available to identify and monitor HCV infection.

  View details for Web of Science ID A1996VK78700043

  View details for PubMedID 8880519

• Molecular determinants of immunity and pathogenicity of rotavirus infection in the mouse model 5th Rotavirus Vaccine Workshop Franco, M. A., Feng, N. G., Greenberg, H. B. UNIV CHICAGO PRESS. 1996: S47–S50

  Abstract

  Rotavirus-induced diarrhea is responsible for the death of approximately 800,000 children per year. Jennerian and modified Jennerian anti-rotavirus vaccines currently being tested have a moderate protective effect against rotavirus disease. Optimization of these vaccines, development of more efficient rationally designed vaccines, and identification of other strategies to prevent or treat rotavirus disease will rely on a thorough understanding of the host-virus relationship and especially on the immune mechanisms developed by the host against rotavirus. Recent advances in our understanding of the mouse model of rotavirus infection will enhance the understanding of human rotavirus disease.

  View details for Web of Science ID A1996VE91000009

  View details for PubMedID 8752290

• Protection against rotavirus infections by DNA vaccination 5th Rotavirus Vaccine Workshop Herrmann, J. E., Chen, S. C., Fynan, E. F., Santoro, J. C., Greenberg, H. B., Wang, S. X., Robinson, H. L. UNIV CHICAGO PRESS. 1996: S93–S97

  Abstract

  DNA vaccines encoding for murine rotavirus proteins VP4, VP6, or VP7 were tested in adult BALB/c mice for their ability to induce immune responses and protect against rotavirus challenge. A gene gun was used to inoculate vaccines into the epidermis. Rotavirus-specific serum antibodies, as measured by ELISA, and virus-specific cytotoxic T lymphocyte responses were generated by each of the three vaccines, but virus-neutralizing antibodies were detected only in mice that were inoculated with DNA vaccines encoding for VP4 and VP7. Efficacy of the vaccines was determined by challenge with 100 ID50 of homotypic rotavirus. Each of the three vaccines was effective in protecting mice against infection after rotavirus challenge as determined by reduction (P < .001) in virus excretion in mice receiving the DNA vaccines. These results demonstrate that DNA vaccination has potential as a new approach for control of rotavirus infections.

  View details for Web of Science ID A1996VE91000016

  View details for PubMedID 8752297

• Quantification of systemic and local immune responses to individual rotavirus proteins during rotavirus infection in mice JOURNAL OF CLINICAL MICROBIOLOGY Ishida, S., Feng, N. G., Tang, B. Z., Gilbert, J. M., Greenberg, H. B. 1996; 34 (7): 1694-1700

  Abstract

  The purpose of the present study was to develop a quantitative assay that could be used to measure the local and systemic immune responses to specific rotavirus proteins following rotavirus infection of adult mice. To measure these responses, we used an immunocytochemical staining assay of Spodoptera frugiperda (Sf-9) cells which were infected with recombinant baculovirus expressing selected rotavirus proteins. The specificity of the assay was documented by using a series of monoclonal antibodies to individual rotavirus proteins. We observed that the assay had high levels of sensitivity and specificity for a series of VP7- and VP4-specific neutralizing monoclonal antibodies which recognized conformation-dependent epitopes on their target proteins. We also studied immunoglobulin G (IgG) immune responses in serum and IgA immune responses in the stools of mice infected with wild-type murine rotavirus strain EHPw. In both sera and stools, the most immunogenic proteins were VP6 and VP4. VP2 was less immunogenic than VP6 or VP4, and the immune responses to VP7, NSP2, and NSP4 were very low in serum and undetectable in stools.

  View details for Web of Science ID A1996UR41600020

  View details for PubMedID 8784572

• Protective effect of rotavirus VP6-specific IgA monoclonal antibodies that lack neutralizing activity SCIENCE Burns, J. W., SIADATPAJOUH, M., Krishnaney, A. A., Greenberg, H. B. 1996; 272 (5258): 104-107

  Abstract

  Rotaviruses are the leading cause of severe gastroenteritis and dehydrating diarrhea in young children and animals worldwide. A murine model and "backpack tumor" transplantation were used to determine the protective effect of antibodies against VP4(an outer capsid viral protein) and VP6(a major inner capsid viral protein). Only two non-neutralizing immunoglobulin A (IgA) antibodies to VP6 were capable of preventing primary and resolving chronic murine rotavirus infections. These antibodies were not active, however, when presented directly to the luminal side of the intestinal tract. These findings support the hypothesis that in vivo intracellular viral inactivation by secretory IgA during transcytosis is a mechanism of host defense against rotavirus infection.

  View details for Web of Science ID A1996UD59700048

  View details for PubMedID 8600516

• Cleavage of rotavirus VP4 in vivo JOURNAL OF GENERAL VIROLOGY Ludert, J. E., Krishnaney, A. A., Burns, J. W., Vo, P. T., Greenberg, H. B. 1996; 77: 391-395

  Abstract

  The infectivity of rotavirus particles is dependent on proteolytic cleavage of the outer capsid protein, VP4, at a specific site. This cleavage event yields two fragments, identified as VP5* and VP8*. It has been hypothesized that the particle is more stable, but non-infectious, when VP4 is in the uncleaved state. Uncleaved VP4 and the resultant increased stability might be advantageous for the virus to resist environmental degradation until it infects a susceptible host. When VP4 is cleaved in the lumen of the host's gastrointestinal tract, the virus particle would become less stable but more infectious. To test this hypothesis, a series of experiments was undertaken to analyse the cleavage state of VP4 on virus shed by an infected host into the environment. Immunoblots of intestinal wash solutions derived from infant and adult BALB/c mice infected with a virulent cell culture-adapted variant of the EDIM virus (EW) or wild-type murine rotavirus EDIM-Cambridge were analysed. Virtually all of the VP4 in these samples was in the cleaved form. Moreover, cell culture titration of trypsin-treated and untreated intestinal contents from pups infected with EW indicated that excreted virus is fully activated prior to trypsin addition. It was also observed that trypsin-activated virus has no disadvantage in initiating infection in naive animals over virions containing an intact VP4. These studies indicate that VP4 is cleaved upon release from the intestinal cell and that virus shed into the environment does not have an intact VP4.

  View details for Web of Science ID A1996TW91600002

  View details for PubMedID 8601772

• DNA vaccines against rotavirus infections Sapporo International Symposium on Viral Gastroenteritis Herrmann, J. E., Chen, S. C., Fynan, E. F., Santoro, J. C., Greenberg, H. B., Robinson, H. L. SPRINGER WIEN. 1996: 207–215

  Abstract

  Plasmid DNA vaccines encoding for murine rotaviral proteins VP4, VP6, and VP7 were tested in adult BALB/c mice for their ability to induce immune responses and provide protection against rotavirus challenge. Serum antibodies were measured by virus neutralization and by ELISA. Cellular immunity was assessed by measuring cytotoxic T cell (CTL) responses. The vaccines were administered by inoculation into cells of the epidermis with an Accell gene gun (Auragen, Inc., Middleton, WI, USA). Each of the three vaccines elicited rotavirus-specific serum antibodies as measured by ELISA. Virus neutralizing antibodies were detected in mice receiving DNA vaccines encoding for VP4 and VP7, but not in those which received the plasmid encoding for VP6. Vaccines encoding for VP4, VP6, or VP7 generated virus-specific CTL responses in recipient mice. Efficacy of the vaccines was determined by challenge with homotypic rotaviruses. Each of the three vaccines was effective in protecting mice against infection after rotavirus (100 ID50) challenge. Significant reductions (p < 0.0002, analysis of variance) in viral excretion measured over a 9 day period were seen in mice receiving the DNA vaccines compared with mice that received control plasmids.

  View details for Web of Science ID A1996WE21200023

  View details for PubMedID 9015117

• Rotavirus immunity in the mouse Sapporo International Symposium on Viral Gastroenteritis Franco, M. A., Feng, N., Greenberg, H. B. SPRINGER WIEN. 1996: 141–152

  Abstract

  Naturally attenuated animal rotaviruses have been tested as antirotavirus vaccines with moderate success. The development of improved vaccines will rely on our understanding of the immune mechanism that mediate clearance and protection from rotaviral reinfection. The mouse model of rotavirus infection is a versatile tool for studying these mechanisms: mice have a relative low cost and there is a rapidly increasing number of immunological reagents to study rotavirus immunology. This review covers recent data on the mouse model of rotavirus infection. We show that both effector arms of the immune system (CD8 + T cells and B cells) mediate anti-rotavirus effects in vivo.

  View details for Web of Science ID A1996WE21200017

  View details for PubMedID 9015111

• Epidemiology of symptomatic human rotaviruses in Bangalore and Mysore, India, from 1988 to 1994 as determined by electropherotype, subgroup and serotype analysis ARCHIVES OF VIROLOGY Aijaz, S., Gowda, K., Jagannath, H. V., Reddy, R. R., Maiya, P. P., Ward, R. L., Greenberg, H. B., Raju, M., Babu, A., Rao, C. D. 1996; 141 (3-4): 715-726

  Abstract

  Epidemiology of symptomatic rotaviruses from Bangalore and Mysore in Southern India was investigated. While serotype G3 predominated throughout the 7-year study period from 1988 to 1994 in Bangalore, serotype G1 was more predominant than serotype G3 in Mysore during 1993 and 1994. Serotype G2 strains were either not detected or infrequently observed in both the cities. However, several strains with subgroup I and 'short' RNA pattern that exhibited high reactivity with typing MAbs specific for serotype 2 as well as other serotypes were detected throughout the period. Among the nonserotypeable strains from both cities, several exhibited dual subgroup (SGI + II) or subgroup I specificity and 'long' RNA pattern indicating their probable animal origin. Notably, a gradual, yet highly significant reduction in rotavirus gastroenteritis, from 45.3% in 1988 to 1.8% during 1994, was observed in Bangalore in stark contrast to the consistently high (about 34%) incidence of asymptomatic infections among neonates by I321-like G10P11 type strains during the same period. Moreover, I321-like asymptomatic strains were not detected in children with diarrhea.

  View details for Web of Science ID A1996UF51300025

  View details for PubMedID 8645107

• Genetic mapping indicates that VP4 is the rotavirus cell attachment protein in vitro and in vivo JOURNAL OF VIROLOGY Ludert, J. E., Feng, N. G., Yu, J. H., Broome, R. L., Hoshino, Y., Greenberg, H. B. 1996; 70 (1): 487-493

  Abstract

  To identify the rotavirus protein which mediates attachment to cells in culture, viral reassortants between the simian rotavirus strain RRV and the murine strains EHP and EW or between the simian strain SA-11 and the human strain DS-1 were isolated. These parental strains differ in the requirement for sialic acid to bind and infect cells in culture. Infectivity and binding assays with the parental and reassortant rotaviruses indicate that gene 4 encodes the rotavirus protein which mediates attachment to cells in culture for both sialic acid-dependent and -independent strains. Using ligated intestinal segments of newborn mice and reassortants obtained between the murine strain EW and RRV, we developed an in vivo infectivity assay. In this system, the infectivity of EW was not affected by prior treatment of the enterocytes with neuraminidase, while neuraminidase treatment reduced the infectivity of a reassortant carrying gene 4 from RRV on an EW background more than 80% relative to the controls. Thus, VP4 appears to function as the cell attachment protein in vivo as well as in vitro.

  View details for Web of Science ID A1996TJ65000058

  View details for PubMedID 8523562

• ROLE OF B-CELLS AND CYTOTOXIC T-LYMPHOCYTES IN CLEARANCE OF AND IMMUNITY TO ROTAVIRUS INFECTION IN MICE JOURNAL OF VIROLOGY Franco, M. A., Greenberg, H. B. 1995; 69 (12): 7800-7806

  Abstract

  The immune mechanisms involved in clearance of and immunity to rotavirus infection are poorly understood. Although mice with severe combined immunodeficiency (SCID mice) become chronically infected, nude mice have been reported to clear rotavirus infection similarly to immunocompetent controls. To better characterize the role of cytotoxic T lymphocytes (CTLs) in clearance of and immunity to rotavirus infection, we infected naive or previously infected beta 2-microglobulin (beta 2m) knockout mice with murine rotavirus. Naive beta 2m knockout mice shed rotavirus antigen 2 days longer than did normal control mice but completely resolved primary infection. beta 2m knockout naive mice treated with depleting doses of an anti-CD8 monoclonal antibody before infection shed viral antigen for an additional day. Upon rechallenge, beta 2m knockout mice, either treated with the anti-CD8 antibody or not treated, were completely resistant to reinfection. Clearance of rotavirus infection in naive beta 2m knockout mice correlated with the development of intestinal rotavirus-specific immunoglobulin A. Before rechallenge, beta 2m knockout mice had high levels of intestinal rotavirus-specific immunoglobulin A. These findings suggest that CTLs mediate rotavirus clearance but are not required for this function and that CTLs are not necessary for development of immunity to rotavirus reinfection. To further characterize the effector mechanisms involved in clearance and prevention of rotavirus infection, similar studies were performed with B-cell-deficient JHD knockout mice. After primary infection, most naive JHD mice had similar virus-shedding clearance curves as did control mice and completely resolved primary infection. However, 2 of 29 became chronically infected. All JHD mice treated with anti-CD8 antibody became chronically infected with murine rotavirus. Upon rechallenge, JHD mice which had cleared primary infection were all susceptible to reinfection. These findings suggest that B cells also play a role in clearance of primary infection but are absolutely necessary for development of immunity against rotavirus reinfection.

  View details for PubMedID 7494291

• ROTAVIRUS-INDUCED FUSION FROM WITHOUT IN TISSUE-CULTURE CELLS JOURNAL OF VIROLOGY FALCONER, M. M., Gilbert, J. M., ROPER, A. M., Greenberg, H. B., Gavora, J. S. 1995; 69 (9): 5582-5591

  Abstract

  We present the first evidence of fusion from without induced in tissue culture cells by a nonenveloped virus. Electron micrographs of two strains of rotavirus, bovine rotavirus C486 and rhesus rotavirus, show that virally mediated cell-cell fusion occurs within 1 h postinfection. Trypsin activation is necessary for rotavirus to mediate cell-cell fusion. The extent of fusion is relative to the amount of virus used, and maximum fusion occurs between pHs 6.5 and 7.5. Fusion does not require virus-induced protein synthesis, as virus from both an empty capsid preparation and from an EDTA-treated preparation, which is noninfectious, can induce fusion. Incubation of rotavirus with neutralizing and nonneutralizing monoclonal antibodies before addition to cells indicates that viral protein 4 (VP4; in the form of VP5* and VP8*) and VP7 are involved in fusion. Light and electron micrographs document this fusion, including the formation of pores or channels between adjacent fused cells. These data support direct membrane penetration as a possible route of infection. Moreover, the assay should be useful in determining the mechanisms of cell entry by rotavirus.

  View details for Web of Science ID A1995RN98600047

  View details for PubMedID 7637004

• DETECTION OF HEPATITIS-C VIRUS-RNA BY METHODS USING PHENOL-CHLOROFORM OR SILICA PARTICLES - REPLY JOURNAL OF CLINICAL MICROBIOLOGY Cheung, R. C., Matsui, S. M., Greenberg, H. B. 1995; 33 (9): 2522-2523

  View details for Web of Science ID A1995RP75500062

• ANTIGENICITY, IMMUNOGENICITY AND PASSIVE PROTECTION INDUCED BY IMMUNIZATION OF MICE WITH BACULOVIRUS-EXPRESSED VP7 PROTEIN FROM RHESUS ROTAVIRUS JOURNAL OF GENERAL VIROLOGY Fiore, L., Dunn, S. J., Ridolfi, B., Ruggeri, F. M., MACKOW, E. R., Greenberg, H. B. 1995; 76: 1981-1988

  Abstract

  The major neutralization antigen VP7 of rhesus rotavirus (RRV) was expressed in a baculovirus recombinant system. The expressed VP7 showed the same molecular mass as native VP7, and was recognized by hyperimmune sera as well as neutralizing and non-neutralizing monoclonal antibodies (MAbs) raised against RRV. Intraperitoneal administration of the expressed VP7 in mice elicited the production of serum antibodies which were able to immunoprecipitate VP7 from RRV-infected cell lysates and to neutralize the virus in vitro. Sera from immunized mice competed for binding to RRV in an ELISA with both neutralizing and non-neutralizing MAbs specific for VP7. Using a passive protection model of rotavirus disease, vaccination of mice with the recombinant VP7 induced partial protection from infection. These results suggest that the baculovirus-expressed VP7 may be useful in priming a protective immune response to rotavirus infection.

  View details for Web of Science ID A1995RN95000011

  View details for PubMedID 7636478

• HETEROGENEITY OF VP4 NEUTRALIZATION EPITOPES AMONG SEROTYPE P1A HUMAN ROTAVIRUS STRAINS CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY Contreras, J. F., MENCHACA, G. E., PADILLANORIEGA, L., TAMEZ, R. S., Greenberg, H. B., Lopez, S., Arias, C. F. 1995; 2 (4): 506-508

  Abstract

  We have used serotype-specific VP4 and VP7 neutralizing monoclonal antibodies (Nt-MAbs), as well as subgroup (SG)-specific MAbs, to characterize by enzyme immunoassay rotavirus strains isolated from diarrheic infants in the city of Monterrey, Mexico, from July 1993 to March 1994. Of a total of 465 children studied, 140 were rotavirus positive, including 3 patients infected with non-group A rotaviruses. The SG and VP7 (G) serotype specificities could be determined for 118 (84%) of the 140 rotavirus-positive stool specimens; 4 rotavirus strains were serotype G1 and SGII; 1 strain was serotype G2 and SGI+II; 112 strains were serotype G3 and SGII; 1 strain was serotype G3 and SGI; and none of the strains was serotype G4. Fifty-eight specimens, representing the 13 different group A rotavirus electropherotypes detected, were chosen for VP4 (P) serotyping. Of these, 48 (83%) strains reacted with the P1A serotype-specific Nt-MAb 1A10. None of the strains reacted with the serotype P2-specific Nt-MAbs tested. Not all viruses that reacted with Nt-MAb 1A10 were recognized by Nt-MAbs 2A3 and 2G1, which also recognize P1A strains, indicating heterogeneity of neutralization epitopes among serotype P1A human rotaviruses. This heterogeneity could be relevant for the specificity of the VP4-mediated neutralizing antibody immune response and indicates the need for antigenic characterization, in addition to genomic typing, of the VP4 proteins of circulating human rotavirus field strains.

  View details for Web of Science ID A1995RJ98700026

  View details for PubMedID 7583936

• EXPRESSION OF THE ROTAVIRUS SA11 PROTEIN VP7 IN THE SIMPLE EUKARYOTE DICTYOSTELIUM-DISCOIDEUM JOURNAL OF VIROLOGY Emslie, K. R., MILLER, J. M., Slade, M. B., Dormitzer, P. R., Greenberg, H. B., Williams, K. L. 1995; 69 (3): 1747-1754

  Abstract

  The outer capsid protein of rotavirus, VP7, is a major neutralization antigen and is considered a necessary component of any subunit vaccine developed against rotavirus infection. For this reason, significant effort has been directed towards producing recombinant VP7 that maintains the antigenic characteristics of the native molecule. Using a relatively new expression system, the simple eukaryote Dictyostelium discoideum, we have cloned the portion of simian rotavirus SA11 genome segment 9, encoding the mature VP7 protein, downstream of a native D. discoideum secretion signal sequence in a high-copy-number extrachromosomal vector. The majority of the recombinant VP7 expressed by transformants was intracellular and was detected by Western immunoblot following gel electrophoresis as two or three bands with an apparent molecular mass of 35.5 to 37.5 kDa. A small amount of VP7 having an apparent molecular mass of 37.5 kDa was secreted. Both the intracellular VP7 and the secreted VP7 were N glycosylated and sensitive to endoglycosidase H digestion. Under nonreducing electrophoresis conditions, over half the intracellular VP7 migrated as a monomer while the remainder migrated with an apparent molecular mass approximately twice that of the monomeric form. In an enzyme-linked immunosorbent assay, intracellular VP7 reacted with both nonneutralizing and neutralizing antibodies. The monoclonal antibody recognition pattern paralleled that found with VP7 expressed in either vaccinia virus or herpes simplex virus type 1 and confirms that D. discoideum-expressed VP7 is able to form the major neutralization domains present on viral VP7. Because D. discoideum cells are easy and cheap to grow, this expression system provides a valuable alternative for the large-scale production of recombinant VP7 protein.

  View details for Web of Science ID A1995QG07300045

  View details for PubMedID 7853513

  View details for PubMedCentralID PMC188780

• ANALYSES OF HOMOLOGOUS ROTAVIRUS INFECTION IN THE MOUSE MODEL VIROLOGY Burns, J. W., Krishnaney, A. A., Vo, P. T., Rouse, R. V., Anderson, L. J., Greenberg, H. B. 1995; 207 (1): 143-153

  Abstract

  The group A rotaviruses are significant human and veterinary pathogens in terms of morbidity, mortality, and economic loss. Despite its importance, an effective vaccine remains elusive due at least in part to our incomplete understanding of rotavirus immunity and protection. Both large and small animal model systems have been established to address these issues. One significant drawback of these models is the lack of well-characterized wild-type homologous viruses and their cell culture-adapted variants. We have characterized four strains of murine rotaviruses, EC, EHP, EL, and EW, in the infant and adult mouse model using wild-type isolates and cell culture-adapted variants of each strain. Wild-type murine rotaviruses appear to be equally infectious in infant and adult mice in terms of the intensity and duration of virus shedding following primary infection. Spread of infection to naive cagemates is seen in both age groups. Clearance of shedding following primary infection appears to correlate with the development of virus-specific intestinal IgA. Protective immunity is developed in both infant and adult mice following oral infection as demonstrated by a lack of shedding after subsequent wild-type virus challenge. Cell culture-adapted murine rotaviruses appear to be highly attenuated when administered to naive animals and do not spread efficiently to nonimmune cagemates. The availability of these wild-type and cell culture-adapted virus preparations should allow a more systematic evaluation of rotavirus infection and immunity. Furthermore, future vaccine strategies can be evaluated in the mouse model using several fully virulent homologous viruses for challenge.

  View details for Web of Science ID A1995QJ65700015

  View details for PubMedID 7871723

• IDENTIFICATION OF 2 INDEPENDENT NEUTRALIZATION DOMAINS ON THE VP4 TRYPSIN CLEAVAGE PRODUCTS VP5-ASTERISK AND VP8-ASTERISK OF HUMAN ROTAVIRUS ST3 VIROLOGY PADILLANORIEGA, L., Dunn, S. J., Lopez, S., Greenberg, H. B., Arias, C. F. 1995; 206 (1): 148-154

  Abstract

  The antigenic structure of the VP4 protein of human rotavirus (HRV) strains Wa and ST3 was studied by using a panel of Wa- and ST3-derived VP4-specific neutralizing monoclonal antibodies (NMAbs) and NMAb-resistant variants. The VP4-coding genes from three Wa and three ST3 variants were sequenced. For Wa VP4, one homotypic and one heterotypic neutralization site, at amino acids 458 and 392, respectively, were identified. For ST3 VP4, three neutralization sites were found at amino acids 72, 217, and 385 that are either homotypic or associated with limited cross-reactivity. Cross-neutralization assays using several pairs of NMAbs and resistant variants showed that Wa VP4 has at least one large neutralization domain on its larger trypsin cleavage product, VP5*, consisting of several operationally related epitopes. VP4 of ST3 has at least two neutralization domains, one located on VP5* that is operationally related to the large neutralization domains on VP5* from HRVs Wa and KU, as well as an independent neutralization domain on VP8*, the smaller trypsin cleavage product of VP4.

  View details for Web of Science ID A1995QD14900017

  View details for PubMedID 7530390

• ADULT NUDE-MICE ARE RELATIVELY RESISTANT TO INFECTION WITH MURINE ROTAVIRUS BUT DO NOT DEVELOP IMMUNITY AFTER A PRIMARY INFECTION Franco, M. A., LUDERT, J., Greenberg, H. B. WILEY-BLACKWELL. 1995: 285–285

  View details for Web of Science ID A1995QQ99700979

• IMMUNOGENICITY, ANTIGENICITY, AND PROTECTION EFFICACY OF BACULOVIRUS-EXPRESSED VP4 TRYPSIN CLEAVAGE PRODUCTS, VP5(1) AND VP8 FROM RHESUS ROTAVIRUS ARCHIVES OF VIROLOGY Dunn, S. J., Fiore, L., WERNER, R. L., Cross, T. L., Broome, R. L., Ruggeri, F. M., Greenberg, H. B. 1995; 140 (11): 1969-1978

  Abstract

  Rhesus rotavirus (RRV) VP4 trypsin cleavage product VP5(1)*, a truncated form of VP5*, was expressed in baculovirus and found by immunoprecipitation to be antigenically similar to VP5* on the virion. Immunization of mice with VP5(1)* elicited neutralizing antibody that was found to be cross-reactive with viruses representing P genotypes 1, 3, 4, 6, 7, and 8. Baculovirus expressed trypsin cleavage products, VP8* (amino acids 1-246) and VP5(1)* (amino acids 247-474), were tested for their ability to elicit a protective response in a murine model of passive protection. These results were compared to those obtained with baculovirus expressed RRV VP4. Dams immunized with baculovirus expressed RRV VP4 gave birth to pups protected from RRV virus challenge. Neither VP5(1)* nor VP8* was as effective at generating protective immunity as full length VP4. However, antibody to VP5(1)* was more effective than antibody to VP8* at mediating protection even though the neutralizing antibody titers as measured by hemagglutination inhibition and focus reduction neutralization were similar.

  View details for Web of Science ID A1995TG91000007

  View details for PubMedID 7503695

• COMPARISON OF MUCOSAL AND SYSTEMIC HUMORAL IMMUNE-RESPONSES AND SUBSEQUENT PROTECTION IN MICE ORALLY INOCULATED WITH A HOMOLOGOUS OR A HETEROLOGOUS ROTAVIRUS JOURNAL OF VIROLOGY Feng, N. G., Burns, J. W., BRACY, L., Greenberg, H. B. 1994; 68 (12): 7766-7773

  Abstract

  Rotaviruses are the single most important cause of severe diarrhea in young children worldwide, and vaccination is probably the most effective way to control the disease. Most current live virus vaccine candidates are based on the host range-restricted attenuation of heterologous animal rotaviruses in humans. The protective efficacy of these vaccine candidates has been variable. To better understand the nature of the heterologous rotavirus-induced active immune response, we compared the differences in the mucosal and systemic immune responses generated by heterologous (nonmurine) and homologous (murine) rotaviruses as well as the ability of these infections to produce subsequent protective immunity in a mouse model. Sucking mice were orally inoculated with a heterologous simian or bovine rotavirus (strain RRV or NCDV) or a homologous murine rotavirus (wild-type or tissue culture-adapted) strain EHP at various doses. Six weeks later, mice were challenged with a virulent murine rotavirus (wild-type strain ECW) and the shedding of viral antigen in feces was quantitated. Levels of rotavirus-specific serum immunoglobulin G (IgG) and fecal IgA prior to challenge were measured and correlated with subsequent viral shedding or protection. Heterologous rotavirus-induced active protection was highly dependent on the strain and dose of the virus tested. Mice inoculated with a high dose (10(7) PFU per mouse) of RRV were completely protected, while the protection was diminished in animals inoculated with NCDV or lower doses of RRV. The ability of a heterologous rotavirus to stimulate a detectable intestinal IgA response correlated with the ability of the virus to generate protective immunity. Serum IgG titer did not correlate with protection. Homologous rotavirus infection, on the other hand, was much more efficient at inducing both mucosal and systemic immune responses as well as protection regardless of the virulence of the virus strain or the size of the immunizing dose.

  View details for Web of Science ID A1994PR43200013

  View details for PubMedID 7966566

• VIRAL GASTROENTERITIS INFECTIOUS DISEASES IN CLINICAL PRACTICE Burns, J. W., Greenberg, H. B. 1994; 3 (6): 411-417

  View details for Web of Science ID A1994PU08200002

• FORMATION AND INTRACELLULAR-LOCALIZATION OF HEPATITIS-C VIRUS ENVELOPE GLYCOPROTEIN COMPLEXES EXPRESSED BY RECOMBINANT VACCINIA AND SINDBIS VIRUSES JOURNAL OF VIROLOGY Dubuisson, J., Hsu, H. H., Cheung, R. C., Greenberg, H. B., Russell, D. G., Rice, C. M. 1994; 68 (10): 6147-6160

  Abstract

  Hepatitis C virus (HCV) encodes two putative virion glycoproteins (E1 and E2) which are released from the polyprotein by signal peptidase cleavage. In this report, we have characterized the complexes formed between E1 and E2 (called E1E2) for two different HCV strains (H and BK) and studied their intracellular localization. Vaccinia virus and Sindbis virus vectors were used to express the HCV structural proteins in three different cell lines (HepG2, BHK-21, and PK-15). The kinetics of association between E1 and E2, as studied by pulse-chase analysis and coprecipitation of E2 with an anti-E1 monoclonal antibody, indicated that formation of stable E1E2 complexes is slow. The times required for half-maximal association between E1 and E2 were 60 to 85 min for the H strain and more than 165 min for the BK strain. In the presence of nonionic detergents, two forms of E1E2 complexes were detected. The predominant form was a heterodimer of E1 and E2 stabilized by noncovalent interactions. A minor fraction consisted of heterogeneous disulfide-linked aggregates, which most likely represent misfolded complexes. Posttranslational processing and localization of the HCV glycoproteins were examined by acquisition of endoglycosidase H resistance, subcellular fractionation, immunofluorescence, cell surface immunostaining, and immunoelectron microscopy. HCV glycoproteins containing complex N-linked glycans were not observed, and the proteins were not detected at the cell surface. Rather, the proteins localized predominantly to the endoplasmic reticular network, suggesting that some mechanism exists for their retention in this compartment.

  View details for Web of Science ID A1994PG54100002

  View details for PubMedID 8083956

  View details for PubMedCentralID PMC237034

• RAPID AND SENSITIVE METHOD FOR DETECTION OF HEPATITIS-C VIRUS-RNA BY USING SILICA PARTICLES JOURNAL OF CLINICAL MICROBIOLOGY Cheung, R. C., Matsui, S. M., Greenberg, H. B. 1994; 32 (10): 2593-2597

  Abstract

  We describe a rapid, sensitive, and economic method for detection of hepatitis C virus (HCV) RNA. This method uses silica particles for purification of nucleic acid and then a modified reverse transcription-PCR that minimizes the risk of contamination and reduces the amount of reagents used. We found purification by silica particles to be at least as sensitive and in certain circumstances more sensitive than that by traditional phenol-chloroform extraction. This improved sensitivity may be due to more efficient recovery of HCV RNA by silica particles. HCV RNA appears to bind to silica particles in a saturable fashion, and the addition of extraneous nucleic acids (salmon sperm DNA or tRNA) decreases the binding in a dose-related fashion. The reverse transcription-PCR is performed by using a modified single tube method which further simplifies and reduces the cost of this assay. Finally, this method may be applied to clinical specimens such as liver tissue.

  View details for Web of Science ID A1994PG91200050

  View details for PubMedID 7529243

  View details for PubMedCentralID PMC264112

• PRESENTATION OF NEUTRALIZING EPITOPES BY ENGINEERED ROTAVIRUS VP7S EXPRESSED BY RECOMBINANT VACCINIA VIRUSES VIROLOGY Dormitzer, P. R., Both, G. W., Greenberg, H. B. 1994; 204 (1): 391-402

  Abstract

  Previous studies showed that a calcium-dependent neutralization domain forms on the rotavirus glycoprotein VP7 during assembly into particles. Here, we demonstrate that expressed, recombinant VP7 is capable of forming this neutralization domain in the absence of other rotavirus proteins, but that the domain is unstable. High calcium environments, incorporation into particles, and binding of neutralizing antibodies stabilize the neutralization domain on expressed VP7. A chimeric, cell surface-anchored molecule, VP7sc, has an enhanced ability to react with neutralizing antibodies. This may explain why immunization of mice with expressed native VP7 has had limited success while immunization with VP7sc efficiently induced neutralizing antibodies and passively protected pups from diarrhea. A model of VP7 folding consistent with these results is presented.

