Investigation Of Arbovirus And Its Molecular Characteristics In Border Areas Of Western Yunnan

Background:Arboviruses are a class of viruses that replicate in blood-sucking arthropods and transmit viruses to host animals through the blood-sucking process.Fever,bleeding and encephalitis are among the diseases and symptoms that humans,poultry,domestic and wild animals experience after being bitten.Arboviruses pose a significant threat to human and animal health worldwide,particularly in developing countries,it’s a major global public health problem.Yunnan Province is located on the southwest border of China,bordering Myanmar,Laos and Vietnam,and has a tropical and subtropical climate.Due to its unique geographical location and climatic characteristics,Yunnan Province is not only has the largest number of imported arboviruses,but also one of the provinces with the largest number of newly discovered arboviruses in China.Therefore,the systematic investigation of arboviruses in the western border of Yunnan Province is helpful to provide scientific basis for the prevention and control of local arboviruses.Objective:A survey of vector insects(mosquitos and midges)and their arboviruses was conducted to understand the species and distribution of vectors and viruses carried by the vectors in the western border area of Yunnan Province.The molecular identification of the newly isolated virus was carried out to clarify the taxonomic status of the newly isolated virus.On this basis,the whole gene sequencing and sequence analysis of a newly isolated Tibet orbivirus were carried out to identify the molecular characteristics and phylogenetic relationship of the newly isolated virus at the genomic level.At the same time,animal seroepidemiological investigation was conducted in the local area to understand the infection and prevalence status of the newly isolated virus in domestic animals,so as to provide a scientific basis for the prevention and control of insect-borne diseases in this area.Methods:1.In July 2018,mosquito and midge were collected in Mangshi,Yingjiang,Lushui and Tengchong,Yunnan Province,and the collected vectors were classified and identified by morphological classification.2.Grind the collected vectors and inoculate them into BHK-21 and C6/36 cells for virus isolation.After the cytopathy,the viral nucleic acid was extracted.The specific primers of arboviruses were used for RT-PCR.The positive products were sent to Biotech for sequencing.Biological software such as Cluster X,DNAStar,and Mega-X are used for sequence analysis.3.The newly isolated Tibet orbivirus named MS 18C217-16 genome was amplified by full-length amplification of cDNA(FLAC).Use bioinformatics software to analyze its genome structure and characteristics.At the same time,observe the proliferation of TIBOV MS18C217-16 and DH13C120 strains with different serotypes in BHK-21,C6/36,VERO,PK15,HELA,MDBK cells,draw the proliferation curve,and compare the biological characteristics of the two TIBOV strains with different serotypes.4.Select 680 serum samples collected in four regions of Yunnan Province since 2018,and use the microneutralization test to detect the neutralizing antibody of the newly isolated TIBOV MS18C217-16 strain in these animal seraum.At the same time,the neutralizing antibody titers of different serum types of TIBOV(MS18C217-16 strain and DH13C120 strain)in animal serum were compared by microneutralization test.The specific immune ascites prepared by different serum types of TIBOV virus strains DH13C120,SZ18 and JCC637-2 were used to carry out the cross immunoneutralization test on the newly isolated TIBOV MS18C217-16 strain.Results:1.In 2018,a total of 8581 mosquitoes collected from Mangshi,Yingjiang,Lushui and Tengchong were subjected to 4 genera 8 species(Culex,Anopheles,Armigeres and Aedes),Culex tritaeniorhynchus was the dominant species in Mangshi and Yingjiang,while Anopheles sinensis was the dominant mosquito in Lushui and Tengchong.A total of 18296 midges were collected,among which 7396 culicoides of 19 species were collected in Mangshi,and Culicoides striata was the dominant midge,with a composition ratio of 59.79%;A total of 4602 Culicoides of 15 species were collected in Lushui,and Culicoides Arakawa was the dominant midge,with a composition ratio of 48.11%.2.Mosquitoes were divided into 170 batches,and midges were divided into 203 batches for arbovirus isolation,112 positive isolates,49 from mosquitoes and 63 from midges were obtained.The batch positive rates of mosquito and midge virus isolation were 28.82%and 31.03%respectively.The batch positive rates of virus isolation in the four regions were Mangshi(51.45%),Lushui(22.73%),Yingjiang(7.41%),Tengchong(5.61%).3.After molecular biological identification,112 batches of positive isolates belonged to 10 kinds of viruses belonging to 8 families and 9 genera,including 33 strains of YUOV,83 strains of TIBOV,6 strains of BAV,5 strains of NDIV,1 strain of AKAV,53 strains of MAV,2 strains of CxFV,4 strains of CppDNV,1 strain of CuTLV,1 strain of ArIFV.4.Separation by PAGE revealed that MS 18C217-16 is a 10-segment dsRNA virus,with a segment pattern of 3-3-3-1.The genome has 19247nt nucleotide sequences,encoding 6616 amino acid,the amino acid homology of T3 and OC1 proteins of MS18C217-16 and 7 strains of TIBOV were 93.9%～97.7%and 27.7%～58%,respectively,suggesting that MS18C217-16 strain may be a novel TIBOV.Studies on the biological characteristics of the virus suggest that both MS18C217-16 and DH13C120 can proliferate in BHK-21,VERO,PK15,HELA,MDBK and C6/36 cells,among which virus plaque can be produced in BHK-21,VERO and PK15 cells.5.The animal seroepidemiological survey showed that neutralizing antibodies of MS 18C217-16 were detected in bovine serum,goat serum and swine serum of Mengla and Mangshi,swine serum of Jinghong and Jiangcheng.The antibody positive rates in the four regions were Mengla(23.86%),Jinghong(12.73%),Mangshi(8.11%),Jiangcheng(7.41%).The antibody positive rate of three kinds of animal serum was goat serum(38.46%),swine serum(12.86%),and bovine serum(3.72%).The comparison of antibody titers between MS 18C217-16 strain and DH13C120 strain suggests that nineteen out of twenty MS18C217-16 strain antibody positive sera were also positive for DH13C120 strain antibody,and one Mengla goat serum(ML 19)was negative for DH13C120 strain antibody.Compared with MS18C217-16 strain,the antibody titer of DH13C120 strain showed a four-fold increase in four sera,a two-fold increase in one sera,a two-fold decrease in four sera,and a four-fold decrease in five sera,and five sera had the same antibody titer.Indirect immunofluorescence tests were performed on the antigen tablets prepared from MS18C217-16 strain and the specific immune ascites prepared from TIBOV DH13C120 strain,SZ18 strain,and JCC637-2 strain,all three showed positive results,with antibody titers of 1:80,1:40,and 1:40,respectively.Conclusion:The results of this study suggest that there are many kinds of mosquitoes(8 species)and midges(29 species)in the border area of western Yunnan,the density of mosquitoes and midges is high.Ten kinds of viruses,nine genera and eight families,were isolated from these vectors,and the positive rate of virus isolation was 30%,indicating that there were many kinds of arboviruses and the vector carrying rate was high in the border areas of western Yunnan.The genome sequence analysis and serum cross neutralization test results of a newly isolated TIBOV suggest that the virus may be a new serotype strain,which can proliferate in many mammalian cells such as swine,cattle,monkeys,humans,mice,etc.At the same time,a relatively high level of neutralizing antibody of the virus has been detected in cattle,goat,swine in many regions of Yunnan Province,suggesting that the virus can infect humans or a variety of mammals,and may be a potential pathogen of animals.