Diet-induced obesity affects influenza disease severity and transmission dynamics in ferrets.

Obesity, and the associated metabolic syndrome, is a risk factor for increased disease severity with a variety of infectious agents, including influenza virus. Yet, the mechanisms are only partially understood. As the number of people, particularly children, living with obesity continues to rise, it is critical to understand the role of host status on disease pathogenesis. In these studies, we use a diet-induced obese ferret model and tools to demonstrate that, like humans, obesity resulted in notable changes to the lung microenvironment, leading to increased clinical disease and viral spread to the lower respiratory tract. The decreased antiviral responses also resulted in obese animals shedding higher infectious virus for a longer period, making them more likely to transmit to contacts. These data suggest that the obese ferret model may be crucial to understanding obesity's impact on influenza disease severity and community transmission and a key tool for therapeutic and intervention development for this high-risk population.

Necroptosis blockade prevents lung injury in severe influenza.

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies.

Establishment of Swine Primary Nasal, Tracheal, and Bronchial Epithelial Cell Culture Models for the Study of Influenza Virus Infection.

We established primary porcine nasal, tracheal, and bronchial epithelial cells that recapitulate the physical and functional properties of the respiratory tract and have the ability to fully differentiate. Trans-well cultures demonstrated increased transepithelial electrical resistance over time the presence of tight junctions as demonstrated by immunohistochemistry. The nasal, tracheal, and bronchial epithelial cells developed cilia, secreted mucus, and expressed sialic acids on surface glycoproteins, the latter which are required for influenza A virus infection. Swine influenza viruses were shown to replicate efficiently in the primary epithelial cell cultures, supporting the use of these culture models to assess swine influenza and other virus infection. Primary porcine nasal, tracheal, and bronchial epithelial cell culture models enable assessment of emerging and novel influenza viruses for pandemic potential as well as mechanistic studies to understand mechanisms of infection, reassortment, and generation of novel virus. As swine are susceptible to infection with multiple viral and bacterial respiratory pathogens, these primary airway cell models may enable study of the cellular response to infection by pathogens associated with Porcine Respiratory Disease Complex.

Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication and pathogenicity.

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibits stress granule assembly and interacts with G3BP1/2 via an ITFG motif, including residue F17, in the N protein. Prior studies examining the impact of the G3PB1-N interaction on SARS-CoV-2 replication have produced inconsistent findings, and the role of this interaction in pathogenesis is unknown. Here, we use structural and biochemical analyses to define the residues required for G3BP1-N interaction and structure-guided mutagenesis to selectively disrupt this interaction. We find that N-F17A mutation causes highly specific loss of interaction with G3BP1/2. SARS-CoV-2 N-F17A fails to inhibit stress granule assembly in cells, has decreased viral replication, and causes decreased pathology in vivo. Further mechanistic studies indicate that the N-F17-mediated G3BP1-N interaction promotes infection by limiting sequestration of viral genomic RNA (gRNA) into stress granules.

Maternal immunization with distinct influenza vaccine platforms elicits unique antibody profiles that impact the protection of offspring.

Pregnant women and infants are considered high-risk groups for increased influenza disease severity. While influenza virus vaccines are recommended during pregnancy, infants cannot be vaccinated until at least six months of age. Passive transfer of maternal antibodies (matAbs) becomes vital for the infant's protection. Here, we employed an ultrasound-based timed-pregnancy murine model and examined matAb responses to distinct influenza vaccine platforms and influenza A virus (IAV) infection in dams and their offspring. We demonstrate vaccinating dams with a live-attenuated influenza virus (LAIV) vaccine or recombinant hemagglutinin (rHA) proteins administered with adjuvant resulted in enhanced and long-lasting immunity and protection from influenza in offspring. In contrast, a trivalent split-inactivated vaccine (TIV) afforded limited protection in our model. By cross-fostering pups, we show the timing of antibody transfer from vaccinated dams to their offspring (prenatal versus postnatal) can shape the antibody profile depending on the vaccine platform. Our studies provide information on how distinct influenza vaccines lead to immunogenicity and efficacy during pregnancy, impact the protection of their offspring, and detail roles for IgG1 and IgG2c in the development of vaccine administration during pregnancy that stimulate and measure expression of both antibody subclasses.