  View details for Web of Science ID A1994PG06700040

  View details for PubMedID 7522373

• MAPPING THE SUBGROUP EPITOPES OF ROTAVIRUS PROTEIN VP6 VIROLOGY Lopez, S., ESPINOSA, R., Greenberg, H. B., Arias, C. F. 1994; 204 (1): 153-162

  Abstract

  VP6, the most abundant protein of rotaviruses, contains epitopes that allow the classification of these viruses into four subgroups (SG), depending on the presence or absence of two epitopes called I and II. The subgroup-specific epitopes are conformational and appear to be present on trimeric but not monomeric VP6. We have identified on VP6 some of the amino acids that determine the reactivity of the subgroup-specific mAbs 255/60 and 631/9. A single amino acid mutation at positions 172 (Met to Ala) or 305 (Asn to Ala) was sufficient to change the subgroup specificity of the human rotavirus Wa VP6 protein from SGII to SGI/II, since either of these mutations allowed the protein to be recognized by the SGI mAb 255/60, while retaining its capacity to interact with the SGII mAb 631/9. In the case of the SGII epitope, the mutation of two contiguous amino acids (Ala305 Asn306 to Asn305 Ala306) in the porcine rotavirus YM VP6 protein (SGI) enabled the protein to be efficiently recognized by the SGII mAb 631/9, while causing the YM VP6 protein to lose its capacity to interact with mAb 255/60. These results suggest that both subgroup Abs interact with an antigenic domain in VP6 that is composed of at least two regions of the protein that, although distant in the linear sequence, might be in close proximity in the structured VP6 trimer.

  View details for Web of Science ID A1994PG06700016

  View details for PubMedID 7522369

• COMPARISON OF VP4 AND VP7 OF 5 MURINE ROTAVIRUS STRAINS VIROLOGY Dunn, S. J., Burns, J. W., Cross, T. L., Vo, P. T., Ward, R. L., Bremont, M., Greenberg, H. B. 1994; 203 (2): 250-259

  Abstract

  The nucleotide and deduced amino acid sequences of outer capsid proteins VP4 and VP7 of five murine rotavirus strains (EW, EB, EC, EL, EHP) were determined. Comparisons of the VP7 amino acid sequences of the five murine rotavirus strains with rotavirus strains representative of G serotypes 1-14 showed that the murine strains were highly homologous to one another and more closely related to strains representing G3 than to any other G type. Analysis of the VP4 amino acid sequences of the murine strains revealed the presence of at least two murine P types. Therefore, sequence analysis would predict that the murine rotaviruses are G3 or G3-like and represent at least two unique P types (tentatively P17 for EW, EB, EC, and EL and P18 for EHP). When we attempted to categorize the murine strains by serum neutralization tests, our results were less clear. Serum to two murine strains, EHP and EW, displayed one-way reactivity by focus-reduction neutralization assays using several prototype G3 strains. The G3 serotyping monoclonal antibody 159 failed to neutralize EHP and EW, while the G3-specific monoclonal YO-1E2 neutralized EHP, but not EW. A reassortant (A15) containing VP7 from EW and VP4 from RRV was neutralized by these two G3-specific monoclonal antibodies to a level 8- to 20-fold more than EW, but was still 64- to 250-fold less than either SA11 and RRV. These results suggest that VP4 can influence the antigenicity of VP7.

  View details for Web of Science ID A1994PB65800006

  View details for PubMedID 8053149

• COMPARISON OF THE ROTAVIRUS NONSTRUCTURAL PROTEIN NSP1 (NS53) FROM DIFFERENT SPECIES BY SEQUENCE-ANALYSIS AND NORTHERN BLOT HYBRIDIZATION VIROLOGY Dunn, S. J., Cross, T. L., Greenberg, H. B. 1994; 203 (1): 178-183

  Abstract

  The nucleotide sequence of gene 5 encoding the rotavirus nonstructural protein NSP1 (NS53) of 6 strains (EW, EHP, RRV, I321, OSU, and Gottfried) was determined and compared to 6 previously reported strains (SA11, UK, RF, Hu803, DS-1, and Wa). The 12 rotavirus strains were derived from a total of five separate species (murine, bovine, simian, porcine, and human). Gene sizes ranged from 1564 to 1611 nucleotides in length and the deduced protein sequences were found to be 486 to 495 amino acids in length. Comparisons of NSP1 amino acid sequences showed identities ranging from 36 to 92%. This diversity was most evident between strains from different species. Phylogenetic analysis revealed a clustering of NSP1 sequences according to species origin with the exception that the human and porcine strains were included in a single grouping. Northern blot hybridizations using additional rotavirus strains from the five species confirmed the grouping found by sequence analysis. The species specificity of NSP1 is consistent with the hypothesis that NSP1 plays a role in host range restriction.

  View details for Web of Science ID A1994NZ23600024

  View details for PubMedID 8030275

• INTRACELLULAR MANIPULATION OF DISULFIDE BOND FORMATION IN ROTAVIRUS PROTEINS DURING ASSEMBLY JOURNAL OF VIROLOGY Svensson, L., Dormitzer, P. R., VONBONSDORFF, C. H., Maunula, L., Greenberg, H. B. 1994; 68 (8): 5204-5215

  Abstract

  Rotavirus undergoes a unique mode of assembly in the rough endoplasmic reticulum (RER) of infected cells. Luminal RER proteins undergo significant cotranslational and posttranslational modifications, including disulfide bond formation. Addition of a reducing agent (dithiothreitol [DTT]) to rotavirus-infected cells did not significantly inhibit translation or disrupt established disulfide bonds in rotavirus proteins but prevented the formation of new disulfide bonds and infectious viral progeny. In DTT-treated, rotavirus-infected cells, all vp4, vp6, and ns28 epitopes but no vp7 epitopes were detected by immunohistochemical staining with a panel of monoclonal antibodies. When oxidizing conditions were reestablished in DTT-treated cells, intramolecular disulfide bonds in vp7 were rapidly and correctly established with the restoration of antigenicity, although prolonged DTT treatment led to the accumulation of permanently misfolded vp7. Electron microscopy revealed that cytosolic assembly of single-shelled particles and budding into the ER was not affected by DTT treatment but that outer capsid assembly was blocked, leading to the accumulation of single-shelled and enveloped intermediate subviral particles in the RER lumen.

  View details for Web of Science ID A1994NW97800055

  View details for PubMedID 8035518

• ANTIBODY-RESPONSE TO HEPATITIS-C VIRUS-INFECTION AFTER LIVER-TRANSPLANTATION AMERICAN JOURNAL OF GASTROENTEROLOGY Hsu, H. H., Wright, T. L., Tsao, S. C., Combs, C., DONETS, M., Feinstone, S. M., Greenberg, H. B. 1994; 89 (8): 1169-1174

  Abstract

  To determine whether liver transplantation and the subsequent immunosuppression affect the antibody response to hepatitis C virus (HCV) infection.Sera from 46 patients were compared before and after liver transplantation for markers of HCV infection. Serum HCV RNA was determined by polymerase chain reaction (PCR). Anti-HCV antibody was determined by first- and second-generation immunoassays as well as a quantitative assay of the titer of anti-HCV core antibody.Among individuals who acquired hepatitis C infection in association with liver transplantation, only 15% (3/12) developed antibody to the core antigen and only 25% (3/12) reacted to any antigen present on the second-generation recombinant immunoblot assay after a mean follow-up period of 18 months. Thirty-eight percent (5/13) were positive, by the second-generation enzyme immunoassay (EIA-2) Whereas 94% (16/17) of the individuals who had detectable anti-HCV core antibodies pretransplant continued to have such antibodies after transplant, the titer of these antibodies declined an average of 4-fold. No significant change was seen in the antibody titer toward rotavirus, a common viral pathogen. Patients who acquired HCV infection or in whom the allograft became reinfected had a significantly increased incidence of posttransplant hepatitis (61% vs. 33%, respectively).Liver transplantation and posttransplant immunosuppression lead to an attenuated antibody response to hepatitis C viral infection. Currently available assays for anti-HCV antibodies may be unreliable in the posttransplant setting.

  View details for Web of Science ID A1994PB41000009

  View details for PubMedID 7519818

• HELICOBACTER-PYLORI IN PEPTIC-ULCER DISEASE JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION Yamada, T., Ahnen, D., Alpers, D. H., Greenberg, H. B., Gray, L., JOSCELYN, K. B., Kauffman, G., Podolsky, D. K., Ray, W. A., Schaberg, D., Silverstein, F. E., Sivak, M. V., WILLIAMS, A. L., Yolken, R. 1994; 272 (1): 65-69

  View details for Web of Science ID A1994NU48500032

• A MONOTYPE OF HUMAN ROTAVIRUS SEROTYPE-1 INVOLVED IN A DIARRHEA OUTBREAK IN A PEDIATRIC WARD ISRAEL JOURNAL OF MEDICAL SCIENCES Shif, I., Silberstein, I., Ashkenazi, S., Greenberg, H. B., Samra, Z. 1994; 30 (3): 240-242

  View details for Web of Science ID A1994NC79600007

  View details for PubMedID 7514161

• Rotavirus pathology and pathophysiology. Current topics in microbiology and immunology Greenberg, H. B., Clark, H. F., Offit, P. A. 1994; 185: 255-283

  View details for PubMedID 8050281

• ANALYSIS OF ASTROVIRUS SEROTYPE-1 RNA, IDENTIFICATION OF THE VIRAL RNA-DEPENDENT RNA-POLYMERASE MOTIF, AND EXPRESSION OF A VIRAL STRUCTURAL PROTEIN JOURNAL OF VIROLOGY Lewis, T. L., Greenberg, H. B., Herrmann, J. E., Smith, L. S., Matsui, S. M. 1994; 68 (1): 77-83

  Abstract

  We report the results from sequence analysis and expression studies of the gastroenteritis agent astrovirus serotype 1. We have cloned and sequenced 5,944 nucleotides (nt) of the estimated 7.2-kb RNA genome and have identified three open reading frames (ORFs). ORF-3, at the 3' end, is 2,361 nt in length and is fully encoded in both the genomic and subgenomic viral RNAs. Expression of ORF-3 in vitro yields an 87-kDa protein that is immunoprecipitated with a monoclonal antibody specific for viral capsids. This protein comigrates with an authentic 87-kDa astrovirus protein immunoprecipitated from infected cells, indicating that this region encodes a viral structural protein. The adjacent upstream ORF (ORF-2) is 1,557 nt in length and contains a viral RNA-dependent RNA polymerase motif. The viral RNA-dependent RNA polymerase motifs from four astrovirus serotypes are compared. Partial sequence (2,018 nt) of the most 5' ORF (ORF-1) reveals a 3C-like serine protease motif. The ORF-1 sequence is incomplete. These results indicate that the astrovirus genome is organized with nonstructural proteins encoded at the 5' end and structural proteins at the 3' end. ORF-2 has no start methionine and is in the -1 frame compared with ORF-1. We present sequence evidence for a ribosomal frameshift mechanism for expression of the viral polymerase.

  View details for Web of Science ID A1994MW25100010

  View details for PubMedID 8254779

• IDENTIFICATION OF A NEW NEUTRALIZATION EPITOPE ON VP7 OF HUMAN SEROTYPE-2 ROTAVIRUS AND EVIDENCE FOR ELECTROPHEROTYPE DIFFERENCES CAUSED BY SINGLE NUCLEOTIDE SUBSTITUTIONS VIROLOGY Dunn, S. J., Ward, R. L., McNeal, M. M., Cross, T. L., Greenberg, H. B. 1993; 197 (1): 397-404

  Abstract

  Neutralizing monoclonal antibodies 2F1 and 1C10, which are specific for VP7 of serotype 2 rotaviruses (G2), were used to select neutralization escape variants of the human serotype 2 rotavirus, DS-1. Neutralization survival patterns generated by monoclonal antibodies 2F1, 1C10, and RV5:3 indicated that 2F1 and 1C10 did not recognize identical epitopes. Direct sequencing of PCR products of gene 8, encoding VP7, revealed that each escape variant possessed only a single nucleotide mutation which resulted in a single amino acid substitution. In one variant, a second nucleotide change occurred, but did not result in an amino acid change. Four independently selected 2F1 mutants showed mutations in four separate sites in antigenic regions A, C, and D. Two independently selected 1C10 variants had mutations in either the A region or an unreported site at amino acid 190. Two of the mutations resulted in the creation of new glycosylation sites which were utilized, but did not appear to greatly affect antigenicity. Of note, three of the mutants also demonstrated alterations in the migration patterns of gene 8 on PAGE. Such electrophoretic mobility shifts caused by single base neutralization escape mutations have not previously been reported for rotavirus. Since multiple mutations were selected with the same monoclonal antibody it appears that the antigenic regions of VP7, although widely separated in the linear sequence, are parts of a single, large and complex neutralization domain which includes amino acid 190 as well as the other previously reported epitopes.

  View details for Web of Science ID A1993MB62100041

  View details for PubMedID 7692670

• THE ENDOPLASMIC RETICULUM-ASSOCIATED VP7 OF ROTAVIRUS IS TARGETED TO AXONS AND DENDRITES IN POLARIZED NEURONS JOURNAL OF NEUROCYTOLOGY Weclewicz, K., Kristensson, K., Greenberg, H. B., Svensson, L. 1993; 22 (8): 616-626

  Abstract

  Rotavirus, which matures and is retained in the endoplasmic reticulum, was used to examine how polarized dorsal root ganglion and spinal cord neurons distributed cytoplasmic and endoplasmic reticulum-associated proteins. A remarkable observation was that NS28, a trans-endoplasmic reticulum-membrane protein which functions as a receptor for budding particles, remained in the cell body during the whole course of infection (48 h) while the VP7 glycoprotein, which is endoplasmic reticulum associated and usually retained in the endoplasmic reticulum, was targeted to axons already 4 h post infection. VP7 was furthermore transported in an endo-beta-N-acetylglucosaminidase H sensitive form through the secretory pathway. The segregated appearances of NS28 and the endo-beta-N-acetylglucosaminidase H sensitive VP7 indicate that VP7 enters a transport compartment separate from NS28. Brefeldin A treatment rapidly disintegrated the Golgi apparatus of the neurons and rapidly blocked axonal transport of Sendai virus glycoproteins, while axonal transport of rotavirus VP7 was not blocked, suggesting that VP7 uses an intracellular pathway in neurons which does not involve the Golgi apparatus.

  View details for Web of Science ID A1993LT95800004

  View details for PubMedID 8229088

• BOTH SURFACE-PROTEINS (VP4 AND VP7) OF AN ASYMPTOMATIC NEONATAL ROTAVIRUS STRAIN (I321) HAVE HIGH-LEVELS OF SEQUENCE IDENTITY WITH THE HOMOLOGOUS PROTEINS OF A SEROTYPE-10 BOVINE ROTAVIRUS VIROLOGY Das, M., Dunn, S. J., WOODE, G. N., Greenberg, H. B., Rao, C. D. 1993; 194 (1): 374-379

  Abstract

  The nucleotide sequence of genes 4 and 9, encoding the outer capsid proteins VP4 and VP7 of a serotype 10 tissue culture-adapted strain, I321, representative of asymptomatic neonatal rotaviruses isolated from neonates in Bangalore, India, were determined. Comparison of nucleotide and deduced amino acid sequences of I321 VP4 and VP7 with previously published sequences of various serotypes revealed that both genes were highly homologous to the respective genes of serotype 10 bovine rotavirus, B223. The VP4 of I321 represents a new human P serotype and the I321 and related strains represent the first description of neonatal rotaviruses that appear to derive both surface proteins from an animal rotavirus.

  View details for Web of Science ID A1993KY34400044

  View details for PubMedID 8386881

• CHARACTERIZATION OF HEPATITIS-C VIRUS STRUCTURAL PROTEINS WITH A RECOMBINANT BACULOVIRUS EXPRESSION SYSTEM HEPATOLOGY Hsu, H. H., DONETS, M., Greenberg, H. B., Feinstone, S. M. 1993; 17 (5): 763-771

  Abstract

  We cloned and expressed the sequences encoding the structural proteins of the hepatitis C virus in a baculovirus eukaryotic expression system. Four recombinant constructs expressed sufficient hepatitis C virus-specific proteins in insect cell culture to allow analysis of protein cleavage, glycosylation and immunoreactivity. Using immunoblot analysis, we detected a 22-kD protein corresponding to the hepatitis C virus capsid protein cleaved from a larger precursor. Recombinant constructs encoding the presumptive envelope (E1) protein produced products ranging from 30 to 35 kD, whereas constructs encoding the presumptive E2/NS1 protein expressed products ranging in size from 68 to 73 kD. The recombinant envelope proteins were glycosylated, as shown by sensitivity to endoglycosidase F digestion, whereas the capsid was not. We examined the immunoreactivity of these recombinant proteins using sera from 50 patients chronically infected with HCV. Forty-seven of 50 of these sera contained antibodies against the capsid, 14 (28%) also had antibodies against E1 and at least 5 (10%) had antibody against E2/NS1. Forty-seven of 50 sera (94%) were viremic, as determined on hepatitis C virus polymerase chain reaction. The three sera that were hepatitis C virus polymerase chain reaction negative did not have envelope antibodies, whereas all sera that had envelope antibodies were also hepatitis C virus polymerase chain reaction positive. Thus antibodies to baculovirus-expressed hepatitis C virus structural proteins, including E1 and E2/NS1, are found in the presence of viremia.

  View details for Web of Science ID A1993LC82500002

  View details for PubMedID 8387945

• MURINE ROTAVIRUS GENES ENCODING OUTER CAPSID PROTEINS VP4 AND VP7 ARE NOT MAJOR DETERMINANTS OF HOST RANGE RESTRICTION AND VIRULENCE JOURNAL OF VIROLOGY Broome, R. L., Vo, P. T., Ward, R. L., Clark, H. F., Greenberg, H. B. 1993; 67 (5): 2448-2455

  Abstract

  Simian rotavirus (RRV) and murine rotavirus (EDIM-RW) differ dramatically in the oral inoculum required to cause diarrheal disease in neonatal mouse pups and in their ability to spread and cause disease in uninoculated littermates. A genetic approach was used to explore the molecular basis of these differences. Reassortant viruses were produced in vivo by coinfecting infant mice with RRV and EDIM-RW. Reassortant viruses were isolated by plaque purification of progeny virus obtained from mouse pup intestines on MA104 cells. The plaque-purified reassortants were evaluated for 50% diarrhea dose (DD50) and for the ability to spread and cause diarrhea in uninoculated littermates. The parental RRV strain had a DD50 of 10(5) PFU per animal, while the EDIM-RW parental strain had a DD50 of less than 1 PFU per animal. RRV never spreads from inoculated to uninoculated littermates and causes disease. Twenty-three reassortants were tested. Of great interest were the reassortants D1/5 and C3/2, which derived genes 4 and 7 (encoding VP4 and VP7) from RRV. These viruses had a DD50 similar or identical to that of EDIM-RW and spread efficiently from inoculated mouse pups to uninoculated pups. We conclude that the major outer capsid proteins VP4 and VP7 are not primarily responsible for virulence or host range restriction in the mouse model using a homologous murine rotavirus.

  View details for Web of Science ID A1993KX33500004

  View details for PubMedID 8386262

• CHARACTERIZATION AND SEROEPIDEMIOLOGY OF A TYPE-5 ASTROVIRUS ASSOCIATED WITH AN OUTBREAK OF GASTROENTERITIS IN MARIN-COUNTY, CALIFORNIA JOURNAL OF CLINICAL MICROBIOLOGY Midthun, K., Greenberg, H. B., Kurtz, J. B., GARY, G. W., Lin, F. Y., KAPIKIAN, A. Z. 1993; 31 (4): 955-962

  Abstract

  The Marin County strain of type 5 astrovirus was associated with two separate outbreaks of nonbacterial gastroenteritis in California in 1978. A safety-tested, bacterium-free filtrate prepared from a stool specimen of an individual who was ill during the original outbreak was given orally to 19 adult volunteers. One volunteer developed a gastrointestinal illness, and nine had serologic responses. Several diarrheal stool specimens from the ill volunteer contained a large number of 27-nm particles. By using immune electron microscopy with acute- and convalescent-phase sera from the original outbreak, these 27-nm particles were shown to be identical to the viral inoculum. The Marin County virus, purified from the stool of the ill volunteer, was shown by immunoprecipitation and polyacrylamide gel electrophoresis to contain a single structural protein with a molecular weight of 30,000. The buoyant density of the virion was 1.39 g/cm3 in cesium chloride. By electron microscopy, approximately 5% of the particles had the characteristic stellate configuration of astrovirus, and serologic studies by immunofluorescence technique confirmed previous classification of the Marin County virus as a type 5 astrovirus. Radioimmunoassay and biotin-avidin immunoassay were used to detect antibody to the Marin County virus in paired acute- and convalescent-phase sera from 32 outbreaks of nonbacterial gastroenteritis, but none of these outbreaks could be attributed to this virus. Prevalence of antibody to this strain of astrovirus was approximately 13% in children 6 months to 3 years of age and increased to 41% in older children and young adults.

  View details for Web of Science ID A1993KT44500034

  View details for PubMedID 8385155

• CLONING AND CHARACTERIZATION OF HUMAN ASTROVIRUS IMMUNOREACTIVE EPITOPES JOURNAL OF VIROLOGY Matsui, S. M., Kim, J. P., Greenberg, H. B., Young, L. M., Smith, L. S., Lewis, T. L., Herrmann, J. E., Blacklow, N. R., Dupuis, K., Reyes, G. R. 1993; 67 (3): 1712-1715

  Abstract

  We report the cloning of antigenic, protein-coding regions of human astrovirus serotype 1 that appear to be common to most, if not all, serotypes of human astrovirus. Screening of lambda gt11 libraries identified three different but overlapping clones (A43, A35, and A1) and one independent clone (A14) that reacted with serum from a rabbit repeatedly immunized with purified astrovirus particles but not with its preimmunization serum. These clones were shown to be astrovirus specific. Of note, a radiolabeled probe representing the immunoreactive clones A43-A35-A1 hybridized exclusively to the 7.2-kb astrovirus genomic RNA, while a clone A14-specific probe hybridized with both the genomic and the 2.8-kb astrovirus subgenomic RNAs. This suggests that the immunoreactive epitopes, selected by antiserum to purified astrovirus particles, are encoded by the subgenomic RNA as well as other regions of the genomic RNA.

  View details for Web of Science ID A1993KM61300072

  View details for PubMedID 7679752

• SEROLOGIC ANALYSIS OF HUMAN ROTAVIRUS SEROTYPES P1A AND P2 BY USING MONOCLONAL-ANTIBODIES JOURNAL OF CLINICAL MICROBIOLOGY PADILLANORIEGA, L., WERNERECKERT, R., MACKOW, E. R., GORZIGLIA, M., LARRALDE, G., Taniguchi, K., Greenberg, H. B. 1993; 31 (3): 622-628

  Abstract

  Three human rotavirus (HRV) VP4 serotypes and one subtype have been described on the basis of a fourfold or an eightfold-or-greater difference in neutralization titer when tested with hyperimmune antisera to recombinant VP4 or VP8* (serotypes P1A, P1B, P2, and P3). To start to analyze the antigenic basis underlying serotype specificity, we produced a library of 13 VP4-specific neutralizing monoclonal antibodies (NMAbs) to two HRVs, the serotype P1A strain Wa and the serotype P2 strain ST3, and characterized the reactivity of these NMAbs with a panel of serotypically diverse HRV strains by neutralization assay and enzyme-linked immunosorbent assay (ELISA). We characterized the serotypic specificity of the NMAbs by using a fourfold or an eightfold-or-greater difference in titer against the homologous (i.e., immunogen) and heterologous strains as a criterion for serotype. Some ST3-derived NMAbs reacted specifically with serotype P2 HRVs by ELISA and/or neutralization assay, while some Wa-derived NMAbs reacted specifically by ELISA and/or neutralization assay with some or all serotype P1A HRVs. Other Wa- and ST3-derived NMAbs reacted with some or all serotype P1A and P2 HRV strains by neutralization assay and ELISA. Most NMAbs did not react with serotype P1B or P3 strains. In previous studies, three distinct operationally defined epitopes have been identified on VP4 by examining the reactivity patterns of selected antigenic variants of HRV strain KU. At least one of the NMAbs described here recognizes an epitope unrelated to these previously identified epitopes, since it neutralized both KU and its variants.

  View details for Web of Science ID A1993KM81000028

  View details for PubMedID 7681440

• IMMUNODOMINANCE OF THE VP4 NEUTRALIZATION PROTEIN OF ROTAVIRUS IN PROTECTIVE NATURAL INFECTIONS OF YOUNG-CHILDREN JOURNAL OF VIROLOGY Ward, R. L., McNeal, M. M., Sander, D. S., Greenberg, H. B., BERNSTEIN, D. I. 1993; 67 (1): 464-468

  Abstract

  Natural infection by very similar strains of rotavirus during the 1988-1989 rotavirus season in Cincinnati, Ohio, provided complete protection of young children against subsequent rotavirus illnesses for a period of at least 2 years. Using this limited strain variability, we characterized the association between the titers of antibody to either the VP4 or the VP7 neutralization protein and protection against subsequent rotavirus disease. This was done by using reassortants that contained only one of the two rotavirus neutralization proteins of 89-12, a culture-adapted isolate representative of the protective rotavirus strains. The other neutralization protein in these reassortants was derived from a heterologous rotavirus (WC3 or EDIM) to which the infected subjects made little or no neutralizing antibody (titers, < or = 20). The geometric mean titer (GMT) of antibody to 89-12 in convalescent-phase sera from the 21 subjects analyzed was 2,323. The GMT of antibody to a reassortant (strain WC-4) that contained the VP7 protein of 89-12 and VP4 of WC3 was 387. In contrast, the GMT of antibody to a reassortant (strain EDIM-7) that contained the VP4 protein of 89-12 and the VP7 protein of EDIM was 1,078. Thus, the major neutralization response was directed against VP4 rather than VP7, a finding that has important implications for development of appropriate rotavirus vaccines.

  View details for Web of Science ID A1993KB97000053

  View details for PubMedID 8416378

• ROTAVIRUS VACCINATION - CURRENT STATUS - A BRIEF SUMMARY Conference on Biotechnology R&D Trends: Science Policy for Development Greenberg, H. B. NEW YORK ACAD SCIENCES. 1993: 32–35

  View details for Web of Science ID A1993BZ60L00004

  View details for PubMedID 8109868

• SEROTYPIC AND GENOTYPIC CHARACTERIZATION OF HUMAN SEROTYPE-10 ROTAVIRUSES FROM ASYMPTOMATIC NEONATES JOURNAL OF CLINICAL MICROBIOLOGY Dunn, S. J., Greenberg, H. B., Ward, R. L., Nakagomi, O., Burns, J. W., Vo, P. T., PAX, K. A., Das, M., Gowda, K., Rao, C. D. 1993; 31 (1): 165-169

  Abstract

  Human rotaviruses were isolated from asymptomatic neonates at various hospitals and clinics in the city of Bangalore, India, and were found to be subgroup I specific and possess long RNA patterns (M. Sukumaran, K. Gowda, P. P. Maiya, T. P. Srinivas, M. S. Kumar, S. Aijaz, R. R. Reddy, L. Padilla, H. B. Greenberg, and C. D. Rao, Arch. Virol. 126:239-251, 1992). Three of these strains were adapted to tissue culture and found by serotype analysis and neutralization assays to be of serotype 10, a serotype commonly found in cattle but infrequently found in humans and not previously identified in neonates. By RNA-RNA hybridization, a high level of relatedness to a serotype 10 bovine rotavirus strain and a low-to-medium level of relatedness to a human rotavirus strain were observed. Since this human isolate shares a genogroup with bovine rotavirus, it is likely that it originated by interspecies transmission. A human rotavirus strain isolated from asymptomatic neonates and similar to bovine rotavirus might represent a good vaccine candidate.

  View details for Web of Science ID A1993KC71800036

  View details for PubMedID 8380181

• DETECTION OF NORWALK VIRUS IN STOOL SPECIMENS BY REVERSE TRANSCRIPTASE-POLYMERASE CHAIN-REACTION AND NONRADIOACTIVE OLIGOPROBES JOURNAL OF CLINICAL MICROBIOLOGY DeLeon, R., Matsui, S. M., Baric, R. S., Herrmann, J. E., Blacklow, N. R., Greenberg, H. B., SOBSEY, M. D. 1992; 30 (12): 3151-3157

  Abstract

  A reverse transcriptase (RT)-polymerase chain reaction (PCR)-oligoprobe (OP), or RT-PCR-OP, method was developed for the detection of the Norwalk virus, which causes acute, epidemic gastroenteritis, in stool specimens. The Norwalk virus genome regions encoding the following two proteins were amplified by RT-PCR: the RNA polymerase (260-bp product) and a putative immunogenic protein (224-bp product). The resulting DNA fragments (amplicons) were hybridized to a digoxigenin-labeled internal OP specific to each amplicon. The detection limit of Norwalk virus, as determined by the endpoint of RT-PCR amplification for serially diluted, positive stool specimens, was similar to the actual virion titer as estimated by electron microscopy and at least 100-fold greater than the titer determined by radioimmunoassay (RIA). The RT-PCR-OP assay was specific for Norwalk virus and negative for other enteric viruses, including human and animal caliciviruses, hepatitis E virus, Snow Mountain agent, astroviruses, 16 human enteroviruses, and 5 human rotaviruses. Components of fecal specimens that interfere with RT-PCR were removed successfully by Sephadex G-200 gel chromatography. Of 20 stool specimens from human volunteers that were positive for Norwalk virus by RIA, a specific RT-PCR-OP result was obtained in 95% (19 of 20) of the samples by using the immunogenic protein primers and 75% (15 of 20) by using the polymerase primers. Twenty-six stool specimens from asymptomatic children and adults were negative by the Norwalk virus RT-PCR-OP. RT-PCR-OP detected Norwalk virus in the 4 of 21 coded fecal specimens that were also positive by enzyme immunoassay. Two samples that were positive by RIA or enzyme immunoassay were negative by RT-PCR, perhaps because viral RNA was not present or RT-PCR inhibitors were not adequately removed.

  View details for Web of Science ID A1992JZ91100021

  View details for PubMedID 1280649

  View details for PubMedCentralID PMC270605

• LIPOSOME-MEDIATED TRANSFECTION OF INTACT VIRAL PARTICLES REVEALS THAT PLASMA-MEMBRANE PENETRATION DETERMINES PERMISSIVITY OF TISSUE-CULTURE CELLS TO ROTAVIRUS JOURNAL OF CLINICAL INVESTIGATION Bass, D. M., Baylor, M. R., Chen, C., Mackow, E. M., Bremont, M., Greenberg, H. B. 1992; 90 (6): 2313-2320

  Abstract

  Rotaviruses are an important cause of gastroenteritis in human infants. In vivo, rotavirus displays striking cell tropism with viral replication generally restricted to the villus tip enterocytes of the small intestine. We studied a panel of cell lines that vary significantly in their permissivity to rotavirus infection. L cells and HEp2 cells were relatively resistant to rotavirus infection compared with permissive Ma104 cells and HT29 cells. RNA transcription among the cell lines was proportional to antigen synthesis making a translational or posttranslational block an unlikely source of observed differences in susceptibility. All of the cell lines bound and internalized radiolabeled virus equally well, as measured by escape from surface protease treatment. Analysis of the escape of cell bound virus from neutralizing monoclonal antibody revealed that rotavirus did not immediately enter an eclipse phase in nonpermissive cells, but was internalized in an infectious form for several hours, possibly sequestered within endocytic vacuoles. L cells and HEp2 cells were as permissive as Ma104 and HT29 cells when rotavirus infection was mediated by transfection of single- or double-shelled rotavirus particles with cationic liposomes (Lipofectin). Rotavirus cell tropism in tissue culture cells is determined by the ability of infecting virions to traverse the plasma membrane of the cells into the cytoplasmic compartment.

