摘要
犬瘟热是由犬瘟热病毒(CDV)引起的犬科、鼬科和浣熊科等动物的一种急性、高度接触性传染病,对养犬业和毛皮动物养殖业危害严重。为了解当前在我国部分地区流行CDV的遗传特征,本研究从黑龙江、北京和河南3个地区采集245份犬临床样品,利用RT-PCR检测并统计CDV的阳性率,结果显示CDV的阳性率为2.04%(5/245)。将5份阳性样品进行病毒分离,经细胞病变(CPE)观察、电镜(EM)形态学鉴定,以及基于CDV特异性抗体的间接免疫荧光试验(IFA)等确定分离到2株CDV,分别命名为CDV BJ-YE和CDV LY株。采用PCR分段扩增分离株的全基因组序列;采用MEGA 11软件构建2株分离株全基因组、H基因等序列的遗传进化树;采用DNAStar中的Megalign程序分析2株分离株与疫苗株全基因组核苷酸序列的同源性,分析分离株与国内11年间CDV分离株H、F基因序列及其氨基酸序列的同源性,以及国内CDV分离株与疫苗株H、F基因编码氨基酸序列的同源性;采用BioEdit软件分析国内CDV分离株与疫苗株H、F基因氨基酸位点的变化,采用NetNGlyc 1.0 Server在线软件预测分离株的N-糖基化位点。结果显示,本研究分别获得CDV BJ-YE和CDV LY株15690 bp的全长基因组序列;2株分离株的全基因组和H基因均属于Asia-1基因型,2014年~2024年11年间国内主要流行的CDV为Asia-1基因型,2株分离株与疫苗株全基因组核苷酸序列的同源性为92.2%,且H、F基因的核苷酸和氨基酸序列的同源性分别为(H)94.9%~99.5%和95.1%~99.8%,(F)94.7%~99.1%和93.2%~98.6%。本实验分离株、国内CDV分离株、疫苗株的H、F基因编码氨基酸序列的同源性仅为88.2%~90.6%和88.2%~89.7%,国内CDV分离株与疫苗株H基因存在51个变异位点,F基因存在78个变异位点;相比疫苗株的潜在N-糖基化位点,本实验分离株H基因编码的氨基酸序列增加了aa309和aa584两个糖基化位点,F基因编码的氨基酸序列增加了aa1081个糖基化位点。上述结果表明,当前国内流行的CDV与现有疫苗株处于两个不同的分支,亲缘性较远,存在一定的抗原差异。本研究为了解当前我国CDV的流行状况和CDV疫苗株的筛选提供了参考依据。
Canine distemper,caused by the canine distemper virus(CDV),is an acute and highly contagious disease affecting animals in the families of Canidae,Mustelidae,and Procyonidae.It poses a severe threat to dog breeding and fur animal farming industries.To understand the genetic characteristics of CDV currently circulating in certain regions of China,this study collected 245 canine clinical samples from Heilongjiang,Beijing,and Henan Provinces.The positive rate of CDV was detected using RT-PCR,and the results showed a positivity rate of 2.04%(5/245).Five positive samples were subjected to virus isolation,and two strains were identified based on cytopathic effect(CPE)observation,electron microscopy(EM)morphological identification,and indirect immunofluorescence assay(IFA)using CDV-specific antibodies.These two strains were named CDV BJ-YE and CDV LY.The complete genome sequences of the isolated strains were amplified using PCR in segments.MEGA 11 software was used to construct genetic phylogenetic trees based on the full genomes and H genes of the two isolates and 30 reference strains from GenBank.Megalign in DNAStar was employed to analyze the homology of the full genome sequences of the isolates and vaccine strains.MEGA 11 software was used to construct a phylogenetic tree based on the H and F gene sequences of CDV strains in China from 2014 to 2024,and Megalign in DNAStar was used to analyze the homology of the H and F gene sequences between domestic strains over the past 11 years and with the Onderstepoort vaccine strain.BioEdit in DNAStar was used to analyze amino acid changes at the H and F gene loci,and the NetNGlyc 1.0 Server online tool was used to predict N-glycosylation sites.Both isolated strains belonged to the Asia-1 genotype based on their full genomes and H genes.From 2014 to 2024,the predominant CDV strains circulating in China were of the Asia-1 genotype.The results showed that the complete genome sequences of the CDV BJ-YE and CDV LY strains obtained in this study were 15690bp in length.Both isolated strains belonged to the Asia-1 genotype based on their full genomes and H genes.From 2014 to 2024,the predominant CDV strains circulating in China were of the Asia-1 genotype.The nucleotide sequence homology between the two isolated strains and the vaccine strain was 92.2%across the full genome.Both isolated strains belonged to the Asia-1 genotype based on their full genomes and H genes.From 2014 to 2024,the predominant CDV strains circulating in China were of the Asia-1 genotype.The nucleotide sequence homology between the two isolated strains and the vaccine strain was 92.2%across the full genome.The nucleotide and amino acid sequence homologies of the H gene were 94.9%-99.5%and 95.1%-99.8%,respectively,while those of the F gene were 94.7%-99.1%and 93.2%-98.6%,respectively.Compared with the vaccine strain,domestic strains exhibited 51 variation sites in the H gene and 78 variation sites in the F gene.In terms of potential Nglycosylation sites,the isolated strains showed additional sites at aa309 and aa584 in the H gene,and at aa108 in the F gene,compared to the vaccine strain.These findings suggest that the current circulating strain and the existing vaccine strain belong to two different branches,with a distant phylogenetic relationship and certain antigenic differences.This study provides a reference for understanding the current CDV epidemic in some regions of China and for the selection of CDV vaccine strains.
作者
姚荣荣
杜炳辰
姚鑫鑫
王世达
杨德成
王靖飞
YAO Rong-rong;DU Bing-chen;YAO Xin-xin;WANG Shi-da;YANG De-cheng;WANG Jing-fei(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;State Key Laboratory of Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China)
出处
《中国预防兽医学报》
北大核心
2025年第11期1199-1205,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金面上项目(32272980)。
关键词
犬瘟热病毒
H基因
F基因
分离鉴定
遗传进化分析
canine distemper virus
H gene
F gene
isolation and identification
genetic evolution analysis