Diet-induced obesity impacts influenza disease severity and transmission dynamics in ferrets.

Obesity, and the associated metabolic syndrome, is a risk factor for increased disease severity with a variety of infectious agents, including influenza virus. Yet the mechanisms are only partially understood. As the number of people, particularly children, living with obesity continues to rise, it is critical to understand the role of host status on disease pathogenesis. In these studies, we use a novel diet-induced obese ferret model and new tools to demonstrate that like humans, obesity resulted in significant changes to the lung microenvironment leading to increased clinical disease and viral spread to the lower respiratory tract. The decreased antiviral responses also resulted in obese animals shedding higher infectious virus for longer making them more likely to transmit to contacts. These data suggest the obese ferret model may be crucial to understanding obesity's impact on influenza disease severity and community transmission, and a key tool for therapeutic and intervention development for this high-risk population. Teaser: A new ferret model and tools to explore obesity's impact on respiratory virus infection, susceptibility, and community transmission.

The ß6 Integrin Negatively Regulates TLR7-Mediated Epithelial Immunity via Autophagy During Influenza A Virus Infection.

Integrins are essential surface receptors that sense extracellular changes to initiate various intracellular signaling cascades. The rapid activation of the epithelial-intrinsic ß6 integrin during influenza A virus (IAV) infection has been linked to innate immune impairments. Yet, how ß6 regulates epithelial immunity remains undefined. Here, we identify the role of ß6 in mediating the Toll-like receptor 7 (TLR7) through the regulation of intracellular trafficking. We demonstrate that deletion of the ß6 integrin in lung epithelial cells significantly enhances the TLR7-mediated activation of the type I interferon (IFN) response during homeostasis and respiratory infection. IAV-induced ß6 facilitates TLR7 trafficking to lysosome-associated membrane protein (LAMP2a) components, leading to a reduction in endosomal compartments and associated TLR7 signaling. Our findings reveal an unappreciated role of ß6-induced autophagy in influencing epithelial immune responses during influenza virus infection.

Astrovirus replication is dependent on induction of double-membrane vesicles through a PI3K-dependent, LC3-independent pathway.

Human astrovirus is a positive-sense, single-stranded RNA virus. Astrovirus infection causes gastrointestinal symptoms and can lead to encephalitis in immunocompromised patients. Positive-strand RNA viruses typically utilize host intracellular membranes to form replication organelles, which are potential antiviral targets. Many of these replication organelles are double-membrane vesicles (DMVs). Here, we show that astrovirus infection leads to an increase in DMV formation through a replication-dependent mechanism that requires some early components of the autophagy machinery. Results indicate that the upstream class III phosphatidylinositol 3-kinase (PI3K) complex, but not LC3 conjugation machinery, is utilized in DMV formation. Both chemical and genetic inhibition of the PI3K complex lead to significant reduction in DMVs, as well as viral replication. Elucidating the role of autophagy machinery in DMV formation during astrovirus infection reveals a potential target for therapeutic intervention for immunocompromised patients. IMPORTANCE These studies provide critical new evidence that astrovirus replication requires formation of double-membrane vesicles, which utilize class III phosphatidylinositol 3-kinase (PI3K), but not LC3 conjugation autophagy machinery, for biogenesis. These results are consistent with replication mechanisms for other positive-sense RNA viruses suggesting that targeting PI3K could be a promising therapeutic option for not only astrovirus, but other positive-sense RNA virus infections.

Host obesity impacts genetic variation in influenza A viral populations.

