一株J亚型禽白血病病毒的分离鉴定及其gp85基因的序列分析

Isolation, identification and phylogenetic analysis on gp85 gene of subgroup J avian leukosis virus

为确定安徽巢湖某地方品种养鸡场的疑似禽白血病(avian leukosis,AL)的病原,以DF-1细胞从肿瘤组织中分离病毒,采用RT-PCR方法进行鉴定,并对其感染细胞上清进行透射电镜观察。同时对送检病鸡的心、肝、脾及腺胃等进行组织病理学观察,最后将扩增出的gp85基因与GenBank收录的其他ALV序列进行比对分析。结果表明:PCR扩增产物大小约为928 bp,经测序证实为ALV gp85基因,将该分离株命名为AHCH-2018株。透射电镜观察到具有囊膜的大小约100 nm的病毒粒子。组织病理学结果显示,病鸡的心、肝、脾及腺胃中有大量成灶淋巴细胞增殖。遗传进化分析结果表明,分离株的gp85基因与多株ALV J参考毒株核苷酸序列的同源性为94.1%～99.5%,且与GD1401J株的核苷酸同源性最高,达99.5%,与A、B、C、D、E亚群同源性为46.5%～49.7%。该分离株与J亚群原型毒株处于同一大分支。本研究为了解安徽土鸡种群中禽白血病的分子流行病学特点提供了一定的数据参考。

To identify the avian leukosis virus (ALV) in chicken farm in Chaohu, Anhui Province, the virus was isolated from the tumor tissue by using DF-1 cells. The results were confirmed by RT-PCR and transmission electron microscopy. The diseased chicken organs such as liver, heart, spleen and glandular stomach were analyzed by clinical observation, pathological anatomy and histopathological examination. Finally, the gene of envelope protein gp85 were cloned and sequenced by designing specific primers for gp 85 motif from ALV reference strains in GenBank, and their nucleotide homologies were compared. The results showed that a virus strain, AHCH-2018, was confirmed by sequencing the 928 bp PCR amplification product. The virus about 100 nm in diameter appeared under the transmission electron microscope, which was enveloped and spherical in shape. Histopathological observation showed that larger areas of the pericardial fat, myocardium, spleen, liver and glandular stomach were infiltrated by lymphocytes and lymphoid cells. The gp 85 gene nucleotide sequence analysis showed that the identity of AHCH-2018 strain and other ALV J reference strains ranged from 94.1%-99.5%, amongst which the highest identity (99.5%) was shown with GD1401J strain, when compared with ALV A, B, C, D and E, the nucleotide identity ranged from 46.5%-49.7%. The phylogenetic analysis showed that AHCH-2018 and J subgroup prototype strain were present in the same branch. This study provided a data reference for understanding the molecular epidemiological characteristics of avian leukemia in Anhui Province, China.