H5N1-infected Cells Research Articles

Inactivation efficacy of H5N1 avian influenza virus by commonly used sample preparation reagents for safe laboratory practices

The objective of this study was to determine the inactivation efficiency of common sample preparation reagents against highly pathogenic avian influenza A (HPAI) H5N1 virus. HPAI H5N1 virus has caused infections in humans with a mortality rate of over 50%. Due to the high mortality and the risk of aerosol transmission of that virus to humans and birds, infectious HPAI H5N1 viruses are contained in a biosafety level 3 laboratory. However, many procedures for further molecular analyses would be easier in lower biosafety conditions. To ensure the laboratory safety the successful inactivation procedures should be demonstrated before the samples are transferred to a lower containment facility. We tested the inactivation capacity of commonly used cell lysis buffer radio-immuno precipitation assay (RIPA) buffer for protein samples, cell fixatives methanol (MeOH) and paraformaldehyde (PFA) and guanidine isothiocyanate-containing lysis buffer for RNA isolation (RLT, Qiagen) in H5N1-infected cells. Based on our results RLT buffer, 90% MeOH (20 min, −20 °C) and 4% PFA (30 min, RT) all completely inactivated the HPAI H5N1 virus. However, RIPA buffer alone was not sufficient to inactivate the HPAI H5N1 virus in infected cell samples but, instead, combining RIPA lysis buffer and boiling for 10 min the samples in Laemmli buffer led to complete inactivation of the virus.

Defective Viral Particles Produced in Mast Cells Can Effectively Fight Against Lethal Influenza A Virus.

Mast cells play an important role in the pathogenesis of highly pathogenic H5N1 avian influenza virus (H5N1-HPAIV) infection. Defective viral particles (DPs) can interfere with the replication of infectious viruses and stimulate the innate immune response of host cells. However, DPs arising from mast cells during HPAIV replication and their potent antiviral actions has not been reported. Here, we showed that the human mastocytoma cell line, HMC-1, allowed for the productive replication of the H5N1-HPAIV. Compared with alveolar cell line A549, DPs were propagated preferentially and abundantly in mast cells following IAV infection, which can be attributed to the wide existence of Argonaute 2 (AGO2) in HMC-1 cells. In addition, DPs generated in H5N1-infected cells could provide great therapeutic protection on mice to fight against various influenza A viruses, which included not only homologous H5N1-HPAIV, but also heterologous H1N1, H3N2, H7N2, and H9N2. Importantly, DPs generated in H5N1-infected HMC-1 cells could diminish viral virulence in vivo and in vitro by triggering a robust antiviral response through type II interferon signaling pathways. This study is the first to illustrate the arising of DPs in H5N1-HPAIV infected mast cells and explore their favorable ability to protect mice from influenza A viruses infection, which provides a novel insight and valuable information for the progress of new strategies to fight influenza A viruses infection, especially highly pathogenic avian influenza virus infection by focusing on the DPs generated in mast cells.

Highly pathogenic avian influenza H5N1 virus delays apoptotic responses via activation of STAT3.

Highly pathogenic avian influenza (HPAI) H5N1 virus continues to pose pandemic threat, but there is a lack of understanding of its pathogenesis. We compared the apoptotic responses triggered by HPAI H5N1 and low pathogenic H1N1 viruses using physiologically relevant respiratory epithelial cells. We demonstrated that H5N1 viruses delayed apoptosis in primary human bronchial and alveolar epithelial cells (AECs) compared to H1N1 virus. Both caspase-8 and -9 were activated by H5N1 and H1N1 viruses in AECs, while H5N1 differentially up-regulated TRAIL. H5N1-induced apoptosis was reduced by TRAIL receptor silencing. More importantly, STAT3 knock-down increased apoptosis by H5N1 infection suggesting that H5N1 virus delays apoptosis through activation of STAT3. Taken together, we demonstrate that STAT3 is involved in H5N1-delayed apoptosis compared to H1N1. Since delay in apoptosis prolongs the duration of virus replication and production of pro-inflammatory cytokines and TRAIL from H5N1-infected cells, which contribute to orchestrate cytokine storm and tissue damage, our results suggest that STAT3 may play a previously unsuspected role in H5N1 pathogenesis.

