INTRODUCTION:Influenza A virus (IAV) is a zoonotic pathogen with pandemic potential that infects a wide range of species, including companion animals. Although surveillance efforts have primarily focused on North America, Europe and Asia, data from South America remain scarce. This study evaluated the molecular and serological evidence of IAV circulation in dogs and cats from shelters and multi-pet households in central Chile. METHODS:Between June and November 2020, oropharyngeal swabs and serum samples were collected from dogs and cats in shelters and multi-pet households in central Chile. Samples were analysed by RT-qPCR, NP-ELISA and hemagglutination inhibition assay. RESULTS:IAV was detected by RT-qPCR in 3.2% (11/342) of dogs (95% CI: 1.3%-5.1%) and 5.8% (4/69) of cats (95% CI: 0.3%-11.3%). Serological analysis revealed IAV seropositivity in 55.5% (96/173) of dogs (95% CI: 48.1%-62.9%) and 50.0% (10/20) of cats (95% CI: 28.1%-71.9%). Additionally, pdmH1N1 antibodies were detected in 26 animals (25.7%; 95% CI: 17.2%-34.2%) out of 101 NP-ELISA-positive serum samples, with higher median titres for cats (median = 160) compared to dogs (median = 20). Low-level antibody titres against Canine/H3N2 (range = 10-20) were identified in three dogs (3.0%; 95% CI: 0.0%-6.3%), whereas no antibodies were detected against Canine/H3N8 or Avian/H3N6. No significant associations were observed between seropositivity and animal age, sex, origin or area. CONCLUSIONS:This study presents the first report of IAV detection in cats in South America and highlights a high level of IAV exposure among companion animals in central Chile. These findings underscore the importance of including pets in IAV surveillance efforts under a One Health approach and highlight the need for expanded monitoring and genetic characterization of circulating strains to assess zoonotic risk.

The outbreak of highly pathogenic avian H5 influenza (HPAI) clade 2.3.4.4b in cattle has spread across the United States. Mice with pre-existing immunity to H1N1 virus or with a live-attenuated influenza vaccine showed protection against a lethal bovine-derived HPAI H5N1 viral challenge. Notably, ferrets with mixed immunity also demonstrated protection against a feline-derived H5N1 virus, independent of cross-reactive neutralization titers, but antibodies to whole virus were observed. To investigate protective factors, we conducted T cell epitope mapping using published H1N1 viral sequences and found high conservation of key T cell epitopes in the bovine HPAI H5N1 strain. Depletion of T cells in mice prior to and during primary H1N1 infection impacted cross-protective antibodies to H5N1 virus, with CD4 depletion increasing mortality and CD8 depletion mildly impacting morbidity upon H5N1 viral challenge. This underscores the need to investigate memory T cell responses alongside antibodies in assessing preexisting cross-protection to HPAI H5N1 viruses.IMPORTANCEThe rapid spread of highly pathogenic avian H5 influenza (HPAI) clade 2.3.4.4b in U.S. cattle represents an urgent and evolving public health threat. Our findings reveal that pre-existing immunity, whether from seasonal H1N1 infection or live-attenuated vaccination, can confer substantial protection against lethal bovine- and feline-derived HPAI H5N1 viruses, even in the absence of strong cross-neutralizing antibody titers. By integrating T cell epitope mapping with mechanistic depletion studies, we demonstrate that conserved CD4 and CD8 T cell epitopes across H1N1 and H5N1 strains underpin this cross-protection. Critically, loss of CD4 T cell help during primary H1N1 infection disrupts the development of cross-reactive antibody responses and markedly worsens outcomes after H5N1 challenge. These results identify memory T cell responses as important determinants of heterosubtypic immunity and highlight the need to incorporate T cell-focused metrics into risk assessment, vaccine evaluation, and preparedness strategies for emerging HPAI H5N1 viruses.

In March 2024, a highly pathogenic avian influenza H5N1 (HPAI) clade 2.3.4.4b virus was identified in US dairy cows, with spillover to cats, poultry, and humans. Up to 30% of commercial pasteurized milk tested contained viral genome copies. The impact of residual viral remnants on host immunity is unknown. Orally ingested proteins can stimulate gut-associated lymphoid tissues, potentially inducing tolerance and altering responses to later infection. We found that milk pasteurization fully inactivated pandemic H1N1 and bovine H5N1 influenza viruses yet preserved hemagglutinin (HA) protein integrity. In mice, repeated oral exposure to inactivated virus did not alter mortality after H5N1 virus challenge. Preliminary data showed that naïve mice exposed to improperly pasteurized milk containing live H5N1 virus developed lethal infection, whereas prior H1N1 infection conferred protection. Mice with preexisting H1N1 immunity remained protected when challenged with bovine H5N1 virus after exposure to H5N1 pasteurized in milk. These findings suggest that pasteurized milk containing inactivated H5N1 virus poses minimal health risks.

