Isolation And Immunogenic Evaluation Of Raccoon Dog Parvovirus

Raccoon dog parvovirus-derived enteritis is an acute, highly contagious disease, which is one of theimportant infectious diseases endangering the raccoon dog industry. RDPV firstly recognized inHeilongjiang province in1984. Three strains were isolated from different regional enteritis raccoondogs by Xianzhu Xia in1988. In recent years, we are facing a growing trend of the prevalence ofraccoon parvovirus enteritis. As there is no specific raccoon parvovirus vaccine controling it, minkenteritis vaccine or canine parvovirus vaccine were usually chosen to substitute to control the RDPV.However, the immune effect of the Vaccines is so bad that the vaccine development of raccoon dogparvovirus with high security and efficiency is imperative.To develop vaccine for Raccoon Dog enteritis caused by Raccoon Dog Parvovirus, Parvovirusstrains with high immunogenicity and high efficacy were selected as vaccine candidates. Liveparvovirus was isolated from feces of clinical raccoon dog form LiaoNing and HeBei province byCrandall-Reese feline kidney (CRFK) cells. The virus strains were isolated then identified withmorphological methods, serological methods, molecular biological methods, animal regressionexperiment and immunogenic evaluation. Two RDPV strains named LN10-1and HeB10-1wereisolated successfully. The93AA residues characterized as determination of host range in LN10-1werechanged (Asn-Lys). The phylogenetic analysis of VP2gene sequences reveales that the LN10-1isolation is presented between the two genetic clusters, one is composed with CPV, and the other clusteris consist of carnivores parvovirus (FPLV, MEV and BFPV). It is supposed that LN10-1is evolvingbetween FPV and CPV, or there may be a new virus that becomes adapted to its new host(raccoondog).The Immunological assay result shows the neutralizing antibody titre of parvovirus was higherthan challenge protection level on21day post-immunization(HI≥1:80；SN≥1:64) and was higher than256folds on28day post-immunization. And the two strains could be used as inactivated vaccinecandidate for preventing haemorrhagic enteritis in Raccoon dogs.To explore the correlation between the parvovirus genome copy number and the virus content, thestudy established the use of fluorescent quantitative PCR to detect the raccoon dog parvovirus genomecopy numbers quantitatively. Standard recombinant plasmid was bult through amplifying a92bpconservitive fragment in VP2gene fragment from RDPV LN10-1strain. In brief, the gene copy numberof the target gene was firstly calculated from the stardard curve of RDPV LN10-1.Correlation analysisof the virus genome copy number and TCID50showed that there are very significant positive correlation(P <0.01) between the genome copy numbers and TCID50.Compared to the hemagglutination, the genecopy numbers are more representative of the live virus content. Taken together, the study providesalternative experimental technology, qPCR technology, for determining the parvovirus content and somepractical significance to vaccine production and testing of animal biotechnology.