Influenza Virus-infected Cells Research Articles

Mouse Models Reveal Role of T-Cytotoxic and T-Reg Cells in Immune Response to Influenza: Implications for Vaccine Design.

Immunopathologic examination of the lungs of mouse models of experimental influenza virus infection provides new insights into the immune response in this disease. First, there is rapidly developing perivascular and peribronchial infiltration of the lung with T-cells. This is followed by invasion of T-cells into the bronchiolar epithelium, and separation of epithelial cells from each other and from the basement membrane leading to defoliation of the bronchial epithelium. The intraepithelial reaction may involve either CD8 or CD4 T-cytotoxic cells and is analogous to a viral exanthema of the skin, such as measles and smallpox, which occur when the immune response against these infections is activated and the infected cells are attacked by T-cytotoxic cells. Then there is formation of B-cell follicles adjacent to bronchi, i.e., induced bronchial associated lymphoid tissue (iBALT). iBALT reacts like the cortex of a lymph node and is a site for a local immune response not only to the original viral infection, but also related viral infections (heterologous immunity). Proliferation of Type II pneumocytes and/or terminal bronchial epithelial cells may extend into the adjacent lung leading to large zones filled with tumor-like epithelial cells. The effective killing of influenza virus infected epithelial cells by T-cytotoxic cells and induction of iBALT suggests that adding the induction of these components might greatly increase the efficacy of influenza vaccination.

Host Lipid Rafts Play a Major Role in Binding and Endocytosis of Influenza A Virus.

Influenza still remains one of the most challenging diseases, posing a significant threat to public health. Host lipid rafts play a critical role in influenza A virus (IAV) assembly and budding, however, their role in polyvalent IAV host binding and endocytosis had remained elusive until now. In the present study, we observed co-localization of IAV with a lipid raft marker ganglioside, GM1, on the host surface. Further, we isolated the lipid raft micro-domains from IAV infected cells and detected IAV protein in the raft fraction. Finally, raft disruption using Methyl-β-Cyclodextrin revealed significant reduction in IAV host binding, suggesting utilization of host rafts for polyvalent binding on the host cell surface. In addition to this, cyclodextrin mediated inhibition of raft-dependent endocytosis showed significantly reduced IAV internalization. Interestingly, exposure of cells to cyclodextrin two hours post-IAV binding showed no such reduction in IAV entry, indicating use of raft-dependent endocytosis for host entry. In summary, this study demonstrates that host lipid rafts are selected by IAV as a host attachment factors for multivalent binding, and IAV utilizes these micro-domains to exploit raft-dependent endocytosis for host internalization, a virus entry route previously unknown for IAV.

234 - Lung epithelial PDIA3 plays a critical role in influenza infection

Protein disulfide isomerases (PDI) are a family of redox chaperones that catalyze formation or isomerization of disulfide bonds in proteins. Previous studies have shown that one member, PDIA3, interacts with influenza A virus (IAV) hemagglutinin (HA), and that this interaction is required for efficient oxidative folding of HA in vitro. However it is unknown whether these host-viral protein interactions occur during active infection and whether this interaction represent a putative target for the treatment of influenza infection. Current therapies targeting viral proteins are often limited in their effectiveness due to rapid development of viral resistance; targeting host proteins may avoid these limitations. Here we show that PDIA3 is specifically upregulated in IAV infected mouse or human lung epithelial cells and PDIA3 directly interacts with IAV-HA. Among PDI inhibitors tested, we found LOC14 inhibits PDIA3 at an IC50 of 5μM. Treatment with LOC14 inhibits PDIA3 activity in lung epithelial cells and subsequently decreases disulfide bonds, and oligomerization (maturation) of HA in both pandemic H1N1 and seasonal H3N2 infected lung epithelial cells. Disulfide labeling analysis indicated that inhibition of PDIA3 activity also decreased disulfide bonds in IAV-neuraminidase (NA) and these differences in oxidative folding correspond to a subsequent decrease in NA activity. Furthermore, these decreases in disulfide bonds significantly decrease viral load, and pro-inflammatory response from primary lung epithelial cells. Lung epithelial specific deletion of PDIA3 in mice results in a significant decreases in viral burden and levels of inflammatory-immune markers in mouse lung, as well as significantly improved airway mechanics (function). Taken together, these results suggest PDIA3 is required for effective influenza pathogenesis in vivo, and inhibition of PDIA3 may aid future development anti-influenza therapies based on inhibition of host PDIs to circumvent antigenic drifts and shifts of influenza and may lend critically needed help during pandemic and severe influenza seasons.

