Michelle Jade Lin

All Publications

• Cutaneous Neurofibromas and Quality of Life in Adults With Neurofibromatosis Type 1. JAMA dermatology Lin, M. J., Yao, H., Vera, K., Patel, E., Johnson, M., Caroline, P., Ramos, J., Mehta, J., Hu, X., Blakeley, J. O., Romo, C. G., Sarin, K. Y. 2024

  Abstract

  There is a burgeoning interest in therapeutic development for cutaneous neurofibromas (cNFs), a major cause of morbidity in persons with neurofibromatosis type 1 (NF1). To determine meaningful clinical trial outcomes, deeper understanding is needed regarding how cNFs are associated with quality of life (QoL). However, this understanding has been hampered by challenges in recruiting participants with this rare genetic disease.To develop a large, crowdsourced validated registry of persons with NF1 and determine the association of specific cNF features with QoL, pain, and itch.From May 2021 to December 2023, a decentralized platform was developed and recruited persons 40 years or older with NF1 and at least 1 cNF from 49 states and 12 countries, who provided clinical survey data, detailed photographs, and genetic sequencing data. Photographs from 583 participants were scored on 12 features of cNFs, including general severity, number, size, facial severity, color, and subtypes.cNF features derived from participant-supplied photographs.Total Skindex scores and subdomain scores (symptoms, emotion, function, pain, and itch).Of 583 participants, 384 (65.9%) were female, and the mean (range) age was 51.7 (40.0-83.0) years. Female sex, general severity, number, size, and facial severity of cNFs were negatively associated with QoL, as demonstrated by increased total Skindex scores. QoL had the largest association with the number of cNFs and presence of facial cNFs. Increasing number of cNFs was associated with worse QoL, and even individuals with a low cNF burden (<10 total cNFs) experienced a decrease in QoL.The results of this study suggest that reducing cNF number, particularly on the face, may be associated with improved QoL in individuals with NF1. In addition, early intervention before the development of numerous tumors may lead to the highest benefit in QoL. These data potentially provide insight into which individuals and cNF tumors may benefit most from therapy and highlights the utility of a completely decentralized, photograph-validated and age-controlled study for rare genetic disease. This cohort will allow analysis of disease and tumor heterogeneity after full phenotypic expression is achieved in NF1 and potentially serves as an example in its design for other rare diseases that struggle from poor recruitment.

  View details for DOI 10.1001/jamadermatol.2024.2912

  View details for PubMedID 39196570

• Clinical performance characteristics of the Swift Normalase Amplicon Panel for sensitive recovery of SARS-CoV-2 genomes. The Journal of molecular diagnostics : JMD Shrestha, L., Lin, M. J., Xie, H., Mills, M. G., Mohamed Bakhash, S. A., Gaur, V. P., Livingston, R. J., Castor, J., Bruce, E. A., Botten, J. W., Huang, M. L., Jerome, K. R., Greninger, A. L., Roychoudhury, P. 2022

  Abstract

  Amplicon-based sequencing methods have been central in characterizing the diversity, transmission, and evolution of SARS-CoV-2, but need to be rigorously assessed for clinical utility. Here, we validated the Swift Biosciences' SARS-CoV-2 Swift Normalase Amplicon Panels using remnant clinical specimens. High quality genomes meeting our established library and sequence quality criteria were recovered from positive specimens with 95% limit of detection of 40.08 SARS-CoV-2 copies/PCR reaction. Breadth of genome recovery ≥was evaluated across a range of Ct values (11.3 - 36.7, median 21.6). Out of 428 positive samples, 413 (96.5%) generated genomes with < 10% Ns, with a mean genome coverage of 13,545X ± SD 8,382X. No genomes were recovered from PCR-negative specimens (n = 30), or from specimens positive for non-SARS-CoV-2 respiratory viruses (n = 20). Compared to whole-genome shotgun metagenomic sequencing (n = 14) or Sanger sequencing for the spike gene (n = 11), pairwise identity between consensus sequences was 100% in all cases, with highly concordant allele frequencies (R2 = 0.99) between Swift and shotgun libraries. When samples from different clades were mixed at varying ratios, expected variants were detected even in 1:99 mixtures. When deployed as a clinical test, 268 tests were performed in the first 23 weeks with a median turnaround time of 11 days, ordered primarily for outbreak investigations and infection control.

  View details for DOI 10.1016/j.jmoldx.2022.05.007

  View details for PubMedID 35863699

  View details for PubMedCentralID PMC9290336

• Fragment Size-Based Enrichment of Viral Sequences in Plasma Cell-Free DNA JOURNAL OF MOLECULAR DIAGNOSTICS Phung, Q., Lin, M. J., Xie, H., Greninger, A. L. 2022; 24 (5): 476-484

  Abstract

  Sequencing of plasma cell-free DNA (cfDNA) is a promising milieu for broad-based cancer and infectious disease diagnostics. The performance of cfDNA sequencing for infectious disease diagnostics is chiefly limited by inadequate analytical sensitivity. The current study investigated whether the analytical sensitivity of cfDNA sequencing for viral diagnostics could be improved by selective sequencing of short cfDNA fragments, given prior observations of shorter fragment size distribution in microbial and cytomegalovirus-derived cfDNA compared with human-derived cfDNA. It shows that the shorter plasma cfDNA fragment size distribution is a general feature of multiple DNA viruses, including adenovirus [interquartile range (IQR), 87 to 165 bp], herpes simplex virus 2 (IQR, 114 to 195 bp), human herpesvirus 6 (IQR, 145 to 176 bp), and varicella zoster virus (IQR, 98 to 182 bp), compared with human (IQR, 148 to 178 bp). It was used to further optimize a size selection-based cfDNA sequencing method, demonstrating an enrichment of viral sequences up to 16.6-fold, with a median fold enrichment of 6.7×, 4.6×, 2.2×, and 10.3× for adenovirus, herpes simplex virus 2, human herpesvirus 6, and varicella zoster virus, respectively. These findings demonstrate a simple yet scalable method for enhanced detection of DNA viremia that maintains the unbiased nature of cfDNA sequencing.

