Study On The Spatial And Temporal Dynamics Of Japanese Encephalitis Virus (JEV)

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 JEVIn 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 ProvinceIn 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 studyThis 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.