关键词： 蝙蝠冠状病毒   S蛋白表达   抗体制备   假病毒感染  

摘要： 2002-2003年暴发的SARS(Severe Acute Respiratory Syndrome)是由一类新型冠状病毒引起的高致死率的呼吸道疾病，在SARS冠状病毒溯源工作中，在蝙蝠体内发现一类与SARS冠状病毒在基因组上有很高相似性的冠状病毒，命名为SARS样冠状病毒(SL-CoV)。随后又在蝙蝠体内发现了多种冠状病毒的存在。本论文对蝙蝠冠状病毒S基因进行研究，内容如下:　　 1，构建了两株分别属于Alphacoronavirus属和Betacoronavirus属的蝙蝠冠状病毒672株和1292株的S蛋白全长真核表达克隆(pCDNA3.1-672S和pCDNA3.1-1292S)及两株蝙蝠冠状病毒RP3株和1292株截短真核表达克隆(pHCMV-RP3S1-his,pHCMV-1292S1-his,pET32a-1292S1)，并以BJ01株SARS-CoV和RP3株SL-CoV为骨架构建了其与BM48-31株RBD区域的嵌合体S蛋白(pCDNA3.1-RP3-BM48S和pCDNA3.1-BJ01-BM48S)　　 2，选取了中华菊头蝠和中华鼠耳蝠两种蝙蝠制备蝙蝠原代细胞，成功制备了来源于肾、肺、心、脑、肠组织的原代细胞共九株，并将其中七株永生化，进行了三种蝙蝠冠状病毒S蛋白的假病毒感染实验，结果显示制备的蝙蝠细胞并不能被所用的假病毒感染。

关键词： Lyssavirus   Rabies virus   Lagos bat virus   Pseudotype   Bat   Africa   Ghana   Antibody   Neutralisation   Serology  

摘要： It is likely that phylogroup 2 lyssaviruses circulate within bat reservoirs. We adapted a pseudotype (pt) neutralisation assay (PNA) to a multiplex format enabling serosurveillance for Lagos bat virus (LBV), Mokola virus (MOKV) and West Caucasian bat virus (WCBV) in a potential reservoir, the African straw-coloured fruit bat, Eidolon helvum. Highly correlated titres were observed between single and multiplex PNAs using ptLBV and ptMOKV (r=0.97, p<0.0001), validating its use for bat serosurveillance. Of the bat serum samples screened 56% neutralised ptLBV, 27% ptMOKV and 1% ptWCBV. Mean VNAb titres were 1:266, 1:35 and 1:7 against ptLBV, ptMOKV and ptWCBV respectively. The high seroprevalence estimates suggest that the infection rate of LBV in E. helvum remains high enough to persist in this species. This supports the hypothesis that LBV is endemic in Ghanaian E. helvum and we speculate that LBV may have co-evolved with African megachiroptera. (C) 2010 Elsevier Inc. All rights reserved.

关键词： LETTERS to the editor   RABIES virus   VAMPIRE bats  

摘要： A letter to the editor is presented about the outbreak of rabies virus RNA in vampire bats in Northeastern Brazil.

关键词： Bat   Cellular immune response   Phytohemagglutinin  

摘要： Bats are known reservoirs for numerous emerging infectious diseases, occupy unique ecological niches, and occur globally except for Antarctica. Given their impact on human and agricultural health, it is critical to understand the mechanisms underlying immunocompetence in this reservoir host. To date, few studies have examined immune function in the Order Chiroptera, particularly among natural colonies of bats. The phytohemagglutinin (PHA) skin test has been widely used to measure delayed-type cellular immune response in a wide variety of vertebrates, and has been routinely employed in immunoecological studies. Although this test is frequently described as a measure of T cell proliferation, recent studies indicate it may represent a combination of immune responses. In mammals, the immune response is differentially, temporally and spatially regulated, therefore, we characterized the infiltrating leukocyte response to the PHA skin test in bats by examining a time-series of histological sections from PHA and saline injection areas in 41 Brazilian free-tailed bats (Tadarida brasiliensis). Results suggest that bats exhibit diverse leukocyte traffic within 6 h, and up to 24 h following subcutaneous PHA injection. There was a significant presence of lymphocytes and neutrophils, as well as eosinophils, basophils, and macrophages observed in the PHA-injected tissues, compared with saline-injected control tissues. We observed a highly significant negative correlation between the number of lymphocytes and neutrophils in PHA-injected tissue, with peak lymphocyte response at 12 h, and peak neutrophil response at 24 h post-injection. These results indicate substantial variation in the immune response of individuals, and may aid our understanding of disease emergence in natural populations of bats.

