关键词： 流行性乙型脑炎病毒   蝙蝠   RT-PCR   SYBR GreenⅠ实时RT-PCR   细胞培养   基因序列分析  

摘要： 研究背景和目的 流行性乙型脑炎（epidemic encephalitis B）,也称为日本脑炎（Japaneseencephalitis,JE）,简称乙脑,是重要的虫媒病毒传染病,其病原体为乙型脑炎病毒（Japanese encephalitis virus,JEV）,简称乙脑病毒。本病1871年首先由日本报道,1924年首次分离出乙脑病毒,中国于1940年用血清学和病毒分离的方法确诊了乙脑病例。多数国家每2～15年出现1次乙脑流行。世界卫生组织（WHO）估计,目前每年乙脑发病约5万例,其中约1/3的病例死亡,存活者20%～40%留有神经麻痹和心理改变等严重后遗症,曾一度被称为“东方的瘟疫”,给社会和家庭造成了巨大的负担。 一般认为,JEV在库蚊和野生水鸟之间的循环,猪是重要的扩散宿主。乙脑的传播媒介是蚊虫,在世界范围内已报道从30种不同的蚊虫体内分离出乙脑病毒,国内也有20余种,三带喙库蚊是其中对JEV易感性最高的蚊种,JEV通过三带喙库蚊在野生鸟类、人和家畜之间传播。 一般来说虫媒病毒都具有宿主泛嗜性,除了鸟类和猪以外,JEV还可以感染绵羊、家燕、猴子、金黄地鼠、小白鼠并可致病。国内学者曾报道从云南、广西、广东、海南、安徽、贵州的蝙蝠检测到JEV血清抗体。但尚没有分离到JEV或检测到JEV核酸的报道。 湖南省是我国乙脑发病较多的地区之一,湖南省的蝙蝠能否携带JEV?如果携带该病毒,与人类乙脑有无关系?为此我们进行了本调查。 研究方法 1.标本来源与制备 2007年至2008年期间4次收集湖南部分地区的蝙蝠共851只。收集的蝙蝠鉴定种类后无菌解剖取脑组织标本,所取得标本放入1.5ml含RNAlater的冻存管中4℃运输送回实验室进一步处理。脑组织标本加Hanks缓冲溶液用高压消毒过的微量研磨器无菌研磨,研磨液离心后取部分上清进行PCR检测和细胞培养,剩余标本放置-80℃保存。 ***-PCR 以JEV的E蛋白基因区和C/prM蛋白基因区为靶序列设计了一对特异RT-PCR引物和两对通用RT-PCR引物。先用QIAamp Viral RNA Mini Kit（QIAGEN公司）提取脑组织研磨液中的核糖核酸（RNA）,用SUPERSCRIPTⅢ1ST STRAND（invitrogen公司）逆转录酶体系和随机引物进行逆转录（RT）合成cDNA;以逆转录产物cDNA为模板进行聚合酶链反应（PCR）检测所采集蝙蝠标本中的JEV有关基因片段。 *** GreenⅠ实时RT-PCR ①引用已得到证实和公认的JEV基因特异性引物,采用SYBR GreenⅠ实时RT-PCR方法对蝙蝠脑组织进行JEV特异基因片段检测。 ②将SYBR GreenⅠ实时RT-PCR判定为JEV阳性或可疑为阳性的脑组织上清液接种Vero细胞逐日观察细胞病变,并盲传3代,继续观察细胞变化情况。 ③取细胞液或脑组织上清液脑腔接种1～3天龄的BALB/c乳鼠,观察乳鼠发病情况,有可疑发病者,取脑组织继续盲传3代,如出现明显发病者再进行SYBR GreenⅠ实时RT-PCR鉴定。 4.细胞培养 将脑组织研磨液上清用每ml含有100单位青霉素和硫酸链霉素的Hanks缓冲溶液1:10稀释,将已培养好长成单层的Vero细胞弃去原培养基,加入稀释好的接种液置于37℃、5%CO2恒温细胞培养箱内吸附2 h,然后弃去吸附液,加入含2%小牛血清的RPMI-1640培养基（Vero细胞）继续培养。观察细胞变化,根据细胞是否发生病变（***）判断病毒培养阴性或者阳性,若细胞形态有明显变化,如细胞圆缩、颗粒增多、融合成片或碎裂死亡判断为阳性,阳性者做进一步培养分离及分子生物学检测;若没有细胞形态变化做RT-PCR检测是否有病毒增殖,连续盲传三代,若仍未有形态变化且RT-PCR产物未扩增出目的基因片断就判断为阴性。 5.乳鼠接种实验 将检测到的阳性样本离心取上清液,颅内接种乳鼠,每只接种0.03 ml。每天观察2～3次,连续观察1周。接种后48h内死亡的乳鼠弃去,视为非特异死亡。乳鼠发病症状为:拒乳、抽搐、侧卧、弓背、直至死亡。若观察1周后无症状出现,则连续盲传,若盲传三代,仍未出现发病症状,且经RT-PCR检测未扩增出目的基因片断则判断为阴性。 6.测序和基因分析 ①对RT-PCR扩增获得的目的基因进行测序; ②测序结果利用GeneBank的BLAST软件进行在线同源性分析; ③在GeneBank下载其他JEV序列,利用Clustal W进行多序列比对分析; ④用MEGA4.0软件,采用Neighbor-Joining法

