Distribution And Molecular Characterization Of Arboviruses In Yunnan Province Near The China-Myanmar-Laos Border

An investigation was conducted to identify mosquito species and mosquito-borne arboviruses near the border of China-Myanmar-Laos in consecutive year. Several arboviruses have been isolated from the mosquito sample. Identification and molecular characteristics of virus isolated from Yunnan province were preformed using molecular biological methods. The results were reported as follows.1 Distribution of mosquitoes and mosquito-borne arboviruses in Yunnan Province near the China-Myanmar-Laos borderIn July 2005 and July 2006, an investigation was conducted to identify mosquito species and mosquito-borne arboviruses at three villages in Yunnan Province (Manxi, Manguo and Manen) on the border of China-Myanmar-Laos border. A total of 14,723 mosquitoes representing four genera (Culex, Anopheles, Armigeres, Lufzia) and seventeen species were collected. Culex tritaeniorhynchus was the dominant mosquito species in Manxi village (62.71%/1838/2931)and Manen village(90.98%/5924/6511), while Anopheles sinensis was the most abundant species in Manguo village (67. 11%/3544/5281). A total of 14,723 mosquitoes were divided into 152 mosquitoe pools. Fourteen strains of mosquito-borne viruses were isolated from Culex tritaeniorhynchus, representing six different viruses in five genera 4 familys:1 strain Sindbis virus (SINV) of the genus Alphavirus, family Togaviridae; 2 strains Japanese Encephalitis virus (JEV) of the genus Flavivirus, family flaviviridae; 1 strain Novel Seadornavirus (NSEAV) of the genus Seadornavirus, family Reoviridae; 6 strains Yunnan orbivirus (YUOV) and 1 strain Novel orbivirus (NOBV) of the genus Orbivirus, family Reoviridae; 3 strains Culex pipiens pallens Densovirus (CPPDenV) of the genus Densovirus, family Parvoviridae. In addition, IgM antibodies against Japanese Encephalitis virus, Sindbis virus, Yunan orbivirus and novel seadornavirus irus were detected in acute serum samples collected from hospitalized patients with fever and encephalitis near the areas where the viruses were isolated, and the total positive rate was 36.1%. This investigation suggests that JEV and SINV as well as lesser-known arboviruses circulate and are causing human disease in the China-Myanmar-Laos border region. Detection and monitoring of multiple arboviruses besides JEV should be strengthened in local areas.2. Molecular Identification of Arboviruses isolated from Yunnan ProvinceA total of 26 stains arboviruses isolated from mosquitoes, bats and patients in Yunnan Province since 1980s were identified using the method of molecular biology, including 19 strains of JEV (of which 17 strains are geneⅢ, two belong to genotypeⅠ),3 strains of Dengue Virus 4 (DENV-4) (belong to genotypeⅠ),1 strain of Kyasanur Forest disease virus (KFDV) and 3 strains of Theilovirus.3. Molecular Characteriistics of Full-genome of two JEVs isolated from bats collected from Yunnan ProvinceTo understand Molecular Characterization of Full-genome of two JEVs isolated from bats collected from Yunnan Province, the full-genome sequence of them was determined and analyzed. The results indicated that the two virus genomes have the same length at 10,977 nt each with a 95-nt 5'non-translated region (NTR) and a 583-nt 3'NTR. The two bat JEV isolates have an overall sequence identity of 99.9% both at nucleotide and protein levels. When compared to 55 known JEV isolates of known full-genome sequences, the nucleotide sequence identity varies from 88.6 to 99.3%, and aa sequence identity from 97.0 to 99.3%. Phylogenetic trees derived from full-length genome or the most variable E gene of selected JEV strains indicated that both JEV isolates are placed within the genotypeⅢ. A more detailed analysis indicated that the bat JEVs are most closely related to human isolate LiYujie and mosquito isolate BN19 within cluster. These data indicate that bats may play an important role as a reservoir and/or transmitting host of JEV in certain parts of the world. Sequence analysis of the full-genome of two JEVs isolated from bats was firstly performed in this study.4. Molecular Characteristics of Full-genome of sindbis virus (MX10) isolated from Yunnan ProvincePrevious investigation shows that MX10 virus recently isolated from Yunnan of China belongs to Oriental-Australian (O/A) genotype of Sindbis virus (SINV). Similar to MRE16 isolate, the prototype O/A genotype of SINV, two virus strains with obviously different plaque morphology were derived from MX10 virus by using plaque purification, and the diameters of larger plaque and small plaque were (1.92±0.08) mm (n=10) and (0.29±0.07) mm (n=10) respectively, which were accordingly denominated as MX10-LP and MX10-SP. The reproduction capacity of MX10-SP virus in BHK-21 