【作者】 郑雅匀；

【导师】 梁国栋；

【作者基本信息】 山东大学 ， 卫生检验学， 2014， 博士

【摘要】 背景山西省地处黄河流域中部、华北平原的西侧,四周分别与内蒙古、河北、河南、陕西四省相邻。省内四周山环水绕,大部分地区海拔在1000米左右,全省自北向南分布有6大盆地,地形复杂、气候多样,促成了媒介生物种类的多样性和繁杂性。河南省位于我国中部偏东、黄河中下游地区,属于北亚热带、暖温带气候,这里日照充足、降水丰沛,同样存在种类丰富的媒介昆虫。对山西省和河南省的病毒性脑炎流行病学监测结果显示,在蚊虫活动高峰的夏秋季节,当地存在病毒性脑炎患者。两个省份也一直存在乙型脑炎的病例,其中河南省至今仍为乙脑病例的高度流行区。上一个十年,我国科学工作者分别对山西和河南省进行虫媒病毒病原学调查,分离到乙脑,盖塔,版纳病毒以及发热伴血小板减少综合征病毒(SFTSV)等,对于解释当地虫媒病毒感染提供了重要的基础数据。但是10年来,经济的快速发展,农村城镇化,农业生产方式的改变等使得当地虫媒病毒滋生环境发生较大变化,为此,再次开始调查对于了解当地存虫媒病毒种类及分布情况,预测相关病原潜在致病性和致病能力,预防和控制当地虫媒病毒病等方面均具有重要意义。本实验室于2009年在西藏林芝地区墨脱县和米林县开展蚊虫及蚊传虫媒病毒调查工作,在蚊虫标本中分离到两株可以引起BHK-21细胞病变的病毒,本实验对以上两个病毒分离物开展生物学表型、病毒分子鉴定、病毒分子遗传进化研究,以及病毒与当地动物感染性关系等研究。目的本课题通过在山西、河南省、西藏自治区蚊传虫媒病毒的调查研究,掌握当地蚊媒及其携带病毒的种类和分布信息,开展相关蚊传病毒的深入研究,为当地蚊传虫媒病毒病的预防和控制提供本底信息和理论依据。研究方法2012年夏季在山西省、河南省多地采集蚊虫标本,将标本现场分类鉴定后液氮保存,运送至实验室备用。将蚊虫标本,分批研磨离心,取上清接种组织细胞进行培养,对阳性分离物首先使用种属特异引物进行扩增测序,对于未知病毒,使用454高通量测序等技术获得序列信息,使用Clustal X2.1、MegAlign、Genedoc3.2和Mega v5.1生物信息学软件进行病毒核苷酸和氨基酸序列比对及系统进化分析；使用马尔科夫蒙特卡洛方法,对我国新分离双链RNA病毒(环状病毒和Seadornavirus属病毒)进行系统进化分析及病毒共同进化祖先分析(tMRCA).结果1、山西省蚊传病毒分离鉴定本研究于2012年夏季,在山西省运城市临猗县和永济市8个乡镇设立19个采集点,共采集蚊虫10455,经鉴定分为4属7种,其中库蚊属两种：淡色库蚊、三带喙库蚊；伊蚊属3种：背点伊蚊、白蚊伊蚊、刺扰伊蚊；按蚊属1种：中华按蚊；阿蚊属1种：骚扰阿蚊。不同地区的蚊种构成和优势蚊种存在明显差异,临猗县优势蚊种为淡色库蚊(91.96%,3911/4253)；永济市优势蚊种为三带喙库蚊(72.85%,4518/6202)。本研究中共使用5种诱蚊灯(器材),其中功夫小帅和CDC诱蚊灯采蚊效果最佳,能够满足捕捉蚊虫数量和种类的多样性。采用组织细胞培养法,将10455只蚊虫标本按照蚊种、采集工具和采集时间地点分190批进行研磨,上清接种细胞连续传代3次,结果分离到23株阳性分离物,来自于4个蚊种。包括三带喙库蚊、淡色库蚊、白蚊伊蚊和骚扰阿蚊。经过分子生物学鉴定,共得到15株乙脑病毒(Japanese encephalitis virus, JEV)、4株库蚊黄病毒(Culex Flavivirus, CxFV)、3株淡色库蚊浓核病毒(Culex pipiens pallens densovirus, CppDNV)和1株盖塔病毒(Getah virus, GETV)从三带喙库蚊和淡色库蚊中分离到病毒最多,分别是12株和10株,其中三带喙库蚊中乙脑病毒的批次阳性率和现场最低感染率最高,分别为14.1%和1：405。所有JEV病毒均为经过C6/36细胞扩增培养后,转接BHK21、Vero细胞可导致二者出现细胞病变效应(Cytopathic effect, CPE)。对新分离病毒进行分子生物学特征分析显示,15株JEV均为基因Ⅰ型病毒,各分离株之间E基因核苷酸和氨基酸序列同源性为99.6%～100%和99.80%～100%。新分离株与疫苗株SA14-14-2在E基因区段存在11处共同的氨基酸差异,但差异位点均不处在决定抗原性的关键氨基酸位点。首次从山西省分离到4株CxFV,各分离株之间核苷酸和氨基酸序列同源性分别为99.9%-100%和99.8%-100%,系统进化分析显示山西分离株与我国山东分离株亲缘关系最接近,其次与辽宁株较为接近。本研究中GETV亦为山西省首次分离,该毒株与我国上海2005年GETV分离株遗传关系相近,二者E2基因之间的核苷酸和氨基酸序列同源性在99%以上。2、河南省蚊传病毒分离鉴定本研究于2012年5月～8月在河南省洛阳市新安县、信阳市息县采集蚊虫标本,共采集淡色库蚊、骚扰阿蚊、三带喙库蚊、中华按蚊、白蚊伊蚊4属5种共计7149只蚊虫标本。其中洛阳市新安县的优势蚊种为骚扰阿蚊,淡色库蚊是信阳市息县优势蚊种。对所有标本分170批进行研磨和病毒分离,结果分离到5个阳性分离物均为JEV,其中2株来自于三带喙库蚊,3株分自于淡色库蚊。新分离病毒的分子生物学特征分析显示,所有5株JEV均为基因Ⅰ型病毒,分离株之间E基因核苷酸和氨基酸序列同源性为98.6%～99.9%和99.8%～100%。新分离JEV与疫苗株SA14-14-2在E基因区段存在11处共同的氨基酸差异,差异位点均未处于决定抗原性的关键氨基酸位点。与山西省JEV分离株相同,该5株JEV是经过C6/36细胞培养扩增后再接种BHK21,Vero细胞才可增殖并导致哺乳动物细胞CPE。3、西藏新分离环状病毒的鉴定及系统进化分析在中国西藏采集的多斑按蚊中分离到两株病毒(XZ0906、XZ0923),可引起哺乳动物细胞(BHK-21)圆缩、破碎；不能致昆虫细胞(C6/36)明显病变但可以在该种细胞中复制。聚丙烯酰胺凝胶电泳(Polyacrylamide gel electrophoresis, PAGE)结果提示两株分离物均为10节段双链RNA病毒,RNA带型完全一致,均为3-3-3-1布局。使用454高通量测序技术初步测定两株病毒各节段核酸序列,结合5’RACE、3’RACE及Sanger测序技术完成毒株XZ0906全基因组(Seg1-Seg10)核苷酸序列测定(GenBank登录号：KF746187to KF746196),序列分析显示XZ0906基因组全部10个节段RNA序列的5’、3’非编码区末端6个核苷酸序列均各自保守(5’-GUAAAA-----ACUUAC-3’)。与其他已报道环状病毒代表株相比较,XZ0906各基因节段氨基酸序列均差异较大,其中VP3(T2)蛋白氨基酸序列同源性仅为22.9-75.9%。基于呼肠孤病毒VP1蛋白氨基酸序列分子遗传进化分析结果,XZ0906病毒属于环状病毒属成员,进一步对环状病毒属病毒T2蛋白氨基酸序列进行系统进化分析,结果显示XZ0906不属于任何一种已知环状病毒,为呼肠孤病毒科环状病毒属新成员,定名为西藏环状病毒(Tibet orbivirus, TIBOV).对XZ0906和XZ0923的10个基因节段比较分析,发现除第6、2节段氨基酸同源性仅为80.39%和39.16%以外,其余各节段氨基酸同源性均大于94%,其中第3、5节段达到100%,T2蛋白氨基酸同源性为100%,提示两株病毒为同一种环状病毒。