马立克病病毒1型LZ1309株的分离鉴定与生物特性分析

Isolation,identification and biological characterization of LZ1309 strain of Marek's disease virus serotype 1 in vaccinated poultry farm

从免疫马立克病(MD)HVT和CVI988疫苗的甘肃某规模化鸡场采集MD阳性病鸡抗凝全血,分离淋巴细胞,接种于鸡胚成纤维细胞,分离出1株马立克病病毒(MDV)。病毒蚀斑纯化后经PCR和间接免疫荧光鉴定,该毒株为MDV1型毒株,命名为LZ1309株。PCR扩增并测序致瘤基因meq。核苷酸同源性分析结果显示,与国内分离株LS株的同源性最高(99.9%),与CVI988株的同源性最低(98.8%)。氨基酸序列分析显示,氨基酸突变位点符合国内强毒株的特征。为掌握LZ1309株的致病力,进行了LZ1309株攻毒试验。结果显示,LZ1309株攻毒组鸡的死亡率为13.3%,攻毒组鸡体重显著降低,肝、脾、腺胃乳头和心脏均出现肿瘤结节,胸腺和法氏囊严重萎缩。PCR检测攻毒鸡的器官组织,均能扩增到MDV1型强毒株meq基因的特异性条带。本研究成功分离鉴定出1株MDV强毒株,此结果为研究国内MDV毒力演化、致病机理和疫苗研发奠定了重要基础。

A local field isolate of Marek's disease virus(MDV)was isolated from a commercial Hylinebrown chicken flock,which had been vaccinated with HVT and CVI988/Rispens vaccine,by transfecting monolayer of chicken embryo fibroblast with the heparin anticoagulant blood of the bird.After purification by plaque assay and identification by polymerase chain reaction(PCR)and immunofluorescence assay,we isolated MDV1 strain,named as LZ1309.The oncogenicity-related gene meq of the isolate was amplified by PCR,and was sequenced and compared with that of the other reference strains of MDV1.Nucleotide comparisons of meq gene of LZ1309 with those of reference strains showed LZ1309 had high homology with LS strain isolated from China of 99.9%,and CVI988 strain for aminimum of 98.8%.The amino acid mutation of meqgene had the characteristics of virulent MDV strains isolated in China.To study the pathogenicity of LZ1309 strain,LZ1309strain-challenge test was carried out.In result,the mortality of LZ1309-infected group was 13.3%,the body weight of LZ1309-challenged chickens decreased significantly,the liver,spleen,glandular stomach and heart showed the tumor nodules,thymus and bursa of fabricius atrophied severely.PCR was used to detect the DNA prepared from tissues of the challenged birds,and the characteristic patterns of electrophoresis of the meq gene specific bands of virulent MDV1 strain were observed from all the samples detected.The above-mentioned results showed that this study successfully isolated and identified a strain of virulent MDV LZ1309,which might provide an excellent material for researching the virulence evolution of MDV,pathogenic mechanism and vaccine development.