  View details for Web of Science ID A1992KE76300020

  View details for PubMedID 1334974

• ISOLATION AND CHARACTERIZATION OF A NOVEL REASSORTANT BETWEEN AVIAN TY-1 AND SIMIAN RRV ROTAVIRUSES JOURNAL OF VIROLOGY KOOL, D. A., Matsui, S. M., Greenberg, H. B., HOLMES, I. H. 1992; 66 (11): 6836-6839

  Abstract

  A reassortant, TyRh, was isolated after coinfection of MA104 cells with avian Ty-1 and simian RRV rotaviruses. Hybridization and serological studies showed that the reassortant's 4th gene, which encodes Vp4, was derived from the simian RRV rotavirus parent, whereas the remaining 10 genes were derived from the avian Ty-1 rotavirus parent.

  View details for Web of Science ID A1992JT70000074

  View details for PubMedID 1328691

• CALCIUM CHELATION INDUCES A CONFORMATIONAL CHANGE IN RECOMBINANT HERPES-SIMPLEX VIRUS-1-EXPRESSED ROTAVIRUS-VP7 VIROLOGY Dormitzer, P. R., Greenberg, H. B. 1992; 189 (2): 828-832

  Abstract

  Rotavirus, strain SA11, glycoprotein VP7 that was expressed by a recombinant herpes simplex virus-1 or contained in purified rotavirus particles lost reactivity with the neutralizing monoclonal antibody (mAb) 159, but not with nonneutralizing mAbs, upon chelation of calcium by EGTA. Exposing VP7, but not the neutralizing mAbs, to a transient excess of EGTA over calcium eliminated VP7 neutralizing epitopes. Therefore, a calcium chelation-induced conformational change in VP7, not in the neutralizing mAbs, caused the epitope loss. Addition of excess calcium or strontium, but not magnesium or barium, to EGTA-treated VP7 restored its 159 epitope. These results suggest that VP7 binds calcium in the absence of other rotavirus proteins and that the calcium chelation-induced conformational change in VP7 may mediate uncoating of double-shelled rotavirus particles.

  View details for Web of Science ID A1992JF68400053

  View details for PubMedID 1322608

• RECURRENT AND ACQUIRED HEPATITIS-C VIRAL-INFECTION IN LIVER-TRANSPLANT RECIPIENTS GASTROENTEROLOGY Wright, T. L., Donegan, E., Hsu, H. H., Ferrell, L., Lake, J. R., Kim, M., Combs, C., FENNESSY, S., Roberts, J. P., Ascher, N. L., Greenberg, H. B. 1992; 103 (1): 317-322

  Abstract

  To examine the postliver transplant recurrence of hepatitis C virus (HCV) infection in patients with pretransplant infection, as well as its acquisition in patients without prior infection, we used the polymerase chain reaction to amplify HCV RNA in serum and/or liver samples of 89 patients with alcoholic and cryptogenic cirrhosis undergoing liver transplantation. Results were correlated with histologic findings from posttransplant liver biopsies. Ninety-five percent of patients with pretransplant infection had posttransplant viremia. In contrast, 35% of patients without pretransplant infection acquired the virus (P less than 0.0001). Pretransplant HCV infection predisposed patients to hepatitis in the new graft. HCV RNA was present in serum of 96% of patients with posttransplant hepatitis. Fifty-six percent of patients with posttransplant HCV infection had no evidence of liver damage at least 1 year posttransplant. However, of those patients with histologic hepatitis, chronic active hepatitis was common. It is concluded that although HCV infection recurs posttransplant in almost all infected patients, acquisition of the HCV infection with transplant is common. Pretransplant HCV infection is an independent risk factor for the development of posttransplant hepatitis. HCV infection accounts for the majority of posttransplant hepatitis not due to cytomegalovirus, and although many patients with posttransplant viremia have little evidence of histologic hepatitis, significant hepatic damage may occur.

  View details for Web of Science ID A1992JA83500042

  View details for PubMedID 1377143

• MOLECULAR-BASIS OF AGE-DEPENDENT GASTRIC INACTIVATION OF RHESUS ROTAVIRUS IN THE MOUSE JOURNAL OF CLINICAL INVESTIGATION Bass, D. M., Baylor, M., Broome, R., Greenberg, H. B. 1992; 89 (6): 1741-1745

  Abstract

  Rotavirus requires specific proteolytic activation by trypsin for efficient replication in tissue culture. To observe the nature of intestinal proteolytic activation of rotavirus in vivo, metabolically labeled rhesus rotavirus (RRV) grown in the presence of trypsin inhibitors was administered to adult and 10-d-old suckling mice by gavage. In the adult stomach, vp4 was cleaved in a manner distinct from in vitro trypsin cleavage. In the suckling stomach, RRV vp4 remains largely uncleaved. The alternative cleavage in the adult stomach was associated with a profound decrease in viral infectivity. vp4 from RRV recovered from the suckling small intestinal lumen was cleaved in a pattern similar or identical to in vitro trypsin-activated virus with bands comigrating with vp5* and vp8*. In contrast, vp4 was not observed in any recognizable form in RRV recovered from adult intestines. Comparison of infectivity of virus recovered from suckling and adult intestines revealed a 10,000-fold decrease in titer in the virus recovered from the adult intestine. In vitro digestions of RRV revealed that pepsin digestion can cleave RRV vp4 and markedly enhance acid-induced loss of rotavirus infectivity. Subsequent digestion with chymotrypsin removes most of the pepsin cleavage products of vp4. Virus injected directly into jejunal loops of adult mice and virus administered orally to adult mice pretreated with antiacid drugs retained infectivity. These studies indicate the development of gastric acid and pepsin secretion may be an important host defense factor in rotavirus gastroenteritis.

  View details for Web of Science ID A1992HX97400007

  View details for PubMedID 1318323

• HUMORAL IMMUNE-RESPONSES TO VP4 AND ITS CLEAVAGE PRODUCTS VP5-ASTERISK AND VP8-ASTERISK IN INFANTS VACCINATED WITH RHESUS ROTAVIRUS JOURNAL OF CLINICAL MICROBIOLOGY PADILLANORIEGA, L., Fiore, L., Rennels, M. B., Losonsky, G. A., MACKOW, E. R., Greenberg, H. B. 1992; 30 (6): 1392-1397

  Abstract

  The humoral immune response to rhesus rotavirus (RRV) VP4 and its cleavage products VP5* and VP8* was determined in paired serum samples from 44 infants vaccinated with RRV or human rotavirus-RRV reassortants and 5 placebo recipients. Our aim was to try to measure the response to those regions of VP4 most closely related to protection. An enzyme-linked immunosorbent assay (ELISA) was used to measure the immunoglobulin G immune response to baculovirus-expressed full-length RRV VP4, full-length VP8*, and the amino-terminal polypeptide of VP5* called VP5*(1) (amino acids 248 to 474). The two antigenic regions of VP4 selected for study, VP5*(1) and VP8*, have previously been shown to contain most of the cross-reactive and strain-specific neutralization epitopes, respectively, while the remaining carboxy-terminal half of VP5* (amino acids 475 to 776) has not been clearly associated with neutralization. All three recombinant proteins were antigenically conserved, since they reacted with a library of neutralizing monoclonal antibodies directed at VP4. There was a high percentage of seroresponders to VP4 (61%) or to VP8* (52%), but fewer infants seroresponded to VP5*(1) (11%). In addition, infants responding to VP5*(1) had considerably lower titers than to VP4 or VP8*. Immune response to VP4 correlated strongly with the responses detected by the plaque reduction neutralization assay but did not correlate with the responses detected by the ELISA to whole RRV. These data imply that the VP5*(1) region is less immunogenic than the VP8* region of VP4 in infants immunized with RRV or RRV reassortants. The low immunogenicity of VP5* might adversely affect the efficacy of RRV vaccine candidates.

  View details for Web of Science ID A1992HU60300005

  View details for PubMedID 1320626

• ASTROVIRUSES AND CALICIVIRUSES - EMERGING ENTERIC PATHOGENS INFECTIOUS AGENTS AND DISEASE-REVIEWS ISSUES AND COMMENTARY Greenberg, H. B., Matsui, S. M. 1992; 1 (2): 71-91

  Abstract

  Acute, infectious gastroenteritis is an extremely common disease that contributes significantly to morbidity and mortality worldwide. In the United States, it is the second most frequent illness encountered in families. While this illness generally runs a self-limited course, it may be temporarily incapacitating and impact substantially on numbers of days lost from work or school. At present, 30-40% of infectious gastroenteritis cases in the United States are attributable to viral agents, while 20-30% are due to bacteria and parasites. These estimates are almost certainly low, since the cause of gastroenteritis is not discernible in approximately 40% of the cases, and gastroenteritis may be caused by viruses or other pathogens that cannot be identified at this time. Rotavirus and enteric adenovirus are two of the most prevalent and well-studied of the viral agents and have been reviewed extensively elsewhere. This review focuses on two broad groups of small round structured viruses (SRSV), astroviruses and caliciviruses (classic, Norwalk, and Norwalk-like). Although recognized in association with acute, nonbacterial gastroenteritis since the early 1970s, the study of these viruses has been hampered by the relatively low levels of viral shedding in feces, difficulty in propagating the virus in cell or organ culture, and the lack of widely available, well-standardized reagents for their detection. In spite of these obstacles, much has been learned about these viruses using standard virologic (electron microscopy, biophysical characterization, immunoassays) and epidemiologic methods. More recently, substantial progress has been made in studying astroviruses and caliciviruses at the molecular level. Molecular techniques are now being used as diagnostic aids to characterize the epidemiology of these agents in greater detail.

  View details for Web of Science ID A1992JT67000002

  View details for PubMedID 1365533

• NEUTRALIZING EPITOPES ON HERPES-SIMPLEX VIRUS-1-EXPRESSED ROTAVIRUS-VP7 ARE DEPENDENT ON COEXPRESSION OF OTHER ROTAVIRUS PROTEINS VIROLOGY Dormitzer, P. R., Ho, D. Y., MACKOW, E. R., Mocarski, E. S., Greenberg, H. B. 1992; 187 (1): 18-32

  Abstract

  We constructed a recombinant thymidine kinase-negative herpes simplex virus type 1 (HSV-1) that expressed the rotavirus major outer capsid glycoprotein, VP7. In the recombinant HSV-1, a promoter from the 5' noncoding region of the HSV-1 glycoprotein B locus regulated the expression of VP7 as a HSV-1 gamma 1 gene product. HSV-1-expressed VP7 resembled rotavirus-expressed VP7 in its SDS-PAGE mobility, high mannose-type glycosylation, disulfide bonding, perinuclear to cytoplasmic localization, intracellular retention, and reactivity with polyclonal antisera and nonneutralizing antibodies. Unlike rotavirus-expressed VP7, HSV-1-expressed VP7 lacked several neutralizing epitopes by immuno-histochemical staining and by ELISA. One neutralizing epitope identified on HSV-1-expressed VP7 by ELISA was masked by paraformaldehyde fixation of recombinant HSV-1- but not rotavirus-infected cells. Neutralizing epitopes were restored to HSV-1-expressed VP7 by coinfection of cells with the HSV-1 recombinant and a heterologous rotavirus that lack the neutralizing epitopes. The recovered neutralizing epitopes were detected on double-shelled rotavirus particles produced in the coinfected cells. This study indicates that the formation of several neutralizing epitopes on rotavirus VP7 requires interaction of VP7 with other rotavirus proteins. In addition, HSV-1 was a useful vector for studying the localization, processing, and antigenicity of an RNA virus glycoprotein.

  View details for Web of Science ID A1992HD62800003

  View details for PubMedID 1371025

• COMPARISONS OF ROTAVIRUS VP7-TYPING MONOCLONAL-ANTIBODIES BY COMPETITION BINDING ASSAY JOURNAL OF CLINICAL MICROBIOLOGY Raj, P., Matson, D. O., Coulson, B. S., BISHOP, R. F., Taniguchi, K., Urasawa, S., Greenberg, H. B., Estes, M. K. 1992; 30 (3): 704-711

  Abstract

  Three sets of neutralizing monoclonal antibodies (MAbs) used to type the outer capsid protein VP7 of four group A rotavirus serotypes (1 through 4) were compared in competition immunoassays. Reciprocal competition was observed for each of the VP7 type 2-, 3-, and 4-specific MAbs. The VP7 type 1 MAbs exhibited variable competition patterns with other VP7 type 1 MAbs. MAb RV4:3, which has been used to recognize antigenic variants within VP7 type 1 strains, showed reciprocal competition with the four VP7 type 3 MAbs (RV3:1, YO-1E2, 4F8, and 159) using a VP7 type 3 virus (SA11) as antigen. MAb 2C9, also prepared against VP7 type 1, reacted with VP7 type 3 strains and competed with a VP7 type 3 MAb, 159, using RRV as antigen. Use of the different sets of VP7 type-specific MAbs in the enzyme-linked immunosorbent assay permitted the recognition of six antigenic variants within VP7 types 1, 2, and 3 among specimens whose VP7 type could not be determined previously with only one set of typing MAbs. These results demonstrate differences of typing ability among these VP7-specific MAbs and emphasize the need to improve the sensitivity of typing systems by incorporating panels of MAbs reacting with several neutralizing epitopes.

  View details for Web of Science ID A1992HE08700033

  View details for PubMedID 1372622

• STRATEGIES FOR THE IDENTIFICATION OF ICOSAHEDRAL VIRUS RECEPTORS JOURNAL OF CLINICAL INVESTIGATION Bass, D. M., Greenberg, H. B. 1992; 89 (1): 3-9

  View details for Web of Science ID A1992GY56100001

  View details for PubMedID 1309536

• EXCLUSIVE ASYMPTOMATIC NEONATAL INFECTIONS BY HUMAN ROTAVIRUS STRAINS HAVING SUBGROUP-I SPECIFICITY AND LONG RNA ELECTROPHEROTYPE ARCHIVES OF VIROLOGY Sukumaran, M., Gowda, K., Maiya, P. P., SRINIVAS, T. P., Kumar, M. S., Aijaz, S., Reddy, R. R., Padilla, L., Greenberg, H. B., Rao, C. D. 1992; 126 (1-4): 239-251

  Abstract

  A large number of stool specimens, of healthy newborn infants, collected from various hospitals and clinics in Bangalore City, India, have been examined for the presence of asymptomatic rotaviral excretion. Out of 370 samples analysed during a three year period from 1988 to 1991, 133 specimens (36%) were positive for rotavirus RNA. All these asymptomatic neonatal strains, without exception, showed "long" RNA pattern, but subgroup I specificity. Serotype analysis by ELISA or by hybridization with serotype-specific probes indicated that these strains probably represent a new serotype in newborn children. We find an exclusive association of human rotaviruses having "long" RNA pattern and subgroup I specificity with asymptomatic neonatal infections in contrast to the earlier observations of association of such unusual strains with acute gastroenteritis in young children.

  View details for Web of Science ID A1992JL66600020

  View details for PubMedID 1326262

• ANTIBODIES TO HEPATITIS-C VIRUS IN LOW-RISK BLOOD-DONORS - IMPLICATIONS FOR COUNSELING POSITIVE DONORS GASTROENTEROLOGY Hsu, H. H., Gonzalez, M., Foung, S. K., Feinstone, S. M., Greenberg, H. B. 1991; 101 (6): 1724-1727

  Abstract

  The significance of antibodies to hepatitis C virus (HCV) found by screening enzyme-linked immunoassay testing in a low-risk blood donor population is unclear. The rate of false positivity in this group as well as the usefulness of supplemental testing were examined by correlating the results of two screening enzyme-linked immunoassays (Ortho Diagnostics, Raritan, NJ, and Abbott Laboratories, North Chicago, IL) with supplemental antibody testing by the recombinant immunoblot assays (1 and 2) (Ortho) and neutralization assay (Abbott). Polymerase chain reaction was used to detect HCV genomic RNA to confirm viremia. Among 11.243 volunteer donors who were screened for the presence of antibodies to HCV by enzyme-linked immunoassay, 60 (0.53%) sera were repeatedly reactive. Twenty-five of these 60 sera were available for further testing. Seven sera were reactive by both screening enzyme-linked immunoassays, as well as by both recombinant immunoblot and neutralization assays. Six of these seven sera had detectable HCV genomic RNA by polymerase chain reaction. Among the remaining 18 sera, none were reactive by either recombinant immunoblot assays, whereas two sera were reactive by the neutralization assay only. None of the 18 samples had detectable HCV genomic RNA. Five of the six sera with elevated aminotransferase levels were among the seven sera reactive by all immunoassays. It is concluded that there is a significant false positivity rate associated with screening enzyme-linked immunoassay testing in a low-risk blood donor population. Supplemental testing correlates well with detection of hepatitis C genomic material by polymerase chain reaction and identifies donors who are truly infected.

  View details for Web of Science ID A1991GT00700035

  View details for PubMedID 1720106

• FAILURE TO DETECT HEPATITIS-C VIRUS GENOME IN HUMAN SECRETIONS WITH THE POLYMERASE CHAIN-REACTION HEPATOLOGY Hsu, H. H., Wright, T. L., LUBA, D., Martin, M., Feinstone, S. M., Garcia, G., Greenberg, H. B. 1991; 14 (5): 763-767

  Abstract

  Although hepatitis C infection has been clearly demonstrated to be transmitted through blood products or blood contamination, most cases of sporadic hepatitis C infection are unassociated with parenteral risk factors, and it is unclear how infection might be acquired by nonparenteral means. One potential mode of nonparenteral transmission is through body secretions. We used a highly sensitive and specific polymerase chain reaction assay to determine whether hepatitis C viral genomic RNA could be detected in secretions obtained from nineteen individuals with chronic hepatitis C virus infection. Although hepatitis C genomic RNA was found in all 19 sera, hepatitis C virus RNA was not detected in any samples of saliva, semen, urine, stool or vaginal secretions from these patients. Viral titers in serum ranged from 10(2) to 10(7) polymerase chain reaction units/ml. The sensitivity of our polymerase chain reaction assay indicates that, if hepatitis C virus were in secretions, it would be present in amounts less than 1 to 4 polymerase chain reaction units/ml. This contrasts with hepatitis B virus infection, in which serum titers frequently are in excess of 10(9) copies of hepatitis B genomes/ml. Body secretions have been found to contain up to 10(6) copies of hepatitis B genomes/ml. Our findings support seroepidemiological studies indicating that nonparenteral transmission of hepatitis C through secretions is uncommon and probably much less efficient than hepatitis B virus infection.

  View details for Web of Science ID A1991GN09800003

  View details for PubMedID 1657752

• IMMUNIZATION WITH BACULOVIRUS-EXPRESSED RECOMBINANT ROTAVIRUS PROTEIN-VP1, PROTEIN-VP4, PROTEIN-VP6, AND PROTEIN-VP7 INDUCES CD8+ LYMPHOCYTES-T THAT MEDIATE CLEARANCE OF CHRONIC ROTAVIRUS INFECTION IN SCID MICE JOURNAL OF VIROLOGY Dharakul, T., Labbe, M., Cohen, J., Bellamy, A. R., Street, J. E., MACKOW, E. R., Fiore, L., Rott, L., Greenberg, H. B. 1991; 65 (11): 5928-5932

  Abstract

  Clearance of chronic murine rotavirus infection in SCID mice can be demonstrated by adoptive transfer of immune CD8+ T lymphocytes from histocompatible donor mice immunized with a murine homotypic rotavirus (T. Dharakul, L. Rott, and H.B. Greenberg, J. Virol 64:4375-4382, 1990). The present study focuses on the protein specificity and heterotypic nature of cell-mediated clearance of chronic murine rotavirus infection in SCID mice. Heterotypic cell-mediated clearance was demonstrated in SCID mice infected with EDIM (murine) rotavirus after adoptive transfer of CD8+ T lymphocytes from BALB/c mice that were immunized with a variety of heterologous (nonmurine) rotaviruses including Wa (human, serotype 1), SA11 and RRV (simian, serotype 3), and NCDV and RF (bovine, serotype 6). This finding indicates the serotypic independence of T-cell-mediated rotavirus clearance. To further identify the rotavirus proteins that are capable of generating CD8+ T cells that mediate virus clearance, donor mice were immunized with SF-9 cells infected with a baculovirus recombinant expressing one of the following rotavirus proteins: VP1, VP2, NS53 (from RF), VP4, VP7, NS35 (from RRV), VP6, and NS28 (from SA11). SCID mice stopped shedding rotavirus after receiving CD8+ T cells from mice immunized with VP1, VP4, VP6, and VP7 but not with VP2, NS53, NS35, NS28, or wild-type baculovirus. These results suggest that heterotypic cell-mediated clearance of rotavirus in SCID mice is mediated by three of the major rotavirus structural proteins and by a putative polymerase protein.

  View details for Web of Science ID A1991GL29600032

  View details for PubMedID 1656079

• IDENTIFICATION AND PARTIAL CHARACTERIZATION OF A RHESUS ROTAVIRUS BINDING GLYCOPROTEIN ON MURINE ENTEROCYTES VIROLOGY Bass, D. M., MACKOW, E. R., Greenberg, H. B. 1991; 183 (2): 602-610

  Abstract

  In order to assess the possibility that rotavirus binds to a specific cellular receptor on enterocytes, we have used a viral overlay protein blot assay to study viral binding to murine intestinal brush border membranes (BBM). Infectious double-shelled particles of rhesus rotavirus bound specifically to two approximately 300- and 330-kDa glycoproteins from BBM prepared from suckling mice. Significantly less rotavirus binding was observed when adult BBM were examined. Rats have never been shown to harbor natural group A rotavirus infection and correspondingly, rat BBM showed no rotavirus binding activity. In suckling mice, rotavirus was found to bind to villus tip membranes to a much greater extent than to crypt preparations. Rotavirus binding activity was abolished by treatment of membrane preparations with protease. Analysis by glycolytic digestion of BBM with N- and O-glyconases revealed evidence for both N- and O-linked glycosylation of the rotavirus binding protein. Also neuraminidase digestion showed that O-linked sialic acid residues were required for virus binding. Monoclonal antibodies which immunoprecipitate the 300-kDa viral binding glycoprotein react with the apical surface of suckling but not adult enterocytes by Western blot. Baculovirus-expressed vp4, the rotavirus outer capsid spike protein, bound to the 300- and 330-kDa proteins and competed with rotavirus particles for binding sites. The ability of rotavirus to bind via vp4 to large BBM glycoproteins correlates with in vivo rotavirus cell tropism and host range restriction. Specific host cell receptor expression may be important in rotavirus pathogenesis.

  View details for Web of Science ID A1991FW36100016

  View details for PubMedID 1649504

• SYMMETRICAL INFECTION OF ROTAVIRUS ON POLARIZED HUMAN INTESTINAL EPITHELIAL (CACO-2) CELLS JOURNAL OF VIROLOGY Svensson, L., Finlay, B. B., Bass, D., VONBONSDORFF, C. H., Greenberg, H. B. 1991; 65 (8): 4190-4197

  Abstract

  When rotavirus infects the mature villus tip cells of the small intestine, it encounters a highly polarized epithelium. In order to understand this virus-cell interaction more completely, we utilized a cell culture-adapted rhesus rotavirus (RRV) to infect human intestinal (Caco-2) and Madin-Darby canine kidney (MDCK-1) polarized epithelial cells grown on a permeable support. Filter-grown Caco-2 cells and MDCK-1 cells, producing a transepithelial resistance of 300 to 500 and greater than 1,000 omega . cm2, respectively, were infected from either the apical or basolateral domain with RRV or Semliki Forest virus. Whereas Semliki Forest virus infection only occurred when input virions had access to the basolateral domain of MDCK-1 or Caco-2 cells, RRV infected MDCK-1 and Caco-2 monolayers in a symmetric manner. The effect of rotavirus infection on monolayer permeability was analyzed by measuring the transepithelial electrical resistance. Rotavirus infection on filter-grown Caco-2 cells caused a transmembrane leak at 18 h postinfection, before the development of the cytopathic effect (CPE) and extensive virus release. Electrical resistance was completely abolished between 24 and 36 h postinfection. Although no CPE could be detected on RRV-infected MDCK cells, the infection caused a transmembrane leak that totally abolished the electrical resistance at 18 to 24 h postinfection. Cell viability and the CPE analysis together with immunohistochemistry and immunofluorescence data indicated that the abolishment of resistance across the monolayer was due not to an effect on the plasma membrane of the cells but to an effect on the paracellular pathway limited by tight junctions. Attachment and penetration of rotavirus onto Caco-2 cells caused no measurable transmembrane leak during the first hour of infection.

  View details for Web of Science ID A1991FY29700028

  View details for PubMedID 1649325

• Viral gastroenteritis. New England journal of medicine Blacklow, N. R., Greenberg, H. B. 1991; 325 (4): 252-264

  View details for PubMedID 1647494

• MEDICAL PROGRESS - VIRAL GASTROENTERITIS NEW ENGLAND JOURNAL OF MEDICINE Blacklow, N. R., Greenberg, H. B. 1991; 325 (4): 252-264

  View details for Web of Science ID A1991FX62700006

• ANTIBODIES TO THE TRYPSIN CLEAVAGE PEPTIDE VP8-STAR NEUTRALIZE ROTAVIRUS BY INHIBITING BINDING OF VIRIONS TO TARGET-CELLS IN CULTURE JOURNAL OF VIROLOGY Ruggeri, F. M., Greenberg, H. B. 1991; 65 (5): 2211-2219

  Abstract

  Two distinct patterns of neutralization were identified by comparing the neutralization curves of monoclonal antibodies (MAbs) directed at the two surface proteins, VP4 and VP7, of rhesus rotavirus. VP7-specific MAbs were able to neutralize virus efficiently, and slight increases in antibody concentration resulted in a sharp decline in infectivity. On the other hand, MAbs to VP4 proved much less efficient at neutralizing rhesus rotavirus, and the fraction of infectious virus decreased gradually throughout a wide range of antibody concentrations. MAbs directed at VP8*, the smaller trypsin cleavage fragment of VP4, were shown to efficiently prevent binding of radiolabeled virions to MA104 cell monolayers, to an extent and at concentrations comparable to those required for neutralization of infectivity. Conversely, MAbs recognizing VP7 or the larger VP4 trypsin cleavage product, VP5*, showed little or no inhibitory effect on virus binding to cells. All MAbs studied were able to neutralize rotavirus that was already bound to the surface of cells. The MAbs directed at VP8*, but not those recognizing VP5* or VP7, were shown to mediate release of radiolabeled virus from the surface of the cells. With MAbs directed at VP7, papain digestion of virus-bound antibody molecules led to an almost complete recovery of infectivity. Neutralization could be fully restored by incubation of virus-Fab complexes with anti-mouse immunoglobulin G antiserum. Neutralization with MAbs directed at VP8* proved insensitive to digestion with papain as well as to the addition of anti-immunoglobulin antibodies.

  View details for Web of Science ID A1991FG77700007

  View details for PubMedID 1850007

• THE ISOLATION AND CHARACTERIZATION OF A NORWALK VIRUS-SPECIFIC CDNA JOURNAL OF CLINICAL INVESTIGATION Matsui, S. M., Kim, J. P., Greenberg, H. B., Su, W. C., Sun, Q. M., Johnson, P. C., DuPont, H. L., OSHIRO, L. S., Reyes, G. R. 1991; 87 (4): 1456-1461

  Abstract

  Norwalk virus, an important cause of epidemic, acute, nonbacterial gastroenteritis in adults and children, has eluded adaptation to tissue culture, the development of an animal model, and molecular cloning. In this study, a portion of the Norwalk viral genome encoding an immunoreactive region was cloned from very small quantities of infected stool using sequence-independent single primer amplification. Six overlapping complementary DNA (cDNA) clones were isolated by immunologic screening. The expressed recombinant protein from a representative clone reacted with six of seven high titer. Norwalk-specific, postinfection sera but not with corresponding preinfection sera. Nucleic acid sequence for all clones defined a single open reading frame contiguous with the lambda gt11-expressed beta-galactosidase protein. Only oligonucleotide probes specific for the positive strand (defined by the open reading frame) hybridized to an RNaseA-sensitive, DNaseI-resistant nucleic acid sequence extracted from Norwalk-infected stool. Furthermore, RNA extracted from serial postinfection, but not preinfection, stools from three of five volunteers hybridized to a Norwalk virus cDNA probe. Clone-specific oligonucleotide probes hybridized with cesium chloride gradient fractions containing purified Norwalk virion. In conclusion, an antigenic, protein-coding region of the Norwalk virus genome has been identified. This epitope has potential utility in future sero- and molecular epidemiologic studies of Norwalk viral gastroenteritis.

  View details for Web of Science ID A1991FE72200044

  View details for PubMedID 2010555

• THE VP8 FRAGMENT OF VP4 IS THE RHESUS ROTAVIRUS HEMAGGLUTININ VIROLOGY Fiore, L., Greenberg, H. B., MACKOW, E. R. 1991; 181 (2): 553-563

  Abstract

  The amino-terminal trypsin cleavage fragment of VP4, called VP8, was expressed from a recombinant baculovirus in Sf-9 cells. The baculovirus-expressed VP8 protein is antigenically conserved as demonstrated by its recognition by a library of neutralizing monoclonal antibodies. In Sf-9 cell sonicates, the expressed VP8 protein is capable of agglutinating human type O erythrocytes, indicating that the functionally intact rhesus rotavirus viral hemagglutinin is contained in the 247-amino acid VP8 trypsin cleavage fragment. Amino acid similarities between VP8 and the amino-terminal 282 amino acids of the reovirus sigma 1 protein suggests that the sigma 1 hemagglutination function resides within these amino-terminal amino acids as well. When the expressed VP8 protein was used to immunize mice, a broadly cross-reactive neutralizing antibody response was obtained. Antibodies elicited to the expressed VP8 protein neutralized viruses of serotypes 1-4 and 6 but not porcine strains OSU (st5) or Gottfried (st4). The neutralizing antibody response to VP8 appeared to be more cross-reactive than the immune response to expressed VP4 or to whole RRV virion. This suggests that subunit protein immunizations may broaden the neutralizing antibody immune responses to rotaviruses and enhance protective immunity to serotypically distinct strains.

  View details for Web of Science ID A1991FB49600014

  View details for PubMedID 1849677

• REACTIVITIES OF SEROTYPING MONOCLONAL-ANTIBODIES WITH CULTURE-ADAPTED HUMAN ROTAVIRUSES JOURNAL OF CLINICAL MICROBIOLOGY Ward, R. L., McNeal, M. M., Clemens, J. D., Sack, D. A., Rao, M., Huda, N., GREEN, K. Y., KAPIKIAN, A. Z., Coulson, B. S., BISHOP, R. F., Greenberg, H. B., Gerna, G., Schiff, G. M. 1991; 29 (3): 449-456

  Abstract

  Rotaviruses collected in Bangladesh during 1985 to 1986 were culture adapted and used in a comparative serotyping study with three groups of monoclonal antibodies, all of which reacted with the major neutralization protein (VP7) of serotype 1, 2, 3, or 4. The goals were to determine which monoclonal antibodies most accurately predicted the serotype and why large variations in serotyping efficiencies have occurred with these monoclonal antibodies in previous studies. The 143 rotavirus isolates used in this study belonged to 69 different electropherotypes; and 44, 23, 21, and 55 isolates were identified as serotype 1 through 4, respectively, by neutralization with serotype-specific hyperimmune antisera. Serotyping specificity by enzyme-linked immunosorbent assay with monoclonal antibodies was 100% consistent with results found by neutralization with polyclonal antisera, but large differences were observed in the sensitivities of the different monoclonal antibodies. Monoclonal antibodies 5E8 (serotype 1), 1C10 (serotype 2), 159 (serotype 3), RV3:1 (serotype 3), ST-3:1 (serotype 4), and ST-2G7 (serotype 4) reacted with all the isolates of the corresponding serotype for which there were sufficient infectious particles. Monoclonal antibody 2F1 (serotype 2) was much less sensitive and reacted with only five serotype 2 isolates, but these were among those with the highest titers. Monoclonal antibodies RV4:2 (serotype 1), KU6BG (serotype 1), RV5:3 (serotype 2), and S2-2G10 (serotype 2), on the other hand, failed to react with between one and three isolates of the corresponding serotypes which had high titers, apparently because of epitope changes in these isolates. Effects of epitope variation were, however, most apparent with monoclonal antibodies 2C9 (serotype 1) and YO-1E2 (serotype 3), which reacted with one and no isolates of the corresponding serotypes, respectively. Cross-neutralization of escape mutants indicated that the serotype 1 monoclonal antibodies 5E8, 2C9, and RV4:2 reacted with different but probably overlapping epitopes, as did serotype 2 monoclonal antibodies 2F1, 1C10, and RV5:3, finding that were consistent with the enzyme-linked immunosorbent assay data. Because of epitope variations between rotavirus strains, serotyping with several monoclonal antibodies directed at different epitopes may increase the sensitivity of the method.

