狂犬病病毒浙江街毒株糖蛋白基因序列分析

目的 测定狂犬病病毒浙江株(鼬獾源和犬源)糖蛋白(GP)基因组全序列,从分子水平比较狂犬病病毒浙江株与其他地区代表性街毒株和疫苗株GP之间的差异.方法 乳鼠接种分离狂犬病病毒,RT-PCR反应测定狂犬病病毒浙江株GP基因核苷酸序列,并进行序列和编码蛋白的比较分析.结果测序获得浙江5株鼬獾源狂犬病病毒和9株犬源狂犬病病毒GP基因序列,全长1575个核苷酸,编码524个氨基酸.浙江病毒株与其他地区街毒株、疫苗株核苷酸和氨基酸序列相似性在82.3%～99.9%和85.1%～99.8%之间,种系分析显示浙汀株均为基因1型,GP编码蛋白无重组,主要抗原位点未出现较大的变异.结论 对GP基因一级结构综合分析表明,在某些区域存在优势抗原表位的可能性较大,可能出现潜在的蛋白质抗原决定簇,浙江株GP基因变异较小,与国内其他地区流行的代表性街毒株相似,但高于其他疫苗株,在基因结构及种系进化关系上存在一定的距离.     

浙江省不同宿主来源狂犬病病毒N基因分子特征分析

目的 测定浙江省不同宿主（人、鼬獾、犬）来源的狂犬病病毒街毒株N基因序列, 分析病毒遗传变异特征及其与流行的关系。方法 采用直接免疫荧光试验和反转录聚合酶链式反应检测狂犬病病毒阳性标本N基因核苷酸序列, 利用生物信息学软件分析基因序列和编码蛋白。结果 共获得浙江省2 个人源、5 个鼬獾源和11 个犬源狂犬病病毒街毒株N基因核苷酸序列, 18 个核苷酸和氨基酸序列同源性在89.7%～100.0%和98.4%～100.0%, N蛋白一级结构上绝大部分为稳定区域, 编码基因的核苷酸变异多为无义突变, 系统发育分析显示18 个街毒株均属于传统的基因1 型。结论 浙江省不同宿主来源狂犬病病毒的流行具有地域性特征, 同类宿主动物病毒株或来自同一县域/相邻县域的毒株在地理位置上最为近缘, 但人源株病毒更具有复杂性。浙江省狂犬病病毒街毒株的流行具有通过犬向鼬獾和人传播, 并在犬、鼬獾中跨区域循环传播的特点。     

湖南省武岗市洞口县狂犬病流行病学研究

目的分析2003-2004年间在湖南省武岗市与洞口县发生的狂犬病并探讨局部地区流行的因素.方法对狂犬病进行个案调查,用直接免疫荧光法检测犬脑组织中的狂犬病毒抗原,用RT-PCR法扩增N基因片段,测定核苷酸序列并构建系统发生树进行遗传特征分析.结果自1991-2002年武岗市与洞口县仅各报告1例人狂犬病,但2003-2004年间,分别报告30例人狂犬病.62例狂犬病患者中61例被犬所伤,1例被猫所伤.在有完整记录的50例患者中,平均潜伏期为44.18天.其中7例（14%）狂犬病患者的潜伏期≤15天,16例（32%）潜伏期≤20天.在疫点周围采集的99只犬脑组织中,13只犬脑组织狂犬病毒抗原阳性,阳性率为13.13%.将Wg13与Dk13株病毒的部分N基因核苷酸序列与已报道中国病毒株比较,发现2株病毒与广西及安徽的毒株有最高的同源性,构建的系统发生树也分在同一分支.用全N基因核苷酸序列分析发现,Wg13与Dk13毒株全为Ⅰ型狂犬病毒,2株病毒之间的同源性为99.4%以上.比较分析2株病毒N基因编码的氨基酸序列,发现包括第Ⅳ抗原位点区在内的多个氨基酸位点发生了氨基酸的替代.结论引起武岗市与洞口县的狂犬病流行的病毒不是新型狂犬病毒,犬饲养量大与病毒携带率高是狂犬病暴发的直接原因.     

