Trisha Barnard

All Publications

• NK Cell-Monocyte Cross-talk Underlies NK Cell Activation in Severe COVID-19. Journal of immunology (Baltimore, Md. : 1950) Lee, M. J., de Los Rios Kobara, I., Barnard, T. R., Vales Torres, X., Tobin, N. H., Ferbas, K. G., Rimoin, A. W., Yang, O. O., Aldrovandi, G. M., Wilk, A. J., Fulcher, J. A., Blish, C. A. 2024

  Abstract

  NK cells in the peripheral blood of severe COVID-19 patients exhibit a unique profile characterized by activation and dysfunction. Previous studies have identified soluble factors, including type I IFN and TGF-β, that underlie this dysregulation. However, the role of cell-cell interactions in modulating NK cell function during COVID-19 remains unclear. To address this question, we combined cell-cell communication analysis on existing single-cell RNA sequencing data with in vitro primary cell coculture experiments to dissect the mechanisms underlying NK cell dysfunction in COVID-19. We found that NK cells are predicted to interact most strongly with monocytes and that this occurs via both soluble factors and direct interactions. To validate these findings, we performed in vitro cocultures in which NK cells from healthy human donors were incubated with monocytes from COVID-19+ or healthy donors. Coculture of healthy NK cells with monocytes from COVID-19 patients recapitulated aspects of the NK cell phenotype observed in severe COVID-19, including decreased expression of NKG2D, increased expression of activation markers, and increased proliferation. When these experiments were performed in a Transwell setting, we found that only CD56bright CD16- NK cells were activated in the presence of severe COVID-19 patient monocytes. O-link analysis of supernatants from Transwell cocultures revealed that cultures containing severe COVID-19 patient monocytes had significantly elevated levels of proinflammatory cytokines and chemokines, as well as TGF-β. Collectively, these results demonstrate that interactions between NK cells and monocytes in the peripheral blood of COVID-19 patients contribute to NK cell activation and dysfunction in severe COVID-19.

  View details for DOI 10.4049/jimmunol.2300731

  View details for PubMedID 38578283

• Zika virus NS3 and NS5 proteins determine strain-dependent differences in dsRNA accumulation in a host cell type-dependent manner. The Journal of general virology Barnard, T. R., Landry, B. N., Wang, A. B., Sagan, S. M. 2023; 104 (6)

  Abstract

  For positive-sense RNA viruses, initiation of viral RNA replication represents a major target of antiviral responses to infection. Despite this, the interplay between viral replication and the innate antiviral response at early steps in the Zika virus (ZIKV) life cycle is not well understood. We have previously identified ZIKV isolates with differing levels of dsRNA accumulation, ZIKVPR (high dsRNA per infected cell) and ZIKVCDN (low dsRNA per infected cell), and we hypothesized that we could use reverse genetics to investigate how host and viral factors contribute to the establishment of viral RNA replication. We found that both the ZIKV NS3 and NS5 proteins as well as host factors were necessary to determine the dsRNA accumulation phenotype. Additionally, we show that dsRNA correlates with viral negative-strand RNA measured by strand-specific RT-qPCR, suggesting that dsRNA is an accurate readout of viral RNA replication. Interestingly, although we did not observe NS3- and NS5-dependent differences in cells with defects in interferon (IFN) production, differences in RNA accumulation precede induction of the IFN response, suggesting that RNA sensing pathways or intrinsic restriction factors may differentially restrict ZIKV in an NS3- and NS5-dependent manner. This work expands our understanding of the interplay of early steps of viral RNA replication and the induction of the innate antiviral response to ZIKV infection.