  View details for Web of Science ID A1991EY46300008

  View details for PubMedID 1709945

• NORWALK VIRUS - ISOLATION AND CHARACTERIZATION OF A VIRUS-SPECIFIC CDNA Matsui, S. M., Kim, J. P., Greenberg, H. B., Johnson, P. C., DuPont, H. L., Reyes, G. R. SLACK INC. 1991: A27–A27

  View details for Web of Science ID A1991ET78600147

• EFFECT OF MALNUTRITION OF EXTRAINTESTINAL SPREAD OF ROTAVIRUS AND DEVELOPMENT OF HEPATITIS IN MICE NUTRITION RESEARCH Uhnoo, I., RIEPENHOFFTALTY, M., CHEGAS, P., Fisher, J. E., Greenberg, H. B., Ogra, P. L. 1990; 10 (12): 1419-1429

  View details for Web of Science ID A1990EP79200008

• RECOVERY FROM CHRONIC ROTAVIRUS INFECTION IN MICE WITH SEVERE COMBINED IMMUNODEFICIENCY - VIRUS CLEARANCE MEDIATED BY ADOPTIVE TRANSFER OF IMMUNE CD8+ LYMPHOCYTES-T JOURNAL OF VIROLOGY Dharakul, T., Rott, L., Greenberg, H. B. 1990; 64 (9): 4375-4382

  Abstract

  Severe combined immunodeficient (SCID) mice lack both functional T and B cells. These mice develop chronic rotavirus infection following an oral inoculation with the epizootic diarrhea of infant mice (EDIM) rotavirus. Reconstitution of rotavirus-infected SCID mice with T lymphocytes from immunocompetent mice allows an evaluation of a role of T-cell-mediated immunity in clearing chronic rotavirus infection. Complete rotavirus clearance was demonstrated in C.B-17/scid mice 7 to 9 days after the transfer of immune CD8+ splenic T lymphocytes from histocompatible BALB/c mice previously immunized intraperitoneally with the EDIM-w strain of murine rotavirus. The virus clearance mediated by T-cell transfer was restricted to H-2d-bearing T cells and occurred in the absence of rotavirus-specific antibody as determined by enzyme-linked immunosorbent assay, neutralization, immunohistochemistry, and radioimmunoprecipitation. Temporary clearance of rotavirus was observed after the transfer of immune CD8+ T cells isolated from the intestinal mucosa (intraepithelial lymphocytes [IELs]) or the spleens of BALB/c mice previously infected with EDIM by the oral route. Chronic virus shedding was transiently eliminated 7 to 11 days after spleen cell transfer and 11 to 12 days after IEL transfer. However, recurrence of rotavirus infection was detected 1 to 8 days later in all but one SCID recipient receiving cells from orally immunized donors. The viral clearance was mediated by IELs that were both Thy1+ and CD8+. These data demonstrated that the clearance of chronic rotavirus infection in SCID mice can be mediated by immune CD8+ T lymphocytes and that this clearance can occur in the absence of virus-specific antibodies.

  View details for Web of Science ID A1990DU75300041

  View details for PubMedID 1974652

• SEROTYPE VARIATION OF HUMAN GROUP-A ROTAVIRUSES IN 2 REGIONS OF THE USA JOURNAL OF INFECTIOUS DISEASES Matson, D. O., Estes, M. K., Burns, J. W., Greenberg, H. B., Taniguchi, K., Urasawa, S. 1990; 162 (3): 605-614

  Abstract

  The first longitudinal study of group A rotavirus serotype distribution in the USA is reported. ELISAs incorporating neutralizing monoclonal antibodies specific for the VP7 protein of serotypes 1, 2, 3, and 4 were used to determine the antigenic variation of group A rotaviruses in two collections of stool specimens. Stool samples were collected from children hospitalized during 1979-1989 at Texas Children's Hospital, Houston, and from children from a more rural population hospitalized during 1981-1989 in the north-central USA. The predominant serotype varied from year to year in Houston, with serotypes 1, 3, and 4 each predominant in 1 or more years. In the north-central population only serotype 1 was predominant each year. Within a single rotavirus season in the Houston area, serotypes were not equally distributed by week of the season or county of residence. These differences in the distribution of serotypes have broad implications for the design and interpretation of vaccine programs.

  View details for Web of Science ID A1990DV90300005

  View details for PubMedID 2167332

• EPITOPE-SPECIFIC ANTIBODY-RESPONSE TO THE SURFACE-ANTIGEN OF DUCK HEPATITIS-B VIRUS IN INFECTED DUCKS VIROLOGY Cheung, R. C., TRUJILLO, D. E., Robinson, W. S., Greenberg, H. B., Marion, P. L. 1990; 176 (2): 546-552

  Abstract

  In order to investigate the immune response to duck hepatitis B virus (DHBV) infection, newly hatched DHBV DNA negative ducklings were injected with infectious serum of sufficiently low DHBV-DNA titer to allow clearance of viremia. Of 20 injected ducklings, 13 (65%) became viremic. Of these, 6 (46%) cleared virus from the serum 3 to 22 weeks postinjection. The convalescent sera of these 6 animals were tested for an epitope-specific antibody response in a highly specific competitive inhibition assay using a panel of monoclonal antibodies against duck hepatitis B surface antigen (DHBsAg) that had been well-characterized. All 6 animals recovering from DHBV infection developed antibodies to epitopes on the preS and S proteins of DHBV. Antibody responses were highly variable with marked differences between animals in the extent and specificity of the antibody response. The humoral response to DHBsAg was prolonged in some animals but transient in others. No antibody to preS or S was detected in either preimmune sera or sera of control animals from an uninfected flock. Infected animals that did not clear viremia also remained antibody negative. The humoral responses to neutralizing preS epitopes III and V were weak but antibodies to two immunodominant epitopes on the preS region (II and B) were present in all 6 animals. The humoral response to the two epitopes in the S region was transient and of lower titer when compared to the two immunodominant preS epitopes. The two immunodominant preS epitopes may play an important role in clearance of DHBV infection in ducks.

  View details for Web of Science ID A1990DF12300025

  View details for PubMedID 1693247

• 3-DIMENSIONAL STRUCTURE OF RHESUS ROTAVIRUS BY CRYOELECTRON MICROSCOPY AND IMAGE-RECONSTRUCTION JOURNAL OF CELL BIOLOGY Yeager, M., Dryden, K. A., Olson, N. H., Greenberg, H. B., BAKER, T. S. 1990; 110 (6): 2133-2144

  Abstract

  The structure of rhesus rotavirus was examined by cryoelectron microscopy and image analysis. Three-dimensional reconstructions of infectious virions were computed at 26- and 37-A resolution from electron micrographs recorded at two different levels of defocus. The major features revealed by the reconstructions are (a) both outer and inner capsids are constructed with T = 13l icosahedral lattice symmetry; (b) 60 spikelike projections, attributed to VP4, extend at least 100 A from the outer capsid surface; (c) the outer capsid, attributed primarily to VP7, has a smoothly rippled surface at a mean radius of 377 A and is perforated by 132 aqueous holes ranging from 40-65 A in diameter; (d) the inner capsid has a "bristled" outer surface composed of 260 trimeric-shaped columns of density, attributed to VP6, which merge with a smooth, spherical shell of density at a lower, mean radius of 299 A, and which is perforated by holes in register with those in the outer capsid; (e) a "core" region contains a third, nonspherical shell of density at a mean radius of 225 A that encapsidates the double-stranded RNA genome; and (f) the space between the outer and inner capsids forms an open aqueous network that may provide pathways for the diffusion of ions and small regulatory molecules as well as the extrusion of RNA. The assignment of different viral structural proteins to specific features of the reconstruction has been tentatively made on the basis of excluded volume estimates and previous biochemical characterizations of rotavirus.

  View details for Web of Science ID A1990DH01900027

  View details for PubMedID 2161857

• DIVERSITY OF ROTAVIRUS SEROTYPES IN MEXICAN INFANTS WITH GASTROENTERITIS JOURNAL OF CLINICAL MICROBIOLOGY PADILLANORIEGA, L., Arias, C. F., Lopez, S., PUERTO, F., Snodgrass, D. R., Taniguchi, K., Greenberg, H. B. 1990; 28 (6): 1114-1119

  Abstract

  One hundred thirty-two stool specimens from infants with rotavirus gastroenteritis hospitalized in two Mexican cities (Mexico City and Mérida) were examined by serotype- and subgroup-specific enzyme immunoassays. Among them, 38 (29%) were serotype 1, 15 (11%) were serotype 2, 13 (10%) were serotype 3, 22 (17%) were serotype 4, none was serotype 5 or 6, and 44 (33%) could not be serotyped. By subgrouping, 121 specimens were characterized as follows: 24 (18%) were subgroup 1, 97 (74%) were subgroup 2, and none had both subgroup specificities. While serotype 1 rotavirus predominated in the Mexico City area for 4 consecutive years (1984 to 1987), serotype 4 predominated in Mérida during the single epidemic season studied (1985). These data demonstrate that all four primary human rotavirus serotypes circulated in Mexico, with serotype 1 being the most prevalent. The seroneutralization responses of 14 of the 22 patients infected with serotype 4 strains had been previously studied. Of these 14 infants, 11 appeared to have primary infections, as indicated by absence of neutralizing antibodies in the acute-phase sera and their young age (8 months on average) at the time of illness. Seven patients seroresponded to serotypes 1 and 4; two seroresponded to serotypes 1, 3, and 4; three seroresponded to serotype 1; and two had low-level seroresponses to serotype 3 or 4. These data indicate that heterotypic neutralizing antibody responses occur frequently following infection with serotype 4 rotaviruses.

  View details for Web of Science ID A1990DE15200007

  View details for PubMedID 2166073

• SUBGROUP CLASSIFICATION OF PORCINE GROUP-A ROTAVIRUSES, USING MONOCLONAL-ANTIBODIES IN AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY AMERICAN JOURNAL OF VETERINARY RESEARCH KASSUBA, A., SAIF, L. J., Greenberg, H. B. 1990; 51 (6): 938-944

  Abstract

  Fifty-six samples of feces and intestinal contents from nonvaccinated diarrheal pigs with rotavirus infections were tested, using a subgroup (SGP)-specific ELISA, to determine rotavirus SGP classification. Forty-one percent (23/56) were SGP 1, 25% (14/56) were SGP 2, and 34% (19/56) were not classifiable. For classifiable samples, the geographic distribution for SGP 1 and SGP 2, respectively was: 60%/40% from Ohio (n = 15), 63%/37% from other midwestern states (Iowa, Minnesota, Nebraska, South Dakota; n = 16), and 67%/33% from Canada (n = 6). Thirty-seven SGP-classifiable samples were categorized according to age of pigs. Of pigs less than or equal to 1 week old, 22% of samples were SGP 1 (n = 8), and 14% (n = 5) were SGP 2. Of samples from 1- to 2-week-old pigs, 8% were SGP 1 (n = 3), and 5% were SGP 2 (n = 2). Of samples from 2- to 3-week-old pigs, 5% were SGP 1 (n = 2), and 8% were SGP 2 (n = 3). Of samples from 3- to 4-week-old pigs, 5% were SGP 1 (n = 2), and 3% were SGP 2 (n = 1). Of samples from pigs greater than 4 weeks old, 22% were SGP 1 (n = 8) and 8% were SGP 2 (n = 3). Double-stranded RNA extracted from positive controls and from 10 selected field samples (5 from SGP 1 and 5 from SGP 2) was electrophoresed in polyacrylamide gels to detect correlation between subgroup classification by ELISA and long or short double-stranded RNA electrophoretic-migration patterns. All SGP-1 and -2 rotavirus samples tested had typical long double-stranded RNA electrophoretic-migration patterns.

  View details for Web of Science ID A1990DG35100019

  View details for PubMedID 1695069

• SERUM-NEUTRALIZING ANTIBODY TO VP4 AND VP7 PROTEINS IN INFANTS FOLLOWING VACCINATION WITH WC3 BOVINE ROTAVIRUS JOURNAL OF VIROLOGY Ward, R. L., KNOWLTON, D. R., Greenberg, H. B., Schiff, G. M., BERNSTEIN, D. I. 1990; 64 (6): 2687-2691

  Abstract

  Serum specimens from infants 2 to 12 months old vaccinated with the WC3 bovine rotavirus were analyzed to determine the relative concentrations of neutralizing antibody to the VP4 and VP7 proteins of the vaccine virus. To do this, reassortant rotaviruses that contained the WC3 genome segment for only one of these two neutralization proteins were made. The segment for the other neutralization protein in these reassortants was from heterotypic rotaviruses that were serotypically distinct from WC3. Sera were examined from 31 infants who had no evidence of a previous rotavirus infection and the highest postvaccination WC3-neutralizing antibody titers (i.e., 160 to 600) of the 103 subjects administered the vaccine. A reassortant (3/17) that contained both neutralization proteins from the heterotypic rotaviruses, i.e., EDIM (EW strain of mouse rotavirus) VP7 and rhesus rotavirus VP4, was not neutralized by these sera (geometric mean titer [GMT], less than 20). A reassortant (E19) that contained EDIM VP7 and WC3 VP4 was also very poorly neutralized by these antisera (GMT = 20). In contrast, antibody titers to a reassortant (R20) that contained WC3 VP7 and rhesus rotavirus VP4 were higher than those against WC3 (GMTs of 458 and 313, respectively). Thus, VP7 appeared to be the dominant immunogen for production of neutralizing antibody after intestinal infection of previously uninfected infants vaccinated with WC3 bovine rotavirus.

  View details for Web of Science ID A1990DD73500028

  View details for PubMedID 2159538

• IMMUNIZATION WITH BACULOVIRUS-EXPRESSED VP4 PROTEIN PASSIVELY PROTECTS AGAINST SIMIAN AND MURINE ROTAVIRUS CHALLENGE JOURNAL OF VIROLOGY MACKOW, E. R., Vo, P. T., Broome, R., Bass, D., Greenberg, H. B. 1990; 64 (4): 1698-1703

  Abstract

  A baculovirus-expressed VP4 protein derived from the simian rhesus rotavirus (RRV) was used to parenterally immunize murine dams. VP4-immunized dams developed high levels of neutralizing antibodies against RRV and low levels of cross-reactive neutralizing antibodies against human strains Wa, ST3, and S2 and animal strains SA-11, NCDV, and Eb. Newborn mice suckled on VP4-immunized dams were protected against a virulent challenge dose of the simian strain RRV and against murine rotavirus Eb. The cross-reactive nature of the serum-neutralizing response generated by VP4 immunization and the protective efficacy of the immunization suggest that recombinant-expressed VP4 proteins should be considered as viable vaccine candidates.

  View details for Web of Science ID A1990CT84500034

  View details for PubMedID 2157052

• FOODBORNE SNOW MOUNTAIN AGENT GASTROENTERITIS WITH SECONDARY PERSON-TO-PERSON SPREAD IN A RETIREMENT COMMUNITY AMERICAN JOURNAL OF EPIDEMIOLOGY Gordon, S. M., OSHIRO, L. S., Jarvis, W. R., DONENFELD, D., Ho, M. S., Taylor, F., Greenberg, H. B., Glass, R., Madore, H. P., Dolin, R., TABLAN, O. 1990; 131 (4): 702-710

  Abstract

  A variety of small round-structured viruses are being recognized with increasing frequency as a cause of gastroenteritis in the community, but have rarely been reported to cause outbreaks in hospitals or extended-care facilities. From March 20 through April 15, 1988, an outbreak of gastroenteritis occurred in a retirement facility in the San Francisco Bay area. Illness was characterized by diarrhea, nausea, and vomiting; two residents died. Attack rates were 46% (155 of 336) in residents and 37% (28 of 75) in employees. During the initial outbreak period, illness among residents was associated with two shrimp meals served in the facility dining hall (odds ratio = 6.7). Person-to-person transmission probably occurred: The risk of becoming ill one or two days after a roommate became ill was significantly greater than that of becoming ill at other times during the outbreak (risk ratio = 6.5). Microbiologic examinations for bacterial and parasitic enteric pathogens were negative; however, 27-nm viral particles were detected by immune electron microscopy and by blocking enzyme immunoassay to Snow Mountain agent in stools obtained at the onset of illness from one of six ill residents. Seroconversion (greater than fourfold antibody rise) to Snow Mountain agent was detected in acute- and convalescent-phase serum specimens from five of six ill residents as measured by enzyme immunoassay, but not for Norwalk agent as measured by radioimmunoassay. This report of an outbreak of Snow Mountain agent gastroenteritis in an extended-care facility documents that these difficult-to-identify 27-nm viruses can cause outbreaks in inpatient settings.

  View details for Web of Science ID A1990CX76200013

  View details for PubMedID 2107737

• ROTAVIRUS SEROTYPE-6 AND SEROTYPE-10 PREDOMINATE IN CATTLE JOURNAL OF CLINICAL MICROBIOLOGY Snodgrass, D. R., Fitzgerald, T., Campbell, I., SCOTT, F. M., Browning, G. F., MILLER, D. L., Herring, A. J., Greenberg, H. B. 1990; 28 (3): 504-507

  Abstract

  Calf fecal rotavirus strains were serotyped in enzyme-linked immunosorbent assays, using monoclonal antibodies to the VP7s of serotypes 1, 2, 3, 5, and 6 and to the VP4 of B223 (designated serotype 10). Sixty-six percent of 162 samples were typed as serotype 6, and 7% were serotyped as serotype 10. Most of the untyped strains did not react with a monoclonal antibody directed to a common VP7 epitope, indicating insufficient virus present in the samples. However, seven untyped samples that did react with this antibody were adapted to culture and typed, and six of these also proved to belong to serotype 6 or 10. Two of these viruses belonged to a monotype within serotype 6 that did not react with the serotype 6 monoclonal antibody. The seventh isolate reacted in cross-neutralization tests with serotype 8 viruses. Bovine rotaviruses from the United Kingdom, Federal Republic of Germany, and Japan that had been shown previously to be distinct from serotype 6 were compared in neutralization tests with B223 from the United States. These viruses proved to be a closely reacting group distinct from all other rotavirus serotypes, justifying the establishment of serotype 10 as the second major type of bovine rotavirus.

  View details for Web of Science ID A1990CP03500022

  View details for PubMedID 2157736

  View details for PubMedCentralID PMC269652

• LACK OF COSEGREGATION OF THE SUBGROUP-II ANTIGENS ON GENES-2 AND GENES-6 IN PORCINE ROTAVIRUSES JOURNAL OF VIROLOGY Svensson, L., PADILLANORIEGA, L., Taniguchi, K., Greenberg, H. B. 1990; 64 (1): 411-413

  Abstract

  The rotavirus subgroup I and II specificities associated with gene 2 and 6 products (vp2 and vp6, respectively) were shown not to cosegregate in a number of porcine rotavirus strains. The porcine OSU rotavirus strain and OSU-vp7-like strains were all found to possess a subgroup II-specific region on vp2 and a subgroup I-specific region on vp6. Of interest is the observation that the subgroup II-specific epitope on vp2 appears to be present only in human and porcine rotavirus strains, suggesting a possible human-pig ancestral lineage for gene 2.

  View details for Web of Science ID A1990CE14700048

  View details for PubMedID 1688386

• EFFECT OF INTESTINAL MUCOSAL ENVIRONMENT ON THE HEPATOPATHOGENIC POTENTIAL OF ROTAVIRUS IN NORMAL AND IMMUNOCOMPROMISED HOSTS 5TH INTERNATIONAL CONGRESS OF MUCOSAL IMMUNOLOGY Uhnoo, I., RIEPENHOFFTALTY, M., CHEGAS, P., Greenberg, H. B., Ogra, P. L. KLUWER ACADEMIC PUBL. 1990: 847–848

  View details for Web of Science ID A1990BR27J00270

• EXTRAMUCOSAL SPREAD AND DEVELOPMENT OF HEPATITIS IN IMMUNODEFICIENT AND NORMAL MICE INFECTED WITH RHESUS ROTAVIRUS JOURNAL OF VIROLOGY Uhnoo, I., RIEPENHOFFTALTY, M., Dharakul, T., CHEGAS, P., Fisher, J. E., Greenberg, H. B., Ogra, P. L. 1990; 64 (1): 361-368

  Abstract

  The pathogenic profiles of two heterologous animal rotaviruses, rhesus rotavirus strain MMU 18006 and bovine rotavirus strain WC3, were evaluated in mice with severe combined immunodeficiency (SCID mice) and normal BALB/c mice. Control animals were inoculated with homologous murine strain EDIM 5099 or a tissue culture-adapted murine rotavirus. Heterologous infection with rhesus rotavirus resulted in hepatitis in 84% of SCID and 21% of BALB/c mice, with mortality rates of 27 and 0%, respectively. Surviving SCID animals developed chronic liver disease, while symptoms in BALB/c mice resolved in 2 to 4 weeks after onset. Histopathologic examination revealed a diffuse hepatitis with focal areas of parenchymal necrosis. Rotavirus was detected in liver tissue from 100% of 29 SCID and 85% (11 of 13) BALB/c animals tested by cell culture infectivity, immunofluorescence, or electron microscopy. No extramucosal spread of virus or hepatitis was observed after infection with heterologous bovine strain WC3 or homologous murine rotaviruses. This finding of a novel rotavirus-induced disease manifestation suggests altered tissue tropism in a heterologous host for a group of viruses previously shown to replicate exclusively in the gut mucosa. The implications of our observations suggest that in human vaccine trials utilizing heterologous rotavirus strains, special attention should be paid to children with immunodeficiency disorders, and screening for hepatic function should be included in vaccine protocols.

  View details for Web of Science ID A1990CE14700041

  View details for PubMedID 2152822

• MULTIPLE-CHALLENGE STUDY OF HOST SUSCEPTIBILITY TO NORWALK GASTROENTERITIS IN UNITED-STATES ADULTS JOURNAL OF INFECTIOUS DISEASES Johnson, P. C., MATHEWSON, J. J., DuPont, H. L., Greenberg, H. B. 1990; 161 (1): 18-21

  Abstract

  In a multiple-challenge study of US adult volunteers with low or high levels of serum antibody to Norwalk virus, Norwalk inoculum 8FIIa was administered to 42 subjects, 22 were challenged 6 months later, and 19 received a third challenge after 6 more months. All 12 with high (greater than or equal to 1:200) but only 19 of 30 with low (less than 1:100) prechallenge titers experienced illness or a fourfold increase in titer after the first challenge (P less than .025). Only 4 of those challenged twice became ill; all had low initial titers but 3 had high titers before the second challenge. None became ill after a third challenge. Nine (47%) had high titers immediately before the third challenge; 3 had low titers before and after each challenge and remained asymptomatic. Thus, preexisting serum antibody to Norwalk virus does not seem to be associated with protective immunity, but antibody levels become associated with protection after repetitive exposure. Short-term resistance lasts greater than or equal to 6 months after challenge, and a small percentage of resistant individuals maintain low antibody titers even after multiple challenges.

  View details for Web of Science ID A1990CH73900004

  View details for PubMedID 2153184

• DNA AMPLIFICATION-RESTRICTED TRANSCRIPTION TRANSLATION - RAPID ANALYSIS OF RHESUS ROTAVIRUS NEUTRALIZATION SITES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA MACKOW, E. R., YAMANAKA, M. Y., Dang, M. N., Greenberg, H. B. 1990; 87 (2): 518-522

  Abstract

  DNA amplification-restricted transcription-translation (DARTT), is based on DNA amplification by the polymerase chain reaction (PCR) and uses PCR to truncate protein-encoding DNA while adding transcriptional and translational initiation signals to the segment. The amplified DNA segments are transcribed into RNA and translated into protein in vitro and the synthesized proteins are used to define functional sites. DARTT was applied to rhesus rotavirus gene segment 4 cDNA in order to create a series of carboxyl-terminal truncations and new amino termini in the encoded VP4 capsid protein. The truncated VP4 polypeptides were tested for reaction with 11 VP4-specific neutralizing monoclonal antibodies to identify the minimum polypeptides required for antibody recognition. Monoclonal antibodies 2G4, M2, and M7, which neutralize a number of serologically distinct rotaviruses, required amino acids 247-474 of VP4 for binding. DARTT is potentially applicable to the identification of discontinuous epitopes and functional domains on a variety of proteins.

  View details for Web of Science ID A1990CK37800004

  View details for PubMedID 2153955

• SEQUENCE-ANALYSIS OF GENE-11 EQUIVALENTS FROM SHORT AND SUPER SHORT STRAINS OF ROTAVIRUS JOURNAL OF VIROLOGY Matsui, S. M., MACKOW, E. R., Matsuno, S., Paul, P. S., Greenberg, H. B. 1990; 64 (1): 120-124

  Abstract

  The molecular basis for the aberrant migration pattern of the gene 11 equivalent in rotaviruses with "short" (human DS-1) and "super short" (human 69M and bovine VMRI) electropherotypes was investigated. The mRNAs of these viruses were synthesized in vitro, and the entire gene 11 equivalent of each of these viruses was sequenced with specific synthetic oligonucleotide primers. These sequences were compared with previously published sequences of "long" pattern rotavirus gene 11 segments. The increased lengths of the gene 11 equivalents of DS-1, 69M, and VMRI are due to a prolonged, 3' untranslated region in this gene segment. The 3' untranslated region of the VMRI gene 11 equivalent contains a clear duplication of a portion of its coding sequence. A stretch of 18 consecutive nucleotides within the 330-nucleotide, 3' untranslated region of 69M is identical to a section of UK coding sequence. The DS-1 and the remainder of the 69M 3'-end additional sequences are similar to each other, but neither is similar to any other currently available rotavirus gene sequence. This finding suggests that a process other than homologous duplication is involved in the evolution of these sequences. The widespread occurrence of human and animal rotaviruses with short and super short electropherotypes provides evidence that intragenic and possibly intergenic recombinational events associated with an error-prone viral RNA polymerase may play a role in increasing the genetic repertoire of rotaviruses.

  View details for PubMedID 2152809

• NS35 AND NOT VP7 IS THE SOLUBLE ROTAVIRUS PROTEIN WHICH BINDS TO TARGET-CELLS JOURNAL OF VIROLOGY Bass, D. M., MACKOW, E. R., Greenberg, H. B. 1990; 64 (1): 322-330

  Abstract

  Recent studies using radiolabeled rotavirus lysates have demonstrated a 35-kilodalton viral protein that binds specifically to the surface of MA104 cells (N. Fukuhara, O. Yoshie, S. Kitakoa, and T. Konno, J. Virol. 62:2209-2218, 1988; M. Sabara, J. Gilchrist, G.R. Hudson, and L.A. Babiuk, J. Virol. 53:58-66, 1985). The binding protein was identified as vp7, an outer capsid glycoprotein and the product of rotavirus gene 9. These studies concluded that vp7 mediated viral attachment to MA104 cells and that the binding of a soluble viral protein to a cell monolayer mirrored the attachment of infectious rotavirus to permissive tissue culture cells. In the process of determining which viral protein adheres to the in vivo target cell in rotavirus infection, the mammalian enterocyte, we found that a similar 35-kilodalton rhesus rotavirus (RRV) protein bound to both MA104 cells and murine enterocytes. However, further analysis of this protein by immunoprecipitation, inhibition of glycosylation, and partial proteolysis showed that it was not the RRV gene 9 product, vp7, but the gene 8 product, NS35. Similar results were obtained by using porcine rotavirus (OSU) and bovine rotavirus (NCDV) strains. Binding studies using the in vitro-expressed products of RRV genes 8 and 9 confirmed these results. Since double-shelled virions inhibited the binding of NS35 to cells, we looked for the presence of this protein in preparations of purified virus. Examination of density gradient-purified virus preparations revealed biochemical and immunological evidence that NS35 copurifies in small amounts with double-shelled virions. Thus, these studies clearly demonstrated that when rotavirus proteins are prepared in a soluble form from infected cells, NS35, and not vp7, binds to the surfaces of MA104 cells and murine enterocytes. The observations do not confirm previous experimental results which supported the hypothesis that vp7 was the viral attachment protein. They are consistent with but do not prove the hypothesis that NS35 functions as the rotavirus attachment protein.

  View details for Web of Science ID A1990CE14700036

  View details for PubMedID 2152820

• PREVALENCE OF ACUTE ENTERIC VIRAL PATHOGENS IN ACQUIRED IMMUNODEFICIENCY SYNDROME PATIENTS WITH DIARRHEA GASTROENTEROLOGY KALJOT, K. T., LING, J. P., GOLD, J. W., LAUGHON, B. E., Bartlett, J. G., Kotler, D. P., OSHIRO, L. S., Greenberg, H. B. 1989; 97 (4): 1031-1032

  Abstract

  Diarrhea due to enteric pathogens is an important complication of advanced human immunodeficiency virus infection. Whereas numerous bacterial and parasitic agents have been implicated, the role of pathogenic enteric viruses is less clear. Stools from 153 human immunodeficiency virus seropositive men were tested by electrophoresis, enzyme-linked immunosorbent assay, and immune electron microscopy for the presence of rotaviruses (group A and non-group A), adenoviruses, and Norwalk agent. Virus was detected in 9% of the patients with acquired immunodeficiency syndrome, 3% of the patients with acquired immunodeficiency syndrome-related complex, and none of the seropositive men without these diagnoses. Virus detection was not more likely in stool from patients with diarrhea.

  View details for Web of Science ID A1989AQ28800034

  View details for PubMedID 2777028

• ROTAVIRUS-SPECIFIC PROTEIN-SYNTHESIS IS NOT NECESSARY FOR RECOGNITION OF INFECTED-CELLS BY VIRUS-SPECIFIC CYTO-TOXIC LYMPHOCYTES-T JOURNAL OF VIROLOGY Offit, P. A., Greenberg, H. B., DUDZIK, K. I. 1989; 63 (8): 3279-3283

  Abstract

  We found that rotavirus-specific protein synthesis was not necessary for recognition by virus-specific cytotoxic T lymphocytes (CTLs). In addition, CTLs lysed rotavirus-infected target cells prior to production of infectious virus. Target cell processing of rotavirus antigens for presentation to CTLs was enhanced by treatment of rotavirus with trypsin prior to infection; trypsin-induced cleavage of the viral hemagglutinin (vp4) has previously been found to facilitate rotavirus entry into target cells by direct penetration of virions through the plasma membrane. We conclude that sufficient quantities of exogenous viral proteins may be introduced into the cytoplasm for processing by target cells. The mechanism by which rotavirus proteins are processed for presentation to the target cell surface remains to be determined.

  View details for Web of Science ID A1989AF40600010

  View details for PubMedID 2545904

• ROTAVIRUS VP7 NEUTRALIZATION EPITOPES OF SEROTYPE-3 STRAINS VIROLOGY Nishikawa, K., Hoshino, Y., Taniguchi, K., GREEN, K. Y., Greenberg, H. B., KAPIKIAN, A. Z., CHANOCK, R. M., GORZIGLIA, M. 1989; 171 (2): 503-515

  Abstract

  Sequence analysis of the gene encoding the major neutralization glycoprotein (VP7) was performed on 27 human and animal rotavirus strains of serotype 3 in order to examine genetic variation within strains of identical serotype. Comparisons of the deduced amino acid sequences of the VP7s showed overall sequence identities of 85% or higher. A higher degree of overall VP7 sequence similarity was observed among strains from the same animal species when compared to strains from different animal species, suggesting that there are species-specific sequences in the VP7 protein. Alignment of the amino acid sequences demonstrated that amino acid sequence divergence among serotype 3 strains from different species was located primarily in previously established VP7 serotype-specific regions where genetic variation was identified among strains of different serotype. These regions were highly conserved among serotype 3 strains derived from the same species. The varying reactivities of three anti-VP7 monoclonal antibodies with the 27 strains was consistent with the occurrence of antigenic variation among serotype 3 strains. Moreover the reactivity of monoclonal antibodies correlated with the amino acid sequence found in two serotype-specific regions (VR5 and VR8). A computer-derived predicted phylogenetic tree suggests that rotavirus strains from different animal species belonging to serotype 3 are more closely related to each other than to rotavirus strains of different serotypes.

