Epidemiological Investigation Of Equid Herpesvirus In Large-scale Donkey Farm And Preparation Of GD Protein Polyclonal Antibody Of EHV-8 Isolate

Equid herpesvirus（EHV）mainly causes nasal pneumonia,encephalomyelitis and abortion and other diseases,which can lead to death,causing serious economic losses to the production of equine animals.With the development of the donkey industry,the donkey breeding has gradually become an intensive and large-scale development,but the donkey respiratory diseases had seriously restricted the healthy development of the industry.EHV-8 has been isolated from large-scale donkey farms in China,but the prevalence of EHV herpes viruses such as EHV-8 in donkeys needs to be further studied.1.Molecular epidemiological investigation of large-scale donkey farm equid herpesvirus in some parts of Shandong provinceTo investigate the current status and characteristics of donkey’s infection with equid herpesvirus（EHV）,1834 nasal swabs were collected from 76 intensive donkey farms in some parts of Shandong Province.The pathogens of EHV-1,EHV-4 and EHV-8 in nasal swabs were detected by PCR.The pathogens of EHV-1 and EHV-4 were not detected in all nasal swab samples;the total positive rate of EHV-8 pathogen was 6.65%（122/1834）,and the positive rate of EHV-8 pathogen farms was 76.32%（58/76）.The isolated EHV-8 strain had the highest homology with domestic EHV-8/wh strain,which was 99.7%.After statistical analysis,EHV-8 infection rates varied significantly between different regions（p<0.05）,highest in Dezhou district,at 13.36%（29/217）;EHV-8 infection rates did not significantly between ages（p>0.05）;The EHV-8 infection rate of donkey in winter was14.57%（73/501）,which was significantly higher than that in other seasons（p<0.05）.This study shows that the prevalence of EHV in large-scale donkey farms in Shandong Province is widespread and should be paid attention to,which provides an important reference for early warning the epidemic risk of EHV.2.Isolation and identification of the EHV-8 donkey derived isolatesIn order to carry out the basic research related to EHV-8,the EHV-8 virus LCDC01strain（Genbank:PRJNA787358）was isolated from the donkey nasal swabs based onmolecular epidemiological surve.The EHV-8 isolate had the highest homology to the EHV-8 Wh strain,ranging from 99.7%.RK-13 cells and BHK-21 cells challenged by LCDC01 strain can induced typical cellular lesions such as cell rounding and cell fusion,with an TCID50 of 1×10^3.75.EHV-8 inoculation with BABL/c accompanied that mice with nasal inoculation accompanied clinical symptoms of weight loss,observed alveolar wall thickening and local bleeding in histopathological sections;challenged amimals had no obvious clinical manifestations and no tissue lesions,with no nucleic acid component of EHV-8 was detected in the organs and blood samples of mice.The successful isolation of EHV-8-scale donkey farm epidemic strains in this study,which proved the foundation for subsequent EHV-8 studies.3.Prokaryotic expression of EHV-8 gD gene and i-ELISA method establishmentIn order to study the serological prevalence of EHV in Liaocheng area,this experiment analyzed and predicted the hydrophilicity,antigenic epitope,signal peptide and transmembrane region of a mino acid molecule encoded by EHV-8 gD gene,cloned the target fragment suitable for prokaryotic expression of gD gene,constructed pet-32a-gD960prokaryotic expression vector,and successfully expressed it in E.coli BL21 strain,which was analyzed by SDS-PAGE electrophoresis.The recombinant protein was expressed as inclusion body.After purification by inclusion body,the recombinant protein with specificity was obtained.Using the purified recombinant protein as the detection antigen coated enzyme label plate,an indirect ELISA detection method was established.Combined with the commercial kit,the serological epidemiology of EHV in the surrounding areas of Liaocheng was investigated.The results showed that the individual antibody positive rate of EHV was 27.74%,and the field positive rate was 73.68%;Statistical analysis showed that there was no significant difference in antibody positive rate between different regions（p>0.05）.This study shows that the prevalence of EHV is widespread in large-scale donkey farms and donkey groups in Liaocheng area,which should be paid attention to.4.Preparation of polyclonal antibody against EHV-8 gD glycoproteinTo prepare polyclonal antibody to EHV-8 gD protein,purified p ET-32a-gD960recombinant protein was used as antigen and high immunity serum was collected after immunizing mice.The results showed that the polyclonal antibody titer of p ET-32a-gD960recombinant protein was 1.6×10⁴and had good specificity;The animal protection test showed that the recombinant protein polyclonal antibody could play a certain protective role against EHV-8 virus infection.This study provides ideas for EHV-8 vaccine development and demonstrates the possibility of EHV-8 gD protein as a vaccine candidate antigen.