40周龄三黄种鸡爆发禽白血病的诊断与病原分析

Diagnosis and pathogen analysis of the avian leukosis outbreak in Three-Yellow Chicken breeders of 40-week-old

为了解鸡群中禽白血病(Avian leukosis,AL)发生的原因以及感染的禽白血病病毒(Avian leukosis virus,ALV)的亚群,研究对发病鸡进行临床诊断、病理剖检和病理组织学观察,并采集肿瘤样病变组织处理后接种DF-1细胞,培养后分别进行禽白血病病毒p27群特异性抗原ELISA检测、亚群特异性PCR检测以及亚群特异性间接免疫荧光法(IFA)鉴定,确定了40周龄三黄种鸡群感染了J亚群禽白血病病毒(ALV-J,分离株命名为GX16YL02);进一步对分离株env基因进行PCR扩增、克隆和序列测定与比较分析。结果表明:分离株GX16YL02的env基因与ALV-J英国原型株HPRS-103的核苷酸及氨基酸的相似性分别为93.9%和91.9%,与其他8株国内外参考株的核苷酸及氨基酸的相似性分别为90.7%~95.5%和89.4%~95.2%,其中gp85基因与其他参考株的核苷酸和氨基酸相似性相对较低,分别为86.9%~94.9%和85.1%~95.4%,整个env基因有义突变和沉默突变的比例分析显示,与gp37基因相比,gp85基因变异较大,尤其是免疫选择压在高变区hr1区域有一定的作用。

To understand the etiology of Avian Leukosis（ AL） and the subgroup of avian leukosis virus in the flocks,in this study,clinical diagnosis,pathologic examination and histopathological observation were performed in diseased chickens. Furthermore,tissue samples with tumor-like lesions were cultured with DF-1 cells and identified by ELISA detection of avian leucosis virus p27 group specific antigen,PCR amplification and indirect immunofluorescence assay（ IFA）. The results confirmed that the 40-week-old Three-Yellow Chickens were infected with subgroup J ALV（ ALV-J） and named as GX16YL02. PCR amplification,cloning and sequencing analysis of env gene of the isolates were further performed and compared. Results revealed that the similarity of nucleotide and amino acid between isolated GX16YL02 env gene and HPRS-103 of the British ALV-J prototype strains were 93. 9% and 91. 9% respectively,and the similarity of nucleotide and amino acid with other 8 strains of domestic and foreign reference strains were 90. 7% ~ 95. 5% and 89. 4% ~ 95. 2%. In addition,the similarity comparison on gp85 gene showed that the isolate had less similarities with the reference strains,which were 86. 9% ~ 94. 9% and 85. 1% ~ 95. 4%,respectively.The proportion of nonsynonymous and synonymous changes in env gene indicated that the mutations in gp85 gene were significantly higher than those in gp37 gene; especially the immune selective pressure which had certain effects on the hyper-variable region of hr1.