Isolation And Identification For Arboviruses Isolated In Mosquitoes Collected In Shanxi Gansu And Jiangxi Provinces

Arboviruses are a class of viruses that can spread diseases by blood-sucking arthropod bites vertebrates. Now more than 500 species of Arbovirus have been found, of which 100 kinds has a close relationship with human and animal diseases. They can cause fever, rash, arthritis, hemorrhage and meningoencephaliris, even cause death. Many emerging infectious diseases such as West Nile Fever and Rift Valley Fever are also arbovirus disease. Therefore, arbovirus and arbovirus diseases have become the public health issue that cause attention of international community.Since the 50's in the 20th century, four kinds of arboviruses and arbovirus related diseases have been confirmed in our country. That is Japanese Encephaitis Virus, Spring-Summer Encephalitis Virus, Xinjiang Haemonhagic Fever Virus and Dengue Fever Virus serotype 1～4. The area of China is vast and the number of insects is large. Arboviruses in our country are far more than the number of currently known, but lack of background investigation. In this study, arbovirus investigation was carried out in Shanxi, Gansu and Jiangxi provinces in the year of 2006～2008. Mosquitoes were collected from July to August, virus isolates were isolated by cell culture and the virus isolates were identified by serological and molecular biology to make it clear that the type and distribution of arbovirus in the local. This study has an important epidemiological significance in understanding background information of arbovirus in our country and provide evidence for arbovirus diseases control and prevention.1. Arbovirus investigation in Shanxi provinceMosquito samples were collected in the villages of 7 counties belong to Yuncheng, Linfen, Yangquan and Xinzhou cities during 2006 and 2007. 17860 mosquitoes had been collected. The mosquitoes were sorted into 144 pools for virus isolation. Total 15 virus isolates were obtained by inoculating the grinded supernatants onto C6/36 and BHK-21 cells and followed by observing cytopathic effect (CPE). 15 virus strains were identified by using morphological, serological and molecular biological methods. They are 10 Banna viruses (BAV), 2 Liaoning viruses(LNV) and 3 Culex pipiens pallens densovirus(CppDNV). In addition, there are 2 virus isolates need further identification. 6 BAV strains were isolated from Culex pipiens pallens, 1 BAV strain isolated from Aedes dorsalis while the remaining isolated from mosquito species mixed. 2 LNV strains were isolated from Culex pipiens pallens and Aedes dorsalis respectively. 2 CppDNVs were isolated from Culex pipiens pallens and 1 CppDNV was isolated from Armigeres obturbans.Sequence analysis about the 10 BAVs 12^th fragment found that the homology is high between 6 BAV strains, the homology is 98.7%～100% in nucleotides sequence and 98.1%～100% in amino acids sequence respectively. Compared with Banna virus standard strain(BANNA), the homology is 90.5%～91.2% in nucleotides sequence and 93.2%～94.2% in amino acids sequence respectively. Phylogenetic analysis showed that the Shanxi BAV isolates possess distinct regionally differentia. Molecular analysis of 2 LNV strains 10^th fragment partial nucleotides sequence showed that the homology between 2 LNVs is 100%. Compared with Liaoning virus standard strain(LNV-NE97-31), the homology is the lowest which is only 76.7%. Phylogenetic tree showed that except LNV-NE97-31, all the Chese LNV strains are located one branch. Sequence analysis about CppDNV non-structural protein 1 (NS1), and non-structural protein 2 (NS2) partial nucleotides sequence showed that 3 CppDNVs have high homology with other Chinese CppDNV strains, which is between99.9% and 100%. Phylogenetic tree showed that all the Chese LNV strains are located one distinct branch and has a close phylogenetic relationship with AaeDNV.In this study, BAV, LNV and CppDNV virus were first reported in Shanxi Province.2. Arbovirus investigation in Gansu provinceMosquito samples were collected in the villages of 5 counties belong to Zhangye, Jiuquan, Jiayuguan, Baiyin and Pingliang cities during July to August in 2008. 13839 mosquitoes had been collected. The mosquitoes were sorted into 280 pools for virus isolation. Total 6 virus isolates were obtained by inoculating the grinded supernatants onto C6/36 and BHK-21 cells and followed by observing cytopathic effect (CPE). 6 virus strains were identified JEV by using serological and molecular biological methods. 6 JEV are all isolated from Culicine.Molecular analysis of JEVshowed that 6 JEVs are all clustered into genotype I. The homology is high between 6 JEV strains in E gene, the homology is 98.1%～99.9% in nucleotides sequence and 99.6%～100% in amino acids sequence respectively. Compared with SA14-14-2 in E gene, the homology is 87.5%～87.9% in nucleotides sequence and 96.8%～97.2% in amino acids sequence respectively. There are 11 sites of differences were found between the 6 JEV strains and the vaccine strains SA14-14-2 in E protein, but these sites were not the key sites that can affect the antigenic and virulence of JEV. The new strains showed a close phylogenetic relationship with JEV strain isolated from Sichuan in 2004 and JEV strain isolated from Shanghai in 2005.The past arbovirus investigation in Gansu province is limited in the southern areas, not involved in the northwestern region. In this study, it is the first time to carried out arbovirus investigation in Zhangye, Jiuquan and Jiayuguan City of northwestern region and Baiyin City of eastern part of Gansu province. 6 genotype I Japanese encephalitis viruses were isolated from Baiyin City, this is the first time that isolated Japanese encephalitis virus from this region and indicated that JEV has spread to inland areas in Northwest China.3. Arbovirus investigation in Jiangxi provinceMosquito samples were collected in the villages of 5 counties belong to Ji'an, Ganzhou, Yichun, Fuzhou and Shangrao cities during July to August in 2008. 11916 mosquitoes had been collected. The mosquitoes were sorted into 240 pools for virus isolation. Total 14 virus isolates were obtained by inoculating the grinded supernatants onto C6/36 and BHK-21 cells and followed by observing cytopathic effect (CPE). The 14 virus isolates can only caused CPE on BHK-21 cells. By using serological and molecular biological methods can rule out Flavivirus, Alpahvirus, Bunyavirus and dsRNA virus. Further identification for 14 virus isolates is doing.331 serum samples from patients with fever were collected in the local hospital. To detect Japanese encephalitis IgM antibodies by ELISA kit. 4 positive samples were detected and the research of other pathogen which can cause encephalitis and fever is in progress.In summary, isolation and identification for arboviruses isolated in mosquitoes collected in Shanxi Gansu and Jiangxi provinces were carried out in this study during 2006 and 2008. Total 21 virus isolates were obtained. There are 10 BAVs, 2 LNVs, 3 CppDNVs and 6 JEVs. These result not only complete the baseline data of arbovirus in these area but also provide evidence for arbovirus diseases control and prevention and can accumulate information for distribution of arbovirus and arbovirus diseases.