关键词： ELISA   Yokose virus   Bat   Experimental infection   Anti-bat IgG   Chiroptera  

摘要： To reveal whether bats serve as an amplifying host for Yokose virus (YOKV), we conducted a serological survey and experimentally infected fruit bats with YOKV isolated from microbats in Japan. YOKV belongs to the Entebbe bat virus group of vector unknown group within the genus Flavivirus and family Flaviviridae. To detect antibodies against YOKV, we developed an enzyme-linked immunosorbent assay (ELISA) using biotinylated anti-bat IgG rabbit sera. Serological surveillance was conducted with samples collected in the Philippines and the sera supplied from Malaysia. One of the 36 samples from the Philippines (2.7%) and 5 of the 26 samples from Malaysia (19%) had detectable ELISA antibodies. In the experimental infections, no clinical signs of disease were observed. Moreover, no significant viral genome amplification was detected. These findings revealed that YOKV replicates poorly in the fruit bat, suggesting that fruit bats do not seem to serve as an amplifying host for YOKV. (C) 2008 Elsevier Ltd. All rights reserved.

关键词： 蝙蝠SARS样冠状病毒   S蛋白   免疫显性区   ORF3b蛋白   抗干扰素   功能鉴定  

摘要： SARS-CoV(Severe Acute Respiratory Syndrome Coronavirus)是2002-2003年在我国暴发的SARS疫情的病原。在追踪SARS-CoV溯源的过程中，我们发现了蝙蝠SARS样冠状病毒(SL-CoV)，证明蝙蝠是SL-CoV的自然宿主。序列分析表明人SARS-CoV与蝙蝠SL-CoV具有相似的基因组结构，基因组核苷酸同源性为88-92％，其关键的spike(S)蛋白同源性只有76-78％。关于SARS-CoV免疫原性及其抗干扰素功能的报道已经很多，然而对于蝙蝠SL-CoV这个同源但又不同的病毒，相关研究几乎没有。本论文着重于探讨蝙蝠SL-CoV的S蛋白免疫显形区以及ORF3b蛋白的抗干扰素功能，以期深入了解该病毒的免疫相关特征，为将来设计针对该病毒的特异性诊断方法、疫苗和药物等提供了科学依据。本论文将从以下几方面进行探讨:　　 第一章以综述的方式介绍了SARS-CoV与蝙蝠SL-CoV的一些生物学功能，以及目前SARS-CoV相关的免疫学研究进展。　　 第二章将介绍我们在蝙蝠SL-CoV的S蛋白免疫显性区鉴定方面取得的进展。我们首先利用HIV假病毒系统构建了含有SARS-CoVBJ01株S(HIV/BJ01-S)、蝙蝠SL-CoVRp3株S(HIV/Rp3-S)以及一个SL-CoVRp3株S受体结合相应区（RBD）被BJ01株SRBD区替换的嵌合体假病毒(HIV/CS310-518)。随后我们发现小鼠抗Rp3-S不能中和HIV/BJ01-S和HIV/CS310-518，并且ELISA反应性很弱。而反之，小鼠抗BJ01-S与HIV/Rp3-S反应性很差。据此我们推断Rp3株S受体结合相应区可能是其主要的免疫显性区。随后蝙蝠SL-CoV阳性血清不能与HIV/BJ01-S、HIV/CS310-518反应而只与HIV/Rp3-S反应阳性这一事实也印证了我们的推论。接下来，我们用五个Rp3-S原核截短表达的蛋白与用Rp3S蛋白制得的小鼠多抗和单抗反应，以期找到Rp3S蛋白确切的免疫显性区。实验数据表明，我们找到了两个免疫决定区，其中截短表达的S280-455具有最好的反应性。据此，我们认为Rp3S的氨基酸位点280-455极可能包括主要免疫显性区。　　 第三章中描述了我们在SL-CoV的ORF3b蛋白抗干扰素功能方面的研究进展。在SARS-CoV的研究中，有报道描述了其ORF3b蛋白的抗干扰素活性。于是我们试图对不同株蝙蝠SL-CoV的ORF3b蛋白的抗干扰素活性进行研究。发现SARS-CoVUrbani、SL-CoVRf1、Rp3及Rm1株ORF3b呈现不同的C端截短形式，而且这些截短造成了他们不同的细胞定位。随后利用含干扰素β启动子的报告基因质粒(p125Luc)，我们研究了不同的ORF3b不同的抗干扰素活性。发现在人源细胞中，和UrbaniORF3b蛋白一样，Rm1ORF3b也具有抑制干扰素产生的功能，尽管其细胞定位不同于UrbaniORF3b蛋白。进一步研究证实，Rm1ORF3b是通过抑制IRF3的入核从而实现其抑制干扰素产生的功能。

