2株云南H9N2亚型禽流感病毒HA和NA基因序列分析

在2019年1月-2019年6月对云南出现呼吸道疫病的57个鸡场进行H9亚型禽流感检测的基础上，选取石林和楚雄2个H9亚型禽流感阳性样品进行病毒分离。从分离的H9N2亚型禽流感病毒感染鸡胚尿囊液中提取总RNA，采用特异性引物经反转录PCR分别扩增HA和NA基因，PCR产物纯化后进行测序。序列比对及系统发育分析结果表明，云南2株H9N2毒株HA基因核苷酸序列同源性为94.2%，NA基因核苷酸序列同源性为93.6%，系统进化分析表明云南H9N2亚型禽流感病毒HA和NA基因均属于欧亚谱系中的类ADKHKY28097分支（Y280-like），ACKYN12019和ACKYN72019 HA基因之间的同源性为94.3%，与参考毒株ACKJX2448的同源性最高，为95.6%~98.5%，与中国流行的H9N2代表株和疫苗株同源性较低。HA蛋白333-340位裂解位点为PSRSSR↓GLF，具有低致病性禽流感病毒分子特征，受体结合位点均发生E198T和Q234L的突变，具有人样受体结合特征，在29、141、298、305、313、492位氨基酸有6个糖基化位点。ACKYN12019和ACKYN72019 NA基因同源性为93.6%，与Y280-like代表毒株的同源性分别为97.1%~97.5%和93.7%~94.6%，NA蛋白缺失63、64、65位氨基酸，在44、69、86、146、200、234位氨基酸处存在6个潜在的糖基化位点，NA蛋白红细胞结合（HB）位点分析发现，368-369、399-403、432位氨基酸处存在变异。研究结果显示，H9N2亚型禽流感病毒一直处于不断的变异之中，故应加强其监测与防控。

Sequence Analysis of HA and NA Genes of Two H9N2 Subtype Avian Influenza Virus in Yunnan

Based on the detection of H9 subtype avian influenza in 57 chicken farms in Yunnan province from January to June 2019,two H9 subtype avian influenza positive samples from Shilin and Chuxiong were selected for virus isolation.Total RNA was extracted from the isolated embryonic allantoic fluid of H9N2 subtype avian influenza virus,and HA and NA genes were amplified by RT-PCR with specific primers.The PCR products were purified and sequenced.Sequence alignment and phylogenetic analysis showed that the nucleotide sequence homology of HA and NA genes for two H9N2 of Yunnan strain in 2019 was 94.2% and 93.6%,respectively.Phylogenetic analysis showed that HA and NA genes of Yunnan H9N2 subtype avian influenza virus in 2019 belonged to ADKHKY28097 branch (Y280-like) of Eurasian lineage.The homology of HA gene of ACKYN12019 and ACKYN72019 was 94.3%,and the highest homology with the reference strain ACKJX2448 was 95.6% to 98.5%,the low homology with the popular H9N2 representative strains and vaccine strains in China.The cleavage site of HA protein 333-340 was PSRSSR↓GLF,which had the molecular characteristics of low pathogenicity avian influenza virus.The receptor binding sites all had mutations of E198T and Q234L,and had the characteristics of human receptor binding.There were 6 glycosylation sites at 29,141,298,305,313 and 492 amino acids.The homology of NA gene of ACKYN12019 and ACKYN72019 was 93.6%,and the homology with the representative strain of Y280-like were 97.1% to 97.5% and 93.7% to 94.6%,respectively.There were 6 potential glycosylation sites at 44,69,86,146,200 and 234 amino acids deleted in NA protein,and 368-369,399-403 were found by analysis of RBC binding sites of NA protein.There were variations in amino acids at position 432.The results showed that H9N2 subtype avian influenza virus had been mutating continuously,so its monitoring and control should be strengthened.