Isolation Of Canine Parvovirus BJ Strains And Model Challenge Of Dogs

Five virus isolates were obtained from bloody stool of dead dogs infected with suspect canine parvovirus in Beijing, and were characterized using various procedures and assays. Fragments of CPV VP2 gene were amplified by RT-PCR and sequenced for the 5 isolates. One isolate was used as challenge virus in in dogs for challenge model development.After 2 passages in CrFK cells, the isolates induced normal hemagglutination (HA), which was specifically inhibited by the antibody against CPV. Direct immunofluorescent assay (FA), using CPV specific monoclonal antibody, showed fluorescent staining in the cytoplasm of the infected cells. PCR amplification of the VP2 region, using CPV specific primer, was conducted to the 5 isolates and CPV specific gene fragment was produced. The results suggest that the 5 isolates are canine parvoviruses.The VP2 gene sequences of those isolates were compared with the VP2 sequences of CPV strains for the homology and relations by using DNAStar software. The amplified VP2 gene was 1755bp long, and had 99.0%~ 99.5% nucleotide homology, to the comparing American strains CPV-b (type 2), CPV-15 (type 2a), and CPV-39 (type 2b), respectively. The homology in amino acid sequences was 98.6%~99.5%, when compared to the 3 American strains. These results showed less variation in the VP2 gene among these different strains, and the isolated CPV-BJ2, CPV-BJ4, CPV-BJ5, CPV-BJ7, and CPV-BJ11 strains belong to CPV-2a. Although there was some variations in the bases of VP2 gene regarding CPV-2a and CPV-2b, the homology of amino acid sequences was still >98% between the isolated strains and the other reported reference CPV strains in abroad. The evolution process of the isolated Beijing strains seems the same as reported in literatures for othe CPV strains, and obviously no unique Chinese CPV strains have been yet found to exist. Phylogenetic analysis of complete VP2 sequence indicated no significant differences between Chinese CPV strains and the foreign strains.Totally sixteen healthy dogs, 6-week old and HI serum titres <1:10, were used in the challenge model development studies to determine the challenge route and dose with CPV-BJ5. In challenge route study, 6 dogs were randomized into 3 groups, with 2 per group. Two pups were subcutaneously injected with 1 mL of the virus, and 2 pups were orally inoculated with 1 mL of the virus, and the other 2 dogs were controls and subcutaneously injected with 1 mL of CrFK cell preparation. After challenge, the dogs were observed for clinical signs of disease, including malaise, lack of appetite, depression, and bloody faces on days -2 to 14 post-challenge. Rectal temperatures were measured daily for the same period. From day -2 to day 14 post-challenge, nasal swabs were taken daily for virus isolation, and rectal swabs were taken daily for virus isolation and HA test. Blood samples for serum were collected at days 7 and 14 after challenge to evaluate antibody response. Typical clinical symptoms of CPV infection, including fever, loss of appetite, depression, vomiting and hemorrhagic diarrhea, were observed during the 14 days course after challenge. The results indicated that clinical signs of disease were observed earlier and more severe in dogs challenged via subcutaneous route than by oral route. The challenge dose study was conducted using 10 animals, with 2 dogs in each of the 5 groups (A, B, C, D and E). The dogs in each group were challenged subcutaneously with different dose of virus: 103.5 TCID50 / mL (group A), 102.5 TCID50 / mL (group B), 101.5 TCID50 / mL (group C), and 100.5 TCID50 / mL (group D). Group E was a control group and each dog received 1 mL of CrFK cell preparation. Clinical signs were observed in groups A, B and C. The challenged dogs showed fever, loss of appetite, depression, vomiting and hemorrhagic diarrhea as evaluated for 14 days after challenge. Active CPV replication was demonstrated in dogs (Group C) with titres 101.5 TCID50, however, it occurred later. The group D and control group (E) did not show any clinical signs. In conclusion, the best challenge condition for CPV-BJ-5-F3 virus is subcutaneous injecting 102.5 TCID50 in 1 mL volume.