A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication

Influenza A virus causes annual epidemics and occasional pandemics in humans. Here, we investigated four members of the fibroblast growth factor receptor (FGFR) family; FGFR1 to 4, and examined their expression patterns in human lung epithelial cells A549 with influenza A virus infection. We identified a functional role of FGFR1 in influenza A/Puerto Rico/8/1934 (PR8) and A/Anhui/01/2005 (H5N1) virus replication. Our results showed that FGFR1 silencing by siRNA interference promoted influenza A/PR8 and H5N1 virus replication in A549 cells, while lentivirus-mediated exogenous FGFR1 expression significantly suppressed influenza A virus replication; however, FGFR4 did not have the same effects. Moreover, FGFR1 phosphorylation levels were downregulated in A549 cells by influenza A virus infection, while the repression of FGFR1 kinase using PD173074, a potent and selective FGFR1 inhibitor, could enhance virus replication. Furthermore, we found that FGFR1 inhibits influenza virus internalization, but not binding, during viral entry. These results suggested that FGFR1 specifically antagonizes influenza A virus replication, probably by blocking viral entry.     

The highly pathogenic H5N1 influenza A virus down‐regulated several cellular MicroRNAs which target viral genome

Higher and prolonged viral replication is critical for the increased pathogenesis of the highly pathogenic avian influenza (HPAI) subtype of H5N1 influenza A virus (IAV) over the lowly pathogenic H1N1 IAV strain. Recent studies highlighted the considerable roles of cellular miRNAs in host defence against viral infection. In this report, using a 3′UTR reporter system, we identified several putative miRNA target sites buried in the H5N1 virus genome. We found two miRNAs, miR‐584‐5p and miR‐1249, that matched with the PB2 binding sequence. Moreover, we showed that these miRNAs dramatically down‐regulated PB2 expression, and inhibited replication of H5N1 and H1N1 IAVs in A549 cells. Intriguingly, these miRNAs expression was differently regulated in A549 cells infected with the H5N1 and H1N1 viruses. Furthermore, transfection of miR‐1249 inhibitor enhanced the PB2 expression and promoted the replication of H5N1 and H1N1 IAVs. These results suggest that H5N1 virus may have evolved a mechanism to escape host‐mediated inhibition of viral replication through down‐regulation of cellular miRNAs, which target its viral genome.     

A novel genetic locus linked to pro-inflammatory cytokines after virulent H5N1 virus infection in mice

Background

Genetic variation in the human population is a key determinant of influenza disease severity. A single nucleotide polymorphism in the antiviral gene IFITM3 was linked to outcomes during the 2009 H1N1 pandemic. To identify variant host genes associated with increased virus replication and severe disease, we performed a quantitative trait locus analysis on pro-inflammatory cytokine production 48 hours after intranasal infection with highly pathogenic H5N1 influenza virus.

Results

Pro-inflammatory cytokines CCL2, TNFα and IFN-α, were measured by ELISA in lung homogenates of DBA/2J (D2), C57BL/6J (B6) and 44 different BXD recombinant inbred mouse strains. Virus titer was also assessed in a subset of these animals. CCL2 (8-fold), TNFα (24-fold) and IFN-α (8-fold) concentrations varied significantly among the different BXD RI strains. Importantly, cytokine concentration correlated very well (r =0.86-0.96, P <0.0001) with virus titer suggesting that early cytokine production is due to increased virus infection and replication. Linkage analysis of cytokine concentration revealed a significant locus on chromosome 6 associated with differences in TNFα, IFN-α and CCL2 cytokine concentration (LRS =26). This locus accounted for nearly 20% of the observed phenotypic variation in the BXD population studied. Sequence and RNA expression analysis identified several candidate host genes containing missense mutations or deletions; Samd9l, Ica1, and Slc25a13. To study the role of Slc25a13, we obtained Slc25a13 knockout line, but upon challenge with H5N1 influenza virus observed no effect on CCL2 production, or morbidity and mortality.

