Journal of Virology

Infection with Human Cytomegalovirus (HCMV) can result in a significant burden of disease in those that are immunocompromised or immunonaive. HCMV encodes a repertoire of glycoproteins that facilitate its extensive viral tropism, some of which remain to be characterized. Currently, there is no effective vaccine or cure for HCMV, therefore emphasizing the need to identify viral proteins of critical function. UL14 was selected as an open reading frame of interest due to its high scoring on an in-silico prediction algorithm, as well as its conservation amongst CMVs. Our goal was to elucidate the function of this uncharacterized viral open reading frame. We hypothesized that UL14 functions in the establishment of infection in epithelial cells, due to its predicted structural similarity to UL141. This study demonstrates that HCMV UL14 is a glycosylated viral protein packaged with the virion. Importantly, the deletion of UL14 resulted in a significant reduction of viral growth in epithelial cells, whereas no growth defect was observed in fibroblasts. Mechanistically, we found this defect to be a result of post entry, pre-IE transcription in the establishment of infection, consistent with a defect endosomal escape. Taken together, our results suggest that UL14 functions in the establishment of infection in an epithelial cell-specific manner and may be a novel target for future vaccines or antiviral therapies. Author SummaryHCMV is found in a wide variety of human cells during the course of viral infection. As such, HCMV encodes several glycoprotein complexes that dictate tropism. In this work we report the identification of a novel glycoprotein, UL14, that is involved in establishing productive infections of epithelial cells, a common site of HCMV induced disease. We report that deletion of UL14 from the viral genome impacts its ability to infect ARPE19 cells at a stage indicative of viral events post viral entry but prior to viral transcriptional activation. Further, trans complementation of UL14 by expansion of mutant virus in cells expressing the viral glycoprotein, restore viral infectivity suggesting that UL14 mediates events early in viral infection. Importantly, the characterization of this viral envelope protein provides key insights into viral tropism and identifies a novel target for vaccine design and antiviral therapies.

Highly pathogenic avian influenza H5N1 2.3.4.4b genotype D1.1 lineage continues to predominate in the United States wild bird population and has spilled over into dairy cattle three independent times. To assess the transmission risk of this sublineage, we performed direct-contact transmission experiments for three distinct D1.1 strains in ferrets. Two of these strains were isolated from humans and one from a lethal cat infection. We found that only one human isolate (A/NV/10/2025) was able to transmit efficiently between ferrets. Compared to the other strains, this isolate harbored the mammalian adaptive PB2 D701N mutation, suggesting this mutation may be critical for D1.1 transmission as opposed to the PB2 E627K substitution present in the lethal cat isolate. Based on these data we conclude that the transmission fitness of D1.1 strains is modest but that special attention should be paid to emergence of adaptation at the PB2 701 position.

The guinea pig with guinea pig cytomegalovirus (GPCMV) is the only small animal model for congenital CMV (cCMV). GPCMV cell entry is dictated by specific viral gH/gL-based complexes: gH/gL/gO trimer (direct entry); pentamer complex, PC (endocytic entry). GPCMV gB as the fusogenic protein is also essential for all entry pathways. PDGFRA and NRP2 are receptors for direct and endocytic virus entry respectively based on strain 13 animal fibroblast ATCC cell line studies. All non-fibroblast guinea pig cell lines are derived from Dunkin-Hartley animals, the focus of cCMV studies. GPCMV infection of Dunkin-Hartley embryo fibroblasts (GEFh) and epithelial cells were compared. Knockout of PDGFRA on GEFh cells prevented GPCMV(PC-) direct entry but not endocytic GPCMV(PC+) infection, demonstrating both pathways of infection. Fibroblast generated virus poorly infected epithelial cells compared to epithelial virus stock, which exhibited full tropism to all cell types. Guinea pig epithelial cell lines are NRP2-positive and PDGFRA-negative requiring PC for GPCMV infection. Epithelial and GEFh cells, but not strain 13 fibroblasts, additionally expressed ThBD. In immunoprecipitation assays, PC and ThBD interacted unlike CD46 receptor candidate targeting gH/gL. Double-knockout of NRP2/ThBD in epithelial cells impaired infection unlike single knockouts. Individual ectopic species-specific receptor expression restored infection on double-knockout epithelial (NRP2/ThBD) and fibroblast (PDGFRA/NRP2) cell lines. Knockout of NRP2/ThBD receptors did not enhance GPCMV neutralization by gB antibodies on PDGFRA-negative cells demonstrating a limitation of a gB vaccine strategy. Overall, GPCMV and HCMV similarity for receptors and cell tropism maintains the translational importance of this model.

