乙脑病毒时空动力学分析
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摘要
乙型脑炎(Japanese Encephalitis, JE),简称乙脑,是目前全世界无可争议的最严重的病毒性脑炎,发病率和病死率高,后遗症严重。乙脑病毒(Japanese Encephalitis virus, JEV)是蚊传虫媒病毒,可以被多种蚊虫携带和传播,猪是其主要的储存宿主,鸟被认为是乙脑病毒的传播宿主,人和马是乙脑病毒的终末宿主。本研究的目的是通过对优势基因型别乙脑病毒进行时空动力学研究分析,寻找乙脑病毒的播散规律和播散特点以及可能影响乙脑病毒传播的因素,预测乙脑病毒将来的分布变化趋势,为全球乙脑的预防控制提供依据。
湖北省位于我国中南部,长江中游,每年降水充沛,具有丰富的蚊虫媒介种类。湖北省曾是蚊传虫媒病毒病乙脑的高发地区,湖北省是否还存在其他蚊传虫媒病毒,至今缺乏系统的调查研究。为系统了解湖北省蚊传虫媒病毒的分布情况及其种类,为当地虫媒病毒病的预防控制提供理论依据,本研究在2009年和2010年7-8月份,分别在湖北省的东西南北中挑选武穴市、通城县、恩施州、神农架林区、江陵县和随州市等6个地区进行蚊虫采集,并通过病毒分离和序列分析确定该地区的虫媒病毒种类、分布及分子生物学特征。
1.乙脑病毒时空动力学分析
本研究首先测定了中国2005-2010年在湖北、重庆、江西、山东、辽宁和云南省新分离的22株基因1型乙脑病毒的E基因序列,其中包括本研究2010年在湖北省新分离的乙脑病毒株,填补了湖北、重庆、江西等亚洲中部区域乙脑病毒的空白,为乙脑病毒时空动力学分析提供了条件。继而利用GenBank下载乙脑病毒序列,最终构建了本研究时空动力学分析所用的涵盖所有乙脑流行区域和多种媒介宿主的优势基因型别乙脑病毒序列数据集,共包括359株基因1型乙脑病毒的E基因序列。
本研究采用贝叶斯马尔科夫蒙特卡洛方法对上述数据集进行乙脑病毒的分子进化分析,结果显示分布在不同区域的乙脑病毒形成了相对独立的病毒进化特征,并分别在亚洲最南部地区、亚洲东部沿海地区、亚洲西部地区和亚洲中部地区形成循环。进一步分析发现在以上4个地区形成的独立进化分支中都具有来自最南部地区的病毒,且最南部地区的病毒大都是每个分支中最早分化出来的病毒；另外,每个独立进化分支中最南部地区分离的毒株具有广泛的时间跨度,以上结果说明亚洲最南部地区分离的乙脑病毒既体现了种群多样性又维持了种群稳定性,提示亚洲大陆最南部地区在乙脑病毒向亚洲大陆传播的过程中担当了重要的源泉地区作用。本研究还讨论了乙脑病毒的传播路线,发现乙脑病毒在亚洲存在由南向北的传播特点,主要通过3条传播路线,而三条传播路线分别与候鸟在亚洲的迁徙路线吻合,提示候鸟在乙脑病毒远距离传播中发挥重要的作用。
优势基因型别乙脑病毒的种群动态分析显示乙脑病毒在传播和地域播散过程中经历了平缓—快速增长—下降—平缓的种群变化。基于种群动态Skyline plots的时间节点对4个地域循环中毒株的分离时间进行分析,进一步证实了亚洲大陆最南部地区是乙脑病毒的源泉地区。
乙脑病毒系统发生分析的结果显示乙脑病毒具有地域分布特征,但也存在毒株地域混合现象,提示乙脑病毒的分布分化机制除了族群结构分化外,还可能存在迁移事件(基因漂移)。因此,为详细地了解乙脑病毒的空间动力学特征,本研究还采用Migraphyla软件对乙脑病毒传播的空间动力学特征进行分析,结果显示乙脑病毒在亚洲的播散存在频繁的迁徙事件,迁徙事件也是促成乙脑病毒地域分布分化的重要方式。更进一步的分析发现,泰国、上海、山东、四川和越南是乙脑病毒重要的迁徙源泉地区。泰国是乙脑病毒向亚洲西部、东亚传播的主要源泉地区；上海维持了乙脑病毒在东亚沿海地区的循环；山东是乙脑病毒从沿海地区向中国内陆地区传播的源泉地区；四川维持了乙脑病毒在中国内陆的循环；而越南则是乙脑病毒从东南亚地区向中国内陆省份传播的源泉。
2.湖北省虫媒病毒调查