关键词： Animals   Antibodies   Viral   Chiroptera   Dengue   Dengue Virus   Enzyme-Linked Immunosorbent Assay   动物   抗体   病毒   翼手目   登革热   登革热病毒   酶联免疫吸附测定   墨西哥   逆转录聚合酶链反应   Artibeus jamaicensis   Dengue Virus   Dengue Fever   Carollia brevicauda   Chiroptera   Reverse Transcriptase Polymerase Chain Reaction   Enzyme-Linked Immunosorbent Assay   Natalus stramineus   Gulf Coastal Plain   Pteronotus parnellii   Mexico  

摘要： Individuals belonging to five families, 12 genera, and 19 different species of bats from dengue endemic areas in the Gulf and Pacific coasts of Mexico were examined by ELISA, RT-PCR, and for the presence of dengue virus (DV) NSI protein. Nine individuals from four species were seropositive by ELISA: three insectivorous, Myotis nigricans (four positives/12 examined), Pteronotus parnellii (3/19), and Natalus stramineus (1/4), and one frugivorous Artibeus jamaicensis (1/35) (12.86% seroprevalence in positive species). DV serotype 2 was detected by RT-PCR in four samples from three species (all from the Gulf coast - rainy season): two frugivorous, A. jamaicensis (2/9). and Carollia brevicauda (1/2), and one insectivorous, M. nigricans (1/11). The latter was simultaneously positive for NSI protein. DV RT-PCR positive animals were all antibody seronegative. M. nigricans showed positive individuals for all three tests. This is the first evidence suggesting the presence of DV in bats from Mexico.

关键词： LETTERS to the editor   NIPAH virus  

摘要： A letter to the editor is presented about Hendra and Nipah viruses in bats in China.

关键词： BATS   DISEASES   VETERINARY virology   VIRUS diseases   IMMUNOGLOBULINS   VIRAL antibodies   MAMMALS  

摘要： The prevalence of neutralizing antibody against West Caucasian bat virus (WCBV) in Miniopterus bats collected in Kenya ranged from 17% to 26%. Seropositive bats were detected in 4 of 5 locations sampled across the country. These findings provide evidence that WCBV, originally isolated in Europe, may emerge in other continents.