  View details for Web of Science ID A1989AH83100022

  View details for PubMedID 2474892

• EPITOPE MAPPING OF NEUTRALIZING MONOCLONAL-ANTIBODIES AGAINST DUCK HEPATITIS-B VIRUS JOURNAL OF VIROLOGY Cheung, R. C., Robinson, W. S., Marion, P. L., Greenberg, H. B. 1989; 63 (6): 2445-2451

  Abstract

  In this article we report the first topological mapping of neutralizing epitopes of a hepadnavirus. Duck hepatitis B virus is the only hepadnavirus that can replicate and spread from cell to cell in tissue culture. As a result, it is possible to study hepadnaviral neutralization in vitro with this system. To accomplish this goal, we produced a library of monoclonal antibodies against duck hepatitis B virus and identified 12 neutralizing monoclonal antibodies by using an in vitro neutralization assay. The characteristics of six of the neutralizing monoclonal antibodies were further studied by epitope mapping. From the results of competitive binding studies, three distinct neutralizing epitopes were identified on the pre-S polypeptides and one was identified on the S polypeptide. Our findings suggest that antibodies to both the pre-S and S gene products of duck hepatitis B virus can neutralize viral infection in vitro. The pre-S gene product is at least as important as the S gene product in eliciting neutralizing antibodies.

  View details for Web of Science ID A1989U609600005

  View details for PubMedID 2470915

• PASSIVE PROTECTION AGAINST ROTAVIRUS-INDUCED DIARRHEA BY MONOCLONAL-ANTIBODIES TO THE HETEROTYPIC NEUTRALIZATION DOMAIN OF VP7 AND THE VP8 FRAGMENT OF VP4 JOURNAL OF CLINICAL MICROBIOLOGY Matsui, S. M., Offit, P. A., Vo, P. T., MACKOW, E. R., Benfield, D. A., Shaw, R. D., PADILLANORIEGA, L., Greenberg, H. B. 1989; 27 (4): 780-782

  Abstract

  A murine model was used to determine whether neutralizing monoclonal antibodies (MAbs) with heterotypic specificity directed to VP7 (MAb 57-8) or to the VP8 fragment of VP4 (MAb M14) passively protect mice against challenge with various strains of rotavirus. (The gene 4 product, an outer capsid protein, has traditionally been called VP3. It has been proposed, however, that the rotavirus gene 4 product be named VP4. The gene 3 product, a core protein, has been identified recently and named VP3 [M. Liu, P. A. Offit, and M. K. Estes, Virology 163:28-32, 1988]). Suckling mice orally inoculated with MAb 57-8 did not develop diarrhea when challenged with virulent serotype 3, 4, or 6 rotaviruses, while those inoculated with MAb M14 were passively protected from challenge with serotype 3 or 6 rotaviruses, as predicted by in vitro neutralization tests. These MAbs, however, did not protect mice from infection when the mice were challenged with rotaviruses of other serotypes. We conclude that specific neutralization epitopes on each surface protein are capable of mediating protection against one or several rotavirus serotypes.

  View details for Web of Science ID A1989T812700040

  View details for PubMedID 2470774

• THE RHESUS ROTAVIRUS OUTER CAPSID PROTEIN VP4 FUNCTIONS AS A HEMAGGLUTININ AND IS ANTIGENICALLY CONSERVED WHEN EXPRESSED BY A BACULOVIRUS RECOMBINANT JOURNAL OF VIROLOGY MACKOW, E. R., Barnett, J. W., Chan, H., Greenberg, H. B. 1989; 63 (4): 1661-1668

  Abstract

  Rhesus rotavirus (RRV) gene 4 was cloned into lambda bacteriophage, inserted into a polyhedrin promoter shuttle plasmid, and expressed in Sf9 cells by a recombinant baculovirus. The baculovirus-expressed VP4 protein made up approximately 5% of the Spodoptera frugiperda-infected cell protein. Monoclonal antibodies that neutralize the virus bound to the expressed VP4 polypeptide, indicating that the expressed VP4 protein was antigenically indistinguishable from viral VP4. In addition, we have determined that the baculovirus-expressed VP4 protein bound to erythrocytes and functions as the RRV hemagglutinin. The endogenous hemagglutinating activity of the VP4 protein, like the virus, was inhibited by guinea pig antirotavirus hyperimmune serum and by VP4-specific neutralizing monoclonal antibodies. The human erythrocyte protein, glycophorin, also inhibited hemagglutination by RRV or the expressed VP4 protein and appears to be the rotavirus erythrocyte receptor. The baculovirus-expressed VP4 protein was conserved functionally and antigenically in the absence of other outer or inner capsid rotavirus components and represents a logical candidate for future immunological studies.

  View details for Web of Science ID A1989T678700019

  View details for PubMedID 2538649

• ROTAVIRUS SEROTYPE-IV - NUCLEOTIDE-SEQUENCE OF GENOMIC SEGMENT 9 OF THE ST-THOMAS-3 STRAIN NUCLEIC ACIDS RESEARCH Reddy, D. A., Greenberg, H. B., Bellamy, A. R. 1989; 17 (1): 449-449

  View details for Web of Science ID A1989R859800040

  View details for PubMedID 2536151

• MOLECULAR DETERMINANT OF ROTAVIRUS NEUTRALIZATION AND PROTECTION ADVANCES IN VIRUS RESEARCH Matsui, S. M., MACKOW, E. R., Greenberg, H. B. 1989; 36: 181-214

  View details for Web of Science ID A1989AH74400005

  View details for PubMedID 2472045

• SEQUENCE-ANALYSIS OF GENE-11 EQUIVALENTS FROM SHORT AND SUPER-SHORT STRAINS OF ROTAVIRUS Matsui, S. M., MACKOW, E. R., Matsuno, S., Paul, P. S., Greenberg, H. B. SLACK INC. 1989: A113–A113

  View details for Web of Science ID A1989R585100657

• REACTOGENICITY AND ANTIGENICITY OF RHESUS ROTAVIRUS VACCINE (MMU-18006) IN NEWBORN-INFANTS IN VENEZUELA PEDIATRIC INFECTIOUS DISEASE JOURNAL Flores, J., DAOUD, G., Daoud, N., Puig, M., Martinez, M., PEREZSCHAEL, I., Shaw, R., Greenberg, H. B., Midthun, K., KAPIKIAN, A. Z. 1988; 7 (11): 776-780

  Abstract

  The reactogenicity and antigenicity of the rhesus rotavirus vaccine, strain MMU18006, developed at the Laboratory of Infectious Diseases (National Institute of Allergy and Infectious Diseases, National Institutes of Health) were examined in a double blind, placebo-controlled study of 40 newborn infants in Caracas, Venezuela. The children were observed for the first few days after birth in the hospital nursery and by home visits for 10 days after vaccination to detect any adverse reactions. No reactions could be attributed to the vaccine. Serologic responses to the vaccine were evaluated in paired sera obtained at birth (cord blood) and 4 weeks after vaccination. Serologic responses to the vaccine were not observed by complement fixation, neutralization or a rhesus rotavirus VP7 epitope-specific competition assay. However, such responses were found in 9 of 14 tested infants by an immunoglobulin A-specific enzyme-linked immunosorbent assay. Seventeen of the 20 vaccinees also shed rhesus rotavirus vaccine in stool during the postvaccination period.

  View details for Web of Science ID A1988Q947100006

  View details for PubMedID 2852792

• PHENOTYPIC MIXING DURING COINFECTION OF CELLS WITH 2 STRAINS OF HUMAN ROTAVIRUS JOURNAL OF VIROLOGY Ward, R. L., KNOWLTON, D. R., Greenberg, H. B. 1988; 62 (11): 4358-4361

  Abstract

  Coinfection of MA-104 cells with serotype 1 (Wa strain) and serotype 3 (P strain) human rotaviruses resulted in progeny viruses that were phenotypically mixed in their major outer-shell structural-protein VP7 and in the genome segment that encodes this protein (segment 9). Segments from the Wa virus predominated in these progeny whether they were of parental or reassortant genotype. Neutralization with monoclonal antibodies specific for VP7 proteins of the coinfecting viruses caused an alteration in genomic distribution favoring the strain heterologous to the neutralizing monoclonal antibody. Because the percentage of change in distribution of segment 9 was similar to that of the other combined segments, there appeared to be no greater association between VP7 and segment 9 than with other segments of the homologous virus during encapsulation. From these results, it was calculated that the progeny of coinfection with P segment 9 were 77.4% mosaic structures and 14.8% pseudotypes; progeny of coinfection with Wa segment 9 were 40.2% mosaic structures and 1.3% pseudotypes. Similar determinations were made for the reassortant progeny alone.

  View details for Web of Science ID A1988Q489900057

  View details for PubMedID 2845148

• ISOLATION OF A BOVINE ROTAVIRUS WITH A SUPER-SHORT RNA ELECTROPHORETIC PATTERN FROM A CALF WITH DIARRHEA JOURNAL OF CLINICAL MICROBIOLOGY Paul, P. S., Lyoo, Y. S., WOODE, G. N., Zheng, S., Greenberg, H. B., Matsui, S., Schwartz, K. J., HILL, H. T. 1988; 26 (10): 2139-2143

  Abstract

  A rotavirus with a "super-short" RNA electropherotype was isolated from a calf with diarrhea and was designated VMRI strain. Segments 10 and 11 of this rotavirus migrated more slowly than did those of bovine rotavirus strains NCDV, B641, and B223. The electrophoretic pattern of the VMRI strain was similar to that reported for rotaviruses with super-short RNA electropherotypes from humans and rabbits. Northern (RNA) blot hybridization indicated that gene 11 of the VMRI strain was altered and migrated between gene segments 9 and 10. The subgroup of the VMRI strain was shown to be subgroup I. The VMRI strain of bovine rotavirus was neutralized by antisera containing polyclonal antibodies to rotavirus serotype 6 (bovine rotavirus serotype I) strains NCDV and B641 and by ascitic fluid containing monoclonal antibodies directed to VP7 of serotype 6 rotavirus. The VMRI strain was not neutralized by either polyclonal or monoclonal antibodies to strain B223 (bovine rotavirus serotype II). Collective data on the neutralization of the VMRI strain with monoclonal antibodies and polyclonal antibodies suggest that this virus is a member of the NCDV group (serotype 6) of rotaviruses (bovine rotavirus serotype I).

  View details for Web of Science ID A1988Q160000041

  View details for PubMedID 2846639

• SEROTYPIC ANALYSIS OF VP3 AND VP7 NEUTRALIZATION ESCAPE MUTANTS OF RHESUS ROTAVIRUS JOURNAL OF VIROLOGY Shaw, R. D., MACKOW, E. R., DYALLSMITH, M. L., LAZDINS, I., HOLMES, I. H., Greenberg, H. B. 1988; 62 (9): 3509-3512

  Abstract

  Neutralization escape mutants of simian rotaviruses (rhesus rotavirus and SA11) were tested in hemagglutination inhibition and neutralization assays against hyperimmune and infection sera to determine if mutation in an immunodominant epitope could enable neutralization escape. An SA11 mutant with a new glycosylation site at amino acid 211 of VP7 was shown to escape neutralization by hyperimmune but not infection sera.

  View details for Web of Science ID A1988P689000057

  View details for PubMedID 2457117

• 2 FORMS OF VP7 ARE INVOLVED IN ASSEMBLY OF SA11 ROTAVIRUS IN ENDOPLASMIC-RETICULUM JOURNAL OF VIROLOGY Kabcenell, A. K., Poruchynsky, M. S., Bellamy, A. R., Greenberg, H. B., Atkinson, P. H. 1988; 62 (8): 2929-2941

  Abstract

  Two pools of the glycoprotein VP7 were detected in the endoplasmic reticulum (ER) of SA11 rotavirus-infected cells. One portion of the newly synthesized protein with VP3 composed the virus outer capsid, while the rest remained associated with the membrane. The two populations could be separated biochemically by fluorocarbon extraction or by immunological methods which used two classes of antibodies. A monoclonal antibody with neutralizing activity recognized VP7 only as displayed on intact virus particles, while a polyclonal antiserum precipitated predominantly the unassembled ER form of the protein and precipitated virus-assembled VP7 poorly. Virus-associated VP7 was localized by immunofluorescence to small punctate structures, presumably corresponding to accumulated virus particles, and to regions of the ER surrounding viroplasmic inclusions, whereas the membrane-associated molecules were distributed in an arborizing reticular pattern throughout the ER. VP3 and the nonstructural glycoprotein NCVP5 displayed a localization similar to that of virus-associated VP7. Intracellular virus particles were isolated from infected cells to determine the kinetics of assembly of VP7 and of the other structural proteins into virions. It was found that incorporation of the inner capsid proteins into single-shelled particles occurred rapidly, while VP7 and VP3 appeared in mature double-shelled particles with a lag time of 10 to 15 min. In addition, the alpha-mannosidase processing kinetics of virus-associated VP7 oligosaccharides showed a 15-min lag compared with that of the membrane-associated form, suggesting that the latter is the precursor to virion VP7. This lag may represent the time required for virus budding and outer capsid assembly.

  View details for Web of Science ID A1988P319500051

  View details for PubMedID 2839712

• CHARACTERIZATION OF HOMOTYPIC AND HETEROTYPIC VP7 NEUTRALIZATION SITES OF RHESUS ROTAVIRUS VIROLOGY MACKOW, E. R., Shaw, R. D., Matsui, S. M., Vo, P. T., Benfield, D. A., Greenberg, H. B. 1988; 165 (2): 511-517

  Abstract

  The gene 9 nucleotide sequence was determined for rhesus rotavirus and each of 14 viral variants selected for their resistance to neutralizing monoclonal antibodies. Each variant contains a single gene 9, VP7, mutation which permits viral growth in the presence of the antibody. Variant mutations were identified in two distinct neutralization regions. Region A was identified by monoclonal antibodies that are involved in both serotype-specific and serotype cross-reactive neutralization. Region C was identified by serotype-specific neutralizing monoclonal antibodies. Heterotypic neutralizing monoclonal antibody 57-8 selected variants with a mutation at amino acid 94 in the A region, the same amino acid location selected by serotype-specific monoclonal antibodies. Monoclonal antibody 3 selected a VP7 mutation at amino acid 99 resulting in additional N-linked glycosylation of the VP7 protein. Despite the added VP7 glycosylation, variant v3 was not broadly resistant to additional VP7-specific neutralizing monoclonal antibodies.

  View details for Web of Science ID A1988P719300019

  View details for PubMedID 2457279

• DETECTION OF HUMAN ROTAVIRUSES WHICH DO NOT REACT WITH SUBGROUP-I-SPECIFIC AND SUBGROUP-II-SPECIFIC MONOCLONAL-ANTIBODIES JOURNAL OF CLINICAL MICROBIOLOGY Svensson, L., Grahnquist, L., PETTERSSON, C. A., GRANDIEN, M., STINTZING, G., Greenberg, H. B. 1988; 26 (6): 1238-1240

  Abstract

  Of 126 rotavirus-positive specimens, 7 could not be subgrouped (I or II). These strains showed a distinct reaction with a monoclonal antibody recognizing a common region on VP6, but they did not react with VP6 subgroup-specific monoclonal antibodies although they contained as much viral antigen as the subgrouped strains.

  View details for Web of Science ID A1988N520400040

  View details for PubMedID 2838518

• FUNCTIONAL AND TOPOGRAPHICAL ANALYSES OF EPITOPES ON THE HEMAGGLUTININ (VP4) OF THE SIMIAN ROTAVIRUS-SA11 JOURNAL OF VIROLOGY Burns, J. W., Greenberg, H. B., Shaw, R. D., Estes, M. K. 1988; 62 (6): 2164-2172

  Abstract

  An immunochemical analysis of the hemagglutinin (VP4) of the simian rotavirus SA11 was performed to better understand the structure and function of this molecule. Following immunization of mice with double-shelled virus particles and VP4-enriched fractions from CsCl gradients, a battery of anti-SA11 hybridomas was generated. A total of 13 clones secreting high levels of anti-VP4 monoclonal antibody (MAb) was characterized and compared with two cross-reactive anti-VP4 MAbs generated against heterologous rhesus (RRV) and porcine (OSU) rotavirus strains. These cross-reactive MAbs effectively neutralized SA11 infectivity in vitro. The epitopes recognized by these 15 MAbs were grouped into six antigenic sites on the SA11 hemagglutinin. These sites were identified following analysis of the MAbs by using a simple competitive binding enzyme-linked immunosorbent assay (ELISA) and biological assays. Three of the antigenic sites were involved in neutralization of virus infectivity in vitro. All the MAbs with neutralization activity and two nonneutralizing MAbs were able to inhibit viral hemagglutination of human erythrocytes. Competitive binding ELISA data showed a positive cooperative binding effect with some pairs of the anti-VP4 MAbs, apparently due to a conformational change induced by the binding of the first MAb. Some of the MAbs also bound better to trypsin-treated virus than to non-trypsin-treated virus. A topographic map for VP4 is proposed on the basis of the observed properties of each antigenic site.

  View details for Web of Science ID A1988N407000040

  View details for PubMedID 2452900

• RELATIVE CONCENTRATIONS OF SERUM NEUTRALIZING ANTIBODY TO VP3 AND VP7 PROTEINS IN ADULTS INFECTED WITH A HUMAN ROTAVIRUS JOURNAL OF VIROLOGY Ward, R. L., KNOWLTON, D. R., Schiff, G. M., Hoshino, Y., Greenberg, H. B. 1988; 62 (5): 1543-1549

  Abstract

  Two outer capsid rotavirus proteins, VP3 and VP7, have been found to elicit neutralizing-antibody production, but the immunogenicity of these proteins during human rotavirus infection has not been determined. The relative amounts of serum neutralizing antibody against the VP3 and VP7 proteins of the CJN strain of human rotavirus were, therefore, determined in adult subjects before and after infection with this virus. Reassortant strains of rotavirus that contained the CJN gene segment for only one of these two neutralization proteins were isolated and used for this study. The geometric mean titer of serum neutralizing antibody to a reassortant virus (CJN-M) that contained VP7 of CJN and VP3 of another human rotavirus was 12.7 times less than that of antibody to CJN before infection and 20.3 times less after infection. This indicated that most neutralizing antibody was against the VP3 rather than the VP7 protein of CJN. This result was confirmed with other reassortants between CJN and animal rotavirus strains (EDIM and rhesus rotavirus). These findings suggest that VP3 is the primary immunogen that stimulates neutralizing antibody during at least some rotavirus infections of humans.

  View details for Web of Science ID A1988N027700010

  View details for PubMedID 3357205

• CONSERVATION AND HOMOLOGY WITH A FUSION PROTEIN-SEQUENCE OF THE MAJOR HETEROTYPIC NEUTRALIZATION SITE ON ROTAVIRUS SURFACE PROTEIN VP3 Matsui, S. M., MACKOW, E. R., Greenberg, H. B. W B SAUNDERS CO-ELSEVIER INC. 1988: A290–A290

  View details for Web of Science ID A1988M939401152

• INFECTIOUS ROTAVIRUS ENTERS CELLS BY DIRECT CELL-MEMBRANE PENETRATION, NOT BY ENDOCYTOSIS JOURNAL OF VIROLOGY KALJOT, K. T., Shaw, R. D., Rubin, D. H., Greenberg, H. B. 1988; 62 (4): 1136-1144

  Abstract

  Rotaviruses are icosahedral viruses with a segmented, double-stranded RNA genome. They are the major cause of severe infantile infectious diarrhea. Rotavirus growth in tissue culture is markedly enhanced by pretreatment of virus with trypsin. Trypsin activation is associated with cleavage of the viral hemagglutinin (viral protein 3 [VP3]; 88 kilodaltons) into two fragments (60 and 28 kilodaltons). The mechanism by which proteolytic cleavage leads to enhanced growth is unknown. Cleavage of VP3 does not alter viral binding to cell monolayers. In previous electron microscopic studies of infected cell cultures, it has been demonstrated that rotavirus particles enter cells by both endocytosis and direct cell membrane penetration. To determine whether trypsin treatment affected rotavirus internalization, we studied the kinetics of entry of infectious rhesus rotavirus (RRV) into MA104 cells. Trypsin-activated RRV was internalized with a half-time of 3 to 5 min, while nonactivated virus disappeared from the cell surface with a half-time of 30 to 50 min. In contrast to trypsin-activated RRV, loss of nonactivated RRV from the cell surface did not result in the appearance of infection, as measured by plaque formation. Endocytosis inhibitors (sodium azide, dinitrophenol) and lysosomotropic agents (ammonium chloride, chloroquine) had a limited effect on the entry of infectious virus into cells. Purified trypsin-activated RRV added to cell monolayers at pH 7.4 medicated 51Cr, [14C]choline, and [3H]inositol released from prelabeled MA104 cells. This release could be specifically blocked by neutralizing antibodies to VP3. These results suggest that MA104 cell infection follows the rapid entry of trypsin-activated RRV by direct cell membrane penetration. Cell membrane penetration of infectious RRV is initiated by trypsin cleavage of VP3. Neutralizing antibodies can inhibit this direct membrane penetration.

  View details for Web of Science ID A1988M444000007

  View details for PubMedID 2831376

• THE RHESUS ROTAVIRUS GENE ENCODING PROTEIN VP3 - LOCATION OF AMINO-ACIDS INVOLVED IN HOMOLOGOUS AND HETEROLOGOUS ROTAVIRUS NEUTRALIZATION AND IDENTIFICATION OF A PUTATIVE FUSION REGION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA MACKOW, E. R., Shaw, R. D., Matsui, S. M., Vo, P. T., Dang, M. N., Greenberg, H. B. 1988; 85 (3): 645-649

  Abstract

  The complete gene 4 nucleotide sequence was determined for rhesus rotavirus and each of 11 viral variants selected by neutralizing monoclonal antibodies. Gene 4 is 2362 bases in length and encodes a protein, VP3, of 776 amino acids with a calculated Mr of 86,500. A conserved trypsin cleavage site, located at amino acid 247, divides VP3 into VP8 and VP5. Neutralizing monoclonal antibodies directed at VP3 were used to select variants that escaped neutralization. Each variant contains a single gene 4 mutation that permits viral growth in the presence of the antibody. Variant mutations were identified in six distinct neutralization regions in VP8 and VP5. Five of the six neutralization regions were found in VP8. The VP8 regions were primarily associated with strain-specific or limited heterotypic rotavirus neutralization. One region was identified in VP5 by three monoclonal antibodies that neutralize a broad range of rotavirus serotypes. The VP5 neutralization region is largely hydrophobic and is similar to putative fusion sequences of Sindbis and Semliki Forest viruses.

  View details for Web of Science ID A1988L992200002

  View details for PubMedID 2829198

• LOCATION AND CONSERVATION OF THE MAJOR OF HETEROTYPIC NEUTRALIZATION SITE IN RHESUS ROTAVIRUS VP3 Matsui, S. M., MACKOW, E. R., Shaw, R. D., Vo, P. T., Dang, M. N., Greenberg, H. B. SLACK INC. 1988: A133–A133

  View details for Web of Science ID A1988L516800777

• EPITOPE-SPECIFIC IMMUNE-RESPONSES TO ROTAVIRUS VACCINATION GASTROENTEROLOGY Shaw, R. D., Fong, K. J., Losonsky, G. A., LEVINE, M. M., Maldonado, Y., Yolken, R., Flores, J., KAPIKIAN, A. Z., Vo, P. T., Greenberg, H. B. 1987; 93 (5): 941-950

  Abstract

  Rotavirus gastroenteritis is a leading cause of infant mortality in developing countries and an important cause of morbidity in children under 2 yr of age in the United States. Vaccine programs have evaluated animal rotavirus strains that are attenuated in humans but antigenically similar to some human strains. Whether a single vaccine strain can elicit protective immunity in humans to rotaviruses of the same or different serotypes is an important question in determining vaccine efficacy. We used characterized serotype-specific monoclonal antibodies directed at VP7 in a competitive solid-phase immunoassay to measure epitope-specific immune responses to serotypes 1, 2, and 3 in sera of children who received a candidate serotype-3 rotavirus vaccine. Antibodies to serotype 3 were detected in 72% of sera samples, and to serotype 1 and 2 in only 11% each. Also, a VP3-specific monoclonal antibody which neutralizes three serotypically distinct strains of rotavirus was used to detect the presence of similar antibodies in 56% of the test sera. This finding suggests a mechanism of heterotypic immunity.

  View details for Web of Science ID A1987K557000004

  View details for PubMedID 2443417

• DIRECT SEROTYPING OF HUMAN ROTAVIRUS IN STOOLS BY AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY USING SEROTYPE 1-SPECIFIC, 2-SPECIFIC, 3-SPECIFIC, AND 4-SPECIFIC MONOCLONAL-ANTIBODIES TO VP7 JOURNAL OF INFECTIOUS DISEASES Taniguchi, K., Urasawa, T., Morita, Y., Greenberg, H. B., Urasawa, S. 1987; 155 (6): 1159-1166

  Abstract

  Four serotypes of human rotaviruses that can be differentiated by neutralization tests have been described so far. We prepared serotype 1-, 2-, 3-, and 4-specific, neutralizing monoclonal antibodies to human rotaviruses. All were directed to VP7, a glycoprotein that carries a major serotype specificity. An enzyme-linked immunosorbent assay using these monoclonal antibodies has been developed for serotyping human rotavirus isolates. The sensitivity and specificity of this method were verified by using various strains of human rotavirus that were adapted to cell culture. Furthermore, it was shown that the method was applicable to serotyping human rotaviruses directly in stool specimens.

  View details for Web of Science ID A1987H341300010

  View details for PubMedID 3033090

• CELLULAR DETERMINANTS OF ROTAVIRUS TISSUE TROPISM KALJOT, K. T., Greenberg, H. B. W B SAUNDERS CO-ELSEVIER INC. 1987: 1458–58

  View details for Web of Science ID A1987G966000683

• CELLULAR DETERMINANTS OF ROTAVIRUS TISSUE TROPISM KALJOT, K. T., Greenberg, H. B. SLACK INC. 1987: A409–A409

  View details for Web of Science ID A1987G986200933

• CELLULAR DETERMINANTS OF ROTAVIRUS TISSUE TROPISM KALJOT, K. T., Greenberg, H. B. SLACK INC. 1987: A590–A590

  View details for Web of Science ID A1987G986202012

• CRITERIA FOR NON-GROUP-A ROTAVIRUSES CIBA FOUNDATION SYMPOSIA BISHOP, R. F., McCrae, M. A., Cubitt, W. D., Greenberg, H. B., KAPIKIAN, A. Z. 1987; 128: 250-251

  View details for Web of Science ID A1987K911800015

• DETERMINANTS OF ROTAVIRUS TISSUE TROPISM KALJOT, K. T., Greenberg, H. B. SLACK INC. 1987: A184–A184

  View details for Web of Science ID A1987F528501061

• ANTIGENIC MAPPING OF THE SURFACE-PROTEINS OF RHESUS ROTAVIRUS VIROLOGY Shaw, R. D., Vo, P. T., Offit, P. A., Coulson, B. S., Greenberg, H. B. 1986; 155 (2): 434-451

  Abstract

  Monoclonal antibodies have been produced and used to map the functional topography of the surface proteins of rhesus rotavirus (RRV) that mediate viral neutralization. Ten monoclonal antibodies directed to VP7 were studied in neutralization assays and competitive binding studies. A large neutralization domain with several interrelated epitopes on VP7 was apparent. Twelve monoclonal antibodies directed to VP3 were used in similar studies and delineated at least 2 distinct neutralization domains on that protein. Neutralizing monoclonal antibodies directed at both VP3 and VP7 were used to isolate viral antigenic variants, which were than studied in neutralization and hemagglutination inhibition assays. The viral variant studies, while confirming the general conclusions obtained from the competitive binding studies, allowed the apparent distinction of two separate neutralization domains on VP7 and three on VP3. All VP7-specific monoclonal antibodies (mAb) mediated serotype-specific neutralization, but a VP3-specific mAb was identified that neutralized rotaviruses of three distinct serotypes. No alteration of viral virulence was apparent in studies of suckling mice orally inoculated with antigenic variant viruses selected with our panel of neutralizing VP3 or VP7-specific mAbs.

  View details for Web of Science ID A1986F343400012

  View details for PubMedID 2431540

• REASSORTANT ROTAVIRUSES CONTAINING STRUCTURAL PROTEINS VP3 AND VP7 FROM DIFFERENT PARENTS INDUCE ANTIBODIES PROTECTIVE AGAINST EACH PARENTAL SEROTYPE JOURNAL OF VIROLOGY Offit, P. A., Clark, H. F., BLAVAT, G., Greenberg, H. B. 1986; 60 (2): 491-496

  Abstract

  Genetic studies of reassortant rotaviruses have demonstrated that gene segments 4 and 9 each segregate with the serotype-specific neutralization phenotype in vitro. Reassortant rotaviruses derived by coinfection of MA-104 cells with the simian strain SA11 and the antigenically distinct bovine strain NCDV were used to determine which viral genes coded for proteins which induced a protective immune response in vivo. In addition, reassortant rotaviruses containing only the gene segment 4 or 9 protein products (vp3 and vp7, respectively) from SA11 or NCDV were used to determine the serotypic specificities of both vp3 and vp7 in several mammalian rotavirus strains. vp3 and vp7 from the murine strain Eb were shown to be indistinguishable from the corresponding proteins from strain SA11. Adult mice orally inoculated with strain Eb developed neutralizing antibodies to both vp3 and vp7. The two naturally occurring bovine rotavirus strains NCDV and UK were shown to contain antigenically similar vp7 but distinct vp3 proteins. Mouse dams orally immunized with a reassortant virus containing only gene 9 from NCDV passively protected their progeny against UK challenge, whereas mouse dams orally immunized with a reassortant virus containing only gene 4 from NCDV did not. Finally, we constructed reassortant viruses that immunized against rotaviruses of two distinct serotypes. SA11 X NCDV reassortants that contained vp3 and vp7 from different parents induced a protective immune response against both parental serotypes. vp3 and vp7 were independently capable of inducing a protective immune response after oral immunization. An understanding of the serotypic specificities of both vp3 and vp7 of human rotavirus isolates will be necessary for the development of successful strategies to protect infants against severe rotavirus infections.

  View details for Web of Science ID A1986E444900020

  View details for PubMedID 3021983

• A FOODBORNE OUTBREAK OF NORWALK VIRUS GASTROENTERITIS - EVIDENCE FOR POST-RECOVERY TRANSMISSION AMERICAN JOURNAL OF EPIDEMIOLOGY White, K. E., Osterholm, M. T., Mariotti, J. A., Korlath, J. A., LAWRENCE, D. H., RISTINEN, T. L., Greenberg, H. B. 1986; 124 (1): 120-126

  Abstract

  From November 10-16, 1982, 220 (57%) of 383 attendees at eight banquets for which food had been prepared at a single hotel restaurant and the employees of the hotel had onset of Norwalk virus gastroenteritis. Epidemiologic investigation of the three largest banquets confirmed consumption of potato and fruit salads (banquet A), coleslaw (banquet B), and tossed salad (banquet C) to be significantly associated with illness. Between November 8-19, similar illness occurred in seven (54%) of 13 hotel kitchen employees. The foods implicated in banquets A and B were prepared by one salad worker during her acute illness and up to 48 hours following her recovery. A second salad worker prepared the implicated tossed salad for banquet C 24 hours following her recovery. To the authors' knowledge, this is the first foodborne outbreak investigation demonstrating Norwalk viral excretion and transmission by a food handler after recovery from illness and either person-to-person or vehicle-borne transmission between food handlers with subsequent transmission by more than one food handler to patrons.