本研究在2009年和2010年的7-8月在分别在湖北省武穴市、通城县、恩施州、神农架林区、江陵县和随州市等6个地区采集蚊虫标本,共采集三带喙库蚊、中华按蚊、骚扰阿蚊、致倦库蚊和未分类杂蚊等3属5种共计22,269只蚊虫标本。三带喙库蚊是武穴市、通城县、随州市和江陵县的优势蚊种,致倦库蚊是恩施州的优势蚊种,在神农架林区主要采集到骚扰阿蚊。对所有标本分272批进行研磨和病毒分离,结果分离到42个阳性分离物,来自4个蚊种——三带喙库蚊、致倦库蚊、骚扰阿蚊和中华按蚊。经过系统鉴定,共得到33株乙脑病毒(Japanese encephalitis virus, JEV)、6株盖塔病毒(Getah virus, GETV)(?)口4株版纳病毒(Banna virus, BAV)。从三带喙库蚊和致倦库蚊中分离到的病毒最多,分别是21株和15株,而且3种病毒都从三带喙库蚊中分离到；从恩施州分离的病毒最多,为31株；恩施州致倦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53.6%和1：103.3,其次是恩施州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44.4%和1：128.8。
新分离病毒分子生物学特征分析显示,所有33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98.7%-100%和99.8%-100%。新分离JEV与疫苗株SA14-14-2在E基因区段存在14处共同的氨基酸差异,但差异位点均不处于决定抗原性的关键氨基酸位点。新分离GETV与我国2002年分离株HB0234、YN0540和韩国2004年分离株Korea的进化关系最近,位于同一个进化分支中；而与俄罗斯分离株LEIV/16275的进化关系较远。新分离BAV第12片段核苷酸之间的同源性为100%,第12片段核苷酸和VP12蛋白序列中存在多个不同于其它地域分离株的共同差异位点,进化分析显示所有新分离株与中国北京和云南的分离株以及越南分离株处于同一亚群。
本研究的意义
本研究首次在全球范围内开展了乙脑病毒的时空动力学分析,阐明了乙脑病毒在亚洲的播散特征和播散方式,首次发现乙脑病毒的传播存在地域分布特征和播散源泉地区,首次发现迁徙事件是乙脑病毒重要的分布分化机制,并首次从系统进化角度揭示候鸟在乙脑病毒远距离传播中具有重要的作用。研究结果还提示乙脑病毒存在向欧洲等传统非流行区传播的可能性,为全球乙脑的预防控制提供了重要依据。
本研究还对湖北省部分地区进行了虫媒病毒调查,分离到乙脑病毒、盖塔病毒和版纳病毒等3种虫媒病毒,并发现库蚊是湖北市携带病毒最多的蚊种。盖塔病毒和版纳病毒为湖北省首次分离；乙脑病毒在湖北的分离也填补了乙脑病毒在亚洲中部的空白,为乙脑病毒时空动力学分析提供了条件。本研究还明确了湖北省新分离病毒与国内外分离株之间的分子生物学差异,丰富了我国和湖北省虫媒病毒信息,为深入开展研究提供了基础数据,也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资料信息。
Japanese encephalitis (JE) is arguably one of the most serious viral encephalitic diseases worldwide with high morbidity and mortality and serious sequela. Japanese encephalitis virus (JEV) is a kind of mosquito-borne arbovirus which can be carried and spread by a variety of mosquitoes. Pigs are the main reservoir host. Migrating birds are thought to be an important factor in the dispersion of JEV to new geographical areas. Horses and humans are incidental hosts of JEV. The objective of this study is to find out the dispersal patterns and characteristics of Japanese encephalitis virus as well as the potential factors that may affect the spread of JEV through study on the spatial and temporal dynamics of the predominant genotypes of JEV, in order to predict the distribution trend of the JEVs in the future and provide the basis for the global prevention and control of JE.
