Early appearance of neutralizing antibodies during acute hepatitis C virus (HCV) infection is associated with spontaneous viral clearance. However, the longitudinal changes in antigen-specific memory B cell (MBCs) associated with divergent HCV infection outcomes remain undefined. We characterized longitudinal changes in E2 glycoprotein-specific MBCs from subjects who either spontaneously resolved acute HCV infection or progressed to chronic infection, using single-cell RNA-seq and functional assays. HCV-specific antibodies in plasma from chronically infected subjects recognized multiple E2 genotypes, while those from spontaneous resolvers exhibited variable cross-reactivity to heterotypic E2. E2-specific MBCs from spontaneous resolvers peaked early after infection (4–6 months), following expansion of activated circulating T follicular helper cells (cTfh) expressing interleukin 21. In contrast, E2-specific MBCs from chronically infected subjects, enriched in VH1-69, expanded during persistent infection (> 1 year), in the absence of significantly activated cTfh expansion. Early E2-specific MBCs from spontaneous resolvers produced monoclonal antibodies (mAbs) with fewer somatic hypermutations and lower E2 binding but similar neutralization as mAbs from late E2-specific MBCs of chronically infected subjects. These findings indicate that early cTfh activity accelerates expansion of E2-specific MBCs during acute infection, which might contribute to spontaneous clearance of HCV.

Gammaherpesviruses are DNA tumor viruses that establish lifelong latent infections in lymphocytes. For viruses such as Epstein-Barr virus and murine gammaherpesvirus 68, this is accomplished through a viral gene-expression program that promotes cellular proliferation and differentiation, especially of germinal center B cells. Intrinsic host mechanisms that control virus-driven cellular expansion are incompletely defined. Using a small-animal model of gammaherpesvirus pathogenesis, we demonstrate in vivo that the tumor suppressor p53 is activated specifically in B cells latently infected by murine gammaherpesvirus 68. In the absence of p53, the early expansion of murine gammaherpesvirus 68 latency greatly increases, especially in germinal center B cells, a cell type whose proliferation is conversely restricted by p53. We identify the B cell-specific latency gene M2, a viral promoter of germinal center B cell differentiation, as a viral protein sufficient to elicit a p53-dependent anti-proliferative response caused by Src-family kinase activation. We further demonstrate that Epstein-Barr virus-encoded latent membrane protein 1 similarly triggers a p53 response in primary B cells. Our data highlight a model in which gammaherpesvirus latency gene-expression programs that promote B cell proliferation and differentiation to facilitate viral colonization of the host trigger aberrant cellular proliferation that is controlled by p53.

SARS-CoV-2 is the causative agent of COVID-19 and continues to pose a significant public health threat throughout the world. Following SARS-CoV-2 infection, virus-specific CD4+ and CD8+ T cells are rapidly generated to form effector and memory cells and persist in the blood for several months. However, the contribution of T cells in controlling SARS-CoV-2 infection within the respiratory tract are not well understood. Using C57BL/6 mice infected with a naturally occurring SARS-CoV-2 variant (B.1.351), we evaluated the role of T cells in the upper and lower respiratory tract. Following infection, SARS-CoV-2-specific CD4+ and CD8+ T cells are recruited to the respiratory tract and a vast proportion secrete the cytotoxic molecule Granzyme B. Using antibodies to deplete T cells prior to infection, we found that CD4+ and CD8+ T cells play distinct roles in the upper and lower respiratory tract. In the lungs, T cells play a minimal role in viral control with viral clearance occurring in the absence of both CD4+ and CD8+ T cells through 28 days post-infection. In the nasal compartment, depletion of both CD4+ and CD8+ T cells, but not individually, results in persistent and culturable virus replicating in the nasal compartment through 28 days post-infection. Using in situ hybridization, we found that SARS-CoV-2 infection persisted in the nasal epithelial layer of tandem CD4+ and CD8+ T cell-depleted mice. Sequence analysis of virus isolates from persistently infected mice revealed mutations spanning across the genome, including a deletion in ORF6. Overall, our findings highlight the importance of T cells in controlling virus replication within the respiratory tract during SARS-CoV-2 infection.