Obesity is a chronic health condition characterized by excess adiposity leading to a systemic increase in inflammation and dysregulation of metabolic hormones and immune cell populations. Obesity is well established as a risk factor for many noncommunicable diseases; however, its consequences for infectious disease are poorly understood. Influenza A virus (IAV) is a highly infectious pathogen responsible for seasonal and pandemic influenza. Host risk factors, including compromised immunity and pre-existing health conditions, can contribute to increased infection susceptibility and disease severity. During viral replication in a host, the negative sense single stranded RNA genome of IAV accumulates genetic diversity that may have important consequences for viral evolution and transmission. Here, we investigated the impact of host obesity on IAV genetic variation using a diet induced obesity ferret model. We infected obese and lean male ferrets with the A/Hong Kong/1073/1999 (H9N2) IAV strain. Using a co-caging study design, we investigated the maintenance, generation, and transmission of intrahost IAV genetic variation by sequencing viral genomic RNA obtained from nasal wash samples over multiple days of infection. We found evidence for an enhanced role of positive selection acting on de novo mutations in obese hosts that led to nonsynonymous changes that rose to high frequency. In addition, we identified numerous cases of recurrent low-frequency mutations throughout the genome that were specific to obese hosts. Despite these obese-specific variants, overall viral genetic diversity did not differ significantly between obese and lean hosts. This is likely due to the high supply rate of de novo variation and common evolutionary adaptations to the ferret host regardless of obesity status, which we show are mediated by variation in the hemagglutinin (HA) and polymerase genes (PB2 and PB1). As with single nucleotide variants, we identified a class of defective viral genomes (DVGs) that were found uniquely in either obese or lean hosts, but overall DVG diversity and dynamics did not differ between the two groups. Our study provides the first insight into the consequences of host obesity on viral genetic diversity and adaptation, suggesting that host factors associated with obesity alter the selective environment experienced by a viral population, thereby impacting the spectrum of genetic variation.

Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication.

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. G3BP1/2 are prominent interactors of the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the functional consequences of the G3BP1-N interaction in the context of viral infection remain unclear. Here we used structural and biochemical analyses to define the residues required for G3BP1-N interaction, followed by structure-guided mutagenesis of G3BP1 and N to selectively and reciprocally disrupt their interaction. We found that mutation of F17 within the N protein led to selective loss of interaction with G3BP1 and consequent failure of the N protein to disrupt stress granule assembly. Introduction of SARS-CoV-2 bearing an F17A mutation resulted in a significant decrease in viral replication and pathogenesis in vivo, indicating that the G3BP1-N interaction promotes infection by suppressing the ability of G3BP1 to form stress granules.

Efficacy of oseltamivir treatment in influenza virus-infected obese mice.

Obesity has been epidemiologically and empirically linked with more severe diseases upon influenza infection. To ameliorate severe disease, treatment with antivirals, such as the neuraminidase inhibitor oseltamivir, is suggested to begin within days of infection especially in high-risk hosts. However, this treatment can be poorly effective and may generate resistance variants within the treated host. Here, we hypothesized that obesity would reduce oseltamivir treatment effectiveness in the genetically obese mouse model. We demonstrated that oseltamivir treatment does not improve viral clearance in obese mice. While no traditional variants associated with oseltamivir resistance emerged, we did note that drug treatment failed to quench the viral population and did lead to phenotypic drug resistance in vitro. Together, these studies suggest that the unique pathogenesis and immune responses in obese mice could have implications for pharmaceutical interventions and the within-host dynamics of the influenza virus population. IMPORTANCE Influenza virus infections, while typically resolving within days to weeks, can turn critical, especially in high-risk populations. Prompt antiviral administration is crucial to mitigating these severe sequalae, yet concerns remain if antiviral treatment is effective in hosts with obesity. Here, we show that oseltamivir does not improve viral clearance in genetically obese or type I interferon receptor-deficient mice. This suggests a blunted immune response may impair oseltamivir efficacy and render a host more susceptible to severe disease. This study furthers our understanding of oseltamivir treatment dynamics both systemically and in the lungs of obese mice, as well as the consequences of oseltamivir treatment for the within-host emergence of drug-resistant variants.

Astrovirus replication is dependent on induction of double membrane vesicles through a PI3K-dependent, LC3-independent pathway.