Targeting of Influenza Viral Epitopes to Antigen-Presenting Cells by Genetically Engineered Chimeric Molecules in a Humanized NOD SCID Gamma Transfer Model.

Antiviral DNA vaccines are a novel strategy in the vaccine development field, which basically consists of the administration of expression vectors coding viral antigen sequences into the host's cells. Targeting of conserved viral epitopes by antibody fragments specific to activating cell surface co-receptor molecules on antigen-presenting cells could be an alternative approach for inducing protective immunity. It has been shown that FcγRI on human monocytes enhances antigen presentation in vivo. Various DNA constructs, encoding a Single-chain variable antibodies (scFv) from mouse anti-human FcγRI monoclonal antibody, coupled to a sequence encoding a T- and B-cell epitope-containing influenza A virus hemagglutinin inter-subunit peptide were inserted into the eukaryotic expression vector system pTriEx-3 Neo. The constructed chimeric DNA molecules were expressed by transfected Chinese hamster ovary cells and the ability of the engineered proteins to interact with FcγRI-expressing cells was confirmed by flow cytometry. The fusion protein induced a strong signal transduction on human monocytes via FcγRI. The expression vector pTriEx-3 Neo containing the described construct was used as a naked DNA vaccine and introduced directly to experimental humanized NOD SCID gamma mice with or without boosting with the expressed fusion protein. Immunization with the generated DNA chimeric molecules and prime-boost with the expressed recombinant proteins induced significant serum levels of anti-influenza immunoglobulin G antibodies and strong cytotoxic T lymphocyte activity against influenza virus-infected cells in humanized animals.

Influenza virus replication raises the temperature of cells

Influenza virus invades the cell by binding sialic acid on the cell membrane through haemagglutinin (HA), and then genome replication and transcription are carried out in the nucleus to produce progeny virus.Multiplication of influenza virus requires metabolites, such as nucleotides and amino acids, as well as cellular machinery to synthesize its genome and proteins, thereby producing viral particles. Influenza virus infection forces the start of several metabolic systems in the cell, which consume or generate large amounts of energy. Thus, the viral multiplication processes involved in both genome replication and transcription are considered to require large numbers of nucleotides. The high-level consumption of nucleotides generates large amounts of energy, some of which is converted into heat, and this heat may increase the temperature of cells. To address this question, we prepared a tool based on rhodamine B fluorescence, which we used to measure the temperatures of influenza virus-infected and uninfected cells. The results indicated that influenza virus multiplication increased the temperature of cells by approximately 4 °C – 5 °C, ATP levels in the cells decreased at 3 h after infection, and mitochondrial membrane potential decreased with multiplication level. Thus, the increase in cellular temperature during influenza virus infection appears to be due to the massive consumption of ATP over a short period.

Variation at Extra-epitopic Amino Acid Residues Influences Suppression of Influenza Virus Replication by M158-66 Epitope-Specific CD8+ T Lymphocytes.

