The type I alpha/beta interferons (IFN-␣/␤) are known to play an important role in host defense against influenza A virus infection, but we have now discovered that the recently identified type III IFNs (IFN-) constitute the major response to intranasal infection with this virus. Type III IFNs were present at much higher levels than type I IFNs in the lungs of infected mice, and the enhanced susceptibility of STAT2 ؊/؊ animals demonstrated that only signaling through the IFN-␣/␤ or IFN-pathways was sufficient to mediate protection. This finding offers a possible explanation for the similar levels of antiviral protection found in wild-type (WT) mice and in animals lacking a functional type I IFN receptor (IFNAR ؊/؊ ) but also argues that our current understanding of type III IFN induction is incomplete. While murine IFN-production is thought to depend on signaling through the type I IFN receptor, we demonstrate that intranasal influenza A virus infection leads to the robust type III IFN induction in the lungs of both WT and IFNAR ؊/؊ mice. This is consistent with previous studies showing that IFNAR-mediated protection is redundant for mucosal influenza virus infection and with data showing that the type III IFN receptor is expressed primarily by epithelial cells. However, the overlapping effects of these two cytokine families are limited by their differential receptor expression, with a requirement for IFN-␣/␤ signaling in combating systemic disease.Type I interferons (IFNs) were first recognized for their ability to interfere with influenza virus replication (31) and are now recognized as an early and powerful host defense against virus infection. In all cell types that have been investigated, virus infection results in the synthesis and secretion of the type I alpha/beta interferons (IFN-␣/␤). Once secreted, IFN-␣/␤ acts in an autocrine or paracrine manner by binding the ubiquitously expressed IFN-␣/␤ receptor (IFNAR). Receptor binding activates the Jak-STAT signaling cascade leading to transcriptional upregulation of the IFN-stimulated genes which mediate the biological effects of IFN (10,18).IFN induction by influenza A virus involves recognition of viral components by both cytoplasmic receptors and TLR7, although the precise mechanism used depends upon the infected cell type. In fibroblasts, epithelial cells, and conventional dendritic cells (cDCs), IFN-␤ gene expression is largely dependent upon virus activation of the RNA helicase retinoic acid-induced gene I (RIG-I) (26), with the subsequent phosphorylation of IFN regulatory factor 3 (IRF3) by IB kinase ε (IKKε)/TANK-binding kinase 1 (TBK1). Once IFN-␤ (as well as IFN-␣4 in mouse) has been synthesized and secreted, signaling through the Jak-STAT pathway upregulates production of IRF7, which then mediates the transcription of additional 33,45). In this way, an amplification pathway is established wherein early, IRF3-mediated production of IFN-␤ promotes the synthesis of multiple IFN-␣ subtypes.Type III IFNs were very recently discovered and are designated ...

Transforming growth factor-beta (TGF-β), a multifunctional cytokine regulating several immunologic processes, is expressed by virtually all cells as a biologically inactive molecule termed latent TGF-β (LTGF-β). We have previously shown that TGF-β activity increases during influenza virus infection in mice and suggested that the neuraminidase (NA) protein mediates this activation. In the current study, we determined the mechanism of activation of LTGF-β by NA from the influenza virus A/Gray Teal/Australia/2/1979 by mobility shift and enzyme inhibition assays. We also investigated whether exogenous TGF-β administered via a replication-deficient adenovirus vector provides protection from H5N1 influenza pathogenesis and whether depletion of TGF-β during virus infection increases morbidity in mice. We found that both the influenza and bacterial NA activate LTGF-β by removing sialic acid motifs from LTGF-β, each NA being specific for the sialic acid linkages cleaved. Further, NA likely activates LTGF-β primarily via its enzymatic activity, but proteases might also play a role in this process. Several influenza A virus subtypes (H1N1, H1N2, H3N2, H5N9, H6N1, and H7N3) except the highly pathogenic H5N1 strains activated LTGF-β in vitro and in vivo. Addition of exogenous TGF-β to H5N1 influenza virus–infected mice delayed mortality and reduced viral titers whereas neutralization of TGF-β during H5N1 and pandemic 2009 H1N1 infection increased morbidity. Together, these data show that microbe-associated NAs can directly activate LTGF-β and that TGF-β plays a pivotal role protecting the host from influenza pathogenesis.

Macrophages are essential for protection against influenza A virus infection, but are also implicated in the morbidity and mortality associated with severe influenza disease, particularly during infection with highly pathogenic avian influenza (HPAI) H5N1 virus. While influenza virus infection of macrophages was once thought to be abortive, it is now clear that certain virus strains can replicate productively in macrophages. This may have important consequences for the antiviral functions of macrophages, the course of disease and the outcome of infection for the host. In this article, we review findings related to influenza virus replication in macrophages and the impact of productive replication on macrophage antiviral functions. A clear understanding of the interactions between influenza viruses and macrophages may lead to new antiviral therapies to relieve the burden of severe disease associated with influenza viruses.

Macrophages are known to be one of the first lines of defense against influenza virus infection. However, they may also contribute to severe disease caused by the highly pathogenic avian (HPAI) H5N1 influenza viruses. One reason for this may be the ability of certain influenza virus strains to productively replicate in macrophages. However, studies investigating the productive replication of influenza viruses in macrophages have been contradictory, and the results may depend on both the type of macrophages used and the specific viral strain. In this work, we investigated the ability of H1 to H16 viruses to productively replicate in primary murine alveolar macrophages and RAW264.7 macrophages. We show that only a subset of HPAI H5N1 viruses, those that cause high morbidity and mortality in mammals, can productively replicate in macrophages, as measured by the release of newly synthesized virus particles into the cell supernatant. Mechanistically, we found that these H5 strains can overcome a block early in the viral life cycle leading to efficient nuclear entry, viral transcription, translation, and ultimately replication. Studies with reassortant viruses demonstrated that expression of the hemagglutinin gene from an H5N1 virus rescued replication of H1N1 influenza virus in macrophages. This study is the first to characterize H5N1 influenza viruses as the only subtype of influenza virus capable of productive replication in macrophages and establishes the viral gene that is required for this characteristic. The ability to productively replicate in macrophages is unique to H5N1 influenza viruses and may contribute to their increased pathogenesis.

