Clinical Diagnosis,Treatment Of Canine Parvovirus(CPV) Disease And Cloning And Expression Of Major CPV Genes

ObjectivesCanine parvovirus(CPV)disease has been one of the most serious infectious disease endangering the health of dogs in the world,especially for 2 ~ 6 months puppies.The clinical characters of this disease are emesis,diarrhea and hemorrhagic gastroenteritis.With the continuous variation of the virus,it has brought new challenges to the treatment and prevention of CPV disease.In this study,anal swabs of dogs suffering from CPV disease in animal hospital of Guiyang were collected from September 2015 to September 2016.Clinical diagnosis and treatment of sick dogs were done to find a best way to improve the CPV disease cure rate.Moreover,the isolation and identification of CPV,the sequence analysis and prokaryotic expression of CPV major genes were performed to investigate the variation of CPV in Guiyang area,which will provide basic data for grasping epidemic genotype of CPV and developing new vaccines in the future.MethodsAccording to the treatment of sick dogs in pet hospital,first of all,make a clinical diagnosis,and then make a definite diagnosis by colloidal gold test paper.According to the client requirements for treatment of dogs suffering from CPV disease,sick dogs were divided into the symptomatic support treatment group,the specific treatment group and comprehension treatment group.By comparing three different treatment methods to find a best way to the treatment of CPV disease.Twenty sick dogs were randomly selected to collected their anal swab,which were dealt with to inoculate F81 cell through adopts synchronous inoculation method to isolate the viruses.The isolated viruses were firstly identified by the hemagglutination and hemagglutination inhibition test,and then verified using the PCR method to identify the isolated CPV.Two pairs of specific primers targeting the VP1 and VP2 genes were designed to amplify the VP1 gene and VP2 gene,respectively.The obtained VP1 and VP2 genes were cloned and sequenced to analyze the genetic variation of the isolated CPV..The VP1 and VP2 gene were sucloned into the pET-32 a to construct the recombinant prokaryotic expression vector pET-32a-VP1 and pET-32a-VP2,respectively.The recombinant plasmids were transformed into component cell BL21(DE3)to express the indicated proteins under the induction of IPTG.The expression temperature and IPTG concentration were optimized to increase the expression level of the proteins.The expressed proteins were further verified by Western blot analysis.Results1.43 dogs were initially diagnosed by clinical symptoms of CPV and 37 dogs were confirmed by colloidal gold test paper of CPV.The accuracy of CPV detection was 86%.The results of three treatment methods showed that the highest cure rate was the comprehensive treatment group(84.6%),the specificity of the treatment group was 50%.,and the symptomatic support treatment group had the lowest cure rate(14.3%).2.Ten CPV strains were isolated from the anal swab-inoculated F81 cells.The isolated CPV viruses could make the F81 cells to produce cytopathic effect,including cell round,aggregation,fall off and so on.Through the hemagglutination assay,the virus titer of isolated strains were above 1:25.Meanwhile,the hemagglutination inhibition test showed that the isolated viruses could be neutralized by CPV monoclonal antibody.Moreover,the specific bands of CPV gene was amplified from the genome of the 10 isolated strains by PCR method.3.The results of sequence analysis showed that the nucleotide homologies of VP1 gene between the 10 isolated strains and 16 reference strains were 98.6% ~ 100%,closed with domestic reference strains of genetic relationship,with foreign reference strains far away;sequence analysis of 10 strains VP2 gene,seven isolated strains genotype were CPV-2a and others were CPV-2c,the VP2 gene compare with 30 reference strains nucleotide homologies were 97.7 % ~ 99.9 %,close with domestic reference strains of genetic relationship,with foreign reference strains far away,and 10 isolated strains genetic relationship far away from three vaccine strains.4.The optimal condition for VP1 protein expression was at 37 ℃,and IPTG concentration over 1 mM,,and VP2 protein was at 37 ℃,and 2 mM IPTG concentration showed that both The expressed VP1 and VP2 protein could be detected by Western blot analysis using the His tag antibody.