Screening And Preliminary Integration Analysis Of MiRNA And MRNA In Lung Tissue Of Mice Infected With Low Pathogenic H7N9 Influenza Virus

The low pathogenic H7N9 subtype influenza virus is a type of zoonotic influenza A virus.In February 2013,the first incident of H7N9 infection in China was occurred,causing extreme panic.Although the current influenza pandemic has subsided,the outbreak of the next influenza pandemic has become a possibility due to the widespread presence of natural hosts such as birds,so it is particularly important to quickly explore its pathogenesis.One of the important reasons for the avian influenza virus to infect humans is that its 627 site is mutated from glutamic acid(E)to lysine acid(K).Influenza virus polymerase genes,especially the PB2 gene,are important determinants of virulence and host range.The 627th position of the PB2 gene of all avian influenza viruses is E,and the vast majority of human influenza viruses are K.The E627K mutation can increase the adaptability of the influenza virus to mammals and enhance pathogenicity.Similarly,the D701N mutation has the function of compensating for the E627K mutation to a certain extent.Although the pathogenesis of influenza virus has been studied intensively,the specific mechanism for the adaptive evolution of the host mutations at positions 627 and 701 on the PB2 gene has not been fully understood.miRNAs are a novel regulatory factor that functions through post-transcriptional regulatory mechanisms.miRNAs regulatory networks play an important role in regulating immune homeostasis or pathological immune responses.More and more studies have confirmed that many miRNAs also play a regulatory role in the interaction between virus and host.miRNA microarray is a rapid method for detecting the expression of thousands of miRNAs in a host.Transcriptome sequencing(RNA-Seq)is a high-throughput sequencing technology that can quickly obtain all information of transcripts in a specific state.It is a method to quickly determine the expression levels of certain genes under different physiological conditions.In this paper,miRNA microarray and transcriptome sequencing technology were used to analyze the miRNA and mRNA expression profiles of low pathogenic H7N9 A/Anhui/1/2103(Ah1)influenza virus and its mutant viruses rAh1-PB2-K627E and rAhl-PB2-D701N,and took comparative analysis.It provides a theoretical basis for the study of immune mechanism of influenza virus.In this study,we first determined the pathogenicity of Ahl and its mutant viruses rAhl-PB2-K627E and rAhl-PB2-D701N by mouse pathogenicity test and virus titer of infected lungs,and then hematoxylin and eosin staining(HE)and immunofluorescence technique(IF)were used to observe the extent of lung lesions and the location of antigens.The miRNA and mRNA expression profiles of Ah1 and its mutant virus were analyzed by miRNA microarray and transcriptome sequencing technology,and comparative analysis was performed.The accuracy of the sequencing results was verified by qRT-PCR.Experiment Ⅰ MiRNA expression profiling of lung tissue of mice infected with low pathogenic H7N9 influenza virus Firstly,the inbreeding BALB/c mice were infected with Ahl influenza virus,and the body weight showed a significant downward trend.The lung tissue sections showed that the lungs of the mice showed obvious symptoms of interstitial pneumonia after infection.The miRNA and mRNA expression profiles of the lung tissue of mice infected with Ahl influenza virus were detected by miRNA microarray and transcriptome sequencing technology.The results showed that 265 differentially expressed miRNAs and 5577 differentially expressed mRNAs significantly changed.Using miRNAorg,Targetscan and PITA software,265 differentially expressed miRNA target genes were predicted,and GO and KEGG analysis were used to screen out seven antiviral immune response signaling pathways that may be associated with low pathogenic Ah1 influenza virus infection:Rapl signal Pathway,PI3K-Akt signaling pathway,Hippo signaling pathway,MAPK signaling pathway,Wnt signaling pathway,focal adhesion,and autophagy-related pathways.The target genes enriched in the seven signaling pathways intersected with the mRNA information,and the miRNA-mRNA negative regulation relationship pair was combined.The final target gene was analyzed by miRNA regulation network,and 15 differentially expressed miRNAs and 31 differentially expressed target gene were enriched into this miRNA regulatory network.qRT-PCR results confirmed that the expression trends of these miRNAs were in good agreement with the sequencing results.The results of this study indicate that miRNA can directly or indirectly regulate the expression of downstream genes during Ahl influenza infection.Experiment Ⅱ MiRNA-mRNA integration analysis of mouse lung tissue infected with low pathogenic H7N9 influenza virus and its mutant viruses Body weight changes,mortality,and organ virus titers of mice infected with the Ah1 influenza virus and its mutant viruses rAhl-PB2-627E and rAh1-PB2-627E+701N were analyzed by comparison.It was proved that the avian-derived rAhl-PB2-627E virus had the weakest pathogenicity to mice,followed by rAhl-PB2-627E+701N,and the Ah1 influenza virus had the strongest pathogenicity to mice.H&E results showed that Ah1 infected mice had the most severe symptoms of interstitial pneumonia,while rAhl-PB2-K627E virus infected mice had the mildest lung tissue symptoms.The results of immunofluorescence technique were consistent with the above results,and it was observed that the antigen was mainly distributed around the bronchi.The miRNA and mRNA expression profiles of mouse lung tissues infected with Ahl influenza virus and its two mutant viruses were detected by miRNA microarray and transcriptome sequencing technology respectively,and comparative analysis was performed.It was found that 220 common differentially expressed miRNAs and 2142 common differentially expressed mRNAs were significantly changed in mice infected with three different viruses.Besides,34,13 and 12 strain-specific differentially expressed miRNAs and 1503,3712 and 3144 specific differentially expressed mRNAs were identified in avian rAhl-PB2-627E influenza virus,mammalian adaptive virus Ahl(627K)and rAhl-PB2-627E+701N infected mice,respectively.mmu-miR-188-5p,mmu-miR-511-5p,mmu-miR-483-5p and mmu-miR-690 play an important role in regulating the infection of rAh1-PB2-627E mutant virus,while mmu-miR-691,mmu-miR-329-3p,mmu-miR-144-3p in rAhl-PB2-627E+D701N virus infected group and mmu-miR-98-5p,mmu-miR-103-3p,mmu-miR-199a-5p and mmu-miR-378a-3p in Ahl infected group also have significant effect.Functional enrichment analysis showed that these miRNAs involved in the regulation of many immune-related signaling pathways.In addition,only mmu-miR-449a-5p showed significant difference in the lung of both avian-derived rAhl-PB2-627E and human-derived Ahl virus infected mice,but the differential expression trend was completely different,and it was significantly up-regulated when infected rAhl-PB2-627E virus but significantly down-regulated after infection with human Ahl virus,its regulatory mechanism needs further research.Therefore,this study revealed the molecular mechanism of avian influenza virus cross-host propagation from the host level by analyzing changes in miRNA and mRNA expression profiles in lung tissues of mice infected with three different influenza virus.For the first time,the miRNA and mRNA expression profiles of mice infected with three different influenza viruses with different virulence(627E,627K or 701N)were analyzed,which laid a foundation for exploring the prevention strategy of influenza virus.