Study On Isolation And Identification As Well As Phylogenetic Analysis In H9N2 Subtype Avian Influenza Virus

Avian Influenza is caused by the avian influenza virus, which can infect poultry and cause many diseases, including respiratory tract, enteron, egg production decline or immunosuppression disease. Avian Influenza can be divided into three categories, including highly pathogenic avian influenza,low pathogenic avian influenza and none pathogenic avian influenza. Though H9N2 subtype avian influenza virus is low pathogenic, it is popular in the whole country and can infect people. Due to the personalities of frequent mutation and recombination, H9N2 AIV not only causes huge losses to farming, but also serious threats to human. The most effective means of prevention and control of avian influenza is vaccination. The current domestic common used for the preparation of H9N2 subtype avian influenza vaccine strain were mainly A/Chicken/Shanghai/F/98 and A/Chicken/Shanghai/6/96 and so on. These strains were isolated before 2008, Whose antigenicity were significantly different from those of the popular stains. Thus, the vaccine virus is urgently needed upgrading.In this study, eight H9 AIV isolates were isolatedfrom the live poultry markets in Zhejiang, Anhui and Hunan provinces in the year of 2013.10 fold dilution of these H9N2 avian influenza strains were inoculated with chicken embryonated eggs respectively.Then the highest of the hemagglutination titer from the highest dilution was selected into the next round of purification. After three purification, the highest dilution and hemagglutination titer of allantoic fluid was collected and stored at -80 degree freezer.After the extraction of RNA with allantoic fluid, reverse transcription and PCR were performedin which PB2, PB1, PA, HA, NP genes were amplified by divided in two parts and the rest of the genes arewhole fragements.The purified PCR products were sent to be sequenced in Shanghai Majorbio Co., Ltd.The sequences were matched by the SeqMan software (DNAstar) and homology analysis were performed by MegAlign.Phylogenetic tree was drawed by using Mega6 in order to analyze the genetic evolution of the subtype AIV pandemic strains of H9N2 in different years and regions.The results demonstrated that:the nucleotide homology of HA among the eight strains is 95.5-100% and its amino acid homology is 96.4-99.6%, NA nucleotide homology is 95.3%-100% and its amino acid homology is 93.1%-99.8%, NS nucleotide homology is 94.1%-94.1%, M nucleotide homology of 98.2%-100%, NP nucleotide homology is 95.3%-100%, PA nucleotide homology is 97.2%-100%, PB1 nucleotide homology is 96.1%-100%, PB2 nucleotide homology is 96.1%-100%. Genetic evolution analysis results show that the HA, NA, NS, M, NP, PA, PB1 and PB2 gene of eight strains of H9N2 subtype avian influenza viruses, all belonged to the same sublineagewith high identities..However, the distance between these H9N2 AIVs and vaccine strains were far away and they were not in the same branch.conclusion:The present H9N2 AIV epidemic strains were much more similar with respect to antigenicity, but were far different from those in the past; The acquisitions of H9N2 AIV epidemic strains were a solid foundation for the new vaccine development of H9N2 AIV.