Isolation And Identification Of Epizootic Haemorrhagic Disease Virus From The Blood Of Cattle

[Objective]Epizootic haemorrhagic disease (EHD) is cased by the epizootic haemorrhagic disease virus (EHDV) which belongs to the genus Orbivirusan of family Reoviridae, which is an infectious non-contagious viral disease transmitted by insects of the genus Culicoides,this virus mainly affects wild and domestic ruminants.This studuy aimed to observe the virus infection condition of sentinel animal in monitoring EHDV of the cattle and sheep in Yangcheng County in Shanxi province. The serum and blood samples of sheep and cattle were collected in Yangcheng from May in 2013 to June in 2014.[Method]The positive blood sample detected by C-ELISA was inoculated in BHK-21 cells and C6/36 cells to isolate virus. The suspected isolate was detected from the sentinel cattle.It was identified by molecular biology techniques and titers of virus antibodies in cattle serum samples were tested by the mirco neutralization test(VN).Besides,RT-PCR was used to identify virus.[Results]One suspected virus strain was isolated and designated as SX218, and the suspected virus strain only causes regular CPE in BHK cells,but no CPE in C6/36 cells. The supernatant of the suspected virus strain was filtered and determined the content of TCID50, and the titer of the suspected virus strain was 104.5TCID50/100μL. By the amplification of RT-PCR using the circovirus genus-specific primer,the suspected virus strain was similar with amplified fragment of the positive control. The suspected virus strain was amplified by random amplification of RT-PCR and followed by sequencing.The result showed that the nucleotide sequence homology of VP2 and VP7 sequence by BLAST in NBCI is up to 99%.The results of VN and the molecular biotechnology experiments showed EHDV-1.[Conclusion]This is the first report that EHDV virus was isolated from the blood of cattle in Shanxi Province,which provides a important reference for future on isolation and identification of arboviruses of Shanxi and lays a theoretical foundation for the prevention and control of arboviruses.