犬腺病毒CAV-BJ02株的分离与鉴定

本研究旨在分离犬腺病毒（CAV）的北京地区流行株，采集北京地区某宠物医院2~4月龄发生咳嗽等呼吸道症状犬的鼻咽拭子，经胶体金和PCR检测，初筛为犬腺病毒阳性。经过处理后，将其处理液接种于MDCK细胞，进行连续传代培养，出现变圆、脱落、葡萄串样等特征性细胞病变。通过形态学观察、PCR鉴定及动物回归试验等方法对其进行鉴定。PCR扩增片段测序结果表明，该分离株为犬腺病毒Ⅱ型，遂将其命名为CAV-BJ02（GenBank：MN744708）株。用Reed-Muench法测得CAV-BJ02株的TCID₅₀为10^6.7·（100 μL）^-1。电镜下可清晰地观察到呈正六边形的二十面体、直径在86 nm左右的犬腺病毒颗粒。遗传进化分析结果表明，本株病毒为CAV-Ⅱ型。动物回归试验结果表明，CAV-BJ02株可引起犬发热等轻微临床症状，以口鼻分泌物为主要排毒方式，排毒期为5~6 d，不导致犬死亡。上述研究结果为深入了解北京地区CAV的流行情况，为犬腺病毒病的诊断、防治及后续相关研究奠定了理论基础。

Isolation and Identification of Canine Adenovirus CAV-BJ02 Strain

This study aimed to isolate an epidemic strain of the canine adenovirus (CAV) from Beijing. Nasopharyngeal swabs were collected from 2 to 4 months dogs with cough and other respiratory symptoms in a pet hospital in Beijing. The sample showing positive with canine adenovirus after initial screening by colloidal gold and PCR, was propagated in MDCK cells after processing. Continuous subculture was carried out, and typical cytopathic lesions such as rounding, shedding, and grape stringing appeared. The sample was identified by morphological observation, PCR identification and animal regression test. The PCR amplification results indicated that the isolate was a canine adenovirus type II, and it was named as CAV-BJ02 (GenBank：MN744708) strain. Virus titers were determined according to the method of Reed and Muench, and TCID₅₀ of CAV-BJ02 isolated was 10^6.7·(100 μL)^-1 in MDCK cells. Canine adenovirus particles with a regular hexagonal icosahedron and a diameter of about 86 nm can be observed clearly under electron microscope. Phylogenetic analysis showed that the sequence of the CAV-BJ02 strain was consistent with that of CAV type Ⅱ. Animal regression test results showed that CAV-BJ02 strain can cause mild clinical symptoms such as fever, with oral and nasal secretions as the main routes for viral shedding, and the viral shedding period is about 5-6 days, which does not cause dogs to die. The results above provided insights into the prevalence of CAV in Beijing and laid a theoretical foundation for the diagnosis, prevention and treatment of canine adenovirus disease.