  View details for DOI 10.1016/j.jmoldx.2022.01.007

  View details for Web of Science ID 000806495700007

  View details for PubMedID 35569878

  View details for PubMedCentralID PMC9127460

• Host-pathogen dynamics in longitudinal clinical specimens from patients with COVID-19. Scientific reports Lin, M. J., Rachleff, V. M., Xie, H., Shrestha, L., Lieberman, N. A., Peddu, V., Addetia, A., Casto, A. M., Breit, N., Mathias, P. C., Huang, M. L., Jerome, K. R., Greninger, A. L., Roychoudhury, P. 2022; 12 (1): 5856

  Abstract

  Rapid dissemination of SARS-CoV-2 sequencing data to public repositories has enabled widespread study of viral genomes, but studies of longitudinal specimens from infected persons are relatively limited. Analysis of longitudinal specimens enables understanding of how host immune pressures drive viral evolution in vivo. Here we performed sequencing of 49 longitudinal SARS-CoV-2-positive samples from 20 patients in Washington State collected between March and September of 2020. Viral loads declined over time with an average increase in RT-QPCR cycle threshold of 0.87 per day. We found that there was negligible change in SARS-CoV-2 consensus sequences over time, but identified a number of nonsynonymous variants at low frequencies across the genome. We observed enrichment for a relatively small number of these variants, all of which are now seen in consensus genomes across the globe at low prevalence. In one patient, we saw rapid emergence of various low-level deletion variants at the N-terminal domain of the spike glycoprotein, some of which have previously been shown to be associated with reduced neutralization potency from sera. In a subset of samples that were sequenced using metagenomic methods, differential gene expression analysis showed a downregulation of cytoskeletal genes that was consistent with a loss of ciliated epithelium during infection and recovery. We also identified co-occurrence of bacterial species in samples from multiple hospitalized individuals. These results demonstrate that the intrahost genetic composition of SARS-CoV-2 is dynamic during the course of COVID-19, and highlight the need for continued surveillance and deep sequencing of minor variants.

  View details for DOI 10.1038/s41598-022-09752-2

  View details for PubMedID 35393464

  View details for PubMedCentralID PMC8987511

• De novo emergence of a remdesivir resistance mutation during treatment of persistent SARS-CoV-2 infection in an immunocompromised patient: a case report NATURE COMMUNICATIONS Gandhi, S., Klein, J., Robertson, A., Pena-Hernandez, M. A., Lin, M. J., Roychoudhury, P., Lu, P., Fournier, J., Ferguson, D., Bakhash, S., Muenker, M., Srivathsan, A., Wunder, E. A., Kerantzas, N., Wang, W., Lindenbach, B., Pyle, A., Wilen, C. B., Ogbuagu, O., Greninger, A. L., Iwasaki, A., Schulz, W. L., Ko, A. 2022; 13 (1): 1547

  Abstract

  SARS-CoV-2 remdesivir resistance mutations have been generated in vitro but have not been reported in patients receiving treatment with the antiviral agent. We present a case of an immunocompromised patient with acquired B-cell deficiency who developed an indolent, protracted course of SARS-CoV-2 infection. Remdesivir therapy alleviated symptoms and produced a transient virologic response, but her course was complicated by recrudescence of high-grade viral shedding. Whole genome sequencing identified a mutation, E802D, in the nsp12 RNA-dependent RNA polymerase, which was not present in pre-treatment specimens. In vitro experiments demonstrated that the mutation conferred a ~6-fold increase in remdesivir IC50 but resulted in a fitness cost in the absence of remdesivir. Sustained clinical and virologic response was achieved after treatment with casirivimab-imdevimab. Although the fitness cost observed in vitro may limit the risk posed by E802D, this case illustrates the importance of monitoring for remdesivir resistance and the potential benefit of combinatorial therapies in immunocompromised patients with SARS-CoV-2 infection.

  View details for DOI 10.1038/s41467-022-29104-y

  View details for Web of Science ID 000770426200040

  View details for PubMedID 35301314

  View details for PubMedCentralID PMC8930970

• Retrospective Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Symptomatic Patients Prior to Widespread Diagnostic Testing in Southern California CLINICAL INFECTIOUS DISEASES Hilt, E. E., Boocock, J., Trejo, M., Le, C. Q., Guo, L., Zhang, Y., Sathe, L., Arboleda, V. A., Yin, Y., Bloom, J. S., Wang, P., Elmore, J. G., Kruglyak, L., Shrestha, L., Bakhash, S., Lin, M., Xie, H., Huang, M., Roychoudhury, P., Greninger, A., Chandrasekaran, S., Yang, S., Garner, O. B. 2022; 74 (2): 271-277

  Abstract

  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused one of the worst pandemics in recent history. Few reports have revealed that SARS-CoV-2 was spreading in the United States as early as the end of January. In this study, we aimed to determine if SARS-CoV-2 had been circulating in the Los Angeles (LA) area at a time when access to diagnostic testing for coronavirus disease 2019 (COVID-19) was severely limited.We used a pooling strategy to look for SARS-CoV-2 in remnant respiratory samples submitted for regular respiratory pathogen testing from symptomatic patients from November 2019 to early March 2020. We then performed sequencing on the positive samples.We detected SARS-CoV-2 in 7 specimens from 6 patients, dating back to mid-January. The earliest positive patient, with a sample collected on January 13, 2020 had no relevant travel history but did have a sibling with similar symptoms. Sequencing of these SARS-CoV-2 genomes revealed that the virus was introduced into the LA area from both domestic and international sources as early as January.We present strong evidence of community spread of SARS-CoV-2 in the LA area well before widespread diagnostic testing was being performed in early 2020. These genomic data demonstrate that SARS-CoV-2 was being introduced into Los Angeles County from both international and domestic sources in January 2020.

