Jl09-11 Strain Of Canine Parvovirus Isolation And Identification And Immunological Studies

Canine parvovirus(CPV)infection is a kind of acute, infectious disease of domestic dogs and wild canidae, which causes large loss to the worldwide dog industry. As a result, the immunfaction for this disease is a compulsory immune progress for puppies. CPV is a kind of virus with high mutation rate; several antigenic variants have been identified after the isolation of the first canine parvovirus isolate CPV-2, including CPV-2a, CPV-2b, CPV-2c and so on. Those variants were labeled by the variety of the amino acid sequences in the key antigenic sites of the VP2, which is the main protein constructing the nucleocapsid of this virus. The mutations of the amino acid in those antigenic sites brought various antigenic properties to different variants, which can be identified by Mabs. Although the cross immune protection among different variants has been confirmed, several cases of immune failure due to the difference of the vaccine strains and the prevalent strains were reported. As a result, the prime task for the prevention and control of this disease is to identify the antigenic property of the prevalent virus, as well as exploit vaccines.In this research, the bowel content of a dog in suspension of canine parvovirus infection was collected and detected by PCR to confirm the CPV infection. After grinding and filing, the bowel content was inoculate to F81 cell line for virus isolation. The specific pathological change of the CPV on F81 cell line was appeared 4 days after inoculation. After three cycles of alternative freezing and thawing, the cell culture supernatant was inoculate on F81 cell for another two generations. At each generation, the typical pathological change of the CPV infecting F81 cell, such as rounding, connecting among each other and shedding were appeared. PCR, HA and HI test were performed to identify the isolated CPV in the cell culture supernatant. The PCR identification employed the universal primers of carnivorous animal CPV got positive result; the results of the HA test indicated the HA titer of the cell culture supernatant was 212To get a better understand to the evolutional relationship of the JL09-11 with that exist in China mainland, as well as the amino acid mutations at the key antigenic sites ; the results of the HI test showed the hemagglutination of the cell culture supernatant could be inhibited by the CPV antibody positive serum stored at our lab, which confirmed that the pathogen we isolated from the bowel content was CPV. The isolated CPV strain was named JL09-11. of the main nucleocapsid protein of this strain, limiting-dilution assay was performed for virus purification. The gene of the main nucleocapsid protein VP2 was amplified through PCR and sequenced. The results showed the JL09-11 is a strain of CPV with new antigenic property, as the amino acid sequences in the key epitopes were not identical with any variant as former published. The VP2 sequences of CPV isolated in China mainland were collected from the NCBI database and involved in the phylogenetic tree reconstruction. In the phylogenetic tree, the JL09-11 strain clustered together with strains which were defined as CPV-2a, although the amino acid analysis indicated the amino acids at site 297 and 555 were A and V orderly. Further analysis indicated those mutations were character of the CPV isolated in China mainland. Then the strain JL09-11 was involved in the development of an inactivated vaccine against canine parvovirus.Before exploiting the inactivated canine parvovirus vaccine, the neutralizing test was established on F81 cell line, which was used in the test of the neutralizing antibody against the CPV in the canine serum samples.The cell culture supernatant of the CPV JL09-11 was inactivated with 1% formalin at 37℃for 24 hours. Then the TCID50The immune effect assessment test was performed on health domestic dogs with no or low titters of neutralizing antibody against CPV in the serum. Each experimental dog was vaccinated with 1.5ml inactivated vaccine, containing about 5.0×10test was performed on the inactivated samples to detect if they were inactivated completely. The results showed the CPV in the cell culture supernatant was inactivated completely. At last the inactivated CPV was mixed with nanometer adjuvant with the proportion of 4:1 to form the inactivated canine parvovirus vaccine. 5 TCID50This study isolated a new CPV strain JL09-11. Phylogenic analysis indicated the JL09-11 shared high phylogenic homology with strains of CPV-2a variant isolated in China mainland. Amino acid analysis was performed and focused on the VP2 of the JL09-11. The result showed the 297 site of this protein was A(alanine), which is a inactivated CPV. We collected the serum samples before and 14 days after vaccination. The neutralizing antibody against the CPV in the serum samples were tested through neutralizing test as former established. The results indicated that the neutralizing antibody against CPV in the serum of experimental dogs was elevated significantly 14 days after vaccination. specific amino acid of S297A variant, at the same time the amino acid at site 555 was also vary from variant S297A variant. There was a retrieve mutation at site 555 from I (isoleucine) to V (valine). Then we recognized that most CPV strains isolated in China mainland and labeled as CPV-2a have this character. Although cross immune protection among different CPV variants has been reported, exception cases happened from time to time. As a result, it is necessary to develop a vaccine based on the endemic CPV variant of China mainland to prevent and control the canine parvovirus infection. So a kind of inactivated vaccine based on the CPV JL09-11 strain with high immune effect was developed in this study. Assessment test showed this vaccine has the potential to be employed in clinical CPV prevention.