Influenza virus-specific CD8+ T lymphocytes (CTLs) contribute to clearance of influenza virus infections and reduce disease severity. Variation at amino acid residues located in or outside CTL epitopes has been shown to affect viral recognition by virus-specific CTLs. In the present study, we investigated the effect of naturally occurring variation at residues outside the conserved immunodominant and HLA*0201-restricted M158-66 epitope, located in the influenza virus M1 protein, on the extent of virus replication in the presence of CTLs specific for the epitope. To this end, we used isogenic viruses with an M1 gene segment derived from either an avian or a human influenza virus, HLA-transgenic human epithelial cells, human T cell clones specific for the M158-66 epitope or a control epitope, and a novel, purposely developed in vitro system to coculture influenza virus-infected cells with T cells. We found that the M gene segment of a human influenza A/H3N2 virus afforded the virus the capacity to replicate better in the presence of M158-66-specific CTLs than the M gene segment of avian viruses. These findings are in concordance with previously observed differential CTL activation, caused by variation at extra-epitopic residues, and may reflect an immune adaptation strategy of human influenza viruses that allows them to cope with potent CTL immunity to the M158-66 epitope in HLA-A*0201-positive individuals, resulting in increased virus replication and shedding and possibly increasing disease severity.IMPORTANCE Influenza viruses are among the leading causes of acute respiratory tract infections. CD8+ T lymphocytes display a high degree of cross-reactivity with influenza A viruses of various subtypes and are considered an important correlate of protection. Unraveling viral immune evasion strategies and identifying signs of immune adaptation are important for defining the role of CD8+ T lymphocytes in affording protection more accurately. Improving our insight into the interaction between influenza viruses and virus-specific CD8+ T lymphocyte immunity may help to advance our understanding of influenza virus epidemiology, aid in risk assessment of potentially pandemic influenza virus strains, and benefit the design of vaccines that induce more broadly protective immunity.

Heatr9 is upregulated during influenza virus infection in lung alveolar epithelial cells

Abstract Influenza virus infection poses a serious threat to public health. Influenza virus infects lung alveolar epithelial cells in the respiratory tract and utilizes them to produce more virus, spreading the infection. Understanding genes that are dynamically regulated during influenza virus infection may help elucidate essential genes and pathways that affect influenza virus infection. We examined by RNAseq the in vivo gene expression profiles of alveolar epithelial cells sorted from A/Puerto Rico/8/1934-GFP (PR8-GFP) expressing influenza virus infected mice. We identified a novel influenza virus-induced gene, Heatr9 (also known as Gm11435), that was upregulated by >200-fold in vivo in mouse alveolar epithelial cells. The upregulation of Heatr9 by influenza virus was further confirmed in vitro in PR8-GFP influenza virus infected human lung A549 cells. In vivo Heatr9 upregulation was found to be an indirect effect of influenza virus infection as bystander alveolar epithelial cells in lungs exhibited similar levels of Heatr9 induction as directly infected cells. Furthermore, supernatants of influenza virus infected A549 cells were capable of potently inducing Heatr9 mRNA even in the absence of infection. To identify factors that upregulate Heatr9 we examined the effect of cytokines on Heatr9 expression in vitro. Heatr9 expression was not induced by type I IFN, TNFα, and IL-1β alone in A549 cell, however when used in combination IFNβ and TNFα or IFNβ and IL-1β potently induced Heatr9 mRNA. Currently, we are generating Heatr9 deficient cell lines to examine the function of this gene in infection. In summary, we have identified Heatr9 as a cytokine-induced gene during influenza virus infection, the function of which has a yet to be determined.

Antiviral effects and mechanisms of Yinhuapinggan granule against H1N1 influenza virus infection in RAW264.7 cells.

Yinhuapinggan granule (YHPG), a modified prescription based on Ma-Huang-Tang (MHT), is used in traditional Chinese medicine (TCM) to treat influenza, cough, and viral pneumonia. In this study, we investigated the antiviral effects of YHPG by means of pre-, post-, and co-treatment, and its underlying mechanisms on regulating the levels of inflammatory-related cytokines, modulating the mRNA expressions of interferon-stimulated genes in influenza virus-infected murine macrophage cells (RAW264.7), and evaluating the protein expressions of key effectors in the Type I IFN and pattern recognition receptor (PRRs) signaling pathways. The results showed that YHPG markedly inhibited influenza virus (IFV) replication in pre-, post- and co-treatment assay, especially in post-treatment assay. Antiviral mechanisms studies revealed that YHPG (500 and 250μg/mL) significantly up-regulated levels of IFN-β, IFN-stimulated genes (Mx-1, ISG-15 and ISG-56) compared with the IFV control group, while the levels of IL-6 and TNF-α were significantly down-regulated. Furthermore, western blot analysis results revealed that the protein expressions of the phosphorylated forms of TBK1, IRF3, ERK1/2, P38 MAPK and NF-κB p65 were significantly down-regulated in RAW264.7 cells with the YHPG (500 and 250μg/mL) treatment, while the expression of the phosphorylated form of STAT1 was significantly enhanced. Based on these results, YHPG had antiviral effects in IFV-infected RAW264.7 cells, which might be associated with regulation of the inflammatory cytokines production, evaluation of the levels of IFN-stimulated genes, and modulation of the protein expressions of key effectors in the Type I IFN and PRRs signaling pathways.

