Isolation And Identification Of Canine Distemper Virus And Canine Parvovirus And Development Of Their Monoclonal Antibodies

Objective In order to offer early rapid diagnosis and preventive therapy for the canine distemper(CD) and canine parvovirus disease(CP). a strain of canine distemper virus and a strain of canine parvovirus were isolated and identified from fecal swabs of dogs successfully, which were suspectedly infected by canine distemper virus and canine parvovirus in Ningxia. The study developed two monoclonal antibodies against the viruses, lay a foundation for early diagnosis and prevention treatment of canine distemper and canine parvovirus disease.Methods 1. We collected fecal swabs from dogs suspectedly infected by canine distemper virus and a fecal swab from dog suspectedly infected by canine parvovirus. After we pretreated the suspicious materials and synchronous vaccinated their sensitive cell line respectively, which were namely the African green monkey kidney cell line(Vero), cat embryo kidney cell line(FK81) and isolated virus. We observed cytopathic effect(CPE).2. The frozen viruses at-20? were gotten out and thawed at 37?. After cultured,CDV and CPV virus were identified respectively. Then the toxicity measurement, electron microscope observation, specific identification of PCR, N gene of CDV gene amplification of PCR and sequence of analysis, VP2 gene of CPV gene amplification of PCR and sequence of analysis, indirect immunofluorescent assay were carried out on the isolated virus.3. After the separated CDV virus and CPV virus were concentrated and purified, we used them to immunize 6-8 weeks old female BALB/c mice respectively. After two weeks in the secondary immune, we sheared tails of the immunized mice and collected blood. We tested the titer of serum antibodies and prepared for cell fusion. At the same time, we established a method of indirect ELISA.4. Preparation of mouse myeloma cells(SP20), BALB/c feed cells, CDV-immunized spleen cells and CPV-immunized spleen cells and did cell fusion respectively. After fusion we added HAT culture medium to screen cells and added HT culture medium after 12 days. We early used limited dilution method to screen positive clones when we found them by indirect ELISA and we detected the secreting of monoclonal antibodies at any time after cloning. Expanding and preserving positive cloning holes which were screened by ELISA monoclonal indirect detection of more than three times.5. Identified monoclonal antibodies against CDV or CPV. The tests as follows: the cell count of hybridoma chromosomes, the test of antibodies titer, subtype identification of monoclonal antibodies, characterization of monoclonal antibodies specificity, the detection of monoclonal antibodies secretion and stability, the analysis of antibodies antigen epitope and monoclonal antibodies were purified by affinity chromatography column.Results 1. The Vero cells had 90% stable toxicity changes after blindly passage to the fourth generation which were inoculated with suspectedly infected materials. The result of electron microscopy showed that virus particles were irregular shape, at about 200 nm in diameter and envelope. The results of specific RT-PCR identification and the length of N gene amplification were consistent with the expected size pieces, one was 484 bp another was 1572 bp. IFA test showed that punctiform with specificity green fluorescence can be observed in the cell cytoplasm.2. The FK81 cells had 90% stable toxicity changes after blindly passage to the third generation which were inoculated with suspectedly infected materials. Virus particles were three-dimensional symmetrical structure, circular, no capsule membrane, the diameter between 20 nm and 23 nm and solid. Specific PCR identification and the length of VP2 gene amplification were consistent with the expected size pieces, one was 209 bp another was 1755 bp. The specific green fluorescent can be observed around the nucleus by IFA.3. Three CDV monoclonal hybrid tumor cells were screened successfully, and they were C6 strain, G10 strain and H2 strain. Besides three CPV monoclonal hybrid tumor cells were also obtained, and they were B8 strain, E10 strain and F12 strain.4. The chromosome number of three CDV monoclonal hybrid tumor cells were more than 90. They can secrete low level of IgG1 monoclonal antibody. Finally they were found to be the same strain. The chromosome number of CPV monoclonal hybrid tumor cells were more than 95. They can secrete a relatively high titer of monoclonal antibody. Both B8 strain and F12 strain can produce IgG1, and E10 strain can secrete IgM. Three monoclonal antibodies could recognize different antigen epitope, and have high specificity and good stability.Conclusions a CDV epidemic strain and a CPV epidemic strain in Ningxia were separated and identified successfully, three strains of CDV monoclonal antibodies were screened and identified by detection of their characteristics and biological activities. Finally we found that they were the same strain. Besides, three strains of CPV monoclonal antibodies were screened successfully and they had different epitopes. This study laid the foundation for the early diagnosis and treatment of canine parvovirus disease will have a certain application in the filed in the future.