Isolation And Characterization Of Canine Herpeavirus From Puppies And Bioninformatics Analysis Of US6 Gene

Canine herpesvirus（CHV）,only infected dogs,is an acute,non-febrile,fatal disease of newborn puppies.The gD glycoprotein encoded by US6 gene is an important component of the herpesvirus envelope,and it has the ability to recognize red blood cells,which make its own characteristics.In the current report,the pathogen was confirmed by clinical symptoms,polymerase chain reaction（PCR）,virus isolation and purification and animal experiment.In addition,the main antigenic region of the gD glycoprotein encoded by the US6 gene was analyzed and an expression vector was constructed.The results as follows.1.Extracted DNA form the liver of dog.A 357 bp gD gene was amplified by PCR and confirmed by BLAST analysis,and the strain was named CHV-JX strain.And measured the TCID₅₀ of the virus was 10^-4.39/0.1mL.Additionly,isolated virus-infected canine kidney cells（MDK）can see round virions under electron microscope.Characteristic CHV cytopathic effects（CPEs）including cataplasia and finally fall off were observed at 24 h after inoculation of virus.The 15-day-old puppies infected with CHV had typical clinical signs of herpes on the abdomen.At necropsy,significant gross lesions including necrotic lesions in the kidneys and so on.2.The results showed that the apoptosis rate were significantly increased（P<0.05）in 48 h,and 96 h groups compared to control group;meanwhile the mitochondrial membrane potential were remarkably increased（P<0.01）in 24h,48h and 96h groups;Compared to control group,the ROS of cells was increased in 24 h group,but it decreased in 48 h,72 h and 96 h groups;Compared to control group,except 24 h group,the pH was decreased in other groups.3.A about 1200 bp gD gene of CHV-JX strain was successfully amplified by PCR.The results of homology analysis between the CHV-JX strain and domestic and foreign CHV strains show that the nucleotide homology rate is 99.9%between CHV-JX strain and Australian,British and American CHV strains,meanwhile,the amino acid homology rate is 100%,however,compared with non-canine herpes virus strains,the homology rate is low.The results of DNA sequence analysis show the number of amino acds（aa）encoded by US6 gene is 346 aa.The gene fragment was synthesized by whole-genome synthesis then cloned into the expression vector pET32a（+）to construct a recombinant plasmid CHV-3F-3R.In this study,a strain of CHV-JX was obtained.After CHV-JX infected dogs,typical herpes virus symptoms were observed around the repeoductive organs;CHV-JX can cause MDCK cells apoptosis;Recombinant plasmid of the major antigenic region of the US6 gene was successfully constructed.In the current report,the bioinformatics analysis of the membrane protein US6 gene in the CHV genome was carried out for the first time,which provided a reference for establish an indirect ELISA method for detecting CHV antibody levels and developing DNA vaccines or subunit vaccines for the prevention of canine herpes virus diseases.