关键词： Rabies   Vampire bat   Human   Brazil   Ecuador  

摘要： Since 2004, the main transmitter of human rabies in Latin America has been the vampire bat (Desmodus rotundus). Based on the nucleoprotein of the rabies virus (RV), we analyzed antigenic and genetic profiles of isolates from 29 samples taken from humans living in different areas of the Amazon region. Two isolates were from Ecuador and 27 from the Northern and Northeastern regions of Brazil, which were obtained during outbreaks in various municipalities in the states of Para and Maranhao in the years 2004 and 2005. The partial N gene (nt 104-1477) of the 29 isolates was sequenced, and the sequences were used to build a neighbor-joining tree with the Kimura-2 parameter model. All 29 human RV isolates were identified as belonging to antigenic variant 3 (AgV3) and were genetically grouped into the D. rotundus cluster, which was divided into two subclusters (A and B), subcluster A in turn being divided into four genetic groups (Al, A2, A3 and A4). Genetic and molecular markers characterizing these genetic lineages were also identified. The results of this study show that the isolates belong to the same rabies cycle as that of the vampire bat D. rotundus. However, the division of clusters within the lineage associated with D. rotundus shows that different genetic sublineages of the virus were circulating in the Amazon region during the study period. Our findings suggest that there are phylogeographic differences between isolates obtained over a short period. Published by Elsevier B.V.

关键词： Rabies   Bats   Glycoprotein   Nucleoprotein   Genealogy   Molecular epidemiology  

摘要： Insectivorous bats are the main reservoirs of rabies virus (RABV) in various regions of the world. The aims of this study were to (a) establish genealogies for RABV strains from different species of Brazilian insectivorous bats based on the nucleoprotein (N) and glycoprotein (G) genes, (b) investigate specific RABV lineages associated with certain genera of bats and (c) identify molecular markers that can distinguish between these lineages. The genealogic analysis of N and G from 57 RABV strains revealed seven genus-specific clusters related to the insectivorous bats Myotis, Eptesicus, Nyctinomops, Molossus, Tadarida, Histiotus and Lasiurus. Molecular markers in the amino acid sequences were identified which were specific to the seven clusters. These results, which constitute a novel finding for this pathogen, show that there are at least seven independent epidemiological rabies cycles maintained by seven genera of insectivorous bats in Brazil. (C) 2010 Elsevier Inc. All rights reserved.

关键词： Brazil   Dog   RT-LAMP   Rabies   Vampire bat  

摘要： At present, the sporadic occurrence of human rabies in Brazil can be attributed primarily to dog- and vampire bat-related rabies viruses. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was employed as a simultaneous detection method for both rabies field variants within 60 min. Vampire bat-related rabies viruses could be distinguished from dog variants by digesting amplicons of the RT-LAMP reaction using the restriction enzyme Alwl. Amplification and digestion could both be completed within 120 min after RNA extraction. In addition, the RI-LAMP assay also detected rabies virus in isolates from Brazilian frugivorous bats and Ugandan dog, bovine and goat samples. In contrast, there were false negative results from several Brazilian insectivorous bats and all of Chinese dog, pig, and bovine samples using the RI-LAMP assay. This study showed that the RT-LAMP assay is effective for the rapid detection of rabies virus isolates from the primary reservoir in Brazil. Further improvements are necessary so that the RT-LAMP assay can be employed for the universal detection of genetic variants of rabies virus in the field. (C) 2010 Elsevier B.V. All rights reserved.

