| H9N2 subtype avian influenza is a low pathogenic disease caused by H9N2 subtype avian influenza virus,which can infect poultry, wild birds and partial mammals, including humans. It may result in egg production decreased, respiratory symptoms and immunosuppressive diseases in flocks, especially, mixed infection was often found in commercial broilers. Since 1994, it has been popular for 20 years in our country from the frist time this disease discovered in Guangdong province, and now it has become a common disease in chicken farms.What’s more, the continuous immune pressure of these years led to H9N2 viral genomic variation itself, generated many immune evasion phenomena, also it played an important role in the generation of new type AIVs as gene donor. Our research including the following three parts:Firstly, isolation and identification of eight H9N2 subtype AIVs and its whole genome sequences analysisWe collected trachea, lung and other tissues of poultry which had typical symptom of H9N2 disease in Shandong, Hebei and other provinces, and then isolated virus by egg inoculation, identification these viruses by HA-HI test and RT-PCR assay, finally we isolated8 H9N2 subtype AIVs and they were named A/chicken/SD/YT07/2010(abbreviate it was YT07), A/Chicken/SD/SIX/2010(SIX), A/Chicken/SD/S2/2005(S2), A/Chicken/SD//2009(DY), A/Chicken/SD/FX/2013(FX), A/Chicken/SD/HK01/2013(HK01),A/Chicken/SD/905F1/2013(905F1) and A/Chicken/ HB/CZ/2013(CZ).According to the sequences of H9N2 genes from Genebank, 10 pairs of primers were designed for amplification of whole genome sequences of 8 strains. Phylogenetic tree analysis of the eight each genes using MEGA5.05 software, also, the Eurasian classical strains(BJ94,Y280, F98 and G1) and North America strains( Wisconsin66 and Y439) sequences were uesd to compare with this strains at amino acid level.Phylogenetic studies show that: compared to NA, NS, PB1 gene, other genes were more conservative, and show the genetic evolution. NA, NS, PB1 gene of eight strains belong to F98 lineage; HA gene belong to the Eurasian lineage, including BJ94(2 strains), S2-like(6strains). They were belonged to F98 lineage except for S2 in NP and PA genes; in PB2 gene,CZ was F98-like, S2 was BJ94-like and other six strains were belonged G1-like; in M gene,S2 was BJ94-like, the others seven strains were G1-like. All of them indicated that the rearrangement of 8 genes of H9N2 subtype AIVs produced a variety of genotype H9N2 virus.Secondly, research of HA gene variation rate of H9N2 AIVsTo make clear the genetic evolution trend of H9N2 avian influenza virus under immune pressure of vaccine by analyzing molecular characteristic and variation regulation of its HA gene in different years. We collected 183 H9N2 HA gene sequences sequenced by our research group and downloaded from Gen Bank,including 7 sequences of classical strains,analyzed its systematic evolution, molecular characteristic and variant frequency using Lasergen7.1 and Mega5.1 softwares. Fristly, phylogenetic analysis suggested that H9N2 strains could be divided into BJ94, Y280 and S2 lineages at least,and there were major epidemic strains in seperate years. Secondly,amino acid variation had its feature in different lineage and there were accumulation characteristic by amino acid sequences comparison.Thirdly, during 1994~2014, the average evolution rates of nucleotide and variation rate of amino acid were 5.74×10-3and 4.25×10-3, respectively. In 2011~2014, they were 6.35×10-3and 5.32×10-3, it was higher than 2006~2010( 5.024.4 × 10-3 and 3.7 × 10-3) obviously.Furthermore,it was significantly higher than 1998~2005( 0.74×10-3 and 0.50×10-3), which was the initial period of the vaccine promotion. The mismatch of H9N2 vaccine strains and pandemic strains was the most important reason of accelerating virus mutation.Thirdly, exploration the function of H9N2 internal genes in novel H5N2 recombinant viruses as donor geneTo explore the function of H9N2 subtype AIV genes in novel H5N2 recombinant viruses, three H5N2 strains(A/chicken/Shijiazhuang/1209/2013,A/chicken/Chiping/0321/2014 and A/chicken/Laiwu/0313/2014) isolated in our laboratory were sequenced and analysed, the results showed that, NA gene had the nucleotide homologous rate of 98.6%-98.9% with A/chicken/Qianzhou/12/2010(H9N2); the other 5internal genes(PB2, PB1, PA, NP and NS)had the highly homologous with A/chicken/Qianzhou/12/2010(H9N2), A/chicken/ Jiangsu/Q3/2010(H9N2),A/chicken/Shandong/397/2010(H9N2), A/chicken/Zhejiang/611/2011(H9N2) and A/chicken/Qianzhou/12/2010(H9N2), respectively. This study demonstrated that the H9N2 genes played a key role in the recombinant viruses as the donor gene. |
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