河南省两株狂犬病毒核蛋白和糖蛋白基因的序列分析

目的分析河南省2株狂犬病毒核蛋白及糖蛋白的基因序列,了解狂犬病毒街毒株与人用及兽用狂犬病疫苗株间的差异。方法以免疫荧光法检测2004年采自河南省的100只犬脑标本,取阳性犬脑组织悬液接种鼠脑分离病毒。以RT-PCR法扩增病毒核蛋白及糖蛋白全基因,克隆测序后进行遗传学分析。结果分离到2株狂犬病毒,分别命名为Henan Hb1与Henan Sq1,序列分析表明2株病毒均为基因1型狂犬病毒,2株病毒的N基因与G基因核苷酸的同源性分别为99.3%和98.9%,氨基酸的同源性分别为98.7%和98.4%。2株病毒与CTN疫苗株同源性较高,N基因与G基因的核苷酸同源性分别为89.1%和85.6%～85.7%,氨基酸同源性分别为97.6%～98.0%和92.3%。与已知的基因1型狂犬病毒相比,2株病毒与印度尼西亚从犬中分离到的2株病毒同源性最高,N基因与G基因核苷酸同源性分别为92.1%～93.2%和91.9%～92.1%,氨基酸同源性分别为97.5%～98.6%和96.0%～96.2%。Henan Sq1株病毒在糖蛋白关键的毒力位点333位由W替换了R。系统发育分析表明Henan Hb1与Henan Sq1与印度尼西亚株、疫苗株CTN、中国分离株,以及泰国与马来西亚分离株进化关系最近,而与疫苗株3aG、PV、ERA及攻击毒CVS株等进化关系较远。结论2株狂犬病毒是基因1型狂犬病毒,但无论是N基因还是G基因的核苷酸序列,以及推导出来的氨基酸序列与已知的1型狂犬病毒株及疫苗株均有一定的差异。     

广西16株狂犬病毒P基因序列分析

目的分析广西16株狂犬病毒P基因序列特点,探讨广西狂犬病高发的可能原因。方法 2005-2008年在广西14个市采集健康犬脑标本3 961份,用直接免疫荧光法（DFA）和RT-PCR法检测狂犬病毒,P基因PCR扩增阳性者进行序列测定和分析。结果 16株狂犬病毒完成P基因序列测定,核苷酸及氨基酸同源性分别为86%-100%和93%-100%,属于基因Ⅰ型狂犬病毒;广西境内至少存在3条狂犬病毒传播链;16株狂犬病毒P基因序列推导的氨基酸序列多个位点发生变异。结论广西存在多个Ⅰ型狂犬病毒株系的流行,可能是近几年广西狂犬病持续高发的原因之一。     

1株驴源狂犬病毒的分离鉴定及N, G基因序列分析

目的 对武汉市汉南区新分离的1株驴源狂犬病毒(RABV)街毒株的N、G基因进行遗传学分析,比较其与近年来湖北省及周边地区分离的代表性街毒株以及人用和兽用狂犬病疫苗病毒株之间的差异.方法 以直接免疫荧光法检测驴脑组织中的RABV,并将驴脑海马组织研磨后接种乳鼠,观察其发病情况,采用双抗体夹心ELISA法检测驴脑组织及发病乳鼠脑组织悬液中的RABV,然后提取发病乳鼠脑组织RNA,利用RT-PCR扩增RABV的N、G基因,测序后进行遗传学分析.结果 在驴脑组织及发病乳鼠脑组织中检出RABV,该病毒株与近年来湖北省及周边地区分离的代表性街毒株以及国内外人用和兽用狂犬病疫苗病毒株相比,N、G基因核苷酸序列的同源性分别为85.7％～99.1％和82.2％～99.7％,推导的氨基酸序列同源性分别为95.6％-99.8％和87.8％～99.4％,与国内分离街毒株核苷酸和氨基酸的同源性高于疫苗株,且与国内疫苗株CTN-181的同源性要高于国外疫苗株.结论 该病毒株鉴定为驴源RABV,与湖北省及周边地区分离的代表性街毒株及中国人用狂犬病疫苗株CTN-181处于同一个亚群,有较近的系统进化关系.     

