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Influenza A viruses (IAV) have caused more documented global pandemics in human history than any other pathogen 1,2 . High pathogenicity avian influenza (HPAI) viruses belonging to the H5N1 subtype are a leading pandemic risk. Two decades after H5N1 "bird flu" became established in poultry in Southeast Asia, its descendants have resurged 3 , setting off an H5N1 panzootic in wild birds that is fueled by (a) rapid intercontinental spread, reaching South America and Antarctica for the first time 4,5 ; (b) fast evolution via genomic reassortment 6 ; and (c) frequent spillover into terrestrial 7,8 and marine mammals 9 . The virus has sustained mammal-to-mammal transmission in multiple settings, including European fur farms 10,11 , South American marine mammals 12-15 , and US dairy cattle 16-19 , raising questions about whether humans are next. Historically, swine are considered optimal intermediary hosts that help avian influenza viruses (AIV) adapt to mammals before jumping to humans 20 . However, the altered ecology of H5N1 has opened the door to new evolutionary pathways. Could dairy cattle, farmed mink, or South American sea lions serve as new mammalian gateways to humans? Here we explore the molecular and ecological factors driving H5N1's sudden expansion in host range and assess the likelihood of different zoonotic pathways leading to an H5N1 pandemic., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Sustained community spread of influenza viruses relies on efficient person-to-person transmission. Current experimental transmission systems do not mimic environmental conditions (e.g., air exchange rates, flow patterns), host behaviors, or exposure durations relevant to real-world settings. Therefore, results from these traditional systems may not be representative of influenza virus transmission in humans. To address this pitfall, we developed a close-range transmission setup that implements a play-based scenario and used it to investigate the impact of ventilation rates on transmission. In this setup, four immunologically naive recipient ferrets were exposed to a donor ferret infected with a genetically barcoded 2009 H1N1 virus (H1N1pdm09) for 4 h. The ferrets interacted in a shared space that included toys, similar to a childcare setting. Transmission efficiency was assessed under low and high ventilation, with air exchange rates of ~1.3 h -1 and 23 h -1 , respectively. Transmission efficiencies observed in three independent replicate studies were similar between ventilation conditions. The presence of infectious virus or viral RNA on surfaces and in air throughout the exposure area was also not impacted by the ventilation rate. While high viral genetic diversity in donor ferret nasal washes was maintained during infection, recipient ferret nasal washes displayed low diversity, revealing a narrow transmission bottleneck regardless of ventilation rate. Examining the frequency and duration of ferret physical touches revealed no link between these interactions and a successful transmission event. Our findings indicate that exposures characterized by frequent, close-range interactions and the presence of fomites can overcome the benefits of increased ventilation., Competing Interests: Competing interests statement:The authors declare no competing interest.

The menstrual cycle influences the risk of acquiring sexually transmitted infections (STIs), including Chlamydia trachomatis ( C. trachomatis ), although the underlying immune contributions are poorly defined. A mouse model simulating the immune-mediated process of menstruation could provide valuable insights into tissue-specific determinants of protection against chlamydial infection within the cervicovaginal and uterine mucosae comprising the female reproductive tract (FRT). Here, we used the pseudopregnancy approach in naïve C57Bl/6 mice and performed vaginal challenge with Chlamydia muridarum ( C. muridarum ) at decidualization, endometrial tissue remodeling, or uterine repair. This strategy identified that the time frame comprising uterine repair correlated with robust infection and greater bacterial burden as compared with mice on hormonal contraception, while challenges during endometrial remodeling were least likely to result in a productive infection. By comparing the infection site at early time points following chlamydial challenge, we found that a greater abundance of innate effector populations and proinflammatory signaling, including IFNγ correlated with protection. FRT immune profiling in uninfected mice over pseudopregnancy or in pig-tailed macaques over the menstrual cycle identified NK cell infiltration into the cervicovaginal tissues and lumen over the course of endometrial remodeling. Notably, NK cell depletion over this time frame reversed protection, with mice now productively infected with C. muridarum following challenge. This study shows that the pseudopregnancy murine menstruation model recapitulates immune changes in the FRT as a result of endometrial remodeling and identifies NK cell localization at the FRT as essential for immune protection against primary C. muridarum infection.

Outbreaks of highly pathogenic H5N1 clade 2.3.4.4b viruses in farmed mink and seals combined with isolated human infections suggest these viruses pose a pandemic threat. To assess this threat, using the ferret model, we show an H5N1 isolate derived from mink transmits by direct contact to 75% of exposed ferrets and, in airborne transmission studies, the virus transmits to 37.5% of contacts. Sequence analyses show no mutations were associated with transmission. The H5N1 virus also has a low infectious dose and remains virulent at low doses. This isolate carries the adaptive mutation, PB2 T271A, and reversing this mutation reduces mortality and airborne transmission. This is the first report of a H5N1 clade 2.3.4.4b virus exhibiting direct contact and airborne transmissibility in ferrets. These data indicate heightened pandemic potential of the panzootic H5N1 viruses and emphasize the need for continued efforts to control outbreaks and monitor viral evolution., (© 2024. The Author(s).)