Hubei Province is located in the south-central China, and the middle reaches of the Yangtze River, where the annual precipitation is high and has an abundant of mosquito species. Hubei province was a JE high prevailing area historically and is still epidemic in recent years. Because of lack of systematic investigation, it is still unknown whether there are other mosquito-borne arboviruses existing in Hubei Province, hi order to systematically understand the species and distribution of mosquito-borne arboviruses in Hubei Province and provide a theoretical basis for local arboviral disease prevention and control, mosquito samples were collected in Wuxue City, Tongcheng County, Enshi Prefecture, Shennongjia, Jiangling County and Suizhou City in July and August of2009and2010, respectively. After determination of mosquito species, virus isolation was conducted and virus isolates were identified and characterized.
1. Study on the spatial and temporal dynamics of JEV
In this study, first of all, we sequenced the E gene of22genotype I Japanese encephalitis viruses newly isolated in Hubei, Chongqing, Jiangxi, Shandong, Liaoning and Yunnan Province from2005to2010. These JEV isolates filled the gap of JEV records in Hubei, Chongqing, Jiangxi and other Asian central regions; furthermore, it provided a basis for spatial and temporal dynamics analysis of JEV. Then, by downloading all available JEV E gene sequences from GenBank, a sequence dataset including E gene sequences of359genotype I JEVs was ultimately established, which covers all JE endemic regions and a variety of media hosts.
The Bayesian Markov chain Monte Carlo method was used to analyze the phylogeny of JEV based on the JEV sequence datasets. The results demonstrated that JEV lineages can be divided into four endemic cycles, comprising of southern Asia endemic cycle, eastern coastal Asia endemic cycle, western Asia endemic cycle, and central Asia endemic cycle. The isolation places of the viruses in each endemic cycle were geographically independent. Analysis of the viruses in each endemic cycle showed that JEV strains isolated from the southernmost region were present in the other three endemic cycles in addition to the southern Asia endemic cycle. Furthermore, these strains were rooted those in each endemic cycle, indicating that southernmost Asia maintains the viral population diversity and stability, and acts as a source from which JEV gradually disperses throughout the Asian continent. We also found that JEV has a dispersal pattern from south to north on the Asian continent through three likely transmission routes which correlates closely with the recognized eastern, central, and western flight paths of migratory birds in Asia. This finding indicates that the migratory birds might play an important role in the long-distance dispersal of the JEV.
The population dynamics analysis of dominant genotype JEV showed that there was minimal fluctuation during the first half of the plot, and it was followed by a major population increase, then a relatively stable period and a marked decrease, and again a relatively stable period. According to the time nodes of change in population dynamics identified in the Skyline plot, the isolation times of the JEV in the four endemic cycles were analyzed. The results further confirmed that the southernmost regions of the Asian continent play a key role as the source for evolution and dispersal of the JEV.
