小鹅瘟病毒常州株的鉴定及其致弱研究

Virulence Identification and Attenuation of Goose Parvovirus Changzhou Strain

从具有小鹅瘟典型症状、病变的病死雏鹅病料中分离到疑似小鹅瘟病毒常州株GPV/JSCZ/2004/01。通过磷钨酸负染经透射电镜观察可见细小病毒样病毒粒子,取死亡胚尿囊液浓缩后与GPV阳性多抗血清作用显示有阳性琼扩线出现,通过GPV特异性引物和相应的PCR试验,扩增出了特异性的DNA条带。研究结果表明分离到小鹅瘟病毒常州株。对常州株第3代毒进行番鸭胚半数致死量(ELD50)测定,为10-3.1/0.2mL。将分离毒GPV/JSCZ/2004/01感染鹅胚成纤维细胞(GEF)并连续传代培养,结果显示该毒株在细胞培养传代到第9代时能用单抗介导的间接免疫荧光试验检测到病毒在GEF的感染,表明产生了细胞适应毒(CZ-ca)。取第14代细胞毒(CZ-ca14)10倍比稀释测定TCID50,同时进行雏鹅致病性试验,结果显示细胞毒的TCID50为10-9.4/0.2mL,对雏鹅的发病率和死亡率为0,但原始分离株的发病率和死亡率为100%。研究结果进一步证明小鹅瘟强毒株通过连续细胞传代培养可以达到致弱效果,为小鹅瘟病毒致弱研究提供了参考。

The suspected goose parvovirus （GPV）Changzhou strain GPV/JSCZ/2004/01 was isolated from the died goslings with typical symptoms and lesions. The virus from the allantoic fluid presented as parvovirus-like virus under the electronic microscope. The AGP was done to verify the isolates. And then, one specific hand was amplified by PCR using the extracted viral genomic DNA as template DNA and with a pair of oligo-primers specific to the parvovirus genome. All results of electronic microscope observation,AGP test,PCR test proved that the isolated virus was GPV. ELD50 of Changzhou strain GPV/JSCZ/2004/01 was 10^-31/0.2 mL. The isolate GPV/JSCZ/2004/01 was infected with goose embryo fibroblasts （GEF）for passage and it was showed that the ninth passage virus was adapted to propagate in GEF and wouhl be detected with monoclonal antibody-mediated indirect immune Fluorescent test. The TCID50 and pathogenisis to goslings of the 14^th passage （：ell adapted virus （CZ-ca14） were tested. The results showed its TCID50 was 10^-9.4/0.2 mL, morbidity and mortality were zero,while the morbidity and mortality of origiral isolate strain,were 100%. This study verified that attenuated GPV would be yielded by infection with GEF for passage.