【作者】 杜林峰；

【导师】 涂长春；

【作者基本信息】 吉林大学 ， 预防兽医学， 2008， 硕士

【摘要】 我们对采自我国广东省西江的150只蝙蝠进行了病毒的分离鉴定工作,本研究采用Vero-E6细胞和BHK-21细胞培养的方法进行病毒分离。在150只蝙蝠共30组样品中,从肺脏组织样品中分离出2株病毒,50只蝙蝠共10组肠道组织样品中分离出1株病毒,对其中一株从棕果蝠( Rousettus leschenaulti)肺脏组织中分离出的病毒进行了鉴定工作。该病毒命名为XJ ,XJ株引起的细胞病变迅速,形态学观察,病毒粒子呈圆球形,直径约为75nm,正二十面体对称,无囊膜,有双层衣壳,病毒具有典型的正呼肠孤病毒科成员的特性。随后对该病毒进行了克隆纯化、滴度测定、形态发生学观察、动物实验,动物实验显示XJ株对老鼠有致病性,为进一步明确其分类学地位,我们将XJ株与兔抗NBV、Pulau Virus全病毒和Melaka VirusσC蛋白阳性血清进行间接免疫荧光试验,结果显示病毒和兔抗Pulau Virus全病毒阳性血清有较特异免疫学反应。本研究对阐明我国呼肠孤病毒的病原生态学具有重要的参考价值,同时丰富了我国蝙蝠携带病原种类数据。对开展蝙蝠的病原监测,有效的预防蝙蝠作为病毒宿主可能导致的疾病传播具有重要的意义。更多还原

【Abstract】 The globe spread of SARS has caused devastating loss of human life and economy. The search for the primary reservoir host of SARS-CoV led to the finding of novel coronaviruses among bats in Hong Kong. One of these was detected in Chinese horseshoe bats (Rhinolophus sinicus (R.rouxii)) in Hong Kong which is currently named bat SARS-CoV (bat-SARS-CoV).As a natural reservoir of manifold zoonotic viruses, bats have been involved in several emerging zoonoses in recent years,Such as Hendra virus、Nipah virus which has caused devastating loss of human life and economy。Bats belong to the Order Chiroprera, which is the second large Order in mammalia. At present there are about 1000 recognized species worldwide, making up almost a quarter of all known mammal species. There are 11 species of fruit bats in China, which are Rousettus leschenaulti, Ramplexicaudatus, Pteropus dasymallus, P. lylei, P.vampyrus, P. giganteus, Cynopterus sphinx, C. brachotis,Sphaerias blanfordi, Eonycteris spelaea, E. sobrinus.However, the ecology and behaviour of these bats remain,despite some very patchy preliminary work, largely unknown。It is obvious then that the lives of humans and fruit bats become more and more intertwined, which as a consequence exposes us to manifold zoonotic viruses. The time has come to begin including fruit bats in the surveillance of pathogen-carrying reservoirs.We have isolated 3 strains virus from 150 bats,Which obtained from Guan Dong province,by Vero-E6 cells and BHK-21 cells.A virus from lung of Rousettus leschenaulti ,named XJ Virus (XJV) ,Which is the subject of this study.XJV causing a syncytial cytopathic effect(CPE)in Vero-E6 cells. When examined by negative contrast electron microscopy, XJV in the culture medium of infected cells contained naked isometric particles approximately 75 nm in diameter. The outer capsid displayed ringlike structures, the centres of which were penetrated with stain. Examination of ultrathin sections of syncytia revealed the presence of viral particles within the cytoplasm and associated with cytoplasmic inclusion bodies. XJV replicted and caused syncytial CPE in all type of mammalian cell lines available in laboratory. Two styles of CPE on different cells,Vero-E6 cells and BHK-21 cells represent respectively.The first reovirus of bat origin, NBV, was isolated in 1968 from the heart blood of a flying fox (Pteropus poliocephalus) in New South Wales, Australia. NBV was the first mammalian orthoreovirus to display the fusogenic properties characteristic of avian orthoreoviruses. Pulau virus (PulV), was isolated from Pteropus hypomelanus.Serological and sequence characterization revealed that PulV was closely related to NBV. of Melaka virus (MelV), the third virus in the NBV species group, and its isolation from a human patient with fever and acute respiratory illness. It is not known whether these bat orthoreoviruses are capable of infecting and causing disease in animals or humans. Data indicate that not only was the MelV capable of human-to-human transmission, but also that its origin was probably bats. Furthermore, serological survey data suggest that the related bat-derived PulV is also capable of infecting humans,raising the possibility that PulV might have caused respiratory illness that was undiagnosed or misdiagnosed in the past.The defining characteristics for inclusion within the genus include: (1) a genome comprising 10 segments of dsRNA grouped into large, medium, and small size-classes which encode the correspondingλ,μ, andσ-class viral proteins; (2) a polycistronic S-class genome segment encoding 2–3 distinct gene products; (3) a double-shelled capsid of approximately 85-nm diameter with turret-like structures projecting from the vertices of the core particle; and (4)isolation from vertebrate hosts .XJV isolated from fruit bat, SDS-PAGE show the virus contain 10 segments,When XJV compared to NBV、Pulau Virus、Melaka Virus ,the S segment nucleotide sequences identity S1:60-49.8%;S2:86.1-95.3%;S3:86.3-96.0%;S4:81.3-88.3%f,while the deduced protein sequence encoded by the S2 and S4 gene had sequence identityσA:97.8-99.3%;σB:90.6-95.3%.So we suggest that XJV as the forth virus in the NBV species group.更多还原