Influenza A Research Articles

Replication kinetics of novel swine influenza A viruses: an approach to vaccine production

Novel swine Influenza A viruses (IAVs) have been described in South America. The objective of this study was to evaluate the replication kinetics of novel swine IAVs as a first step for vaccine production. Different swine IAV lineages (H1N1, H1N2, and H3N2), infection doses (MOI: 1, 0.1, 0.01, 0.001, 0.0001, and 0.00001), harvest times (every 12 h), and substrates (MDCK and Vero cells) were used. For all IAV strains, MDCK cells were the most efficient substrate, generating titers greater than or equal to 128 HAU/50 µL with an MOI of 0.00001 at 60 h post-infection. These data could be useful for vaccine-producing laboratories.

Wastewater surveillance together with metaviromic data revealed the unusual resurgence of infectious diseases after the first wave of the COVID-19 outbreak

How to address public health priorities after COVID-19 is becoming a critical task. To this end, we conducted wastewater surveillance for six leading pathogens, namely, SARS-CoV-2, norovirus, rotavirus, influenza A virus (IAV), enteroviruses and respiratory syncytial virus (RSV), in Nanchang city from January to April 2023. Metaviromic sequencing was conducted at the 1st, 4th, 7th, 9th, 12th and 14th weeks to reveal the dynamics of viral pathogens that were not covered by qPCR. Amplicon sequencing of the conserved region of norovirus GI and GII and the rotavirus and region encoding nonstructural protein of RSV was also conducted weekly. The results showed that after a rapid decrease in SARS-CoV-2 sewage concentrations occurred in January 2023, surges of norovirus, rotavirus, IAV and RSV started at the 6th, 7th, 8th and 11th weeks, respectively. The dynamics of the sewage concentrations of norovirus, rotavirus, IAV and RSV were consistent with the off-season resurgence of the above infectious diseases. Notably, peak sewage concentrations of norovirus GI, GII, rotavirus, IAV and RSV were found at the 6th, 3rd, 7th, 7th and 8th weeks, respectively. Astroviruses also resurge after the 7th week, as revealed by metaviromic data, suggesting that wastewater surveillance together with metaviromic data provides an essential early warning tool for revealing patterns of infectious disease resurgence.

An aggregated dataset of serial morbidity and titer measurements from influenza A virus-infected ferrets

Data from influenza A virus (IAV) infected ferrets provides invaluable information towards the study of novel and emerging viruses that pose a threat to human health. This gold standard model can recapitulate many clinical signs of infection present in IAV-infected humans, support virus replication of human, avian, swine, and other zoonotic strains without prior adaptation, and permit evaluation of virus transmissibility by multiple modes. While ferrets have been employed in risk assessment settings for >20 years, results from this work are typically reported in discrete stand-alone publications, making aggregation of raw data from this work over time nearly impossible. Here, we describe a dataset of 728 ferrets inoculated with 126 unique IAV, conducted by a single research group under a uniform experimental protocol. This collection of morbidity, mortality, and viral titer data represents the largest publicly available dataset to date of in vivo-generated IAV infection outcomes on a per-ferret level.

Identifying cellular RNA-binding proteins during infection uncovers a role for MKRN2 in influenza mRNA trafficking.

Utilisation of RNA-binding proteins (RBPs) is an important aspect of post-transcriptional regulation of viral RNA. Viruses such as influenza A viruses (IAV) interact with RBPs to regulate processes including splicing, nuclear export and trafficking, while also encoding RBPs within their genomes, such as NP and NS1. But with almost 1000 RBPs encoded within the human genome it is still unclear what role, if any, many of these proteins play during viral replication. Using the RNA interactome capture (RIC) technique, we isolated RBPs from IAV infected cells to unravel the RBPome of mRNAs from IAV infected human cells. This led to the identification of one particular RBP, MKRN2, that associates with and positively regulates IAV mRNA. Through further validation, we determined that MKRN2 is involved in the nuclear-cytoplasmic trafficking of IAV mRNA potentially through an association with the RNA export mediator GLE1. In the absence of MKRN2, IAV mRNAs accumulate in the nucleus of infected cells, which may lead to their degradation by the nuclear RNA exosome complex. MKRN2, therefore, appears to be required for the efficient nuclear export of IAV mRNAs in human cells.

Virus versus host: influenza A virus circumvents the immune responses.