  View details for DOI 10.1093/cid/ciab360

  View details for Web of Science ID 000771063600013

  View details for PubMedID 33939799

  View details for PubMedCentralID PMC8135745

• Treponema pallidum genome sequencing from six continents reveals variability in vaccine candidate genes and dominance of Nichols clade strains in Madagascar PLOS NEGLECTED TROPICAL DISEASES Lieberman, N. P., Lin, M. J., Xie, H., Shrestha, L., Nguyen, T., Huang, M., Haynes, A. M., Romeis, E., Wang, Q., Zhang, R., Kou, C., Ciccarese, G., Dal Conte, I., Cusini, M., Drago, F., Nakayama, S., Lee, K., Ohnishi, M., Konda, K. A., Vargas, S. K., Eguiluz, M., Caceres, C. F., Klausner, J. D., Mitja, O., Rompalo, A., Mulcahy, F., Hook, E. W., Lukehart, S. A., Casto, A. M., Roychoudhury, P., DiMaio, F., Giacani, L., Greninger, A. L. 2021; 15 (12): e0010063

  Abstract

  In spite of its immutable susceptibility to penicillin, Treponema pallidum (T. pallidum) subsp. pallidum continues to cause millions of cases of syphilis each year worldwide, resulting in significant morbidity and mortality and underscoring the urgency of developing an effective vaccine to curtail the spread of the infection. Several technical challenges, including absence of an in vitro culture system until very recently, have hampered efforts to catalog the diversity of strains collected worldwide. Here, we provide near-complete genomes from 196 T. pallidum strains-including 191 T. pallidum subsp. pallidum-sequenced directly from patient samples collected from 8 countries and 6 continents. Maximum likelihood phylogeny revealed that samples from most sites were predominantly SS14 clade. However, 99% (84/85) of the samples from Madagascar formed two of the five distinct Nichols subclades. Although recombination was uncommon in the evolution of modern circulating strains, we found multiple putative recombination events between T. pallidum subsp. pallidum and subsp. endemicum, shaping the genomes of several subclades. Temporal analysis dated the most recent common ancestor of Nichols and SS14 clades to 1717 (95% HPD: 1543-1869), in agreement with other recent studies. Rates of SNP accumulation varied significantly among subclades, particularly among different Nichols subclades, and was associated in the Nichols A subclade with a C394F substitution in TP0380, a ERCC3-like DNA repair helicase. Our data highlight the role played by variation in genes encoding putative surface-exposed outer membrane proteins in defining separate lineages, and provide a critical resource for the design of broadly protective syphilis vaccines targeting surface antigens.

  View details for DOI 10.1371/journal.pntd.0010063

  View details for Web of Science ID 000733436300002

  View details for PubMedID 34936652

  View details for PubMedCentralID PMC8735616

• Human parainfluenza virus evolution during lung infection of immunocompromised individuals promotes viral persistence JOURNAL OF CLINICAL INVESTIGATION Greninger, A. L., Rybkina, K., Lin, M. J., Drew-Bear, J., Marcink, T. C., Shean, R. C., Makhsous, N., Boeckh, M., Harder, O., Bovier, F., Burstein, S. R., Niewiesk, S., Rima, B. K., Porotto, M., Moscona, A. 2021; 131 (23)

  Abstract

  The capacity of respiratory viruses to undergo evolution within the respiratory tract raises the possibility of evolution under the selective pressure of the host environment or drug treatment. Long-term infections in immunocompromised hosts are potential drivers of viral evolution and development of infectious variants. We showed that intrahost evolution in chronic human parainfluenza virus 3 (HPIV3) infection in immunocompromised individuals elicited mutations that favored viral entry and persistence, suggesting that similar processes may operate across enveloped respiratory viruses. We profiled longitudinal HPIV3 infections from 2 immunocompromised individuals that persisted for 278 and 98 days. Mutations accrued in the HPIV3 attachment protein hemagglutinin-neuraminidase (HN), including the first in vivo mutation in HN's receptor binding site responsible for activating the viral fusion process. Fixation of this mutation was associated with exposure to a drug that cleaves host-cell sialic acid moieties. Longitudinal adaptation of HN was associated with features that promote viral entry and persistence in cells, including greater avidity for sialic acid and more active fusion activity in vitro, but not with antibody escape. Long-term infection thus led to mutations promoting viral persistence, suggesting that host-directed therapeutics may support the evolution of viruses that alter their biophysical characteristics to persist in the face of these agents in vivo.

  View details for DOI 10.1172/JCI150506

  View details for Web of Science ID 000726765900002

  View details for PubMedID 34609969

  View details for PubMedCentralID PMC8631596

• Oral prodrug of remdesivir parent GS-441524 is efficacious against SARS-CoV-2 in ferrets NATURE COMMUNICATIONS Cox, R. M., Wolf, J. D., Lieber, C. M., Sourimant, J., Lin, M. J., Babusis, D., DuPont, V., Chan, J., Barrett, K. T., Lye, D., Kalla, R., Chun, K., Mackman, R. L., Ye, C., Cihlar, T., Martinez-Sobrido, L., Greninger, A. L., Bilello, J. P., Plemper, R. K. 2021; 12 (1): 6415

  Abstract

  Remdesivir is an antiviral approved for COVID-19 treatment, but its wider use is limited by intravenous delivery. An orally bioavailable remdesivir analog may boost therapeutic benefit by facilitating early administration to non-hospitalized patients. This study characterizes the anti-SARS-CoV-2 efficacy of GS-621763, an oral prodrug of remdesivir parent nucleoside GS-441524. Both GS-621763 and GS-441524 inhibit SARS-CoV-2, including variants of concern (VOC) in cell culture and human airway epithelium organoids. Oral GS-621763 is efficiently converted to plasma metabolite GS-441524, and in lungs to the triphosphate metabolite identical to that generated by remdesivir, demonstrating a consistent mechanism of activity. Twice-daily oral administration of 10 mg/kg GS-621763 reduces SARS-CoV-2 burden to near-undetectable levels in ferrets. When dosed therapeutically against VOC P.1 gamma γ, oral GS-621763 blocks virus replication and prevents transmission to untreated contact animals. These results demonstrate therapeutic efficacy of a much-needed orally bioavailable analog of remdesivir in a relevant animal model of SARS-CoV-2 infection.