Bax Inhibitor-1 Acts as an Anti-Influenza Factor by Inhibiting ROS Mediated Cell Death and Augmenting Heme-Oxygenase 1 Expression in Influenza Virus Infected Cells.

Influenza virus remains a major health concern worldwide, and there have been continuous efforts to develop effective antivirals despite the use of annual vaccination programs. The purpose of this study was to determine the anti-influenza activity of Bax inhibitor-1 (BI-1). Madin-Darby Canine Kidney (MDCK) cells expressing wild type BI-1 and a non-functional BI-1 mutant, BI-1 ∆C (with the C-terminal 14 amino acids deleted) were prepared and infected with A/PR/8/34 influenza virus. BI-1 overexpression led to the suppression of virus-induced cell death and virus production compared to control Mock or BI-1 ∆C overexpression. In contrast to BI-1 ∆C-overexpressing cells, BI-1-overexpressing cells exhibited markedly reduced virus-induced expression of several viral genes, accompanied by a substantial decrease in ROS production. We found that treatment with a ROS scavenging agent, N-acetyl cysteine (NAC), led to a dramatic decrease in virus production and viral gene expression in control MDCK and BI-1 ∆C-overexpressing cells. In contrast, NAC treatment resulted in the slight additional suppression of virus production and viral gene expression in BI-1-overexpressing cells but was statistically significant. Moreover, the expression of heme oxygenase-1 (HO-1) was also significantly increased following virus infection in BI-1-overexpressing cells compared to control cells. Taken together, our data suggest that BI-1 may act as an anti-influenza protein through the suppression of ROS mediated cell death and upregulation of HO-1 expression in influenza virus infected MDCK cells.

Sialic acid-dependent interaction of group B streptococci with influenza virus-infected cells reveals a novel adherence and invasion mechanism.

Group B streptococci (GBS) contain a capsular polysaccharide with side chains terminating in α2,3-linked sialic acids. Because of this linkage type, the sialic acids of GBS are recognised by lectins of immune cells. This interaction results in a dampening of the host immune response and thus promotes immune evasion. As several influenza A viruses (IAV) use α2,3-linked sialic acid as a receptor determinant for binding to host cells, we analysed whether GBS and influenza viruses can interact with each other and how this interaction affects viral replication and bacterial adherence to and invasion of host cells. A co-sedimentation assay revealed that viruses with a preference for α2,3-linked sialic acids bind to GBS in a sialic acid-dependent manner. There is, however, a large variation in the efficiency of binding among avian influenza viruses of different subtypes as shown by a hemagglutination-inhibition assay. A delay in the growth curve of IAV indicated that GBS has an inhibitory effect on virus replication. On the other hand, both the adherence and invasion efficiency of GBS were enhanced when the cells were pre-infected by IAV with appropriate receptor specificity. Our results suggest that GBS infection may result in a more severe disease when patients are co-infected by influenza viruses. This co-infection mechanism may have relevance also to other human diseases, as there are more bacterial pathogens with α2,3-linked sialic acids and human viruses binding to this linkage type.

3D Electron Microscopy (EM) and Correlative Light Electron Microscopy (CLEM) Methods to Study Virus-Host Interactions.

Viruses use different strategies to interact with their host and perform a successful viral infection that results in the formation of new infectious viral particles and their propagation to new hosts. Understanding how viruses interact with their hosts requires the use of high-resolution techniques for the direct visualization of these interactions. Here electron microscopy (EM) methods are described that allow the 3D ultrastructural analysis of virus-infected cells. These methods can be implemented with light microscopy (LM) to certainly allocate virus-infected cells or cells displaying a specific/interesting phenotypecaused by the interaction of viral proteins with the cellular machinery. Somesample preparation procedures where LM is integrated, known as correlative light electron microscopy (CLEM), are also explained in this chapter. All of these methods are applicable to any kind of cultured cells, including influenza virus-infected cells.

