Whole Genome Sequencing And Genetic Analysis Of H9 Avian Influenza Virus Isolated In Fujian Provice

Two strains of the Avian Influenza virus(AIV)were islotated from the H9 positive samples of the epidemiology investigation of AIV in Fujian in 2009-2010. Hemagglutination inhibition test(HI)test and sequence analysis was indicated that the two strains were AIV H9 subtype.Based on the 8 reported gene sequences of AIV H9 subtypes in GenBank,using Primer 5.0 software design respectively corresponding reverse transcription Primers and scale-up Primers, which HA,PB1,PB2,PA gene into two segments amplification. Extracting allantoic liquid viral RNA,using RT-PCR,it were succeed to respectively amplificated 2 strains all the genes of AIV H9 subtype.Then connected to the pMD 18-T vertor,transformed to DH5αcompetent cell,extracted plasmids,PCR appraisal and sequence analysis.Results showed that:1,In this research,the two strains of AIV H9 subtype are typical low pathogenic avian influenza virus mode,and all were inclined to combine with person influenza virus receptor.2,The research had successfully amplificated and cloned all gene sequences of two strains of AIV H9 subtype in our province.The HA,NA,M,NS,NP,PB1,PB2,PA genes were 1742bp,1463bp(CK 2 FJ 2010H9 isolates strains is 1454bp), 1027bp,890bp,1565bp,2341bp,2341bp and 2233 bp respectively in length nucle- Otide,and all contained the integrity of the corresponding gene open reading frame,respectively encoding for 560,469(CK 2 FJ 2010H9 isolates strains has 466),350,339,498,757,759 and 716 amino acids.The Cleavage site of HA gene were PSRSSR↓G,which were the low pathogenic avian influenza virus.In the HA peptide chain,CK 1 FJ 2010H9 isolates strains have seven conservative potential glycosyl sites and the CK 2 FJ 2010H9 isolates strains have six potential glycols- yl sites.The amino acids on the binding receptor site of 226 which was decided to host specific were L,so they were inclined to combine with person influenza virus receptor.But CK 2 FJ 2010H9 isolates strains was more inclined to combi- ne with person influenza virus receptor,which had More predisposition on people.In the NA peptide chains,CK 1 FJ 2010H9 isolates strains did not exist neck flaw and had seven conservative potential glycosyl sites,but CK 2 FJ 2010H9 isolates strains only had six potential glycosyl turn site,because there was three amino acids lack on 61 sites,leading to miss a glycosyl sites.3,The whole gene homology and genetic evolution analysis to two isolates strains of AIV H9 subtypes in our province,the results showed that the nucleotide homology of HA,NA,M,NS,NP,PB1,PB2,PA gene of two isolates strains of AIV H9 subtypes was r 94.6%,86.8%,95.6%,94.6%,94.9%,88.5%,95.5%,88.0% espectively,which the nucleotide homology of HA,M,NS,NP,PB2 gene had higher homology.Genetic evolution analysis results,showed that the HA gene of the two isolates strains of AIV H9 subtypes belonged to A/Duck/Hong Kong/ Y280/97,accounting for the H9 subtypes AIV in Fujian in 2010 may originated from the same ancestor.PB2 gene was alone in one branch,not in Eurasian represe- ntative strain subsets of branches,deduced that the gene may originate from unknown wild strains.CK 1 FJ 2010H9 and CK 2 FJ 2010H9 isolates strains were isolated from different regions of our province in 2010,which their geogra- phical relations were far.The whose every eight genes did not have unity in genetic evolution tree branches,explained that the two isolates strains Happened gene rearrangement because of obtain different AIV strain from some gene segments.