对同期在当地采集的66份健康猪血清进行IFA检测,结果提示当地家猪血清标本TIBOV IgG抗体阳性率为14.0%(8/57)。本研究采用贝叶斯马尔科夫蒙特卡洛方法,对包括TIBOV在内的12种、78株环状病毒属成员的VP1蛋白氨基酸序列数据集进行分子遗传进化分析,通过对Orbivirus病毒属病毒分歧时间分析发现,Orbivirus病毒属最近共同祖先(Time to most recent common ancestor, tMRCA)约出现在5051年(置信度1984～8893年)以前,其中西藏环状病毒TIBOV出现在大约距今43年前,与分离自澳大利亚的EUBV病毒具有较近的亲缘关系,并与主要由蠓携带传播的EHDV,BTV, EEV等能够引起家畜疾病和死亡的的虫媒病毒处于同一进化分支。4、其他工作对我国新分离Seadornavirus病毒属进行病毒分子遗传进化分析。基于LNV第10节段碱基替换值推算,LNV最近共同进化祖先出现时间分别在距今约316(95%HPD为70-800)年前,其中最早进化出现的为吉林分离株NE9731；基于BAV第12节段碱基替换值推算其最近共同进化祖先出现时间约在距今241年(95%HPD为89-500)前,我国最古老的BAV应当属于中国南方分离株,即中国云南分离株。结论及意义本课题对山西、河南省部分地区开展蚊传虫媒病毒调查。在山西省分离到4种病毒,其中CxFV和GETV均为山西省首次分离到；在河南省蚊虫标本分离到5株乙脑病毒,山西和河南两省蚊虫标本中分离JEV均为基因Ⅰ型。本研究结果显示,乙脑病毒仍然是山西、河南两地蚊虫携带的最主要的虫媒病毒,以上研究结果对当地虫媒病毒病的防控具有重要意义。同时,本课题通过病毒生物学和病毒分子遗传进化分析首次阐明我国西藏分离的两株病毒为环状病毒新种(TIBOV),病毒基因组信息已经提交至基因库(GenBank)注册。研究结果还提示,西藏当地家猪血清标本中存在西藏新分离环状病毒的IgG抗体,提示该病毒与当地动物感染有关,本结果为当地动物疾病的预防控制提供了基础数据。更多还原

【Abstract】 BackgroundShanxi Province is located in the middle of the Yellow River on the west of the North China Plain, which is surrounded by Inner Mongolia, Hebei, Henan and Shannxi Province. There are mountains and rivers around the province and most of the area is about1000meters above sea level. There are six major basins in the province from north to south so that complicated topography and various climate contribute to diversity and complexity of vector species. Henan Province is located in east-central China on the Yellow River region, which belongs to the north subtropical and warm climate where sunshine and rainfall are abundant. There are also a wide variety of insect vectors. The results of viral encephalitis epidemiological surveillance in Shanxi and Henan Province showed that in summer and autumn at mosquito activity peaks, there were some patients with viral encephalitis in the locality in two provinces. There always exist Japanese encephalitis cases in two provinces, in which Henan has remained highly endemic areas of JE cases. In the last decade, Chinese researchers had finished arboviruses surveillance in both province, and lots of arboviruses have been isolated including JEV, GETV, BAV and SFTSV. Ten years later, environment for arbovirus breeding has changed greatly as the development of economics, the rural urbanization and changing of agricultural practices. So it’s significant to move with the time to investigate the species and distribution of local arboviruses, which could help to predict emerging pathogens and their pathogenicity, prevent and control related arbovirus diseases. Mosquito samples were collected in Medog and Mainling County (altitude1000m) in the Nyingchi area of Tibet, China during the summer of2009. Two isolates which could cause BHK-21cytopathic effect has been got. In this study, we will finish the identification of two isolates, and discuss their biological phenotype, molecular evolutionary characteristics and relationship with local diseases.ObjectiveTo understand the species and distribution of mosquito-borne arboviruses in Shanxi, Henan and Tibet Autonomous Region, and focus on molecular