Although phylogenetic analysis of JEV shows that JEV had a characterization of geographical distribution, there was also geographical mixing phenomenon. It indicated that migration events (like genetic drift) may also contribute to the differentiation of JEV's distribution besides the population structure differentiation. Therefore, in order to better understand the spatial dynamics characteristic of JEV, the Migraphyla software was used to conduct the analysis. The results showed that there were frequent migration events during the dispersal of JEV in Asia, and these migration events also contributed to differentiation in the geographical distribution of JEV. Further analysis revealed that Thailand, Shanghai, Shandong, Sichuan and Vietnam were the important migration source region of JEV. Thailand is the source of JEV spreading to West Asia and East Asia region; Shanghai maintains the JEV circulating in the coastal areas of East Asia; Shandong is the source of JEV spreading from coastal areas to inland areas of China; Sichuan maintains the JEV circulating in the inland China; Vietnam is the source of JEV spreading from Southeast Asia to China's inland provinces.
2. Mosquito-borne arbovirus investigation in Hubei Province
In this study,22,269mosquito samples including Culex tritaeniorhynchus, Anopheles sinensis, Armigeres subalbatus, Culex pipiens pallens and unidentified mosquitos which belongs to5species in3genuses were collected in Wuxue City, Tongcheng County, Enshi Prefecture, Shennongjia, Jiangling County and Suizhou from July to August in2009and2010. Culex tritaeniorhynchus was the dominant species in Wuxue City, Tongcheng, Suizhou and Jiangling County; Culex pipiens pallens was the dominant species in Enshi; Armigeres subalbatus was mainly collected in Shennongjia. All samples were divided into272pools according to species, locations and collection time. After grinding and virus isolation,42positive isolates were isolated from four mosquito species including Culex tritaeniorhynchus, Anopheles sinensis, Armigeres subalbatus and Culex pipiens pallens. Through systematic identification, totally42virus strains were obtained including33JEVs,6Getah virus, and4Banna virus. Viruses isolated from Culex tritaeniorhynchus and Culex pipiens pallens accounted for the largest proportion, with21strains and15strains isolated respectively; and all three kinds of viruses can be isolated from Culex tritaeniorhynchus. Up to31strains virus were isolated from Enshi Prefecture. The pool positive rate and minimal virus infection rate in Culex pipiens pallens collected in Enshi was the highest, with53.6%and1:103.3respectively. The pool positive rate and minimal virus infection rate in Culex tritaeniorhynchus collected in Enshi was also high, with44.4%and1:128.8respectively.
The molecular characterization analysis of these newly virus strains showed that all33JEVs belonged to genotype I and the nucleotide and amino acid sequence homology between E gene was98.7%to100%and99.8%to100%respectively. There are14amino acid differences between the new JEV strains and SA14-14-2vaccine strain in the E gene, but the differences are not in the critical amino acid sites where determine the antigenicity. The newly isolated GETV has a close evolutionary relationship with strain HB0234and YN0540isolated in China and strain Korea isolated in South Korea in2004, and a far evolutionary relationship with LEIV/16275isolated in Russia. The sequences of segment12of newly isolated BAVs share a100%homology. Several sites were found in new BAVs which were different from strains isolated from other regions. Phylogenetic analysis revealed that all the new isolates can be grouped in the same subset with isolates from Beijing, Yunnan and Vietnam.
The significance of this study
This is the first time to carry out a spatial and temporal dynamics analysis of JEV in global scale. It clarified the dispersal characteristics and patterns of JEV in Asia and find out the geographical distribution characteristics and transmission source region in the dispersal of JEV. Also the migration events were found to contribute to distribution and differentiation of JEV. Migratory birds were speculated for the first time from the perspective of phylegenetic evolution to play an important role in the long-distance spread of JEV. The results also suggest the possibility that JEV can be transmitted to Europe and other non-endemic areas and provide an important basis for the global prevention and control of JE.
