【作者】 曹玉玺；

【导师】 梁国栋；

【作者基本信息】 中国疾病预防控制中心 ， 病原生物学， 2013， 博士

【摘要】 湖南省位于我国长江下游,夏季高温多雨,适宜蚊虫滋生和繁殖,从而利于虫媒病毒传播和扩散。长期以来湖南省是我国虫媒传染病—乙型脑炎的流行疫区,特别是西部、南部的山区和丘陵区的乙型脑炎发病率一直较高。湖南省开展的登革热血清学调查表明当地健康人群存在登革病毒抗体阳性,提示当地存在乙脑以外虫媒病毒性疾病的可能性。西藏自治区位于青藏高原西南部,与缅甸、印度、尼泊尔等国接壤,是我国西南边陲的重要门户。在藏东的高山峡谷区,海拔较低,气温较高,湿度较大,适于蚊虫滋生,对当地正常人群的血清学调查结果显示人群中存在乙脑病毒、登革病毒、基孔肯雅病毒等抗体阳性,提示藏东地区可能存在虫媒病毒的流行。伴随西藏旅游业的发展和西部大开发,将有大量人员和物资的来往和流动,可能会造成虫媒病毒病的传入和传出、甚至爆发流行。因此,对湖南省和西藏自治区进行虫媒病毒病原学的调查,全面了解当地存在和循环的虫媒病毒种类与分布情况,对于研究我国虫媒病毒生物学及分子生物学特征、预防和控制当地虫媒病毒病等具有重要意义。本课题对湖南省和西藏自治区开展虫媒病毒病原学调查,以明确当地存在的虫媒病毒种类与分布,并对分离到的虫媒病毒进行详细鉴定和序列特征分析,以了解这些新分离病毒株的系统分类地位、分子生物学特征及其与国内外分离株间的分子生物学差异。1.湖南省虫媒病毒调查本研究分别于2010和2011年的7-8月在湖南省怀化、张家界、岳阳、郴州、湘潭、邵阳、株洲和长沙市的16个区县采集蚊虫标本4属7种16076只,按形态学分类分装347批。蚊种包括库蚊属的致倦库蚊、淡色库蚊、三带喙库蚊、凶小库蚊,按蚊属的中华按蚊,阿蚊属的骚扰阿蚊及伊蚊属的白纹伊蚊。2年内采集最多的蚊种为致倦库蚊(8140只,50.63%),其次为中华按蚊(3900只,24.26%),采集最少的蚊种为白纹伊蚊(14只,0.09%)。蚊虫标本研磨后利用C6/36和BHK-21等细胞培养开展病毒分离,结果得到6株阳性分离物。经过系统鉴定,分别为2株乙脑病毒(Japanese encephalitis virus, JEV)、2株环状病毒(Orbivirus)、2株病毒需要进一步鉴定。6株分离物分别来自4个蚊种一致倦库蚊、淡色库蚊、三带喙库蚊和中华按蚊；致倦库蚊分离病毒最多,为3株,其余3株由淡色库蚊、三带喙库蚊和中华按蚊分离;并且3类病毒均能从致倦库蚊中分离到。2株乙脑病毒的分子生物学特征分析显示,2株JEV均为基因Ⅰ型病毒,2株病毒间E基因核苷酸和氨基酸序列同源性为100%,与疫苗株P3、Beijing-1和SA14-14-2株在E基因核苷酸和氨基酸序列同源性分别为87.7-88.1%和97.2-98.2%。2株JEV与疫苗株SA14-14-2株在E基因区段存在14处的氨基酸差异,但差异位点均不处于决定抗原性的关键氨基酸位点。2株JEV在E基因区段决定病毒毒力的8个氨基酸位点未见改变。2株在湖南新分离的环状病毒在致细胞病变效应上,可引起BHK-21细胞病变；在PAGE电泳中,环状病毒的带型为3-6-1型；基于第1片段和第10片段基因的系统进化分析表明,2株环状病毒在进化树中具有相似的拓扑结果,与家畜流行性出血热病毒(Epizootic hemorrhagic disease virus, EHDV)有较近的亲缘关系,而与云南环状病毒的亲缘关系较远。在湖南分离的2株环状病毒与以往在蚊虫中分离的云南环状病毒不同,为环状病毒属的一种新病毒。另外2株病毒可引起BHK-21细胞病变,空斑形成实验中可见直径约为3.0mm的空斑。利用针对黄病毒科、布尼亚病毒科、乙脑病毒、盖塔病毒等特异引物的RT-PCR实验鉴定病毒为阴性结果；利用甲病毒缺陷性复制子鉴定未知病毒实验为阴性结果。以上结果初步排除这2株病毒为我国常见的虫媒病毒种类,需要进一步鉴定。2.西藏自治区虫媒病毒调查2009年8月在西藏自治区林芝地区的墨脱县和米林县采集4089只蚊虫,在墨脱县采集的三带喙库蚊中分离到1株乙脑病毒(XZ0934株)对XZ0934株的分子生物学特征分析表明,该病毒基因组全长为10983nt;从第96-10397位是一个单一开放读码框(ORF),长度为10302nt,编码3433个氨基酸。乙脑病毒全基因组系统进化分析表明,XZ0934株独立于目前已知的4个基因型别的乙脑病毒之外,是基因Ⅴ型乙脑病毒。与目前已知的4个基因型别乙脑病毒全序相比,XZ0934株在开放读码框NS4a区段比其他乙脑病毒都要多3个核苷酸(AGC),相应地多编码1个氨基酸(丝氨酸)。XZ0934毒株在E基因区段决定病毒毒力的8个氨基酸位点未见改变；与疫苗株SA14-14-2株在E基因区段存在24处的氨基酸差异,其中E340位点的氨基酸突变可能会导致其抗原性的改变。采用贝斯马尔科夫蒙特卡洛算法,构建基于全世界100株乙脑病毒全基因组最大可信树(Maximum clade credibility tree, MCC Tree)的结果显示,乙脑病毒的最近共同进化祖先出现于3255年前,5个基因型别的乙脑病毒进化分歧时间按照Ⅴ,Ⅳ,Ⅲ,Ⅱ,Ⅰ的顺序排列,基因Ⅴ型乙脑病毒最先从乙脑病毒种群中分化出来,是最古老的乙脑病毒种系。本课题的意义及创新性本课题对湖南省和西藏部分地区进行了虫媒病毒调查,在湖南省分离到乙脑病毒和环状病毒等,在西藏分离到乙脑病毒。在湖南省分离到基因Ⅰ型JEV属国内首次报道,同时发现湖南省分离的环状病毒为环状病毒属的一种新病毒属国际上首次报道。在西藏自治区分离到的基因Ⅴ型乙脑病毒是时隔57年后该基因型乙脑病毒的再次出现,并在国际上首次利用生物信息学软件推算出5个型别乙脑病毒的最近共同进化祖先为3255年前。这些研究数据不但丰富了我国湖南省、西藏自治区虫媒病毒的种类,而且为了解当地传染病病因以及积极预防和控制相关虫媒病毒疾病提供了重要的信息。对新分离到的JEV、环状病毒等分离物的鉴定和分子生物学特征分析,明确了这些病毒的分类地位及其与国内外分离株间的分子生物学差异,丰富了我国虫媒病毒分子生物学研究内容,为深入研究我国存在和流行的虫媒病毒积累了科学数据。更多还原