  View details for DOI 10.1038/s41467-021-26760-4

  View details for Web of Science ID 000714972500023

  View details for PubMedID 34741049

  View details for PubMedCentralID PMC8571282

• A Bifluorescent-Based Assay for the Identification of Neutralizing Antibodies against SARS-CoV-2 Variants of Concern In Vitro and In Vivo JOURNAL OF VIROLOGY Chiem, K., Vasquez, D., Silvas, J. A., Park, J., Piepenbrink, M. S., Sourimant, J., Lin, M. J., Greninger, A. L., Plemper, R. K., Torrelles, J. B., Walter, M. R., de la Torre, J. C., Kobie, J. K., Ye, C., Martinez-Sobrido, L. 2021; 95 (22): e0112621

  Abstract

  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and has been responsible for the still ongoing coronavirus disease 2019 (COVID-19) pandemic. Prophylactic vaccines have been authorized by the U.S. Food and Drug Administration (FDA) for the prevention of COVID-19. Identification of SARS-CoV-2-neutralizing antibodies (NAbs) is important to assess vaccine protection efficacy, including their ability to protect against emerging SARS-CoV-2 variants of concern (VoC). Here, we report the generation and use of a recombinant (r)SARS-CoV-2 USA/WA1/2020 (WA-1) strain expressing Venus and an rSARS-CoV-2 strain expressing mCherry and containing mutations K417N, E484K, and N501Y found in the receptor binding domain (RBD) of the spike (S) glycoprotein of the South African (SA) B.1.351 (beta [β]) VoC in bifluorescent-based assays to rapidly and accurately identify human monoclonal antibodies (hMAbs) able to neutralize both viral infections in vitro and in vivo. Importantly, our bifluorescent-based system accurately recapitulated findings observed using individual viruses. Moreover, fluorescent-expressing rSARS-CoV-2 strain and the parental wild-type (WT) rSARS-CoV-2 WA-1 strain had similar viral fitness in vitro, as well as similar virulence and pathogenicity in vivo in the K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 infection. We demonstrate that these new fluorescent-expressing rSARS-CoV-2 can be used in vitro and in vivo to easily identify hMAbs that simultaneously neutralize different SARS-CoV-2 strains, including VoC, for the rapid assessment of vaccine efficacy or the identification of prophylactic and/or therapeutic broadly NAbs for the treatment of SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 is responsible of the COVID-19 pandemic that has warped daily routines and socioeconomics. There is still an urgent need for prophylactics and therapeutics to treat SARS-CoV-2 infections. In this study, we demonstrate the feasibility of using bifluorescent-based assays for the rapid identification of hMAbs with neutralizing activity against SARS-CoV-2, including VoC in vitro and in vivo. Importantly, results obtained with these bifluorescent-based assays recapitulate those observed with individual viruses, demonstrating their feasibility to rapidly advance our understanding of vaccine efficacy and to identify broadly protective human NAbs for the therapeutic treatment of SARS-CoV-2.

  View details for DOI 10.1128/JVI.01126-21

  View details for Web of Science ID 000718342500001

  View details for PubMedID 34495697

  View details for PubMedCentralID PMC8549516

• Analysis of SARS-CoV-2 infection dynamic in vivo using reporter-expressing viruses PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ye, C., Chiem, K., Park, J., Silvas, J. A., Vasquez, D., Sourimant, J., Lin, M. J., Greninger, A. L., Plemper, R. K., Torrelles, J. B., Kobie, J. J., Walter, M. R., de la Torre, J., Martinez-Sobrido, L. 2021; 118 (41)

  Abstract

  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, is one of the biggest threats to public health. However, the dynamic of SARS-CoV-2 infection remains poorly understood. Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing recombinant (r)SARS-CoV-2 in the locus of the open reading frame (ORF)7a protein have jeopardized their use to monitor the dynamic of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the highly expressed viral nucleocapsid (N) gene followed by a porcine tescherovirus (PTV-1) 2A proteolytic cleavage site. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and excised lungs or whole organism of infected K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice. Importantly, real-time viral infection was readily tracked using a noninvasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2, in which a viral gene was not deleted, not only retained wild-type (WT) virus-like pathogenicity in vivo but also exhibited high stability in vitro and in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis, and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

  View details for DOI 10.1073/pnas.2111593118

  View details for Web of Science ID 000707948500014

  View details for PubMedID 34561300

  View details for PubMedCentralID PMC8521683

• Longitudinal TprK profiling of in vivo and in vitro-propagated Treponema pallidum subsp. pallidum reveals accumulation of antigenic variants in absence of immune pressure PLOS NEGLECTED TROPICAL DISEASES Lin, M. J., Haynes, A. M., Addetia, A., Lieberman, N. P., Phung, Q., Xie, H., Nguyen, T. V., Molini, B. J., Lukehart, S. A., Giacani, L., Greninger, A. L. 2021; 15 (9): e0009753