  View details for Web of Science ID A1986C817300014

  View details for PubMedID 3013001

• PASSIVE PROTECTION AGAINST ROTAVIRUS-INDUCED DIARRHEA BY MONOCLONAL-ANTIBODIES TO SURFACE PROTEIN-VP3 AND PROTEIN-VP7 JOURNAL OF VIROLOGY Offit, P. A., Shaw, R. D., Greenberg, H. B. 1986; 58 (2): 700-703

  Abstract

  Monoclonal antibodies directed against two rotavirus surface proteins (vp3 and vp7) as well as a rotavirus inner capsid protein (vp6) were tested for their ability to protect suckling mice against virulent rotavirus challenge. Monoclonal antibodies to two distinct epitopes of vp7 of simian rotavirus strain RRV neutralized RRV in vitro and passively protected suckling mice against RRV challenge. A monoclonal antibody directed against vp3 of porcine rotavirus strain OSU neutralized three distinct serotypes in vitro (OSU, RRV, and UK) and passively protected suckling mice against OSU, RRV, and UK virus-induced diarrhea. The role of vp3 in eliciting protection against heterotypic rotavirus challenge should be considered when developing a vaccine with cloned rotavirus genes. Alternatively, immunization with a reassortant rotavirus containing vp3 and vp7 from two antigenically distinct rotavirus parents might protect against diarrhea induced by two or more rotavirus serotypes.

  View details for Web of Science ID A1986A983900062

  View details for PubMedID 2422398

• INFECTIOUS ROTAVIRUS ENTERS CELLS BY DIRECT PENETRATION OF THE PLASMA-MEMBRANE, NOT BY RECEPTOR MEDIATED ENDOCYTOSIS KALJOT, K. T., Shaw, R. D., Greenberg, H. B. SLACK INC. 1986: A677–A677

  View details for Web of Science ID A1986C539802397

• CULTIVATION AND CHARACTERIZATION OF 3 STRAINS OF MURINE ROTAVIRUS JOURNAL OF VIROLOGY Greenberg, H. B., Vo, P. T., Jones, R. 1986; 57 (2): 585-590

  Abstract

  Three distinct strains of murine rotavirus were adapted to growth in cell culture. These strains are genetically related but not identical; they are serotypically heterogeneous. The cultivatable strains were substantially more infectious (approximately 10(6)-fold) for suckling mice than heterologous simian rotaviruses were. Homologous murine rotavirus strains spread from inoculated to uninoculated litter mates and caused diarrhea, while heterologous rotaviruses did not spread and cause illness.

  View details for Web of Science ID A1986AXZ1900022

  View details for PubMedID 3003390

• MOLECULAR-BASIS OF ROTAVIRUS VIRULENCE - ROLE OF GENE SEGMENT-4 JOURNAL OF VIROLOGY Offit, P. A., BLAVAT, G., Greenberg, H. B., Clark, H. F. 1986; 57 (1): 46-49

  Abstract

  Bovine rotavirus NCDV and simian rotavirus SA-11 exhibited markedly different patterns of gastrointestinal tract disease when inoculated orally into newborn mice. A genetic approach was used to define the molecular basis of these differences. The SA-11 strain of rotavirus was more virulent than the NCDV strain when inoculated orally into newborn mice; the dose of SA-11 required to cause diarrhea in 50% of infant mice was 50-fold less than that required for NCDV. Nineteen reassortant viruses were derived by coinfection of MA-104 cells in vitro with the SA-11 and NCDV strains. The parental origin of reassortant virus double-stranded RNA segments was determined by gene segment migration differences in polyacrylamide gels and hybridization with radioactively labeled parental viral transcripts. The neutralization antigen phenotype of reassortant viruses was determined by plaque reduction neutralization. We found that the dose of SA-11 and NCDV rotavirus required to induce gastroenteritis in newborn mice was determined by gene segment 4. The results suggest that rotavirus virulence may be manipulated by modification or reassortment of gene segment 4.

  View details for Web of Science ID A1986AWG7700007

  View details for PubMedID 3001364

• FUNCTIONAL AND ANTIGENIC EPITOPE MAPPING OF ROTAVIRUS-VP7 AND ROTAVIRUS-VP3 WITH MONOCLONAL-ANTIBODIES Shaw, R. D., KALJOT, K., Offit, P., Greenberg, H. B. SLACK INC. 1986: A33–A33

  View details for Web of Science ID A1986C748300189

• SPECIFIC ENZYME-LINKED IMMUNOASSAY FOR ROTAVIRUS SEROTYPE-1 AND SEROTYPE-3 JOURNAL OF CLINICAL MICROBIOLOGY Shaw, R. D., STONERMA, D. L., Estes, M. K., Greenberg, H. B. 1985; 22 (2): 286-291

  Abstract

  We prepared monoclonal antibodies against two serotypically distinct rotavirus strains: Wa, a serotype 1 virus of human origin, and rhesus rotavirus, a simian serotype 3 virus. Monoclonal antibodies which react specifically with VP7 of each serotype were identified by hemagglutination inhibition tests, plaque reduction neutralization studies, and solid-phase immunoassays which used wild-type and reassortant strains of rotavirus. An enzyme-linked immunoassay was designed which utilizes two of these antibodies to correctly identify serotype 1 and serotype 3 viruses.

  View details for Web of Science ID A1985ANV3700032

  View details for PubMedID 2993354

• REASSORTANT ROTAVIRUSES AS POTENTIAL LIVE ROTAVIRUS VACCINE CANDIDATES JOURNAL OF VIROLOGY Midthun, K., Greenberg, H. B., Hoshino, Y., KAPIKIAN, A. Z., WYATT, R. G., CHANOCK, R. M. 1985; 53 (3): 949-954

  Abstract

  A series of reassortants was isolated from coinfection of cell cultures with a wild-type animal rotavirus and a "noncultivatable" human rotavirus. Wild-type bovine rotavirus (UK strain) was reassorted with human rotavirus strains D, DS-1, and P; wild-type rhesus rotavirus was reassorted with human rotavirus strains D and DS-1. The D, DS-1, and P strains represent human rotavirus serotypes 1, 2, and 3, respectively. Monospecific antiserum (to bovine rotavirus, NCDV strain) or a set of monoclonal antibodies to the major outer capsid neutralization glycoprotein, VP7 (of the rhesus rotavirus), was used to select for reassortants with human rotavirus neutralization specificity. This selection technique yielded many reassortants which received only the gene segment coding for the major neutralization protein from the human rotavirus parent, whereas the remaining genes were derived from the animal rotavirus parent. Single human rotavirus gene substitution reassortants of this sort represent potential live vaccine strains.

  View details for Web of Science ID A1985ABV7400034

  View details for PubMedID 2983101

• EVIDENCE OF IMMUNITY INDUCED BY NATURALLY ACQUIRED ROTAVIRUS AND NORWALK VIRUS-INFECTION ON 2 REMOTE PANAMANIAN ISLANDS JOURNAL OF INFECTIOUS DISEASES Ryder, R. W., Singh, N., Reeves, W. C., KAPIKIAN, A. Z., Greenberg, H. B., Sack, R. B. 1985; 151 (1): 99-105

  Abstract

  For better understanding of the role of humoral immunity in ameliorating infections with rotavirus (RV) and Norwalk virus (NW), 305 Cuna Indians living on two isolated islands located off Panama's Carribean coast were surveyed daily for diarrhea over a seven-month period. Nine (8%) of 108 persons with a baseline RV antibody titer of greater than 1:4 developed RV infection compared with 70 (46%) of 151 persons with a baseline RV antibody titer of less than 1:4 (P less than .001). Thirty-eight (25%) of 151 persons of all ages with baseline RV antibody titer of less than 1:4 had at least one episode of RV diarrhea compared with 6 (6%) of 108 persons who had baseline RV antibody titers of greater than 1:4 (P less than .001). Thirty-two (47%) of 68 persons of all ages who had a baseline NW antibody titer of less than 1:100 developed NW infection compared with 30 (13%) of 237 persons with a baseline NW titer of greater than 1:100 (P less than .001). The high NW and RV infection rates and the excellent levels of protection provided by specific preexisting humoral antibody to these agents should promote activities aimed at developing vaccines for preventing these infections.

  View details for Web of Science ID A1985TY92400013

  View details for PubMedID 2981278

• NORWALK GASTROENTERITIS - A COMMUNITY OUTBREAK ASSOCIATED WITH BAKERY PRODUCT CONSUMPTION ANNALS OF INTERNAL MEDICINE Kuritsky, J. N., Osterholm, M. T., Greenberg, H. B., Korlath, J. A., GODES, J. R., Hedberg, C. W., FORFANG, J. C., KAPIKIAN, A. Z., McCullough, J. C., White, K. E. 1984; 100 (4): 519-521

  Abstract

  From 23 to 26 August 1982, a gastrointestinal illness occurred among 129 of 248 (52%) persons interviewed who had attended four social events in the Minneapolis-St. Paul area. The median incubation period was 36 hours, and symptoms included diarrhea, nausea, headache, and vomiting. Findings of a food-specific questionnaire given to attendants of the four events confirmed that consumption of cake and frosting was significantly associated with development of the illness (odds ratio, 7.9 to 48.3; p = 0.006 to 0.00001). All cake items were purchased from a single bakery, where the employee who had prepared the frosting had had onset of diarrhea and vomiting on August 20. Given an approximate 60% attack rate among persons who ate frosted items, we estimate that 3000 outbreak-associated cases occurred. Serologic analysis confirmed that 17 of 25 ill persons had fourfold or greater rises in their antibody titer to Norwalk virus. Thus, foodborne transmission of Norwalk virus can result from contamination by a single foodhandler.

  View details for Web of Science ID A1984SL21900010

  View details for PubMedID 6322631

• RELATIVE FREQUENCY OF ROTAVIRUS SUBGROUP-1 AND SUBGROUP-2 IN VENEZUELAN CHILDREN WITH GASTROENTERITIS AS ASSAYED WITH MONOCLONAL-ANTIBODIES JOURNAL OF CLINICAL MICROBIOLOGY White, L., Perez, I., Perez, M., URBINA, G., Greenberg, H., KAPIKIAN, A., Flores, J. 1984; 19 (4): 516-520

  Abstract

  Monoclonal antibodies recently developed against the 42,000-dalton protein of two rotavirus strains were used in an enzyme-linked immunosorbent assay to determine the subgroup specificity of 252 specimens collected during a 45-month period from Venezuelan children with rotavirus gastroenteritis. Subgroup 2 rotavirus was shed by 85% of the children, whereas only 14% shed subgroup 1 rotavirus (one-half of them in a 3-month period). No differences were found in the occurrence of fever and vomiting between children shedding either rotavirus subgroup, but it appeared that the syndrome tended to last longer in children shedding subgroup 2 rotavirus. The monoclonal subgrouping enzyme-linked immunosorbent assay seemed to be more sensitive than an immune adherence hemagglutination assay, an enzyme-linked immunosorbent assay with polyclonal antibodies, or the electrophoretic analysis of RNA extracted from the virus. Overall, 99% of the specimens could be subgrouped by this assay.

  View details for Web of Science ID A1984SJ21700016

  View details for PubMedID 6325496

• SEROLOGICAL RESPONSES AMONG TEENAGERS AFTER NATURAL EXPOSURE TO NORWALK VIRUS JOURNAL OF INFECTIOUS DISEASES Baron, R. C., Greenberg, H. B., Cukor, G., Blacklow, N. R. 1984; 150 (4): 531-534

  Abstract

  Twenty-one teenagers exposed to a contaminated water supply during an outbreak of gastroenteritis were tested for seroconversion to Norwalk virus. Serum specimens were collected within 72 hr of exposure and four weeks later. Each of the 11 individuals who developed symptoms and five of the 10 who remained well had a whole-antibody response in serum. None of the remaining five teenagers became ill or seroconverted. Neither seroconversion nor susceptibility to illness was associated with an absence of detectable antibody from acute-phase serum specimens. These findings support the view that immunity to Norwalk virus is not determined by serum antibody. Furthermore, the results are consistent with the possibility, suggested by previous studies in volunteers, that susceptibility is determined by Norwalk virus-specific intestinal receptor sites. IgM responses to the Norwalk virus were detected in only seven persons who became ill (64%) and nine who seroconverted (56%). The seroassay for the Norwalk IgM component might have proved a more sensitive diagnostic tool in this outbreak if convalescent-phase specimens had been collected sooner than four weeks after the onset of illness.

  View details for Web of Science ID A1984TM26900010

  View details for PubMedID 6092484

• SEROTYPIC SIMILARITY AND DIVERSITY OF ROTAVIRUSES OF MAMMALIAN AND AVIAN ORIGIN AS STUDIED BY PLAQUE-REDUCTION NEUTRALIZATION JOURNAL OF INFECTIOUS DISEASES Hoshino, Y., WYATT, R. G., Greenberg, H. B., Flores, J., KAPIKIAN, A. Z. 1984; 149 (5): 694-702

  Abstract

  A total of 16 different strains of rotavirus derived from seven mammalian species (four each from human and porcine species, two each from equine and simian species, and one each from canine and bovine species) and two avian species (one each from turkeys and chickens) were examined in plaque-reduction neutralization tests. Seven antigenically distinct serotypes were established on the basis of a greater than or equal to 20-fold difference between titers of homologous and heterologous reciprocal neutralizing antibodies. Serotypes 1 (strain Wa) and 2 (strain DS-1) were recovered only from humans. Serotype 3 included human rotavirus strain WALK 57/14, rhesus monkey rotavirus strain MMU18006 , vervet monkey rotavirus strain SA-11, dog rotavirus strain CU-1, and horse rotavirus strain H-2. The newly established serotype 4 was identified in both humans (strain St. Thomas no. 4) and pigs (strains Gottfried , SB-1A, and SB-2). Porcine (strain OSU ) and equine (strain H-1) rotaviruses made up a possible fifth serotype. Bovine rotavirus (strain NCDV) constituted a sixth serotype, and chicken rotavirus (strain Ch 2), which had a prime-strain relation with turkey rotavirus (strain Ty 1), was designated serotype 7. A surprising observation that emerged from this study was the existence of a rotavirus (porcine strain SB-1A) bridging serotypes 4 and 5.

  View details for Web of Science ID A1984SU31600007

  View details for PubMedID 6202807

• ULTRASTRUCTURAL-LOCALIZATION OF ROTAVIRUS ANTIGENS USING COLLOIDAL GOLD VIRUS RESEARCH PETRIE, B. L., Greenberg, H. B., Graham, D. Y., Estes, M. K. 1984; 1 (2): 133-152

  Abstract

  Colloidal gold was used to localize six of the ten known proteins of the simian rotavirus SA11 within infected cells by ultrastructural immunocytochemistry. Monospecific or monoclonal antibodies to selected structural and nonstructural proteins were the primary antisera. The major outer capsid glycoprotein, VP7, was associated with nonenveloped particles, with particles de-enveloped by Triton X-100 and with both nuclear and cytoplasmic inclusions. The protease-sensitive outer capsid protein, VP3, was also found on nonenveloped and de-enveloped particles. The major inner capsid protein, VP6, was accessible to antibodies on some of the nonenveloped particles (presumably single-shelled particles) and on the de-enveloped particles. A monospecific antibody to the gene 11 product, believed to be a precursor to a minor structural protein, VP9, reacted strongly with viroplasmic inclusions. Virus particles were weakly labeled by this antibody. NS35, a nonstructural SA11 protein, was found only in the viroplasms. NS29, a nonstructural glycoprotein, was localized to the cytoplasmic side of the endoplasmic reticulum membrane and to the inside of enveloped virus particles. These data support the hypothesis that NS29 facilitates budding of the virus particles and acquisition of the outer capsid layer.

  View details for Web of Science ID A1984TF93000004

  View details for PubMedID 6099654

• ISOLATION AND CHARACTERIZATION OF AN EQUINE ROTAVIRUS JOURNAL OF CLINICAL MICROBIOLOGY Hoshino, Y., WYATT, R. G., Greenberg, H. B., KALICA, A. R., Flores, J., KAPIKIAN, A. Z. 1983; 18 (3): 585-591

  Abstract

  A rotavirus, designated as the H-1 strain, was isolated from a diarrheic foal in primary African green monkey kidney cells and MA104 cells. This cell culture-adapted strain hemagglutinated erythrocytes of human group O, rhesus monkeys, guinea pigs, and sheep. It was found to be similar, if not identical, to porcine rotaviruses (strains OSU, EE, and A-580) by plaque reduction neutralization and hemagglutination inhibition tests, and, in addition, it was found to belong to subgroup 1. This equine rotavirus has an RNA electrophoretic migration pattern which was distinct from those of the three strains of porcine rotavirus. The serological relationship established by plaque reduction neutralization and hemagglutination inhibition tests between the equine (H-1) and porcine (OSU, EE, and A-580) rotaviruses is an example of a rotavirus of the same serotype being isolated from different species. The H-1 strain was distinct from four human rotavirus serotypes (Wa, DS-1, P, and St. Thomas 4) as well as from bovine rotavirus NCDV, simian rotavirus MMU18006, and canine rotavirus CU-1 by plaque reduction neutralization tests. This equine isolate (H-1) was found to be related antigenically to canine CU-1 and bovine NCDV rotaviruses in a one-way fashion by hemagglutination inhibition tests.

  View details for Web of Science ID A1983RE57500028

  View details for PubMedID 6313746

• ROTAVIRUS - SPECIFIC BINDING AND PROCESSING BY BRUSH-BORDER MEMBRANES KALJOT, K. T., Greenberg, H. B., Gray, G. M. W B SAUNDERS CO-ELSEVIER INC. 1983: 1201–

  View details for Web of Science ID A1983QN29000466

• ROTAVIRUS - SPECIFIC BINDING TO INTESTINAL BRUSH-BORDER KALJOT, K. T., Greenberg, H. B., Gray, G. M. SLACK INC. 1983: A86–A86

  View details for Web of Science ID A1983PU88600509

• SEROLOGICAL ANALYSIS OF THE SUBGROUP PROTEIN OF ROTAVIRUS, USING MONOCLONAL-ANTIBODIES INFECTION AND IMMUNITY Greenberg, H., McAuliffe, V., VALDESUSO, J., Wyatt, R., Flores, J., KALICA, A., Hoshino, Y., Singh, N. 1983; 39 (1): 91-99

  Abstract

  Ten monoclones directed to the 42,000-dalton inner structural protein of rotavirus were analyzed. Eight monoclones reacted broadly with antigenic domains common to virtually all mammalian rotaviruses. Two monoclones had specificities similar or identical to previously characterized subgroup specificities. These subgroup monoclones were more efficient in detecting subgroup antigen than either hyperimmune or postinfection antisera. Using the subgroup monoclones, we determined that some animal as well as human rotavirus strains carry subgroup 2 specificity and that epizootic diarrhea of infant mice virus and turkey rotavirus are antigenically distinct from other mammalian rotavirus strains.

  View details for Web of Science ID A1983PV89900011

  View details for PubMedID 6185436

• DIRECT ISOLATION IN CELL-CULTURE OF HUMAN ROTAVIRUSES AND THEIR CHARACTERIZATION INTO 4 SEROTYPES JOURNAL OF CLINICAL MICROBIOLOGY WYATT, R. G., JAMES, H. D., Pittman, A. L., Hoshino, Y., Greenberg, H. B., KALICA, A. R., Flores, J., KAPIKIAN, A. Z. 1983; 18 (2): 310-317

  Abstract

  Of 73 rotavirus-positive fecal specimens tested, 39 yielded a human rotavirus that could be cultivated serially in MA104 or primary African green monkey kidney cells or both; 18 were serotyped. Four distinct serotypes were identified by plaque reduction or tube neutralization assay or both, and three of these serotypes were the same as those established previously by plaque reduction, using human rotaviruses cultivated by genetic reassortment with a cultivable bovine rotavirus. Ten human rotavirus strains received from Japan were found to be similar, if not identical, to our candidate prototype strains representing these four human rotavirus serotypes.

  View details for Web of Science ID A1983RA53400015

  View details for PubMedID 6311872

• ISOLATION, PROPAGATION, AND CHARACTERIZATION OF A 2ND EQUINE ROTAVIRUS SEROTYPE INFECTION AND IMMUNITY Hoshino, Y., WYATT, R. G., Greenberg, H. B., KALICA, A. R., Flores, J., KAPIKIAN, A. Z. 1983; 41 (3): 1031-1037

  Abstract

  A rotavirus designated strain H-2 was isolated in primary African green monkey kidney cells from a foal with diarrhea. This cell culture-adapted strain was found to be similar, if not identical, to simian rotavirus (strains MMU18006 and SA-11) and canine rotavirus (strain CU-1) and, in addition, demonstrated a one-way antigenic relationship with five human rotavirus strains (P, B, no. 14, no. 15, and YO) of the third human rotavirus serotype by the plaque reduction neutralization test. This is the fifth example of an animal rotavirus which shares serotypic specificity with a human rotavirus. The H-2 strain is distinct from the H-1 strain (Y. Hoshino et al., J. Clin. Microbiol., in press) of equine rotavirus not only in serotypic specificity by neutralization but also in subgroup specificity, hemagglutinating activity, and RNA electrophoretic migration pattern, thus establishing the existence of a second equine rotavirus serotype. This H-2 isolate is also distinct by neutralization from three other human rotavirus serotypes, 1 (Wa), 2 (DS-1), and 4 (St. Thomas no. 4), as well as bovine (NCDV), and porcine (OSU) rotaviruses.

  View details for Web of Science ID A1983RE61100022

  View details for PubMedID 6309657

• IDENTIFICATION OF THE ROTAVIRAL GENE THAT CODES FOR HEMAGGLUTINATION AND PROTEASE-ENHANCED PLAQUE-FORMATION VIROLOGY KALICA, A. R., Flores, J., Greenberg, H. B. 1983; 125 (1): 194-205

  Abstract

  Temperature-sensitive mutants of bovine rotavirus, UK Compton strain, and rhesus monkey rotavirus, MMU18006 strain, were used to derive 16 reassortants by coinfection of MA104 cells. The parental viruses differed phenotypically in their neutralization specificity, their ability to hemagglutinate, and their requirement for exogenous trypsin for infectivity. When the reassortants were assayed for neutralization specificity and hemagglutination, four phenotypes were observed, indicating that these two rotaviral functions segregated independently. Protease-enhanced infectivity phenotype segregated with the HA phenotype indicating that these two functions were manifestations of the same gene product. In order to determine the gene responsible for these rotaviral functions, the reassortants were genotyped by hybridizing 32P-labeled parental transcripts and denatured reassortant genomic RNAs and analyzing the resulting hybrids by gel electrophoresis. The fourth RNA segment was clearly shown to code for HA and protease enhanced plaque formation in MA104 cells. The neutralization antigen was linked to the eighth and ninth RNA segments that comigrated during gel electrophoresis and thus could not be differentiated.

  View details for Web of Science ID A1983QE52200016

  View details for PubMedID 6299006

• DEVELOPMENT OF VACCINES AGAINST ROTAVIRUS DISEASE PROGRESS IN FOOD AND NUTRITION SCIENCE WYATT, R. G., KAPIKIAN, A. Z., Greenberg, H. B., KALICA, A. R., Flores, J., Hoshino, Y., CHANOCK, R. M., LEVINE, M. M. 1983; 7 (3-4): 189-192

  View details for Web of Science ID A1983RT50300021

  View details for PubMedID 6318264

• GENE CODING ASSIGNMENTS FOR GROWTH RESTRICTION, NEUTRALIZATION AND SUBGROUP SPECIFICITIES OF THE W-STRAINS AND DS-1-STRAINS OF HUMAN ROTAVIRUS JOURNAL OF GENERAL VIROLOGY Greenberg, H. B., Flores, J., KALICA, A. R., WYATT, R. G., Jones, R. 1983; 64 (FEB): 313-320

  Abstract

  Gene coding assignments for growth restriction, neutralization and subgroup specificities were determined for two human rotavirus strains, DS-1 and W, which represent two distinct serotypes. The 4th gene segment of both viruses was associated with restriction of growth in cell culture. The 9th gene segment of W virus and 8th segment of DS-1 were associated with serotype specificity, while the 6th gene segment of W virus was associated with subgroup specificity.

  View details for Web of Science ID A1983QD35900007

  View details for PubMedID 6300286

• ORAL-ADMINISTRATION OF HUMAN ROTAVIRUS TO VOLUNTEERS - INDUCTION OF ILLNESS AND CORRELATES OF RESISTANCE JOURNAL OF INFECTIOUS DISEASES KAPIKIAN, A. Z., WYATT, R. G., LEVINE, M. M., Yolken, R. H., VANKIRK, D. H., Dolin, R., Greenberg, H. B., CHANOCK, R. M. 1983; 147 (1): 95-106

  Abstract

  Four of 18 volunteers challenged orally with human rotavirus strain D (subgroup 2, serotype Wa) developed a diarrheal illness two to four days after inoculation. Viral shedding was detected in five of the 18 volunteers, whereas 12 (67%) developed serologic evidence of infection. Two volunteers who developed diarrheal illness after the initial inoculation were given the same inoculum 19 months later; neither developed diarrhea, although one developed constitutional and gastrointestinal symptoms. The presence of preinoculation serum immunofluorescent antibody to rotavirus strain D or high levels of neutralizing antibody to Wa or reassortant DS-1 human rotavirus correlated with resistance to diarrheal illness. Although prechallenge serum antibody correlated with resistance to diarrhea and/or shedding of rotavirus, the relationship of preexisting local neutralizing activity in intestinal fluid was less clear-cut.

  View details for Web of Science ID A1983QA17200011

  View details for PubMedID 6296243

• PRODUCTION AND PRELIMINARY CHARACTERIZATION OF MONOCLONAL-ANTIBODIES DIRECTED AT 2 SURFACE-PROTEINS OF RHESUS ROTAVIRUS JOURNAL OF VIROLOGY Greenberg, H. B., VALDESUSO, J., VANWYKE, K., Midthun, K., Walsh, M., McAuliffe, V., WYATT, R. G., KALICA, A. R., Flores, J., Hoshino, Y. 1983; 47 (2): 267-275

  Abstract

  A series of monoclonal antibodies was isolated which reacted with one of two major surface proteins of rhesus rotavirus. Thirty-six monoclonal antibodies immunoprecipitated the 82-kilodalton outer capsid protein, the product of the fourth gene, the viral hemagglutinin. These monoclonal antibodies exhibited hemagglutination inhibition activity and neutralized rhesus rotavirus to moderate or high titer. Three monoclonal antibodies immunoprecipitated the 38-kilodalton outer capsid glycoprotein, the eighth or ninth gene product. These three monoclonal antibodies neutralized rhesus rotavirus to high titer and also inhibited viral hemagglutination.

  View details for Web of Science ID A1983QZ28300001

  View details for PubMedID 6312065

• SEROLOGICAL COMPARISON OF CANINE ROTAVIRUS WITH VARIOUS SIMIAN AND HUMAN ROTAVIRUSES BY PLAQUE REDUCTION NEUTRALIZATION AND HEMAGGLUTINATION INHIBITION TESTS INFECTION AND IMMUNITY Hoshino, Y., WYATT, R. G., Greenberg, H. B., KALICA, A. R., Flores, J., KAPIKIAN, A. Z. 1983; 41 (1): 169-173

  Abstract

  By the plaque reduction neutralization test, the CU-1 strain of canine rotavirus was similar, if not identical, to three strains (no. 14, no. 15, and P) of the tentatively designated third human rotavirus serotype. In addition, strain CU-1 demonstrated a one-way antigenic relationship with two other strains (M and B) of the third human rotavirus serotype. The CU-1 strain of canine rotavirus hemagglutinated human group O, rhesus monkey, dog, sheep, and guinea pig erythrocytes. A two-way antigenic relationship between canine (CU-1) and simian (MMU 18006 and SA11) rotaviruses demonstrated previously by the plaque reduction neutralization test was confirmed further with two additional isolates (A79-10 and LSU 79C-36) of canine rotavirus by the plaque reduction neutralization test and the hemagglutination inhibition test. The CU-1 strain of canine rotavirus, which is known to be distinct from two well-characterized human rotavirus serotypes (Wa and DS-1), was also found to be distinct from the St. Thomas no. 4 strain, which is a newly defined fourth human rotavirus serotype. Thus, this canine strain, which is related antigenically to one of four human rotavirus serotypes, is another example of an animal rotavirus which shares serotype specificity with a human rotavirus.

  View details for Web of Science ID A1983QW41200026

  View details for PubMedID 6190752

• Studies in volunteers with human rotaviruses. Developments in biological standardization KAPIKIAN, A. Z., WYATT, R. G., LEVINE, M. M., BLACK, R. E., Greenberg, H. B., Flores, J., KALICA, A. R., Hoshino, Y., CHANOCK, R. M. 1983; 53: 209-218

  View details for PubMedID 6307776

• WATERBORNE GASTROENTERITIS DUE TO THE NORWALK AGENT - CLINICAL AND EPIDEMIOLOGIC INVESTIGATION AMERICAN JOURNAL OF PUBLIC HEALTH Wilson, R., Anderson, L. J., Holman, R. C., GARY, G. W., Greenberg, H. B. 1982; 72 (1): 72-74

  Abstract

  An outbreak of gastroenteritis occurred at a Pennsylvania summer camp in July 1978. Symptoms included abdominal pain (81 per cent), nausea (72 per cent), and vomiting (53 per cent); upper respiratory infection symptoms occurred in 35 per cent of the campers. Illness was associated with consumption of five or more glasses of water or water-containing beverages. Stool cultures from affected persons were negative for bacterial pathogens; however, a fourfold or greater rise to the Norwalk agent was demonstrated in serum samples of three of three ill persons tested and in none of eight controls (p < .02). Campers ill during the first session who were also present during the second session did not become ill during the second session (p < .001). (Am J Public Health 1982; 72:72-74.)

  View details for Web of Science ID A1982MW07700017

  View details for PubMedID 6274208

• FOODBORNE NORWALK VIRUS AMERICAN JOURNAL OF EPIDEMIOLOGY Griffin, M. R., SUROWIEC, J. J., McCloskey, D. I., Capuano, B., PIERZYNSKI, B., Quinn, M., WOJNARSKI, R., PARKIN, W. E., Greenberg, H., GARY, G. W. 1982; 115 (2): 178-184

  Abstract

  On December 6, 1979, three luncheon banquets were served in a New Jersey restaurant. Thirty-eight of 41 members (92.7%) of the first group became ill as did 25 of 31 members (80.6%) of the second group. None of 12 members of the third group were ill. Illness consisted primarily of diarrhea (76%), nausea, (73%), vomiting (67%), cramps (46%) and fever (18%); the median incubation period was 31 hours and median duration 24 hours. The same foods were served to all three groups, except that cole slaw was substituted for a green salad and mixed vegetables for lima beans for the third group. Consumption of green salad was associated with illness (p less than 0.0001). A total of 118 other persons who ate at the restaurant on the same day were interviewed, 60% of whom reported being ill. Green salad was significantly associated with illness for this group at lunch (p = 0.005) and dinner (p = 0.00007). Serologic studies on seven of 12 patients and on one of four exposed controls showed a fourfold or greater rise in antibody titer to Norwalk virus.

  View details for Web of Science ID A1982NC07300005

  View details for PubMedID 6277186

• STUDIES IN VOLUNTEERS WITH HUMAN ROTAVIRUSES DEVELOPMENTS IN BIOLOGICALS KAPIKIAN, A. Z., WYATT, R. G., LEVINE, M. M., BLACK, R. E., Greenberg, H. B., Flores, J., KALICA, A. R., Hoshino, Y., CHANOCK, R. M. 1982; 53: 209-218

  View details for Web of Science ID A1982QV05000031

• AN OUTBREAK OF NORWALK GASTROENTERITIS ASSOCIATED WITH SWIMMING IN A POOL AND SECONDARY PERSON-TO-PERSON TRANSMISSION AMERICAN JOURNAL OF EPIDEMIOLOGY KAPPUS, K. D., Marks, J. S., Holman, R. C., BRYANT, J. K., Baker, C., GARY, G. W., Greenberg, H. B. 1982; 116 (5): 834-839

  Abstract

  In June 1977 an outbreak of acute gastroenteritis affected 103 students and teachers at an elementary school in Ohio. The illness typically lasted 24 hours or less and was characterized by vomiting (86%) and cramping (70%), but more than half of the persons involved also reported having nausea, diarrhea, and headache. Similar illness frequently followed in household members (29%) of families with primary cases. Investigation revealed that 70% of the children and teachers who swam in a pool at an all day outing June 1 (4 classrooms) and 55% of those who swam during a similar outing June 2 (2 classrooms) had the onset of acute illness from 12--48 hours later. None of the children who attended the outings but did not swim had a similar illness. The evidence suggested that the primary outbreak was caused by contaminated water in the pool and that person-to-person spread of illness followed. Results of a microbiologic study of pool water were negative for bacterial and viral pathogens. Throat washings, stool specimens, and paired blood samples studied for evidence of pathogens were negative initially, but subsequent serologic studies suggested that infection by Norwalk virus was the cause of the outbreak. The pool chlorinator which was inadvertently unconnected at the time of the school visits was reconnected and an underground leak in the water supply pipes was corrected. No more cases were reported after the pool was drained, cleaned, and reopened.

