Isolation And Identification Of Equine Herpesvirus 1 And Sequencing Analyze Of GB And GD Gene In Ili Of Xinjiang

Equine herpesvirus type 1(EHV-1) is a member of Alpha subfamily herpesvirus, and is the agent causing equine rhinopneumonitis(ER) which is characterised by abortion, acute respiratory disease, ataxia, and viremia in equine. ER is widely distributed in our country and seriously threatens the healthy development of the horse industry, leading to great economic loss. This study is aimed to isolate and identify equine rhinopneumonitis virus circulating in Ili district of Xinjiang province from the horses suspected with equine rhinopneumonitis, providing experimental basis for epidemiological investigation and vaccine development.In this study, we collected the lung of aborted fetuses suspected with equine rhinopneumonitis cases in Ili, Xinjiang. Lung homogenates were inoculated into BHK-21 and MDBK cells for virus isolation and culture. Etiologic and serological methods were used to identify the isolated virus. The conservative regions of EHV-1 gB gene were amplificated by PCR, followed bysequencing.These results showed that BHK-21 and MDBK cells generated cytopathic effect, such as shrinkage, fusion, desquamated and clustering into grape bunches at 48 h post-inoculation. A 622 bp fragment was obtained by PCR amplification, the result of partial gene sequencing showed that the homology between the isolated virus and EHV-1(Gen Bank accession number KF644579.1) was 100%. The titer of the isolated virus TCID50 was 105.8/0.1 m L. The isolated virus was intolerance to acid and heat, and was sensitive to ultraviolet, chloroform, ether treatment. Transmission electron microscopy(TEM) showed that viral particles were round, capsuled, and its diameter was about 130 nm. The result of serum neutralization test showed that EHV-1 positive serum can neutralize the isolated virus and inhibited cytopathic effect, and the neutralized titer of EHV-1 positive serum was l:24. Indirect immunofluorescence assay test showed green fluorescence. In conclusion, the equine herpesvirus 1 strain was isolated and identified by above mentioned experiments, named it as EHV-1-XJ2015 strain.In order to further research genetic constitution and biology function of the strain, we used specific primers to amplif gB and gD genes The gB and gD genes of the isolated virus were cloned respectively into pMD19-T vector. Then selected positive clones and tested the nucleic acid sequence. Using bioinformatics software analyzed homology and hereditary of nucleic acid of gB and gD gene and forecasted secondary structure and tertiary structure of encoding protein. The results showed that EHV-1-XJ2015 belonged to equine herpesvirus type 1(EHV-1). The conservatism of gB and gD gene were good and variation of isolates were small.Through sequence analysis of gB and gD gene of this strain, provide technical support for screening conventional vaccines and genetic engineering candidate vaccine.