Isolating And Sequencing Of The Bluetongue From Cattle In Xinjiang

Blue Tongue(BT)is an acute viral vector disease which is transmitted by the virus of Bluetongue(BTV)through the bites of insects such as midges and Aedes albopictus.The world Organization for Animal Health(OIE)stipulates that the Bluetongue disease is a OIE listed diseases and one of Class A diseases in China.The mortality rate of this disease is generally around 10,because of the increasing incidence of the disease in recent years,the animal trade has been seriously affected.Based on the epidemiological history of Bluetongue disease in Xinjiang and the habits of vector insects,combined with factors such as geographical location,climate characteristics,and pastoral distribution,the project selected a site in the town of Dunkuo Township,Yuli County as a monitoring point for the establishment of a monitoring group.In the year of screening animals aged less than 1 year,10cattle,10 goats and 10 sheep with negative BT test results were selected as sentinel animals to establish monitoring groups.Blood samples were collected once a week from a monitoring animal group.Each blood collection tube(separated serum),heparin sodium anticoagulation blood collection tube and EDTA anticoagulation blood collection tube was used for 3 tubes.From June 28,2016 to December 6,2016.The Japanese sample was collected 21 times and 492 serum samples were collected.Serum BTV antibodies were detected by BTV competition ELISA kit.Blood samples of all the collected monitoring animals were inoculated into BHK-21 cells,Vero cells and C6/36 cells for BTV isolation.The cells were observed for lesions on a daily basis.Identification of cell fluids with cytopathic effect(CPE);group-specific identification using BTV antigen capture ELISA(AC-ELISA),RT-PCR amplification of BTV VP7fragments and immunoelectron microscopy;type-specific identification using virus neutralization Experiments;whole-genome sequencing and preliminary analysis of the isolated BTV(4630 sample).492 serum samples were tested by BTV competitive ELISA.The total positive rate was 0.41%(2/492),and the goat positive rate was 1.29%(2/155).The conversion time was October 10 and October 17.The sheep and cattle in the monitoring group did not turn positive.After inoculating cells with 492 blood samples,CPE occurred in 18 samples.Among them,12 samples showed CPE on the 3rd day of blind passage to the 3rd generation,and 6 samples appeared on the 3rd day of blind passage to 4th generation.In CPE,4 samples from 18 samples of CPE originated from cattle.Eighteen CPE samples were tested by AC-ELISA and all showed BTV positive.4 cattle samples were amplified by BTV VP7 RT-PCR and a single band of target bands appeared at 1100 bp.The TCID₅₀ was determined on 4 bovine samples with TCID₅₀ between 10^-3.25/0.1 m L and 10^-4.33/0.1 m L.Neutralization experiments were performed on sample No.4630.The results showed that No.4630 samples and BTV-1?BTV-24 type serum had no neutral reaction and did not belong to BTV-1?BTV-24 type.Immunoelectron microscopy was performed.Viral particles with typical characteristics of the Orthomyxovirus were observed.The whole genome of the isolate BTV/XJYL/2016/01 was sequenced.The sequencing results showed that the isolated strain BTV/XJYL/2016/01 had the highest similarity to the BTV V196 strain isolated from Xinjiang and the XJ1407 strain,and the isolate BTV/XJYL/2016/01 was indeed a Bluetongue virus and a new serotype.It is the first Bluetongue virus isolated from cattle in Xinjiang.