H1N1亚型猪流感病毒HA蛋白225位氨基酸对病毒致病性的影响  

Amino Acid of 225 in the HA Protein Affects the Pathogenicities of H1N1 Subtype Swine Influenza Viruses

作　　者：杨时鳗 许程志 许榜丰[1] 吴运谱 贾云慧 乔传玲[1] 陈化兰[1] YANG ShiMan;XU ChengZhi;XU BangFeng;WU YunPu;JIA YunHui;QIAO ChuanLing;CHEN HuaLan(Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences/State Key Laboratory of Veterinary Biotechnology,Harbin 150069;Laboratory Animal Center,Liaoning University of Traditional Chinese Medicine,Shenyang 110847)

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,哈尔滨150069 [2]辽宁中医药大学实验动物中心,沈阳110847

出　　处：《中国农业科学》2022年第4期816-824,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金面上项目(31872472)。

摘　　要：【目的】流感病毒的致病性是由多个基因共同决定的,笔者之前的研究结果发现当两株具有相似基因特征的H1N1亚型猪流感病毒进行HA基因替换以后,病毒对小鼠的致病性发生了改变。通过确定影响病毒致病性的关键氨基酸位点,为进一步揭示流感病毒致病力差异奠定了基础。【方法】对ZD71和SY130的HA蛋白氨基酸序列进行比对,确认氨基酸差异位点并设计定点突变引物,构建单点突变的重组病毒并测定其鸡胚半数感染量(EID50)。将拯救的亲本病毒、HA单基因替换重组病毒及单点突变重组病毒以感染复数(MOI)为0.001和0.1的病毒量分别接种于MDCK和A549细胞,测定病毒的体外复制能力;将上述各株病毒以50μL含106EID50的剂量经滴鼻途径接种BALB/c小鼠,分别采集小鼠的脑、鼻甲、肺、脾、肾等脏器,匀浆处理后接种鸡胚分析病毒在小鼠体内各脏器的复制情况;将病毒分别以50μL含101-106 EID50的剂量经鼻腔感染BALB/c小鼠,感染后每天称量小鼠体重,当小鼠体重下降比率超过25%时判定为死亡,统计死亡情况,测定病毒的小鼠半数致死量(MLD_(50)),评估各突变病毒对小鼠的致病性,与亲本病毒进行分析比较后,获知决定病毒致病性的关键氨基酸位点。【结果】ZD71和SY130病毒HA蛋白共存在4个氨基酸差异位点,分别为4、138、144和225位(H3 Numbering)。经过定点突变、病毒拯救及序列测定确认,分别获得含有单个位点突变的重组病毒。通过测定病毒在MDCK和A549细胞上的复制情况,结果发现与亲本病毒rZD71相比较,突变病毒rZD71-HA/G225E在感染MDCK细胞的各检测时间点、及感染A549细胞24—48 h后,复制滴度均显著提高;与rSY130相比较,突变病毒rSY130-HA/E225G在感染MDCK细胞12—36 h后、及感染A549细胞36—72 h后,复制滴度均显著降低。而4、138、144位点的突变对病毒的复制影响较小。进一步的小鼠感染试验发现HA 225位�【Objective】The pathogenicities of influenza viruses are determined by multiple viral genes.The results of our previous study indicated that hemagglutinin(HA)gene substitutions of the two genetically similar H1 N1 swine influenza viruses altered their pathogenicities in mice.This study aimed to further identify the key amino acids affecting viral pathogenicity.【Method】After analyzing the amino acid differences of HA protein between the two H1 N1 viruses,the reassortant viruses bearing the single amino acid mutations were constructed using the site-directed mutagenesis primers,and their EID50 values were determined.To determine the growth of the parental,reassortant and mutant viruses in vitro,MDCK cells and A549 cells were infected with the indicated viruses at a multiplicity of infection(MOI)of 0.001 and 0.1,respectively.The BALB/c mice was further intranasally(i.n.)inoculated with 106 EID50 of each virus,and three mice were euthanized at 3 days post-infection(dpi).The organs,including brain,nasal turbinate,lung,kidney and spleen,were collected from the mice and titrated in eggs to evaluate the viral replication abilities in vivo.The MLD_(50) values of the indicated viruses were determined by inoculating i.n.groups of five mice with 101-106 EID50 of viruses.The body weight was measured daily for 14 dpi,and the mice that lost more than 25%of their original weight were euthanized for humane reasons.【Result】The HA proteins of the ZD71 and SY130 viruses differed at four amino acids at positions 4,138,144,and 225(H3 numbering).Four reassortants were rescued,followed by whole-genome sequencing to ensure the absence of unwanted mutations.The viral replication abilities of the reassortant viruses(rZD71-HA/G225 E and rSY130-HA/E225 G)were significantly affected in MDCK,as well as in A549 cells,when G225 E and E225 G substitutions were introduced into the rZD71 and rSY130 virus,respectively.In contrast,the mutations of the other three amino acids had little effect on viral replication in vitro.Further mouse inf

关 键 词：猪流感病毒 HA蛋白 氨基酸 致病性 

分 类 号：S852.651[农业科学—基础兽医学]