Human adenovirus serotype 4 (Ad4) is used as a replication-competent oral vaccine that safely and effectively prevents Ad4 respiratory illness in US military personnel. Recombinant Ad4 vaccine candidates elicit mucosal and systemic immune responses against respiratory viruses in hamsters, nonhuman primates, and humans. Although evaluation of Ad4 vaccine candidates in mice would be extremely useful given the large number of immunologic tools available, this has been limited by concerns about a lack of viral replication in these animals. Here we generated recombinant Ad4 vectors that express either luciferase (Ad4-Luc) or herpes simplex virus type 2 (HSV-2) glycoprotein D (Ad4-gD2) to identify transgene expression kinetics, the presence of Ad4 vector replication, and HSV-2 immune responses and protection against HSV-2 infection. Local luciferase activity was observed from 7 hours to 20 days after intranasal inoculation of BALB/c and humanized mice. Subsequent inoculations with Ad4-Luc showed reduced luciferase expression in BALB/c mice, but robust expression in humanized mice, suggesting an immune response to the vector in wild-type mice. Ad4 DNA, but not luciferase activity, was reduced in the lungs of BALB/c mice treated with cidofovir before inoculation with Ad4, implying that Ad4 replicated, albeit at a low level, in the lungs. Intranasal vaccination of mice with Ad4-gD2 resulted in HSV-2 neutralizing antibody in the serum, and after HSV-2 intravaginal challenge reduced disease scores, increased survival, and reduced shedding. Overall, the BALB/c mouse model is semi-permissive to Ad4 mucosal infection, but transgene expression is sufficient for the study of Ad4-based vaccine candidates. ImportanceMucosal surfaces serve as the primary site of infection and shedding for many viral pathogens. Immune responses at mucosal sites provide protection, but few mucosal vaccines are licensed. The oral replication-competent adenovirus serotype 4 (Ad4) vaccine is used to prevent respiratory illness in military recruits, has an extraordinary record of safety and efficacy and has been tested as a recombinant platform for other viruses. Further development of this vaccine platform has been partially hindered by the perceived inability to evaluate vaccine candidates in mice. Here we characterize recombinant Ad4 transgene expression kinetics and viral replication after inoculation at various sites and show protection against herpes simplex virus type 2 (HSV-2) genital disease in mice after intranasal vaccination. We show that Ad4 can induce protective efficacy, even in a semi-permissive mouse model, suggesting this is a promising vector for HSV-2 and potentially other viral pathogens.

Many mammalian cells restrict viral replication by utilizing various host restriction factors. We recently demonstrated that CCHC-type zinc-finger-containing protein 3 (ZCCHC3) suppresses human immunodeficiency virus type 1 (HIV-1) replication through multiple mechanisms. We also revealed that single-nucleotide polymorphisms (SNPs) in human ZCCHC3 affect its antiviral function; however, whether similar genetic and functional diversity is present in other species remains unknown. In this study, we investigated the genetic and functional diversity of ZCCHC3 in cynomolgus macaques, a critical animal model for HIV-1-related research. Sequencing analysis of eight independent ZCCHC3 clones per animal revealed substantial amino acid diversity among cynomolgus macaques. We selected 12 representative variants and examined their antiviral activity against several retroviral vectors derived from HIV-1, simian immunodeficiency virus, feline immunodeficiency virus, and murine leukemia virus. Moreover, using replication-competent HIV-1, we showed that selected cynomolgus macaque ZCCHC3 variants can affect both viral production and viral infectivity. These results suggest that the genetic and functional diversity of ZCCHC3 is not limited to humans and underscore the importance of considering ZCCHC3 variation in cynomolgus macaques when using them as animal models for HIV-1-related research.