A mosquito-borne arbovirus investigation was also conducted in part of Hubei Province. JEV, Getah virus, and Banna virus were isolated from the mosquito samples collected. And Culex was found to be the species that carried the most viruses in Hubei province. Getah virus and Banna virus were isolated in Hubei Province for the first time; the isolation of JEV in Hubei also filled the blank of JEV's distribution in central Asia, which provided the conditions for the JEV spatiotemporal dynamics analysis. The study also carried out study on molecular biological differences between newly isolated virus in Hubei and other available isolates. It enriched the information about arboviruses in Hubei Province and China and provided the basic data for carrying out a further study. Moreover, it provided important information for understanding the etiology of local infectious diseases as well as conducting active prevention and control of arbovirus-related diseases.
湖北省位于我国中南部,长江中游,每年降水充沛,具有丰富的蚊虫媒介种类。湖北省曾是蚊传虫媒病毒病乙脑的高发地区,湖北省是否还存在其他蚊传虫媒病毒,至今缺乏系统的调查研究。为系统了解湖北省蚊传虫媒病毒的分布情况及其种类,为当地虫媒病毒病的预防控制提供理论依据,本研究在2009年和2010年7-8月份,分别在湖北省的东西南北中挑选武穴市、通城县、恩施州、神农架林区、江陵县和随州市等6个地区进行蚊虫采集,并通过病毒分离和序列分析确定该地区的虫媒病毒种类、分布及分子生物学特征。
1.乙脑病毒时空动力学分析
本研究首先测定了中国2005-2010年在湖北、重庆、江西、山东、辽宁和云南省新分离的22株基因1型乙脑病毒的E基因序列,其中包括本研究2010年在湖北省新分离的乙脑病毒株,填补了湖北、重庆、江西等亚洲中部区域乙脑病毒的空白,为乙脑病毒时空动力学分析提供了条件。继而利用GenBank下载乙脑病毒序列,最终构建了本研究时空动力学分析所用的涵盖所有乙脑流行区域和多种媒介宿主的优势基因型别乙脑病毒序列数据集,共包括359株基因1型乙脑病毒的E基因序列。
本研究采用贝叶斯马尔科夫蒙特卡洛方法对上述数据集进行乙脑病毒的分子进化分析,结果显示分布在不同区域的乙脑病毒形成了相对独立的病毒进化特征,并分别在亚洲最南部地区、亚洲东部沿海地区、亚洲西部地区和亚洲中部地区形成循环。进一步分析发现在以上4个地区形成的独立进化分支中都具有来自最南部地区的病毒,且最南部地区的病毒大都是每个分支中最早分化出来的病毒；另外,每个独立进化分支中最南部地区分离的毒株具有广泛的时间跨度,以上结果说明亚洲最南部地区分离的乙脑病毒既体现了种群多样性又维持了种群稳定性,提示亚洲大陆最南部地区在乙脑病毒向亚洲大陆传播的过程中担当了重要的源泉地区作用。本研究还讨论了乙脑病毒的传播路线,发现乙脑病毒在亚洲存在由南向北的传播特点,主要通过3条传播路线,而三条传播路线分别与候鸟在亚洲的迁徙路线吻合,提示候鸟在乙脑病毒远距离传播中发挥重要的作用。
优势基因型别乙脑病毒的种群动态分析显示乙脑病毒在传播和地域播散过程中经历了平缓—快速增长—下降—平缓的种群变化。基于种群动态Skyline plots的时间节点对4个地域循环中毒株的分离时间进行分析,进一步证实了亚洲大陆最南部地区是乙脑病毒的源泉地区。
乙脑病毒系统发生分析的结果显示乙脑病毒具有地域分布特征,但也存在毒株地域混合现象,提示乙脑病毒的分布分化机制除了族群结构分化外,还可能存在迁移事件(基因漂移)。因此,为详细地了解乙脑病毒的空间动力学特征,本研究还采用Migraphyla软件对乙脑病毒传播的空间动力学特征进行分析,结果显示乙脑病毒在亚洲的播散存在频繁的迁徙事件,迁徙事件也是促成乙脑病毒地域分布分化的重要方式。更进一步的分析发现,泰国、上海、山东、四川和越南是乙脑病毒重要的迁徙源泉地区。泰国是乙脑病毒向亚洲西部、东亚传播的主要源泉地区；上海维持了乙脑病毒在东亚沿海地区的循环；山东是乙脑病毒从沿海地区向中国内陆地区传播的源泉地区；四川维持了乙脑病毒在中国内陆的循环；而越南则是乙脑病毒从东南亚地区向中国内陆省份传播的源泉。