【Abstract】 Hunan Province is located in the lower reaches of the Yangtze River, which is hot and rainy in the summer. The weather there is suitable for mosquito breeding and reproduction, so it is conducive to the spread of arboviruses. Hunan is still the epidemic area of Japanese encephalitis (JE) for many years, especially in the western and southern mountainous and hilly region. In addition, dengue serological survey in Hunan showed that local health people presented dengue virus antibodies, suggesting the possibility of arboviral disease. Tibet Autonomous Region is located in the southwest of the Qinghai-Tibet Plateau, bordering with Myanmar, India, Nepal and other countries, so Tibet is an important gateway in the southwestern border. In the mountains and canyons of eastern Tibet, the circumstance with the low altitude, high temperatures, high humidity, is suitable for mosquito breeding. Serological survey in the local population results showed that the antibodies to Japanese encephalitis virus, dengue virus, Chikungunya virus were positive.This study conducted arboviruses investigation in Hunan in2010,2011and Tibet in2009. The aim of this work is to know the arboviruses species and their distributions in these two provinces. This work had also identified and studied the molecular characterizations of arbovirus isolates in these two provinces.1. Arbovirus investigation in Hunan provinceMosquito samples were collected in the villages of16counties belonging to8cities in Hunan during July to August in2010and2011.16,076individuals covering4genus and7species had been collected, which represent Culex quinquefasciatus, Culex pipiens pallens, Culex tritaeniorhynchus, Culex modestus, Armigeres subalbatus, Anopheles sinensis and Aedes albopictus. The mosquitoes were sorted into347pools for virus isolation. Total of6virus isolates were obtained by inoculating the grinded supernatants onto C6/36and BHK-21cells and by observing cytopathic effect (CPE). The virus isolates were from4species including Culex quinquefasciatus, Culex pipiens pallens, Culex tritaeniorhynchus, and Anopheles sinensis.4isolates were observed to cause CPE in BHK-21cells only, while the other2isolates caused CPE both in C6/36cells and BHK-21cells.6isolates were identified by using morphological, serological and molecular biological methods.2isolates are Japanese encephalitis viruses (JEV),2isolates are orbiviruses, and the other two isolates are in the process of identification.Molecular analysis of the new isolates showed that2JEVs are all clustered into genotype I. The new strains showed very high identities in nucleotides and amino acids of E gene, which are87.7～88.1%and97.2～98.2%respectively.14sites of differences were found between the new strains and the vaccine strains SA14-14-2in E protein. These sites were not the key sites that affect the antigenic and virulence of JEV.Two isolates of orbivirus from Hunan Province can cause CPE in BHK-21cell. The profile of PAGE for2isolates showed the pattern of3-6-1. Phylogenetic analysis of the nucleotide sequences of the S and M segment of orbiviruses generated highly comparable topologies, which indicate that HN11066and HN11121have a relative high level of sequence homology with Epizootic hemorrhagic disease virus (EHDV). These results suggested that two isolates probably are a new species of orbivirus.The other two isolates could cause CPE in BHK-21, not in C6/36cell