Influenza A virus (IAV) is a highly contagious pathogen causing dreadful losses to humans and animals around the globe. As is known, immune escape is a strategy that benefits the proliferation of IAVs by antagonizing, blocking, and suppressing immune surveillance. The HA protein binds to the sialic acid (SA) receptor to enter the cytoplasm and initiate viral infection. The conserved components of the viral genome produced during replication, known as the pathogen-associated molecular patterns (PAMPs), are thought to be critical factors for the activation of effective innate immunity by triggering dependent signaling pathways after recognition by pattern recognition receptors (PRRs), followed by a cascade of adaptive immunity. Viral infection-induced immune responses establish an antiviral state in the host to effectively inhibit virus replication and enhance viral clearance. However, IAV has evolved multiple mechanisms that allow it to synthesize and transport viral components by "playing games" with the host. At its heart, this review will describe how host and viral factors interact to facilitate the viral evasion of host immune responses.

Structures of H5N1 influenza polymerase with ANP32B reveal mechanisms of genome replication and host adaptation

Avian influenza A viruses (IAVs) pose a public health threat, as they are capable of triggering pandemics by crossing species barriers. Replication of avian IAVs in mammalian cells is hindered by species-specific variation in acidic nuclear phosphoprotein 32 (ANP32) proteins, which are essential for viral RNA genome replication. Adaptive mutations enable the IAV RNA polymerase (FluPolA) to surmount this barrier. Here, we present cryo-electron microscopy structures of monomeric and dimeric avian H5N1 FluPolA with human ANP32B. ANP32B interacts with the PA subunit of FluPolA in the monomeric form, at the site used for its docking onto the C-terminal domain of host RNA polymerase II during viral transcription. ANP32B acts as a chaperone, guiding FluPolA towards a ribonucleoprotein-associated FluPolA to form an asymmetric dimer—the replication platform for the viral genome. These findings offer insights into the molecular mechanisms governing IAV genome replication, while enhancing our understanding of the molecular processes underpinning mammalian adaptations in avian-origin FluPolA.

Infectivity of Wild-Bird Origin Influenza A Viruses in Minnesota Wetlands across Seasons.

The environmental tenacity of influenza A viruses (IAVs) in the environment likely plays a role in their transmission; IAVs are able to remain infectious in aquatic habitats and may have the capacity to seed outbreaks when susceptible wild bird hosts utilize these same environments months or even seasons later. Here, we aimed to assess the persistence of low-pathogenicity IAVs from naturally infected ducks in Northwestern Minnesota through a field experiment. Viral infectivity was measured using replicate samples maintained in distilled water in a laboratory setting as well as in filtered water from four natural water bodies maintained in steel perforated drums (hereafter, mesocosms) within the field from autumn 2020 to spring 2021. There was limited evidence for the extended persistence of IAVs held in mesocosms; from 65 initial IAV-positive samples, only six IAVs persisted to at least 202 days in the mesocosms compared to 17 viruses persisting at least this long when held under temperature-controlled laboratory settings in distilled water. When accounting for the initial titer of samples, viruses detected at a higher concentration at the initiation of the experiment persisted longer than those with a lower starting titer. A parallel experimental laboratory model was used to further explore the effects of water type on viral persistence, and the results supported the finding of reduced tenacity of IAVs held in mesocosms compared to distilled water. The results of this investigation provide evidence that many factors, including temperature and physicochemical properties, impact the duration of viral infectivity in natural settings, further extending our understanding of the potential and limitations of environmental-based methodologies to recover infectious IAVs.

Influenza A virus NS1 effector domain is required for PA-X-mediated host shutoff in infected cells.