Patterns of within-host influenza A virus (IAV) diversity and evolution have been described in natural human infections, but these patterns remain poorly characterized in non-human hosts. Elucidating these dynamics is important to better understand IAV biology and the evolutionary processes that govern spillover into humans. Here, we sampled an IAV outbreak in pigs during a week-long county fair to characterize viral diversity and evolution in this important reservoir host. Nasal wipes were collected on a daily basis from all pigs present at the fair, yielding up to 421 samples per day. Subtyping of PCR-positive samples revealed the co-circulation of H1N1 and H3N2 subtype swine IAVs. PCR-positive samples with robust Ct values were deep-sequenced, yielding 506 sequenced samples from a total of 253 pigs. Based on higher-depth re-sequenced data from a subset of these initially sequenced samples (260 samples from 168 pigs), we characterized patterns of within-host IAV genetic diversity and evolution. We find that IAV genetic diversity in single-subtype infected pigs is low, with the majority of intrahost Single Nucleotide Variants (iSNVs) present at frequencies of <10%. The ratio of the number of nonsynonymous to the number of synonymous iSNVs is significantly lower than under the neutral expectation, indicating that purifying selection shapes patterns of within-host viral diversity in swine. The dynamic turnover of iSNVs and their pronounced frequency changes further indicate that genetic drift also plays an important role in shaping IAV populations within pigs. Taken together, our results highlight similarities in patterns of IAV genetic diversity and evolution between humans and swine, including the role of stochastic processes in shaping within-host IAV dynamics., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Competing Interests: The authors declare no conflict of interest.

An ideal vaccine both attenuates virus growth and disease in infected individuals and reduces the spread of infections in the population, thereby generating herd immunity. Although this strategy has proved successful by generating humoral immunity to measles, yellow fever and polio, many respiratory viruses evolve to evade pre-existing antibodies 1 . One approach for improving the breadth of antiviral immunity against escape variants is through the generation of memory T cells in the respiratory tract, which are positioned to respond rapidly to respiratory virus infections 2-6 . However, it is unknown whether memory T cells alone can effectively surveil the respiratory tract to the extent that they eliminate or greatly reduce viral transmission following exposure of an individual to infection. Here we use a mouse model of natural parainfluenza virus transmission to quantify the extent to which memory CD8 + T cells resident in the respiratory tract can provide herd immunity by reducing both the susceptibility of acquiring infection and the extent of transmission, even in the absence of virus-specific antibodies. We demonstrate that protection by resident memory CD8 + T cells requires the antiviral cytokine interferon-γ (IFNγ) and leads to altered transcriptional programming of epithelial cells within the respiratory tract. These results suggest that tissue-resident CD8 + T cells in the respiratory tract can have important roles in protecting the host against viral disease and limiting viral spread throughout the population., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

One potential advantage of live attenuated influenza vaccines (LAIVs) is their ability to establish both virus-specific Ab and tissue-resident memory T cells (TRM) in the respiratory mucosa. However, it is hypothesized that pre-existing immunity from past infections and/or immunizations prevents LAIV from boosting or generating de novo CD8+ T cell responses. To determine whether we can overcome this limitation, we generated a series of drifted influenza A/PR8 LAIVs with successive mutations in the hemagglutinin protein, allowing for increasing levels of escape from pre-existing Ab. We also inserted a CD8+ T cell epitope from the Sendai virus nucleoprotein (NP) to assess both generation of a de novo T cell response and boosting of pre-existing influenza-specific CD8+ T cells following LAIV immunization. Increasing the level of escape from Ab enabled boosting of pre-existing TRM, but we were unable to generate de novo Sendai virus NP+ CD8+ TRM following LAIV immunization in PR8 influenza-immune mice, even with LAIV strains that can fully escape pre-existing Ab. As these data suggested a role for cell-mediated immunity in limiting LAIV efficacy, we investigated several scenarios to assess the impact of pre-existing LAIV-specific TRM in the upper and lower respiratory tract. Ultimately, we found that deletion of the immunodominant influenza NP366-374 epitope allowed for sufficient escape from cellular immunity to establish de novo CD8+ TRM. When combined, these studies demonstrate that both pre-existing humoral and cellular immunity can limit the effectiveness of LAIV, which is an important consideration for future design of vaccine vectors against respiratory pathogens., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Respiratory viruses, such as influenza viruses, cause significant morbidity and mortality worldwide through seasonal epidemics and sporadic pandemics. Influenza viruses transmit through multiple modes including contact (either direct or through a contaminated surface) and inhalation of expelled aerosols. Successful human to human transmission requires an infected donor who expels virus into the environment, a susceptible recipient, and persistence of the expelled virus within the environment. The relative efficiency of each mode can be altered by viral features, environmental parameters, donor and recipient host characteristics, and viral persistence. Interventions to mitigate transmission of influenza viruses can target any of these factors. In this review, we discuss many aspects of influenza virus transmission, including the systems to study it, as well as the impact of natural barriers and various nonpharmaceutical and pharmaceutical interventions.

The interface between humans and wildlife is changing and, with it, the potential for pathogen introduction into humans has increased. Avian influenza is a prominent example, with an ongoing outbreak showing the unprecedented expansion of both geographic and host ranges. Research in the field is essential to understand this and other zoonotic threats. Only by monitoring dynamic viral populations and defining their biology in situ can we gather the information needed to ensure effective pandemic preparation., Competing Interests: The authors declare a conflict of interest. We wish to disclose funding received by authors to support research on viruses, from the National Institutes of Health and other funding agencies.