Respiratory viral-bacterial co-infections cause severe disease across species, yet the molecular mechanisms underlying enhanced pathogenesis remain poorly understood. This study characterised H3N8 equine influenza A virus (IAV) and Streptococcus equi subspecies zooepidemicus (SEZ) co-infections using complementary ultrastructural and transcriptomic approaches. Transmission electron microscopy demonstrated direct physical binding between spherical (A/equine/Miami/63) and filamentous (A/equine/Sussex/89 and A/equine/Newmarket/5/2003) IAV isolates and SEZ, including when SEZ was heat-inactivated ({theta}SEZ). Lectin staining revealed that SEZ expresses predominantly 2,3-linked sialic acids, the receptor for equine IAV. However, virus-bacteria binding persisted despite neuraminidase treatment. Scanning electron microscopy quantification demonstrated that viral pre-infection significantly enhanced bacterial adherence to cells of the DH82 canine macrophage-like cell line (2-fold increase, p<0.01) but not ExtEqFL (equine lung-derived) cells, revealing cell-type-specific enhancement. RNA-sequencing analysis showed that bacterial infection drove most transcriptional changes during co-infection with little difference in the number of differentially expressed genes (DEGs) between infection with SEZ alone (146 DEGS) or after pre-infection with either A/equine/Sussex/89 (166 DEGS) or A/equine/Newmarket/5/2003 (149 DEGS). Validation of upregulation of selected cytokines by RT-qPCR and ELISA demonstrated that SEZ infection drives dramatic cytokine upregulation compared to mock or {theta}SEZ controls. Viral pre-infection did not alter the SEZ-induced pro-inflammatory cytokine responses (IL-6, IL-8, TNF-) but significantly reduced IFN-{beta} expression compared to SEZ infection alone. These findings suggest that direct virus-bacteria physical interactions may drive cell-type-specific enhancement of bacterial colonisation, fundamentally advancing our understanding of respiratory co-infection pathogenesis.

Dengue virus (DENV) is the most prevalent mosquito-borne viral disease with over ten million cases worldwide. There are four antigenically distinct, co-circulating DENV serotypes (DENV 1-4) capable of infecting humans. Given the lack of immunocompetent animal models and the limitations of known cell culture models, more physiologically relevant experimental models are needed to recapitulate DENV infection and host immune responses. Building on previous observations that DENV-2 and DENV-4 serotypes elicit distinct innate immune responses in monocyte-derived dendritic cells (moDC) in vitro, we utilized a human tonsil histoculture (HC) model to further investigate serotype-specific differences within the physiologically relevant human lymphoid environment. We show that human tonsil HCs preserved their tissue cytoarchitecture for up to 6 days in culture, including maintaining functional germinal centers and diverse immune populations within T and B cell compartments. Exposure of tonsil HCs to DENV-2 and DENV-4 showed that DENV-4 replication peaked earlier and induced enhanced innate immune activation compared to DENV-2, consistent with previous observations in human DCs. Moreover, by leveraging the structural and cellular complexity of the HC system, we further identified that DENV E protein co-localized with HLA-DR+ antigen-presenting cells, confirming that DENV-infected cells within tonsil HCs predominantly express antigen-presenting cell markers. Altogether, these findings demonstrate that different DENV serotypes can exhibit different viral replication dynamics and induce distinct immune responses within human lymphoid tissue. This establishes human tonsil HCs as human model system with intact cytoarchitecture that closely mirrors lymph node structure and function, providing a powerful platform to study antigen-driven and virus-specific human immune responses and ultimately to evaluate vaccine candidates and antiviral therapeutics.

Like most herpesviruses, KSHV encodes multiple microRNAs (miRNAs). Collectively, they comprise an important mechanism through which the virus maintains latency and persists in cells. At the same time, individual miRNAs can also play distinct, nonredundant roles. Past experiments with single miRNA knockout viruses showed that miR-K12-9, in particular, filled a unique niche. Endothelial cells latently infected with the miR-K12-9 knockout grew to be many times larger than WT-infected cells and proliferated at a significantly slower rate. Their ability to migrate was slowed as well. RNA-seq identified nearly 8,500 differentially expressed genes between miR-K12-9 knockout- and WT-infected cells. To further study miR-K12-9, we generated Telomerase-Immortalized Microvascular Endothelial (TIME) cells expressing either miR-K12-9 or a control miRNA from a lentivirus. Unexpectedly, after approximately one month in culture, unmistakable morphological changes began to occur in two of the three miR-K12-9-expressing cell lines. These smaller, more rounded cells proliferated rapidly and swiftly took over the two cultures. Given this result, we proceeded to characterize all the lentivirus-transduced cell lines in various assays focused on oncogenesis. When looking at colony formation in soft agar, only those two miR-K12-9-expressing cell lines produced colonies, indicating a loss of contact inhibition. NOD/SCID mice injected with the two cell miR-K12-9-expressing cell lines developed tumors while those receiving other cell lines did not. To confirm reproducibility of these results, we transduced both TIME and primary endothelial cells (HUVECs) with the miR-K12-9 and control lentiviruses. Once again, approximately half of the cell lines expressing miR-K12-9 showed hallmark phenotypes of transformation. We are currently characterizing the miR-K12-9 targetome in the transduced cell lines and mouse tumors using bulk and single-cell RNA-seq. This should yield insights into the underlying mechanism and required cofactors of miR-K12-9-induced transformation. To our knowledge, this is the first description of transformation of endothelial cells by a viral miRNA.

