Arboviruses Investigation In Shanxi And Henan Provinces And Molecular Evolutionary Genetics Analysis Of Tibet Novel Orbiviruses

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.