Obesity is a global health issue, increasing susceptibility to viral infections. Obese mice have increased mortality and morbidity from influenza (IAV) infection due to increased viral spread and lung damage. However, the specific innate immune responses contributing to increased disease severity in obesity remain unknown. NK cells are crucial for defense against viral infections, including IAV. Depletion of NK cells during IAV infection increases mortality with higher viral loads in lean but not obese mice, indicating NK cell dysfunction in obesity. In response to IAV, NK cells in obese mice displayed significantly reduced function, as evidenced by diminished activation (CD69+), cytokine production (IFN-γ), and cytolytic activity (granzyme B). We observed defects in the mTOR-p6SK pathway in obese NK cells that are crucial for antiviral function alongside decreased mitochondrial mass, indicating impaired metabolic function. Obese NK cells maintained high expression of the lipid transporter CD36 post-IAV infection, resulting in an accumulation of intracellular lipids (LipidTOX+), which is known to hinder NK cell function. Preliminary lipidomics studies show elevated and altered species of phospholipids in the bronchoalveolar lavage and lungs of obese mice compared to lean, resulting in a lipid-rich environment that might drive NK cell dysfunction in obesity. These findings suggest that targeting immunometabolism could lead to new antiviral therapies in high-risk populations. 1F32AI183804, 5T32AI106700, CEIRR 75N93021C000016 Viral Immunology (VIR)

Zoonotic transmission of avian influenza viruses into mammals is relatively rare due to anatomical differences in the respiratory tract between species. Recently, clade 2.3.4.4b highly pathogenic H5N1 avian influenza viruses were detected circulating in North American cattle. Sporadic transmission between cattle, humans, and other animals proximal to cattle or after consuming products from infected cattle has occurred, but thus far there is no evidence of human-to-human transmission. However, the virus has the potential to adapt to the mammalian respiratory tract with every transmission event that occurs, making it crucial to understand cellular and species tropism of the H5N1 2.3.4.4b viruses. We compared viral kinetics of clade 2.3.4.4b viruses isolated from birds and mammals in respiratory epithelial cells derived from cattle, human, swine, and ferret. We found that avian derived viruses could replicate in swine cells only, yet mammalian derived strains could replicate efficiently in all tracheal and nasal epithelial cells tested. Interestingly, only bovine mammary epithelial cells (MEC) and swine respiratory epithelial cells were permissive to both avian and mammalian derived strains, possibly due to increased sialic acid expression on bovine MEC compared to bovine tracheal epithelial cells (TEC). However, sialic acid expression differed between dairy and beef cows: TEC derived from a dairy cow had increased expression of α2,3 sialic acid receptors compared to TEC from a beef-dairy cow cross. This study highlights the ability of clade 2.3.4.4b H5N1 viruses derived from mammals but not wild birds to infect the respiratory epithelium of other mammalian hosts.

Educational farms provide students with hands-on experience in agricultural and animal practices. However, the close contact between humans and farm animals creates a significant interface for zoonotic disease transmission, yet research on infectious diseases in such settings remains limited. This study investigates the ongoing spillovers of human-origin influenza A virus (IAV) into swine at an educational farm in central Chile, describing IAV prevalence, outbreak dynamics, and the genomic characterization of detected strains. The Menesianos educational farm, located in Melipilla, central Chile, houses approximately 40 swine alongside other domestic animals, such as horses and cows. As part of an active IAV surveillance project, monthly nasal swab samples were collected from pigs between June 2019 and September 2023 for IAV detection via RT-qPCR targeting the M gene, with positive samples subsequently sequenced. During the study period, monthly IAV prevalence ranged from 0% to 52.5%, with a notable outbreak detected between May and June 2023. The outbreak lasted 5 weeks, peaking at 52.5% prevalence during week 3. Nine IAV strains were isolated over the study period, eight of which were obtained during weeks 2 and 3 of the outbreak. Phylogenetic analysis revealed that all strains were closely related to the pandemic H1N1 2009 influenza virus, with the closest related strains being those circulating in humans in Chile during the same years. These findings highlight the importance of conducting regular IAV surveillance on educational farms, where close interactions between animals and individuals—particularly children and young people—can facilitate viral spillovers and potential reverse zoonosis events.