  View details for DOI 10.1099/jgv.0.001855

  View details for PubMedID 37289497

• A highly sensitive strand-specific multiplex RT-qPCR assay for quantitation of Zika virus replication JOURNAL OF VIROLOGICAL METHODS Barnard, T. R., Wang, A. B., Sagan, S. M. 2022; 307: 114556

  Abstract

  Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is widely used to quantify viral RNA genomes for diagnostics and research, yet conventional RT-qPCR protocols are unable to accurately distinguish between the different viral RNA species that exist during infection. Here we show that false-priming and self-priming occur during reverse transcription with several published Zika virus (ZIKV) primer sets. We developed a RT-qPCR assay using tagged primers and thermostable reverse transcriptase, which greatly reduced the occurrence of nonspecific cDNA products. Furthermore, we optimized the assay for use in multiplex qPCR which allows for simultaneous quantitative detection of positive-strand and negative-strand ZIKV RNA along with an internal control from both human and mosquito cells. Importantly, this assay is sensitive enough to study early stages of virus infection in vitro. Strikingly, using this assay, we detected ZIKV negative-strand RNA as early as 3 h post-infection in mammalian cell culture, at a time point prior to the onset of positive-strand RNA synthesis. Overall, the strand-specific RT-qPCR assay developed herein is a valuable tool to quantify ZIKV RNA and to study viral replication dynamics during infection. The application of these findings has the potential to increase accuracy of RNA detection methods for a variety of viral pathogens.

  View details for DOI 10.1016/j.jviromet.2022.114556

  View details for Web of Science ID 000814744700002

  View details for PubMedID 35654259

• Molecular Determinants of Flavivirus Virion Assembly TRENDS IN BIOCHEMICAL SCIENCES Barnard, T. R., Abram, Q. H., Lin, Q., Wang, A. B., Sagan, S. M. 2021; 46 (5): 378-390

  Abstract

  Virion assembly is an important step in the life cycle of all viruses. For viruses of the Flavivirus genus, a group of enveloped positive-sense RNA viruses, the assembly step represents one of the least understood processes in the viral life cycle. While assembly is primarily driven by the viral structural proteins, recent studies suggest that several nonstructural proteins also play key roles in coordinating the assembly and packaging of the viral genome. This review focuses on describing recent advances in our understanding of flavivirus virion assembly, including the intermolecular interactions between the viral structural (capsid) and nonstructural proteins (NS2A and NS2B-NS3), host factors, as well as features of the viral genomic RNA required for efficient flavivirus virion assembly.

  View details for DOI 10.1016/j.tibs.2020.12.007

  View details for Web of Science ID 000641462600004

  View details for PubMedID 33423940

• Hepatitis B Virus Therapeutic Agent ARB-1740 Has Inhibitory Effect on Hepatitis Delta Virus in a New Dually-Infected Humanized Mouse Model ACS INFECTIOUS DISEASES Ye, X., Tateno, C., Thi, E. P., Kakuni, M., Snead, N. M., Ishida, Y., Barnard, T. R., Sofia, M. J., Shimada, T., Lee, A. H. 2019; 5 (5): 738-749

  Abstract

  Hepatitis delta virus (HDV) infects 10-20 million individuals worldwide and causes severe fulminant hepatitis with high likelihood of cirrhosis and hepatocellular carcinoma. HDV infection cannot occur in the absence of the surface antigen (HBsAg) of the hepatitis B virus. RNA interference is an effective mechanism by which to inhibit viral transcripts, and siRNA therapeutics sharing this mechanism have begun to demonstrate clinical efficacy. Here we assessed the outcome of HBV-targeting siRNA intervention against HDV and compared it to a direct anti-HDV siRNA approach in dually infected humanized mice. Treatment with ARB-1740, a clinical stage HBV-targeting siRNA agent delivered using lipid nanoparticle (LNP) technology, effectively reduced HBV viremia by 2.3 log10 and serum HBsAg by 2.6 log10, leading to 1.6 log10 reduction of HDV viremia. In contrast, HDV-targeting siRNA inhibited HDV in both blood and liver compartments without affecting HBV and PEGylated interferon-alpha reduced HBV viremia by 2.0 log10 but had no effect on HDV viremia under these study conditions. These results illustrate the inhibitory effects of siRNAs against these two viral infections and suggest that ARB-1740 may be of therapeutic benefit for hepatitis delta patients, a subpopulation with high unmet medical need.