关键词： Intergenic Region   Rabies   Rabies Virus   Mountain Ridge   Phylogenetic Lineage  

摘要： Background. Vampire bat related rabies harms both livestock industry and public health sector in central Brazil. The geographical distributions of vampire bat-transmitted rabies virus variants are delimited by mountain chains. These findings were elucidated by analyzing a high conserved nucleoprotein gene. This study aims to elucidate the detailed epidemiological characters of vampire bat-transmitted rabies virus by phylogenetic methods based on 619-nt sequence including unconserved G-L intergenic region. Findings. The vampire bat-transmitted rabies virus isolates divided into 8 phylogenetic lineages in the previous nucleoprotein gene analysis were divided into 10 phylogenetic lineages with significant bootstrap values. The distributions of most variants were reconfirmed to be delimited by mountain chains. Furthermore, variants in undulating areas have narrow distributions and are apparently separated by mountain ridges. Conclusions. This study demonstrates that the 619-nt sequence including G-L intergenic region is more useful for a state-level phylogenetic analysis of rabies virus than the partial nucleoprotein gene, and simultaneously that the distribution of vampire bat-transmitted RABV variants tends to be separated not only by mountain chains but also by mountain ridges, thus suggesting that the diversity of vampire bat-transmitted RABV variants was delimited by geographical undulations. © 2010 Itou et al;licensee BioMed Central Ltd.

关键词： 环介导等温核酸扩增   乙脑病毒   蝙蝠   实时荧光定量PCR法   携带状况  

摘要： 研究背景和目的：　　 乙型脑炎病毒(Japanese encephalitis virus，JEV，简称乙脑病毒)是流行性乙型脑炎(简称乙脑)的病原体，属于黄病毒属。乙脑是经蚊虫媒介叮咬传播而引起的一种中枢神经系统感染的急性传染病，是对人畜危害较大的一种自然疫源性疾病，并可造成严重的神经系统后遗症。“蚊-动物-蚊”是乙型脑炎病毒在自然循环的基本环节。乙脑在世界许多国家均有流行的报道，我国是高发区，全国除东北北部、青海、新疆、西藏外均有乙脑流行，局部地区有爆发或流行。　　 目前乙脑病毒的检测主要有病毒分离法、血清学方法和分子生物学检测方法等。病毒分离法是金标准方法，但敏感性较差，且操作步骤繁琐复杂，耗时较长。血清学方法操作相对简便、快速，但血清学方法的不足是不能获得病原体的详细生物学信息。分子生物学检测方法因为其敏感、特异并能得到病原体的基因做进一步分析，因此得到广泛应用。PCR方法有多种，各有利弊，因此须根据实际情况选择。2000年日本学者Notomi等发明了一种新颖的恒温核酸扩增方法，即环介导等温扩增反应(Loop-mediated isothermal amplification，LAMP)，其特点是针对靶基因的6个区域设计4种特异引物，利用链置换DNA聚合酶(Bst DNA polymerase)在等温条件保温数十分钟，即可完成核酸扩增反应。该方法步骤简单，扩增RNA只要在反应体系中加入逆转录酶，就能完全像DNA基因扩增那样，一步实现RNA扩增;灵敏度高，扩增模板可达几个拷贝;快速、高效扩增，整个扩增反应在不到1h即可完成;高特异性，4条引物对靶序列的6个特异序列区的识别，可根据是否扩增来判断目标基因的存在与否：结果鉴定简便，可以采用肉眼观察反应液的颜色变化来迅速判定，也可以通过琼脂糖凝胶电泳，紫外灯下观察有无特异性梯状条带来判别结果。目前很多实验室已经建立LAMP方法检测病原体，有两个实验室也报道了建立逆转录LAMP(RT-LAMP)方法检测乙脑病毒，表明敏感性和特异性均能达到常规RT-PCR水平，但未见与逆转录实时荧光定量PCR(Real-time RT-PCR)比较的报道，而Real-time PCR是目前公认的敏感性最高的PCR方法。　　 蝙蝠是哺乳类中分布最广、数量最多的动物之一，属翼手目，分大蝙蝠亚目和小蝙蝠亚目，全世界约有1107种。除南北极外，蝙蝠在世界各地都有分布，包括在高纬度地区、孤立的岛屿和荒凉的沙漠等，然而大部分种类生活在热带和亚热带地区。迄今为止已在蝙蝠体内分离出80多种病毒，其中一些是人兽共患病病毒，人类一些新病毒传染病的爆发也被证实与蝙蝠有关，如1994年澳大利亚爆发的亨德拉病毒(Hendra virus)感染、1998年马来西亚爆发的尼巴病毒(Nipah virus)感染等。国内外学者也有从蝙蝠体内分离出乙型脑炎病毒的报道，一些种类蝙蝠因栖息地点或觅食等生活习性，如捕食蚊、蝇等，与人类接触密切，因而这些蝙蝠可能在乙脑病毒的保存和扩散中起重要作用。过去两年中，我们已经在我国南方一些省份发现一些种类的蝙蝠可携带乙脑病毒，但研究的内容和范围仍很局限，仍然需要更多的研究和科学证据。　　 为深入研究蝙蝠在传播乙脑病毒中的作用，在本研究中，我们建立了乙脑病毒RT-LAMP检测方法，并与RT-PCR及Real-time RT-PCR检测方法进行了比较，以期为蝙蝠乙脑病毒的快速检测提供一种更为简便的方法。同时，我们继续从海南、广东和湖南省部分地区收集蝙蝠标本，调查蝙蝠携带乙脑病毒的状况。　　 研究方法　　 1.乙脑病毒RT-LA肝检测方法的建立　　 1.1 LAMP引物设计　　 从GeneBank中获得乙脑病毒(strain JaOArS982)E基因区序列，利用在线Primer Explorer version 3.0软件，根据LAMP引物设计的原则，设计RT-LAMP检测方法所用的6条引物，包括内引物(FIP、BIP)，外引物(F3、B3)和环引物(LoopF、Loop B)各1对。　　 1.2乙脑病毒RT-LAMP检测方法反应体系及反应条件　　 根据反应体系优化策略，对Bst DNA聚合酶(8U/μL)、SuperScdptⅢ逆转录酶(200U/μL)、dNTP mix(10mM)、Betaine(5M)、MgCl2(50mM)、6条引物等成分进行优化和调整。　　 依据Tm值，将温度按60℃、61℃、62℃、63℃、64℃、65℃依次递增，从多次重复试验中确定最佳退火温度。　　 1.3乙脑病毒RT-LAMP检测方法特异性测试　　 以登革2型病毒、诺如病毒、EV71病毒、星状病毒、犬五联减毒活疫苗的核酸为阴性对照样品，同时设立提取的乙型脑炎病毒核酸为阳性对照，DEPC-Wat

