犬细小病毒河南株的分离鉴定及全基因组序列分析  

Isolation,Identification and Genomic Analysis of Canine Parvovirus-HN Strain

作　　者：唐磊 张弛 秦亚 孙浩杰 张楠[1] 张百惠 蔡亚南[1] 魏战勇[2] TANG Lei;ZHANG Chi;QIN Ya;SUN Haojie;ZHANG Nan;ZHANG Baihui;CAI Ya’nan;WEI Zhanyong(Jilin Agricultural University,Changchun 130118,China;Henan Agricultural University,Zhengzhou 450002,China)

机构地区：[1]吉林农业大学,吉林长春130118 [2]河南农业大学,河南郑州450002

出　　处：《河南农业科学》2019年第5期130-136,共7页Journal of Henan Agricultural Sciences

基　　金：国家自然科学基金项目(31772722);中国博士后科学基金面上项目(2014M561308)

摘　　要：为了研究河南省当前犬细小病毒(Canine parvovirus,CPV)的流行情况,收集经CPV胶体金试纸检测阳性的病犬血便病料,对其处理后进行特异性PCR扩增,然后将粪便处理物经滤器过滤后接种于F81细胞,进行细胞盲传并观察细胞的病变情况,同时对病毒进行理化性质及血凝试验等检测,最后对病毒的全基因组进行测序。结果表明,该病毒经特异性PCR扩增初步鉴定为CP V,接种于F81细胞盲传2代后,出现明显细胞病变。全基因组测序显示,该病毒分离株基因组全长5 052 bp ,其中包含U型发卡结构188 bp,Y型发卡结构120 bp。此外,其ORF1全长2 007 bp,能够编码非结构蛋白NS1,ORF2全长2 257 bp,能够编码结构蛋白VP1、VP2。经VP2氨基酸序列分析,鉴定其为New CPV-2b亚型,命名为CPV/HN1,该病毒分离株TCID 50 为10 -4.85 /0.1 mL,血凝效价为1∶ 256。与国内的17株参考毒株全基因组序列核苷酸同源性为96.4%~99.9%,其中与北京分离毒株同源性为99.9%。In order to study the current epidemic situation of canine parvovirus (CPV) in Henan province,we collected the stool of dogs which was positive after testing by CPV colloidal gold test paper,and amplified two genes by specific PCR after treatment. Then the fecal treatment content was filtered through a filter and inoculated in F81 cells,and blind transmission of F81 cells was performed to observe whether there was obvious cytopathic effect.Meanwhile,the physical and chemical properties and hemagglutination tests of the virus were carried out,and finally the whole genome of the virus was sequenced.The results showed that the virus was identified as CPV by specific PCR amplification.After being inoculated in F81 cells for two generations of blind transmission,the cytopathic effect appeared.The whole genome sequencing showed that the genome of the virus isolate was 5 052 bp in length,including 188 bp U-type hairpin structure and 120 bp Y-type hairpin structure.ORF1 was 2 007 bp and encoded non-structural protein NS1,ORF2 was 2 257 bp and encoded structural protein VP1 and structural protein VP2.After the VP2 amino acid sequence analysis,we identified it as New CPV-2b subtype and named CPV/HN1.The TCID 50 of this virus isolate strain was 10 -4.85 /0.1 mL and its coagulation titre was 1∶ 256.Besides,it showed 96.4%- 99.9% nucleotide sequence identity with 17 domestic reference virus strains and its homology with the virus strain isolated from Beijing was up to 99.9%.

关 键 词：犬细小病毒 分离鉴定 全基因组测序 序列分析 河南省 

分 类 号：S855.3[农业科学—临床兽医学]