  Abstract

  Immune evasion by Treponema pallidum subspecies pallidum (T. pallidum) has been attributed to antigenic variation of its putative outer-membrane protein TprK. In TprK, amino acid diversity is confined to seven variable (V) regions, and generation of sequence diversity within the V regions occurs via a non-reciprocal segmental gene conversion mechanism where donor cassettes recombine into the tprK expression site. Although previous studies have shown the significant role of immune selection in driving accumulation of TprK variants, the contribution of baseline gene conversion activity to variant diversity is less clear. Here, combining longitudinal tprK deep sequencing of near clonal Chicago C from immunocompetent and immunosuppressed rabbits along with the newly developed in vitro cultivation system for T. pallidum, we directly characterized TprK alleles in the presence and absence of immune selection. Our data confirm significantly greater sequence diversity over time within the V6 region during syphilis infection in immunocompetent rabbits compared to immunosuppressed rabbits, consistent with previous studies on the role of TprK in evasion of the host immune response. Compared to strains grown in immunocompetent rabbits, strains passaged in vitro displayed low level changes in allele frequencies of TprK variable region sequences similar to that of strains passaged in immunosuppressed rabbits. Notably, we found significantly increased rates of V6 allele generation relative to other variable regions in in vitro cultivated T, pallidum strains, illustrating that the diversity within these hypervariable regions occurs in the complete absence of immune selection. Together, our results demonstrate antigenic variation in T. pallidum can be studied in vitro and occurs even in the complete absence of immune pressure, allowing the T. pallidum population to continuously evade the immune system of the infected host.

  View details for DOI 10.1371/journal.pntd.0009753

  View details for Web of Science ID 000729033700003

  View details for PubMedID 34492041

  View details for PubMedCentralID PMC8480903

• Specific allelic discrimination of N501Y and other SARS-CoV-2 mutations by ddPCR detects B.1.1.7 lineage in Washington State JOURNAL OF MEDICAL VIROLOGY Perchetti, G. A., Zhu, H., Mills, M. G., Shrestha, L., Wagner, C., Bakhash, S., Lin, M. J., Xie, H., Huang, M., Mathias, P., Bedford, T., Jerome, K. R., Greninger, A. L., Roychoudhury, P. 2021; 93 (10): 5931-5941

  Abstract

  Real-time epidemiological tracking of variants of concern (VOCs) can help limit the spread of more contagious forms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), such as those containing the N501Y mutation. Typically, genetic sequencing is required to be able to track VOCs in real-time. However, sequencing can take time and may not be accessible in all laboratories. Genotyping by RT-ddPCR offers an alternative to rapidly detect VOCs through discrimination of specific alleles such as N501Y, which is associated with increased transmissibility and virulence. Here we describe the first cases of the B.1.1.7 lineage of SARS-CoV-2 detected in Washington State by using a combination of reverse-transcription polymerase chain reaction (RT-PCR), RT-ddPCR, and next-generation sequencing. We initially screened 1035 samples positive for SARS-CoV-2 by our CDC-based laboratory-developed assay using ThermoFisher's multiplex RT-PCR COVID-19 assay over four weeks from late December 2020 to early January 2021. S gene target failures (SGTF) were subsequently assayed by RT-ddPCR to confirm four mutations within the S gene associated with the B.1.1.7 lineage: a deletion at amino acid (AA) 69-70 (ACATGT), deletion at AA 145, (TTA), N501Y mutation (TAT), and S982A mutation (GCA). All four targets were detected in two specimens; follow-up sequencing revealed a total of 9 mutations in the S gene and phylogenetic clustering within the B.1.1.7 lineage. Next, we continued screening samples for SGTF detecting 23 additional B.1.1.7 variants by RT-ddPCR and confirmed by sequencing. As VOCs become increasingly prevalent, molecular diagnostic tools like RT-ddPCR can be utilized to quickly, accurately, and sensitively distinguish more contagious lineages of SARS-CoV-2.

  View details for DOI 10.1002/jmv.27155

  View details for Web of Science ID 000669242200001

  View details for PubMedID 34170525

  View details for PubMedCentralID PMC8427099

• A SARS-CoV-2 Nucleocapsid Variant that Affects Antigen Test Performance JOURNAL OF CLINICAL VIROLOGY Bourassa, L., Perchetti, G. A., Phung, Q., Lin, M. J., Mills, M. G., Roychoudhury, P., Harmon, K. G., Reed, J. C., Greninger, A. L. 2021; 141: 104900

  Abstract

  More than one year into a global pandemic, SARS-CoV-2 is now defined by a variety of rapidly evolving variant lineages. Several FDA authorized molecular diagnostic tests have been impacted by viral variation, while no reports of viral variation affecting antigen test performance have occurred to date. While determining the analytical sensitivity of the Quidel Sofia SARS Antigen FIA test (Sofia 2), we uncovered a high viral load specimen that repeatedly tested negative by this antigen test. Whole genome sequencing of the specimen uncovered two mutations, T205I and D399N, present in the nucleocapsid protein of the isolate. All six SARS-CoV-2 positive clinical specimens available in our laboratory with a D399N nucleocapsid mutation and CT < 31 were not detected by the Sofia 2 but detected by the Abbott BinaxNOW COVID-19 Ag Card, while clinical specimens with the T205I mutation were detected by both assays. Testing of recombinant SARS-CoV-2 nucleocapsid with these variants demonstrated an approximate 1000-fold loss in sensitivity for the Quidel Sofia SARS Antigen FIA test associated with the D399N mutation, while the BinaxNOW and Quidel Quickvue SARS Antigen tests were unaffected by the mutation. The D399N nucleocapsid mutation has been relatively uncommon to date, appearing in only 0.02% of genomes worldwide at time of writing. Our results demonstrate how routine pathogen genomics can be integrated into the clinical microbiology laboratory to investigate diagnostic edge cases, as well as the importance of profiling antigenic diversity outside of the spike protein for SARS-CoV-2 diagnostics.

