| Objectives:1.To understand the species,distribution and prevalence of arboviruses in Yuxi,Yunnan Province,provide a basis for the prevention and control of mosquito-borne arboviruses in this area.2.To understand the whole genome molecular characteristics and genetic evolution characteristics of 2 strains of Japanese encephalitis virus(JEV)in Tonghai County,Yuxi City,provide data for the prevention and vaccination of Japanese Encephalitis(JE)in this area.3.To understand the situation of arbovirus infection in domestic animals in Yuxi area.Methods:1.In 2015,mosquitos were captured overnight using mosquito traps in Tonghai County,Huaning County,Jiangchuan County and Chengjiang County,Yuxi City,Yunnan Province.Grounded mosquitoes were inoculated in BHK-21 and C6/36 cells for virus isolation.Extraction of RNA from positive isolates,Reverse transcription synthesis of first strand cDNA,using multiple pairs of arbovirus general primers or virus specific primers for RT-PCR amplification、Agarose gel electrophoresis analysis,Positive isolates were sent to sequencing companies for sequencing.Using bioinformatics software such as DNAstar and Mega X for sequence analysis.2.With reference to the complete genome sequence of GI JEV,a set of JEV full-gene primers was designed by using Primer 5.0 software.2 strains of JEV(HL-3,HL-32)in Tonghai County were amplified by RT-PCR and sequenced.Sequence splicing,homology analysis,differential sites and phylogenetic analysis were carried out by using software such as Seqman and Megalign,GeneDoc,Mega X.3.The serum of domestic animals collected from Tonghai County,Huanning County and Yimen County of Yuxi in 2019 and 2020 were detected for neutralizing antibodies against JEV(HL-3 strain)and Getah virus(GETV)(NM20 strain)by TCID-50.Results:1.A total of 13050 mosquitoes were collected from 4 counties of Yuxi city and ground in 261 batches.95 batches produced different cytopathic effects on golden hamster kidney cells(BHK-21 cells)and Aedes albopictus cells(C6/36 cells).A variety of arbovirus primers were used to identify the virus.The results showed that they were JEV,Banna virus(BAV),GETV,Tibet orbivirus(TIBOV),Nam Dinh virus(NiDV),Akaban virus(AKAV),Ngewotan virus(NWTV),Quang binh virus(QBV)and Culex pipiens pallens densovirus(CppDNV).Among them,there were 35 strains of 6 arbovirus in Tonghai County,88 strains of 6 arbovirus in Huaning County and 5 strains of 2 arbovirus in Chengjiang County.TIBOV and NWTV was identified in 3 counties,while NDiV was isolated in 2 counties.JEV,AKAV and QBV were isolated only in Tonghai county.BAV,GETV and CppDNV were isolated only in Huanning county.2.2 strains of JEV(HL-3 and HL-32)isolated in Tonghai county were amplified with 16 pairs of primers.After sequencing and splicing,the gene sequences of 10965 nucleotides(nt)were obtained,including a 10299nt open reading frame(ORF),encoding 3432 amino acid(aa).The nucleotide and amino acid homology of newly isolated HL-3 and HL-32 strains with GI JEV were 93.6%～98.9%、98.0%～99.7%.Compared with GII～GV,the JEV nucleotide homology was less than 90%,and the amino acid homology was less than 98.0%.Genetic evolution analysis showed that HL-3,HL-32 and GI-b JEV strains were in the same evolutionary cluster,suggesting that the 2 newly isolates were GI-b JEV.Further analysis showed that the 2 newly isolates were in the same smaller evolutionary branch with the epidemic isolates in Yunnan Province in recent years,suggesting that there was a close genetic evolution relationship between them.There were 56 amino acid difference sites between HL-3,HL-32and the JE attenuated live vaccine strain SA14-14-2,there were 12 amino acid difference sites on the E gene,and 8 virulence related sites(E107,E138,E176,E177,E264,E279,E315,E439),there were 6 different sites.3.A total of 275 serum samples from pigs,cattle and sheep were collected in Huaining,Tonghai and Yimen counties.The JEV virulence of HL-3 strain was 10-4.5/100μL,and the GETV virulence of NM20 strain was 10-7.3/100 μL.JEV and GETV neutralizing antibodies in serum collected from Yuxi area were negative.Conclusion:1.A total of 128 strains of 9 kinds of arbovirus were isolated and identified in Tonghai County,Huaning County,Jiangchuan District and Chengjiang County of Yuxi,which distributed in different districts and counties,and the types of arboviruses prevalent in different districts and counties were different,indicating that there are many kinds of arboviruses in this area and they are widely distributed.2.The 2 JEV strains isolated from Tonghai County,Yunnan Province had the highest nucleotide and amino acid homology with GI JEV,and were located in the same evolutionary cluster with GI-b strains,which belonged to GI-b JEV.There were 2 mutations in the key sites related to virulence of the JEV E gene,but the key antigens of domain Ⅲ did not change.SA14-14-2 attenuated vaccine strain can still be used for immunization of people in this area to prevent the occurrence and epidemic of JE.3.The neutralization antibodies of JEV and GETV were not detected in the animal serum collected from Yuxi area,but the 2 arbovirus still pose a potential threat to people and animals in this area.It is necessary to strengthen the surveillance and detection of these arboviruses in people or domestic animals in this area,so as to provide scientific basis for the prevention and control of arbovirus diseases in this area and even in Yunnan Province. |
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