2.湖北省虫媒病毒调查
本研究在2009年和2010年的7-8月在分别在湖北省武穴市、通城县、恩施州、神农架林区、江陵县和随州市等6个地区采集蚊虫标本,共采集三带喙库蚊、中华按蚊、骚扰阿蚊、致倦库蚊和未分类杂蚊等3属5种共计22,269只蚊虫标本。三带喙库蚊是武穴市、通城县、随州市和江陵县的优势蚊种,致倦库蚊是恩施州的优势蚊种,在神农架林区主要采集到骚扰阿蚊。对所有标本分272批进行研磨和病毒分离,结果分离到42个阳性分离物,来自4个蚊种——三带喙库蚊、致倦库蚊、骚扰阿蚊和中华按蚊。经过系统鉴定,共得到33株乙脑病毒(Japanese encephalitis virus, JEV)、6株盖塔病毒(Getah virus, GETV)(?)口4株版纳病毒(Banna virus, BAV)。从三带喙库蚊和致倦库蚊中分离到的病毒最多,分别是21株和15株,而且3种病毒都从三带喙库蚊中分离到；从恩施州分离的病毒最多,为31株；恩施州致倦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53.6%和1：103.3,其次是恩施州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44.4%和1：128.8。
新分离病毒分子生物学特征分析显示,所有33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98.7%-100%和99.8%-100%。新分离JEV与疫苗株SA14-14-2在E基因区段存在14处共同的氨基酸差异,但差异位点均不处于决定抗原性的关键氨基酸位点。新分离GETV与我国2002年分离株HB0234、YN0540和韩国2004年分离株Korea的进化关系最近,位于同一个进化分支中；而与俄罗斯分离株LEIV/16275的进化关系较远。新分离BAV第12片段核苷酸之间的同源性为100%,第12片段核苷酸和VP12蛋白序列中存在多个不同于其它地域分离株的共同差异位点,进化分析显示所有新分离株与中国北京和云南的分离株以及越南分离株处于同一亚群。
本研究的意义
本研究首次在全球范围内开展了乙脑病毒的时空动力学分析,阐明了乙脑病毒在亚洲的播散特征和播散方式,首次发现乙脑病毒的传播存在地域分布特征和播散源泉地区,首次发现迁徙事件是乙脑病毒重要的分布分化机制,并首次从系统进化角度揭示候鸟在乙脑病毒远距离传播中具有重要的作用。研究结果还提示乙脑病毒存在向欧洲等传统非流行区传播的可能性,为全球乙脑的预防控制提供了重要依据。
本研究还对湖北省部分地区进行了虫媒病毒调查,分离到乙脑病毒、盖塔病毒和版纳病毒等3种虫媒病毒,并发现库蚊是湖北市携带病毒最多的蚊种。盖塔病毒和版纳病毒为湖北省首次分离；乙脑病毒在湖北的分离也填补了乙脑病毒在亚洲中部的空白,为乙脑病毒时空动力学分析提供了条件。本研究还明确了湖北省新分离病毒与国内外分离株之间的分子生物学差异,丰富了我国和湖北省虫媒病毒信息,为深入开展研究提供了基础数据,也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资料信息。
Japanese encephalitis (JE) is arguably one of the most serious viral encephalitic diseases worldwide with high morbidity and mortality and serious sequela. Japanese encephalitis virus (JEV) is a kind of mosquito-borne arbovirus which can be carried and spread by a variety of mosquitoes. Pigs are the main reservoir host. Migrating birds are thought to be an important factor in the dispersion of JEV to new geographical areas. Horses and humans are incidental hosts of JEV. The objective of this study is to find out the dispersal patterns and characteristics of Japanese encephalitis virus as well as the potential factors that may affect the spread of JEV through study on the spatial and temporal dynamics of the predominant genotypes of JEV, in order to predict the distribution trend of the JEVs in the future and provide the basis for the global prevention and control of JE.
