Replication Of Influenza Virus In Different Cell Lines

Influenza(IA)is a highly contagious,acute respiratory infection with high morbidity.Viral variability,frequent global outbreaks,and uncertain regional epidemics threaten human health.The death toll caused by this disease around the world reached up to 300,000 people each year,and it caused huge losses to the global economy.Infection with Influenza virus(IAV)provokes activation of host immune response contributing to defending against and clearing the viral infection.Innate immune system is comprised of physical barriers,various phagocytic cells,cytokines,interferons and interferon stimulated genes(ISGs),which provide the first line of defense against IAV infection.Upon infection with IAV,the virus can be recognized by host pathogen recognition receptors(PRRs),leading to activation of innate immune signaling,which results in the rapid induction of inflammatory cytokines and acute inflammation.Influenza A virus has evolved several strategies to promote their own replication through utilizing and disrupting cellular proteins and signal pathways.At present,deficiency in the understanding of the process of virus infection and replication,interaction between virus and host factor and host antiviral immune response limit the development of specific and effective antiviral drugs and vaccines.On the other hand,IAVs have developed multiple strategies to escape from host immune surveillance for effective replication in host cells.Therefore,understanding the mechanisms of virus to escape the host immune response will provide the guidance for virus control and antiviral drug design.In this study,the IAV virus strains displaying the different virus replication in human cells were used as the model virus,the mechanisms how virus infect human cells were investigated.On the other hand,the high-throughput RNA sequencing technology(RNA-seq)was used to identify differentially gene expression upon infection with IAV in A549 and 293T cells.Furthermore,the functions of IL17A during viral infection were identified.This study is mainly divided into two parts.Part 1:The critical role of NS1 amino acid substitutions during virus replication.Highly pathogenic avian influenza(HPAI)H5N1 virus,a highly infectious respiratory pathogen,can evolve from an animal infected pathogen to a human infected pathogen.H5N1 viruses have infected hundreds of people with a fatality rate approaching 60%,pose a continual threat to public health.In this study,three viruses belonging to clades 2.3.4(A/wild duck/Human/021/2005(HN021)and A/chicken/Hunan/1/2009(HN01))and 2.3.2.1c(A/tiger/Jiangsu/01/2013(JS01))were used as model viruses.The A549 cells and human macrophages derived from human PBMC were infected were separately infected with the three viruses.The samples were harvested at indicated time points and their replication kinetics was determined by TCID50.In both cell lines,HN021 displayed similar replication kinetics to JS01.In contrast,HN01 replicated to lower titers than HN021 and JS01.To further confirm the infection capability of the three H5N1 viruses in human cells,the infected human macrophages and A549 cells were also harvested for detection of viral NP expression using immunofluorescence microscopy and Western blotting.Consistently,HN01 infected cells displayed lower NP expression.To identify the reasons for differential virus replication,the real-time RT-PCR methods were developed to detect the PPR,interferon and ISGs gene expression.Infected A549 and macrophage cells were harvested,and a series of host genes were analyzed by real time RT-PCR.The results showed that the expression of pattern receptor recognition(PRR),RIG-I,IFN-? and the interferon response-related genes Mxl and GBP-1 were significantly increased in HN01-infected A549 cells compared to HN021-and JS01-infected cells.Similar results were also observed in HN01-infected macrophages.These observations revealed a strong induction of the host interferon response in HN01-infected human cells,indicating that the differential virus replication of three viruses is associated with the different induction activation of interferon response in human cells.To test the ability of the three H5N1 NS1 proteins to inhibit IFN-? promoter activity in human cells.The luciferase assay was used to detect IFN-? promoter activity.The results showed that Sev infection induced enhanced IFN-? promoter activity in HN01 NS protein,while HN021 and JS01 failed to do so.In addition,to identify the potential residues that lead the differential virus replication among these three viruses,we analyze the sequence alignment of NS1 proteins.According to the alignment analysis,the NS1 amino acid residues at positions K55,K66 and C133 of HN01 virus displayed difference.Interestingly,NS 1-K55E,K66E,and C133F triple mutant(TM)resulted in significantly lower IFN-? and ISRE promoter activity compared to wild-type infected with Sev.The NS1 substitutions that efficiently blocked the promoter activity also displayed weak expression of NS1 protein and general gene expression in transfected cells,as detected by western blotting.Furthermore,to determine whether HN01 NS1 mutation can affect the interaction with cellular proteins,we measured the ability of 55E,66E,133F and triple mutant NS1 to bind to cellular proteins by Co-IPs.The results showed that CPSF30 efficiently bound to the triple mutant,suggesting that the H5N1 HN01 NS1-CPSF30 interaction is very weak but can be strengthened by the triple mutant.Differences were not observed between NS1-WT and NS1 triple mutant with regard to interaction with TRIM25,PKR,NF90,or PABPII,indicating the NS1 substitutions in HN01 specifically enhance CPSF30 binding.Finally,the parental HN01 virus and recombinant viruses incorporating K55E,K66E,C133F,and triple mutant were generated by using reverse genetics.The A549 and macrophage cells were infected with recombinant viruses,respectively,and the replication kinetics were determined.The results showed that C133F and TM displayed higher virus titers in both A549 and macrophage cells.Taken together,the binding of HN01 NS1 protein to cellular CPSF30 contributes to the activation of IFN and ISGs,leading to the differential virus replication in human cells.Part 2:The host inflammatory cytokines influence on IAV virus replicationRNA-sequencing(RNA-seq),which is a genome-wide analytical technology,has been extensively used to analyze the transcriptome of various viral infectious disease,including IAV infection.In this study,A549 cells and 293T cells were infected with CA04,PR8 viruses,respectively.The virus titers were determined by plaque assay.The results showed that virus titers in 293T cells showed more than 200-fold lower than infection in A549 cells,indicating the less efficiently H1N1 influenza virus replication in 293T cells.To determine the host response of A549 and 293T cells during influenza virus infection,the high-throughput RNA-seq was used to identify differentially gene expression.The results indicated that the viral sensing signals were significantly activated,and the cytokines were highly expressed in influenza virus(AIV)-infected A549 cells,and all these play an important role in the activation of innate immune and inflammatory responses.In contrast,AIV-infected 293T cells had weak signals in immune response and low cytokine expression.Additionally,the IL-17A was also enriched in 293T cells infected with PR8 virus,indicating the potential role of IL-17A in regulating the virus propagation or inflammatory response in 293T cells.To further confirm the function of IL-17A during IAV virus infection,the A549 cell were treated with IL-17A protein followed by infected with PR8 influenza virus.The results showed that IL-17A can partially promote PR8 virus infection in A549 cells.Furthermore,the IL-17A knockout mice were also developed to detect the function of IL-17A in vivo.The results demonstrated thet PR8-infected IL-17A knockout mice consistently showed decreased body weight and weakened immunopathology in the lungs compared to the control group.These results revealed diverse host responses of various H1N1 influenza virus-infected cells and the potential role of IL-17A in regulating viral infection.