Conclusion

A novel genetic locus on chromosome 6 modulates early pro-inflammatory cytokine production and virus replication after highly pathogenic influenza virus infection. Candidate genes, Samd9l and Ica1, may be important for the control of influenza virus infection and pathogenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1017) contains supplementary material, which is available to authorized users.     

Proinflammatory Cytokine Response and Viral Replication in Mouse Bone Marrow Derived Macrophages Infected with Influenza H1N1 and H5N1 Viruses

The pathogenesis of human influenza H5N1 virus infection remains poorly understood and controversial. Cytokine dysregulation in human infection has been hypothesized to contribute to disease severity. We developed in vitro cultures of mouse bone marrow derived macrophages (BMDMΦ) from C57BL/6N mouse to compare influenza A (H5N1 and H1N1) virus replication and pro-inflammatory cytokine and chemokine responses. While both H1N1 and H5N1 viruses infected the mouse bone marrow derived macrophages, only the H1N1 virus had showed evidence of productive viral replication from the infected cells. In comparison with human seasonal influenza H1N1 (A/HK/54/98) and mouse adapted influenza H1N1 (A/WSN/33) viruses, the highly pathogenic influenza H5N1 virus (A/HK/483/97) was a more potent inducer of the chemokine, CXCL 10 (IP-10), while there was not a clear differential TNF-α protein expression pattern. Although human influenza viruses rarely cause infection in mice without prior adaption, the use of in vitro cell cultures of primary mouse cells is of interest, especially given the availability of gene-defective (knock-out) mice for specific genes.     

H5N1亚型禽流感病毒NS1基因的克隆及其诱导肺腺癌细胞A549凋亡的研究

通过RT-PCR的方法克隆H5N1亚型禽流感病毒NS1基因,并构建了真核表达载体pCMV-Myc/NS1。将此真核表达质粒转染肺腺癌细胞A549,48 h后,经Western印迹检测,NS1基因能在细胞中正确表达。经荧光显微镜、透射电镜观察和流式细胞仪检测,发现该株流感病毒的NS1蛋白可诱导肺腺癌细胞A549凋亡。     

Activation of type I and III interferon signalling pathways occurs in lung epithelial cells infected with low pathogenic avian influenza viruses

The host response to the low pathogenic avian influenza (LPAI) H5N2, H5N3 and H9N2 viruses were examined in A549, MDCK, and CEF cells using a systems-based approach. The H5N2 and H5N3 viruses replicated efficiently in A549 and MDCK cells, while the H9N2 virus replicated least efficiently in these cell types. However, all LPAI viruses exhibited similar and higher replication efficiencies in CEF cells. A comparison of the host responses of these viruses and the H1N1/WSN virus and low passage pH1N1 clinical isolates was performed in A549 cells. The H9N2 and H5N2 virus subtypes exhibited a robust induction of Type I and Type III interferon (IFN) expression, sustained STAT1 activation from between 3 and 6 hpi, which correlated with large increases in IFN-stimulated gene (ISG) expression by 10 hpi. In contrast, cells infected with the pH1N1 or H1N1/WSN virus showed only small increases in Type III IFN signalling, low levels of ISG expression, and down-regulated expression of the IFN type I receptor. JNK activation and increased expression of the pro-apoptotic XAF1 protein was observed in A549 cells infected with all viruses except the H1N1/WSN virus, while MAPK p38 activation was only observed in cells infected with the pH1N1 and the H5 virus subtypes. No IFN expression and low ISG expression levels were generally observed in CEF cells infected with either AIV, while increased IFN and ISG expression was observed in response to the H1N1/WSN infection. These data suggest differences in the replication characteristics and antivirus signalling responses both among the different LPAI viruses, and between these viruses and the H1N1 viruses examined. These virus-specific differences in host cell signalling highlight the importance of examining the host response to avian influenza viruses that have not been extensively adapted to mammalian tissue culture.     

