Molecular Epidemiological Survey Of Bluetongue Virus And Genomics Analysis Of Midge Metagenomics In Yunnan Province

Midges are important insect vectors for various infectious viruses including bluetongue,deer epidemic hemorrhagic disease,vesicular stomatitis,and Schmallenberg disease,and are the vectors of many important animal infectious diseases.In recent years,with the gradual warming of the global climate,the ability of midges to spread diseases has been enhanced to a certain extent,and the distribution of midges has also expanded to high altitudes.Yunnan Province in China has a variety of arboviruses,of which bluetongue virus is one of the major viruses transmitted by mites.Bluetongue virus is an animal-borne infectious disease caused by the genus Rhodopsinvirus of the Reoviridae family.It can infect most domestic and wild ruminants.Bluetongue virus has been found to have 27 serotypes.Bluetongue virus has caused serious economic losses to the domestic animal husbandry industry.Therefore,relevant research on BTV must be done,and necessary testing methods must be developed to monitor the bluetongue virus of midges in Yunnan Province,China.This study aims to establish an effective monitoring tool and detection of bluetongue virus.According to the S10 gene of GenBank registered in GenBank and the S10 gene of BTV16 preserved in our laboratory,two universal primers for BTV were designed and optimal primers were selected to optimize the annealing temperature,cycle number and annealing of RT-PCR method.Time and extension time.The RT-PCR detection method was established and optimized,and sensitivity and specificity evaluation were performed.Testing was carried out to confirm the feasibility of the method.On the basis of establishing the RT-PCR detection method,a total of more than 250,000 Midges were collected from three counties in Zhaotong City,Yunnan Province and the bluetongue virus vector(Cangyu)in Puxian County,Pu’er City.After grinding,they were extracted.The virus RNA in the supernatant was extracted and reverse transcribed,and the blue tongue virus in the sample was detected by the established RT-PCR method.The supernatant was inoculated into BHK cells and C6/36 cells,and the viral RNA of the cell supernatant was extracted,and the S10 gene fragment was amplified by RT-PCR to detect the blue tongue virus carried by the Midges.The established RT-PCR method was used to detect 250,000 samples from Yunnan Province,and no blue tongue virus was detected in the library.The newly established RT-PCR method was used to detect diseased cells,and no blue was detected in the inoculated cells.Tongue virus.The 250,000 Midges collected were combined into three samples by region.Using metagenomic sequencing and bioinformatics analysis,abundant viral sequences were detected and more than 50 virus taxa were annotated,and then phylogenetic analysis was performed.According to the results of the metagenomic sequencing results and the Blastn nucleic acid sequence,the unenomic tree analysis and homology analysis confirmed the un-classified virus Hubei macula-like virus,Hubei permutotetra-like virus,Beihai picorna-like virus,of which Yunan Hubei macula-like virus 1 has 87.6%homology with Hubei invertebrate virus,and Yunan Beihai picorna-like virus 3 has 81.2%homology with Beihai invertebrate virus.Yunan Beihai picorna-like virus 3 and Beihai The homology to parvovirus was 95.4%.The proven La Jolla virus has at least 38%homology with the 2015 Australian La Jolla virus,and this distant relationship indicates that the parental virus may belong to the new La Jolla virus.In addition,many known viruses and some unknown viruses have been detected in midges in Yunnan Province,China.