关键词： Chiroptera   emerging infectious disease   IUCN   population structure   sampling effort   viral richness  

摘要： Historic and contemporary host ecology and evolutionary dynamics have profound impacts on viral diversity, virulence, and associated disease emergence. Bats have been recognized as reservoirs for several emerging viral pathogens, and are unique among mammals in their vagility, potential for long-distance dispersal, and often very large, colonial populations. We investigate the relative influences of host ecology and population genetic structure for predictions of viral richness in relevant reservoir species. We test the hypothesis that host geographic range area, distribution, population genetic structure, migratory behavior, International Union for Conservation of Nature and Natural Resources (IUCN) threat status, body mass, and colony size, are associated with known viral richness in bats. We analyze host traits and viral richness in a generalized linear regression model framework, and include a correction for sampling effort and phylogeny. We find evidence that sampling effort, IUCN status, and population genetic structure correlate with observed viral species richness in bats, and that these associations are independent of phylogeny. This study is an important first step in understanding the mechanisms that promote viral richness in reservoir species, and may aid in predicting the emergence of viral zoonoses from bats.

关键词： European bat lyssavirus 2   United Kingdom   Lyssavirus   first occurrence   Chiroptera   Viral Antigens  

摘要： In August 2007, European bat lyssavirus type 2 (EBLV-2) was isolated from a Daubenton's bat found at Stokesay Castle. In September 2008, another bat from the same vicinity of Stokesay Castle also tested positive for EBLV-2. This is the first occurrence of repeated detection of EBLV-2 from a single site. Here, we report the detection of low levels of viral RNA in various bat organs by qRT-PCR and detection of viral antigen by immunohistochemistry. We also report sequence data from both cases and compare data with those derived from other EBLV-2 isolations in the UK.

关键词： acquired resistance   anticoagulants   common vampire bat   Desmodus rotundus   vampire saliva  

摘要： Ranchers have observed that residual hemorrhage decreases after livestock suffer repeated bites from common vampire bats (Desmodus rotundus). We hypothesized that repeated exposure to the anticoagulants in the bats' saliva generated an immune response. We compared clotting, time of blood mixed with Desmodus saliva between individuals of several species of livestock that were regularly exposed to vampire bats ("batattacked") and individuals living where bat predation was not observed ("bat-naive"). We also compared the bleeding times of domestic sheep following a single defensive bite from a bat, before and after subjecting the animals to a series of feeding bites by Desmodus. Clotting time was significantly shorter in bat-attacked than in bat-naive livestock. Bleeding time after a provoked defensive bat bite was significantly shorter after sheep were exposed to a series of feeding bites from Desmodus. Bat predation induced increased resistance by the prey against the anticoagulants, suggesting an immune response.