  View details for DOI 10.1016/j.jcv.2021.104900

  View details for Web of Science ID 000677682400019

  View details for PubMedID 34171548

  View details for PubMedCentralID PMC8219478

• In Vivo Generation of BK and JC Polyomavirus Defective Viral Genomes in Human Urine Samples Associated with Higher Viral Loads JOURNAL OF VIROLOGY Addetia, A., Phung, Q., Bradley, B. T., Lin, M. J., Zhu, H., Xie, H., Huang, M., Greninger, A. L. 2021; 95 (12)

  Abstract

  Defective viral genomes (DVGs) are parasitic viral sequences containing point mutations, deletions, or duplications that might interfere with replication. DVGs are often associated with viral passage at high multiplicities of infection in culture systems but have been increasingly reported in clinical specimens. To date however, only RNA viruses have been shown to contain DVGs in clinical specimens. Here, using direct deep sequencing with multiple library preparation strategies and confirmatory digital droplet PCR (ddPCR) of urine samples taken from immunosuppressed individuals, we show that clinical BK polyomavirus (BKPyV) and JC polyomavirus (JCPyV) strains contain widespread genomic rearrangements across multiple loci that likely interfere with viral replication. BKPyV DVGs were derived from BKPyV genotypes Ia, Ib-1, and Ic. The presence of DVGs was associated with specimens containing higher viral loads but never reached clonality, consistent with a model of parasitized replication. These DVGs persisted during clinical infection as evidenced in two separate pairs of samples containing BK virus collected from the same individual up to 302 days apart. In a separate individual, we observed the generation of DVGs after a 57.5-fold increase in viral load. In summary, by extending the presence of DVGs in clinical specimens to DNA viruses, we demonstrate the ubiquity of DVGs in clinical virology.IMPORTANCE Defective viral genomes (DVGs) can have a significant impact on the production of infectious virus particles. DVGs have only been identified in cultured viruses passaged at high multiplicities of infection and RNA viruses collected from clinical specimens; no DNA virus in the wild has been shown to contain DVGs. Here, we identified BK and JC polyomavirus DVGs in clinical urine specimens and demonstrated that these DVGs are more frequently identified in samples with higher viral loads. The strains containing DVGs had rearrangements throughout their genomes, with the majority affecting genes required for viral replication. Longitudinal analysis showed that these DVGs can persist during an infection but do not reach clonality within the chronically infected host. Our identification of polyomavirus DVGs suggests that these parasitic sequences exist across the many classes of viruses capable of causing human disease.

  View details for DOI 10.1128/JVI.00250-21

  View details for Web of Science ID 000656996900008

  View details for PubMedID 33827948

  View details for PubMedCentralID PMC8316075

• Molecular Features of the Measles Virus Viral Fusion Complex That Favor Infection and Spread in the Brain MBIO Mathieu, C., Bovier, F. T., Ferren, M., Lieberman, N. P., Predella, C., Lalande, A., Peddu, V., Lin, M. J., Addetia, A., Patel, A., Outlaw, V., Corneo, B., Dorrello, N., Briese, T., Hardie, D., Horvat, B., Moscona, A., Greninger, A. L., Porotto, M. 2021; 12 (3): e0079921

  Abstract

  Measles virus (MeV) bearing a single amino acid change in the fusion protein (F)-L454W-was isolated from two patients who died of MeV central nervous system (CNS) infection. This mutation in F confers an advantage over wild-type virus in the CNS, contributing to disease in these patients. Using murine ex vivo organotypic brain cultures and human induced pluripotent stem cell-derived brain organoids, we show that CNS adaptive mutations in F enhance the spread of virus ex vivo. The spread of virus in human brain organoids is blocked by an inhibitory peptide that targets F, confirming that dissemination in the brain tissue is attributable to F. A single mutation in MeV F thus alters the fusion complex to render MeV more neuropathogenic. IMPORTANCE Measles virus (MeV) infection can cause serious complications in immunocompromised individuals, including measles inclusion body encephalitis (MIBE). In some cases, MeV persistence and subacute sclerosing panencephalitis (SSPE), another severe central nervous system (CNS) complication, develop even in the face of a systemic immune response. Both MIBE and SSPE are relatively rare but lethal. It is unclear how MeV causes CNS infection. We introduced specific mutations that are found in MIBE or SSPE cases into the MeV fusion protein to test the hypothesis that dysregulation of the viral fusion complex-comprising F and the receptor binding protein, H-allows virus to spread in the CNS. Using metagenomic, structural, and biochemical approaches, we demonstrate that altered fusion properties of the MeV H-F fusion complex permit MeV to spread in brain tissue.

  View details for DOI 10.1128/mBio.00799-21

  View details for Web of Science ID 000694797200002

  View details for PubMedID 34061592

  View details for PubMedCentralID PMC8263006

• Sensitive Recovery of Complete SARS-CoV-2 Genomes from Clinical Samples by Use of Swift Biosciences' SARS-CoV-2 Multiplex Amplicon Sequencing Panel JOURNAL OF CLINICAL MICROBIOLOGY Addetia, A., Lin, M. J., Peddu, V., Roychoudhury, P., Jerome, K. R., Greninger, A. L. 2021; 59 (1)

  View details for DOI 10.1128/JCM.02226-20

  View details for Web of Science ID 000600837900031

  View details for PubMedID 33046529

  View details for PubMedCentralID PMC7771467

• Estimation of Full-Length TprK Diversity in Treponema pallidum subsp. pallidum MBIO Addetia, A., Lin, M. J., Phung, Q., Xie, H., Huang, M., Ciccarese, G., Dal Conte, I., Cusini, M., Drago, F., Giacani, L., Greninger, A. L. 2020; 11 (5)

