1株犬副流感病毒的分离鉴定及生物学特性研究

试验旨在分离鉴定犬副流感病毒(canine parainfluenza virus,CPIV),并对其生物学特性进行研究。用Vero细胞接种感染CPIV阳性犬肺脏组织,盲传4代,收集72 h病毒液进行RT-PCR鉴定、电镜观察、血凝试验、热敏性试验、紫外照射试验及病毒一步生长曲线的测定,同时扩增N基因进行序列分析,并构建系统进化树。结果显示,试验成功从出现咳嗽、流鼻涕等呼吸系统疾病症状的病犬肺脏中分离出1株CPIV,命名为CPIV-BJ01;RT-PCR扩增结果发现,在534 bp处有特异性目的条带。病毒电镜观察发现,其超微结构呈圆形、有囊膜、直径在80～200 nm之间;血凝试验显示,病毒在4和37℃均能凝集1%猪红细胞,与报道的CPIV血凝特性一致;病毒对热敏感,长时间高温下病毒毒价会随之下降;紫外照射可使病毒在短时间内对细胞的感染性急剧下降。病毒一步生长曲线测定结果显示,在12～48 h病毒高速增殖,细胞培养液中病毒滴度急剧上升,之后趋于稳定。CPIV N基因序列与19株有代表性的副流感病毒N基因相比,其核苷酸序列同源性为97.1%～99.8%。遗传进化分析表明,CPIV-BJ01与PIV5 1168-1(登录号:KC237064.1)和PIV5 ZJQ 221(登录号:KX100034.1)位于同一分支上,亲缘关系较近。

Isolation,Identification and Biological Characterization Analysis of One Strain Canine Parainfluenza Virus

The aim of this study was to isolate and identify the canine parainfluenza virus (CPIV),and analyze its biological characteristics.CPIV positive canine lung tissue was inoculated into Vero cells.CPE were observed after four blind passages,then the cell culture fluid after 72 h was assayed for further RT-PCR identification,electron microscopy,HA and biological characteristic experiments.At the same time,N gene of CPIV was amplified,and phylogenetic tree was constructed.The result showed that one CPIV was successfully isolated and named as CPIV-BJ01.The result of RT-PCR showed that there was a specific target fragment of 534 bp.The electron microscopic observation showed that the virus were circular,enveloped and between 80 to 200 nm in diameter.It could agglutinate 1% porcine erythrocytes at 4 or 37 ℃.The virus was sensitive to high temperature and UV irradiation.The one-step growth curve showed that the virus replicated at a high speed between 12 to 48 h.Compared with 19 representative CPIV N genes,the nucleotide sequence homology analysis was 97.1% to 99.8%.The CPIV-BJ01 strain was closely genetically related to strain PIV5 1168-1 (GenBank accession No.:KC237064.1) and PIV5 ZJQ 221(GenBank accession No.:KX100034.1).