The human coronavirus HKU1, causing common colds and occasionally severe illness, remains largely uncharacterized because it has not been successfully grown on immortalized cells. Here, we identified Caco2 cells overexpressing TMPRSS2, the HKU1 receptor, as being highly permissive to infection. HKU1 replicated efficiently, formed syncytia and released infectious progeny in these cells at 33{degrees}C, the temperature of the nasal cavity, but was attenuated at 37{degrees}C. Viral entry occurred similarly at both temperatures, but subsequent viral RNA synthesis was enhanced at 33{degrees}C. Released virions displayed higher stability at 33{degrees}C. In Caco2 and primary epithelial nasal cells, HKU1 was sensitive to interferons (IFN), but induction of IFN stimulated genes, such as IFN-Induced Transmembrane Proteins (IFITMs), was delayed at 33{degrees}C. Once expressed, IFITMs comparably inhibited HKU1 fusion at both temperatures. In contrast, SARS-CoV-2 robustly replicated at 37{degrees}C. Thus, cellular permissiveness, innate immunity and viral properties collectively explain why HKU1 replicates more efficiently at nasal temperature. Our results highlight temperature-sensitivity disparities between coronaviruses, likely associated to different pathogenic outcomes.

How host functions affect resistance to antiviral drugs is poorly understood. Ganciclovir, a chain-terminating nucleoside analog, is a first-line therapy against human cytomegalovirus, a widespread herpesvirus that causes life-threatening disease in immunocompromised individuals and newborns. Ganciclovir resistance, which is caused by mutations that affect the viral kinase, UL97 and/or the viral polymerase, UL54, can cause treatment failures. Among these mutations, those reducing the exonuclease activity of the viral DNA polymerase permit ganciclovir incorporation without chain termination. However, the fate of DNA strands containing the incorporated nucleotide analog is unknown. We show here that template DNA containing ganciclovir fails to support DNA synthesis of the complementary strand by exonuclease-mutant polymerase. Moreover, while DNA synthesis and ganciclovir incorporation are limited in drug-treated fibroblasts infected by virus with wild-type polymerase, an exonuclease-resistant mutant virus can better synthesize full-length genomes and incorporate substantially more ganciclovir into DNA. Notably, ganciclovir is lost from DNA when drug is removed, suggesting that ganciclovir-containing templates are repaired. We identify the host nucleotide excision repair component, XPA, and the repair enzyme, polymerase kappa, as each being necessary for mutant virus ganciclovir resistance and polymerase kappa as being required for the mutants cidofovir resistance, demonstrating a role for host DNA repair machinery in a mechanism of antiviral resistance. We propose a model for this mechanism, which has relevance for at least one other antiviral drug and likely other nucleoside analog therapeutics, and highlights the participation of host DNA repair machinery during human cytomegalovirus DNA replication. IMPORTANCENucleoside analogues such as ganciclovir, which is a leading drug for preventing and treating human cytomegalovirus, are a critical defense against viral diseases, but antiviral resistance often results in treatment failures. This study reveals a critical role for host DNA repair in a mechanism of resistance to ganciclovir, and identifies at least one specific repair pathway that permits viral DNA synthesis in the presence of ganciclovir, defining a mechanism by which cellular DNA repair pathways conspire to enable antiviral drug resistance. This mechanism is relevant to at least one other antiviral drug and may apply to other antiviral and anticancer agents. The study also showcases the participation of host DNA repair machinery during human cytomegalovirus DNA synthesis.

Eurasian avian like H1N1 (EAavH1N1) and influenza D viruses (IDV) with their ongoing evolution and zoonotic potential are a serious threat to animal and human health. Using experimental infection of pigs, we characterized and compared their pathogenesis, and immune responses. EAavH1N1 induced rapid viral clearance, early immune activation, including robust systemic and mucosal antibody responses and increased IFN{gamma} and TNF production. This heightened immune response was associated with more severe pathology of the upper and lower respiratory tract. In contrast, IDV infection resulted in prolonged viral shedding and higher viral titres, with delayed and attenuated cellular immune responses. Single cell transcriptomic analysis of lung further indicated early and persistent suppression of antiviral and innate immune pathways during IDV infection. These findings demonstrate that EAavH1N1 and IDV exhibit distinct viral kinetics, immune activation profiles, and lung responses, providing insight into differences in transmission dynamics, disease severity, and immune control among influenza virus types in swine.

