Optimization And Application The Primers For Identification,Amplification And Sequencing Of AIV And Molecular Basis Of Pathogenicity Of H9N2 AIV In Mammals

Subtype identification is the crucial step to study avian influenza virus, the common method for subtype identification is RT-PCR, appropriate and matching primer determine the sensitivity and the specificity of RT-PCR. Avian influenza virus has the variation due to drift and shift easily, so the primers for RT-PCR for avian influenza virus should be adjusted and updated timely.Because H5, H7, H9, H3, H4 and H6 subtype, as well as N1, N2, N6, N8 and N9 subtype are generally existing in China at present, according to the alignments of the sequences in our laboratory and downloaded from Genbank, universal and identification primers were designed by Primer5 software,including the primer for amplification the internal genes. M13 F and M13 R were selected for sequencing all the eight segments of avian influenza viruses.In order to check the sensitivity and specificity of the new primers, we applied them to identify, amply and sequence the HA positive samples from the surveillance in border provinces. 5 isolates of H5N6 subtype, 2 isolates of H5N1 subtype, 10 isolates of H3N2 subtype and 24 isolates of H9N2 subtype influenza viruses were identified and sequenced. The phylogenic analysis indicated that H5 subtype AIVs in the study were belonged to, clade2.3.4.4 and clade 2.3.2.1c, all of them were bearing–RRRKR-polybasic motif in HA cleavage sites, which is the typical characteristics of HPAIV. The genetic evolution of the H3 and H9 subtype viruses demonstrated that they were in the Eurasian lineages, they were LPAIVs due to lack of possessing polybasic motif in the HA connecting peptides. From the homology comparison, we found that H5N1 from tiger in the study were reassortant by H9N2 donating internal genes,one H9N2 from wild mammalian was also isolated.H9N2 was the internal segments donator for human infection of influenza viruses, such as H5N1 in Hongkong in 1997, H7N9 in 2013, H10N8 in 2014, this subtype avian influenza viruses are circulating widely in poultry in China, playing the crucial role in the generation of novel reassortant infecting mammalians, the molecular mechanism of H9N2 infecting mammals is under determined. We selected Civet/GX/S9/2013(H9N2) to assess the pathogenicity and the adaptation in the mammal model of BALB/C mice, the parent virus could replicate limitedly in the lung and turbinate of mice with low virus titer and was not lethal to mammal model. The virulence were increase by leading to mice dead with virus found in brain and higher titer in the other tissues of infected mice after nine sequential passages in mice. Genomic sequence alignment revealed that there were 3 amino acid substitutions in PB2 protein of the viruses as the ninth mice passages different with the initial of Civet/GX/S9/13(H9N2). With reverse genetic technology,the eligible parent virus was rescued, which maintained the original biological characteristics of wild-type virus. Mutant virus with 3 different amino acid will be generated and continuously evaluated the pathogenicity in mice for further study to explore the mechanism of H9N2 infected the mammalians.