The hemagglutinin (HA) stem region is a major target of universal influenza vaccine efforts owing to the presence of highly conserved epitopes across multiple influenza A virus (IAV) strains and subtypes. To explore the potential impact of vaccine-induced immunity targeting the HA stem, we examined the fitness effects of viral escape from stem-binding broadly neutralizing antibodies (stem-bnAbs). Recombinant viruses containing each individual antibody escape substitution showed diminished replication compared to wild-type virus, indicating that stem-bnAb escape incurred fitness costs. A second-site mutation in the HA head domain (N129D; H1 numbering) reduced the fitness effects observed in primary cell cultures and likely enabled the selection of escape mutations. Functionally, this putative permissive mutation increased HA avidity for its receptor. These results suggest a mechanism of epistasis in IAV, wherein modulating the efficiency of attachment eases evolutionary constraints imposed by the requirement for membrane fusion. Taken together, the data indicate that viral escape from stem-bnAbs is costly but highlights the potential for epistatic interactions to enable evolution within the functionally constrained HA stem domain.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Central nervous system (CNS) disease is the most common extra-respiratory tract complication of influenza A virus infections in humans. Remarkably, zoonotic highly pathogenic avian influenza (HPAI) H5N1 virus infections are more often associated with CNS disease than infections with seasonal influenza viruses. Evolution of avian influenza viruses has been extensively studied in the context of respiratory infections, but evolutionary processes in CNS infections remain poorly understood. We have previously observed that the ability of HPAI A/Indonesia/5/2005 (H5N1) virus to replicate in and spread throughout the CNS varies widely between individual ferrets. Based on these observations, we sought to understand the impact of entrance into and replication within the CNS on the evolutionary dynamics of virus populations. First, we identified and characterized three substitutions-PB1 E177G and A652T and NP I119M - detected in the CNS of a ferret infected with influenza A/Indonesia/5/2005 (H5N1) virus that developed a severe meningo-encephalitis. We found that some of these substitutions, individually or collectively, resulted in increased polymerase activity in vitro. Nevertheless, in vivo, the virus bearing the CNS-associated mutations retained its capacity to infect the CNS but showed reduced dispersion to other anatomical sites. Analyses of viral diversity in the nasal turbinate and olfactory bulb revealed the lack of a genetic bottleneck acting on virus populations accessing the CNS via this route. Furthermore, virus populations bearing the CNS-associated mutations showed signs of positive selection in the brainstem. These features of dispersion to the CNS are consistent with the action of selective processes, underlining the potential for H5N1 viruses to adapt to the CNS., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Siegers et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

When multiple viral populations propagate within the same host environment, they often shape each other's dynamics. These interactions can be positive or negative and can occur at multiple scales, from coinfection of a cell to co-circulation at a global population level. For influenza A viruses (IAVs), the delivery of multiple viral genomes to a cell substantially increases burst size. However, despite its relevance for IAV evolution through reassortment, the implications of this positive density dependence for coinfection between distinct IAVs has not been explored. Furthermore, the extent to which these interactions within the cell shape viral dynamics at the level of the host remains unclear. Here we show that, within cells, diverse coinfecting IAVs strongly augment the replication of a focal strain, irrespective of their homology to the focal strain. Coinfecting viruses with a low intrinsic reliance on multiple infection offer the greatest benefit. Nevertheless, virus-virus interactions at the level of the whole host are antagonistic. This antagonism is recapitulated in cell culture when the coinfecting virus is introduced several hours prior to the focal strain or under conditions conducive to multiple rounds of viral replication. Together, these data suggest that beneficial virus-virus interactions within cells are counterbalanced by competition for susceptible cells during viral propagation through a tissue. The integration of virus-virus interactions across scales is critical in defining the outcomes of viral coinfection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Delima et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Viruses arriving late to an individual cell are blocked from replicating, an effect called superinfection exclusion. A study in PLOS Biology indicates that this exclusion at the level of individual cells gives rise to a heterogenous landscape of infection within a host., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lowen, Ferreri. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Influenza A virus (IAV) genetic exchange through reassortment has the potential to accelerate viral evolution and has played a critical role in the generation of multiple pandemic strains. For reassortment to occur, distinct viruses must co-infect the same cell. The spatio-temporal dynamics of viral dissemination within an infected host therefore define opportunity for reassortment. Here, we used wild type and synonymously barcoded variant viruses of a pandemic H1N1 strain to examine the within-host viral dynamics that govern reassortment in guinea pigs, ferrets and swine. The first two species are well-established models of human influenza, while swine are a natural host and a frequent conduit for cross-species transmission and reassortment. Our results show reassortment to be pervasive in all three hosts but less frequent in swine than in ferrets and guinea pigs. In ferrets, tissue-specific differences in the opportunity for reassortment are also evident, with more reassortants detected in the nasal tract than the lower respiratory tract. While temporal trends in viral diversity are limited, spatial patterns are clear, with heterogeneity in the viral genotypes detected at distinct anatomical sites revealing extensive compartmentalization of reassortment and replication. Our data indicate that the dynamics of viral replication in mammals allow diversification through reassortment but that the spatial compartmentalization of variants likely shapes their evolution and onward transmission., (© 2022. The Author(s).)

For diverse viruses, cellular infection with single vs. multiple virions can yield distinct biological outcomes. We previously found that influenza A/guinea fowl/Hong Kong/WF10/99 (H9N2) virus (GFHK99) displays a particularly high reliance on multiple infection in mammalian cells. Here, we sought to uncover the viral processes underlying this phenotype. We found that the need for multiple infection maps to amino acid 26K of the viral PA protein. PA 26K suppresses endonuclease activity and viral transcription, specifically within cells infected at low multiplicity. In the context of the higher functioning PA 26E, inhibition of PA using baloxavir acid augments reliance on multiple infection. Together, these data suggest a model in which sub-optimal activity of the GFHK99 endonuclease results in inefficient priming of viral transcription, an insufficiency which can be overcome with the introduction of additional viral ribonucleoprotein templates to the cell. More broadly, the finding that deficiency in a core viral function is ameliorated through multiple infection suggests that the fitness effects of many viral mutations are likely to be modulated by multiplicity of infection, such that the shape of fitness landscapes varies with viral densities., Competing Interests: The authors have declared that no competing interests exist.