Human astrovirus is a positive sense, single stranded RNA virus. Astrovirus infection causes gastrointestinal symptoms and can lead to encephalitis in immunocompromised patients. Positive strand RNA viruses typically utilize host intracellular membranes to form replication organelles, which are potential antiviral targets. Many of these replication organelles are double membrane vesicles (DMVs). Here we show that astrovirus infection leads to an increase in DMV formation, and this process is replication-dependent. Our data suggest that astrovirus infection induces rearrangement of endoplasmic reticulum fragments, which may become the origin for DMV formation. Transcriptional data suggested that formation of DMVs during astrovirus infection requires some early components of the autophagy machinery. Results indicate that the upstream class III phosphatidylinositol 3-kinase (PI3K) complex, but not LC3 conjugation machinery, is utilized in DMV formation. Inhibition of the PI3K complex leads to significant reduction in viral replication and release from cells. Elucidating the role of autophagy machinery in DMV formation during astrovirus infection reveals a potential target for therapeutic intervention for immunocompromised patients. Importance: These studies provide critical new evidence that astrovirus replication requires formation of double membrane vesicles, which utilize class III PI3K, but not LC3 conjugation autophagy machinery for biogenesis. These results are consistent with replication mechanisms for other positive sense RNA viruses. This suggests that targeting PI3K could be a promising therapeutic option for not only astrovirus, but other positive sense RNA virus infections.

Influenza A virus undergoes compartmentalized replication in vivo dominated by stochastic bottlenecks.

Transmission of influenza A viruses (IAV) between hosts is subject to numerous physical and biological barriers that impose genetic bottlenecks, constraining viral diversity and adaptation. The bottlenecks within hosts and their potential impacts on evolutionary pathways taken during infection are poorly understood. To address this, we created highly diverse IAV libraries bearing molecular barcodes on two gene segments, enabling high-resolution tracking and quantification of unique virus lineages within hosts. Here we show that IAV infection in lungs is characterized by multiple within-host bottlenecks that result in "islands" of infection in lung lobes, each with genetically distinct populations. We perform site-specific inoculation of barcoded IAV in the upper respiratory tract of ferrets and track viral diversity as infection spreads to the trachea and lungs. We detect extensive compartmentalization of discrete populations within lung lobes. Bottleneck events and localized replication stochastically sample individual viruses from the upper respiratory tract or the trachea that become the dominant genotype in a particular lobe. These populations are shaped strongly by founder effects, with limited evidence for positive selection. The segregated sites of replication highlight the jackpot-style events that contribute to within-host influenza virus evolution and may account for low rates of intrahost adaptation.

Innate Antiviral Cytokine Response to Swine Influenza Virus by Swine Respiratory Epithelial Cells.

Swine influenza virus (SIV) can cause respiratory illness in swine. Swine contribute to influenza virus reassortment, as avian, human, and/or swine influenza viruses can infect swine and reassort, and new viruses can emerge. Thus, it is important to determine the host antiviral responses that affect SIV replication. In this study, we examined the innate antiviral cytokine response to SIV by swine respiratory epithelial cells, focusing on the expression of interferon (IFN) and interferon-stimulated genes (ISGs). Both primary and transformed swine nasal and tracheal respiratory epithelial cells were examined following infection with field isolates. The results show that IFN and ISG expression is maximal at 12 h postinfection (hpi) and is dependent on cell type and virus genotype. IMPORTANCE Swine are considered intermediate hosts that have facilitated influenza virus reassortment events that have given rise pandemics or genetically related viruses have become established in swine. In this study, we examine the innate antiviral response to swine influenza virus in primary and immortalized swine nasal and tracheal epithelial cells, and show virus strain- and host cell type-dependent differential expression of key interferons and interferon-stimulated genes.

Evidence of influenza infection in dogs and cats in central Chile.