In March 2024, there was the first reported outbreak of a highly pathogenic avian H5N1 influenza (HPAI) clade 2.3.4.4b virus in dairy cows in the United States. Since then, there have been several spillover events to cats, poultry, and humans. Multiple reports have discovered infectious virus in raw milk from infected dairy cows. Infectious virus can also last over a period on milking machine surfaces as a potential route of spread in cattle and contamination in raw milk. While the U.S. Food and Drug Administration has cleared commercial pasteurized milk as safe for consumption given the lack of infectious virus, there have been numerous reports that up to 30 percent of commercial milk tested were positive for HPAI H5N1 influenza virus genome copies. This is not necessarily unique to the HPAI H5N1 virus, as retrospective studies have identified H1N1 and H3N2 seropositivity in cows linked to decreased milk production. However, it is unknown how repeat exposure to the remaining viral proteins and genomic material in pasteurized milk modulates immune responses once ingested. We developed a successful in-house pasteurization protocol that inactivated high viral loads of the pandemic H1N1 strain A/California/04/2009 (Cal09) or bovine-derived HPAI H5N1 (A/bovine/Ohio.B24OSU-439/2024) viruses in raw milk. Mice were administered this milk daily for five days and rechallenged with each respective virus. We found that repeated oral exposure to inactivated virus was not sufficient to prevent or accelerate mortality from lethal challenge of HPAI H5N1, though it did result in a ~0.5 log10 reduction viral titers in the brain and delayed clinical signs. In contrast, oral gavage of mice with pre existing immunity to H1N1 influenza virus with virus pasteurized in milk were protected from morbidity and mortality upon bovine H5N1 viral challenge. These findings suggest that ingestion of inactivated HPAI H5N1 has limited potential health risk and does not prevent protective immune history-mediated responses to lethal infection. ### Competing Interest Statement The authors have declared no competing interest.

The most recent outbreak of highly pathogenic avian H5 influenza (HPAI) virus in cattle is now widespread across the U.S. with spillover events happening to other mammals, including humans. Several human cases have been reported with clinical signs ranging from conjunctivitis to respiratory illness. However, most of those infected report mild to moderate symptoms, while previously reported HPAI H5Nx infections in humans have had mortality rates upwards of 50%. We recently reported that mice with pre-existing immunity to A/Puerto Rico/08/1934 H1N1 virus were protected from lethal challenge from highly pathogenic clade 2.3.4.4b H5N1 influenza virus. Here, we demonstrate that mice infected with the 2009 pandemic H1N1 virus strain A/California/04/2009 (Cal09) or vaccinated with a live-attenuated influenza vaccine (LAIV) were moderately-to-highly protected against a lethal A/bovine/Ohio/B24OSU-439/2024 H5N1 virus challenge. We also observed that ferrets with mixed pre-existing immunity-either from LAIV vaccination and/or from Cal09 infection-showed protection against a HPAI H5N1 clade 2.3.4.4b virus isolated from a cat. Notably, this protection occurred independently of any detectable hemagglutination inhibition titers (HAIs) against the H5N1 virus. To explore factors that may contribute to protection, we conducted detailed T cell epitope mapping using previously published sequences from H1N1 strains. This analysis revealed a high conservation of amino acid sequences within the internal proteins of our bovine HPAI H5N1 virus strain. These data highlight the necessity to explore additional factors that contribute to protection against HPAI H5N1 viruses, such as memory T cell responses, in addition to HA-inhibition or neutralizing antibodies.

Integrins are essential surface receptors that sense extracellular changes to initiate various intracellular signaling cascades. The rapid activation of the epithelial-intrinsic β6 integrin during influenza A virus (IAV) infection has been linked to innate immune impairments. Yet, how β6 regulates epithelial immunity remains undefined. Here, we identify the role of β6 in mediating the Toll-like receptor 7 (TLR7) through the regulation of intracellular trafficking. We demonstrate that deletion of the β6 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 β6 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 β6-induced autophagy in influencing epithelial immune responses during influenza virus infection.

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.

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.

Astroviruses cause a spectrum of diseases spanning asymptomatic infections to severe diarrhea, but little is understood about their pathogenesis. We previously determined that small intestinal goblet cells were the main cell type infected by murine astrovirus-1. Here, we focused on the host immune response to infection and inadvertently discovered a role for indoleamine 2,3-dioxygenase 1 (Ido1), a host tryptophan catabolizing enzyme, in the cellular tropism of murine and human astroviruses. We identified that Ido1 expression was highly enriched among infected goblet cells, and spatially corresponded to the zonation of infection. Because Ido1 can act as a negative regulator of inflammation, we hypothesized it could dampen host antiviral responses. Despite robust interferon signaling in goblet cells, as well as tuft cell and enterocyte bystanders, we observed delayed cytokine induction and suppressed levels of fecal lipocalin-2. Although we found Ido-/- animals were more resistant to infection, this was not associated with fewer goblet cells nor could it be rescued by knocking out interferon responses, suggesting that IDO1 instead regulates cell permissivity. We characterized IDO1-/- Caco-2 cells and observed significantly reduced human astrovirus-1 infection. Together this study highlights a role for Ido1 in astrovirus infection and epithelial cell maturation.