浙江省1999—2011年麻疹流行株全基因组序列分析

目的 从全基因组层面探讨浙江省1999-2011年麻疹病毒的变异及其与疫苗株S191之间的差异.方法 选取浙江省不同年份分离的麻疹病毒流行株9株,采用RT-PCR方法扩增全基因组序列,并与疫苗株S191及国外主要流行基因型毒株进行全序列比较分析.结果 1999-2011年浙江麻疹流行株间的氨基酸同源性为98.77％ ～ 99.89％,麻疹病毒尚未受到明显的正向压力,各基因的变异仍属随机漂移.与疫苗株S191相比,两者间氨基酸的同源性仅为96.63％～ 98.16％,分别存在135～159个氨基酸的改变,其中共同的氨基酸变异位点113个,导致5个糖基化位点的变异.在核苷酸水平上,N基因的平均变异率最大(5.5％);而在氨基酸水平上,P蛋白的平均变异率最大(7.7％).此外,在全基因组序列上,浙江流行株与各国疫苗株的遗传距离均大于D4、B3基因型流行株与各国疫苗组的遗传距离(t=9.76,P＜0.05;t=-12.39,P＜0.05).结论 浙江麻疹流行株与疫苗株S 191在各基因上均产生了较大的差异,现行疫苗株与H基因型流行株之间的差异远大于疫苗株与国外优势流行株(D4、B(1)基因型)之间的差异,应密切关注这一变化趋势,及早研究后备麻疹免疫株.     

H基因型:中美洲新发现的美洲土著HBV基因型

目前公认而权威的HBV基因分型方法是根据HBV DNA全序列的核苷酸变异,即同基因型其序列的同源性和异型间序列的差异。本文作者所做的HBV DNA全基因组测序,提示一个新的HBV基因型的发现,从而使HBV基因型扩展到A～H八种。 作者对来自世界不同地区的10株HBV毒株进行了全基因组测序。5株来自中美洲,属于F基因型。另外2株分别来自西班牙和瑞典,尽管编码d     

广西狂犬病病毒分子流行病学研究

目的分析广西狂犬病毒的分布和来源,从病原学角度分析广西狂犬病疫情高发的原因。方法2005年9月~2006年4月在广西玉林、贵港、柳州、来宾、南宁和河池等6市共收集健康犬脑标本1 352份,用直接免疫荧光法(DFA)和RT-PCR法检测狂犬病毒,其中22份标本完成狂犬病毒N基因编码区下游720bp核苷酸序列的测定。结果用DFA法检测出阳性标本160份,阳性率为11.83%,RT-PCR检测出阳性标本26份,阳性率1.9%,对其中22份标本完成N基因编码区下游720bp核苷酸序列的测定,核苷酸序列同源性为87.8%~100%,推导氨基酸序列的同源性为97.5%~99.6%,表明广西病毒标本N基因核苷酸序列的变异主要是同义突变。种系发生分析显示,广西病毒标本分为A、B、C群,各群分布范围不同,具有明显地域性特征;中国存在的3群狂犬病毒(CHINA-1、CHINA-2和CHINA-3群)目前在广西均有分布。广西流行的3群病毒来源各不相同:A群可能来源于与广西相邻的湖南、贵州;B群可能由广西北部省区传入;C群是在广西境内循环的毒株。结论广西境内普遍存在狂犬病毒感染犬只,并有外省狂犬病毒的传入,可能是近年广西狂犬病疫情上升的重要原因。     

Molecular Characterization of Cryptically Circulating Rabies Virus from Ferret Badgers,Taiwan