Many viruses inhibit general host gene expression to limit innate immune responses and gain preferential access to the cellular translational apparatus for their protein synthesis. This process is known as host shutoff. Influenza A viruses (IAVs) encode two host shutoff proteins: nonstructural protein 1 (NS1) and polymerase acidic X (PA-X). NS1 inhibits host nuclear pre-messenger RNA maturation and export, and PA-X is an endoribonuclease that preferentially cleaves host spliced nuclear and cytoplasmic messenger RNAs. Emerging evidence suggests that in circulating human IAVs NS1 and PA-X co-evolve to ensure optimal magnitude of general host shutoff without compromising viral replication that relies on host cell metabolism. However, the functional interplay between PA-X and NS1 remains unexplored. In this study, we sought to determine whether NS1 function has a direct effect on PA-X activity by analyzing host shutoff in A549 cells infected with wild-type or mutant IAVs with NS1 effector domain deletion. This was done using conventional quantitative reverse transcription polymerase chain reaction techniques and direct RNA sequencing using nanopore technology. Our previous research on the molecular mechanisms of PA-X function identified two prominent features of IAV-infected cells: nuclear accumulation of cytoplasmic poly(A) binding protein (PABPC1) and increase in nuclear poly(A) RNA abundance relative to the cytoplasm. Here we demonstrate that NS1 effector domain function augments PA-X host shutoff and is necessary for nuclear PABPC1 accumulation. By contrast, nuclear poly(A) RNA accumulation is not dependent on either NS1 or PA-X-mediated host shutoff and is accompanied by nuclear retention of viral transcripts. Our study demonstrates for the first time that NS1 and PA-X may functionally interact in mediating host shutoff.IMPORTANCERespiratory viruses including the influenza A virus continue to cause annual epidemics with high morbidity and mortality due to the limited effectiveness of vaccines and antiviral drugs. Among the strategies evolved by viruses to evade immune responses is host shutoff-a general blockade of host messenger RNA and protein synthesis. Disabling influenza A virus host shutoff is being explored in live attenuated vaccine development as an attractive strategy for increasing their effectiveness by boosting antiviral responses. Influenza A virus encodes two proteins that function in host shutoff: the nonstructural protein 1 (NS1) and the polymerase acidic X (PA-X). We and others have characterized some of the NS1 and PA-X mechanisms of action and the additive effects that these viral proteins may have in ensuring the blockade of host gene expression. In this work, we examined whether NS1 and PA-X functionally interact and discovered that NS1 is required for PA-X to function effectively. This work significantly advances our understanding of influenza A virus host shutoff and identifies new potential targets for therapeutic interventions against influenza and further informs the development of improved live attenuated vaccines.

Modular Biomimetic Strategy Enables Discovery and SAR Exploration of Oxime Macrocycles as Influenza A Virus (H1N1) Inhibitors.

Although vaccination remains the prevalent prophylactic means for controlling Influenza A virus (IAV) infections, novel structural antivirus small-molecule drugs with new mechanisms of action for treating IAV are highly desirable. Herein, we describe a modular biomimetic strategy to expeditiously achieve a new class of macrocycles featuring oxime, which might target the hemagglutinin (HA)-mediated IAV entry into the host cells. SAR analysis revealed that the size and linker of the macrocycles play an important role in improving potency. Particularly, as a 14-membered macrocyclic oxime, 37 exhibited potent inhibitory activity against IAV H1N1 with an EC50 value of 23 nM and low cytotoxicity, which alleviated cytopathic effects and protected cell survival obviously after H1N1 infection. Furthermore, 37 showed significant synergistic activity with neuraminidase inhibitor oseltamivir in vitro.

Broadly Reactive Nanobody Targeting the H3 Hemagglutinin of the Influenza A Virus.

Monoclonal antibodies and recombinant antibody fragments are a very promising therapeutic tool to combat infectious diseases. Due to their unique paratope structure, nanobodies (VHHs) hold several advantages over conventional monoclonal antibodies, especially in relation to viral infections. Influenza A viruses (IAVs) remain a major threat to public health. The hemagglutinin (HA) protein is the main protective and immunodominant antigen of IAVs. In this study, three broadly reactive nanobodies (D9.2, E12.2, and D4.2) to H3N2 influenza strains were isolated and Fc-fusion proteins (VHH-Fcs) were obtained and characterized in vitro. This modification improved the nanobodies' binding activity and allowed for their interaction with a wider range of strains. The D9.2-Fc antibody showed a 100% protection rate against mortality in vivo in a mouse lethal model. Furthermore, we demonstrated that the observed protection has to do with Fc-FcγR interactions. These results indicate that D9.2-Fc can serve as an effective antiviral agent against the H3N2 influenza infection.

Development and characterization of an antibody that recognizes influenza virus N1 neuraminidases

Influenza A viruses (IAVs) continue to pose a huge threat to public health, and their prevention and treatment remain major international issues. Neuraminidase (NA) is the second most abundant surface glycoprotein on influenza viruses, and antibodies to NA have been shown to be effective against influenza infection. In this study, we generated a monoclonal antibody (mAb), named FNA1, directed toward N1 NAs. FNA1 reacted with H1N1 and H5N1 NA, but failed to react with the NA proteins of H3N2 and H7N9. In vitro, FNA1 displayed potent antiviral activity that mediated both NA inhibition (NI) and blocking of pseudovirus release. Moreover, residues 219, 254, 358, and 388 in the NA protein were critical for FNA1 binding to H1N1 NA. However, further validation is necessary to confirm whether FNA1 mAb is indeed a good inhibitor against NA for application against H1N1 and H5N1 viruses.

