| Avian influenza virus(AIV) is a negative-sense, segmented, single-stranded RNA virus which belongs to the Orthomyxoviridae family.There were many new subtypes of avian influenza virus coming out in recent year. H5N1、H6N1、H7N2、H7N3、H7N7、H7N9、H9N2、H10N7 and H10N8 all can infect humans. Avian influenza virus is prevalent in the whole world, especially in Asia which causes great economic losses to poultry industry and also has critical threat to public health. Avian A(H10N8) virus was isolated as early as 1965, among quails in Italy.In China,it has been identified in water samples from Dongting Lake in 2007 and in a duck from Guangdong in 2012. Human infection with a novel influenza A(H10N8) virus was first described in China in December 2013.However,the origin and genetic diversity of this virus is still poorly understood.We performed a phylogenetic analysis and coalescent analysis of two viruses from the first case of influenza A(H10N8)(A/Jiangxi-Donghu/346-1 and A/Jiangxi-Donghu/346-2) and a novel A(H10N8) virus( A/chicken/Jiangxi/102/2013) isolated from a live poultry market that the patient had visited. A comparison of sequences showed that the H10 and N8 gene segments underwent reassortment in wild birds or most likely in ducks,because viruses possessing such H10 and N8 have been isolated from ducks, respectively. This gave rise to a hypothetical A(H10N8) influenza virus in ducks, which continued to reassort with chicken A(H9N2) influenza virus and obtained the A(H9N2) internal genes though a series of reassortment events. The haemagglutinin(HA),neuraminidase(NA), PA subunit of the virus polymerase complex,nucleoprotein(NP),M and nonstructural protein(NS) genes of the three virus strains shared the same genetic origins. And the PB2,PB1,PA,NP,M and NS genes were similar to those of chicken influenza A(H9N2) virus. NA has been classified into two groups, group 1 and group 2,based on primary sequences. N1,N4,N5 and N8 are group1 members and N2,N3,N6,N7 and N9 belong to group2.It is clearly shown that N8 in this article belongs to group 1, whose mechanism of resistance to oseltamivir is much more frequently associated with H275 Y substitution. The report of 292 K in group 1 NA is extremely rare. The oseltamivir-resistance mutation, R292K(R291K in A(H10N8) numbering) in the NA protein, occurred after four days of oseltamivir treatment.Reverse genetics is deal with genes compared with classical genetics. It can determine the impact on phenotype and character of organism. Reverse genetics was commonly called the rescue of virus, It was been established by infectious clone of RNA viruses. We obtained the c DNA by RT-PCR first, and the c DNA was inserted in p HW2000 by restriction enzymes and T4 DNA ligase. Then genome c DNA clonings were transinfected into 293 T cells. In this study, we rescued recombined viruses by reverse genetic, after 5 passages in SPF chicken embryo eggs, and all the recombined viruses could be continuously cultured. The three recombined viruses can continue to be used for follow-up studies in our laboratory. |
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