Influenza A virus H5N1 entry into host cells is through clathrin-dependent endocytosis

Influenza A virus H5N1 presents a major threat to human health. The entry of influenza virus into host cells is believed to be mediated by hemagglutinin (HA), a virus surface glycoprotein that can bind terminal sialic acid residues on host cell glycoproteins and glycolipids. In this study, we elucidated the pathways through which H5N1 enters human lung carcinoma cell line A549. We first proved that H5N1 can enter A549 cells via endocytosis, as lysosomotropic agents, such as bafilomycin A1 and chloroquine, can rescue H5N1-induced A549 cell death. By using specific inhibitors, and siRNAs that target the clathrin pathway, we further found that H5N1 could enter A549 cells via clathrin-mediated endocytosis, while inhibitors targeting caveolae-mediated endocytosis could not inhibit H5N1 cell entry. These findings expand our understanding of H5N1 pathogenesis and provide new information for anti-viral drug research. Supported by the National Natural Science Foundation of China (Grant No. 30623009) and National Basic Research Program of China (Grant No. 2005CB523000)     

Differential suppressive effect of promyelocytic leukemia protein on the replication of different subtypes/strains of influenza A virus

Promyelocytic leukemia protein (PML) plays an important role in the defense against a number of viruses, including influenza A virus. However, the sensitivity of influenza A virus subtypes/strains to PML is unknown. We investigated the role of PML in the replication of different influenza A virus subtypes/strains using pan-PML knock-down A549 cells and PML-VI-overexpressed MDCK cells. We found that (i) depletion of pan-PML by siRNA rendered A549 cells more susceptible to influenza A virus strains PR8(H1N1) and ST364(H3N2), but not to strains ST1233(H1N1), Qa199(H9N2) and Ph2246(H9N2); (ii) overexpression of PML-VI in MDCK cells conferred potent resistance to PR8(H1N1) infection, while lacked inhibitory activity to ST1233(H1N1), ST364(H3N2), Qa199(H9N2) and Ph2246(H9N2). Our results suggest that the antiviral effect of PML on influenza A viruses is viral subtype/strain specific.     

A genome-wide CRISPR/Cas9 gene knockout screen identifies immunoglobulin superfamily DCC subclass member 4 as a key host factor that promotes influenza virus endocytosis

Influenza virus infection is dependent on host cellular factors, and identification of these factors and their underlying mechanisms can provide important information for the development of strategies to inhibit viral infection. Here, we used a highly pathogenic H5N1 influenza virus to perform a genome-wide CRISPR/Cas9 gene knockout screen in human lung epithelial cells (A549 cells), and found that knockout of transmembrane protein immunoglobulin superfamily DCC subclass member 4 (IGDCC4) significantly reduced the replication of the virus in A549 cells. Further studies showed that IGDCC4 interacted with the viral hemagglutinin protein and facilitated virus internalization into host cells. Animal infection studies showed that replication of H5N1 virus in the nasal turbinates, lungs, and kidneys of IGDCC4-knockout mice was significantly lower than that in the corresponding organs of wild-type mice. Half of the IGDCC4-knockout mice survived a lethal H5N1 virus challenge, whereas all of the wild-type mice died within 11 days of infection. Our study identifies a novel host factor that promotes influenza virus infection by facilitating internalization and provides insights that will support the development of antiviral therapies.     

Type 1 responses of human Vγ9Vδ2 T cells to influenza A viruses

γδ T cells are essential constituents of antimicrobial and antitumor defenses. We have recently reported that phosphoantigen isopentenyl pyrophosphate (IPP)-expanded human Vγ9Vδ2 T cells participated in anti-influenza virus immunity by efficiently killing both human and avian influenza virus-infected monocyte-derived macrophages (MDMs) in vitro. However, little is known about the noncytolytic responses and trafficking program of γδ T cells to influenza virus. In this study, we found that Vγ9Vδ2 T cells expressed both type 1 cytokines and chemokine receptors during influenza virus infection, and IPP-expanded cells had a higher capacity to produce gamma interferon (IFN-γ). Besides their potent cytolytic activity against pandemic H1N1 virus-infected cells, IPP-activated γδ T cells also had noncytolytic inhibitory effects on seasonal and pandemic H1N1 viruses via IFN-γ but had no such effects on avian H5N1 or H9N2 virus. Avian H5N1 and H9N2 viruses induced significantly higher CCL3, CCL4, and CCL5 production in Vγ9Vδ2 T cells than human seasonal H1N1 virus. CCR5 mediated the migration of Vγ9Vδ2 T cells toward influenza virus-infected cells. Our findings suggest a novel therapeutic strategy of using phosphoantigens to boost the antiviral activities of human Vγ9Vδ2 T cells against influenza virus infection.     