evolutionary genetics analysis for new isolates. The data will be of great significance to local prevention and control of related mosquito-borne arbovirus diseases.MethodsMosquitoes were collected from Shanxi and Henan provinces in summer2012, and transported to the laboratory in liquid nitrogen containers, following morphological classification and species identification of specimens on-site.All specimens were homogenized and centrifuged and supernatant was then added to monolayers of both C6/36and BHK-21cells to isolate the virus. RT-PCR using specific primer for several common arboviruses was used to identify the isolates which could cause CPE. Then454high throughput sequencing was used to identify unknown virus isolates. After getting sequence information of new isolates, the nucleotides, amino acids analysis and phylogenetic analysis were conducted using software of Clustal X2.1, MegAlign, Genedoc3.2and Mega v5.1. The Bayesian Markov chain Monte Carlo method was used to analyse the phylogeny and calculate the time of most recent common ancestor (tMRCA) for dsRNA viruses, including Orbiviruses and Seadornaviruses which have been newly isolated in China.Results1. Identification of mosquito-borne arboviruses in Shanxi Province Mosquito specimens were collected in19collection sites, which belong to eight towns of Linyi County and Yongji City, Yuncheng City, Shanxi Province. A total of10455mosquitoes which belongs to7species in4genuses were collected, and two kinds of culex (including Culex pipiens pallens, Culex tritaeniorhynchus), three kinds of Aedes (including Aedes dorsalis, Aedes albopictus, Aedes vexans), and one kind of Anopheles (Anopheles sinensis); one kind of armigeres (armigeres subalbatus) were included. There were some significant differences on mosquito species composition and dominant mosquito species between different regions. The predominant mosquito specie in Linyi County was Culex pipiens (91.96%,3911/4253); the predominant mosquito specie in Yongji City was Culex tritaeniorhynchus (72.85%,4518/6202). In this study,5kinds of Traps (equipment for capturing mosquitoes) were used, and UV trap was the best one to capture mosquitoes, which could trap more and diverse mosquitoes samples.Tissue culture method was used to isolate viruses.10,455specimens which were divided into190pools according to the mosquito species, collecting tools and their source were numbered and grind. The supernatant was inoculated into both BHK-21and C6/36cells and23positive isolates were got. All isolates, including15strains of Japanese encephalitis virus (JEV),4strains of Culex Flavivirus (CxFV),3strains of Culex pipiens pallens densovirus (CppDNV) and1strain of Getah virus (GETV) were from four mosquito species (Culex tritaeniorhynchus, Culex pipiens, Armigeres subalbatus and Aedes albopictus). The pool positive rate and minimal virus infection rate in Culex tritaeniorhynchus were the highest, with14.1%and1:405, respectively. All JEV isolates could cause CPE in BHK-21and Vero only after being transferred from their C6/36cell culture to BHK-21or Vero cells.The molecular characterization