Upregulation of miRNA-4776 in Influenza Virus Infected Bronchial Epithelial Cells Is Associated with Downregulation of NFKBIB and Increased Viral Survival.

Influenza A virus (IAV) infection remains a significant cause of morbidity and mortality worldwide. One key transcription factor that is activated upon IAV infection is nuclear factor Kappa B (NF-κB). NF-κB regulation involves the inhibitor proteins NF-κB inhibitor beta (NFKBIB), (also known as IκB β), which form complexes with NF-κB to sequester it in the cytoplasm. In this study, microarray data showed differential expression of several microRNAs (miRNAs) on exposure to IAV. Target scan analysis revealed that miR-4776, miR-4514 and miR-4742 potentially target NFKBIB messenger RNA (mRNA). Time-course analysis of primary bronchial epithelial cells (HBEpCs) showed that miR-4776 expression is increased within 1 h of infection, followed by its downregulation 4 h post-exposure to IAV. NFKBIB upregulation of miR-4776 correlated with a decrease in NFKBIB expression within 1 h of infection and a subsequent increase in NFKBIB expression 4 h post-infection. In addition, miRNA ago-immunoprecipitation studies and the three prime untranslated region (3’ UTR) luciferase assay confirmed that miR-4776 targets NFKBIB mRNA. Furthermore, uninfected HBEpCs transfected with miR-4776 mimic showed decreased expression of NFKBIB mRNA. Overexpression of NFKBIB protein in IAV infected cells led to lower levels of IAV. Taken together, our data suggest that miRNA-4776 modulates IAV production in infected cells through NFKBIB expression, possibly through the modulation of NF-κB.

Metal-chelating properties and antiviral activity of some 2-hydroxyphenyl amides

Influenza virus is an hot topic in medicinal chemistry and great efforts are ongoing for the discover of new antivirals able to overcome problems related to resistant strains and adverse side effects of current drugs. Influenza virus endonuclease is an attractive target for antiviral drug development and in particular the strategy to chelate the metal ion(s) within the active site proved to be an efficient mode to inhibit enzymatic activity. Our previous findings revealed that 2-hydroxyamide derivatives are able to chelate Mg2+ ions, forming complexes with different stoichiometric ratios. Here we report on the activity of the three ligands N-(4-fluorobenzyl)-2-hydroxybenzamide, N-(4-fluorobenzyl)-2,3-dihydroxybenzamide, and N1,N3-bis(4-fluorobenzyl)-2-hydroxyisophthalamide, containing the salicylic group, and their Mg2+ complexes (7)–(9), evaluated by means of virus yield assay in influenza virus-infected MDCK cells and vRNP reconstitution assay in HEK293T cells. In some cases, promising anti-influenza activities with EC50 values in the low micromolar range were found. As a contribute to clarify the activity in cells of the ligands, here we also present a study on the their coordinating properties towards the other essential metal ion Cu(II), carried out by potentiometric and calorimetric measurements.

Influenza Virus Infection Induces Host Pyruvate Kinase M Which Interacts with Viral RNA-Dependent RNA Polymerase.

Influenza virus RNA-dependent RNA polymerase (RdRp) is a heterotrimer of three viral proteins, PB1, PB2, and PA and is involved in both transcription and replication of the negative strand of the viral RNA (vRNA) genome. RdRp is multifunctional, possessing RNA polymerase, cap binding, and endonuclease activities. The enzyme synthesizes three different RNAs, complementary RNA (cRNA) and messenger RNA (mRNA) from vRNA, and vRNA from cRNA. To synthesize these three RNAs, RdRp requires conversion of its function by host factor. Here, we performed yeast two-hybrid screening to identify the relevant host factor, revealing that pyruvate kinase M2 (PKM2) interacted with the PA subunit of influenza virus RdRp. PKM2 is one of two enzymes (PKM1 and PKM2) produced by alternative splicing of the pyruvate kinase M (PKM) pre-mRNA. We determined the interacting regions in both PKM2 and PA, the expression level of PKM by western blotting at different time points after viral infection, and the effects of transfection of siRNA targeting PKM on influenza virus replication. The results demonstrated that the C-terminal region of PKM2 interacted with the C-terminus of the PA subunit, that the expression level of PKM2 increased with influenza virus infection time, and that this enzyme is essential for influenza virus multiplication. Moreover, isoelectric focusing of uninfected and influenza virus infected cell extracts, followed by gradient gel electrophoresis to separate the PKM1 and PKM2 isoforms and western blotting indicated that PKM2 became more acidic after influenza infection. The decreased pH of PKM2 may have been due to phosphorylation, and phosphorylated PKM2 is active as a pyruvate kinase and protein kinase; therefore, it is possible that PKM2 may transfer a phosphate group to PA and consequently transform the function of RdRp from transcriptase to replicase.