As companion animals, dogs and cats live in close contact with humans, generating the possibility of interspecies pathogen transmission events. Equine origin H3N8 and avian origin H5N1 influenza virus have been reported in dogs and cats respectively since 2004 with outbreaks associated with different strains recorded for both species in Asia and North America. To date, there have been no reports of influenza viruses from companion animals in South America. To fill this gap in knowledge, we performed active epidemiological surveillance in shelters that received abandoned animals, backyard production systems and veterinary clinics between May 2017 and January 2019 to estimate the burden of influenza infection in cats and dogs in the central region of Chile. Blood samples, oropharyngeal swabs or both were collected for influenza A virus detection by RT-qPCR, NP-ELISA, and hemagglutination inhibition assay. Logistic regression models were performed to assess the association between NP-ELISA-positivity and variables including sex and animal origin. The percentage of ELISA-positive samples was 43.5 % (95 % CI: 37.0-50.1) and 23.3 % (95 % CI: 10.6-42.7) for dogs and cats, respectively. No association was found between NP-ELISA results and sex or animal origin for either dogs or cats. Two ELISA positive samples showed hemagglutination inhibition titers against pandemic H1N1 influenza. One dog sample tested positive by RT-qPCR, indicating an overall RT-qPCR positivity in dogs of 1.1 % (95 % CI: 0.05-6.7). None of the tested cat samples were positive by this assay.

Influenza virus and SARS-CoV-2: pathogenesis and host responses in the respiratory tract.

Influenza viruses cause annual epidemics and occasional pandemics of respiratory tract infections that produce a wide spectrum of clinical disease severity in humans. The novel betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 and has since caused a pandemic. Both viral and host factors determine the extent and severity of virus-induced lung damage. The host's response to viral infection is necessary for viral clearance but may be deleterious and contribute to severe disease phenotypes. Similarly, tissue repair mechanisms are required for recovery from infection across the spectrum of disease severity; however, dysregulated repair responses may lead to chronic lung dysfunction. Understanding of the mechanisms of immunopathology and tissue repair following viral lower respiratory tract infection may broaden treatment options. In this Review, we discuss the pathogenesis, the contribution of the host response to severe clinical phenotypes and highlight early and late epithelial repair mechanisms following influenza virus infection, each of which has been well characterized. Although we are still learning about SARS-CoV-2 and its disease manifestations in humans, throughout the Review we discuss what is known about SARS-CoV-2 in the context of this broad knowledge of influenza virus, highlighting the similarities and differences between the respiratory viruses.

Equine-Like H3 Avian Influenza Viruses in Wild Birds, Chile.

Since their discovery in the United States in 1963, outbreaks of infection with equine influenza virus (H3N8) have been associated with serious respiratory disease in horses worldwide. Genomic analysis suggests that equine H3 viruses are of an avian lineage, likely originating in wild birds. Equine-like internal genes have been identified in avian influenza viruses isolated from wild birds in the Southern Cone of South America. However, an equine-like H3 hemagglutinin has not been identified. We isolated 6 distinct H3 viruses from wild birds in Chile that have hemagglutinin, nucleoprotein, nonstructural protein 1, and polymerase acidic genes with high nucleotide homology to the 1963 H3N8 equine influenza virus lineage. Despite the nucleotide similarity, viruses from Chile were antigenically more closely related to avian viruses and transmitted effectively in chickens, suggesting adaptation to the avian host. These studies provide the initial demonstration that equine-like H3 hemagglutinin continues to circulate in a wild bird reservoir.

Respiratory Bacteria Stabilize and Promote Airborne Transmission of Influenza A Virus.

