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CPV,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-2 b 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%.
引文
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