Swine harbors a genetically diverse population of swine influenza A viruses (IAV-S), with demonstrated potential to transmit to the human population, causing outbreaks and pandemics. Here, we describe the development of a one-step, triplex real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay that detects and distinguishes the majority of the antigenically distinct influenza A virus hemagglutinin (HA) clades currently circulating in North American swine, including the IAV-S H1 1A.1 (α), 1A.2 (β), 1A.3 (γ), 1B.2.2 (δ1) and 1B.2.1 (δ2) clades, and the IAV-S H3 2010.1 clade. We performed an in-field test at an exhibition swine show using in-field viral concentration and RNA extraction methodologies and a portable real-time PCR instrument, and rapidly identified three distinct IAV-S clades circulating within the N.A. swine population. Portable sequencing is used to further confirm the results of the in-field test of the swine triplex assay. The IAV-S triplex rRT-PCR assay can be easily transported and used in-field to characterize circulating IAV-S clades in North America, allowing for surveillance and early detection of North American IAV-S with human outbreak and pandemic potential.

Influenza A(H3N2) viruses circulate globally and cause respiratory illness. The FDA-cleared CDC Human Influenza Virus Real-Time RT-PCR Influenza A (H3/H1pdm09) Subtyping assay is used to subtype seasonal influenza A viruses. However, emerging mutations in the H3 reverse primer region raised concerns about reduced assay sensitivity for contemporary circulating viruses. We updated the H3 assay by redesigning the reverse primer to target a more conserved region of the hemagglutinin gene, and incorporating minor adjustments into the forward primer and probe. Analytical sensitivity, inclusivity, and exclusivity evaluations were performed using cultured influenza viruses, synthetic RNA, and panels of non-influenza respiratory pathogens. Clinical performance was evaluated using seasonal A(H3N2) residual respiratory specimens. The updated assay (H3_v2) demonstrated analytical sensitivity comparable to or improved over the initial H3 assay, with a limit of detection of approximately 10 ^1.7 –10 ^2.1 EID ₅₀ /mL and five copies of RNA per reaction. Improved performance was observed for viruses containing A21G with T23G, T23C, or T23A double mismatches in the reverse primer, with a measurable Ct value shift between the initial H3 and H3_v2 assay. The assay showed 100% agreement with expected results in 139 clinical specimens, and no cross-reactivity with other influenza subtypes or other respiratory pathogens. These findings indicate the updated H3_v2 assay improves sensitivity for contemporary and emerging seasonal A(H3N2) viruses while maintaining high specificity. This update ensures continued reliable detection of seasonal A(H3N2), including genetically drifted A(H3N2) viruses, such as the recently emerged A(H3N2) subclade K viruses, and supports global influenza surveillance and diagnostic preparedness. IMPORTANCE: Accurate detection and subtyping of influenza A(H3N2) viruses are essential for clinical diagnostics, outbreak response, and global influenza surveillance. The emergence and fixation of mutations leading to primer mismatches in the FDA-cleared CDC A(H3) real-time RT-PCR assay raised concerns about reduced sensitivity for contemporary circulating A(H3N2) viruses. To address this, we redesigned the reverse primer and optimized additional assay components to improve detection of currently circulating seasonal A(H3N2) viruses. The updated H3_v2 assay restores and enhances sensitivity, including for viruses harboring key reverse primer mismatches, while maintaining high specificity and complete agreement with clinical specimens. This update ensures reliable detection of seasonal influenza A(H3N2) and A(H3N2) variant viruses, including recently emerged A(H3N2) subclade K viruses, and strengthens diagnostic preparedness and surveillance capacity nationally and internationally.