Hubei Province is located in the south-central China, and the middle reaches of the Yangtze River, where the annual precipitation is high and has an abundant of mosquito species. Hubei province was a JE high prevailing area historically and is still epidemic in recent years. Because of lack of systematic investigation, it is still unknown whether there are other mosquito-borne arboviruses existing in Hubei Province, hi order to systematically understand the species and distribution of mosquito-borne arboviruses in Hubei Province and provide a theoretical basis for local arboviral disease prevention and control, mosquito samples were collected in Wuxue City, Tongcheng County, Enshi Prefecture, Shennongjia, Jiangling County and Suizhou City in July and August of2009and2010, respectively. After determination of mosquito species, virus isolation was conducted and virus isolates were identified and characterized.
1. Study on the spatial and temporal dynamics of JEV
In this study, first of all, we sequenced the E gene of22genotype I Japanese encephalitis viruses newly isolated in Hubei, Chongqing, Jiangxi, Shandong, Liaoning and Yunnan Province from2005to2010. These JEV isolates filled the gap of JEV records in Hubei, Chongqing, Jiangxi and other Asian central regions; furthermore, it provided a basis for spatial and temporal dynamics analysis of JEV. Then, by downloading all available JEV E gene sequences from GenBank, a sequence dataset including E gene sequences of359genotype I JEVs was ultimately established, which covers all JE endemic regions and a variety of media hosts.
The Bayesian Markov chain Monte Carlo method was used to analyze the phylogeny of JEV based on the JEV sequence datasets. The results demonstrated that JEV lineages can be divided into four endemic cycles, comprising of southern Asia endemic cycle, eastern coastal Asia endemic cycle, western Asia endemic cycle, and central Asia endemic cycle. The isolation places of the viruses in each endemic cycle were geographically independent. Analysis of the viruses in each endemic cycle showed that JEV strains isolated from the southernmost region were present in the other three endemic cycles in addition to the southern Asia endemic cycle. Furthermore, these strains were rooted those in each endemic cycle, indicating that southernmost Asia maintains the viral population diversity and stability, and acts as a source from which JEV gradually disperses throughout the Asian continent. We also found that JEV has a dispersal pattern from south to north on the Asian continent through three likely transmission routes which correlates closely with the recognized eastern, central, and western flight paths of migratory birds in Asia. This finding indicates that the migratory birds might play an important role in the long-distance dispersal of the JEV.
The population dynamics analysis of dominant genotype JEV showed that there was minimal fluctuation during the first half of the plot, and it was followed by a major population increase, then a relatively stable period and a marked decrease, and again a relatively stable period. According to the time nodes of change in population dynamics identified in the Skyline plot, the isolation times of the JEV in the four endemic cycles were analyzed. The results further confirmed that the southernmost regions of the Asian continent play a key role as the source for evolution and dispersal of the JEV.
Although phylogenetic analysis of JEV shows that JEV had a characterization of geographical distribution, there was also geographical mixing phenomenon. It indicated that migration events (like genetic drift) may also contribute to the differentiation of JEV's distribution besides the population structure differentiation. Therefore, in order to better understand the spatial dynamics characteristic of JEV, the Migraphyla software was used to conduct the analysis. The results showed that there were frequent migration events during the dispersal of JEV in Asia, and these migration events also contributed to differentiation in the geographical distribution of JEV. Further analysis revealed that Thailand, Shanghai, Shandong, Sichuan and Vietnam were the important migration source region of JEV. Thailand is the source of JEV spreading to West Asia and East Asia region; Shanghai maintains the JEV circulating in the coastal areas of East Asia; Shandong is the source of JEV spreading from coastal areas to inland areas of China; Sichuan maintains the JEV circulating in the inland China; Vietnam is the source of JEV spreading from Southeast Asia to China's inland provinces.