  Abstract

  Immune evasion and disease progression of Treponema pallidum subsp. pallidum are associated with sequence diversity in the hypervariable outer membrane protein TprK. Previous attempts to study variation within TprK have sequenced at depths insufficient to fully appreciate the hypervariable nature of the protein, failed to establish linkage between the protein's seven variable regions, or were conducted on isolates passed through rabbits. As a consequence, a complete profile of tprK during infection in the human host is still lacking. Furthermore, prior studies examining how T. pallidum subsp. pallidum uses its repertoire of genomic donor sites to generate diversity within the variable regions of the tprK have yielded a partial understanding of this process due to the limited number of tprK alleles examined. In this study, we used short- and long-read deep sequencing to directly characterize full-length tprK alleles from T. pallidum subsp. pallidum collected from early lesions of patients attending two sexually transmitted infection clinics in Italy. We demonstrate that strains collected from cases of secondary syphilis contain significantly more unique variable region sequences and full-length TprK sequences than those from cases of primary syphilis. Our data, combined with recent data available on Chinese T. pallidum subsp. pallidum specimens, show the near-complete absence of overlap in TprK sequences among the 41 specimens profiled to date. We further estimate that the potential antigenic variability carried by TprK rivals that of current estimates of the human adaptive immune system. These data underscore the immunoevasive ability of TprK that allows T. pallidum subsp. pallidum to establish lifelong infection.IMPORTANCE Syphilis continues to be a significant public health issue in both low- and high-income countries, including the United States where the rate of syphilis infection has increased over the past 5 years. Treponema pallidum subsp. pallidum, the causative agent of syphilis, carries the outer membrane protein TprK that undergoes segmental gene conversion to constantly create new sequences. We performed full-length deep sequencing of TprK to examine TprK diversity in clinical T. pallidum subsp. pallidum strains. We then combined our results with data from all samples for which TprK deep sequencing results were available. We found almost no overlap in TprK sequences between different patients. Moreover, our data allowed us to estimate the total number of TprK variants that T. pallidum subsp. pallidum can potentially generate. Our results support how the T. pallidum subsp. pallidum TprK antigenic variation system is an equal adversary of the human immune system leading to pathogen persistence in the host.

  View details for DOI 10.1128/mBio.02726-20

  View details for Web of Science ID 000680958400017

  View details for PubMedID 33109767

  View details for PubMedCentralID PMC7593977

• Inhibition of Coronavirus Entry In Vitro and Ex Vivo by a Lipid-Conjugated Peptide Derived from the SARS-CoV-2 Spike Glycoprotein HRC Domain MBIO Outlaw, V. K., Bovier, F. T., Mears, M. C., Cajimat, M. N., Zhu, Y., Lin, M. J., Addetia, A., Lieberman, N. P., Peddu, V., Xie, X., Shi, P., Greninger, A. L., Gellman, S. H., Bente, D. A., Moscona, A., Porotto, M. 2020; 11 (5)

  Abstract

  The emergence of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the etiological agent of the 2019 coronavirus disease (COVID-19), has erupted into a global pandemic that has led to tens of millions of infections and hundreds of thousands of deaths worldwide. The development of therapeutics to treat infection or as prophylactics to halt viral transmission and spread is urgently needed. SARS-CoV-2 relies on structural rearrangements within a spike (S) glycoprotein to mediate fusion of the viral and host cell membranes. Here, we describe the development of a lipopeptide that is derived from the C-terminal heptad repeat (HRC) domain of SARS-CoV-2 S that potently inhibits infection by SARS-CoV-2. The lipopeptide inhibits cell-cell fusion mediated by SARS-CoV-2 S and blocks infection by live SARS-CoV-2 in Vero E6 cell monolayers more effectively than previously described lipopeptides. The SARS-CoV-2 lipopeptide exhibits broad-spectrum activity by inhibiting cell-cell fusion mediated by SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV) and blocking infection by live MERS-CoV in cell monolayers. We also show that the SARS-CoV-2 HRC-derived lipopeptide potently blocks the spread of SARS-CoV-2 in human airway epithelial (HAE) cultures, an ex vivo model designed to mimic respiratory viral propagation in humans. While viral spread of SARS-CoV-2 infection was widespread in untreated airways, those treated with SARS-CoV-2 HRC lipopeptide showed no detectable evidence of viral spread. These data provide a framework for the development of peptide therapeutics for the treatment of or prophylaxis against SARS-CoV-2 as well as other coronaviruses.IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, continues to spread globally, placing strain on health care systems and resulting in rapidly increasing numbers of cases and mortalities. Despite the growing need for medical intervention, no FDA-approved vaccines are yet available, and treatment has been limited to supportive therapy for the alleviation of symptoms. Entry inhibitors could fill the important role of preventing initial infection and preventing spread. Here, we describe the design, synthesis, and evaluation of a lipopeptide that is derived from the HRC domain of the SARS-CoV-2 S glycoprotein that potently inhibits fusion mediated by SARS-CoV-2 S glycoprotein and blocks infection by live SARS-CoV-2 in both cell monolayers (in vitro) and human airway tissues (ex vivo). Our results highlight the SARS-CoV-2 HRC-derived lipopeptide as a promising therapeutic candidate for SARS-CoV-2 infections.