Chemoenzymatic Synthesis of Tri-antennary N-Glycans Terminating in Sialyl-Lewisx Reveals the Importance of Glycan Complexity for Influenza A Virus Receptor Binding.

Sialyl-Lewisx (SLex) is involved in immune regulation, human fertilization, cancer, and bacterial and viral diseases. The influence of the complex glycan structures, which can present SLex epitopes, on binding is largely unknown. We report here a chemoenzymatic strategy for the preparation of a panel of twenty-two isomeric asymmetrical tri-antennary N-glycans presenting SLex-Lex epitopes on either the MGAT4 or MGAT5 arm that include putative high-affinity ligands for E-selectin. The N-glycans were prepared starting from a sialoglycopeptide isolated from egg yolk powder and took advantage of inherent substrate preferences of glycosyltransferases and the use of 5'-diphospho-N-trifluoracetylglucosamine (UDP-GlcNHTFA) that can be transferred by branching N-acetylglucosaminyltransferases to give, after base treatment, GlcNH2-containing glycans that temporarily disable an antenna from enzymatic modification. Glycan microarray binding studies showed that E-selectin bound equally well to linear glycans and tri-antennary N-glycans presenting SLex-Lex. On the other hand, it was found that hemagglutinins (HA) of H5 influenza A viruses (IAV) preferentially bound the tri-antennary N-glycans. Furthermore, several H5 HAs preferentially bound to N-glycan presenting SLex on the MGAT4 arm. SLex is displayed in the respiratory tract of several avian species, demonstrating the relevance of investigating the binding of, among others IAVs, to complex N-glycans presenting SLex.

Seasonal antigenic prediction of influenza A H3N2 using machine learning

Antigenic characterization of circulating influenza A virus (IAV) isolates is routinely assessed by using the hemagglutination inhibition (HI) assays for surveillance purposes. It is also used to determine the need for annual influenza vaccine updates as well as for pandemic preparedness. Performing antigenic characterization of IAV on a global scale is confronted with high costs, animal availability, and other practical challenges. Here we present a machine learning model that accurately predicts (normalized) outputs of HI assays involving circulating human IAV H3N2 viruses, using their hemagglutinin subunit 1 (HA1) sequences and associated metadata. Each season, the model learns an updated nonlinear mapping of genetic to antigenic changes using data from past seasons only. The model accurately distinguishes antigenic variants from non-variants and adaptively characterizes seasonal dynamics of HA1 sites having the strongest influence on antigenic change. Antigenic predictions produced by the model can aid influenza surveillance, public health management, and vaccine strain selection activities.

Swine T cell responses to influenza A virus following live attenuated influenza virus or replicon particle vaccine

Abstract Influenza A virus (IAV) is a significant economic concern in the swine industry. Live attenuated influenza virus vaccines (LAIV) in swine are not available commercially but experimentally shown to induce mucosal cellular immunity. Non-adjuvanted hemagglutinin (HA) replicon particle (HA-RP) vaccines induce robust HA antibody, but little is reported about induction of T cell responses compared to LAIV. This study compared HA-RP and LAIV induction of T cell responses. Pigs were vaccinated with experimental LAIV or HA-RP, challenged with heterologous IAV, and necropsied at 5 or 21 days post infection. Virus titration was performed on nasal swabs and bronchoalveolar lavage fluid (BALF). T cells isolated from blood, lung, lymph node, and BALF were stimulated ex vivo with IAV and evaluated for cytokine production and proliferation. HA-RP-vaccinated pigs had lower macroscopic lesion scores but similar virus titers in nasal swabs and BALF to non-vaccine challenge (NV/CH) pigs. LAIV-vaccinated pigs had similar macroscopic lesion scores compared to NV/CH pigs but minimal virus titers. Both vaccines induced T cell production of IFNγ and TNFα and T cell proliferation after IAV stimulation, though LAIV responded at greater magnitude. In conclusion, both LAIV and HA-RP vaccines induced T cell responses and variable protection from heterologous IAV challenge. Understanding vaccine induction of cellular immunity as a correlate of protection will aid in improved swine IAV vaccine strategies.