Influenza A virus (IAV) is a major pathogen of the human respiratory tract, where the virus coexists and interacts with bacterial populations comprising the respiratory tract microbiome. Synergies between IAV and respiratory bacterial pathogens promote enhanced inflammation and disease burden that exacerbate morbidity and mortality. We demonstrate that direct interactions between IAV and encapsulated bacteria commonly found in the respiratory tract promote environmental stability and infectivity of IAV. Antibiotic-mediated depletion of the respiratory bacterial flora abrogated IAV transmission in ferret models, indicating that these virus-bacterium interactions are operative for airborne transmission of IAV. Restoring IAV airborne transmission in antibiotic-treated ferrets by coinfection with Streptococcus pneumoniae confirmed a role for specific members of the bacterial respiratory community in promoting IAV transmission. These results implicate a role for the bacterial respiratory flora in promoting airborne transmission of IAV.IMPORTANCE Infection with influenza A virus (IAV), especially when complicated with a secondary bacterial infection, is a leading cause of global mortality and morbidity. Gaining a greater understanding of the transmission dynamics of IAV is important during seasonal IAV epidemics and in the event of a pandemic. Direct bacterium-virus interactions are a recently appreciated aspect of infectious disease biology. Direct interactions between IAV and specific bacterial species of the human upper respiratory tract were found to promote the stability and infectivity of IAV during desiccation stress. Viral environmental stability is an important aspect during transmission, suggesting a potential role for bacterial respiratory communities in IAV transmission. Airborne transmission of IAV was abrogated upon depletion of nasal bacterial flora with topical antibiotics. This defect could be functionally complemented by S. pneumoniae coinfection. These data suggest that bacterial coinfection may be an underappreciated aspect of IAV transmission dynamics.

Primary Swine Respiratory Epithelial Cell Lines for the Efficient Isolation and Propagation of Influenza A Viruses.

Influenza virus isolation from clinical samples is critical for the identification and characterization of circulating and emerging viruses. Yet efficient isolation can be difficult. In these studies, we isolated primary swine nasal and tracheal respiratory epithelial cells and immortalized swine nasal epithelial cells (siNEC) and tracheal epithelial cells (siTEC) that retained the abilities to form tight junctions and cilia and to differentiate at the air-liquid interface like primary cells. Critically, both human and swine influenza viruses replicated in the immortalized cells, which generally yielded higher-titer viral isolates from human and swine nasal swabs, supported the replication of isolates that failed to grow in Madin-Darby canine kidney (MDCK) cells, and resulted in fewer dominating mutations during viral passaging than MDCK cells.IMPORTANCE Robust in vitro culture systems for influenza virus are critically needed. MDCK cells, the most widely used cell line for influenza isolation and propagation, do not adequately model the respiratory tract. Therefore, many clinical isolates, both animal and human, are unable to be isolated and characterized, limiting our understanding of currently circulating influenza viruses. We have developed immortalized swine respiratory epithelial cells that retain the ability to differentiate and can support influenza replication and isolation. These cell lines can be used as additional tools to enhance influenza research and vaccine development.

Obesity-Related Microenvironment Promotes Emergence of Virulent Influenza Virus Strains.

Obesity is associated with increased disease severity, elevated viral titers in exhaled breath, and significantly prolonged viral shed during influenza A virus infection. Due to the mutable nature of RNA viruses, we questioned whether obesity could also influence influenza virus population diversity. Here, we show that minor variants rapidly emerge in obese mice. The variants exhibit increased viral replication, resulting in enhanced virulence in wild-type mice. The increased diversity of the viral population correlated with decreased type I interferon responses, and treatment of obese mice with recombinant interferon reduced viral diversity, suggesting that the delayed antiviral response exhibited in obesity permits the emergence of a more virulent influenza virus population. This is not unique to obese mice. Obesity-derived normal human bronchial epithelial (NHBE) cells also showed decreased interferon responses and increased viral replication, suggesting that viral diversity also was impacted in this increasing population.IMPORTANCE Currently, 50% of the adult population worldwide is overweight or obese. In these studies, we demonstrate that obesity not only enhances the severity of influenza infection but also impacts viral diversity. The altered microenvironment associated with obesity supports a more diverse viral quasispecies and affords the emergence of potentially pathogenic variants capable of inducing greater disease severity in lean hosts. This is likely due to the impaired interferon response, which is seen in both obese mice and obesity-derived human bronchial epithelial cells, suggesting that obesity, aside from its impact on influenza virus pathogenesis, permits the stochastic accumulation of potentially pathogenic viral variants, raising concerns about its public health impact as the prevalence of obesity continues to rise.