大熊猫犬冠状病毒的分离与鉴定

以犬肾传代细胞(MDCK)从1只病死大熊猫肝脏中分离到1株病毒,命名为DXMV。电镜观察磷钨酸负染的病毒细胞培养物，发现冠状病毒样粒子，超薄切片上可见细胞浆内病毒包涵体。理化学和生物学鉴定结果表明，DXMV核酸类型为RNA，对氯仿、乙醚敏感，可耐受1％胰蛋白酶的作用，具有一定的耐酸性和耐热性，可在MDCK、FCWF、F81细胞中增殖，无血凝性和血吸附特性。血清中和试验结果表明，DXMV可被犬冠状病毒阳性血清中和。采用犬冠状病毒间接荧光抗体法染色DXMV细胞飞片，可在细胞浆内见到特异性荧光。以冠状病毒通用引物和犬冠状病毒特异性引物，可从大熊猫肝脏和不同代次的病毒细胞培养物中扩增出与预期值251bp和515bp大小相同的核苷酸片段。由此说明，该病毒为犬冠状病毒，大熊猫可被犬冠状病毒感染。

Isolation and Identification of Canine Coronavirus of Giant panda

With the MDCK cell line,a strain of the virus named DXMV was isolated from the liver of a died giant panda from an animal park in China.The coronavirus like virus particles were observed in the virus culture fluids with negative staining electron microscopy.In the ultrathin sections,the viruses were found to multiply and induce inclusion body forming in the cytoplasm.Physicochemically and biologically,the isolate was sensitive to choroform and ether.resistant to 1% trysin,acid and heat.The viruses could multiply in the FCWF,F81 cell lines,too and could not sorbet and haemagllutinate erythrocytes of human,some animals and chicken.CCV positive serum could neutralized the virus.The specific fluorescent were observed in the section stained with the method of CCV indirect fluorescent antibody.By RT- PCR method with 2 pairs of primers 2 Bp and 4 Bm conserved to 11 coronaviruses and CCVF2 and CCVR2 specific to canine coronavirus,251 bp and 515 bp nucleotide segments were amplified respectively from the giant panda liver and virus culture.The results indicated that the isolate classified into canine coronavirus and disclosed that the giant panda was a new animal infected by the CCV.