cells was only 4 folds lower than that of MX10-LP virus. In contrast, MRE16SP (the small-plaque variant of MRE16 virus) released 100 folds less particles than MRE16 did. Moreover, the neurovirulence of MX10-LP and MX10-SP in neonatal mice showed significantly different. Analysis on whole viral genome suggested that the length of whole genome of MX10-LP and MX10-SP [not including 5'cap and 3'poly (A)] were both 11700nt, and 7 seven nucleotide differences were found between the whole genome of both viruses, whose nucleotide homology was 99.9%. The nucleotide homology between the whole genome of MX10-LP and MX10-SP as well as MRE16 was only 91.3%, and the amino acid (aa) homology was 98.1%. Compared with MRE16 virus, MRE16SP deleted 30 aa (aa 200-229) in E2 gene, which was proved to be the molecular basis for different plaque morphology. However, MX10-SP virus had no deletion of 30 amino acids in E2 gene. Alignment of NS3 gene showed that, compared with Paleoarctic-Ethiopian (P/E) and Western-Australian (W/A) genotype of SINV, O/A genotype of SINV inserts multiple nucleotide in NS3 gene (nt 1027-1107). For example,57 nucleotides of insertion was found in NS3 gene of O/A genotype prototype MRE16 virus, and both of MX10-LP and MX10-SP had 63 nucleotides insertions in corresponding position. These results demonstrate that the O/A genotype of Sindbis virus isolated from China exhibited itself phenotypic and molecular characteristics distinctly different from the prototype MRE16 of this genotype, indicating O/A genotype of SINV might have multiple variants with different molecular basis. It was the first isolate of O/A genotype of SINV from China.5 Identification of three virus isolates from bat of Yunnan Province and its infection in humanIdentification of three virus isolates (B90-121-125,B90-126-130 and B8613) from Rousettus leschenaulti were performed by using the method of molecular biology, such as sequence independent single primer amplification (SISPA) and RT-PCR. The results showed that partial nucleotide sequences of three virus isolates from bat were 94.7% identical to that of Theilovirus DA strain, and phylogenetic analysis indicate that three isolates from bat and Theilovirus were in the same genetic cluster. Analysis on full genome suggested that the length of whole genome of B90-126-130 virus was 8094nt, and encoding 2301 aa. Compared to the full-genome sequences of 46 known Cardiovirus genus virus isolates of, the nucleotide homology between the full genomes of B90-126-130 and Theilovirus DA strain was 90.3%, and aa sequence homology was 99.9%. The nucleotide homology between the full genomes of B90-126-130 and the other 8 Theilovirus isolates varied from 86.9% to 88%, and aa sequence from 94.3% to 96.3% respectively.3 isolates from bat could kill one-day-old neonatal mice on the 5th day post inoculation and could not caused CPE on C6/36, BHK-21, Vero, MDCK or Tubli cells. Nucleotide of B90-126-130 virus was detected in the 3rd passage supernarnt of Tubli cells culture by using RT-PCR by Theilovirus specific primers. A serosurvey focused on B90-126-130 viruse was carried out by ELISA in Xishuangbannan and Dehong Prefecture of Yunnan province. The results showed that the positive rate of IgM or IgG antibody against B90-126-130 virus was 17.14% or 43.82% in 461 cases with fever or encephalitis patients respectively. IgG antibody against B90-126-130 virus (7/32) was also detected from the serum of Bat collected form Dehong Prefecture of Yunnan province. These results suggest that 3 isolates from bat was identified to be Theilovirus, and can replicated in Tubi cell and have high neuroviluence. In addition, the results of serosurvey suggest that B90-126-130 virus was an emerging or potential pathogen causing unknown fever and encephalitis in the location.Conclusion and creationIntegrated entomological-viral-serological surveys were firstly conducted on the border area of China-Myanmar-Laos. Several arboviruses, including known and potential pathogens, were isolated from the border area, some of which were firtly isolated in China. Moreover, the human infection caused by these viruses has been proved in this investigation. These results lay basis for elucidating the pathogen of the local patients with unknown fever or encephalitis, and play a curial role in public health. Identification and classification of 26 stains arboviruses isolated from mosquitoes, bats and patients in Yunnan Province since 1980s were performed by using the method of molecular biology, and the relationship between these viruses and the local infectious disease was also clarified. Our findings contribute to growing information about arboviruses as well as to controlling and preventing the infectious disease caused by arboviruses in Yunan province, or in China, even internationally.