Little is known about intrinsic epithelial cell responses against astrovirus infection. Here we show that human astrovirus type 1 (HAstV-1) infection induces type I interferon (beta interferon [IFN-␤]) production in differentiated Caco2 cells, which not only inhibits viral replication by blocking positive-strand viral RNA and capsid protein synthesis but also protects against HAstV-1-increased barrier permeability. Excitingly, we found similar results in vivo using a murine astrovirus (MuAstV) model, providing new evidence that virus-induced type I IFNs may protect against astrovirus replication and pathogenesis in vivo. IMPORTANCEHuman astroviruses are a major cause of pediatric diarrhea, yet little is known about the immune response. Here we show that type I interferon limits astrovirus infection and preserves barrier permeability both in vitro and in vivo. Importantly, we characterized a new mouse model for studying astrovirus replication and pathogenesis. The successful replication and spread of many enteric viruses depend upon modulating immune factors produced by intestinal epithelial cells (IECs) including interferons (IFNs) (1, 2). For instance, enteric adenoviruses are sensitive to IEC-produced type I IFNs, unlike respiratory adenoviruses (3), while rotavirus exploits type I IFN signaling in IECs to promote early viral replication (4). However, nothing is known about the impact of IFN on astrovirus infection.Astroviruses are small, nonenveloped, RNA viruses that are one of the most important causes of pediatric acute gastroenteritis worldwide (5-8). Infection begins by binding to an unidentified receptor(s) on epithelial cells after fecal-oral transmission followed by entry via endosomes (9). After viral uncoating, the positive-sense, single-stranded RNA genome is translated into a polyprotein precursor that is subsequently cleaved into proteins required for replication and the assembly of progeny virions. The genome contains three open reading frames: ORF1a, ORF1b, and ORF2. ORF1a and ORF1b encode nonstructural proteins involved in transcription and replication of the virus, while ORF2 encodes the capsid protein (10, 11). Negative-strand RNA is produced from the positive genomic strand, which can be detected 6 to 12 h postinfection (hpi) (12). Transcription of the negativestrand genome yields the genomic and subgenomic RNA. Human astrovirus (HAstV) proteins have been associated with membranes in infected cells likely serving as the site for replication and assembly (13-15). After assembly, the progeny virions egress from the cell, a process promoted by caspase activation (16).Recently, Guix et al. found that HAstV type 4 (HAstV-4) replication induces type I IFN production and that pretreatment of Caco2 cells with beta interferon (IFN-␤) reduced HAstV-4 capsid protein synthesis and progeny virion production (17). However, whether the IFN-␤ produced during astrovirus infection is sufficient to limit astrovirus replication, and at what step in the viral life cycle IFN-␤ affects astrovirus, remains...

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Since emerging in 2013, the avian-origin H7N9 influenza viruses have resulted in over 400 human infections, leading to 115 deaths to date. Although the epidemiology differs from human highly pathogenic avian H5N1 influenza virus infections, there is a similar rapid progression to acute respiratory distress syndrome. The aim of these studies was to compare the pathological and immunological characteristics of a panel of human H7N9 and H5N1 viruses in vitro and in vivo. Although there were similarities between particular H5N1 and H7N9 viruses, including association between lethal disease and spread to the alveolar spaces and kidney, there were also strain-specific differences. Both H5N1 and H7N9 viruses are capable of causing lethal infections, with mortality correlating most strongly with wider distribution of viral antigen in the lungs, rather than with traditional measures of virus titer and host responses. Strain-specific differences included hypercytokinemia in H5N1 infections that was not seen with the H7N9 infections regardless of lethality. Conversely, H7N9 viruses showed a greater tropism for respiratory epithelium covering nasal passages and nasopharynx-associated lymphoid tissue than H5N1 viruses, which may explain the enhanced transmission in ferret models. Overall, these studies highlight some distinctive properties of H5N1 and H7N9 viruses in different in vitro and in vivo models. IMPORTANCEThe novel avian-origin H7N9 pandemic represents a serious threat to public health. The ability of H7N9 to cause serious lung pathology, leading in some cases to the development of acute respiratory distress syndrome, is of particular concern. Initial reports of H7N9 infection compared them to infections caused by highly pathogenic avian (HPAI) H5N1 viruses. Thus, it is of critical importance to understand the pathology and immunological response to infection with H7N9 compared to HPAI H5N1 viruses. We compared these responses in both in vitro and in vivo models, and found that H5N1 and H7N9 infections exhibit distinct pathological, immunological, and tissue tropism differences that could explain differences in clinical disease and viral transmission.

Viruses IMPORTANCEWe found that FAK links early activation of PI3K and actin reorganization, thereby regulating influenza virus entry. Surprisingly, we also found that FAK can regulate viral RNA replication independently of its role in entry. Our study addresses a knowledge gap in the understanding of signaling events triggered by influenza virus that mediate its internalization and initiation of the infection cycle. Understanding of these fundamental molecular events will be necessary to identify novel host targets, such as FAK, and development of future anti-influenza virus therapeutics.