Infectivity Of Influenza A Virus From Different Species And Study On Mirnas Associated With Infected A549 Cells

Background:Influenza A virus?IAV?could even trigger a global influenza pandemic while causes seasonal influenza epidemics,which poses a health burden and potential threat to human beings.In recent years,avian influenza virus?AIV?could infect human being directly many times in China,even leads to death,swine influenza virus?SIV?can also infect humans occasionally in other countries.AIV and SIV have always threatened human health,so it is necessary to study the molecular mechanism of influenza A virus from different species.The discovery of miRNAs provides a new idea for the study of anti-influenza targets,because several miRNAs have been demonstrated to be involved in the influenza virus replication cycle,but most studies are focusing on the research of a certain IAVs subtype,and there is rare research on miRNA associated with LAVs from different species.Since these miRNAs are regulating various IAVs infections,they may become the new target of broad-spectrum anti-influenza drugs or vaccines.In this research,the infection ability of IAVs from three different species was studied,and the miRNAs that related in IAV infection were screened and functionally verified to explore the regulatory role of miRNAs in the process of IAV in humans.It provides a theoretical basis for the clarification of the mechanism of IAV infection in mammals,and lay a foundation for the application of miRNAs to anti-influenza drugs and vaccines.Objective:To reveal the infection characteristics of the three IAVs from different species;to investigate the regulatory mechanism of miRNAs involved in IAV infections;to preliminarily confirm whether hsa-miR-371a-5p has regulatory functions on two target genes and to explore the effect of hsa-miR-371a-5p on three viral infections.Methods:1.Using three kinds of IAVs from different species?human seasonal influenza virus H3N2,swine influenza virus H1N1 and avian influenza virus H3N2?to infect mammalian cells and mice respectively to explore the optimal infection conditions,and establish cells and animal model of IAV infection.Using HA,TCID50,plaque formation test,immunofluorescence test and immunohistochemistry test to reveal the infection characteristics of the three viruses and clarify the pathogenesis mechanism of IAV from different species.2.A549 cells were infected by three kinds of IAVs from different species,the expression profiles of miRNA and mRNA were obtained by miRNA microarray detection and high-throughput sequencing technology,and miRNA-mRNA interactions were obtained by bioinformatics methods.In addition,qPCR was also performed to verify the actual expression of 8 miRNAs and 13 mRNAs in infected cells.3.Using iTRAQ to detect the protein expression profiles of A549 cells infected with IAVs from three different species,and the function prediction of differently expressed proteins were performed by GO and KEGG functional enrichment analysis.The miRNA-protein pairs were obtained by bioinformatics methods.In addition,qPCR and Western blot were used to verify the actual expression of two miRNAs and three proteins in infected cells,respectively.4.The hsa-miR-371a-5p and its two potential target genes?IFIT2 and XAF1?were selected for functional verification,and A549 cells were transfected with hsa-miR-371a-5p inhibitor/mimics to simulate the expression/overexpression of hsa-miR-371a-5p.qPCR and Western blot techniques were used to detect the expression of hsa-miR-371a-5p and target protein to explore the interaction between hsa-miR-3 71 a-5p and two target genes.hsa-miR-3 71 a-5p mimics was also used to mimic the overexpression of hsa-miR-371a-5p in infected cells to study the role of hsa-371a-5p in IAV infection.Conclusions:In this study,IAVs from three different species were used as research objects,and the infection characteristics of three viruses and the mechanism of infection-related miRNAs were studied.Initially got the following findings:1.After IAV infection of A549 cells and BALB/c mice,in.three kinds of viruses,AVI₉990 had the strongest biosynthesis ability at the early stage of infection,but the weakest adaptability and weakest virulence;SW₃861 had good adaptability and highest virulence;Human_Br07 virus has the best adaptability,and the virulence is medium.2.There are 1852 pairs of miRNA-mRNA that may be associated with IAV infection,107 pairs of miRNA-mRNA associated with viral defense,and 79 pairs of miRNA-mRNA involved in influenza A-associated pathway.3.There are 455 pairs of miRNA-proteins that may be associated with IAV infection,and 352 pairs of proteins have an interaction relationship.4.In A549 cells,the change in the expression level of hsa-miR-371 a-5p caused a negative change in the expression levels of two potential target proteins?IFIT2 and XAF1?.The expression level of hsa-miR-37 1a-5p is up-regulated,which can promote the replication of three influenza viruses.In summary,miRNA plays an important regulatory role in the process of IAV infection.In this study,miRNAs were integrated analyzed with mRNA and protein,respectively.And miRNAs related to different-species IAV infection were screened by bioinformatics methods.Hsa-miR-371a-5p was found to be involved in IAV infection,and these results suggest the potential value of using miRNAs for anti-influenza target studies.