Current influenza vaccines, while being the best method of managing viral outbreaks, have several major drawbacks that prevent them from being wholly-effective. They need to be updated regularly and require extensive resources to develop. When considering alternatives, the recent deployment of mRNA vaccines for SARS-CoV-2 has created a unique opportunity to evaluate a new platform for seasonal and pandemic influenza vaccines. The mRNA format has previously been examined for application to influenza and promising data suggest it may be a viable format for next-generation influenza vaccines. Here, we discuss the prospect of shifting global influenza vaccination efforts to an mRNA-based system that might allow better control over the product and immune responses and could aid in the development of a universal vaccine.

Influenza A viruses (IAV) remain emerging threats to human public health. Live-attenuated influenza vaccines (LAIV) are one of the most effective prophylactic options to prevent disease caused by influenza infections. However, licensed LAIV remain restricted for use in 2- to 49-year-old healthy and nonpregnant people. Therefore, development of LAIV with increased safety, immunogenicity, and protective efficacy is highly desired. The U.S.-licensed LAIV is based on the master donor virus (MDV) A/Ann Arbor/6/60 H2N2 backbone, which was generated by adaptation of the virus to growth at low temperatures. Introducing the genetic signature of the U.S. MDV into the backbone of other IAV strains resulted in varying levels of attenuation. While the U.S. MDV mutations conferred an attenuated phenotype to other IAV strains, the same amino acid changes did not significantly attenuate the pandemic A/California/04/09 H1N1 (pH1N1) strain. To attenuate pH1N1, we replaced the conserved leucine at position 319 with glutamine (L319Q) in PB1 and analyzed the in vitro and in vivo properties of pH1N1 viruses containing either PB1 L319Q alone or in combination with the U.S. MDV mutations using two animal models of influenza infection and transmission, ferrets and guinea pigs. Our results demonstrated that L319Q substitution in the pH1N1 PB1 alone or in combination with the mutations of the U.S. MDV resulted in reduced pathogenicity (ferrets) and transmission (guinea pigs), and an enhanced temperature sensitive phenotype. These results demonstrate the feasibility of generating an attenuated MDV based on the backbone of a contemporary pH1N1 IAV strain. IMPORTANCE Vaccination represents the most effective strategy to reduce the impact of seasonal IAV infections. Although LAIV are superior in inducing protection and sterilizing immunity, they are not recommended for many individuals who are at high risk for severe disease. Thus, development of safer and more effective LAIV are needed. A concern with the current MDV used to generate the U.S.-licensed LAIV is that it is based on a virus isolated in 1960. Moreover, mutations that confer the temperature-sensitive, cold-adapted, and attenuated phenotype of the U.S. MDV resulted in low level of attenuation in the contemporary pandemic A/California/04/09 H1N1 (pH1N1). Here, we show that introduction of PB1 L319Q substitution, alone or in combination with the U.S. MDV mutations, resulted in pH1N1 attenuation. These findings support the development of a novel LAIV MDV based on a contemporary pH1N1 strain as a medical countermeasure against currently circulating H1N1 IAV.

Transmission efficiency is a critical factor determining the size of an outbreak of infectious disease. Indeed, the propensity of SARS-CoV-2 to transmit among humans precipitated and continues to sustain the COVID-19 pandemic. Nevertheless, the number of new cases among contacts is highly variable and underlying reasons for wide-ranging transmission outcomes remain unclear. Here, we evaluated viral spread in golden Syrian hamsters to define the impact of temporal and environmental conditions on the efficiency of SARS-CoV-2 transmission through the air. Our data show that exposure periods as brief as one hour are sufficient to support robust transmission. However, the timing after infection is critical for transmission success, with the highest frequency of transmission to contacts occurring at times of peak viral load in the donor animals. Relative humidity and temperature had no detectable impact on transmission when exposures were carried out with optimal timing and high inoculation dose. However, contrary to expectation, trends observed with sub-optimal exposure timing and lower inoculation dose suggest improved transmission at high relative humidity or high temperature. In sum, among the conditions tested, our data reveal the timing of exposure to be the strongest determinant of SARS-CoV-2 transmission success and implicate viral load as an important driver of transmission., Competing Interests: The authors have declared that no competing interests exist.

