犬2型腺病毒的分离鉴定

Isolation and identification of canine adenovirus type 2

研究旨在通过对采集自辽宁省辽阳某犬专业合作社犬传染性肝炎患犬病料进行分离和鉴定,了解犬腺病毒(CAV)的生物学特性。试验通过分子生物学鉴定、病毒分离、抗性测定、电镜观察、病毒特异性鉴定和动物回归试验等对分离毒株的生物学特性及致病力进行研究。结果显示,通过PCR扩增可得到1019 bp的特异性的核酸片段,命名为CAV A22株。通过核苷酸序列分析,分离株与GenBank上登录的国内外已发表CAV参考序列的核苷酸序列同源性最高达100%。由遗传进化树可知,分离株与U77082亲缘关系较近,处于同一分支;该分离株可在MDCK细胞上连续传代,病毒含量可达105.5TCID50/0.1 mL以上;电镜观察可见圆形、无囊膜、直径约80 nm的典型病毒粒子。病毒特异性鉴定表明分离株为犬2型腺病毒(CAV-2)。病毒回归试验表明,分离株致病力较弱,加以驯化可成为一株良好的疫苗毒株。

The objective of the study was to isolate and identify canine infectious hepatitis samples collected from a professional dog cooperative in Liaoyang,Liaoning Province,and to understand the biological characteristics of CAV.The biological characteristics and pathogenicity of the isolated virus were studied by molecular biological identification,virus isolation,resistance detection,electron microscopy,virus specificity identification and animal regression test.The results showed that 1019 bp specific nucleic acid fragment was obtained by PCR amplification and named CAV A22 strain.By nucleotide sequence analysis,the nucleotide sequence homology between the isolates and the published CAV reference sequences listed in GenBank was up to 100%.According to the genetic evolution tree,the isolated strain is closely related to U77082 and in the same branch.The virus content of the isolated strain could reach more than 105.5TCID50/0.1 mL after continuous passage on MDCK cells.The typical round,membraneless virions with a diameter of about 80 nm were observed by electron microscopy.Virus specificity indicated that the isolated strain was CAV-2.The virus regression test showed that the strain was weak in pathogenicity and could be a good vaccine strain after domestication.