Clinical Performance of Self-Collected Purified Water Gargle for Detection of Influenza a Virus Infection by Real-Time RT-PCR.

Self-collected specimens are increasingly being used as alternatives to swab-based methods for the detection of respiratory viruses. While saliva is well accepted, gargle specimens are a potential alternative with characteristics that are more favorable for laboratory handling. This study assessed the performance of gargle specimens in the detection of influenza A viruses (IAVs). We performed a prospective head-to-head comparison between combined nasopharyngeal and oropharyngeal swabs (NPS&OPS) and purified water gargle (PWG) among adult outpatients with febrile respiratory symptoms to detect IAVs using real-time RT-PCR during two influenza seasons. During study periods 1 (July 13 to 26, 2022, H3N2 predominated) and 2 (February 25 to March 10, 2023, H1N1 pdm09 predominated), a total of 459 patients were recruited. The overall agreement between the NPS&OPS and PWG was 85.0% (390/459, κ = 0.697), with 88.0% in period 1 and 82.6% in period 2. The detection rate of IAVs in PWG (51.6%, 237/459) was lower than that in NPS&OPS (62.3%, 286/459) (p < 0.0001). The overall sensitivity and specificity were 96.6% (93.7-98.3%) and 100% (97.1-100%) in NPS&OPS and were 80.1% (75.0-84.4%) and 100% (97.1-100%) in PWG, respectively. Among the 227 pairs of concordant positive specimens, cycle threshold (Ct) values were significantly lower in NPS&OPS than in PWG (median Ct values: 24.2, 28.2, p < 0.0001). Although self-collected PWG specimens offer acceptable performance for IAVs molecular testing, NPS&OPS remain a reliable option. Given the convenience of collection, nonviscous gargles are recommended for viral detection during emergencies or under specific conditions.

Longitudinal analysis of IAV-specific CD8+ T cell response to the high dose influenza vaccine (Fluzone) in healthy older adults

Abstract Influenza infection remains a leading cause of death among older adults worldwide. While vaccines offer an effective means of reducing the severity of influenza, their efficacy reduces in this demographic. To gain insight into the adaptive immune response, we conducted an analysis of antigen-specific CD8+ T cells before and after the administration of the high-dose seasonal influenza vaccine (Fluzone) over six seasons (2014, 2018, 2019, 2021, 2022, and 2023) in a cohort of healthy older adults (n=37, M=18, F=19, aged 72-92, spanning visits 1-3). Using the HAI assay, we observed seroconversion (a 4-fold antibody increase post-vaccination) for most strains (&amp;gt;67% of strains) in 46% of donors, for less than half of the strains (25-50% of strains) in 13% of donors, and no seroconversion (0% of strains) occurred in 30% of donors. Notably, there was a twofold higher occurrence of seroconversion for Influenza A virus (IAV) (51%) compared to Influenza B virus (IBV) (25%). As age advanced, 54% of donors exhibited a reduced antibody response, while 23% either experienced no change or saw an increase in their antibody response. Presently, we investigate the frequency and clonotype of influenza A virus (IAV) M1158-166-specific CD8+ T cells, tracking their changes post-vaccination and over a span of 1-5 years. This endeavor aims to uncover new insights into the molecular basis of the reduced antigen-specific CD8+ T cell response observed in certain older adults.

Macrophage expressed TRIM47 is required to control RNA virus infection by targeting MAVS

Abstract In the past half century, there have been multiple RNA virus pandemics (influenza 1957, 1968 and 2009; SARS-CoV and COVID-19), inhabiting 44% of all emerging contagious diseases. Innate immunity is the first line of defense against viral infection. Here we found that TRIM47, a member of the TRIM family E3 ligase, plays a pivotal role in positively regulating host innate immunity in response to Influenza A virus (IAV). Among immune cells, TRIM47 is highly expressed in macrophages. Compared with wild type, TRIM47 deficient bone marrow derived macrophages exhibited a significantly lower level of type I interferon (IFN-I) following RNA mimics infection, encompassing IAV and poly I: C. After in vivo IAV infection, TRIM47 knockout mice showed an extremely lower survival rate, along with a lower level of IFN-I, higher viral load and more inflammatory cell infiltration in lungs compared to wild-type mice. Mechanistically, TRIM47 induces K68-linked ubiquitination of mitochondrial antiviral-signaling protein (MAVS), a key adaptor in RNA-sensing pathway, leading to robust IFN-I production. This K68-linked ubiquitination by TRIM47 is essential for MAVS aggregation and activation in macrophage. These findings provide compelling evidence that TRIM47 interacts with MAVS, enhancing IFN-I production to effectively restrict IAV infection in macrophages. This research not only advances our understanding of influenza pathogenesis but also opens avenues for potential therapeutic interventions.