Tomato fruit blotch virus (ToFBV) is an emerging multipartite positive-sense RNA virus associated with blotchy symptoms on tomato fruits and classified within the genus Blunervirus (family Kitaviridae). Despite its increasing agricultural relevance, the study of ToFBV has been hindered by the lack of mechanical transmissibility and the difficulty in reproducing infections under controlled conditions. In this work, we report a preliminary step toward the development of the first infectious agroclone system for ToFBV, based on full-length cDNA copies of its four genomic RNAs. We demonstrate that the cloned viral genome is capable of initiating cell autonomous replication in Nicotiana benthamiana, as indicated by the accumulation of negative-sense RNA intermediates in infiltrated tissues. To further validate the system, RNA3 was engineered to express GFP, enabling visualization of infection foci and confirming active viral replication in both N. benthamiana and tomato. Functional assays of RNA4-encoded proteins demonstrated that it encodes a movement protein capable of complementing movement-deficient viral vectors and a putative suppressor of post-transcriptional gene silencing (PTGS). Together, these results establish a versatile reverse genetics platform for ToFBV, providing new insights into the replication and functional organization of blunerviruses and enabling future studies on virus-host interactions, pathogenicity, and control strategies.

We previously found that high genome replication fitness of the hepatitis C virus (HCV) was associated with severe disease in immunocompromised patients. Elevated replication fitness was mediated by accumulation of mutations in the replication enhancing domain (ReED) within domain (D) 2 of non-structural protein (NS) 5A. NS5A is a partially unstructured phosphoprotein lacking enzymatic activity but fulfilling a key role in HCV replication due to interacting with various cellular and viral proteins. It can exist in a variety of dimeric and oligomeric conformations mediated by NS5A D1 with clinically approved NS5A inhibitors proposed to exert their antiviral function by fixing these dimers in distinct conformations. In this study, we aimed at elucidating the ReEDs mode of action. AlphaFold modelling indicated a so far unrecognized NS5A dimerization site in the ReED. Indeed, split nano luciferase assays revealed a significantly stronger NS5A dimerization of high replicator ReED variants, suggesting that high replication fitness is mediated by enforcement of NS5A self-interaction. This hypothesis was supported by the effect of low dose (1 pM) NS5A inhibitor treatment, increasing replication fitness and phenocopying the effects of ReED mutations. Furthermore, we found that HCV isolate JFH1, replicating with very high efficiency, is completely resistant to the regulatory function of the ReED. Chimeric replicons composed of ReED resistant JFH1 and the ReED sensitive isolate J6 identified NS3 helicase and NS5B polymerase as critical genetic elements mediating ReED sensitivity/resistance. Our data overall suggest that NS5A is a negative regulator of HCV replication fitness with dimerization releasing the inhibitory interaction with helicase and/or polymerase, thereby likely facilitating initiation of RNA synthesis.

Sarbecoviruses exhibit extensive diversity in host range and zoonotic potential. Although the ectodomain of the viral spike protein has been well characterized, the functional landscape of the cytoplasmic tail (CT) remains poorly defined. To address this gap, we systematically generated CT truncation variants of representative spike proteins from all major ACE2-utilizing clades to define the roles of conserved host factor-interacting motifs associated with COPI, COPII, FERM, and SNX27. Using vesicular stomatitis virus (VSV)- and lentivirus-based pseudotyping systems, we evaluated viral entry in cells expressing varying levels of ACE2 and TMPRSS2. Our results demonstrate that CT-associated motifs differentially regulate viral infectivity. Specifically, truncation of the COPI- or SNX27-binding motifs markedly reduces entry efficiency, whereas disruption of the COPII-binding motif produces the opposite outcome. By contrast, removal of the FERM-binding motif consistently enhances infectivity across lineages. Mechanistically, truncation of this motif increases spike expression, cell surface localization, incorporation into virions, and particle stability. Importantly, despite these pronounced effects on viral infectivity, deletion of the FERM-binding motif does not affect antigenicity, receptor dependence, or sensitivity to protease inhibitors, as demonstrated by neutralization and inhibition assays. In addition, this approach substantially increases spike protein density on virus-like particles (VLPs). Collectively, by extending the analysis beyond SARS-CoV-1 and SARS-CoV-2, our study reveals a generalizable mechanism in which cytoskeletal anchoring mediated by the FERM-binding motif acts as a limiting determinant of viral assembly. These findings provide a practical framework for optimizing pseudovirus platforms and guiding vaccine development against emerging viral threats.