  View details for Web of Science ID A1982PQ74300013

  View details for PubMedID 6293305

• NORWALK GASTROENTERITIS ASSOCIATED WITH A WATER-SYSTEM IN A RURAL GEORGIA COMMUNITY ARCHIVES OF ENVIRONMENTAL HEALTH Goodman, R. A., Buehler, J. W., Greenberg, H. B., MCKINLEY, T. W., Smith, J. D. 1982; 37 (6): 358-360

  Abstract

  An outbreak of acute gastroenteritis occurred during January 4-9, 1982, in a rural community in north Georgia. A systematic telephone survey revealed that 63% of persons living in homes served by the community water system had symptoms of acute gastroenteritis in contrast to 9% of persons in homes served by private wells or other sources (P less than .001). A fourfold rise in antibody titer to the Norwalk virus occurred in 20 of 22 serum pairs obtained from ill persons. Fecal coliforms (greater than 16 MPN/100 ml) were detected in a spring which served as one water source for the community system. Surface runoff from a heavy rainfall, which preceded the outbreak, may have contaminated the system. Outbreaks of gastroenteritis should be promptly reported and investigated to facilitate corrective measures and to identify causative agents such as Norwalk virus.

  View details for Web of Science ID A1982PZ41500006

  View details for PubMedID 6295289

• INVITRO TRANSCRIPTION OF 2 HUMAN ROTAVIRUSES JOURNAL OF VIROLOGY Flores, J., Myslinski, J., KALICA, A. R., Greenberg, H. B., WYATT, R. G., KAPIKIAN, A. Z., CHANOCK, R. M. 1982; 43 (3): 1032-1037

  Abstract

  The RNA polymerase activities of a cultivatable (Wa) and a noncultivatable (DS-1) strain of human rotavirus were studied. Under optimal conditions, transcription of all of their RNA segments occurred, as evidenced by the hybridization of labeled transcripts to genomic RNA. Cross-hybridization between the two viruses showed that none of their 11 genes were completely homologous. The transcription products could be translated in vitro, yielding proteins with an electrophoretic pattern resembling that obtained with proteins labeled in vivo during infection with the Wa virus.

  View details for Web of Science ID A1982PC12000029

  View details for PubMedID 6292446

• DEFINITION OF HUMAN ROTAVIRUS SEROTYPES BY PLAQUE REDUCTION ASSAY INFECTION AND IMMUNITY WYATT, R. G., Greenberg, H. B., James, W. D., Pittman, A. L., KALICA, A. R., Flores, J., CHANOCK, R. M., KAPIKIAN, A. Z. 1982; 37 (1): 110-115

  Abstract

  Twenty different human rotavirus reassortants were characterized serologically by a plaque reduction assay as belonging to one of three distinct serotypes. Fourteen were similar if not identical to our prototype Wa strain; two were like the prototype DS-1 strain, and four belonged to a third serotype for which a prototype has not yet been selected. Hyperimmune sera raised against the three serotypes were required to distinguish among them, since postinfection sera had lower titers and were more cross-reactive than hyperimmune sera. These results confirmed the ability of a qualitative cytopathic neutralization test to predict correctly the Wa or DS-1 serotype. A strain of rhesus rotavirus (MMU 18006) was identified as belonging to the newly defined third serotype. Finally, an attempt was made to correlate previously published serotype analysis by neutralization of fluorescent cell-forming units with the results determined by the plaque reduction neutralization assay.

  View details for Web of Science ID A1982NV66300017

  View details for PubMedID 6286487

• USE OF TRANSCRIPTION PROBES FOR GENOTYPING ROTAVIRUS REASSORTANTS VIROLOGY Flores, J., Greenberg, H. B., Myslinski, J., KALICA, A. R., WYATT, R. G., KAPIKIAN, A. Z., CHANOCK, R. M. 1982; 121 (2): 288-295

  View details for Web of Science ID A1982PH10500007

  View details for PubMedID 6289518

• ACQUISITION OF SERUM ANTIBODY TO NORWALK VIRUS AND ROTAVIRUS AND RELATION TO DIARRHEA IN A LONGITUDINAL-STUDY OF YOUNG-CHILDREN IN RURAL BANGLADESH JOURNAL OF INFECTIOUS DISEASES BLACK, R. E., Greenberg, H. B., KAPIKIAN, A. Z., Brown, K. H., Becker, S. 1982; 145 (4): 483-489

  Abstract

  Serum antibodies to Norwalk virus and to rotavirus were measured during longitudinal studies of infectious diseases and nutrition in rural Bangladesh. Initially, the prevalence of antibody to Norwalk virus was 7% in children younger than six months and increased to 80% in children two to five years of age. The incidence of titer increases was highest in one- and two-year-olds and in children who had low or undetectable levels of antibody. Some Norwalk virus infections appeared to result in diarrhea. Nearly all children had serum antibodies to rotavirus at the beginning of the study; however, children with the lowest levels of antibody to rotavirus had the greatest risk of rotavirus diarrhea. Over half of the children had a fourfold increase in titer of antibody to rotavirus during the year, and 7% had increases in two of the three study periods during the year. Most increases in titer of antibody to rotavirus appeared to result from subclinical infections.

  View details for Web of Science ID A1982NL90200007

  View details for PubMedID 6279734

• ANTIVIRAL TREATMENT OF CHRONIC HEPATITIS-B VIRUS-INFECTION - INFECTIOUS VIRUS CANNOT BE DETECTED IN PATIENT SERUM AFTER PERMANENT RESPONSES TO TREATMENT HEPATOLOGY SCULLARD, G. H., Greenberg, H. B., Smith, J. L., Gregory, P. B., Merigan, T. C., Robinson, W. S. 1982; 2 (1): 39-49

  Abstract

  Fourteen chimpanzees were inoculated with pre- and posttreatment sera from seven patients with persistent hepatitis B virus infection and chronic hepatitis who had permanent responses of their infection to treatment with interferon and/or adenine arabinoside. Inoculation of pretreatment serum at a dilution of 10(-8) from a patient with a Type I response to treatment [disappearance of Dane particle DNA polymerase (DNAP) activity, HBeAg, and HBsAg from serum] resulted in infection, while undiluted posttreatment serum (all markers negative) failed to infect another animal. Pretreatment sera (DNAP, HBeAg, and HBsAg positive) from all six patients with a Type II response to treatment (disappearance of DNAP activity and HBeAg but not HBsAg from serum) led to infection in six chimpanzees after inoculation of serum dilutions varying between 10(-2) and 10(-7). Inoculation of undiluted posttreatment sera (HBsAg positive and DNAP and HBeAg negative) from the same six patients produced no evidence of hepatitis B virus infection in another six animals. These results indicate that a Type I or II response to treatment with these antiviral agents reduces the infectivity in the serum of patients with chronic hepatitis B to below the level of detection by this assay. Such changes should be useful in interrupting spread of the infection between individuals. Our findings suggest that the serum of some patients who, without treatment are HBsAg positive and DNAP and HBeAg negative, may also be free of detectable infectious hepatitis B virus.

  View details for Web of Science ID A1982NL29100006

  View details for PubMedID 6172352

• ACUTE VIRAL-ENTERITIS AND EXACERBATIONS OF INFLAMMATORY BOWEL-DISEASE GASTROENTEROLOGY Gebhard, R. L., Greenberg, H. B., Singh, N., Henry, P., Sharp, H. L., Kaplan, L., KAPIKIAN, A. Z. 1982; 83 (6): 1207-1209

  Abstract

  In order to determine whether or not acute viral gastroenteritis predisposes to exacerbations of inflammatory bowel disease, patients with Crohn's disease and ulcerative colitis were observed longitudinally. Assessment of disease activity was correlated with evidence for viral infection by serology and stool antigen testing. Disease exacerbations were rarely associated with rotavirus, Norwalk agent, or adenovirus infection, primarily because these infection rates were very low. However, the few patients having these viral infections often had relapse symptoms.

  View details for Web of Science ID A1982PQ60800007

  View details for PubMedID 6290307

• NORWALK GASTROINTESTINAL ILLNESS - AN OUTBREAK ASSOCIATED WITH SWIMMING IN A RECREATIONAL LAKE AND SECONDARY PERSON-TO-PERSON TRANSMISSION AMERICAN JOURNAL OF EPIDEMIOLOGY Baron, R. C., Murphy, F. D., Greenberg, H. B., Davis, C. E., BREGMAN, D. J., GARY, G. W., Hughes, J. M., Schonberger, L. B. 1982; 115 (2): 163-172

  Abstract

  An outbreak of gastrointestinal illness in which headache, low grade fever and myalgia were common symptoms occurred among persons who visited a recreational park in Macomb County, Michigan, on July 13-16, 1979. The temporal clustering of onsets of 121 persons who were the first in their households to become ill suggested an incubation period ranging from 4-77 hours. A history of swimming in the park's lake was elicited with significantly greater frequency from these persons than from park visitors who were not ill (age standardized odds ratio = 4.8; 95% confidence interval, 1.8-12.7). One hundred twenty-six park visitors who became ill were household contacts of index patients who had swum in the lake; at least 62 of these 126 cases were probably due to secondary transmission. A secondary attack rate of 19% was observed in household contacts who had not visited the park. Serologic studies identified Norwalk virus as the etiologic agent. The source of the contamination of the lake could not be determined. Although some water samples collected just before and after the epidemic period had high coliform counts, the geometric mean coliform density of all samples collected on those days was within the limits established by the Environmental Protection Agency as acceptable for recreational contact water.

  View details for Web of Science ID A1982NC07300003

  View details for PubMedID 6277184

• EPIDEMIOLOGY OF NORWALK GASTROENTERITIS AND THE ROLE OF NORWALK VIRUS IN OUTBREAKS OF ACUTE NONBACTERIAL GASTROENTERITIS ANNALS OF INTERNAL MEDICINE Kaplan, J. E., GARY, G. W., Baron, R. C., Singh, N., Schonberger, L. B., Feldman, R., Greenberg, H. B. 1982; 96 (6): 756-761

  Abstract

  Outbreaks of Norwalk gastroenteritis, which may involve persons of all ages, occur during all seasons and in various locations. Waterborne, foodborne, and person-to-person modes of transmission have been described, and secondary person-to-person transmission is common. Outbreaks generally end in about 1 week; longer outbreaks occur only when new groups of susceptible persons are introduced, usually in the setting of a persistent common source of infection. The illness is generally mild and characterized by nausea, vomiting, diarrhea, and abdominal cramps. Vomiting is the predominant symptom among children, whereas diarrhea is commoner among adults. Forty-two percent of 74 outbreaks of acute nonbacterial gastroenteritis investigated by the Centers for Disease Control from 1976 to 1980 were attributed to the Norwalk virus. The rest resembled Norwalk outbreaks clinically and epidemiologically and were probably caused by 27-nm viral agents similar to the Norwalk virus.

  View details for Web of Science ID A1982NT96800011

  View details for PubMedID 6283977

• NORWALK VIRUS GASTROENTERITIS FOLLOWING RAW OYSTER CONSUMPTION AMERICAN JOURNAL OF EPIDEMIOLOGY Gunn, R. A., Janowski, H. T., LIEB, S., PRATHER, E. C., Greenberg, H. B. 1982; 115 (3): 348-351

  Abstract

  In January, 1980, six out of 13 persons (46%) attending a party in a small northwest Florida town near the Gulf of Mexico became ill with Norwalk virus gastroenteritis after eating raw oysters. Symptoms experienced by the ill persons were principally nausea (100%), vomiting (83%) and diarrhea (50%) and were of brief duration. The symptom complex and epidemiology of Norwalk virus infection closely resemble the gastrointestinal illness commonly referred to as the 24-hour intestinal flu or "stomach flu." Norwalk virus infection was identified in this outbreak by application of a recently developed sensitive and specific serologic radioimmunoassay. Oysters from the incriminated batch had fecal coliform levels above recommended standards; however, recent studies of oyster-harvesting waters have shown only a weak correlation between fecal coliforms and the presence of enteric viruses. Further studies are needed to determine whether modifications of monitoring modalities for oyster-harvesting waters are needed.

  View details for Web of Science ID A1982NG07400005

  View details for PubMedID 6278928

• RESCUE AND SEROTYPIC CHARACTERIZATION OF NON-CULTIVABLE HUMAN ROTAVIRUS BY GENE REASSORTMENT INFECTION AND IMMUNITY Greenberg, H. B., WYATT, R. G., KAPIKIAN, A. Z., KALICA, A. R., Flores, J., Jones, R. 1982; 37 (1): 104-109

  Abstract

  Thirty-three of 50 noncultivable human rotavirus strains from a variety of locations were successfully rescued by gene reassortment. The serotype of each of the 33 strains was investigated by a qualitative cytopathic effect neutralization assay. Nineteen strains resembled the previously characterized human rotavirus serotype Wa, whereas three strains were serologically related to the DS-1 strain. Eleven strains appeared to be serotypically distinct from the Wa and DS-1 strains and thus apparently represent one or more new human rotavirus serotypes.

  View details for Web of Science ID A1982NV66300016

  View details for PubMedID 6286486

• NORWALK VIRUS ENTERIC ILLNESS ACQUIRED BY SWIMMING EXPOSURE AMERICAN JOURNAL OF EPIDEMIOLOGY Koopman, J. S., Eckert, E. A., Greenberg, H. B., STROHM, B. C., Isaacson, R. E., MONTO, A. S. 1982; 115 (2): 173-177

  Abstract

  In an epidemic of gastrointestinal illness strongly associated with swimming at a recreational park in Macomb County, Michigan, in July, 1979, the authors demonstrated the value of serologic testing to detect Norwalk virus infection. Rises in antibody titer to Norwalk virus were noted in all 11 individuals tested. Electron microscopy on stools from 20 ill individuals revealed only one with Norwalk virus-like particles. This particle was shown by radioimmunoassay and immune electron microscopy not to be Norwalk virus and not to have stimulated detectable antibodies in this individual. These results not only indicate that electron microscopy is insensitive in detecting Norwalk virus, but that it has the potential to mislead. A low rate of respiratory symptoms was associated with gastrointestinal illness in this Norwalk virus outbreak. The route of exposure might have been important for this. The outbreak was also noteworthy in that, although there was evidence of familial clusters of resistance, a very high percentage of the population was proved to be susceptible to the Norwalk virus.

  View details for Web of Science ID A1982NC07300004

  View details for PubMedID 6277185

• GENES OF HUMAN (STRAIN WA) AND BOVINE (STRAIN UK) ROTAVIRUSES THAT CODE FOR NEUTRALIZATION AND SUBGROUP ANTIGENS VIROLOGY KALICA, A. R., Greenberg, H. B., WYATT, R. G., Flores, J., SERENO, M. M., KAPIKIAN, A. Z., CHANOCK, R. M. 1981; 112 (2): 385-390

  View details for Web of Science ID A1981MA19200002

  View details for PubMedID 6266134

• TRAVELERS DIARRHEA IN PANAMANIAN TOURISTS IN MEXICO JOURNAL OF INFECTIOUS DISEASES Ryder, R. W., OQUIST, C. A., Greenberg, H., TAYLOR, D. N., ORSKOV, F., ORSKOV, I., KAPIKIAN, A. Z., Sack, R. B. 1981; 144 (5): 442-448

  Abstract

  To determine whether residents of developing countries are unlikely to acquire travelers' diarrhea, 64 Panamanians of widely divergent socioeconomic strata were studied during a 15-day tour through Mexico. Twenty-three (36%) tourists experienced 27 episodes of travelers' diarrhea that were caused by seven different pathogens. The most commonly identified etiologic agents were rotavirus (26%), Norwalk virus (15%), and Campylobacter fetus (11%), whereas enterotoxigenic Escherichia coli was not frequently associated with travelers' diarrhea. Acquisition of travelers' diarrhea was correlated directly with high socioeconomic status. Varying levels of immunity to enteropathogens that are endemic in Panama may explain the different isolation rates of pathogens.

  View details for Web of Science ID A1981MR66700008

  View details for PubMedID 6273474

• BIWEEKLY PROPHYLACTIC DOXYCYCLINE FOR TRAVELERS DIARRHEA JOURNAL OF INFECTIOUS DISEASES Santosham, M., Sack, R. B., Froehlich, J., Greenberg, H., Yolken, R., KAPIKIAN, A., Javier, C., Medina, C., ORSKOV, F., ORSKOV, I. 1981; 143 (4): 598-602

  Abstract

  A double-blind study to determine the efficacy of biweekly oral doxycycline in the prevention of travelers' diarrhea was conducted among 46 Peace Corps volunteers during their first six weeks in Honduras. The volunteers took either 100 mg of doxycycline per dose or a placebo for three weeks and were observed for an additional three weeks. There was no significant difference in the number of persons with travelers' diarrhea in the two groups (eight of 24 in the doxycycline group and 10 of 22 in the placebo group) in the three weeks when the drug was taken. However, significantly fewer episodes (P less than 0.05) of travelers' diarrhea occurred in the doxycycline group than in the placebo group at the end of the second, third, and fourth weeks. Enterotoxigenic Escherichia coli (ETEC) was the most common pathogen identified. ETEC from 13 (62%) of 21 stool samples were resistant to doxycycline. Biweekly doxycycline was only marginally effective in preventing travelers' diarrhea and did not prevent diarrhea secondary to doxycycline-resistant ETEC.

  View details for Web of Science ID A1981LT69200014

  View details for PubMedID 7017017

• RESCUE OF NON-CULTIVATABLE HUMAN ROTAVIRUS BY GENE REASSORTMENT DURING MIXED INFECTION WITH TS-MUTANTS OF A CULTIVATABLE BOVINE ROTAVIRUS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Greenberg, H. B., KALICA, A. R., WYATT, R. G., Jones, R. W., KAPIKIAN, A. Z., CHANOCK, R. M. 1981; 78 (1): 420-424

  Abstract

  Fastidious human rotaviruses that did not undergo productive infection in tissue culture were rescued by genetic reassortment during mixed infection with a temperature-sensitive (ts) mutant of a cultivatable bovine rotavirus. In this manner, the genes of the fastidious rotavirus that restricted growth in vitro were replaced by the corresponding genes from a tissue culture-adapted rotavirus. We recovered genetically reassorted viruses that grew to high titer and were neutralized specifically by hyperimmune guinea pig type 1 or type 2 human rotavirus antiserum. Preliminary RNA analysis of these clones disclosed that they were indeed viruses with reassorted genes.

  View details for Web of Science ID A1981LA96300081

  View details for PubMedID 6264442

• PROTEINS OF NORWALK VIRUS JOURNAL OF VIROLOGY Greenberg, H. B., VALDESUSO, J. R., KALICA, A. R., WYATT, R. G., MCAULIFFE, V. J., KAPIKIAN, A. Z., CHANOCK, R. M. 1981; 37 (3): 994-999

  Abstract

  The proteins of the Norwalk virus were studied by polyacrylamide gel electrophoresis. Highly purified specifically immunoprecipitated virions appeared to contain a single primary structural protein with a molecular weight of 59,000. In addition, a soluble Norwalk viral protein with a molecular weight of 30,000 was identified in fecal specimens containing Norwalk virus. The protein structure of the virion is similar to that of the Calciviridae family.

  View details for Web of Science ID A1981LF11500019

  View details for PubMedID 6785451

• ANTIGENIC CHARACTERIZATION OF HUMAN AND ANIMAL ROTAVIRUSES BY IMMUNE ADHERENCE HEMAGGLUTINATION ASSAY (IAHA) - EVIDENCE FOR DISTINCTNESS OF IAHA AND NEUTRALIZATION ANTIGENS INFECTION AND IMMUNITY KAPIKIAN, A. Z., CLINE, W. L., Greenberg, H. B., WYATT, R. G., KALICA, A. R., Banks, C. E., JAMES, H. D., Flores, J., CHANOCK, R. M. 1981; 33 (2): 415-425

  Abstract

  An immune adherence hemagglutination assay (IAHA) and a modified enzyme-linked immunosorbent assay for antigenic characterization of human rotaviruses were developed. The designations of type 1 and type 2 were identical to those established previously by specific complement fixation, enzyme-linked immunosorbent assay, and immune electron microscopy. By IAHA (and modified enzyme-linked immunosorbent assay) certain animal rotaviruses were found to be closely related to human rotavirus type 1. The pattern of IAHA reactivity and the cell culture neutralization serotype were found to be distinct properties. The separation of neutralization and IAHA reactivity was apparent when animal rotaviruses which were distinguishable from each other by neutralization assays were found to share IAHA specificity. Further evidence for the dissociation of the neutralization and IAHA specificities was found in studies of human and bovine rotaviruses which underwent genetic reassortment during coinfection. Thus, it appeared that the IAHA and neutralization antigens were coded for by different genes. In view of these findings, we suggest that the term serotype be reversed to identify the antigen that reacts with neutralizing antibodies as is customary for other viruses and that the term subgroup (instead of serotype) be used for the specificity detected by specific complement fixation, enzyme-linked immunosorbent assay, and now IAHA.

  View details for Web of Science ID A1981LZ95100013

  View details for PubMedID 6168588

• NORWALK-RELATED VIRAL GASTROENTERITIS DUE TO CONTAMINATED DRINKING-WATER AMERICAN JOURNAL OF EPIDEMIOLOGY Taylor, J. W., GARY, G. W., Greenberg, H. B. 1981; 114 (4): 584-592

  Abstract

  An explosive outbreak of gastrointestinal illness clinically compatible with infection by an agent serologically related to Norwalk virus agent occurred in an elementary school in May 1978. Seroconversion by radioimmunoassay to the Norwalk antigen was noted in two of three ill persons, but no viral particles were identified in stool. Illness developed in 72% of students and teachers at the school, and 32% of household contacts of these ill persons. Of household contacts of persons exposed at school but not clinically ill, 11% developed illness. This value, however, was not statistically different from the level of illness observed concurrently in household contacts of students at an unaffected school nearby. Epidemiologic investigation implicated water as the mode of transmission. Average consumption of one or more glasses per day was strongly associated with illness (p less than 0.00000001). Among soccer team members with limited school contact, water consumption at the school was associated with a 14-fold greater risk of illness (p less than 0.000001). Drinking water was most likely contaminated by back-siphonage through a cross-connection between the school's well and septic tank. This contamination occurred approximately 24 to 36 hours before the outbreak developed.

  View details for Web of Science ID A1981ML54600014

  View details for PubMedID 6272571

• DISTINCTIVE RIBONUCLEIC-ACID PATTERNS OF HUMAN ROTAVIRUS SUBGROUPS-1 AND SUBGROUPS-2 INFECTION AND IMMUNITY KALICA, A. R., Greenberg, H. B., ESPEJO, R. T., Flores, J., WYATT, R. G., KAPIKIAN, A. Z., CHANOCK, R. M. 1981; 33 (3): 958-961

  Abstract

  The ribonucleic acid migration patterns of 7 subgroup 1 and 16 subgroup 2 human rotaviruses recovered from four geographic areas were compared. The subgroup 1 ribonucleic acid patterns had strikingly slower-moving segments 10 and 11, suggesting a correlation between the ribonucleic acid pattern and the subgroup specificity.

  View details for Web of Science ID A1981MF65000050

  View details for PubMedID 6270002

• ANTIVIRAL TREATMENT OF CHRONIC HEPATITIS-B VIRUS-INFECTION - IMPROVEMENT IN LIVER-DISEASE WITH INTERFERON AND ADENINE-ARABINOSIDE HEPATOLOGY SCULLARD, G. H., ANDRES, L. L., Greenberg, H. B., Smith, J. L., Sawhney, V. K., NEAL, E. A., MAHAL, A. S., Popper, H., Merigan, T. C., Robinson, W. S., Gregory, P. B. 1981; 1 (3): 228-232

  View details for Web of Science ID A1981MP47800005

  View details for PubMedID 6169615

• NORWALK VIRUS GASTROENTERITIS IN VOLUNTEERS CONSUMING DEPURATED OYSTERS AUSTRALIAN JOURNAL OF EXPERIMENTAL BIOLOGY AND MEDICAL SCIENCE GROHMANN, G. S., Murphy, A. M., Christopher, P. J., Auty, E., Greenberg, H. B. 1981; 59 (APR): 219-228

  Abstract

  Following the widespread outbreaks of oyster-associated gastroenteritis which occurred throughout Australia in 1978, several programmes were introduced to minimise the occurrence of further outbreaks. One programme included the depuration (purification) of oysters and the use of human volunteers to test-consume samples from batches of depurated oysters before their sale to the public. Oysters from the Georges River and Brisbane Waters were test-consumed from December, 1978, to August, 1979. None of the volunteers was ill after consuming Brisbane Waters oysters but 52 reported ill after eating Georges River oysters. The predominant symptoms were nausea, vomiting and diarrhoea with an average incubation period of 42 hours. Recovery was usually complete in 36-48 hours. Of the 52 illnesses reported 31 (60%) occurred in two particular weeks ending July 1st and 22nd when rates of 18.3% and 7.8% were reported. The average illness rate for the remainder of the period under study was only 1%. Norwalk virus was found in 8 of 25 (32%) stools, and antibody increases demonstrated in seven of ten paired sera, giving an overall diagnostic rate for Norwalk infection of 37.0% for these two peak periods. Heavy rain preceded these two weeks in which the illnesses occurred. No evidence of Norwalk infection was found at any other time. These studies confirmed the epidemiological findings of the major outbreak of gastroenteritis in 1978, and showed that only Georges River oysters caused Norwalk virus infections and that depuration as carried out in 1979 was not entirely satisfactory.

  View details for Web of Science ID A1981LH91300011

  View details for PubMedID 6789806

• HEPATITIS-B VIRAL MARKERS IN SEVERE VIRAL-HEPATITIS - INFLUENCE OF STEROID-THERAPY HEPATOLOGY Greenberg, H. B., Robinson, W. S., Knauer, C. M., Gregory, P. B. 1981; 1 (1): 54-57

  Abstract

  In our double-blind randomized trial of methylprednisolone vs. placebo in severe viral hepatitis, 16 patients with hepatitis B (8 on steroid, 8 on placebo) were followed for at least 4 weeks. Four of the eight patients receiving methylprednisolone eventually died and all patients on placebo survived. Despite marked reduction in serum IgG in steroid-treated patients, the decline in HBsAg titer and disappearance of Dane particle markers was the same in both treatment groups. A nonspecific depression of anti-HBc was noted in patients given steroid. There is no evidence that corticosteroid therapy accelerates viral replication when the acute hepatitis is severe.

  View details for Web of Science ID A1981MP47500008

  View details for PubMedID 7026400

• HAZEL ELEPHANT IS DEAD (OF TUBERCULOSIS) AMERICAN REVIEW OF RESPIRATORY DISEASE Greenberg, H. B., Jung, R. C., GUTTER, A. E. 1981; 124 (3): 341-341

  View details for Web of Science ID A1981MH21300028

  View details for PubMedID 7283270

• Sampling New Orleans' drug addicts for tuberculosis. journal of the Louisiana State Medical Society Greenberg, H. B., TRACHTMAN, L. 1980; 132 (2): 17-18

  View details for PubMedID 7365310

• INFECTIOUS-DISEASE PATTERNS IN THE WAORANI, AN ISOLATED AMERINDIAN POPULATION AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE Kaplan, J. E., Larrick, J. W., Yost, J., Farrell, L., Greenberg, H. B., Herrmann, K. L., Sulzer, A. J., WALLS, K. W., Pederson, L. 1980; 29 (2): 298-312

  Abstract

  The Waorani Indians of eastern Ecuador provide a unique opportunity for studying exposure of an isolated human population to various infectious disease agents. Using serologic tests to determine antibody prevalence, skin test data, and stool examination for parasites, we have been able to construct a profile of infectious diseases which are endemic, and others which have been introduced into the Waorani population. These findings are compared with similar data reported from elsewhere in the Amazon. Serologic studies demonstrating the presence of antibody to measles and poliovirus type 3 after vaccination indicate that the Waorani respond normally to viral challenge with these agents. The question of genetic inability among aboriginal Amerindians to respond to viral agents is discussed. Finally, general recommendations are made regarding the future health care of the Waorani.

  View details for Web of Science ID A1980JL58500024

  View details for PubMedID 7369449

• APPROACHES TO IMMUNIZATION OF INFANTS AND YOUNG-CHILDREN AGAINST GASTROENTERITIS DUE TO ROTAVIRUSES REVIEWS OF INFECTIOUS DISEASES KAPIKIAN, A. Z., WYATT, R. G., Greenberg, H. B., KALICA, A. R., KIM, H. W., BRANDT, C. D., RODRIGUEZ, W. J., PARROTT, R. H., CHANOCK, R. M. 1980; 2 (3): 459-469

  Abstract

  Recent studies have shown that in developed countries rotaviruses are the single most important etiologic agents of acute gastroenteritis that requires hospitalization of infants and young children. Although deaths from gastroenteritis are, in general, infrequent in the developed countries, an effective rotavirus vaccine would clearly be of benefit to reduce the heavy toll of morbidity from gastroenteritis due to rotavirus. In the developing countries the impact of diarrheal diseases is staggering. It was recently estimated that in Asia, Africa, and Latin-America during a one-year period there would be 3.5 billion cases of diarrhea and 5-10 million deaths associated with diarrhea; in addition, diarrhea was ranked first in freqency in the categories of disease and mortality. In the developing countries rotaviruses are known to cause diarrhea, but their relative role in this high mortality rate is not yet known. epidemiologic data indicate that development of an effective rotavirus vaccine would reduce morbidity, and they suggest that a vaccine would also reduce a portion of the mortality from diarrheal disease. The prospects and approaches for the development of an effective rotavirus vaccine are presented. The recent successful propagation of rotavirus type 2 in cell culture represents an important step in this regard. In addition, the antigenic relation between human and animal strains offers another possible approach. The need for a live attenuated vaccine is indicated by the prime role played by local intestinal immunity in resistance to rotavirus disease.

  View details for Web of Science ID A1980KB47700012

  View details for PubMedID 6251528

• BISMUTH SUB-ALICYLATE THERAPY OF VIRAL GASTROENTERITIS GASTROENTEROLOGY STEINHOFF, M. C., Douglas, R. G., Greenberg, H. B., CALLAHAN, D. R. 1980; 78 (6): 1495-1499

  Abstract

  The therapeutic efficacy of bismuth subsalicylate was examined in a randomized double-blind fashion in 59 volunteers who were inoculated with Norwalk agent. Sixty-eight percent of the volunteers demonstrated seroconversion; 57% became ill. The severity and duration of the illness in 32 volunteers in the treatment and placebo groups were compared. Significant reduction in the severity and duration of abdominal cramps (P less than 0.01) and in the median duration of GI symptoms (P less than 0.05) was noted in the treatment group. There was no difference in the number, weight, or water content of stools, or in the rate of viral excretion between the two groups. The median duration of illness was 20 hr in the treatment group and 27 hr in the placebo group (0.1 greater than P greater than 0.05).