Infection with influenza A viruses causes changes in promoter DNA methylation of inflammatory genes

Replication of influenza virus in the host cells results in production of immune mediators like cytokines. Excessive secretion of cytokines (hypercytokinemia) has been observed during highly pathogenic avian influenza virus (HPAI-H5N1) infections resulting in high fatality rates. The exact mechanism of hypercytokinemia during influenza virus infection is still not known completely. As promoter DNA methylation changes are linked with expression changes in genes, we intend to identify whether changes in promoter DNA methylation have any role in expression of cytokines during influenza A virus infection. A panel of 24 cytokine genes and genes known to be involved in inflammatory response were analyzed for their promoter DNA methylation changes during influenza A virus infections. Four different strains of influenza A viruses, viz. H5N1, H1N1, pandemic (2009) H1N1, and a vaccine strain of H5N1, were used for the study. We found seven of the total 24 inflammatory genes studied, showing significant changes in their promoter methylation levels in response to virus infection. These genes included proinflammatory cytokines CXCL14, CCL25, CXCL6, and interleukines IL13, IL17C, IL4R. The changes in DNA methylation levels varied across different strains of influenza viruses depending upon their virulence. Significant promoter hypomethylation in IL17C and IL13 genes was observed in cells infected with HPAI-H5N1 virus compared with other influenza viruses. This decrease in methylation was found to be positively correlating with the increased expression of these genes. Analysis of IL17C promoter region using bisulfite sequencing resulted in identification of a CpG site within Retinoid X receptor-alpha (RXR-α) transcription factor binding site undergoing demethylation specifically in H5N1-infected cells but not in other influenza-infected cells. Thus, the study could demonstrate that changes in promoter methylation in certain specific cytokine genes actually have a possible role in their expression changes during influenza A virus infection.

Involvement of GRIM-19 in apoptosis induced in H5N1 virus-infected human macrophages

The fatal H5N1 infection has a high mortality rate among infected patients. The pathogenesis of H5N1 viral infection is associated with the ability of the virus to induce apoptotic cell death. However, the molecular mechanism of apoptosis induced by H5N1 remains unclear. In the present study we demonstrate that H5N1 virus is able to up-regulate the expression of gene associated with retinoid and interferon induced mortality-19 (GRIM-19) in human monocyte-derived macrophages (hMDMs). GRIM-19 has been identified as a novel gene with apoptotic effects in virus-infected cells. The percentage of apoptotic cells is significantly decreased in H5N1-infected GRIM-19 depleted hMDMs, which is also associated with a decrease of BH3-interacting domain death agonist cleavage and apoptosis-inducing factor (AIF) release to the cytosol. These results suggested the involvement of GRIM-19 in apoptosis induced by H5N1 virus. Furthermore, neutralizing-IFN-β Ab is able to suppress GRIM-19 expression in H5N1-infected cells resulting in a decrease in apoptotic cell number, indicating that IFN-β secreted by H5N1-infected hMDMs regulates GRIM-19 expression leading to apoptosis. Altogether, the results presented here provide additional insight on the regulatory mechanism of H5N1 viral-induced apoptotic cell death in hMDMs.

Cellular transcriptional profiling in human lung epithelial cells infected by different subtypes of influenza A viruses reveals an overall down-regulation of the host p53 pathway

BackgroundInfluenza viruses can modulate and hijack several cellular signalling pathways to efficiently support their replication. We recently investigated and compared the cellular gene expression profiles of human lung A549 cells infected by five different subtypes of human and avian influenza viruses (Josset et al. Plos One 2010). Using these transcriptomic data, we have focused our analysis on the modulation of the p53 pathway in response to influenza infection.ResultsOur results were supported by both RT-qPCR and western blot analyses and reveal multiple alterations of the p53 pathway during infection. A down-regulation of mRNA expression was observed for the main regulators of p53 protein stability during infection by the complete set of viruses tested, and a significant decrease in p53 mRNA expression was also observed in H5N1 infected cells. In addition, several p53 target genes were also down-regulated by these influenza viruses and the expression of their product reduced.ConclusionsOur data reveal that influenza viruses cause an overall down-regulation of the host p53 pathway and highlight this pathway and p53 protein itself as important viral targets in the altering of apoptotic processes and in cell-cycle regulation.

Response network analysis of differential gene expression in human epithelial lung cells during avian influenza infections

BackgroundThe recent emergence of the H5N1 influenza virus from avian reservoirs has raised concern about future influenza strains of high virulence emerging that could easily infect humans. We analyzed differential gene expression of lung epithelial cells to compare the response to H5N1 infection with a more benign infection with Respiratory Syncytial Virus (RSV). These gene expression data are then used as seeds to find important nodes by using a novel combination of the Gene Ontology database and the Human Network of gene interactions. Additional analysis of the data is conducted by training support vector machines (SVM) with the data and examining the orientations of the optimal hyperplanes generated.ResultsAnalysis of gene clustering in the Gene Ontology shows no significant clustering of genes unique to H5N1 response at 8 hours post infection. At 24 hours post infection, however, a number of significant gene clusters are found for nodes representing "immune response" and "response to virus" terms. There were no significant clusters of genes in the Gene Ontology for the control (Mock) or RSV experiments that were unique relative to the H5N1 response. The genes found to be most important in distinguishing H5N1 infected cells from the controls using SVM showed a large degree of overlap with the list of significantly regulated genes. However, though none of these genes were members of the GO clusters found to be significant.ConclusionsCharacteristics of H5N1 infection compared to RSV infection show several immune response factors that are specific for each of these infections. These include faster timescales within the cell as well as a more focused activation of immunity factors. Many of the genes that are found to be significantly expressed in H5N1 response relative to the control experiments are not found to cluster significantly in the Gene Ontology. These genes are, however, often closely linked to the clustered genes through the Human Network. This may suggest the need for more diverse annotations of these genes and verification of their action in immune response.

