Isolation And Identification Of Canine Parvovirus And Study On The Immunogenicity Of CPV-2c Virus-like Particles

Canine parvovirus（CPV）infection is a serious infectious disease,which is mainly characterized by severe vomiting,hemorrhagic diarrhea,and massive leukopenia.The disease has a fast course and a high mortality.It can cause myocarditis in puppies and is the leading cause of death in puppies worldwide.CPV has maintained a high rate of mutation since its discovery.According to its molecular characteristics of VP2 protein,CPV can be divided into six genotypes（CPV-2,CPV-2a,CPV-2b,New CPV-2a,New CPV-2b,CPV-2c）.Studies have shown that CPV-2c has a wider epidemic range and shows a stronger pathogenicity than other subtypes,and often causes disease in adults and immunized dogs.At present,the commercial vaccines is CPV-2 or CPV-2b strains,which are antigenic different from CPV-2c strains,so the spread of the disease has not been effectively controlled.This study conducted a molecular epidemiological survey of CPV in Yangtze River Delta from 2018 to 2020,analyzed the genetic variation of its VP2 gene,and understood the CPV epidemic situation.The VP2 protein was soluble expressed by prokaryotic expression system,and CPV-2c virus-like particles（VLPs）were assembled in vitro to studied on its immunogenicity.The research contents include the following three parts:1、Molecular epidemiological investigation and virus isolation of CPV in Yangtze River Delta.In order to understand the prevalence of CPV in Yangtze River Delta,130 clinical suspected specimens were collected from Jiangsu,Zhejiang and Shanghai from 2018 to2020,and 71 CPV VP2 gene sequences were detected,16 CPV strains were isolated.CPV strains bank was established and virus titers of all isolates ranged from 2.15×10³to 3.16×10⁵TCID₅₀/0.1m L.The VP2 gene was amplified and analyzed by bioinformatics software.The results showed that the New CPV-2a stains accounted for 4.23%（3/71）,New CPV-2b strains accounted for 33.80%（24/71）,and CPV-2c strains accounted for 61.97%（44/71）.CPV-2c strains has become the main epidemic strains in Yangtze River Delta.The isolates strains had high nucleotide homology with the reference strains（98.2%-100%）.But the CPV-2c isolate is far from the strains in Europe and America,showing regional differences,and the main amino acid difference sites are at the 5th and 370th positions of VP2 protein.2、Expression of recombinant CPV-2c VP2 protein and assembly of CPV-2c-VLPs.The CPV-2c strains（JSLYG-18）was used as the template,and its VP2 gene was connected to the p ET28a vector,where the recombinant expression plasmid was constructed and converted to E.coli BL21（DE3）.The recombinant VP2 protein with molecular weight of about 72 k Da was identified by SDS-PAGE and Western blot.The induction temperature,IPTG concentration and induction time were optimized,and the recombinant VP2 protein was induced for 12 h at 25℃and 0.05 m M IPTG to achieve soluble expression.The ultrasonic fragmentation supernatant was purified using Ni-NTA purification column to obtain the high purity recombinant CPV-2c VP2 protein.CPV-2c-VLPs was self-assembled by dialysis.Under the electron microscope,it was similar to the morphological structure of natural virus particles and had the activity of agglutinating pig red blood cells.The hemagglutination titer reached 12 log₂.3、The immunogenicity of CPV-2c-VLPs.BALB/c mice were immunized with CPV-2c-VLPs at different doses to evaluate their immunogenicity.The results of indirect ELISA showed that CPV-2c-VLPs could stimulate the production of specific antibodies comparable to those of the commercial vaccine group.Spleen lymphocyte proliferation assay showed that the lymphocytes of CPV-2c-VLPs group and commercial vaccine group could produce specific proliferation and differentiation.Spleen lymphocyte proliferation assay showed that the lymphocytes of CPV-2c-VLPs group and commercial vaccine group could produce specific proliferation and differentiation.The identification of splenic lymphocyte subsets showed that the percentages of CD3⁺,CD3⁺CD4⁺and CD3⁺CD8⁺cells in the CPV-2c-VLPs group were significantly higher than those in the control group,and the ratio of CD3⁺CD4⁺/CD3⁺CD8⁺was significantly decreased,suggesting that CPV-2c-VLPs could stimulate specific humoral and cellular immunity in mice.In conclusion,through molecular epidemiological investigation,the epidemic situation of CPV in Yangtze River Delta was known.CPV epidemic strains were isolated and identified,and the strains bank was established.It was found that CPV-2c strains had become the main epidemic strains in Yangtze River Delta.The recombinant CPV-2c VP2protein was prepared and assembled into complete CPV-2c-VLPs.The immunological experiment of mouse model proved that CPV-2c-VLPs has good immunogenicity.This study can provide data reference and new ideas for the prevention and control of CPV and the development of new vaccines.