| Bluetongue disease is a non-contact arbovirus disease transmitted by hematophagous Culicoides,and its pathogen is bluetongue virus of orbivirus of reoviridae.As a kind of animal disease spread in China,bluetongue disease is characterized by strong infectivity,high fatality rate,wide distribution,rich serotypes and frequent cross-border transmission,many animals were susceptible to BTV.It has seriously affected the improvement of epidemiological data of the disease,the establishment of universal diagnostic technology,and the development of broad-spectrum high-efficiency vaccine.BTV has caused a lot of losses to the global animal husbandry.Yunnan province is located in the southwest border area of China with the most abundant BTV serotypes.The torridity and humid climate,abundant animal trade and breeding vector Culicoides provide suitable conditions for the spread of bluetongue disease.The purpose of this study was to investigate the molecular epidemiology of BTV in cattle in Yunnan province border area of China,and to clarify the infection status,dominant serotypes and evolutionary variation of BTV in cattle.At the same time,the isolation and genetic evolution analysis of BTV in Culicoides from yunnan province border area and metagenomic analysis of BTV in Culicoides were carried out to understand the background,species diversity and molecular relationship of Culicoides transmission virus,which lay a foundation for the effective prevention and control of bluetongue disease and the early warning of new and recurrent infectious diseases caused by virus in Culicoides.The main results of this study are as follows:(1)To clarify the prevalence of BTV in yunnan border areas of China,a total of2,259 cattle blood samples from 7 border cities were monitored for BTV from 2018 to2019 and 2021.The total positive rate of BTV in cattle in yunnan border area was17.00%,and the positive rate of BTV in cattle in different border cities was13.26%-20.65%.And the BTV positive rate of cattle in border cities increased from11.86% of 2018 to 23.22% of 2021.The results showed that the BTV infection rate of cattle in 7 border cities of yunnan province maintained high levels and had an increasing trend.To further analysis of the evolution of BTV,S10 gene was amplified in some blood samples,and a total of 28 BTV S10 gene sequences were obtained.The nucleotide homology ranged from 81.2% to 100%,forming an evolutionary branch in the evolutionary tree,which was most similar to the standard strain of BTV in eastern region countries.(2)In order to understand the molecular epidemiological characteristics of BTV in the border area of yunnan,chicken embryos were used to inoculate with BTV from cattle,and a total of 5 BTV strains were harvested.Serotypes of BTV were identified by S2 Gene.2 strains of BTV-1 were isolated from Baoshan city(2019)and Pu ’er city(2021),1 strain of BTV-2 was isolated from Pu ’er city(2019),1 strain of BTV-16 was isolated from Dehong autonomous prefecture(2021),1 strain of BTV-21 was isolated from Banna autonomous prefecture(2019).In order to further analysis the genetic variation of BTV,the homology of S2 gene of BTV in Yunnan border area was compared.The results showed that the BTV strain in yunnan border area had higher genetic stability and had the highest homology with the corresponding serotype reported in China.The amino acid sequence alignment showed that the amino acid mutation sites of VP2 protein of Baoshan city,Pu ’er city and Simao county strains involved antigen epitopes.Genetic evolution analysis of S2 gene showed that there were 5 BTV isolates in the same evolutionary branch with the Chinese strain,and the molecular relationship was similar.These results provide reference for understanding the epidemic situation,vaccine adaptation,cross-border transmission routes and genetic variation of bluetongue disease.(3)In order to understand the abundance of virus in Culicoides,Culicoides abrasive solution was inoculated into insect cells for repeated transmission,and a total of 3 strains of virus were obtained.Serotypes of BTV were identified by S2 gene PCR,and 2 strains(collected in Dehong in 2020)were identified as BTV-16.Homology and evolutionary analysis of S2 gene of the two Culicoides BTV strains showed that the two strains were closely related to BTV-16 strains in China and western regions.The results of viromics research showed that culicoviruses mainly belonged to seven viral families,including flaviviridae,embranviridae,Chuviridae,retroviridae and herpesviridae.The phylogenetic analysis of the glycoprotein coding genes of the representative arbovirus chu virus showed that the chu virus sequences obtained in this study had high homology with the same virus sequences reported,and clustered into an evolutionary branch.Bioinformatics prediction showed that GUTV glycoprotein was a hydrophilic protein with transmembrane structure,no signal peptide,subcellular localization to the nucleus or plasma membrane,72 potential phosphorylation sites and 10 epitopes.Besides,PERV SYBR Green I qPCR method was also established for PERV of retroviridae,which had high sensitivity,good repeatability and strong specificity,and was convenient for the detection of clinical samples.These results provide support for studying the mechanism of adaptation evolution of BTV in Culicoides to ruminant origin,as well as molecular traceability,pathogenicity analysis and disease early warning of viruses carried by Culicoides. |
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