Segmentation of viral genomes provides the potential for genetic exchange within coinfected cells. However, for this potential to be realized, coinfecting genomes must mix during the viral life cycle. The efficiency of reassortment, in turn, dictates its potential to drive evolution. The opportunity for mixing within coinfected cells may vary greatly across virus families, such that the evolutionary implications of genome segmentation differ as a result of core features of the viral life cycle. To investigate the relationship between viral replication compartments and genetic exchange, we quantified reassortment in mammalian orthoreovirus (reovirus). Reoviruses carry a 10-segmented, double-stranded RNA genome, which is replicated within proteinaceous structures termed inclusion bodies. We hypothesized that inclusions impose a barrier to reassortment. We quantified reassortment between wild-type ( wt ) and variant ( var ) reoviruses that differ by one nucleotide per segment. Studies of wt/var systems in both T1L and T3D backgrounds revealed frequent reassortment without bias toward particular genotypes. However, reassortment was more efficient in the T3D serotype. Since T1L and T3D viruses exhibit different inclusion body morphologies, we tested the impact of this phenotype on reassortment. In both serotypes, reassortment levels did not differ by inclusion morphology. Reasoning that the merging of viral inclusions may be critical for genome mixing, we then tested the effect of blocking merging. Reassortment proceeded efficiently even under these conditions. Our findings indicate that reovirus reassortment is highly efficient despite the localization of many viral processes to inclusion bodies, and that the robustness of this genetic exchange is independent of inclusion body structure and fusion. IMPORTANCE Quantification of reassortment in diverse viral systems is critical to elucidate the implications of genome segmentation for viral evolution. In principle, genome segmentation offers a facile means of genetic exchange between coinfecting viruses. In practice, there may be physical barriers within the cell that limit the mixing of viral genomes. Here, we tested the hypothesis that localization of the various stages of the mammalian orthoreovirus life cycle within cytoplasmic inclusion bodies compartmentalizes viral replication and limits genetic exchange. Contrary to this hypothesis, our data indicate that reovirus reassortment occurs readily within coinfected cells and is not strongly affected by the structure or dynamics of viral inclusion bodies. We conclude that the potential for reassortment to contribute to reovirus evolution is high.

The 2'-5'-oligoadenylate synthetases (OAS) are innate immune sensors of cytosolic double-stranded RNA (dsRNA) that play a critical role in limiting viral infection. How these proteins are able to avoid aberrant activation by cellular RNAs is not fully understood, but adenosine-to-inosine (A-to-I) editing has been proposed to limit accumulation of endogenous RNAs that might otherwise cause stimulation of the OAS/RNase L pathway. Here, we aim to uncover whether and how such sequence modifications can restrict the ability of short, defined dsRNAs to activate the single-domain form of OAS, OAS1. Unexpectedly, we find that all tested inosine-containing dsRNAs have an increased capacity to activate OAS1, whether in a destabilizing (I•U) or standard Watson-Crick-like base pairing (I-C) context. Additional variants with strongly destabilizing A•C mismatches or stabilizing G-C pairs also exhibit increased capacity to activate OAS1, eliminating helical stability as a factor in the relative ability of the dsRNAs to activate OAS1. Using thermal difference spectra and molecular dynamics simulations, we identify both increased helical dynamics and specific local changes in helical structure as important factors in the capacity of short dsRNAs to activate OAS1. These helical features may facilitate more ready adoption of the distorted OAS1-bound conformation or stabilize important structures to predispose the dsRNA for optimal binding and activation of OAS1. These studies thus reveal the molecular basis for the greater capacity of some short dsRNAs to activate OAS1 in a sequence-independent manner., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Influenza A viruses (IAVs) are constantly evolving. Crucial steps in the infection cycle, such as sialic acid (SA) receptor binding on the host cell surface, can either promote or hamper the emergence of new variants. We previously assessed the relative fitness in Japanese quail of H9N2 variant viruses differing at a single amino acid position, residue 216 in the hemagglutinin (HA) viral surface protein. This site is known to modulate SA recognition. Our prior study generated a valuable set of longitudinal samples from quail transmission groups where the inoculum comprised different mixed populations of HA 216 variant viruses. Here, we leveraged these samples to examine the evolutionary dynamics of viral populations within and between inoculated and naïve contact quails. We found that positive selection dominated HA gene evolution, but fixation of the fittest variant depended on the competition mixture. Analysis of the whole genome revealed further evidence of positive selection acting both within and between hosts. Positive selection drove fixation of variants in non-HA segments within inoculated and contact quails. Importantly, transmission bottlenecks were modulated by the molecular signature at HA 216, revealing viral receptor usage as a determinant of transmitted diversity. Overall, we show that selection strongly shaped the evolutionary dynamics within and between quails. These findings support the notion that selective processes act effectively on IAV populations in poultry hosts, facilitating rapid viral evolution in this ecological niche., (© The Author(s) 2022. Published by Oxford University Press.)

Influenza A virus [IAV] genomes comprise eight negative strand RNAs packaged into virions in the form of viral ribonucleoproteins [vRNPs]. Rab11a plays a crucial role in the transport of vRNPs from the nucleus to the plasma membrane via microtubules, allowing assembly and virus production. Here, we identify a novel function for Rab11a in the inter-cellular transport of IAV vRNPs using tunneling nanotubes [TNTs]as molecular highways. TNTs are F-Actin rich tubules that link the cytoplasm of nearby cells. In IAV-infected cells, Rab11a was visualized together with vRNPs in these actin-rich intercellular connections. To better examine viral spread via TNTs, we devised an infection system in which conventional, virion-mediated, spread was not possible. Namely, we generated HA-deficient reporter viruses which are unable to produce progeny virions but whose genomes can be replicated and trafficked. In this system, vRNP transfer to neighboring cells was observed and this transfer was found to be dependent on both actin and Rab11a. Generation of infectious virus via TNT transfer was confirmed using donor cells infected with HA-deficient virus and recipient cells stably expressing HA protein. Mixing donor cells infected with genetically distinct IAVs furthermore revealed the potential for Rab11a and TNTs to serve as a conduit for genome mixing and reassortment in IAV infections. These data therefore reveal a novel role for Rab11a in the IAV life cycle, which could have significant implications for within-host spread, genome reassortment and immune evasion., Competing Interests: The authors have declared that no competing interests exist.