关键词： Communicable Diseases  

关键词： Communicable Diseases  

关键词： Lyssavirus   Bats   Rabies   Molecular clock   Evolution  

摘要： The previous study of the evolutionary rates of European bat lyssavirus type 1 (EBLV-1) used a strict molecular clock to estimate substitution rates of the nucleoprotein gene and in turn times of the most recent common ancestor (tMRCA) of the entire genotype and the two major EBLV-1 lineages (EBLV-1 A and EBLV-1B). The results of that study suggested that the evolutionary rate of EBLV-1 was one of the lowest recorded for RNA viruses and that genetic diversity of EBLV-1 arose 500-750 years ago. Here I have shown that the use of a relaxed molecular clock (allowing branch rates to vary within a phylogeny) shows that these previous estimates should be revised. The relaxed clock provides a significantly better fit to all datasets. The substitution rate of EBLV-1B is compatible to that expected given previous estimates for the N gene of rabies virus whilst rate estimations for EBLV-1A appear to be confounded by substantial rate variation within the phylogeny. The relaxed clock substitution rate for EBLV-1 (1.1 X 10(-4)) is higher than had been estimated previously, and closer to that expected for the N gene. Moreover, tMRCA estimates for EBLV-1 are substantially reduced using the relaxed molecular clock (70-300 years) although the differing dynamics of EBLV-1 A and EBLV-1 B confound the confidence in this estimate. Current diversity of both EBLV-1A and EBLV-1B appears to have emerged within the last 100 years. Reconstruction of the population histories suggests that EBLV-1B maybe emerging whilst the signal derived from the EBLV-1A phylogeny may be dampened by clade-specific dynamics. (C) 2008 Elsevier B.V. All rights reserved.

关键词： Immune system   Viral infection   Rabies   Individual heterogeneity   Disease processes and demographics   Bats  

摘要： Many emerging and reemerging viruses, such as rabies, SARS, Marburg, and Ebola have bat populations as disease reservoirs. Understanding the spillover from bats to humans and other animals, and the associated health risks requires an analysis of the disease dynamics in bat populations. Traditional compartmental epizootic models, which are relatively easy to implement and analyze, usually impose unrealistic aggregation assumptions about disease-related structure and depend on parameters that frequently are not measurable in field conditions. We propose a novel combination of computational and adaptive modeling approaches that address the maintenance of emerging diseases in bat colonies through individual (intra-host) models of the response of the host to a viral challenge. The dynamics of the individual models are used to define survival, susceptibility and transmission conditions relevant to epizootics as well as to develop and parametrize models of the disease evolution into uniform and diverse populations. Applications of the proposed approach to modeling the effects of immunological heterogeneity on the dynamics of bat rabies are presented. (C) 2008 Elsevier Ltd. All rights reserved.

关键词： Lyssavirus   Aravan virus   Khujand virus   Irkut virus   West Caucasian bat virus   complete genome  

摘要： The purpose of this study was to generate complete genome sequences of Aravan (ARAV), Khujand (KHUV), Irkut (IRKV) and West Caucasian bat (WCBV) viruses, and to compare them with genomes of other lyssaviruses. We focused on RNA-dependent RNA-polymerase (L) and non-coding regions, because other genes of these viruses have been described previously. The L protein is organized into six conserved blocks (I-VI), previously detected in all Mononegavirales. Furthermore, lyssaviruses have two additional conserved regions, L1 and L2, located in the COOH part of the L L1 may be responsible for methylation of viral mRNA cap structures, whereas the significance of L2 is unclear. Phylogenetic patterns based on the L are similar to those described for the nucleoprotein. The WCBV is the most divergent member of the genus. Besides phylogeny, it has a short trailer region (57 nucleotides versus 69-70 nucleotides in other lyssaviruses) and different intergenic region lengths, including an exceptionally long non-coding region of the glycoprotein (697 nucleotides) containing a potential open reading frame of 180 nucleotides. The absence of a flanking transcription initiation signal, as well as Northern and Western blot data, suggests that this region is not independently transcribed but is a part of G mRNA. (c) Published by Elsevier B.V.

关键词： lyssaviruses   rabies   phylogeny   Lagos bat virus   rabies-related   taxonomy  

摘要： Lagos bat virus (LBV) belongs to genotype 2 of the Lyssavirus genus. The complete nucleoprotein (N), phosphoprotein (P), matrixprotein (M) and glycoprotein (G) genes of 13 LBV isolates were sequenced and phylogenetically compared with other lyssavirus representatives. The results identified three different lineages of LBV. One of these lineages demonstrated sufficient sequence diversity to be considered a new lyssavirus genotype (Dakar bat lyssavirus). The suggested quantitative separation of lyssavirus genotypes using the N, P, M and G genes was also investigated using P-distances matrixes. Results indicated that the current criteria should be revised since overlaps between intergenotypic and intragenotypic variation occur. (c) 2008 Elsevier B.V. All rights reserved.