犬细小病毒北京分离株的鉴定及其全基因组序列分析  

Isolation,identification and genomic analysis of canine parvovirus-BJ strain

作　　者：于永乐 王吉贵[1] 郗冀 毛亚萍 侯蔷[1] 张晓梅[1] 刘维全[1] 

机构地区：[1]中国农业大学生物学院农业生物技术国家重点实验室,北京100193

出　　处：《中国兽医学报》2017年第6期1023-1029,共7页Chinese Journal of Veterinary Science

基　　金：国家863计划资助项目(2011AA10A213)

摘　　要：首先对疑似犬细小病毒(CPV)感染的病犬粪便进行特异性PCR扩增,经目的片段测序分析,初步证明为CPV感染。将该病犬粪便处理物接种F81细胞,自第3代起,细胞出现典型CPE。对该株病毒进行电镜观察、理化特性试验、HA-HI及基因型等方面鉴定,证明该分离株为CPV,命名为CPV/BJ-L1株。该分离株的血凝效价为1∶512,其血凝性能被特异性抗体抑制;Reed-Muench法测定分离株病毒TCID_(50)为10-5.15/0.1 mL;电镜观察可见25nm左右的病毒颗粒;动物回归试验显示,攻毒犬能复制出CPV的典型临床症状和病理变化;对分离株的全基因组(包含两端完整的发夹结构序列)进行克隆、测序与分析。结果表明,BJ-L1为CPV-2a亚型,与GenBank中的21株CPV全基因序列核苷酸同源性为98.8%~99.8%,且与国内近期分离毒株高度同源。对2006-2014年北京市CPV分离株VP2基因进行序列分析,结果显示,CPV流行株的变异具有某种时间相关性。The feces from clinically suspected dogs were used for PCR specific amplification and ca- nine parvovirus （CPV） infection was initially confirmed by sequencing and analysis of target frag- ment. Then,the faecal samples were treated with antibiotics and inoculated into F81 cells. The ey- topathic effect（CPE） was obvious after 3 blind passages. Finally, the isolate,named as CPV/BJ-L1 was confirmed as CPV with physical and chemical properties experiment, electronic microscope, HA-HI test and gene sequencing. The hemagglutination titer to pig erythrocytes was 1 ： 512,and the haemagglutination could be inhibited by monoclonal antibody specific to CPV. The Reed- Muench method for the determination of isolated virus TCID50 was 10^-5.15/0.1 mL. The virus par- tides with a diameter of 25 nm was observed by transmission electronic microscope. The animal regression test showed that challenged dogs reproduced typical clinical symptoms and pathological changes of CPV infection. The full genome was cloned, sequenced and analyzed. The results showed that CPV/BJ-L1 belonged to CPV-2a subtype and comparative analyses revealed that the CPV/IAJ-L1 had high homology with the genomic sequence of 21 representative strains, ranging from 98.8%-99.8% ,and it was higher homologous with the recent domestic CPV isolates. The an- alyses of the VP2 sequences indicated that the canine parvovirus genetic variation was correlated with time during 2006 to 2008 in Beijing.

关 键 词：犬细小病毒 分离鉴定 全基因组 序列分析 

分 类 号：S852.65[农业科学—基础兽医学]