Viral recombination can generate novel genotypes with unique phenotypic characteristics, including transmissibility and virulence. Although the capacity for recombination among betacoronaviruses is well documented, recombination between strains of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has not been characterized in detail. Here, we present a lightweight approach for detecting genomes that are potentially recombinant. This approach relies on identifying the mutations that primarily determine SARS-CoV-2 clade structure and then screening genomes for ones that contain multiple mutational markers from distinct clades. Among the over 537,000 genomes queried that were deposited on GISAID.org prior to 16 February 2021, we detected 1,175 potential recombinant sequences. Using a highly conservative criteria to exclude sequences that may have originated through de novo mutation, we find that at least 30 per cent ( n  = 358) are likely of recombinant origin. An analysis of deep-sequencing data for these putative recombinants, where available, indicated that the majority are high quality. Additional phylogenetic analysis and the observed co-circulation of predicted parent clades in the geographic regions of exposure further support the feasibility of recombination in this subset of potential recombinants. An analysis of these genomes did not reveal evidence for recombination hotspots in the SARS-CoV-2 genome. While most of the putative recombinant sequences we detected were genetic singletons, a small number of genetically identical or highly similar recombinant sequences were identified in the same geographic region, indicative of locally circulating lineages. Recombinant genomes were also found to have originated from parental lineages with substitutions of concern, including D614G, N501Y, E484K, and L452R. Adjusting for an unequal probability of detecting recombinants derived from different parent clades and for geographic variation in clade abundance, we estimate that at most 0.2-2.5 per cent of circulating viruses in the USA and UK are recombinant. Our identification of a small number of putative recombinants within the first year of SARS-CoV-2 circulation underscores the need to sustain efforts to monitor the emergence of new genotypes generated through recombination., (© The Author(s) 2021. Published by Oxford University Press.)

Since its first detection in December 2019, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly spread worldwide, resulting in over 79.2 million documented cases in one year. Lack of pre-existing immunity against this newly emerging virus has pushed the urgent development of anti-viral therapeutics and vaccines to reduce the spread of the virus and alleviate disease. Appropriate animal models recapitulating the pathogenesis of and host responses to SARS-CoV-2 infection in humans have and will continue to accelerate this development process. Several animal models including mice, hamsters, ferrets, and non-human primates have been evaluated and actively applied in preclinical studies. However, since each animal model has unique features, it is necessary to weigh the strengths and weaknesses of each according to the goals of the study. Here, we summarize the key features, strengths and weaknesses of animal models for SARS-CoV-2, focusing on their application in anti-viral therapeutic and vaccine development., (Copyright © 2021 Elsevier B.V. All rights reserved.)

It is well documented that influenza A viruses selectively package 8 distinct viral ribonucleoprotein complexes (vRNPs) into each virion; however, the role of host factors in genome assembly is not completely understood. To evaluate the significance of cellular factors in genome assembly, we generated a reporter virus carrying a tetracysteine tag in the NP gene (NP-Tc virus) and assessed the dynamics of vRNP localization with cellular components by fluorescence microscopy. At early time points, vRNP complexes were preferentially exported to the MTOC; subsequently, vRNPs associated on vesicles positive for cellular factor Rab11a and formed distinct vRNP bundles that trafficked to the plasma membrane on microtubule networks. In Rab11a deficient cells, however, vRNP bundles were smaller in the cytoplasm with less co-localization between different vRNP segments. Furthermore, Rab11a deficiency increased the production of non-infectious particles with higher RNA copy number to PFU ratios, indicative of defects in specific genome assembly. These results indicate that Rab11a+ vesicles serve as hubs for the congregation of vRNP complexes and enable specific genome assembly through vRNP:vRNP interactions, revealing the importance of Rab11a as a critical host factor for influenza A virus genome assembly., Competing Interests: The authors have declared that no competing interests exist.

Efforts to estimate the risk posed by potentially pandemic influenza A viruses (IAV), and to understand the mechanisms governing interspecies transmission, have been hampered by a lack of animal models that yield relevant and statistically robust measures of viral fitness. To address this gap, we monitored several quantitative measures of fitness in a guinea pig model: infectivity, magnitude of replication, kinetics of replication, efficiency of transmission, and kinetics of transmission. With the goal of identifying metrics that distinguish human- and non-human-adapted IAV we compared strains derived from humans to those circulating in swine and canine populations. Influenza A/Panama/2007/99 (H3N2), A/Netherlands/602/2009 (H1N1), A/swine/Kansas/77778/2007 (H1N1), A/swine/Spain/53207/2004 [M1 P41A] (H1N1), and A/canine/Illinois/41915/2015 (H3N2) viruses were evaluated. Our results revealed higher infectivity and faster kinetics of viral replication and transmission for human and canine strains compared to the swine viruses. Conversely, peak viral titers and efficiency of transmission were higher for human strains relative to both swine and canine IAVs. Total viral loads were comparable among all strains tested. When analyzed together, data from all strains point to peak viral load as a key driver of transmission efficiency and replication kinetics as a key driver of transmission kinetics. While the dose initiating infection did not strongly impact peak viral load, dose was found to modulate kinetics of viral replication and, in turn, timing of transmission. Taken together, our results point to peak viral load and transmission efficiency as key metrics differentiating human and non-human IAVs and suggest that high peak viral load precipitates robust transmission. Importance Influenza pandemics occur when an IAV from non-human hosts enters the human population and adapts to give rise to a lineage capable of sustained transmission among humans. Despite recurring zoonotic infections involving avian or swine adapted IAVs, influenza pandemics occur infrequently because IAVs typically exhibit low fitness in a new host species. Anticipating when a zoonosis might lead to a pandemic is both critical for public health preparedness and extremely challenging. The approach to characterizing IAVs reported here is designed to aid risk assessment efforts by generating rigorous and quantitative data on viral phenotypes relevant for emergence. Our data suggest that the ability to replicate to high titers and transmit efficiently irrespective of initial dose are key characteristics distinguishing IAVs that have established sustained circulation in the human population from IAVs that circulate in non-human mammalian hosts., (Copyright © 2021 American Society for Microbiology.)

