用RT-PCR检测海兰褐蛋鸡禽戊型肝炎病毒

Detection of Avian Hepatitis E Virus in Hailan Brown Layer Chickens

为证实我国蛋鸡群中存在禽戊型肝炎病毒（hepatitis E virus,HEV）,从患有大肝大脾病的某海兰褐蛋鸡群采集粪便样品40份、血浆样品10份。提取RNA后,进行禽HEVORF2基因片段的RT-PCR（reverse transcription PCR）检测,阳性样品进行克隆测序。同时采集疑似HEV感染鸡的肝,在两周龄SPF鸡进行了人工接种试验。结果表明,来自海兰褐蛋鸡群的40份粪便和10份血浆样品中,27份粪便样品和4份血浆样品为禽HEV RNA阳性。随机选取两个阳性样品的序列进行分析,其与国内外参考序列相似性为78.1%~98.3%。进化树分析显示与中国、欧洲参考株在同一分支,属于禽HEV基因3型。人工接种试验结果显示,接种后14d的SPF鸡肝中均鉴定出HEV RNA阳性（4/4）。我国蛋鸡群存在基因3型禽HEV。本研究首次发现并报道国内蛋鸡存在禽HEV,这为进一步了解禽HEV在我国鸡群的流行状况及其危害提供了依据。

We aimed to identify the existence of avian hepatitis E virus（HEV）in broiler breeders in China.In the current study,a total of 40 fecal and 10 serum samples were collected from Hailan brown laying hens suffered from the big liver and spleen disease to detect the avian HEV ORF2 gene by reverse transcription PCR（RT-PCR）.The positive PCR products were cloned and sequenced.Artificial inoculation experiment was carried out on two-week-old SPF chickens with the liver of suspected HEV infected chickens.As the result,27 of 40fecal samples and 4of 10 serum samples were detected positive for avian HEV RNA.Sequences analysis revealed that 2 HEV RNA positive samples shared 78.1%-98.3% nucleotide sequence identities to avian HEV strain reported in NCBI.Phylogenetic analysis showed that these avian HEVs related to China and Europe isolates belonged to avian HEV genotype 3.All of four SPF chickens were positive of avian HEV helicase gene RNA at 14 days after intravenous inoculation.These results confirmed the existence of avian HEV infection in laying hens in China,which provide experimental research basis of avian HEV.