O-GlcNAc transferase plays dual antiviral roles by integrating innate immunity and lipid metabolism

Abstract Challenge of host cells with viral pathogens induces robust metabolic reprogramming; however, whether viral component(s) is detected by metabolic enzyme remains completely unknown. Our previous study identified the O-GlcNAc transferase (OGT), an important glucose metabolic enzyme, as a crucial mediator of MAVS-dependent antiviral response. Here, by studying a mouse model with enzyme-inactive OGT, we demonstrate an enzyme-independent function of OGT in limiting influenza A virus (IAV) infection, in addition to its enzyme-dependent effect on MAVS. Biochemical studies reveal a critical antiviral effect of OGT binding with IAV genomic RNA via its N-terminal tetratricopeptide repeats-4 domain. Binding with viral RNA causes a robust translocation of nuclear OGT to cytosolic lipid droplets (LDs) and subsequently antagonizes LD accumulation via destabilizing perilipin 2. This LD-targeting effect of OGT limits IAV replication without altering MAVS signaling. Our findings reveal OGT as a multifaceted metabolic sensor by integrating MAVS-dependent innate immune signaling and lipid metabolism.

Mitigating adverse effects of B cell depletion therapy on acute respiratory virus infections through IFN-β intervention treatment.

Abstract In autoimmune patients, the use of disease-modifying therapies (DMTs) can compromise immunity to viral infections. The COVID-19 pandemic generated interest in the effects of two DMTs on acute viral infections:B cell depletion therapy (α-CD20) and Interferon-beta (IFN-β) therapy. Severe acute viral infections have been associated with B cell depletion therapy, while IFN-β is believed to provide protection due to its antiviral properties. The goal of our study is to elucidate the individual and combined effects of α-CD20 and IFN-β during acute respiratory viral infections in mice. In our study, B cell depletion was achieved by administering α-CD20 three times every 5 days, starting 7 days before influenza A virus (IAV) infection, while IFN-β was given on days 1 and 2 post-infection. When administered alone, α-CD20 resulted in increased weight loss, reduced viral clearance, heightened lung-infiltrating myeloid cells, and elevated inflammatory cytokines in the blood on day 9 post-infection. T cell responses to IAV were not inhibited by α-CD20. While IFN-β alone did not protect against IAV, co-administration of IFN-β with α-CD20 effectively mitigated adverse effects of B cell depletion, by reducing viral load, mitigating morbidity, and slowing the infiltration of myeloid cells into the lung. These data demonstrate IFN-β’s ability to counteract α-CD20’s negative impact on viral infections, providing insights for potential treatment strategies in autoimmunity during viral outbreaks.

ZBP1 regulates cytokine production and cell death in lung fibroblasts following influenza virus infection

Abstract Regulated cell death following influenza viral infection is an important determinant of disease severity and is required for effective viral clearance. However, extensive cell death can lead to severe tissue damage and compromised lung function. Z-DNA binding protein-1 (ZBP1) is a critical sensor of influenza A virus (IAV) that can activate both apoptotic (non-inflammatory) or necrotic (inflammatory) cell death pathways. The cell type-specific effects of ZBP1 activation during influenza virus infection have not been clearly defined. Here, our single-cell gene expression studies identified lung fibroblasts as a cell type expressing high levels of Zbp1 following IAV infection in vivo. Following infection in vitro, we observed that ~20% of primary lung fibroblasts were readily infected by IAV but did not support high levels of spreading infection. Lung fibroblasts from wildtype mice underwent cell death with ~50% reduction in cell survival at 24 hours while lung fibroblasts from Zbp1 knockout mice were almost completely resistant to cell death. Analysis of cytokine production prior to cell death demonstrated that IAV infection in lung fibroblasts triggered an early ZBP1-dependent production of inflammatory proteins. Knockout of ZBP1 reduced inflammatory cytokine/chemokine production, but left interferon pathways intact. These studies identify lung fibroblasts as a key cell type activating Zbp1-mediated cytokine production and cell death during influenza virus infection.