Infectious bursal disease virus (IBDV) and H9N2 avian influenza virus (AIV) are significant global threats to poultry health and production. While IBDV induces severe immunosuppression, undermining host defense and vaccine efficacy, H9N2 AIV is characterized by widespread prevalence, persistent shedding, and substantial economic losses. Conventional inactivated vaccines often fail to elicit robust cellular immunity and necessitate multiple booster doses, underscoring the urgent requirement for advanced multivalent vaccination platforms. To address this, we developed a recombinant herpesvirus of turkey (rHVT BAC-VP2-HA) using a bacterial artificial chromosome (BAC) vector system, engineered to co-express the major protective antigen VP2 of IBDV and the hemagglutinin (HA) of H9N2 AIV. Genetic stability and in vitro characterization confirmed that the recombinant exhibited replication kinetics and plaque morphology comparable to parental HVT, with stable antigen expression. In SPF chickens, rHVT BAC-VP2-HA induced strong humoral immune responses against both target antigens, comparable to those elicited by a commercial inactivated vaccine. Crucially, the recombinant virus significantly enhanced cellular immunity, evidenced by markedly elevated CD3+CD8+ T cell responses. Upon challenge, the recombinant conferred high clinical protection (86%) against virulent IBDV, significantly ameliorating bursal pathology and reducing viral loads. Notably, it provided complete (100%) protection against H9N2 AIV, effectively abolishing viral shedding and suppressing viral replication in respiratory tissues. These results demonstrate that rHVT BAC-VP2-HA is a safe and efficacious candidate capable of eliciting humoral and cellular immune responses, offering a promising strategy for the integrated control of major poultry diseases. ImportanceInfectious bursal disease virus (IBDV) and H9N2 avian influenza virus (AIV) are major pathogens that frequently co-circulate in poultry, where IBDV-induced immunosuppression compromises the efficacy of vaccination against other infectious diseases. Conventional inactivated vaccines primarily induce humoral immunity and are often insufficient to prevent viral shedding or provide broad protection against multiple pathogens. In this study, we developed a recombinant herpesvirus of turkeys (HVT) vaccine co-expressing the IBDV VP2 and H9N2 HA antigens and demonstrated that it induces both robust antibody responses and enhanced CD8+ T cell immunity. Notably, this vaccine not only provided effective protection against IBDV but also completely prevented viral shedding following H9N2 challenge. These findings highlight the advantage of HVT-vectored multivalent vaccines in eliciting balanced immune responses and controlling virus transmission, providing important insights for the development of next-generation vaccines against immunosuppressive and respiratory viral co-infections in poultry.

Avian influenza A viruses (IAV) pose a constant pandemic threat, with the recent 2.3.4.4b clade of the H5 subtype causing high pathogenicity and spreading across animal species and geographic locations. Understanding human pre-existing immunity to avian H5 IAV can inform on population susceptibility, a critical aspect of pandemic preparedness. To that end, we analysed the IAV HA-specific antibodies across individuals born between 1928-1999 with different early life exposures to IAV subtypes. Individuals born prior to 1957 had the highest pre-existing serum antibodies to group 1 HA antigens, including the 2.3.4.4b H5 and a group 1 HA stem antigen. These birth-year-specific patterns were not reflected in the limited pre-existing serum neutralising antibodies detectable against a 2.3.4.4b H5 IAV or in H5-specific memory B cell populations. They were however evident in pre-existing nasal IgG and IgA titres to H5, which were greater in individuals born prior to 1957. Our findings demonstrate that the immunological biases afforded by early life exposure extend to antibodies detected in the nasal mucosa, the site of IAV replication. ImportanceUnderstating pre-existing immunity to influenza A viruses of pandemic potential is an important aspect of pandemic preparedness. This includes an understanding the heterogeneity of pre-existing immunity across the population. Here, we demonstrate that pre-existing antibodies to H5 IAV vary according to year of birth and childhood imprinting. We demonstrate that this is the case for both systemic and nasal antibodies, highlighting the importance of understanding pre-existing mucosal immunity at the sites of influenza virus replication.

