犬蝠源呼肠病毒的分离与鉴定

Isolation and Identification of Reovirus from Short-nosed Fruit Bats (Cynopterus Sphinx)

【目的】从野生蝙蝠体内分离病毒,为开展蝙蝠的病原监测奠定基础。【方法】从广东省韶关采集了30份野生犬蝠样品,研磨后接种Vero E6细胞,从中分离到2株病毒,对其中一株病毒进行研究,使用生物学与分子生物学方法确定其分类学地位。【结果】该毒株在Vero E6细胞上盲传至第4代开始出现细胞病变,表现为细胞内颗粒增多,细胞收缩、变圆,最后细胞从瓶壁脱落。反复冻融3次后,收集细胞及培养液,电镜观察发现,病毒粒子无囊膜,有双层衣壳,呈正二十面体对称,将病毒命名为Bat/China/2003(B/03)。红细胞凝集试验表明该病毒能凝集健康人O型血红细胞,不能凝集SPF鸡、实验用普通级牛、大鼠和豚鼠的红细胞。理化特性试验表明该病毒对氯仿有抵抗力、能耐受pH3.0的酸性环境、50℃水浴1h病毒丧失感染能力、1mol·L-1MgCl2能提高病毒对热的抵抗力。病毒基因组经琼脂糖凝胶电泳分大、中、小3个区段。用哺乳动物呼肠病毒特异性引物进行反转录-聚合酶链式反应(RT-PCR),扩增得到了预期大小的片段。测序结果经NCBI BLAST分析表明,与呼肠病毒科正呼肠病毒属的Ndelle virus核苷酸同源性最高为91.2%。用DNAMAN Multiple Alignment进行序列同源性分析,与哺乳动物呼肠病毒血清1、2、3型的代表株T1L,T2J,T3D的核苷酸同源性分别为89.9%、76.9%、89.9%。【结论】由以上结果推断该病毒为呼肠病毒科的成员。

[ Objective ] We isolated virus from wild bats, it is convenient for pathogen monitoring of bats. [ Method ] Wild short-nosed fruit bats samples were collected from Shaoguan City Guangdong Province. The samples were scrunched and infected Vero E6 cells. Two strains viruses were isolated. Our researches were focused on one of them. We decided the classify of this virus by biological and molecular biological method. [Result] One virus isolated from short-nosed fruit bats hadn＇t caused cytopathic effects （CPE） until fourth-passage on Vero E6 cells. Infected cells emerged granulating, shrinking, rounding and falling off. After thrice freeze-thaw cells and culture medium were harvested for electron microscopy. Virus particles were nonenveloped, double capsids and icosahedral symmetry. The virus was denominated Bat/China/2003 （B/03）. Hemagglutination test indicated this virus could agglutinate healthy human type O red cells, but could not agglutinate SPF chicken, experimental common bovine, rat and guinea pig red cells. This virus was tolerant to chloroform treatment and pH3.0. Virus losed infectivity in water bath 50℃ 1 h. 1M MgCl2 could enhance resistance of virus to heat treatment. Nucleic acid electrophoresed on agarose gels suggested that the genome of this virus was divided into large, medium, small three parts. One pairs of specific primers according to mammalian reovirus were used for Reverse Transcription Polymerase Chain Reaction （RT-PCR）. Appropriate specific products were amplified by RT-PCR. NCBI BLAST analysis indicated that this segment shares the highest identity with Ndelle virus which is a member of the genus Orthoreovirus, and the homology is 91.2 %. DNAMAN Multiple Alignment analysis of nucleotide sequences with mammalianreovirus serotype 1（T1L）, 2（T2J） and 3（T3D）. Homologies were 89.9%, 76.9% and 89.9%, respectively; [Conclusion] So we can deduce this virus is a member of the virus family Reoviridae.