关键词： Pregnant Female   Corrected Optical Density   Marburg Virus   Hendra Virus   Rousettus Aegyptiacus  

摘要： Background: Ebola and Marburg viruses cause highly lethal hemorrhagic fevers in humans. Recently, bats of multiple species have been identified as possible natural hosts of Zaire ebolavirus (ZEBOV) in Gabon and Republic of Congo, and also of marburgvirus (MARV) in Gabon and Democratic Republic of Congo. Methods: We tested 2147 bats belonging to at least nine species sampled between 2003 and 2008 in three regions of Gabon and in the Ebola epidemic region of north Congo for IgG antibodies specific for ZEBOV and MARV. Results: Overall, IgG antibodies to ZEBOV and MARV were found in 4% and 1% of bats, respectively. ZEBOV-specific antibodies were found in six bat species (Epomops franqueti, Hypsignathus monstrosus, Myonycteris torquata, Micropteropus pusillus, Mops condylurus and Rousettus aegyptiacus), while MARV-specific antibodies were only found in Rousettus aegyptiacus and Hypsignathus monstrosus. The prevalence of MARV-specific IgG was significantly higher in R. aegyptiacus members captured inside caves than elsewhere. No significant difference in prevalence was found according to age or gender. A higher prevalence of ZEBOV-specific IgG was found in pregnant females than in non pregnant females. Conclusion: These findings confirm that ZEBOV and MARV co-circulate in Gabon, the only country where bats infected by each virus have been found. IgG antibodies to both viruses were detected only in Rousettus aegyptiacus, suggesting that this bat species may be involved in the natural cycle of both Marburg and Ebola viruses. The presence of MARV in Gabon indicates a potential risk for a first human outbreak. Disease surveillance should be enhanced in areas near caves.

关键词： SL-CoV   Spike protein   DNA immunization   Immunodominant region  

摘要： SARS-like coronavirus (SL-CoV) in bats have a similar genomic organization to the human SARS-CoV. Their cognate gene products are highly conserved with the exception of the N-terminal region of the S proteins, which have only 63-64% sequence identity. The N-terminal region of coronavirus S protein is responsible for virus-receptor interaction. In this study, the immunogenicity of the SL-CoV S protein (S-SL) was studied and compared with that of SARS-CoV (S-SARS). DNA immunization in mice with S-SL elicited a high titer of antibodies against HIV-pseudotyped S-SL. The sera had low cross-reactivity, but no neutralization activity, for the HIV-pseudotyped S-SARS. Studies using wild bat sera revealed that it is highly likely that the immunodominant epitopes overlap with the major neutralizing sites of the SL-CoV S protein. These results demonstrated that SL-CoV and SARS-CoV shared only a limited number of immunogenic epitopes in their S proteins and the major neutralization epitopes are substantially different. This work provides useful information for future development of differential serologic diagnosis and vaccines for coronaviruses with different S protein sequences. (C) 2009 Elsevier Inc. All rights reserved.

关键词： Rabies   Lyssavirus   EBLV   ABLV   RFFIT   PCECV  

摘要： Tissue-culture vaccines like purified chick embryo cell vaccine (PCECV) have been shown to provide protection against classical rabies virus (RABV) via pre-exposure or post-exposure prophylaxis. A cross-neutralization study was conducted using a panel of 100 human sera, to determine, to what extent after vaccination with PCECV protection exists against non-classical bat lyssavirus strains like European bat lyssavirus (EBLV) type 1 and 2 and Australian bat lyssavirus (ABLV). Virus neutralizing antibody (VNA) concentrations against the rabies virus variants CVS-11, ABLV, EBLV-1 and EBLV-2 were determined by using a modified rapid fluorescent focus inhibition test. For ABLV and EBLV-2, the comparison to CVS-11 revealed almost identical results (100% adequate VNA concentrations >= 0.5 IU/mL;correlation coefficient r(2) = 0.69 and 0.77, respectively), while for EBLV-1 more scattering was observed (97% adequate VNA concentrations;r(2) = 0.50). In conclusion, vaccination with PCECV produces adequate VNA concentrations against classical RABV as well as non-classical lyssavirus strains ABLV, EBLV-1, and EBLV-2. (C) 2009 Elsevier Ltd. All rights reserved.