鸭坦布苏病毒分离鉴定及其囊膜蛋白遗传进化分析

Isolation,Identification of Duck Tembusu Virus,and Genetic and Evolutionary Analysis of Its Envelope Protein

【目的】了解并掌握鸭坦布苏病毒(Duck Tembusu virus,DTMUV)流行特点及病毒生物学特性,为DTMUV防治提供理论依据和技术支撑。【方法】通过细胞及鸡胚接毒试验对河北某鸭场10只具有典型临床症状的发病鸭进行病毒分离,用RT-PCR、透射电镜观察、Western blotting及间接免疫荧光试验(IFA)等方法鉴定,进行动物回归试验测定病毒毒力并对其进行囊膜蛋白遗传进化分析。【结果】分离到的病毒可在DF-1细胞上稳定增殖产生典型细胞病变效应(CPE)并致死鸡胚;病毒纯化后经电镜观察可见直径30~60 nm的病毒粒子;RT-PCR结果显示,在约270 bp处可见单一条带,与DTMUV预期大小一致;Western blotting结果显示,在60 ku处有特异性条带,与E蛋白大小一致;IFA结果表明,接种病毒的DF-1细胞胞质中可见明亮的特异性荧光,以上结果均表明分离的病毒为DTMUV。将分离到的病毒命名为AX2020株,AX2020株经肌内注射感染北京鸭后感染率高达100%,发病鸭产生神经症状及腹泻等典型临床症状;经序列比对发现AX2020株和GA株(MK907880.1)相似性最高,与SD14毒株(MH748542.1)亲缘关系较远。与商品灭活疫苗毒株HB2010株(MN649262.1)和活疫苗毒株FX2010株(MH414568.1)相比,AX2020株第93、277和487位氨基酸发生了的突变。【结论】成功分离得到1株DTMUV AX2020株,分离毒株对北京鸭具有较强的致病性,AX2020株的囊膜蛋白与国内疫苗毒株相比,已经发生了氨基酸位点的突变,结果为鸭坦布苏病毒病的流行病学及后续疫苗相关研究奠定了一定的基础。

【Objective】The aim of this study was to understand and grasp the epidemiological and biological characteristics of Duck Tembusu virus(DTMUV),in order to provide theoretical basis and technical support for DTMUV prevention and treatment.【Method】The pathogen was isolated from 10 ducks showing typical clinical signs of DTMUV from duck farm in Hebei province by cell and chicken embryo inoculation method,and was identified by RT-PCR,transmission electron microscopy,Western blotting and indirect immunofluorescence assay(IFA).The animal regression test was performed to detect its virulency,and the inheritance of envelop protein was carried out.【Result】The isolated DTMUV strain could well proliferate in DF-1 cells and produce typical cytopathic effect(CPE)and had ability to kill the chicken embryo.After purification,the virus particles with a diameter of 30-60 nm were observed under electron microscopy.RT-PCR results showed that there was a single band at about 270 bp,which was consistent with the expected size of DTMUV;Western blotting results showed that there was a specific band at 60 ku,which was consistent with the size of E protein;The results of IFA showed that after DF-1 cells were inoculated with virus,bright specific fluorescence appeared in the cytoplasm,the above results showed that the isolated virus was DTMUV.The identified virus strain was named as AX2020 strain.After intramuscular injection,the infection rate of AX2020 strain infected Peking duck was as high as 100%,the diseased ducks had typical clinical symptoms such as neurological symptoms and diarrhea.Through sequence alignment,it was found that AX2020 strain had the highest similarity with GA strain(MK907880.1),and was far away from SD14 strain(MH748542.1).Compared with commercial inactivated vaccine strain HB2010(MN649262.1)and live vaccine strain FX2010(MH414568.1),amino acid site mutations had occurred at positions 93,277 and 487 of AX2020 strain.【Conclusion】A DTMUV strain AX2020 was successfully isolated,which showed strong pathogenicity in Peking ducks.Compared with the domestic vaccine strains,the envelope protein of AX2020 strain had mutation at amino acid site,which laid a certain foundation for epidemiological investigations and vaccine-related studies.