Regulation and functions of NLRP3 inflammasome during influenza virus infection.

The NLRP3 inflammasome constitutes a major antiviral host defense mechanism during influenza virus infection. Inflammasome assembly in virus-infected cells facilitates autocatalytic processing of pro-caspase-1 and subsequent cleavage and secretion of proinflammatory cytokines IL-1β and IL-18. The NLRP3 inflammasome is critical for induction of both innate and adaptive immune responses during influenza virus infection. Inflammasome-dependent antiviral responses also regulate immunopathology and tissue repair in the infected lungs. The regulation of NLRP3 inflammasome assembly is an area of active research and recent studies have unraveled multiple cellular and viral factors involved in inflammasome assembly. Emerging studies have also identified the cross talk between inflammasome activation and programmed cell death pathways in influenza virus-infected cells. Here, we review the current literature regarding regulation and functions of NLRP3 inflammasome during influenza virus infection.

Edible bird\u2019s nest modulate intracellular molecular pathways of influenza A virus infected cells

BackgroundEdible Bird’s Nest (EBN) as a popular traditional Chinese medicine is believed to have health enhancing and antiviral activities against influenza A virus (IAV); however, the molecular mechanism behind therapeutic effects of EBN is not well characterized.MethodsIn this study, EBNs that underwent different enzymatic preparation were tested against IAV infected cells. 50% cytotoxic concentration (CC50) and 50% inhibitory concentration (IC50) of the EBNs against IAV strain A/Puerto Rico/8/1934(H1N1) were determined by HA and MTT assays. Subsequently, the sialic acid content of the used EBNs were analyzed by fluorometric HPLC. Western Blotting and immunofluorescent staining were used to investigate the effects of EBNs on early endosomal trafficking and autophagy process of influenza virus.ResultsThis study showed that post inoculations of EBNs after enzymatic preparations have the highest efficacy to inhibit IAV. While CC50 of the tested EBNs ranged from 27.5–32 mg/ml, the IC50 of these compounds ranged between 2.5–4.9 mg/ml. EBNs could inhibit IAV as efficient as commercial antiviral agents, such as amantadine and oseltamivir with different mechanisms of action against IAV. The antiviral activity of these EBNs correlated with the content of N-acetyl neuraminic acid. EBNs could affect early endosomal trafficking of the virus by reducing Rab5 and RhoA GTPase proteins and also reoriented actin cytoskeleton of IAV infected cells. In addition, for the first time this study showed that EBNs can inhibit intracellular autophagy process of IAV life cycle as evidenced by reduction of LC3-II and increasing of lysosomal degradation.ConclusionsThe results procured in this study support the potential of EBNs as supplementary medication or alternative to antiviral agents to inhibit influenza infections. Evidently, EBNs can be a promising antiviral agent; however, these natural compounds should be screened for their metabolites prior to usage as therapeutic approach.

Targeting a metabolic pathway to fight the flu

Our antiviral arsenal to fight influenza viruses is limited and we need novel anti-flu drugs. Recently, cellular drug targets came into focus and omics analysis were instrumental to suggest candidate factors. In this issue of The FEBS Journal, Kainov and colleagues used transcriptome data to investigate virus-induced changes in tryptophan metabolism that may serve as immunomodulatory approach against viruses.