analysis of newly virus isolates showed that all15JEVs belonged to genotype Ⅰ, and their nucleotide and amino acid sequence identity in E gene ranged from99.6%～100%and99.8%～100%, respectively. There are11amino acid differences between the new JEV isolates and SA14-14-2vaccine strain in E gene, but no difference in critical amino acid sites where determine the antigenicity was found.4CxFV strains were firstly isolated in Shanxi province, and sequence identity for nucleotide and amino acid ranged from99.9%～100%and99.8%-100%, respectively. The new isolated CxFV has a close relationship with Shandong isolate (SDD0611), and then is Liaoning isolate (HLD105). GETV is also the first report to be isolated from Shanxi province. Phylogenetic analysis demonstrate a close relationship between new isolate and Shanghai isolates. Both their identity in nucleotide and amino acid sequence is more than99%.2. Identification of mosquito-borne arboviruses in Henan ProvinceThis study collected mosquito specimens in Xin’an County, Luoyang City and Xi County, Xinyang City, Henan Province in August2012, Total of7,149mosquito specimens including5species in4geneus (Culex pipiens pallens, armigeres subalbatus, Culex tritaeniorhynchus, Anopheles sinenese, Aedes albopictus) were collected. The predominant mosquito specie in Xin’an County is armigeres subalbatus and Culex pipiens pallens in Xi County. All specimens were divided into170pools for grinding and virus isolation. At last, five JEV positive isolates were got, of which two were from Culex tritaeniorhynchus and three were from Culex pipiens pallens. The analysis of molecular biological characterization of newly isolated virus showed that all five JEV were genotype I virus, nucleotide and amino acid sequence homology of E gene in isolates were98.6%-99.9%and99.8%-100%, respectively. There were11amino acid differences in the E gene segments between newly isolated JEV and vaccine strains SA14-14-2, but the different sites were not in the critical amino acid sites that could control virus antigenicity. The same with Shanxi JEV isolates, five JEV isolates from Henan province also need a "transfer" process to cause BHK-21and Vero CPE. Culture by C6/36cells is nessary for new JEV isolates to proliferate and cause CPE in mammalian cells.3. Identification of novel Orbiviruses in Tibet and Phylogenetic analysis of Orbiviruses This study describes two viral strain(XZ0906and XZ0923) isolated from Anopheles maculatus specimens collected in Tibet, China. Initial viral screens identified that the both viruses caused significant cytopathic effect (CPE) in BHK-21cells. Although CPE was not observed in insect cells (C6/36), these cells supported viral replication. Polyacrylamide gel analysis revealed a genome consisting of10segments of double-stranded RNA (dsRNA), with a distribution pattern of3-3-3-1.454high throughput sequencing of culture supernatant was used for viral identification. Together with5’-RACE,3’-RACE and Sanger sequencing, Complete genome sequencing of XZ0906was performed. Sequence analysis demonstrated that all5’-and3’-untranslated