两株H9N2亚型禽流感病毒的遗传演化及其致病性分析  

Phylogenetic analysis and pathogenicity evaluation of two H9N2 subtype avian influenza viruses

作　　者：房敬真 侯玉杰[1] 尹馨 王冬雪 邓国华[1] 崔鹏飞[1] 施建忠[1] 陈化兰[1] FANG Jing-zhen;HOU Yu-jie;YIN Xin;WANG Dong-xue;DENG Guo-hua;CUI Peng-fei;SHI Jian-zhong;CHEN Hua-lan(Influenza Laboratory of the Ministry of Agricultural,State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/农业部动物流感重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2021年第9期913-919,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发项目(2016YFD0500201、2016YFD0500203)。

摘　　要：为了解H9N2亚型禽流感病毒(AIV)的分子特征及其对家禽和哺乳动物小鼠的致病性,本研究对2020年分离到的两株H9N2亚型AIV:A/chicken/Jiangsu/S1625/2020(H9N2)(简称CK/JS/S1625/2020)株和A/duck/Sichuan/S1537/2020(H9N2)(简称DK/SC/S1537/2020)株,进行了基因组测序、序列分析及其对SPF鸡、鸭和BALB/c小鼠的致病力评价。同源性分析结果显示,两株病毒HA和NA基因的同源性分别为93.3%和95.8%,其编码氨基酸序列的同源性分别为95.4%和95.9%,同源性较低。且两株病毒HA蛋白裂解位点分别为^(333)PSRSSR↓GLF^(341)、^(333)PSRSNR↓GLF^(341),均不存在多个连续的碱性氨基酸,符合低致病性禽流感病毒(LPAIV)HA序列特征。两株病毒的HA蛋白均为典型的人源受体结合特征,即^(155)T、^(183)N和^(226)L。遗传演化分析结果显示,两株病毒的HA、NA基因均属于欧亚分支,但二者的亲缘关系较远。鸡感染性试验结果显示,CK/JS/S1625/2020株感染组鸡可通过喉头和泄殖腔向外界环境排毒。虽然在全部鸡的喉头中检测到了DK/SC/S1537/2020株,但仅在部分鸡的泄殖腔检测到了该病毒,且喉头的排毒量明显高于泄殖腔。鸡血清抗体检测结果显示,两株病毒感染鸡血清的HI抗体全部阳转,效价在1:128~1:2048。表明,鸡源H9N2病毒比鸭源H9N2病毒在鸡体内的适应性和复制能力更强;鸭感染性试验结果显示,两株病毒均只能通过鸭的喉头向外界环境排毒,但病毒滴度均较低,且均不能在鸭体内有效复制;小鼠感染性试验结果显示,两株病毒仅能在小鼠的鼻甲中有效复制,而在小鼠的脑、脾脏和肾脏中均未检测到病毒。以上结果表明两株H9N2亚型AIV对家禽和小鼠均呈低致病力。本研究为进一步了解H9N2 AIV提供了一定的参考数据,有必要对自然界中传播的H9N2流感病毒持续监测和进化分析。To investigate the molecular characteristics of H9N2 subtype avian influenza virus(AIV)and its pathogenic risk on birds and mammal mice,two viruses,CK/JS/S1625/2020(H9N2)and DK/SC/S1537/2020(H9N2),were isolated from poultry farms in 2020.The genetic evolution of the two H9N2 viruses was analyzed and the pathogenicity was evaluated in SPF chickens,SPF ducks and BALB/c mice.Homology analysis showed that the homologies of the HA and NA genes between the two viruses were 93.3%and 95.8%respectively,and the corresponding homologies of the encoded amino acid sequences were 95.4%and 95.9%,respectively.HA protein cleavage sites of these two viruses were^(333)PSRSR↓GLF^(341)and^(333)PSRSNR↓GLF^(341),respectively,and there was no multiple consecutive basic amino acids in HA cleavage site motifs of the two viruses,which was identical to the sequence characters of low pathogenic AIV(LPAIV).The HA and NA proteins of the two viruses had 7 and at least 5 potential glycosylation sites respectively.The HA proteins of these two viruses have typical human receptor-binding characteristics,possessing^(155)T,^(183)N and^(226)L.Genetic evolution analysis showed that the HA and NA genes of both viruses belong to the Eurasian branch,although the kinship between the two viruses was far away.Pathogenicity analysis in SPF chickens showed that the animals in CK/JS/S1625/2020-infected group shed viruses from pharynx and cloacae on day 3 post-inoculation.Although viruses were detected in the throats of all DK/SC/S1537/2020-infected chickens,viruses were only detected in the cloacae of some chickens,and the virus titers in pharynx were significantly higher than those in cloacae.All of the chickens inoculated with these two viruses seroconverted with HI titers from 128 to 2048.The replication and virulence test of the two H9N2 viruses in chickens indicate that the adaptability and replication ability of the chicken-origin H9N2 virus were stronger than that of the duck-origin H9N2 virus in chickens.Both viruses do not replicate effectively in

关 键 词：禽流感病毒 H9N2亚型 遗传演化分析 致病性 

分 类 号：S852.65[农业科学—基础兽医学]