Modulation of the innate immune-related genes expression in H9N2 avian influenza virus-infected chicken macrophage-like cells (HD11) in response to Escherichia coli LPS stimulation

Macrophages play important roles in mediating virus-induced innate immune responses and are thought to be involved in the pathogenesis of bacterial superinfections. The innate immune response initiated by both low pathogenicity AIV and bacterial superinfection in their avian host is not fully understood. We therefore determine the transcripts of innate immune-related genes following avian H9N2 AIV virus infection and E. coli LPS co-stimulation of avian macrophage-like cell line HD11 cells. More pronounced expression of pro-inflammatory cytokines (IL-6 and IL-1β) as well as the inflammatory chemokines (CXCLi1 and CXCLi2) was observed in virus infected plus LPS treated HD11 cells compared to H9N2 virus solely infected control. For two superinfection groups, the levels of genes examined in a prior H9N2 virus infection before secondary LPS treatment group were significantly higher as compared with simultaneous virus infection plus LPS stimulation group. Interestingly, similar high levels of IL-6 gene were observed between LPS sole stimulation group and two superinfection groups. Moreover, IL-10 and TGF-β3 mRNA levels in both superinfection groups were moderately upregulated compared to sole LPS stimulation group or virus alone infection group. Although TLR4 and MDA5 levels in virus alone infection group were significantly lower compared to that in both superinfection groups, TLR4 upregulation respond more rapid to virus sole infection compared to LPS plus virus superinfection. Collectively, innate immune-related genes respond more pronounced in LPS stimulation plus H9N2 virus infection HD11 cells compared to sole virus infection or LPS alone stimulation control cells.

Indelible labeling and tracking of influenza virus infected antigen presenting cells

Abstract Influenza A virus is a seasonal pathogen with the potential to unpredictably cause catastrophic pandemics. The virus primarily replicates in epithelial cells of the upper and lower respiratory tract but can also infect a spectrum of other cell lineages, including cells of the immune system. Classically infected cells have been tracked through detection of virus products (RNA or protein) or through virus-derived reporters. However, these methods cannot define any potentially remaining infected cells after active replication has ceased. Using a novel virus expressing Cre recombinase to indelibly label infected cells in reporter mice, we unexpectedly found that epithelial cells were capable of surviving influenza virus infection long-term (Heaton and Langlois et al J. Ex. Med. 2014). We have subsequently discovered that infected CD45+ immune cells are also capable of surviving acute influenza virus infection in vivo. Importantly this virus-reporter system is able to distinguish between immune cells that are directly infected versus those that acquire virus protein/antigen exogenously. We find that both dendritic cells and macrophages, but not B cells, are directly infected within the lung and that the number of these cells increases during the acute infection phase. However, infected dendritic cells and macrophages do not persist in the lungs nor do they migrate systemically. Together these data demonstrate a new tool to track infected cells and defines the migration, turnover, and survival of infected antigen presenting cells.

The hemifusion structure induced by influenza virus haemagglutinin is determined by physical properties of the target membranes.

SUMMARYInfluenza A virus hemagglutinin (HA) changes conformation and drives membrane fusion of viral and endosomal membrane at low pH. Membrane fusion proceeds through an intermediate called hemifusion1,2. For viral fusion the hemifusion structures are not determined3. Here, influenza virus-like particles (VLP)4 carrying wild-type (WT) HA or HA hemifusion mutant G1S5 and liposome mixtures were studied at low pH by Volta phase plate (VPP) cryo-electron tomography (cET) which improves signal-to-noise ratio close to focus. We determined two distinct hemifusion structures: a hemifusion diaphragm (HD) and a novel structure termed lipidic junction. Liposomes with lipidic junctions were ruptured with membrane edges stabilized by HA. The rupture frequency and HD diameter were not affected by G1S mutation, but decreased when the cholesterol level in the liposomes was close to physiological concentrations. We propose that HA induces merger between the viral and a target membrane by one of two independent pathways: rupture-insertion pathway leading to lipidic junction and hemifusion-stalk pathway leading to fusion pore. The latter is relevant under the conditions of influenza virus infection of cells. Cholesterol concentration functions as a pathway switch due to its negative spontaneous curvature in the target bilayer as determined by continuum analysis.