regions (UTRs) for each of the10genome segments of XZ0906contained a series of six highly conserved nucleotides. Phylogenetic analysis performed using the amino acid sequences of Reoviridae VP1showed that XZ0906belonged to the genus Orbivirus. Further phylogenetic analysis of Orbivirus T2proteins revealed that virus XZ0906was not a member of any known species or serotype of Orbivirus, indicating it to be a new species within the genus Orbivirus. This novel virus was subsequently named Tibet Orbivirus, TIBOV to denote the location from which it was isolated. By comparing the coding region of all10segments between XZ0906and XZ0923, we found two isolates share high amino acid identity in most segments except segment6and2(80.39%and39.16%, respectively).100%amino acid identity in T2protein reminder that two isolates belong to the same orbivirus species. The positive rate of TIBOV IgG antibody (14.0%,8/57)) in local healthy swine prompt a certain infection of TIBOV among local livestock.The Bayesian Markov chain Monte Carlo method is used in this study to take the phylogenetic analysis for VP1protein amino acid sequence database for Orbiviruses (including78strains in12species). Based on analysis of divergence time of the genus Orbivirus, the most recent common ancestor of the genus (Time of most recent common ancestor, tMRCA) was calculated to appeared in about5051years ago (from1984to8893), among them TIBOV appeared in about43years ago which had a close genetic relationship with Eubenangee virus (EUBV). TIBOV was also in the same group with orbiviruses that could cause illness and death of livestock (EHDV, BTV, EEV) which was mainly spread by midge.4. Other workAnalysis of population dynamics and evolutionary history based on the sequence database of10th segment of Liaoning viruses and12th segment of Banna viruses show that, the time of most recent ancestor of LNV is-316years, and BAV is-241years. In China, the oldest strain for LNVs is NE9731, and BAVs are strains isolated from Yunnan province.ConclusionIn this study, the investigation of mosquito-borne virus was taken in Shanxi and Henan Province. Four kinds of viruses were obstained from mosquitoes of Shanxi including JEV, GETV, CxFV and CppDNV, in which GETV, CxFV were firstly isolated in Shanxi province.5JEVs were isolated from mosquitoes of Henan province. All the JEVs isolates from both provinces belong to genotype I. The result show that JEV is still the main arbovirus in Shanxi and Henan province.Meanwhile, the Tibet Orbivirus (TIBOV) was Identified and clearly defined as a new member of the genus Orbivirus in this study, which enriching the classification information of arboviruses in our country and even the world, and genome sequence for TIBOV has been submitted to GenBank. Phylogenetic analysis showed that TIBOV was in the same clade with Orbivirus that was strong pathogenic to animals. Together with IFA results for TIBOV in serum of local livestocks, we conclude that TIBOV cycle in local environment. All these results can provide basic information for prevention and control of local arbovirus diseases.更多还原