After the last reported cases of rabies in a human in 1959 and a nonhuman animal in 1961, Taiwan was considered free from rabies. However, during 2012–2013, an outbreak occurred among ferret badgers in Taiwan. To examine the origin of this virus strain, we sequenced 3 complete genomes and acquired multiple rabies virus (RABV) nucleoprotein and glycoprotein sequences. Phylogeographic analyses demonstrated that the RABV affecting the Taiwan ferret badgers (RABV-TWFB) is a distinct lineage within the group of lineages from Asia and that it has been differentiated from its closest lineages, China I (including isolates from Chinese ferret badgers) and the Philippines, 158–210 years ago. The most recent common ancestor of RABV-TWFB originated 91–113 years ago. Our findings indicate that RABV could be cryptically circulating in the environment. An understanding of the underlying mechanism might shed light on the complex interaction between RABV and its host.     

Evidence for inter- and intra-clade recombinations in rabies virus

Homologous recombination is considered rare in negative-strand RNA viruses and has not been reported for rabies virus. In this study, full-length genomes of 44 rabies virus strains were analyzed for potential homologous recombination events. Phylogenetic analysis classified these strains into three clades. By applying six different recombination detection methods, one inter-clade and one intra-clade potential recombination events were identified with high confidence values. Software-predicted recombination break points of the two events were all located within the polymerase gene. This report presents the first evidence suggesting the possibility of homologous recombination in rabies virus, which could provide valuable insights for understanding the diversity and evolution of rabies virus as well as other negative-strand RNA viruses.     

广西狂犬病毒野毒株的基因和抗原分析（英文）

Analyse the genetic and antigenic variant of rabies virus in Guangxi with epidemiological method, monoclonal antibodies reaction pattern and molecular biological technique. Rabies incidence has increase in recent three years in Guangxi, especially in mountainous areas. Investigation into the human rabies found that there are about 20％ vaccinated defeat cases.On the basis of their reactivity to monoclonal antibodies against the viral nucleocapsid protein(mAb-N),4 street strains isolated from different areas where enzootic rabies is shown had different reaction patterns compared with Chinese vaccine strain(3aG). The nucleotide and amino acid sequence of street strain(GX89_1) G and N gene were compared with 3aG, the overall nucleotide homology of GX89_1 with 3aG was 84.5％ and amino acid homology of GX89_1 with 3aG was 89.5％ in G gene.The overall nucleotide homology of GX89_1 with 3aG was 86％ in N gene and they were not in the same group.There are several amino acid replacement in AA 243_268 of G protein that can affect the antigenicity of rabies virus.The findings suggest that there are different street strains of rabies virus in Guangxi.     

Genetic and antigenic analysis of street strains of rabies virus in GuangxiP.R.China

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Genetic characterisation of attenuated SAD rabies virus strains used for oral vaccination of wildlife

The elimination of rabies from the red fox (Vulpes vulpes) in Western Europe has been achieved by the oral rabies vaccination (ORV) of wildlife with a range of attenuated rabies virus strains. With the exception of the vaccinia rabies glycoprotein recombinant vaccine (VRG), all strains were originally derived from a common ancestor; the Street Alabama Dufferin (SAD) field strain. However, after more than 30 years of ORV it is still not possible to distinguish these vaccine strains and there is little information on the genetic basis for their attenuation. We therefore sequenced and compared the full-length genome of five commercially available SAD vaccine viruses (SAD B19, SAD P5/88, SAG2, SAD VA1 and SAD Bern) and four other SAD strains (the original SAD Bern, SAD VA1, ERA and SAD 1-3670 Wistar). Nucleotide sequencing allowed identifying each vaccine strain unambiguously. Phylogenetic analysis revealed that the majority of the currently used commercial attenuated rabies virus vaccines appear to be derived from SAD B19 rather than from SAD Bern. One commercially available vaccine virus did not contain the SAD strain mentioned in the product information of the producer. Two SAD vaccine strains appeared to consist of mixed genomic sequences. Furthermore, in-del events targeting A-rich sequences (in positive strand) within the 3' non-coding regions of M and G genes were observed in SAD-derivates developed in Europe. Our data also supports the idea of a possible recombination that had occurred during the derivation of the European branch of SAD viruses. If confirmed, this recombination event would be the first one reported among RABV vaccine strains.     

