一株犬瘟热病毒H基因T193I突变株的分离鉴定及序列分析

Isolation,Identifi cation and Sequence Analysis of a T193I Mutant of Canine Distemper Virus H Gene

为对吉林省地区犬瘟热病毒(CDV)流行发病情况调查,本研究从吉林省长春市、吉林市、四平市的养犬场、毛皮动物养殖场、宠物医院共收集12份经胶体金试纸条检测为CDV阳性的肺部样品,利用Vero/DogSLAM细胞进行病毒分离培养,并对分离的病毒经PCR鉴定、间接免疫荧光试验确定该病的致病病原,并对该病毒分型鉴定和H基因的遗传进化分析,并且选取1株犬源CDV病毒感染6只2~3月龄的幼犬进行动物回归试验,每天观察试验犬的临床症状、测量体温、采集眼鼻、肛拭子,拭子经PCR检测CDV的粪便排毒情况。结果显示,有1株犬源样品在Vero/DogSLAM细胞上盲传第4代出现明显的CPE,毒价达104.56 TCID50/mL,经PCR鉴定结果表明分离出1株犬源CDV,命名MHK-04;病毒的分型鉴定结果表明,分离的MHK-04为Asia-I型;对MHK-04毒株的H基因的同源性分析结果表明,MHK-04株为Asia-I型,与广东分离株GD1818株的同源性最高,与疫苗株(Onderstepoort和CDV3)差异较大,核苷酸同源率均为90.1%,氨基酸同源率分别为90.0%和90.1%;经SWISS-MODEL预测H蛋白三维立体模型结果表明,MHK-04株H蛋白氨基酸序列T193I突变位点,位于H蛋白的外部结构中β6S4位,是其起始位点,推测T193I点突变与H蛋白的构像改变及SLAM受体亲和力有关,有待进一步验证;动物回归试验结果显示,MHK-04毒株在感染幼犬后,幼犬出现犬瘟热发病症状,体温在第4 d升高到41.4℃,并出现排毒现象。本研究为了解吉林地区CDV的流行规律及疾病的诊断、防治提供参考。

In order to investigate the prevalence of canine distemper virus(CDV)in Jilin province,12 CDV positive anal swabs were collected from dog farms,fur animal farms and pet hospitals in Changchun City,Jilin City and Siping City.These swab samples were inoculated onto Vero/dog SLAM cells for virus isolation.The isolated viruses were identified by PCR.The viruses were also tested in indirect immunofluorescence for their genotyping and genetic evolution of H gene.Afterwards,six 2-3-month-old puppies were infected with a canine CDV virus isolate for animal regression analysis.The clinical signs,body temperature,eye,nose and anus swabs were examined every day.The stool excretion of CDV was detected by PCR.The results showed that a canine CDV strain MHK-04 was isolated using Vero/dog SLAM cells and confirmed by PCR.The CDV strain MHK-04 was determined to be Asia-I type and the homology analysis of its H gene also showed that the MHK-04 was strain belonged to Asia-I type with the highest identity to Guangdong isolate GD1818.On the other hand,there were significant differences between the MHK-04 was strain and vaccine strains(Onderstepoort and CDV3)with the nucleotide homology at 90.1%and amino acid at 90.0%.The 3D model of H protein was predicted using software SWISS-MODEL.The amino acid sequence of H protein of the MHK-04 strain had T193I mutation,which was located in the external structure of H proteinβ.Although the mutation of T193I did not cause the glycosylation site of H protein,it was speculated that T193I mutation was related to the conformation change of H protein and the affinity of slam receptor,which needed further verification;The results of animal regression experiment showed that the puppies infection with the MHK-04 strain developed clinical signs of canine distemper virus disease,high body temperature and virus shedding.This study provided a reference for understanding the epidemic situation,disease diagnosis,prevention and treatment of CDV in Jilin area.