Arboviruses Investigation In Gansu And Shaanxi Provinces And Molecular Characterization Of Virus Isolates

Gansu and Shaanxi provinces are located in the northwest of Mainland China,which is the gateway through northwestern inland and is also the boundary of distinguishing climates.The two provinces are still the epidemic area of Japanese encephalitis(JE) for many years.In addition,unknown viral encephalitis cases were also reported during the JE epidemic time every year.Several investigations showed that many different insect vectors distribute in these two provinces,which suggests that it could exist many arboviruses besides JE virus.This study conducted arboviruses investigation in Gansu and Shaanxi provinces from July to August in 2006 and 2007.The aim of this work is to know the arboviruses species and their distributions in these two provinces.This study had also identified and studied the molecular characterizations of arbovirus isolates isolated during arboviruses investigations in China in recent years.1.Arbovirus investigation in Gansu provinceMosquito samples were collected in the villages of 11 counties belonged to Tianshui, Longnan and Pingliang cities during July to August in 2006 and 2007.13872 individuals covering 4 genus and 6 species had been collected.The mosquitoes were sorted into 206 pools for virus isolation.Total 30 virus isolates were obtained by inoculating the grinded supernatants onto C6/36 and BHK-21 cells and followed by observing cytopathic effect(CPE).The virus isolates were from five species including Culex tritaeniorhynchus,Anopheles sinensis,Armigeres subalbatus,Culex pipiens pallens and Aedes sp.24 isolates were observed to cause CPE in C6/36 cells only, while the other 6 isolates caused CPE both in C6/36 cells and BHK-21 cells.33 virus strains from these 30 isolates were identified by using morphological,serological and molecular biological methods.They are 4 Japanese encephalitis viruses(JEV),2 Getah viruses(GETV),10 Banna viruses(BAV) and 17 Armigeres subalbatus Totiviruses(AsTV).It was found that 20 isolates covering all these 4 different virus species could be isolated from Culex tritaeniorhynchus,which demonstrated that this species is the dominant vector in the local area.Molecular analysis of the new isolates showed that 4 JEVs are all clustered into genotypeâ… .The new strains showed very high identities in nucleotides and amino acids of E gene,which are 98.3%-100%and 99.8%-100%respectively.11 sites of differences were found between the new strains and the vaccine strains SA 14-14-2 in E protein.These sites were not the key sites that affect the antigenic and virulence of JEV.It is the first time to report that genotypeâ… JEV has spread into Gansu province. Analysis of the 2 GETVs found that there are very close genetic relationship between Gansu strains and other GETV isolates.The identities of the Capsid gene between all strains were more than 97%both in nucleotide and amino acid levels.One amino acid insertion was found in the site aa 76 of Capsid protein.In 3'UTR,a ten nucleic acid deletion occurs in position 45-54.The genome electrophoresis profiles of the ten BAV isolates showed small differences from the Beijing strain(BJ95-75).The 6^th fragment showed shorter and the 9^th fragment showed larger than the Beijing strains' in the PAGE profile.Sequence analysis about the 12^th fragment found that one nucleic acid deletion occurred in all of the ten BAVs.The coding region of VP12 was 627 nt, which was different from the other strains including China and Indonesia isolates which had 624 nt long.Two continued initiation codon(ATG) were found only in Gansu strains,while the stop codon(TGA) was the same to Indonesia isolates but different from the others.The nucleotides and amino acids identities of VP12 were 91.4%-93.3%and 91.8%-93.7%between Gansu isolates and genotype A strains,and were 88.6%-89.9%and 85.5%-87.9%between Gansu isolates and genotype B strains, respectively.Sequence and phylogenetic analysis showed that the BAV Gansu isolates possess distinct regionally differentia and should be identified as a novel subtype of this species. 2.Arbovirus investigation in Shaanxi province2765 mosquito samples were collected in the villages of Ankang and Baoji cities during the arbovirus investigation in Shaanxi province in September of 2006.Virus isolation and identification results showed that two viruses were isolated from Armigeres sp.,one is insect densovirus CppDNV and one is AsTV.3.Molecular analysis of arboviruses isolated in China.10 strains of GETV isolated from five provinces during arboviruses investigations in China since 1964 were analyzed.The full-length sequences of three Chinese isolates (M1,HB0234,YN0540) were determined.The full-length sequences of these isolates were 11696nt,11686nt and 11690nt respectively,and showed the identities of 97.4%-98.8%and 98.7%-99.6%in nucleotide and amino acid level,respectively.The E2 gene of the 10 Chinese GETV isolates showed identity values of 98 to 100%for nucleic acids,when compared with other GETV isolates.The 3'UTRs of the Chinese isolates showed a deletion between positions 45 and 54,and nucleotide transition at positions 43,64 and 148.This deletion was the same to Taiwan district,Russia and Mongolia isolates.Sequence and phylogenetic analyses showed that there was a relatively high degree of conservation among GETV isolates,with sequence particularities specific to the Chinese isolates.16 Culex pipiens pallens densovirus isolated from Liaoning,Yunnan,Xinjiang and Guizhou provinces were identified and analyzed.Sequencing results showed that the complete coding region of CppDNV is 3335nt,and that it contains 3 open reading frames(ORFs).These were ORF1(2376 nt) encoding non-structural protein 1(NS1), ORF2(1092 nt) encoding non-structural protein 2(NS2) and ORF3(1071 nt) encoding capsid protein(VP).CppDNV shares 82 to 93%identical nucleotides with isolates of the Aedes albopictus densovirus(isolates AalDNV-1,AalDNV-2 and AalDNV-3),Aedes aegypti densovirus,and Haemagogus equines densovirus.The nucleotide sequence identity among CppDNV isolates exceeds 98%.Phylogenetic trees based on VP,NS1 and NS2 gene show that CppDNV clustered with the species Aedes aegypti densovirus and should be identified as a new variant of this species within genus Brevidensovirus. The complete sequence of AsTV strain SaX06-AK20 was determined and genomic characterization was analyzed.The result showed that AsTV has a genome with 7510 nt long,in which two ORFs were found.ORF1(4443 nt) encodes a structural protein Cote protein,ORF2(2286 nt) encodes a nonstructural protein RNA-dependent-RNA polymerase.Nucleotides and amino acids identities between AsTV and members of family Totiviridae were 17.5%-41.4%and 5.3%-43.1%respectively.Phylogenetic analysis showed that AsTV should be identified as a novel member of the family Totiviridae or the species representation of a novel dsRNA family that infects invertebrates.4.other workIn addition,this study have also identified and / or analyzed 7 strains of virus isolates isolated from Henan province in 2006,one isolate from Xinjiang in 2005 and 6 strains of isolates isolated from Guizhou province in 2006.The result showed that 7 Henan isolates and 3 Guizhou isolates were genotypeâ… JEV,while the Xinjiang isolate M0507JS9 should be identified a new member of the family Tymoviridae.In summary,arbovirus investigation in Gansu and Shaanxi provinces were carried out in this study during 2006 and 2007.Results showed genotypeâ… JEV was the first time to be isolated from Gansu and the BAV isolates was found to possess distinct regionally differentia.The new virus AsTV could be isolated from both Gansu and Shaanxi province.These findings give us a clew to strengthen arbovirus surveillance in order to prevent arbovirus diseases.Identification and / or molecular analysis of GETV,CppDNV,JEV 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.