| 【Objectives】1.To understand the types,distribution and infection of mosquito-borne viruses in Xichang City,southern Sichuan Province,and Dongchuan District,northern Yunnan Province,in order to provide evidence for the prevention and control of mosquito-borne arbovirus diseases in this region.2.To understand the molecular characteristics and genotyping of Japanese encephalitis virus(JEV)and Banna virus(BAV)in Dongchuan from the molecular level of the whole genome,and to provide data for the prevention and control of JEV and BAV in Dongchuan.【Methods】1.In 2018 and 2016,mosquito traps were used to capture mosquitoes from Minzhu Village of Daqing Township,Xichang City,Sichuan Province,Chen Bao Village and Zhonghe Village of Gaocao Hui Township,Xiaoxin Village of Tongdu Subdistrict,Dongchuan District,Yunnan Province,and Batang Village of Oolong Town,respectively.Mosquito samples collected from these two areas were detected and the virus was isolated.The procedure for virus detection is as follows: Total RNA was extracted from the lapping solution,and c DNA was synthesized by reverse transcription.The c DNA was amplified by RT-PCR using the arbovirus specific primers of Flavivirus,Genovirus,Tibetan cyclic virus,Yunnan cyclic virus,Bunyavirus and alphavirus,and the amplified products were analyzed by agarose gel electrophoresis.Positive products were sent to sequencing companies for sequencing and sequence analysis using bioinformatics software such as DNAstar and Mega X.The steps of virus isolation were as follows: BHK-21 and C6/36 cells were respectively used for virus isolation,and the cytopathic changes were observed daily for 7 consecutive days.The positive samples with regular lesions were identified for virus identification,including RNA extraction,reverse transcription and RT-PCR amplification,the products were subjected to agarose gel electrophoresis,and the positive products were sent to sequencing companies.2.The JEV sequences of 2 isolates from Xichang City and 1 isolates from Dongchuan District were compared with 54 isolates from Gen Bank of different ages and regions of genotype I ～ V using Mega X and other software(proximity method)for phylogenetic analysis.3.Whole genome amplification sequencing was performed for the two strains of virus in Dongchuan District(random amplification for 18-1,FLAC amplification for55-33),and the amplified products were sent to the sequencing company for highthroughput sequencing.DNAstar,Mega X and other software were used for sequence splicing,nucleotide and amino acid homology analysis,differential sites and phylogenetic tree analysis.【Results】1.A total of 15,020 mosquitoes of 6 species were collected from Xichang City and Dongchuan District and tested for the virus in 176 batches.Six kinds of viruses were detected in Xichang City,and the positive rate of virus batches was 21.59%,including7.95% for BAV batches,7.95% for TIBOV batches,2.27% for JEV batches and 1.14%for YOUV batches.The positive isolates of XCM72-20 contained MXV and Cpp DNV.The positive rate of MXV and Cpp DNV batches was 1.14% and 1.14% respectively.Two viruses,including JEV and BAV,were detected in positive mosquito isolates from Dongchuan District.The positive rates of total virus batches were 3.41%,JEV batches were 1.14% and BAV batches were 2.27%.2.The two strains of JEV detected in the abrasive liquor of mosquitoes in Xichang City and the JEV identified in Dongchuan 55-33 were identified by phylogenetic tree analysis,and the three strains of JEV were located in a larger clade,all of which were located in the I-b clade.Further analysis showed that the two strains of Xichang City virus were located in a smaller clade,and were closely related to the Yunnan strain.The virus strain in Dongchuan District was closely related to the Yunnan strain in 2010.3.The full-length gene sequence analysis of 18-1 and 55-33 showed that 55-33 contained two virus sequences,namely JEV and BAV.The full length of JEV was10851 nt and the content of G+C was 51.97 %.The nucleotide and amino acid homology of 55-33 with gene I-b was 97.4% ～ 99.1% and 93.1% ～ 98.0%,respectively.The nucleotide and amino acid homology of 55-33 with gene I-b was higher than 95%in C,Pr M and E genes,and the nucleotide and amino acid homology with other genotypes(II ～ V)was lower than 95%.Amino acid homology was 77.2% ～ 95.3%,89.8% ～ 97.6% and 91.0% ～ 98.4%,respectively.The amino acid terminal sequence of55-33 in C gene was the same as that of genotype I to III(A-G-A),and there were 17 different loci in E gene from vaccine strain SA14-14-2(domain I:4;Domain II:7;Domain III:4,extraterritorially: 2),were different at eight virulence related sites(E107,E138,E176,E177,E264,E279,E315,E439).Phylogenetic analysis showed that 55-33 was located in the I-b branch of JEV genotype,which was closely related to the Yunnan strain,suggesting a close relationship with the other strains isolated from Yunnan.In18-1 and 55-33,BAVs contained 12 segments,all of which were in the size of Seg-1(3762 bp)～ Seg-12(861 bp),and the content of G+C was 39.23% and 39.37%,respectively.Based on phylogenetic analysis of whole gene ORF,these two strains of BAVs were located in the clta of genotype A.The nucleotide homology with genotype A BAV was 85.2%-96.8% and 73.9%-84.1%,respectively.The nucleotide homology with Yunnan strain was 95.4%-96.8% and 82.8%-84.1%,respectively.The two strains of Seg-9 were located in the evolutionary branch of genotype A,and had 83.1% ～ 97.9%and 83.0%-97.8% nucleotide homology with genotype A.Further analysis showed the highest nucleotide homology with Yunnan strain,which were 95.5% ～ 97.9% and 95.9%～ 97.8%,respectively.In Seg-12,the two strains were located in the clades of type A,and the nucleotide homology was 97.6%-98.4% and 96.8%-97.6%,respectively.Further analysis showed that the two strains had the highest nucleotide homology with the Yunnan strain A2 type BAV,which was 98.1%-98.4% and 97.4%-97.6%,respectively.【Conclusions】Six viruses were detected in the mosquitoes collected from Xichang City and Dongchuan district(6 in Xichang City and 2 in Dongchuan district).The total positive rate of virus batches was 12.50%,that of JEV batches was 1.70%,that of BAV batches was 5.11% and that of TIBOV batches was 3.98%.The positive rates of YOUV,MXV and Cpp DNV were 0.57%,suggesting the existence of a variety of arbovirus species and mosquito infection in Xichang.Phylogenetic tree analysis of the three JEV isolates from Xichang and Dongchuan showed that the isolates from Xichang and Dongchuan were located in different clades,but all of them were located in the I-b evolutionary clades,and were close to the isolates from southern Yunnan,suggesting that Yunnan strains were still prevalent in Yunnan province and some parts of southern Sichuan.At the genome-wide level,it is suggested that 55-33 belongs to genotype I-b virus,which is closely related to Yunnan strain.In the 8 important amino acid sites of E gene,55-33 is different from vaccine strain SA14-14-2,which is the same as Yunnan strain in recent years.It is suggested that SA4-14-2 can still be used for immunization,which provides scientific basis for vaccination.The two strains of BAV were both A2 subtype and closely related to Yunnan strain,suggesting that there was an epidemic of Yunnan strain in Dongchuan District.The detection of arboviruses should be strengthened as soon as possible to provide scientific data for the prevention and control of arboviruses. |
PDF Full Text Request