Systems-Level Comparison of Host-Responses Elicited by Avian H5N1 and Seasonal H1N1 Influenza Viruses in Primary Human Macrophages

Human disease caused by highly pathogenic avian influenza (HPAI) H5N1 can lead to a rapidly progressive viral pneumonia leading to acute respiratory distress syndrome. There is increasing evidence from clinical, animal models and in vitro data, which suggests a role for virus-induced cytokine dysregulation in contributing to the pathogenesis of human H5N1 disease. The key target cells for the virus in the lung are the alveolar epithelium and alveolar macrophages, and we have shown that, compared to seasonal human influenza viruses, equivalent infecting doses of H5N1 viruses markedly up-regulate pro-inflammatory cytokines in both primary cell types in vitro. Whether this H5N1-induced dysregulation of host responses is driven by qualitative (i.e activation of unique host pathways in response to H5N1) or quantitative differences between seasonal influenza viruses is unclear. Here we used microarrays to analyze and compare the gene expression profiles in primary human macrophages at 1, 3, and 6 h after infection with H5N1 virus or low-pathogenic seasonal influenza A (H1N1) virus. We found that host responses to both viruses are qualitatively similar with the activation of nearly identical biological processes and pathways. However, in comparison to seasonal H1N1 virus, H5N1 infection elicits a quantitatively stronger host inflammatory response including type I interferon (IFN) and tumor necrosis factor (TNF)-α genes. A network-based analysis suggests that the synergy between IFN-β and TNF-α results in an enhanced and sustained IFN and pro-inflammatory cytokine response at the early stage of viral infection that may contribute to the viral pathogenesis and this is of relevance to the design of novel therapeutic strategies for H5N1 induced respiratory disease.     

Non structural protein of avian influenza A (H11N1) virus is a weaker suppressor of immune responses but capable of inducing apoptosis in host cells

ABSTRACT: BACKGROUND: The Non-Structural (NS1) protein of Influenza A viruses is an extensively studied multifunctional protein which is commonly considered as key viral component to fight against host immune responses. Even though there has been a lot of studies on the involvement of NS1 protein in host immune responses there are still ambiguities regarding its role in apoptosis in infected cells. Interactions of NS1 protein with host factors, role of NS1 protein in regulating cellular responses and apoptosis are quiet complicated and further studies are still needed to understand it completely. RESULTS: NS1 genes of influenza A/Chicken/India/WBNIV2664/2008 (H5N1) and A/Aquatic bird/India/NIV-17095/2007(H11N1) were cloned and expressed in Human embryonic kidney (293T) cells. Microarray based approach to study the host cellular responses to NS1 protein of the two influenza A viruses of different pathogenicity showed significant differences in the host gene expression profile. NS1 protein of H5N1 resulted in suppression of IFN-beta mediated innate immune responses in 293T cells, leading to down-regulation of the components of JAK-STAT pathway like STAT1 which further suppressed the expression of pro-inflammatory cytokines like CXCL10 and CCL5. The degree of suppression of host immune genes was found considerable with NS1 protein of H11N1 but was not as prominent as with H5N1-NS1. TUNEL assay analyses were found to be positive in both the NS1 transfected cells indicating both H5N1 as well as H11N1 NS1 proteins were able to induce apoptosis in transfected cells. CONCLUSIONS: We propose that NS1 protein of both H5N1 and H11N1 subtypes of influenza viruses are capable of influencing host immune responses and possess necessary functionality to support apoptosis in host cells. H11N1, a low pathogenic virus without any proven evidence to infect mammals, contains a highly potential NS1 gene which might contribute to greater virus virulence in different gene combinations.     