Genetic diversity and molecular evolution of the rabies virus matrix protein gene in China

To investigate the diversity of rabies virus (RABV) matrix protein (M) gene in the current Chinese rabies epidemic, we fully examined M gene of 63 street RABVs (Virus isolated from naturally infected animals), and performed phylogenetic and mutational analysis. Our results indicate that the Chinese RABV M gene is well conserved with 90.6% to 100% amino acid similarity. Analysis of the mutations indicates that the sequences can be divided into four groups with each group defined by distinct substitutions. The PPxY motif and residue E58, which are essential for efficient virus production and pathogenicity, were completely conserved. The estimated mean rate of nucleotide substitution was 4.6×10⁻⁴ substitutions per site per year, and the estimated average time of the most recent common ancestor (TMRCA) was 265 years ago based on the M gene of Chinese street RABVs, which are similar to previously reported values for the glycoprotein (G) and nucleoprotein (N) gene. This indicates that the genomic RNA of RABVs circulating worldwide is stable; G, N and M genes are evolving at a similar rate. This study showed that although the Chinese RABV strains could be divided into distinct clades based on the phylogenetic analysis, their functional domains of M proteins were highly conserved.     

Efficacy of rabies vaccines against Duvenhage virus isolated from European house bats (Eptesicus serotinus), classic rabies and rabies-related viruses

Isolates of rabies from separate enzootics can be distinguished by their reactions with panels of monoclonal antibodies (mAbs) directed to different sites on the nucleocapsid and glycoproteins of the virus. Estimates of antigenic relatedness can be made by comparing similarities among groups. In this manner it can be shown that while classic strains of rabies react with most of the mAbs, the rabies related Lyssaviruses (Mokola, Lagos and Duvenhage) react with only a few of the mAbs and isolates of rabies from Eptesicus serotinus bats in Europe are intermediate between the two groups. Mice immunized intraperitoneally with human diploid vaccine (HDCV) or animal vaccines (Rabisin and Rabiffa) were protected against a challenge with DBV, DUV-1 and most classic rabies strains. HDCV gave only partial protection against human virus isolates from Finland and Saudi Arabia. The HDCV did not protect mice against challenges with Lagos bat or Mokola virus (rabies-like viruses). The animal vaccines, however, did protect mice against Lagos bat virus, but not against Mokola. Dogs immunized with Rabisin were protected against an intracerebral challenge with DBV. Dogs developed rabies-neutralizing antibody titres after intramuscular or intravenous inoculation with live DBV or DUV-1 virus; these dogs were protected against an intramuscular canine street rabies virus challenge. We conclude that the rabies vaccines tested protect against DBV/DUV-1 and classic street rabies strains, but not Mokola.     

Protein and nucleic acid analyses of influenza C viruses isolated from pigs and man

The virus-coded proteins and the genomes of influenza C virus isolates obtained from Chinese pigs in 1981–1982 and of human influenza C virus strains isolated between 1947 and 1981 were compared. Using SDS polyacrylamide gel electrophoresis and one-dimensional peptide mapping we found the virus-coded proteins of the pig influenza C viruses to be similar to those of human influenza C virus strains. The sizes of the genomes of human and pig influenza C viruses were indistinguishable. Genome analysis by oligonucleotide (ON) mapping revealed that the genomes of the pig influenza C viruses were very similar to but not identical with those of human influenza C virus strains. ON changes were found scattered over the whole genome. ON mapping of isolated segments of several influenza C virus strains suggested that two pig strains (C/P/B/10/81 and C/P/B/32/81) are related by a reassortment event which is likely to have occurred in nature. The rate of genome variation in influenza C viruses seemed to be similar to that seen in influenza B, and slower than that recorded for influenza A viruses.