2013年广西分离株H9N2亚型禽流感病毒全基因组特性分析

Genome sequencing and analysis of one strain of avian influenza virus subtype H9N2 isolated in Guangxi Province

目的对2013年从广西分离的H9N2亚型禽流感病毒进行全基因测序,并分析其基因组和遗传进化特性,为进一步研究H9N2禽流感病毒的致病机制提供科学依据。方法运用RT-PCR方法克隆广西分离株H9N2亚型禽流感病毒8个基因片段,进行全基因测序,对测序结果进行序列比对和遗传进化分析。结果序列比对分析表明,H9N2亚型分离株的HA序列裂解位点为RSSR↓GLF,受体结合位点为YWTNKLY且发生Q226G和G228R双重位点突变,NA耐药相关位点发生3处突变(E119V,R292W和R294G),M2蛋白跨膜区存在S31N变异;编码内部蛋白的6个基因与同年H7N9亚型分离株相似度为90.9%~97.6%。其分子遗传进化关系属于欧亚种系,其中HA基因属于Y280亚系,M基因属于G1亚系,PB2基因属于Korea亚系,PB1、PA、NA、NP和NS属于SH/F/98亚系。结论2013年广西分离株是由4种不同H9N2亚型禽流感病毒亚系发生自然重排的产物,属于低致病性禽流感病毒,但对抗流感药物存在一定耐药性,故应加强对禽农场中H9N2病毒流行株变异趋势和传播风险的监测。

Objective To provide a theoretical basis for better prevention and treatment of avian influenza virus subtype H9N2 by analyzing the characteristics and phylogenetics of the avian influenza virus genome. Methods A gene fragment of the avian influenza virus that was isolated from Guangxi in 2013 was sequenced using a transcription-polymerase chain reaction（RT-PCR）.The whole genome was sequenced,and characteristics such as receptor binding sites,drug resistance,and sites related to pathogenicity were identified using bioinformatics software.A phylogenetic tree was also constructed. Results Sequencing indicated that the hemagglutinin（HA）cleavage site was RSSR↓GLF.The receptor binding site was YWTNKLY.Q226 Gand G228Rmutations in the HA protein were noted.E119 V,R292W,and R294 G mutations in the NA protein were noted.An S31 Nmutation was noted in the M2 transmembrane domain.Six genes encoding internal proteins were similar to those of another H7N9isolate;the sequence similarity of those genes was 90.9%-97.6%.Phylogenetic analysis revealed that gene fragments of the avian influenza virus in this study belonged to different branches of the Eurasian lineage.For example,the HA gene belonged to the Y280 clade,the M gene belonged to the G1 clade,the PB2 gene belonged to the Korea clade,and the PB1,PA,NA,NP,and NS genes belonged to the SH/F/98 clade. Conclusion The H9N2 subtype is a less pathogenic subtype of the avian influenza virus originating from the natural reassortment of the avian influenza virus from different sublineages,and the H9N2 subtype had a certain level of resistance to anti-flu drugs.Therefore,the monitoring of mutations and the monitoring of the potential spread of the H9N2subtype in poultry farms should be enhanced.