Dairy cows have emerged as a reservoir for human infection with highly pathogenic avian influenza (HPAI) H5N1. At the bovine-human interface, H5N1 strains may acquire adaptive mutations that influence their zoonotic potential. Sequence analysis identified a K142E substitution (bovine to human) in the PA and PA-X proteins, with the potential to affect both polymerase activity and host shutoff. Here, we used a loss-of-function approach to investigate how the bovine substitution (E142K) in PA/PA-X impacts viral replication, host shutoff activity, and pathogenicity in the human H5N1 background. Viral growth kinetics demonstrated that the virus containing the E142K substitution is attenuated, with reduced replication compared to wild-type (WT) virus. Consistently, PA-X-mediated host shutoff activity was reduced, resulting in increased induction of interferon (IFN) responses relative to WT. In vivo, mice infected with the E142K mutant virus survived, whereas infection with the WT virus was uniformly lethal. Despite comparable viral titers and inflammation score in mouse lungs, cytokine and chemokine profiling revealed distinct immune responses, with reduced CCL2 and increased CCL5 and IFN-{gamma} in mice infected with the E142K mutant virus compared to mice infected with the WT virus. These findings indicate that increased virulence of the human-adapted strain is driven by a PA-X mutation that modulates inflammatory responses, producing distinct immune signatures linked to host survival or viral lethality rather than changes in polymerase activity by PA. Collectively, these results highlight PA-X as a key determinant of pathogenicity of H5N1 and a potential target for the rational design of antiviral strategies. IMPORTANCEHighly pathogenic avian influenza (HPAI) H5N1 viruses have recently expanded beyond their traditional avian hosts to infect mammals, where they are acquiring mutations associated with mammalian adaptation. These changes raise the concern that influenza H5N1 viruses could evolve the capacity for sustained human infection and human-to-human transmission and pose a pandemic threat. Therefore, it is critical to identify and functionally characterize emerging mutations that influence viral pathogenicity and host interactions. Such studies will enhance our understanding of the requirements for efficient infection and disease in mammalian hosts and inform the rational design of antiviral strategies. In this study, we present data characterizing a bovine-to-human substitution (K142E) in the viral PA-X impacting viral replication, host shutoff activity, and pathogenicity. Our results demonstrate the key role of PA-X in H5N1 viral pathogenicity and the feasibility of targeting PA-X for the rational design of antivirals to control influenza infections.

In the respiratory epithelium, interferon (IFN)-induced antiviral resistance acts as a defense against infection. Influenza A viruses (IAVs) have evolved multiple strategies to counteract these defenses, including expression of the viral protein NS1, which inhibits both IFN production and the IFN-mediated transcription of Interferon Stimulated Genes (ISG) in infected cells. However, experiments show that this inhibition is imperfect, especially at a low multiplicity of infection (MOI). One hypothesis to describe this phenomenon relies on the presence of Semi-infectious Particles (SIPs) that fail to express NS1. In this scenario, the IFN response is incompletely suppressed at low MOI, while it is successfully inhibited at high MOI because most cells are infected by multiple virions, allowing complementation to rescue NS1 expression. To test this hypothesis, we developed a computer simulation that models viral gene defects and complementation. We compared the model outputs with in vitro experiments at different MOIs. To assess inter-host reproducibility and calibrate the model parameters, we measured IFN levels and viral load over time in bronchial epithelial cell cultures from five human donors. We observed no statistically significant heterogeneity in IFN response or virus production between donors, and the calibrated simulation fits the experimental time series for IFN and viral load. Consistent with literature (1,2), the model predicted higher IFN levels at low MOI than at high MOI. Finally, simulations of IFN treatment applied before and during infection showed reduced viral load, in agreement with our experiments. Increasing the viral genome defect rate above the experimentally estimated rate increased IFN levels and reduced viral load. High MOI simulations showed lower cumulative IFN levels, while NS1 knockout recovered high IFN levels. These results demonstrate the ability of mechanistic models of viral dynamics to predict the innate immune response of epithelial cells during viral infection. Author SummaryRespiratory viruses such as influenza A are highly infectious and pose significant challenges for the human immune system. Through laboratory experiments and computer simulations, we investigated how cells in the respiratory epithelium defend themselves and their neighbors against infection. Using cells collected from different donors, we generated 3-dimensional cell cultures that mimic human airways and measured how they respond to IAV. When a tissue was initially exposed to a small amount of virus, cells could successfully slow or stop the spread of the infection. This phenomenon is hypothesized to be due in part to the high error rate in IAV replication, resulting in many viral particles that are not fully functional. We recapitulated this experimental result with our computational model, validating the model design and parameter estimates. We then simulated a scenario in which cells were pre-treated with interferon, a protective cytokine important to early immune response, and showed that this pre-treatment could successfully limit infection. Laboratory experiments subsequently confirmed this predicted behavior. The computational model reproduced key observations across infection conditions and identified nonfunctional viral particles as important drivers of the early immune response.