line. The diameter of plaque formed in BHK-21cell was about3.0mm. RT-PCR tests were negative using the primers of flaviviruses, bunyaviruses, Japanese encephalitis virus and Getah virus. Detection of alphaviruses from cell culture using a replicon-defective reporter gene assay was negative. The two isolates need further tests to identify the species.2. Arbovirus investigation in TibetIn August of2009,4089mosquitoes were collected in Linzhi, Tibet. And one virus was isolated from Culex tritaeniorhynchus collected in Motuo and identified as JEV (XZ0934strain).Compared with the Muar strain, the first isolate of genotype V JEV, the full-length genome of XZ0934and Muar were composed of10,983and10,988nucleotides respectively. And XZ0934strain shared a lower level of identity with JEV genotypes Ⅰ-Ⅳ, ranged from78.4%(G I, KV1899) to79.7%(G Ⅲ, JaGAr01) for the nucleotide sequences, and from90.0%(G I, KV1899) to91.8%(G Ⅲ, JaGAr01) for the amino acid sequences. The open reading frame (ORF) of genotype V JEVs spanned nucleotides96to10397and encoded3,433amino acids. A comparison with genotype Ⅰ-Ⅳ JEV strains revealed that three nucleotides (encoding a Serine residue) were inserted in the NS4A gene of genotype V JEVs, and the insertion of nucleotides were also found downstream of the ORF stop codon in the3’-untranslated region. Moreover, numerous amino acid mutations were observed in three functional domains of the E gene of genotype V JEVs.The Maximum clade credibility tree for the whole genomes of JEV was constructed. The tree contained five distinct clades corresponding to genotypes Ⅴ, Ⅳ, Ⅲ, Ⅱ, Ⅰ. The most recent common ancestor for all genotype was estimated to have occurred3255years ago. The branching of the lineages occurred in the following order:genotypes Ⅴ, Ⅳ, Ⅲ, Ⅱ, Ⅰ.SignificanceIn this study, arboviruses investigations were conducted in Hunan Province and Tibet. Two isolates of Japanese encephalitis virus belonging to Genotype I were isolated in Hunan Province, and it is the first time to report that genotype I JEV has spread into Hunan province. Also, two isolates of orbiviruses were probably the new-species of orbivirus.The JEV isolate from Tibet suggested that Genotype Ⅴ JEV is reemerging after57years. The molecular characterization of genotype Ⅴ Japanese encephalitis virus is significantly divergent from known genotype Ⅰ-Ⅳs. The mutations locate in coding region and non coding region may be molecular markers of genotype V JEVs and warrant further studies to determine their effects on the biology and immunogenicity of the genotype V strains.These findings provide a clue to strengthen arbovirus surveillance in order to prevent arbovirus diseases. Identification and/or molecular analysis of JEV, orbivirus and unknown virus isolates isolated from China can help to know more about the virus characterization, especially the molecular difference from foreign isolates, and can provide basic information for further study of the arboviruses distributed in Chinese territory.更多还原