广西犬源H9N2亚型流感病毒分离株的全基因组测序分析及其对小鼠的致病性实验

Complete genome sequence analysis and mouse pathogenicity of canine H9N2 subtype influenza virus from Guangxi

目的对广西犬只尤其是宠物犬中携带H9N2亚型流感病毒进行全基因组序列特征分析以及小鼠的致病性研究,为流感病毒疫情的防控提供科学依据。方法采用RT-PCR的方法扩增、测序全基因组,DNAStar和MEGA6.0软件分析氨基酸同源性和进化树,通过小鼠感染实验探究其致病性。结果13株H9N2毒株8个基因节段来源于5个不同谱系的毒株,属于新基因型,氨基酸同源性与广西分离株A/equine/Guangxi/3/2011≥99.0%,6个内部基因与2013年自人流感病毒分离株H7N9亲缘关系密切。毒株HA的裂解位点均为RSSR↓GLF,表明低致病力。氨基酸位点HA226、NA119、NP375和PB2的701位等有变化,其中HA226位为亮氨酸L,具有与人SAα2,6-Gal结合的特性。BALB/c小鼠感染健康犬只中分离的H9N2病毒未在体内复制,而源自感冒犬只的毒株Ca/GX/8和Ca/GX/10能够在体内复制,临床症状明显但不致死。病毒经小鼠体内传代后致病力增强,推测是PB2的611位和PA623位氨基酸改变导致。结论从广西各地犬只中获得的13株H9N2亚型流感病毒株经分析发现虽源于禽源,但具备和人源受体特异性结合的能力,且内部基因遗传进化关系复杂,对哺乳动物及人类存在较大威胁,亟需加强防控。

To analyze the whole genome sequence and pathogenicity of H9N2 subtype influenza virus carried by pet dogs in Guangxi,we amplified and sequenced the whole genome by RT-PCR.The amino acid homology and evolutionary tree were analyzed in DNA-Star and MEGA 6.0 software,and pathogenicity was studied through mouse infection experiments.Eight gene segments of 13 H9N2 strains were derived from 5 different lineages,which belonged to the new genotype.The amino acid sequence identity was 99.0%with respect to A/equine/Guangxi/3/2011 isolated from Guangxi.Six internal genes were closely related to the H7N9 strain from human influenza virus in 2013.The HA cleavage site of all strains was RSSR↓GLF,thus indicating low virulence.The amino acid sites HA226,NA119,NP375 and PB2 showed some changes,among which HA226 was changed to leucine L,which binds human SAα2,6-Gal.In BALB/c mice infected with H9N2 virus isolated from healthy dogs,the virus did not replicate in vivo,but the strains Ca/GX/8 and Ca/GX/10 from cold dogs replicated in vivo and caused clear clinical symptoms but not death.The virulence of the virus was enhanced after passage in mice,a finding speculated to be caused by the changes in amino acids at positions PA623 and 611 of PB2.A total of 13 H9N2 subtype influenza virus strains obtained from dogs in Guangxi have avian origin,specifically bind human receptors and have a complex genetic evolution relationship,thus posing a great threat to mammals and humans.Therefore,prevention and control measures must urgently be strengthened.