Highly pathogenic avian influenza (HPAI) A/H5N1 viruses continue to pose a significant threat to animal and human health worldwide. In late 2022, the first confirmed case of HPAI A/H5N1 infection in wild birds in Chile near the Chilean-Peruvian border was reported. Active surveillance by our group in the adyacent Lluta river estuary revealed an increase in A/H5N1 prevalence coinciding with the arrival of migratory birds from the Northern Hemisphere. Genomic analysis of A/H5N1-positive samples demonstrated a close genetic relationship to strains detected in Peru during the same period, which originated from A/H5N1 viruses causing outbreaks in North America. Notably, we identified genetic mutations that did not correlate with known enhanced transmission or binding traits to mammalian receptors. In summary, this study provides valuable genomic insights into the A/H5N1 Clade 2.3.4.4b viruses in wild birds in Chile, emphasizing the need for enhanced surveillance and response strategies to mitigate the threat posed by these highly pathogenic avian influenza viruses in South America.

Little is known about the prevalence of avian influenza viruses (AIV) in wildlife and domestic animals in Polynesia. Here, we present the results of active AIV surveillance performed during two sampling seasons in 2019 on Easter Island (Rapa Nui). Tracheal and cloacal swabs as well as sera samples were obtained from domestic backyard poultry, while fresh faeces were collected from wild birds. In addition to detecting antibodies against AIV in 46% of the domestic chickens in backyard production systems tested, we isolated a novel low pathogenic H6N1 virus from a chicken. Phylogenetic analysis of all genetic segments revealed that the virus was closely related to AIV’s circulating in South America. Our analysis showed different geographical origins of the genetic segments, with the PA, HA, NA, NP, and MP gene segments coming from central Chile and the PB2, PB1, and NS being closely related to viruses isolated in Argentina. While the route of introduction can only be speculated, our analysis shows the persistence and independent evolution of this strain in the island since its putative introduction between 2015 and 2016. The results of this research are the first evidence of AIV circulation in domestic birds on a Polynesian island and increase our understanding of AIV ecology in region, warranting further surveillance on Rapa Nui and beyond.

Persons with obesity are at higher risk for developing severe complications upon influenza virus infection making vaccination a priority. Yet, studies demonstrate vaccine responses are less effective in obese hosts. In these studies, we examined how the timing of weight loss influenced influenza vaccine efficacy in male and female diet- induced obese mice. Here, we show weight loss post-vaccination is insufficient to rescue poor vaccine efficacy; however, weight loss occurring pre-vaccination successfully improves outcomes at viral challenge. Pre-vaccination weight loss improved vaccine immunogenicity and restored a functional recall response at challenge. Through tracking sera metabolic biomarkers, we propose the metabolic state at the time of vaccination is predictive of vaccine immunogenicity. Altogether, these findings highlight how timing of host-directed interventions is vital when seeking to improve influenza vaccine immunogenicity in obese hosts.

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.

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.

Astroviruses are a global cause of pediatric diarrhea, but they are largely understudied, and it is unclear how and where they replicate in the gut. Using an in vivo model, here we report that murine astrovirus preferentially infects actively secreting small intestinal goblet cells, specialized epithelial cells that maintain the mucus barrier. Consequently, virus infection alters mucus production, leading to an increase in mucus-associated bacteria and resistance to enteropathogenic E. coli colonization. These studies establish the main target cell type and region of the gut for productive murine astrovirus infection. They further define a mechanism by which an enteric virus can regulate the mucus barrier, induce functional changes to commensal microbial communities, and alter host susceptibility to pathogenic bacteria.

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.

Human astroviruses (HAstV) are thought to cause between 2 and 9% of acute, nonbacterial diarrhea cases in children worldwide. HAstV infection can be especially problematic in immunocompromised people and infants, where the virus has been associated with necrotizing enterocolitis and severe and persistent diarrhea, as well as rare instances of systemic and fatal disease. And yet, no antivirals have been identified to treat astrovirus infection. Our study provides the first evidence that nitazoxanide may be an effective therapeutic strategy against astrovirus disease.