N-acetyl-l-cysteine (NAC) inhibits virus replication and expression of pro-inflammatory molecules in A549 cells infected with highly pathogenic H5N1 influenza A virus

The antioxidant N-acetyl-l-cysteine (NAC) had been shown to inhibit replication of seasonal human influenza A viruses. Here, the effects of NAC on virus replication, virus-induced pro-inflammatory responses and virus-induced apoptosis were investigated in H5N1-infected lung epithelial (A549) cells. NAC at concentrations ranging from 5 to 15mM reduced H5N1-induced cytopathogenic effects (CPEs), virus-induced apoptosis and infectious viral yields 24h post-infection. NAC also decreased the production of pro-inflammatory molecules (CXCL8, CXCL10, CCL5 and interleukin-6 (IL-6)) in H5N1-infected A549 cells and reduced monocyte migration towards supernatants of H5N1-infected A549 cells. The antiviral and anti-inflammatory mechanisms of NAC included inhibition of activation of oxidant sensitive pathways including transcription factor NF-κB and mitogen activated protein kinase p38. Pharmacological inhibitors of NF-κB (BAY 11-7085) or p38 (SB203580) exerted similar effects like those determined for NAC in H5N1-infected cells. The combination of BAY 11-7085 and SB203580 resulted in increased inhibitory effects on virus replication and production of pro-inflammatory molecules relative to either single treatment. NAC inhibits H5N1 replication and H5N1-induced production of pro-inflammatory molecules. Therefore, antioxidants like NAC represent a potential additional treatment option that could be considered in the case of an influenza A virus pandemic.

Mouse-Passaged Severe Acute Respiratory Syndrome-Associated Coronavirus Leads to Lethal Pulmonary Edema and Diffuse Alveolar Damage in Adult but Not Young Mice

Advanced age is a risk factor of severe acute respiratory syndrome (SARS) in humans. To understand its pathogenesis, we developed an animal model using BALB/c mice and the mouse-passaged Frankfurt 1 isolate of SARS coronavirus (SARS-CoV). We examined the immune responses to SARS-CoV in both young and adult mice. SARS-CoV induced severe respiratory illness in all adult, but not young, mice on day 2 after inoculation with a mortality rate of 30 to 50%. Moribund adult mice showed severe pulmonary edema and diffuse alveolar damage accompanied by virus replication. Adult murine lungs, which had significantly higher interleukin (IL)-4 and lower IL-10 and IL-13 levels before infection than young murine lungs, rapidly produced high levels of proinflammatory chemokines and cytokines known to induce macrophage and neutrophil infiltration and activation (eg, tumor necrosis factor-alpha). On day 2 after inoculation, young murine lungs produced not only proinflammatory cytokines but also IL-2, interferon-gamma, IL-10, and IL-13. Adult mice showed early and acute excessive proinflammatory responses (ie, cytokine storm) in the lungs after SARS-CoV infection, which led to severe pulmonary edema and diffuse alveolar damage. Intravenous injection with anti-tumor necrosis factor-alpha antibody 3 hours after infection had no effect on SARS-CoV infection. However, intraperitoneal interferon-gamma injection protected adult mice from the lethal respiratory illness. The experimental model described here may be useful for elucidating the pathophysiology of SARS and for evaluating therapies to treat SARS-CoV infection.

Hyper-induction of cytokines by avian influenza A (H5N1): The role of signaling pathways and Toll-like receptors (B131)

Abstract Avian influenza virus A/HK/483/97 (H5N1) is associated with unusually severe human disease. H5N1 viruses hyper-induce pro-inflammatory cytokines (tumor necrosis factor alpha [TNF-α] and interferon beta [IFN-β]) and macrophage-tropic chemokines from primary human macrophages in vitro and this is paralleled by elevated levels of these cytokines in patients sera. It is therefore important to understand the mechanisms underlying this H5N1 induced differential hyper-induction of cytokines. Unlike H1N1, H5N1 virus strongly activated interferon regulatory factor 3 (IRF3). siRNA mediated gene silencing of IRF3 led to a dramatic reduction of the gene expression of IFN-β in H5N1-infected cells but only a partial decrease in TNF-α gene expression. Thus the differential activation of IRF3 may explain the hyper-induction of IFN-β, but does not fully explain the hyper-induction of TNF-α. Similarly, gene silencing of toll-like receptor 3 (TLR3) did not completely abrogate the TNF-α response. The significance of these findings in the pathogenesis of human H5N1 disease will be discussed.