一株H5N6亚型高致病性禽流感病毒的遗传演化及生物学特性研究  

Genetic evolution and biological characterization of one H5N6 highly pathogenic avian influenza virus

作　　者：赵志国 田井满 李明慧 白晓利 宋兴栋 李雁冰[1] 陈化兰[1] ZHAO Zhi-guo;TIAN Jing-man;LI Ming-hui;BAI Xiao-li;SONG Xing-dong;LI Yan-bing;CHEN Hua-lan(Animal Influenza Key Laboratory of the Ministry of Agriculture and Rural Affairs,State Key Laboratory for Animal Disease Control and Prevention,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动物疫病防控全国重点实验室/农业农村部动物流感重点开放实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2025年第2期117-123,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金创新研究群体科学基金(31521005);国家重点研发计划“病原学与防疫技术体系研究”重点专项“大流行风险病毒的监测、传播规律及预警研究”项目(2023YFC2307500)。

摘　　要：2023年5月本实验室从家禽的监测样品中分离到一株H5N6亚型高致病性禽流感病毒(HPAIV),命名为A/chicken/Jiangsu/S1/2023(H5N6)株,简称CK/JS/S1/2023(H5N6)株。为进一步了解该病毒的生物学特性,本研究对其进行全基因组的分段PCR扩增与测序分析各基因节段编码的关键氨基酸位点及各基因节段的遗传演化分析,采用Simplot软件分析该病毒全基因组的重配事件,采用交叉血凝抑制试验分析该病毒的抗原性。结果显示,该病毒HA蛋白的裂解位点为PLRERRRKR↓GLF,符合HPAIV的分子特征。关键氨基酸位点分析显示HA蛋白受体结合区域出现S^(133)A和T^(156)A增强病毒与人型受体(α2,6)结合能力的突变;NA蛋白颈部出现了aa59~aa69的缺失;PB1蛋白的D622G、M1蛋白的^(I43)M和T^(215)A及NS1蛋白的I106M,这些位点的突变均可以增强病毒对哺乳动物的致病性。遗传演化分析结果显示,该病毒HA基因属于2.3.4.4b亚分支,NA基因及5个内部基因(PB2、PB1、PA、NP、M)均属于欧亚分支,NS基因属于等位基因A分支(Allele A)。基因重配分析结果显示该病毒是由野鸟源的H5N8和家鸭源的H5N6、H4N6和H3N2AIV形成的一株多元重配病毒。抗原性分析结果显示,该病毒与2.3.4.4b亚分支H5-Re14疫苗株抗原性相匹配。将该病毒以10~1EID_(50)/50μL~10~6EID_(50)/50μL剂量经鼻腔感染小鼠,通过小鼠的存活率和各脏器病毒滴度分析该病毒的致病性。结果显示,感染组小鼠均出现精神沉郁、被毛凌乱、体质量下降等症状,感染该病毒后第4 d开始出现死亡。经计算该病毒对小鼠的半数致死量(MLD_(50))为1.5 log_(10)EID_(50),呈高致病性(MLD_(50)<3 log_(10)EID_(50))。该病毒不经提前适应即可在小鼠的多脏器中有效复制,其在肺脏中的病毒滴度最高,达到7.25 log_(10)EID_(50)/m L。本研究通过对H5N6亚型HPAIV的部分生物学特性研究,为2.3.4.4b亚分支H5亚型HPAI的防控提供数据支持与借鉴。In May 2023,one H5N6 highly pathogenic avian influenza virus(HPAIV),named as A/chicken/Jiangsu/S1/2023(H5N6),abbreviated as CK/JS/S1/2023(H5N6),was isolated from poultry.To further understand the biological characteristics of this virus,we conducted whole genome sequencing,phylogenetic analysis,and reassortment analysis using Simplot software.Antigenicity was assessed via hemagglutination inhibition assays.The results indicated that the HA protein of the virus contains a cleavage site at PLRERRRKR↓GLF,consistent with the molecular characteristics of HPAIVs.Analysis of key amino acid sites revealed that mutations S^(133)A and T^(156)A in the receptor-binding domain of the HA protein enhance the virus's ability to bind to humantype receptors(α2,6).Additionally,a deletion of amino acids aa59 to aa69 was identified in the stalk region of the NA protein.Mutations associated with increased pathogenicity in mammals were also observed,including D622G in the PB1 protein,^(I43)M and T^(215)A in the M1 protein,and I^(106)M in the NS1 protein.Phylogenetic analysis revealed that the HA gene belongs to subclade 2.3.4.4b,while the NA gene and five internal genes(PB2,PB1,PA,NP,M)are of the Eurasian lineage,and the NS gene belongs to the allele A branch.Reassortment analysis showed that this virus is a complex reassortant strain,originating from H5N8 in wild birds and H5N6,H4N6,and H3N2 in domestic ducks.Antigenicity analysis indicated that the virus antigenically matches the antisera induced by the H5-Re14 seed virus of the subclade 2.3.4.4b vaccine.To assess the virus's pathogenecity,mice were intranasally infected with doses ranging from 101EID_(50)/50μL to 106EID_(50)/50μL,and the survival rates and viral titers of each organ of the mice were measured.Infected mice displayed clinical symptoms such as lethargy,ruffled fur,and weight loss,with mortality beginning on the fourth day post-infection.Pathogenicity experiments in mice demonstrated that the virus could efficiently replicate in multiple organs without prior adapta

关 键 词：H5N6亚型 高致病性禽流感病毒 分子特征 遗传演化 抗原性 哺乳动物致病性 

分 类 号：S852.65[农业科学—基础兽医学]