2. Mosquito-borne arbovirus investigation in Hubei Province
In this study,22,269mosquito samples including Culex tritaeniorhynchus, Anopheles sinensis, Armigeres subalbatus, Culex pipiens pallens and unidentified mosquitos which belongs to5species in3genuses were collected in Wuxue City, Tongcheng County, Enshi Prefecture, Shennongjia, Jiangling County and Suizhou from July to August in2009and2010. Culex tritaeniorhynchus was the dominant species in Wuxue City, Tongcheng, Suizhou and Jiangling County; Culex pipiens pallens was the dominant species in Enshi; Armigeres subalbatus was mainly collected in Shennongjia. All samples were divided into272pools according to species, locations and collection time. After grinding and virus isolation,42positive isolates were isolated from four mosquito species including Culex tritaeniorhynchus, Anopheles sinensis, Armigeres subalbatus and Culex pipiens pallens. Through systematic identification, totally42virus strains were obtained including33JEVs,6Getah virus, and4Banna virus. Viruses isolated from Culex tritaeniorhynchus and Culex pipiens pallens accounted for the largest proportion, with21strains and15strains isolated respectively; and all three kinds of viruses can be isolated from Culex tritaeniorhynchus. Up to31strains virus were isolated from Enshi Prefecture. The pool positive rate and minimal virus infection rate in Culex pipiens pallens collected in Enshi was the highest, with53.6%and1:103.3respectively. The pool positive rate and minimal virus infection rate in Culex tritaeniorhynchus collected in Enshi was also high, with44.4%and1:128.8respectively.
The molecular characterization analysis of these newly virus strains showed that all33JEVs belonged to genotype I and the nucleotide and amino acid sequence homology between E gene was98.7%to100%and99.8%to100%respectively. There are14amino acid differences between the new JEV strains and SA14-14-2vaccine strain in the E gene, but the differences are not in the critical amino acid sites where determine the antigenicity. The newly isolated GETV has a close evolutionary relationship with strain HB0234and YN0540isolated in China and strain Korea isolated in South Korea in2004, and a far evolutionary relationship with LEIV/16275isolated in Russia. The sequences of segment12of newly isolated BAVs share a100%homology. Several sites were found in new BAVs which were different from strains isolated from other regions. Phylogenetic analysis revealed that all the new isolates can be grouped in the same subset with isolates from Beijing, Yunnan and Vietnam.
The significance of this study
This is the first time to carry out a spatial and temporal dynamics analysis of JEV in global scale. It clarified the dispersal characteristics and patterns of JEV in Asia and find out the geographical distribution characteristics and transmission source region in the dispersal of JEV. Also the migration events were found to contribute to distribution and differentiation of JEV. Migratory birds were speculated for the first time from the perspective of phylegenetic evolution to play an important role in the long-distance spread of JEV. The results also suggest the possibility that JEV can be transmitted to Europe and other non-endemic areas and provide an important basis for the global prevention and control of JE.
A mosquito-borne arbovirus investigation was also conducted in part of Hubei Province. JEV, Getah virus, and Banna virus were isolated from the mosquito samples collected. And Culex was found to be the species that carried the most viruses in Hubei province. Getah virus and Banna virus were isolated in Hubei Province for the first time; the isolation of JEV in Hubei also filled the blank of JEV's distribution in central Asia, which provided the conditions for the JEV spatiotemporal dynamics analysis. The study also carried out study on molecular biological differences between newly isolated virus in Hubei and other available isolates. It enriched the information about arboviruses in Hubei Province and China and provided the basic data for carrying out a further study. Moreover, it provided important information for understanding the etiology of local infectious diseases as well as conducting active prevention and control of arbovirus-related diseases.
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