一株樱桃谷鸭源鸭腺病毒B2毒株的分离与鉴定

Isolation and identification of a duck adenovirus B2 strain from Cherry Valley ducks

摘要:

【背景】 2014年以来，鸭腺病毒(duck adenovirus, DAdV) B2感染引起的番鸭“白肝病”在我国番鸭群广泛流行，其他品种肉鸭未见发病，本研究发现两起樱桃谷鸭疑似“白肝病”的病例。【目的】 鉴定樱桃谷鸭“白肝病”的病原。【方法】 对送检鸭的肝脏等内脏样品进行PCR或RT-PCR检测、病原分离鉴定、序列测定及动物回归试验。【结果】 内脏组织PCR检测DAdV B2阳性，并成功分离到一株DAdV B2毒株，命名为DAdV B2/BG19。该分离株BG19感染的鸡肝癌细胞(leghorn male hepatoma cell line, LMH)变大变圆、最后崩解，形成特征性细胞病变。分离株BG19有2个fiber基因，主要功能基因如hexon、fiber 1、fiber 2及DNA polymerase基因与国内DAdV B2分离株核苷酸相似性均达98.26%以上，与DAdV B1代表株GR对应基因的核苷酸相似性分别为77.12%、30.8%、34%和99.78%；所有DAdV B1和DAdV B2分离株的DNA polymerase基因处于同一个进化分支；分离株BG19的ORF67基因从起始密码子往后的第133位碱基为T，造成提早产生终止密码子，导致ORF67基因编码蛋白比国内早前流行代表株CH-GD-12-2014少了14个氨基酸。分离株BG19人工感染2日龄樱桃谷鸭和番鸭的发病率分别为50.0%和87.5%，死亡率分别为12.5%和37.5%，病死鸭可见与自然感染病例相似的临床症状和病理变化。【结论】 从樱桃谷鸭白肝病病例成功分离鉴定一株ORF67蛋白截短表达的DAdV B2强毒株，发现DAdV B2能感染樱桃谷鸭。

Abstract:

[Background] Pale liver disease caused by duck adenovirus B2 (DAdV B2) infection has been widely prevalent in Muscovy duck flocks in China since 2014, and no incidence has been observed in other duck breeds. In this study, two suspected cases of pale liver disease were reported in Cherry Valley ducks. [Objective] To identify the causative agent of pale liver disease in Cherry Valley ducks. [Methods] PCR or RT-PCR and pathogen isolation were carried out with the internal organ samples of diseases ducks. The isolate was identified, sequenced, and used for the challenge in ducklings. [Results] The internal organ samples were DAdV B2 positive, and a strain named DAdV B2/BG19 was successfully isolated. The LMH cells infected with BG19 developed a characteristic cytopathic effect (CPE) with cell enlargement, rounding, and final disintegration. The isolate BG19 carried two fiber genes, and its sequences of hexon, fiber-1, fiber 2, and DNA polymerase gene shared the sequence identity above 98.26% with those of the DAdV B2 isolates submitted in GenBank, whereas these genes had the sequence identity of 77.12%, 30.8%, 34%, and 99.78%, respectively, with those of the DAdV B1 isolate GR. All the strains of DAdV B1 and DAdV B2 shared the same clade in the phylogenetic tree built based on DNA polymerase gene. The mutation G133T in the ORF67 gene of BG19 resulted in a premature stop codon, which led to a truncated ORF67 protein 14 residues shorter than that of previous prevalent strain CH-GD-12-2014. BG19 was pathogenic to 2-day-old Cherry Valley ducklings and Muscovy ducklings, with the morbidities of 50.0% and 87.5% and the mortalities of 12.5% and 37.5%, respectively. The clinical symptoms and pathological changes of dead ducks were similar to those of clinical cases. [Conclusion] A highly virulent strain of DAdV B2 with truncated ORF67 protein expression was successfully isolated and identified from a case of pale liver disease in Cherry Valley ducks. This was the first report about the pathogenicity of DAdV B2 in Cherry Valley ducks.