  View details for DOI 10.1021/acsinfecdis.8b00192

  View details for Web of Science ID 000468015100009

  View details for PubMedID 30408957

• Contemporary Zika Virus Isolates Induce More dsRNA and Produce More Negative-Strand Intermediate in Human Astrocytoma Cells VIRUSES-BASEL Barnard, T. R., Rajah, M. M., Sagan, S. M. 2018; 10 (12)

  Abstract

  The recent emergence and rapid geographic expansion of Zika virus (ZIKV) poses a significant challenge for public health. Although historically causing only mild febrile illness, recent ZIKV outbreaks have been associated with more severe neurological complications, such as Guillain-Barré syndrome and fetal microcephaly. Here we demonstrate that two contemporary (2015) ZIKV isolates from Puerto Rico and Brazil may have increased replicative fitness in human astrocytoma cells. Over a single infectious cycle, the Brazilian isolate replicates to higher titers and induces more severe cytopathic effects in human astrocytoma cells than the historical African reference strain or an early Asian lineage isolate. In addition, both contemporary isolates induce significantly more double-stranded RNA in infected astrocytoma cells, despite similar numbers of infected cells across isolates. Moreover, when we quantified positive- and negative-strand viral RNA, we found that the Asian lineage isolates displayed substantially more negative-strand replicative intermediates than the African lineage isolate in human astrocytoma cells. However, over multiple rounds of infection, the contemporary ZIKV isolates appear to be impaired in cell spread, infecting a lower proportion of cells at a low MOI despite replicating to similar or higher titers. Taken together, our data suggests that contemporary ZIKV isolates may have evolved mechanisms that allow them to replicate with increased efficiency in certain cell types, thereby highlighting the importance of cell-intrinsic factors in studies of viral replicative fitness.

  View details for DOI 10.3390/v10120728

  View details for Web of Science ID 000455313100067

  View details for PubMedID 30572570

  View details for PubMedCentralID PMC6316034

• Zika virus infection: induction, restriction and evasion of host interferon responses FUTURE VIROLOGY Barnard, T. R., Rajah, M., Sagan, S. M. 2017; 12 (11): 627-630

  View details for DOI 10.2217/fvl-2017-0099

  View details for Web of Science ID 000416973900001

• Lipid nanoparticle siRNA treatment of Ebola-virus-Makona-infected nonhuman primates NATURE Thi, E. P., Mire, C. E., Lee, A. H., Geisbert, J. B., Zhou, J. Z., Agans, K. N., Snead, N. M., Deer, D. J., Barnard, T. R., Fenton, K. A., MacLachlan, I., Geisbert, T. W. 2015; 521 (7552): 362-+

  Abstract

  The current outbreak of Ebola virus in West Africa is unprecedented, causing more cases and fatalities than all previous outbreaks combined, and has yet to be controlled. Several post-exposure interventions have been employed under compassionate use to treat patients repatriated to Europe and the United States. However, the in vivo efficacy of these interventions against the new outbreak strain of Ebola virus is unknown. Here we show that lipid-nanoparticle-encapsulated short interfering RNAs (siRNAs) rapidly adapted to target the Makona outbreak strain of Ebola virus are able to protect 100% of rhesus monkeys against lethal challenge when treatment was initiated at 3 days after exposure while animals were viraemic and clinically ill. Although all infected animals showed evidence of advanced disease including abnormal haematology, blood chemistry and coagulopathy, siRNA-treated animals had milder clinical features and fully recovered, while the untreated control animals succumbed to the disease. These results represent the first, to our knowledge, successful demonstration of therapeutic anti-Ebola virus efficacy against the new outbreak strain in nonhuman primates and highlight the rapid development of lipid-nanoparticle-delivered siRNA as a countermeasure against this highly lethal human disease.

  View details for DOI 10.1038/nature14442

  View details for Web of Science ID 000354816500058

  View details for PubMedID 25901685

  View details for PubMedCentralID PMC4467030