Reassortment among co-infecting influenza A viruses (IAVs) is an important source of viral diversity and can facilitate expansion into novel host species. Indeed, reassortment played a key role in the evolution of the last three pandemic IAVs. Observed patterns of reassortment within a coinfected host are likely to be shaped by several factors, including viral load, the extent of viral mixing within the host and the stringency of selection. These factors in turn are expected to vary among the diverse host species that IAV infects. To investigate host differences in IAV reassortment, here we examined reassortment of two distinct avian IAVs within their natural host (mallards) and a mammalian model system (guinea pigs). Animals were co-inoculated with A/wildbird/California/187718-36/2008 (H3N8) and A/mallard/Colorado/P66F1-5/2008 (H4N6) viruses. Longitudinal samples were collected from the cloaca of mallards or the nasal tract of guinea pigs and viral genetic exchange was monitored by genotyping clonal isolates from these samples. Relative to those in guinea pigs, viral populations in mallards showed higher frequencies of reassortant genotypes and were characterized by higher genotype richness and diversity. In line with these observations, analysis of pairwise segment combinations revealed lower linkage disequilibrium in mallards as compared to guinea pigs. No clear longitudinal patterns in richness, diversity or linkage disequilibrium were present in either host. Our results reveal mallards to be a highly permissive host for IAV reassortment and suggest that reduced viral mixing limits avian IAV reassortment in a mammalian host.

The newly emerged human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a pandemic of respiratory illness. Current evidence suggests that severe cases of SARS-CoV-2 are associated with a dysregulated immune response. However, little is known about how the innate immune system responds to SARS-CoV-2. In this study, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral, surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by proinflammatory cytokines and chemokine induction, including interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and CXCL8, and identified NF-κB and ATF-4 as key drivers of this proinflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III interferon (IFN) response to SARS-CoV-2 infection. However, pretreatment and posttreatment with type I and III IFNs significantly reduced virus replication in pHAE cultures that correlated with upregulation of antiviral effector genes. Combined, our findings demonstrate that SARS-CoV-2 does not trigger an IFN response but is sensitive to the effects of type I and III IFNs. Our studies demonstrate the utility of pHAE cultures to model SARS-CoV-2 infection and that both type I and III IFNs can serve as therapeutic options to treat COVID-19 patients. IMPORTANCE The current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. Our research identifies an excellent system to model SARS-CoV-2 infection of the human airways that can be used to test various treatments. Analysis of infection in this model system found that human airway epithelial cell cultures induce a strong proinflammatory cytokine response yet block the production of type I and III IFNs to SARS-CoV-2. However, treatment of airway cultures with the immune molecules type I or type III interferon (IFN) was able to inhibit SARS-CoV-2 infection. Thus, our model system identified type I or type III IFN as potential antiviral treatments for COVID-19 patients., (Copyright © 2020 American Society for Microbiology.)

Infection with a single influenza A virus (IAV) is only rarely sufficient to initiate productive infection. Instead, multiple viral genomes are often required in a given cell. Here, we show that the reliance of IAV on multiple infection can form an important species barrier. Namely, we find that avian H9N2 viruses representative of those circulating widely at the poultry-human interface exhibit acute dependence on collective interactions in mammalian systems. This need for multiple infection is greatly reduced in the natural host. Quantification of incomplete viral genomes showed that their complementation accounts for the moderate reliance on multiple infection seen in avian cells but not the added reliance seen in mammalian cells. An additional form of virus-virus interaction is needed in mammals. We find that the PA gene segment is a major driver of this phenotype and that both viral replication and transcription are affected. These data indicate that multiple distinct mechanisms underlie the reliance of IAV on multiple infection and underscore the importance of virus-virus interactions in IAV infection, evolution and emergence.

2'-5'-Oligoadenylate synthetases (OAS) are innate immune sensors of cytosolic double-stranded RNA (dsRNA) and play a critical role in limiting viral infection. dsRNA binding induces allosteric structural changes in OAS1 that reorganize its catalytic center to promote synthesis of 2'-5'-oligoadenylate and thus activation of endoribonuclease L. Specific RNA sequences and structural motifs can also enhance activation of OAS1 through currently undefined mechanisms. To better understand these drivers of OAS activation, we tested the impact of defined sequence changes within a short dsRNA that strongly activates OAS1. Both in vitro and in human A549 cells, appending a 3'-end single-stranded pyrimidine (3'-ssPy) can strongly enhance OAS1 activation or have no effect depending on its location, suggesting that other dsRNA features are necessary for correct presentation of the motif to OAS1. Consistent with this idea, we also find that the dsRNA binding position is dictated by an established consensus sequence (WWN9WG). Unexpectedly, however, not all sequences fitting this consensus activate OAS1 equivalently, with strong dependence on the identity of both partially conserved (W) and non-conserved (N9) residues. A picture thus emerges in which both specific RNA features and the context in which they are presented dictate the ability of short dsRNAs to activate OAS1., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Reassortment of segmented viruses can be an important source of genetic diversity underlying viral evolution and emergence. Methods for the quantification of reassortment have been described but are often cumbersome and best suited for the analysis of reassortment between highly divergent parental strains. While it is useful to understand the potential of divergent parents to reassort, outcomes of such heterologous reassortment are driven by differential selection acting on the progeny and are typically strain specific. To quantify reassortment robustly, a system free of differential selection is needed. We have generated such a system for influenza A virus and for mammalian orthoreovirus by constructing well-matched parental viruses carrying small genetic tags. The method utilizes high-resolution melt technology for the identification of reassortant viruses. Ease of sample preparation and data analysis enables streamlined genotyping of a large number of virus clones. The method described here thereby allows quantification of the efficiency of reassortment and can be applied to diverse segmented viruses., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned., Competing Interests: AG-S. is inventor of patents on influenza virus vaccines owned by the Icahn School for Medicine at Mount Sinai and licensed to Medimmune, BI Vetmedica, Vivaldi Biosciences, Zoetis and Avimex.