关键词： Paramyxovirus   Tuhokovirus   Bat   Genome   Novel   Phylogeny  

摘要： Among 489 bats of 11 species in China, three novel paramyxoviruses [Tuhokovirus 1,2 and 3 (ThkPV-1, ThkPV-2 and ThkPV-3)] were discovered in 15 Leschenault's rousettes. Phylogenetically, the three viruses are most closely related to Menangle and Tioman virus. Genome analysis showed that their 3'-leader sequences are unique by possessing GA instead of AG at the 5th and 6th positions. Unlike Menangle and Tioman virus, key amino acids for neuraminidase activity characteristic of rubulavirus attachment proteins are present. The genome of ThkPV-1 represents the largest rubulavirus genome. Unique features between the three viruses include perfect complementary 5'-trailer and 3'-leader sequence and a unique cysteine pair in attachment protein of ThkPV-1, Cat + 1 position in all predicted mRNA sequences of ThkPV-2, and amino acid substitutions in the conserved N-terminal motif of nucleocapsid of ThkPV-3. Analysis of phosphoprotein gene mRNA products confirmed mRNA editing. Antibodies to the viruses were detected in 48-60% of Leschenault's rousettes. (C) 2010 Elsevier Inc. All rights reserved.

关键词： Animals   Bayes Theorem   Chiroptera/classification   Chiroptera/genetics   Chiroptera/virology   Communicable Diseases, Emerging/transmission   Communicable Diseases, Emerging/veterinary   Communicable Diseases, Emerging/virology   Evolution, Molecular   Evolution, Molecular   Genes, Viral   Genes, Viral   Host-Pathogen Interactions   Likelihood Functions   Molecular Sequence Data   Monte Carlo Method   Nucleocapsid Proteins/genetics   Phylogeny   Phylogeny   Rabies/transmission   Rabies/veterinary   Rabies/virology   Rabies virus/classification   Rabies virus/genetics   Rabies virus/pathogenicity   Rabies virus/physiology   Species Specificity  

摘要： For RNA viruses, rapid viral evolution and the biological similarity of closely related host species have been proposed as key determinants of the occurrence and long-term outcome of cross-species transmission. Using a data set of hundreds of rabies viruses sampled from 23 North American bat species, we present a general framework to quantify per capita rates of cross-species transmission and reconstruct historical patterns of viral establishment in new host species using molecular sequence data. These estimates demonstrate diminishing frequencies of both cross-species transmission and host shifts with increasing phylogenetic distance between bat species. Evolutionary constraints on viral host range indicate that host species barriers may trump the intrinsic mutability of RNA viruses in determining the fate of emerging host-virus interactions.

关键词： Fruit bats   Ebola virus   Bats   Antibodies   Animal sociality   Filoviruses   Animal migration   Lyssavirus  

摘要： Ebolaviruses (EBOV) (family Filoviridae) cause viral hemorrhagic fevers in humans and non-human primates when they spill over from their wildlife reservoir hosts with case fatality rates of up to 90%. Fruit bats may act as reservoirs of the Filoviridae. The migratory fruit bat, Eidolon helvum, is common across sub-Saharan Africa and lives in large colonies, often situated in cities. We screened sera from 262 E. helvum using indirect fluorescent tests for antibodies against EBOV subtype Zaire. We detected a seropositive bat from Accra, Ghana, and confirmed this using western blot analysis. The bat was also seropositive for Lagos bat virus, a Lyssavirus, by virus neutralization test. The bat was fitted with a radio transmitter and was last detected in Accra 13 months after release post-sampling, demonstrating long-term survival. Antibodies to filoviruses have not been previously demonstrated in E. helvum. Radio-telemetry data demonstrates long-term survival of an individual bat following exposure to viruses of families that can be highly pathogenic to other mammal species. Because E. helvum typically lives in large urban colonies and is a source of bushmeat in some regions, further studies should determine if this species forms a reservoir for EBOV from which spillover infections into the human population may occur.