犬细小病毒CPV-YH毒株的分离鉴定及其基因组变异分析

摘要

为分析当前犬细小病毒(canine parvovirus,CPV)基因组遗传变异情况,本研究从细小病毒阳性患犬粪便中分离到一株病毒,经过病毒的形态学观察、血凝试验、动物回归试验以及分子生物学鉴定,分离的病毒确实为犬细小病毒,并命名为CPV-YH.病毒分离结果显示,病料接种A-72细胞能产生典型的细胞病变,病毒能凝集猪红细胞,在电子显微镜下呈圆形或六边形,无囊膜,直径约为20 nm.动物回归试验复制出了犬细小病毒病典型症状:出血性肠炎、肝水肿、肺淤血.全基因组序列测定与分析显示,其基因组全长为4 921 nt,与2011年兰州分离的CPV-LZ2分离株基因组相似性最高,为99％;VP2氨基酸进化树显示,CPV-YH分离株与2013-BJ-P27(中国)分离株和UY306(乌拉圭)分离株在同一个小的分支上.与GenBank上收录的CPV代表株的VP2基因比对,核苷酸和氨基酸相似性分别是98％～99.4％和97.1％～99.5％.以上试验结果表明,成功分离一株CPV变异株,这些数据将为CPV的流行情况和防控提供基础.%To analyze genetic variation of canine parvovirus (CPV),a new virus strain was isolated from the feces of a dog infected by CPV.The virus strain,named as CPV-YH,was identified by red cell agglutination assay,morphology,artificial infection of Beagle dogs and molecular biology.The results showed that the isolate induced specific CPE in A-72 cells,and it could agglutinate porcine red blood cells.The virus were round or hexagonal by the electron microscope,and there was no capsule,with a diameter of about 20 nm.Animal regression test showed that the infected dogs had typical symptoms of CPV disease such as hemorrhagic enteritis,liver edema,pulmonary hyperaemia.The complete genome of CPV-YH was 4 921 nt and it shared the highest nucleotide identity (99％) with CPV-LZ2 isolates isolated from Lanzhou in 2011.The amino acid phylogenetic tree of VP2 showed that CPV-YH isolate,2013-BJ-P27 (China) and UY306 (Uruguay) isolates were on the same small branch.Compared with the CPV strains on GenBank,the homology of VP2 gene nucleotide and amino acid showed 98％-99.4％ and 97.1％-99.5％ identities,respectively.In conclusion,one CPV mutant strain was successfully isolated,and these data would provide valuable theoretical basis for the epidemic situation and prevention and control of CPV.