Human influenza A viruses are known to be transmitted via the air from person to person. It is unknown from which anatomical site of the respiratory tract influenza A virus transmission occurs. Here, pairs of genetically tagged and untagged influenza A/H1N1, A/H3N2 and A/H5N1 viruses that are transmissible via the air are used to co-infect donor ferrets via the intranasal and intratracheal routes to cause an upper and lower respiratory tract infection, respectively. In all transmission cases, we observe that the viruses in the recipient ferrets are of the same genotype as the viruses inoculated intranasally, demonstrating that they are expelled from the upper respiratory tract of ferrets rather than from trachea or the lower airways. Moreover, influenza A viruses that are transmissible via the air preferentially infect ferret and human nasal respiratory epithelium. These results indicate that virus replication in the upper respiratory tract, the nasal respiratory epithelium in particular, of donors is a driver for transmission of influenza A viruses via the air.

The M segment of the 2009 pandemic influenza A virus (IAV) has been implicated in its emergence into human populations. To elucidate the genetic contributions of the M segment to host adaptation, and the underlying mechanisms, we examined a panel of isogenic viruses that carry avian- or human-derived M segments. Avian, but not human, M segments restricted viral growth and transmission in mammalian model systems, and the restricted growth correlated with increased expression of M2 relative to M1. M2 overexpression was associated with intracellular accumulation of autophagosomes, which was alleviated by interference of the viral proton channel activity by amantadine treatment. As M1 and M2 are expressed from the M mRNA through alternative splicing, we separated synonymous and non-synonymous changes that differentiate human and avian M segments and found that dysregulation of gene expression leading to M2 overexpression diminished replication, irrespective of amino acid composition of M1 or M2. Moreover, in spite of efficient replication, virus possessing a human M segment that expressed avian M2 protein at low level did not transmit efficiently. We conclude that (i) determinants of transmission reside in the IAV M2 protein, and that (ii) control of M segment gene expression is a critical aspect of IAV host adaptation needed to prevent M2-mediated dysregulation of vesicular homeostasis., Competing Interests: The authors have declared that no competing interests exist.

Segmentation of viral genomes into multiple RNAs creates the potential for replication of incomplete viral genomes (IVGs). Here we use a single-cell approach to quantify influenza A virus IVGs and examine their fitness implications. We find that each segment of influenza A/Panama/2007/99 (H3N2) virus has a 58% probability of being replicated in a cell infected with a single virion. Theoretical methods predict that IVGs carry high costs in a well-mixed system, as 3.6 virions are required for replication of a full genome. Spatial structure is predicted to mitigate these costs, however, and experimental manipulations of spatial structure indicate that local spread facilitates complementation. A virus entirely dependent on co-infection was used to assess relevance of IVGs in vivo. This virus grows robustly in guinea pigs, but is less infectious and does not transmit. Thus, co-infection allows IVGs to contribute to within-host spread, but complete genomes may be critical for transmission.

The high degree of conservation of CD8 T cell epitopes of influenza A virus (IAV) may allow for the development of T cell-inducing vaccines that provide protection across different strains and subtypes. This conservation is not fully explained by functional constraint, since an additional mutation(s) can compensate for the replicative fitness loss of IAV escape variants. Here, we propose three additional mechanisms that contribute to the conservation of CD8 T cell epitopes of IAV. First, influenza-specific CD8 T cells may protect predominantly against severe pathology rather than infection and may have only a modest effect on transmission. Second, polymorphism of the human major histocompatibility complex class I (MHC-I) gene restricts the advantage of an escape variant to only a small fraction of the human population who carry the relevant MHC-I alleles. Finally, infection with CD8 T cell escape variants may result in a compensatory increase in the responses to other epitopes of IAV. We use a combination of population genetics and epidemiological models to examine how the interplay between these mechanisms affects the rate of invasion of IAV escape variants. We conclude that for a wide range of biologically reasonable parameters, the invasion of an escape variant virus will be slow, with a timescale of a decade or more. The results suggest T cell-inducing vaccines do not engender the rapid evolution of IAV. Finally, we identify key parameters whose measurement will allow for more accurate quantification of the long-term effectiveness and impact of universal T cell-inducing influenza vaccines. IMPORTANCE Universal influenza vaccines against the conserved epitopes of influenza A virus have been proposed to minimize the burden of seasonal outbreaks and prepare for the pandemics. However, it is not clear how rapidly T cell-inducing vaccines will select for viruses that escape these T cell responses. Our mathematical models explore the factors that contribute to the conservation of CD8 T cell epitopes and how rapidly the virus will evolve in response to T cell-inducing vaccines. We identify the key biological parameters to be measured and questions that need to be addressed in future studies., (Copyright © 2019 Li et al.)