Study On Arboviruses Isolated From Mosquitoes Collected In Hunan And Tibet,China

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.