| Backgroud:The 2009 H1N1 pandemic influenza virus(pdm/09)caused the 2009 pandemic to human,then came into pigs and become one of the components in the swine influenza virus gene pool.This swine-origin virus was generated via multiple reassortments of swine,human and avian influenza viruses.Pdm/09 not only infects humans but also infects swine and further reassorts with the enzootic swine influenza virus(SIV).New gene reassortment viruses with gene segments of pdm/09 and other SIVs have introduced repeatedly into pigs raise the possibility of generating novel swine influenza viruses with the potential to infect humans.It is a special process of trans-species infection induce by influenza virus,miRNA may play important roles in it,and a majority of miRNAs in host cells will perform two diverse functions during infection.On the one hand,miRNAs can be up-or downregulated by viruses involved in pathways that can promote viral replication to prevent the host antiviral cellular mechanism.On the other hand,the host cell can respond to viral infection via altered miRNA expression,which can have effects on viral transcription or replication processes to limit viral propagation.However,if there is a unique miRNA and relative gene/protein that influnce virus replication during the infection of cross-species influenza virus,then it is still unknown.Several previous studies have reported an interactional correlation between miRNA profile alterations and influenza virus infections with various models,such as different types of cells,patient serum samples or throat swabs,and multiple kinds of mammals.However,due to the various study methods or models,the results lack a uniform trend and because of a wide range of miRNAs,more evidence is needed.We focus the the miRNAs associated with the cross-species infection to A549 cells of the emergent H3N2 SIV with the methods of miRNA microarrays,transcriptome sequencing and iTRAQ technique.Through the interaction analysis of the miRNA-mRNA-ptotein network investigation to explore special miRNAs and relevant mRNAs'significant roles in influenza virus cross-species infection.This finding demonstrates that global miRNA and related gene/protein is an innovative prompt for uncovering new biomarkers and sensitive measures drug targets of response to novel reassortment swine influenza virus infection in humans.Objctive:From the view of miRNA,mRNA and protein,this study investigates special small RNA in trans-species infection after A549 cells were infected with a novel H3N2 SIV.Methods:1.To compare the difference during the infection of three strains of influenza virus(emergent H3N2 SIV-SW2783,classical SIV-SW3861 and pdm/09 vaccine strain-CA09),we builded in vivo and vitro infection models.The virus replication dynamics were plotted and the different pathogenicity characteristics were studied through HA,TCID50,plaque,IHC and IF test.2.Microarrays and high-throughput sequencing were used to detect differentially expressed miRNAs and mRNAs after A549 cells were infected by three strains of influenza virus at 24h(acute infection period).A miRNA-mRNA network was generated to show the possible correlation of unique miRNAs related to the cross-species infection of humans.3.iTRAQ technique was detected to show specific differential expression of proteins after SW2783 infection of A549 cells,and combined candidate miRNA results to construct the interaction miRNA-protein construct the interaction map.Specific miRNAs associated with trans-species infection were identified and investigated again at the protein level.4.Using knock out and overexpression exprements to testify the interaction between candidate miRNAs and their potencial target gene.Rescue exprements were designed to examine whether candidate miRNAs will influence the virus replication or will regulate SW2783 infection specially.Results:1.Three strains of influenza virus infection model in vitro and vivo were established successfully,and showed different infection capacities.The novel SIV showed efficient replication and proliferation when it infected A549 cells and human lung tissue in vitro,who has the potential to trans-species infection humans;classical SIV had relatively weak infection ability and the possibility of cross-species infection is lower;pdm/09 vaccine strain could establish an effective infection of human cells and tissues.By analyzing the pathogenic characteristics of mice infection,it was found that three strains of influenza virus could infect mice's lung and cause corresponding pathological reactions,but the infection capacity of SW2783 was slightly higher than that of the other two influenza viruses in mice.2.Global cellular miRNA and mRNA expression patterns in A549 cells infected with the influenza virus strains SW2783,SW3 861 and CA09 were compared with those of uninfected controls.Venn diagram was generated to display the differentially expressed miRNAs and mRNAs that were changed uniquely in SW2783 but commonly in SW2783 and CA09 compared to NC were identified as lists of interests.We found that 52 unique miRNAs and 2543 related mRNAs may be related to cross-species infection of A549 cells.The interaction analysis was performed to describe their relationship and potential function by GO term and KEGG pathway analyses.The expression of 3 miRNAs(hsa-miR-140-5p,hsa-miR-30a-3p and hsa-miR-582-5p)and 5 relevant mRNAs(RCC1,ERVFRD-1,RANBP1,SCARB2 and RPS29),which were implicated in viral infection,were verified to be consistent with microarray and high-throughput sequencing results.3.All proteins was detected by iTRAQ technique and a total of 128 differentially expressed proteins may be associated with cross-species infection in A549 cells,70 proteins were associated with 52 candidate miRNAs.The interaction network between these proteins and three miRNAs,a total of 6 proteins were screened to meet the requirements.RPS29,HMGA2,IFIT2 and PPIA were up-regulated distinctively during SW2783 infection of A549 cells and aslo verified by Westein blot,which could believe to be a functional protein significantly expressed by novel SIV trans-species A549 cells.4.The functions and relationships with viruses of all candidate miRNAs,genes and proteins were analyzed comprehensively,we chose hsa-miR-140-5p and target gene or protein ERVFRD-1,PPIA and IFIT2.as objectives for further discussion.The hsa-miR-140-5p has been confirmed to have a targeted regulatory effect on ERVFRD-1,PPIA and IFIT2 through knockout and overexpression experiments,virus rescue experiments showed that hsa-mir-140-5p could specifically affect SW2783 infection,but had little effect on the infectious capacity of SW3861 and CA09.It is preliminarily believed that hsa-miR-140-5p can be used as one of the potential biomarkers or new drug targets for screening influenza viruses with cross-species infection.Conclusions:We studied on the global host miRNA-mRNA-protein network function to cross-species infection caused by novel reassortment SIV in A549 cells.Base on this finding,some preliminary discussions about representation and specificity change between in trans-species infection were further made.The novel H3N2 SIV showed the ability of trans-species infection in vitro and vivo.Though the comprehensive analysis about candidate miRNAs,genes and proteins induced by virus in A549 cells,hsa-miR-140-5p,hsa-miR-30a-3p and hsa-miR-582-5p can be specifically expressed differently during SW2783 infection,which may be closely related to cross-species infection.The hsa-miR-140-5p had regulatory effect on the expression of ERVFRD-1,PPIA and IFIT2,and could specifically affect SW2783 infection,could be one of the potential biomarkers or new drug targets for screening influenza viruses with trans-species infection potency. |
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