  View details for DOI 10.1128/mBio.01935-20

  View details for Web of Science ID 000579503600038

  View details for PubMedID 33082259

  View details for PubMedCentralID PMC7587434

• Orally efficacious broad-spectrum allosteric inhibitor of paramyxovirus polymerase NATURE MICROBIOLOGY Cox, R. M., Sourimant, J., Toots, M., Yoon, J., Ikegame, S., Govindarajan, M., Watkinson, R. E., Thibault, P., Makhsous, N., Lin, M. J., Marengo, J. R., Sticher, Z., Kolykhalov, A. A., Natchus, M. G., Greninger, A. L., Lee, B., Plemper, R. K. 2020; 5 (10): 1232-+

  Abstract

  Paramyxoviruses such as human parainfluenza virus type-3 (HPIV3) and measles virus (MeV) are a substantial health threat. In a high-throughput screen for inhibitors of HPIV3 (a major cause of acute respiratory infection), we identified GHP-88309-a non-nucleoside inhibitor of viral polymerase activity that possesses unusual broad-spectrum activity against diverse paramyxoviruses including respiroviruses (that is, HPIV1 and HPIV3) and morbilliviruses (that is, MeV). Resistance profiles of distinct target viruses overlapped spatially, revealing a conserved binding site in the central cavity of the viral polymerase (L) protein that was validated by photoaffinity labelling-based target mapping. Mechanistic characterization through viral RNA profiling and in vitro MeV polymerase assays identified a block in the initiation phase of the viral polymerase. GHP-88309 showed nanomolar potency against HPIV3 isolates in well-differentiated human airway organoid cultures, was well tolerated (selectivity index > 7,111) and orally bioavailable, and provided complete protection against lethal infection in a Sendai virus mouse surrogate model of human HPIV3 disease when administered therapeutically 48 h after infection. Recoverees had acquired robust immunoprotection against reinfection, and viral resistance coincided with severe attenuation. This study provides proof of the feasibility of a well-behaved broad-spectrum allosteric antiviral and describes a chemotype with high therapeutic potential that addresses major obstacles of anti-paramyxovirus drug development.

  View details for DOI 10.1038/s41564-020-0752-7

  View details for Web of Science ID 000548164600002

  View details for PubMedID 32661315

  View details for PubMedCentralID PMC7529989

• Comparative genomics and full-length Tprk profiling of Treponema pallidum subsp. pallidum reinfection PLOS NEGLECTED TROPICAL DISEASES Addetia, A., Tantalo, L. C., Lin, M. J., Xie, H., Huang, M., Marra, C. M., Greninger, A. L. 2020; 14 (4): e0007921

  Abstract

  Developing a vaccine against Treponema pallidum subspecies pallidum, the causative agent of syphilis, remains a public health priority. Syphilis vaccine design efforts have been complicated by lack of an in vitro T. pallidum culture system, prolific antigenic variation in outer membrane protein TprK, and lack of functional annotation for nearly half of the genes. Understanding the genetic basis of T. pallidum reinfection can provide insights into variation among strains that escape cross-protective immunity. Here, we present comparative genomic sequencing and deep, full-length tprK profiling of two T. pallidum isolates from blood from the same patient that were collected six years apart. Notably, this patient was diagnosed with syphilis four times, with two of these episodes meeting the definition of neurosyphilis, during this interval. Outside of the highly variable tprK gene, we identified 14 coding changes in 13 genes. Nine of these genes putatively localized to the periplasmic or outer membrane spaces, consistent with a potential role in serological immunoevasion. Using a newly developed full-length tprK deep sequencing protocol, we profiled the diversity of this gene that far outpaces the rest of the genome. Intriguingly, we found that the reinfecting isolate demonstrated less diversity across each tprK variable region compared to the isolate from the first infection. Notably, the two isolates did not share any full-length TprK sequences. Our results are consistent with an immunodominant-evasion model in which the diversity of TprK explains the ability of T. pallidum to successfully reinfect individuals, even when they have been infected with the organism multiple times.

  View details for DOI 10.1371/journal.pntd.0007921

  View details for Web of Science ID 000558074700010

  View details for PubMedID 32251462

  View details for PubMedCentralID PMC7162541

• VAPiD: a lightweight cross-platform viral annotation pipeline and identification tool to facilitate virus genome submissions to NCBI GenBank BMC BIOINFORMATICS Shean, R. C., Makhsous, N., Stoddard, G. D., Lin, M. J., Greninger, A. L. 2019; 20: 48

  Abstract

  With sequencing technologies becoming cheaper and easier to use, more groups are able to obtain whole genome sequences of viruses of public health and scientific importance. Submission of genomic data to NCBI GenBank is a requirement prior to publication and plays a critical role in making scientific data publicly available. GenBank currently has automatic prokaryotic and eukaryotic genome annotation pipelines but has no viral annotation pipeline beyond influenza virus. Annotation and submission of viral genome sequence is a non-trivial task, especially for groups that do not routinely interact with GenBank for data submissions.We present Viral Annotation Pipeline and iDentification (VAPiD), a portable and lightweight command-line tool for annotation and GenBank deposition of viral genomes. VAPiD supports annotation of nearly all unsegmented viral genomes. The pipeline has been validated on human immunodeficiency virus, human parainfluenza virus 1-4, human metapneumovirus, human coronaviruses (229E/OC43/NL63/HKU1/SARS/MERS), human enteroviruses/rhinoviruses, measles virus, mumps virus, Hepatitis A-E Virus, Chikungunya virus, dengue virus, and West Nile virus, as well the human polyomaviruses BK/JC/MCV, human adenoviruses, and human papillomaviruses. The program can handle individual or batch submissions of different viruses to GenBank and correctly annotates multiple viruses, including those that contain ribosomal slippage or RNA editing without prior knowledge of the virus to be annotated. VAPiD is programmed in Python and is compatible with Windows, Linux, and Mac OS systems.We have created a portable, lightweight, user-friendly, internet-enabled, open-source, command-line genome annotation and submission package to facilitate virus genome submissions to NCBI GenBank. Instructions for downloading and installing VAPiD can be found at https://github.com/rcs333/VAPiD .

  View details for DOI 10.1186/s12859-019-2606-y

  View details for Web of Science ID 000456522700002

  View details for PubMedID 30674273

  View details for PubMedCentralID PMC6343335