  View details for Web of Science ID A1980JS78800019

  View details for PubMedID 7372069

• DETECTION OF ESCHERICHIA-COLI ENTEROTOXINS IN STOOLS INFECTION AND IMMUNITY Merson, M. H., Yolken, R. H., Sack, R. B., FROEHLICH, J. L., Greenberg, H. B., Huq, I., Black, R. W. 1980; 29 (1): 108-113

  Abstract

  We determined whether enterotoxigenic Escherichia coli diarrhea could be diagnosed by direct examination of stools for heat-labile (LT) and heat-stable (ST) enterotoxins. The Y-1 adrenal cell and an enzyme-linked immunosorbent assay (ELISA) detected LT in 85 and 93%, respectively, of stool specimens obtained from adults with acute diarrhea from whom an LT- and ST-producing organism had been isolated. Furthermore, the ELISA assay detected LT in 8 of 35 stool specimens from which no LT-producing E. coli had been isolated. The infant mouse assay was utilized to detect ST in these stool specimens and was found to be an insensitive method, showing positive results in only 36% of the specimens from which an ST-producing organism was isolated. Further studies are warranted to determine the diagnostic value of direct detection of LT in stools, especially by the ELISA method.

  View details for Web of Science ID A1980JY62900017

  View details for PubMedID 6995331

• HUMAN ROTAVIRUS TYPE-2 - CULTIVATION INVITRO SCIENCE WYATT, R. G., James, W. D., BOHL, E. H., THEIL, K. W., SAIF, L. J., KALICA, A. R., Greenberg, H. B., KAPIKIAN, A. Z., CHANOCK, R. M. 1980; 207 (4427): 189-191

  Abstract

  A strain of type 2 human rotavirus (Wa) was grown to relatively high titer through 14 passages in primary cultures of African green monkey kidney (AGMK) cells. This passage series was initiated with virus that had been passaged 11 times serially in newborn gnotobiotic piglets. In contrast, virus present in the stool of patient Wa as well as virus from the first, second, or third passage in piglets could not be propagated successfully in African green monkey kidney cells. Prior to each passage in cell culture, the virus was treated with trypsin and the inoculated cultures were centrifuged at low speed. Cultivation of a type 2 human rotavirus should aid attempts to characterize this virus and to develop a means of immunoprophylaxis for a serious diarrheal disease of human infants.

  View details for Web of Science ID A1980HZ70500024

  View details for PubMedID 6243190

• OYSTER-ASSOCIATED GASTROENTERITIS IN AUSTRALIA - THE DETECTION OF NORWALK VIRUS AND ITS ANTIBODY BY IMMUNE ELECTRON-MICROSCOPY AND RADIOIMMUNOASSAY JOURNAL OF MEDICAL VIROLOGY GROHMANN, G. S., Greenberg, H. B., Welch, B. M., Murphy, A. M. 1980; 6 (1): 11-19

  Abstract

  Following widespread outbreaks of oyster-associated gastroenteritis in Australia during 1978 in which Norwalk virus was implicated as the causative agent, collaborative studies were undertaken between laboratories in Australia and the United States to confirm the etiology. Immune electron microscopy (IEM) techniques were used in Australia and radioimmunoassay (RIA) methods in the United States. Norwalk virus was detected by IEM in seven of 15 faecal samples, and four were positive by RIA. A much better correlation was found with antibody determinations. Both methods demonstrated significant increases in antibody to Norwalk virus in 22 of 30 sets (73%) of "acute" and "convalescent" sera, confirming that Norwalk virus was responsible for the majority of cases. It is significant that the RIA serology was determined using Norwalk antigen originating in the United States and the IEM serology was determined using 27--30-nm particles originating in Australia.

  View details for Web of Science ID A1980KU73700002

  View details for PubMedID 6785390

• NORWALK VIRUS GASTROENTERITIS ABOARD A CRUISE SHIP - AN OUTBREAK ON 5 CONSECUTIVE CRUISES AMERICAN JOURNAL OF EPIDEMIOLOGY Gunn, R. A., TERRANOVA, W. A., Greenberg, H. B., YASHUK, J., GARY, G. W., Wells, J. G., Taylor, P. R., Feldman, R. A. 1980; 112 (6): 820-827

  Abstract

  An explosive outbreak of gastroenteritis caused by a parvovirus-like (PVL) agent (Norwalk agent) affected 521 (64%) cruise ship passengers in 1977. The illness was characterized as mild-to-moderate and lasted one to two days. Principal symptoms experienced by ill passengers were nausea, vomiting, and diarrhea; fever was reported for approximately 25% of the patients. The outbreak was compatible with a common-source exposure, but no such exposure was identified. On the next four cruises, passengers experienced a similar gastrointestinal illness, but the outbreaks were less explosive, affected fewer persons, and symptoms appeared milder, on each subsequent cruise. No common source was identified, and person-to-person transmission may have occurred. The etiology of these outbreaks would have remained unknown were it not for the recent development of a specific radioimmunoassay (RIA) for the serologic identification of Norwalk virus and the measurement of antibody titers to the virus. A fourfold or greater rise in serum antibody titers to Norwalk antigen in serum from seven of eight ill passengers and the identification of Norwalk antigen in stool specimens from two ill passengers on the first cruise demonstrated that Norwalk virus caused this outbreak. The symptoms experienced by passengers on the four subsequent cruises suggest that a Norwalk agent probably also caused these outbreaks. In addition, a stool specimen from an ill passenger on cruise 3 contained Norwalk antigen.

  View details for Web of Science ID A1980KU88400011

  View details for PubMedID 7457473

• Vomitus new nosocomial infection risk? No standard guidelines for isolation precautions. Hospital infection control Greenberg, H. B. 1979; 6 (3): 29-30

  View details for PubMedID 10240518

• NORWALK VIRUS IN VOMITUS LANCET Greenberg, H. B., WYATT, R. G., KAPIKIAN, A. Z. 1979; 1 (8106): 55-55

  View details for Web of Science ID A1979GC51400063

  View details for PubMedID 83510

• IMMUNE-RESPONSE AND PREVALENCE OF ANTIBODY TO NORWALK ENTERITIS VIRUS AS DETERMINED BY RADIOIMMUNOASSAY JOURNAL OF CLINICAL MICROBIOLOGY Blacklow, N. R., Cukor, G., BEDIGIAN, M. K., Echeverria, P., Greenberg, H. B., Schreiber, D. S., Trier, J. S. 1979; 10 (6): 903-909

  Abstract

  A solid-phase microtiter radioimmunoassay was established for the detection of Norwalk virus and its antibody, with clinical materials from human volunteers previously studied in Massachusetts as reagents. A study of 308 Massachusetts residents showed that serum antibody to Norwalk agent was rarely present during childhood but was detectable in approximately 50% of adults. All volunteers inoculated with Norwalk virus who developed illness seroconverted (10/10), whereas only one-third (5/15) of nonill volunteers seroconverted (P = 0.0009). The 10 nonill, nonseroconverting subjects had undetectable to low preexisting antibody levels. Paradoxically, 10/13 subjects with preexisting antibody became ill, whereas 17/25 lacking antibody did not (P = 0.009). All 3 subjects with preexisting anti-Norwalk radioimmunoassay blocking activity in duodenal intraluminal fluids became ill, whereas only 5/11 lacking such activity developed illness (P = 0.15). These data further support the unique concept that some individuals are susceptible to repeated infections with this agent, whereas others are incapable of developing infection.

  View details for Web of Science ID A1979HY90900025

  View details for PubMedID 118178

• ANTIBODIES TO VIRAL GASTROENTERITIS VIRUSES IN CROHNS-DISEASE GASTROENTEROLOGY Greenberg, H. B., Gebhard, R. L., McClain, C. J., SOLTIS, R. D., KAPIKIAN, A. Z. 1979; 76 (2): 349-350

  Abstract

  Antibody prevalence and titer to rotavirus and Norwalk virus were studied in Crohn's disease patients and in age-, sex-, and time-matched controls. There were no significant antibody differences between the groups studied.

  View details for Web of Science ID A1979GE75900018

  View details for PubMedID 215490

• SOLID-PHASE MICROTITER RADIOIMMUNOASSAY BLOCKING TEST FOR DETECTION OF ANTIBODIES TO ESCHERICHIA-COLI HEAT-LABILE ENTEROTOXIN JOURNAL OF CLINICAL MICROBIOLOGY Greenberg, H. B., LEVINE, M. M., Merson, M. H., Sack, R. B., Sack, D. A., VALDESUSO, J. R., Nalin, D., Hoover, D., CHANOCK, R. M., KAPIKIAN, A. Z. 1979; 9 (1): 60-64

  Abstract

  The development of a solid-phase microtiter radioimmunoassay blocking test to detect serum antibody to Escherichia coli heat-labile enterotoxin is described. The assay is easy to perform and quantitate, and it is sensitive and specific.

  View details for Web of Science ID A1979GN25200011

  View details for PubMedID 372216

• ENHANCEMENT OF ANTIGEN INCORPORATION AND INFECTIVITY OF CELL-CULTURES BY HUMAN ROTAVIRUS JOURNAL OF CLINICAL MICROBIOLOGY Schoub, B. D., KALICA, A. R., Greenberg, H. B., BERTRAN, D. M., SERENO, M. M., WYATT, R. G., CHANOCK, R. M., KAPIKIAN, A. Z. 1979; 9 (4): 488-492

  Abstract

  Infection of cell cultures with human rotavirus preparations was attempted and the effects of trypsin and low-speed centrifugation on antigen incorporation, as demonstrated by immunofluorescence and radioimmunoassay, were determined. In addition, the effect of viral aggregation on antigen incorporation was investigated by filtering viral preparations. Four strains of human rotavirus were employed, and the results were compared to those obtained with two tissue culture-adapted animal rotaviruses. Centrifugation and trypsin appeared to have little or no effect on infectivity of the tissue culture-adapted (simian rotavirus) or -adaptable (Nebraska calf diarrhea virus) strains, whereas centrifugation and viral aggregation appeared to be essential for the human viruses. In addition, trypsin enhanced antigen incorporation of the human strains to some extent. Infectivity for cell cultures and in vitro human rotavirus protein formation was demonstrated by [35S]methionine incorporation, and the specificity of this human viral protein was established by radio-immunoprecipitation.

  View details for Web of Science ID A1979GT13100007

  View details for PubMedID 222805

• PREVALENCE OF ANTIBODY TO THE NORWALK VIRUS IN VARIOUS COUNTRIES INFECTION AND IMMUNITY Greenberg, H. B., VALDESUSO, J., KAPIKIAN, A. Z., CHANOCK, R. M., WYATT, R. G., SZMUNESS, W., LARRICK, J., Kaplan, J., Gilman, R. H., Sack, D. A. 1979; 26 (1): 270-273

  Abstract

  Serum samples from children and adults from several countries were tested by radioimmunoassay for antibody to the Norwalk virus. Antibody was commonly found in adults from all the countries tested. Antibody appears to be acquired more rapidly in children from underdeveloped countries than in children from the United States.

  View details for Web of Science ID A1979HQ70000043

  View details for PubMedID 227798

• DIARRHEA ASSOCIATED WITH ROTAVIRUS IN RURAL GUATEMALA - LONGITUDINAL-STUDY OF 24 INFANTS AND YOUNG-CHILDREN AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE WYATT, R. G., Yolken, R. H., URRUTIA, J. J., Mata, L., Greenberg, H. B., CHANOCK, R. M., KAPIKIAN, A. Z. 1979; 28 (2): 325-328

  Abstract

  A population of 24 infants and young children followed prospectively during the first 3 years of life was studied for the occurrence of rotavirus infection by using enzyme-linked immunosorbent assay to detect virus in stools. Infection with rotavirus was associated with 26 (14.2%) of 183 selected diarrheal episodes. Twenty of the 24 infants and young children had diarrhea associated with rotavirus on at least one occasion and six had two such episodes. Rotavirus infection was documented in over 50% of the dehydrating episodes studied, thus further indicating the importance of rotavirus in this population.

  View details for Web of Science ID A1979GT95200022

  View details for PubMedID 222155

• MYCOPLASMA CONTAMINATION IN CELL-CULTURE OF CROHNS-DISEASE MATERIAL LANCET KAPIKIAN, A. Z., BARILF, M. F., WYATT, R. G., Yolken, R. H., Tully, J. G., Greenberg, H. B., KALICA, A. R., CHANOCK, R. M. 1979; 2 (8140): 466-467

  View details for Web of Science ID A1979HJ63200026

  View details for PubMedID 89521

• ROLE OF NORWALK VIRUS IN OUTBREAKS OF NON-BACTERIAL GASTROENTERITIS JOURNAL OF INFECTIOUS DISEASES Greenberg, H. B., VALDESUSO, J., Yolken, R. H., GANGAROSA, E., GARY, W., WYATT, R. G., Konno, T., Suzuki, H., CHANOCK, R. M., KAPIKIAN, A. Z. 1979; 139 (5): 564-568

  Abstract

  Twenty-five separate outbreaks of nonbacterial gastrointestinal illnesses were studied serologically for evidence of infection with the Norwalk virus and the rotaviruses that affect humans. Eight of 25 outbreaks appeared to be related to the Norwalk virus. In one of the 25 outbreaks, there was evidence of rotavirus infection. These observations suggest that the Norwalk virus or serologically related agents play an important role in epidemic nonbacterial gastroenteritis in adults and older children.

  View details for Web of Science ID A1979GU94100010

  View details for PubMedID 220341

• PROPHYLACTIC DOXYCYCLINE FOR TRAVELERS DIARRHEA - RESULTS OF A PROSPECTIVE DOUBLE-BLIND-STUDY OF PEACE-CORPS VOLUNTEERS IN MOROCCO GASTROENTEROLOGY Sack, R. B., FROEHLICH, J. L., ZULICH, A. W., SIDIHIDI, D., KAPIKIAN, A. Z., ORSKOV, F., ORSKOV, I., Greenberg, H. B. 1979; 76 (6): 1368-1373

  Abstract

  A second randomized double-blind study to determine the efficacy of doxycycline, 100 mg daily, for the prevention of travelers' diarrhea was carried out among 50 Peace Corps Volunteers during their first 10 wk in Morocco. The volunteers took either doxycycline or placebo for 3 wk, and were observed for an additional 7 wk. Eleven of 24 taking the placebo and 2 of 26 taking doxycycline had travelers' diarrhea during the treatment period (P less than 0.01). One week after cessation of the doxycycline, however, persons in that group developed an increase in frequency of travelers' diarrhea (P less than 0.05) so that by 3 wk after the drug was stopped, there were no differences between groups. Enterotoxigenic E. coli, most of which were sensitive to doxycycline, were the most frequently isolated pathogens during the entire study. This study corroborates the effectiveness of doxycycline prophylaxis for travelers' diarrhea.

  View details for Web of Science ID A1979GV91500009

  View details for PubMedID 374178

• EPIDEMIOLOGY OF HUMAN ROTAVIRUS TYPE-1 AND TYPE-2 AS STUDIED BY ENZYME-LINKED IMMUNOSORBENT ASSAY NEW ENGLAND JOURNAL OF MEDICINE Yolken, R. H., WYATT, R. G., ZISSIS, G., BRANDT, C. D., RODRIGUEZ, W. J., KIM, H. W., PARROTT, R. H., URRUTIA, J. J., Mata, L., Greenberg, H. B., KAPIKIAN, A. Z., CHANOCK, R. M. 1978; 299 (21): 1156-1161

  Abstract

  To determine the relative importance of two known serotypes of human rotavirus, we developed an enzyme-linked immunosorbent assay to differentiate serotype-specific rotavirus antigen and antibody. Using this technic, we studied the epidemiology of the two serotypes in acute gastroenteritis. Seventy-seven per cent of 414 rotavirus isolates were Type 2, and the remainder were Type 1. The serotype distribution was similar in specimens from children in Washington, D.C., and other parts of the world. Sero-epidemiologic studies revealed that most children living in the Washington, D.C., area acquired antibody to both types by the age of two years. An analysis of children who were reinfected indicated that sequential infections usually involved different serotypes and that illness caused by one serotype did not provide resistance to illness caused by the other serotype. These results suggest that, to be completely effective, a vaccine must provide resistance to both serotypes.

  View details for Web of Science ID A1978FW86200003

  View details for PubMedID 212678

• SOLID-PHASE MICROTITER RADIOIMMUNOASSAY FOR DETECTION OF NORWALK STRAIN OF ACUTE NON-BACTERIAL, EPIDEMIC GASTROENTERITIS VIRUS AND ITS ANTIBODIES JOURNAL OF MEDICAL VIROLOGY Greenberg, H. B., WYATT, R. G., VALDESUSO, J., KALICA, A. R., London, W. T., CHANOCK, R. M., KAPIKIAN, A. Z. 1978; 2 (2): 97-108

  Abstract

  The development of microtiter solid-phase radioimmunoassays for the detection of Norwalk antigen and its antibody is described. The tests are simple to perform and are sensitive and specific. The test for antigen can be used on crude stool filtrates and suspensions. Both tests are at least as sensitive as immune electron microscopy and more sensitive than immune adherence assay.

  View details for Web of Science ID A1978FC55900003

  View details for PubMedID 97365

• SPONTANEOUS RESOLUTION OF SEVERE APLASTIC-ANEMIA ASSOCIATED WITH VIRAL HEPATITIS-A IN A 6-YEAR-OLD CHILD AMERICAN JOURNAL OF HEMATOLOGY Smith, D., Gribble, T. J., YEAGER, A. S., Greenberg, H. B., Purcell, R. H., Robinson, W., Schwartz, H. C. 1978; 5 (3): 247-252

  Abstract

  Recovery from acquired aplastic anemia associated with hepatitis is rare. This case of a 6-year-old boy with severe aplastic anemia is the first reported association of this disease with a hepatitis A infection. Antibody to hepatitis A (anti-HA) was not detected on admission, but was detected three weeks later. Infection with hepatitis B virus, cytomegalovirus (CMV), and Epstein-Barr virus (EBV) were excluded. The peak titer of anti-HA was higher than would be expected for passive transfer of antibody resulting from transfusions. The persistence of antibody for more than 20 months after the last transfusion was not consistent with passive antibody, which would be expected to disappear over that time. This child had indications for allogeneic marrow transplant and a sibling donor who was histocompatible. However, the transplant was postponed because the prognosis was considered to be poor in the presence of active hepatitis. There was a spontaneous remission without the necessity of the transplantation procedure.

  View details for Web of Science ID A1978GS60000008

  View details for PubMedID 752262

• EXPERIMENTAL-INFECTION OF CHIMPANZEES WITH NORWALK AGENT OF EPIDEMIC VIRAL GASTROENTERITIS JOURNAL OF MEDICAL VIROLOGY WYATT, R. G., Greenberg, H. B., Dalgard, D. W., Allen, W. P., SLY, D. L., Thornhill, T. S., CHANOCK, R. M., KAPIKIAN, A. Z. 1978; 2 (2): 89-96

  Abstract

  A fecal filtrate of human origin containing the Norwalk agent of epidemic viral gastroenteritis was administered by stomach tube to chimpanzees in an attempt to induce diarrheal disease. Significant postchallenge serum antibody rises against Norwalk viral antigens were demonstrated in all animals using the techniques of immune electron microscopy and radioimmunoassay. In addition, viral antigens were detected in feces from five of nine animals using radioimmunoassay. Clinical illness characterized by diarrhea and/or vomiting did not occur. Infection was transmitted subsequently by feeding four additional chimpanzees a fecal filtrate prepared from one of the previously infected animals. Development of an antibody response in four animals and detection of viral antigen in two animals that received this passage filtrate indicated that viral replication had occurred in the absence of clinical illness. The availability of the chimpanzee as an experimental animal host susceptible to infection with the Norwalk agent should facilitate the study of epidemic viral gastroenteritis.

  View details for Web of Science ID A1978FC55900002

  View details for PubMedID 97364

• DETECTION OF NORWALK AGENT ANTIBODY AND ANTIGEN BY SOLID-PHASE RADIOIMMUNOASSAY AND IMMUNE ADHERENCE HEMAGGLUTINATION ASSAY JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION Greenberg, H. B., KAPIKIAN, A. Z. 1978; 173 (5): 620-623

  View details for Web of Science ID A1978FP10100028

  View details for PubMedID 100482

• PREVALENCE OF ANTIBODY TO NORWALK AGENT BY A NEWLY DEVELOPED IMMUNE ADHERENCE HEMAGGLUTINATION ASSAY JOURNAL OF MEDICAL VIROLOGY KAPIKIAN, A. Z., Greenberg, H. B., CLINE, W. L., KALICA, A. R., WYATT, R. G., JAMES, H. D., Lloyd, N. L., CHANOCK, R. M., Ryder, R. W., KIM, H. W. 1978; 2 (4): 281-294

  Abstract

  An immune adherence hemagglutination assay (IAHA) for the detection of antibody to the Norwalk agent of acute epidemic nonbacterial gastroenteritis was developed using as antigen virus purified from stool from an experimentally infected volunteer. The assay was sensitive and specific and was efficient for detecting Norwalk antibody seroresponses. The prevalence of Norwalk antibody in various groups in the United States was studied. Antibody to the Norwalk agent was acquired gradually, beginning slowly in childhood and accelerating in the adult period so that by the fifth decade 50% possessed antibody. This pattern of antibody acquisition contrasted sharply with that for the human rotavirus of infantile gastroenteritis. Rotavirus antibody was acquired during early childhood by almost all individuals in the pediatric groups studied. Antibody to the Norwalk agent was also found in rural Bangladesh; in a small prevalence survey of 39 children and adults 21% possessed Norwalk IAHA antibody, whereas 95% possessed antibody to the human rotavirus.

  View details for Web of Science ID A1978FV58500001

  View details for PubMedID 215713

• STRUCTURE OF HEPATITIS-B DANE PARTICLE DNA AND NATURE OF ENDOGENOUS DNA-POLYMERASE REACTION JOURNAL OF VIROLOGY Landers, T. A., Greenberg, H. B., Robinson, W. S. 1977; 23 (2): 368-376

  Abstract

  The circular DNA of hepatitis B Dane particles, which serves as the primer/template for an endogenous DNA polymerase, was analyzed by electrophoresis before and after a polymerase reaction and after digestion by restriction endonuclease or single-strand-specific endonuclease S1. The unreacted molecules extracted from the particles were electrophoretically heterogeneous, and treatment with S1 nuclease produced double-stranded linear DNA ranging in length from 1,700 to 2,800 base pairs (bp). After an endogenous DNA polymerase reaction, two discrete species of DNA molecules were found: a circular form and a linear form 3,200 bp long. The reaction resulted in a population of molecules with an elongated and more homogeneous double-stranded region. These results suggest that the circular molecules in Dane particles have single-stranded regions of varying lengths that are made double stranded during the DNA polymerase reaction. The endogenous DNA polymerase was found to initiate apparently at random in a region spanning more than a third of the molecule. Analysis of restriction endonuclease cleavage fragments of the fully elongated DNA revealed that although the molecules were of a uniform length, they were somewhat heterogeneous in sequence. The sum of the sizes of the 10 major endonuclease Hae III-generated fragments, detected by ethidium bromide, was 3,880 bp. Two additional fragments (B and G) detected by autoradiography after an endogenous DNA polymerase reaction with (32)P-labeled deoxynucleoside triphosphates made the total 4,910 bp.

  View details for Web of Science ID A1977DP49000017

  View details for PubMedID 69727

• IMMUNOPROPHYLAXIS OF EXPERIMENTAL MYCOPLASMA-PNEUMONIAE DISEASE - EFFECT OF ROUTE OF ADMINISTRATION ON IMMUNOGENICITY AND PROTECTIVE EFFECT OF INACTIVATED MYCOPLASMA-PNEUMONIAE VACCINE INFECTION AND IMMUNITY Greenberg, H., Helms, C. M., GRIZZARD, M. B., James, W. D., HORSWOOD, R. L., CHANOCK, R. M. 1977; 16 (1): 88-92

  Abstract

  Formalin-inactivated Mycoplasma pneumoniae vaccine was administered subcutaneously or intranasally to hamsters to examine the effect of route of administration on immunogenicity and protective effect. Parenterally administered vaccine in the doses employed induced serum complement-fixing antibody formation, but did not significantly decrease the frequency of pneumonia following challenge with virulent M. pneumoniae. Intranasally instilled vaccine was ineffective in stimulating serum antibody, but did diminish the frequency of experimentally induced pneumonia due to M. pneumoniae. However, a greater degree of resistance was induced by intranasal infection with either wild-type organisms or the ts 640 attenuated mutant of M. pneumoniae.

  View details for Web of Science ID A1977DD50500015

  View details for PubMedID 873618

• ENZYME-LINKED IMMUNOSORBENT ASSAY FOR DETECTION OF ESCHERICHIA-COLI HEAT-LABILE ENTEROTOXIN JOURNAL OF CLINICAL MICROBIOLOGY Yolken, R. H., Greenberg, H. B., Merson, M. H., Sack, R. B., KAPIKIAN, A. Z. 1977; 6 (5): 439-444

  Abstract

  The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of heat-labile Escherichia coli enterotoxin is described. The assay, which is based on the immunological similarity between Vibrio cholerae toxin and heat-labile E. coli enterotoxin, is similar in design to a radioimmunoassay but utilizes enzyme-labeled rather than radioactive isotope-labeled reagents. The ELISA system is as sensitive as both radioimmunoassay and the y-1 adrenal cell assay for the detection of heat-labile E. coli enterotoxin but requires neither radioactive reagents nor tissue culture techniques. The ELISA is easy to perform and is adaptable for use in small laboratories.

  View details for Web of Science ID A1977EC23900001

  View details for PubMedID 336638

• MICROTITER SOLID-PHASE RADIOIMMUNOASSAY FOR DETECTION OF ESCHERICHIA-COLI HEAT-LABILE ENTEROTOXIN INFECTION AND IMMUNITY Greenberg, H. B., Sack, D. A., Rodriguez, W., BRADLEYSACK, R., WYATT, R. G., KALICA, A. R., HORSWOOD, R. L., CHANOCK, R. M., KAPIKIAN, A. Z. 1977; 17 (3): 541-545

  Abstract

  The development of a microtiter solid-phase radioimmunoassay for the detection of Vibrio cholerae enterotoxin and heat-labile Escherichia coli enterotoxin is described. The test is based on the immunological similarity between V. cholerae toxin and E. coli heat-labile toxin. The assay is easy to perform, quantitative, and at least as sensitive and specific as the Y-1 adrenal cell system.

  View details for Web of Science ID A1977DV23100012

  View details for PubMedID 332626

• UNEXPECTEDLY HIGH-FREQUENCY OF ANTIBODY TO MYCOPLASMA-PNEUMONIAE IN HUMAN SERA AS MEASURED BY SENSITIVE TECHNIQUES JOURNAL OF INFECTIOUS DISEASES Brunner, H., Prescott, B., Greenberg, H., James, W. D., HORSWOOD, R. L., CHANOCK, R. M. 1977; 135 (4): 524-530

  Abstract

  Mycoplasmacidal and radioimmunoprecipitating antibodies to Mycoplasma pneumoniae were commonly detected in healthy children younger than five years of age, in whom natural disease due to M. pneumoniae is rare. Antibody in serum could also be demonstrated in Marine recruits several weeks or months before development of pneumonia due to M. pneumoniae. The latter observation suggests that serum antibody does not confer protection against disease caused by M. pneumoniae.

  View details for Web of Science ID A1977DD62600005

  View details for PubMedID 323374

• EFFECT OF HUMAN LEUKOCYTE INTERFERON ON HEPATITIS B VIRUS-INFECTION IN PATIENTS WITH CHRONIC ACTIVE HEPATITIS NEW ENGLAND JOURNAL OF MEDICINE Greenberg, H. B., Pollard, R. B., Lutwick, L. I., Gregory, P. B., Robinson, W. S., Merigan, T. C. 1976; 295 (10): 517-522

  Abstract

  Four patients with chronic hepatitis B infection and chronic active hepatitis were treated with human leukocyte interferon. Three of them had consistently elevated levels of circulating Dane-particle markers, including Dane-particle-associated DNA polymerase activity, hepatitis B core antigen and Dane-particle-associated DNA. Parenteral interferon administration at a dosage between 6.0 X 10(3) and 17 X 10(4) U per kilogram per day was associated with a rapid and reproducible fall in all Dane-particle markers in the three patients. The suppressive effect was transient when the interferon was given for 10 days or less but appeared to be more permanent when administration was prolonged for a month or more. In addition, long-term interferon therapy was associated with a marked fall in hepatitis B surface antigen in two of three patients and a disappearance of e antigen in two of two patients. Interferon may be useful in limiting carrier infectivity or eradicating chronic infection.

  View details for Web of Science ID A1976CB97100001

  View details for PubMedID 950957

• ASYMPTOMATIC INFECTION OF ADULT VOLUNTEERS WITH A TEMPERATURE SENSITIVE MUTANT OF MYCOPLASMA-PNEUMONIAE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Greenberg, H., Helms, C. M., Brunner, H., CHANOCK, R. M. 1974; 71 (10): 4015-4019

  Abstract

  Temperature sensitive mutants of Mycoplasma pneumoniae were developed with the expectation that their temperature sensitive defects would restrict replication in vivo at the temperature of the lower respiratory tract, whereas such defects would not seriously impair replication in the cooler environment of the upper respiratory passages. One such ts mutant, ts-H43, which does not replicate at a temperature of 37 degrees or above, although noninfectious for hamsters, infected each of 16 seronegative adult volunteers when given by the intranasal route. The mutant remained genetically stable throughout the course of infection and stimulated a moderate systemic and local antibody response. The mutant was entirely avirulent for the volunteers but appeared to stimulate resistance to subsequent challenge with partially attenuated wild-type (ts(+)) Mycoplasma pneumoniae.

  View details for Web of Science ID A1974U614000050

  View details for PubMedID 4530281

• ANTIBODY TO MYCOPLASMA-PNEUMONIAE IN NASAL SECRETIONS AND SPUTA OF EXPERIMENTALLY INFECTED HUMAN VOLUNTEERS INFECTION AND IMMUNITY Brunner, H., GREENBER, H. B., James, W. D., HORSWOOD, R. L., Couch, R. B., CHANOCK, R. M. 1973; 8 (4): 612-620

  Abstract

  After experimental infection with Mycoplasma pneumoniae, 42% of 67 volunteers developed a threefold or greater rise in antibody in nasal secretions as measured by radioimmunoprecipitation. Development of an antibody increase in sputum was detected more often, i.e., in 73% of the volunteers. Each of the antibody increases involved immunoglobulin (Ig) A. Twelve rises in IgG antibody were detected in the specimens which exhibited a rise in IgA antibody. In almost every instance the rise in IgA antibody exceeded that seen with IgG antibody. Analysis of the response to experimental challenge with M. pneumoniae of volunteers with different levels of preexisting respiratory tract IgA antibody suggested that this secretory antibody was related to host resistance to M. pneumoniae disease. Further, respiratory tract IgA antibody appeared to be more directly related to host resistance than was antibody in serum.

  View details for Web of Science ID A1973Q784800018

  View details for PubMedID 4582638

• DECREASED VIRULENCE AND PROTECTIVE EFFECT OF GENETICALLY STABLE TEMPERATURE-SENSITIVE MUTANTS OF MYCOPLASMA-PNEUMONIAE ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Brunner, H., Greenberg, H., James, W. D., HORSWOOD, R. L., CHANOCK, R. M. 1973; 225 (DEC14): 436-452

  View details for Web of Science ID A1973R916000039

• ANALYSIS OF ANTIBODY RESPONSE TO AUSTRALIA ANTIGEN IN MAN JOURNAL OF INFECTIOUS DISEASES Greenberg, H. B., GOCKE, D. J. 1971; 123 (4): 356-?

  View details for Web of Science ID A1971J002800003

  View details for PubMedID 4999903

• CORRELATION OF AUSTRALIA ANTIGEN WITH POSTTRANSFUSION HEPATITIS JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION GOCKE, D. J., GREENBER, H. B., Kavey, N. B. 1970; 212 (5): 877-?

  View details for Web of Science ID A1970G175000061

  View details for PubMedID 5467384

• HEPATITIS ANTIGEN - DETECTION OF INFECTIOUS BLOOD DONORS LANCET GOCKE, D. J., GREENBER, H. B., Kavey, N. B. 1969; 2 (7614): 248-?

  View details for Web of Science ID A1969D824700011

  View details for PubMedID 4184114