The NS1 protein of influenza a virus interacts with heat shock protein Hsp90 in human alveolar basal epithelial cells: Implication for virus-induced apoptosis

Background

Our previous study showed that the NS1 protein of highly pathogenic avian influenza A virus H5N1 induced caspase-dependent apoptosis in human alveolar basal epithelial cells (A549), supporting its function as a proapoptotic factor during viral infection, but the mechanism is still unknown.

Results

To characterize the mechanism of NS1-induced apoptosis, we used a two-hybrid system to isolate the potential NS1-interacting partners in A549 cells. We found that heat shock protein 90 (Hsp90) was able to interact with the NS1 proteins derived from both H5N1 and H3N2 viruses, which was verified by co-immunoprecitation assays. Significantly, the NS1 expression in the A549 cells dramatically weakened the interaction between Apaf-1 and Hsp90 but enhanced its interaction with cytochrome c (Cyt c), suggesting that the competitive binding of NS1 to Hsp90 might promote the Apaf-1 to associate with Cyt c and thus facilitate the activation of caspase 9 and caspase 3.

Conclusions

The present results demonstrate that NS1 protein of Influenza A Virus interacts with heat hock protein Hsp90 and meidates the apoptosis induced by influenza A virus through the caspase cascade.

     

Vaccination against seasonal influenza A/H3N2 virus reduces the induction of heterosubtypic immunity against influenza A/H5N1 virus infection in ferrets

Infection with seasonal influenza viruses induces a certain extent of protective immunity against potentially pandemic viruses of novel subtypes, also known as heterosubtypic immunity. Here we demonstrate that infection with a recent influenza A/H3N2 virus strain induces robust protection in ferrets against infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Prior H3N2 virus infection reduced H5N1 virus replication in the upper respiratory tract, as well as clinical signs, mortality, and histopathological changes associated with virus replication in the brain. This protective immunity correlated with the induction of T cells that cross-reacted with H5N1 viral antigen. We also demonstrated that prior vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity otherwise induced by infection with the influenza A/H3N2 virus. The implications of these findings are discussed in the context of vaccination strategies and vaccine development aiming at the induction of immunity to pandemic influenza.     

Novel pandemic influenza A (H1N1) virus infection modulates apoptotic pathways that impact its replication in A549 cells

It is not well-known whether apoptosis signaling affects influenza virus infection and reproduction in human lung epithelial cells. Using A549 cell line, we studied the relationship of some apoptosis-associated molecules with novel pandemic influenza A (H1N1) virus, A/California/04/2009. Infected cells displayed upregulated Fas ligand, activated FADD and caspase-8, and downregulated FLIP in the extrinsic apoptotic pathway. p53 expression increased and Bcl-X_L expression decreased in the intrinsic pathway. Expression of pre-apoptotic molecules (FasL, FADD, and p53) increased virus replication, while inhibition of activity of FADD, caspase-8 and caspase-3, and expression of anti-apoptotic proteins (FLIP and Bcl-X_L) decreased virus replication. p38, ERK and JNK from MAPK pathways were activated in infected cells, and inhibition with their inhibitors diminished virus replication. In the p38 superfamily, p38α expression increased viral RNA production, while expression of p38β and p38γ decreased. These data indicated that influenza virus induces apoptotic signaling pathways, which benefit virus replication.     

Characterization of the 2009 Pandemic A/Beijing/501/2009 H1N1 Influenza Strain in Human Airway Epithelial Cells and Ferrets

Background

A novel 2009 swine-origin influenza A H1N1 virus (S-OIV H1N1) has been transmitted among humans worldwide. However, the pathogenesis of this virus in human airway epithelial cells and mammals is not well understood.