Most HIV-1 transmission occurs at mucosal surfaces, which are colonized by the host microbiota. However, interactions between HIV and bacteria or bacterial products derived from the human microbiome are poorly characterized, and their biological consequences are largely unexplored. Here, we evaluated the effects of sialic acid-binding lectins expressed by bacterial species ubiquitous in the human microbiota on HIV-1 infectivity using viruses produced in 293T cells and human primary cells. We demonstrated that these bacterial lectins enhanced HIV-1 infectivity in a sialoglycan-dependent manner. Specifically, Siglec-like binding region lectins (SLBR-N, SLBR-H, and SLBR-B) from Streptococcus gordonii and Staphylococcal superantigen-like lectins (SSL3, SSL4, and SSL11) from Staphylococcus aureus increased HIV-1 infectivity to varying extents, depending on lectin type and virus strain. Among these lectins, SLBR-N exhibited the greatest potency, corresponding with its superior ability to bind virions and promote virus-cell attachment. This enhancing activity was observed for direct infection of TZM-bl reporter cells and primary CD4+ T cells, as well as trans-infection in the presence or absence of the mannose-binding host lectin DC-SIGN. Importantly, these findings were corroborated in vivo using humanized mice, in which pre-exposure to SLBR-N promoted rectal HIV-1 transmission and increased viral burdens in plasma and splenic cells. Collectively, the data show sialoglycan-binding bacterial lectins as microbial factors that can enhance HIV-1 transmission at mucosal surfaces, highlighting a potential direct role for the microbiota in modulating HIV-1 acquisition risk. Author SummaryHIV is commonly transmitted from one person to another across mucosal surfaces, such as those lining the genital and rectal tracts, which are densely populated by bacteria that make up the human microbiota. Yet, surprisingly little is known about how these bacteria and the molecules they produce influence HIV infection. In this study, we investigated a group of bacterial proteins known as sialic acid-binding lectins that are expressed by common members of the human microbiome: Siglec-like binding region lectins from Streptococcus gordonii and superantigen-like lectins from Staphylococcus aureus. Using multiple HIV strains and several types of target cells, we demonstrate that lectin binding to HIV can increase virus attachment to target cells and thereby enhance infection, although the magnitude of this effect varies among lectins and virus strains. Lectin binding also facilitates HIV spread from cell to cell and promotes mucosal HIV infection in a humanized mouse model, resulting in a higher viral burden in the blood and tissues. These findings identify bacterial lectins as important factors that can influence HIV infection and implicate a potential role for the human microbiota in determining susceptibility to HIV infection.

Coronaviruses (CoVs) replicate unusually large RNA genomes that necessitate proofreading by the 3'-to-5' exoribonuclease (ExoN) formed by nonstructural proteins 14 (nsp14) and 10 (nsp10). Previous studies suggested that inactivation of the ExoN catalytic site in severe acute respiratory syndrome CoV 2 (SARS-CoV-2) is lethal, leaving unresolved whether the virus can tolerate impaired proofreading activity. Here, we investigated the functional requirement for ExoN in SARS-CoV-2 replication by combining a continuous fluorescence-based biochemical assay with an optimized single-bacmid reverse genetics system. Mutational analysis of residues involved in RNA binding or catalysis revealed graded effects on ExoN activity in vitro. Alanine substitution of Lys9, a residue positioned near the RNA-binding interface, did not reduce ExoN activity, whereas charge reversal at this position (K9E) impaired activity more strongly than alanine substitutions of the catalytic motif I residues D90 and E92 (D90A/E92A). Correspondingly, recombinant SARS-CoV-2 carrying K9A was readily recovered, whereas the D90A/E92A mutant was recovered only after an extended delay and K9E could not be rescued despite repeated attempts. The D90A/E92A mutant exhibited reduced replication while maintaining the engineered ExoN substitutions during serial passage. Deep sequencing of viral populations revealed a marked increase in genome-wide sequence variation in the D90A/E92A mutant, demonstrating a stable mutator phenotype. Together, these findings indicate that SARS-CoV-2 can tolerate substantial impairment of ExoN activity but depends on a minimal activity threshold for viability. This system provides a platform for defining how SARS-CoV-2 proofreading controls genome stability, viral fitness, and sensitivity to antiviral strategies that exploit reduced replication fidelity. ImportanceCoronaviruses have unusually large RNA genomes because they encode a proofreading enzyme that removes copying errors during replication. It has been unclear whether SARS-CoV-2 can survive when this proofreading function is strongly weakened, because earlier studies suggested that loss of the enzymes catalytic activity is lethal. We show that SARS-CoV-2 can tolerate substantial impairment of proofreading, but only when residual exonuclease activity remains above a minimal threshold. A virus with impaired proofreading replicates less efficiently and accumulates mutations across its genome, whereas a more severe defect prevents virus recovery. These findings clarify how coronavirus proofreading balances genome stability with viral fitness and provide a useful system for studying how reduced replication fidelity affects viral evolution, antiviral sensitivity, and attenuation. Defining this activity threshold may also help guide antiviral strategies that target coronavirus proofreading.