Methodology/Principal Finding

In this study, we showed that a 2009 A (H1N1) influenza virus strain, A/Beijing/501/2009, isolated from a human patient, caused typical influenza-like symptoms including weight loss, fluctuations in body temperature, and pulmonary pathological changes in ferrets. We demonstrated that the human lung adenocarcinoma epithelial cell line A549 was susceptible to infection and that the infected cells underwent apoptosis at 24 h post-infection. In contrast to the seasonal H1N1 influenza virus, the 2009 A (H1N1) influenza virus strain A/Beijing/501/2009 induced more cell death involving caspase-3-dependent apoptosis in A549 cells. Additionally, ferrets infected with the A/Beijing/501/2009 H1N1 virus strain exhibited increased body temperature, greater weight loss, and higher viral titers in the lungs. Therefore, the A/Beijing/501/2009 H1N1 isolate successfully infected the lungs of ferrets and caused more pathological lesions than the seasonal influenza virus. Our findings demonstrate that the difference in virulence of the 2009 pandemic H1N1 influenza virus and the seasonal H1N1 influenza virus in vitro and in vivo may have been mediated by different mechanisms.

Conclusion/Significance

Our understanding of the pathogenesis of the 2009 A (H1N1) influenza virus infection in both humans and animals is broadened by our findings that apoptotic cell death is involved in the cytopathic effect observed in vitro and that the pathological alterations in the lungs of S-OIV H1N1-infected ferrets are much more severe.     

Polygonum cuspidatum and Its Active Components Inhibit Replication of the Influenza Virus through Toll-Like Receptor 9-Induced Interferon Beta Expression

Influenza virus infection is a global public health issue. The effectiveness of antiviral therapies for influenza has been limited by the emergence of drug-resistant viral strains. Therefore, there is an urgent need to identify novel antiviral therapies. Here we tested the effects of 300 traditional Chinese medicines on the replication of various influenza virus strains in a lung cell line, A549, using an influenza-specific luciferase reporter assay. Of the traditional medicines tested, Polygonum cuspidatum (PC) and its active components, resveratrol and emodin, were found to attenuate influenza viral replication in A549 cells. Furthermore, they preferentially inhibited the replication of influenza A virus, including clinical strains isolated in 2009 and 2011 in Taiwan and the laboratory strain A/WSN/33 (H1N1). In addition to inhibiting the expression of hemagglutinin and neuraminidase, PC, emodin, and resveratrol also increased the expression of interferon beta (IFN-β) through Toll-like receptor 9 (TLR9). Moreover, the anti-viral activity of IFN-β or resveratrol was reduced when the A549 cells were treated with neutralizing anti-IFN-β antibodies or a TLR9 inhibitor, suggesting that IFN-β likely acts synergistically with resveratrol to inhibit H1N1 replication. This potential antiviral mechanism, involving direct inhibition of virus replication and simultaneous activation of the host immune response, has not been previously described for a single antiviral molecule. In conclusion, our data support the use of PC, resveratrol or emodin for inhibiting influenza virus replication directly and via TLR-9–induced IFN-β production.     

Investigation of the influence of EPs 7630, a herbal drug preparation from Pelargonium sidoides, on replication of a broad panel of respiratory viruses

The Pelargonium sidoides extract EPs^® 7630 is an approved drug for the treatment of acute bronchitis in Germany. The postulated mechanisms underlying beneficial effects of EPs^® 7630 in bronchitis patients include immunomodulatory and cytoprotective effects, inhibition of interaction between bacteria and host cells, and increase of cilliary beat frequency on respiratory cells. Here, we investigated the influence of EPs^® 7630 on replication of a panel of respiratory viruses. Determination of virus-induced cytopathogenic effects and virus titres revealed that EPs^® 7630 at concentrations up to 100 μg/ml interfered with replication of seasonal influenza A virus strains (H1N1, H3N2), respiratory syncytial virus, human coronavirus, parainfluenza virus, and coxsackie virus but did not affect replication of highly pathogenic avian influenza A virus (H5N1), adenovirus, or rhinovirus. Therefore, antiviral effects may contribute to the beneficial effects exerted by EPs^® 7630 in acute bronchitis patients.