The Molecular Epidemiology Investigation Of Canine Parvovirus Infection In Beijing

With the rapid expansion of China's pets industry, the diseases in pets pose a serious threat to their health and create a great loss to the pets industry. As an acute infective sickness, Canine parvovirus infection has the morbidity rate and mortality rate of 50%¹00% and 10%⁵0% respectively, which is consider to be one of the most serious diseases in pets. As the pathogeny of Canine parvovirus infection, Canine parvovirus (CPV) has a rapid speed of evolution which is rare in DNA virus. The decline of effectiveness of commercial vaccine and failure in its immunity has been more serious at present. In this research, by studying canine parvovirus infection molecular epidemiology and its phylogenic analysis from2010 to 2011 in Beijing, it is very necessary to analysis the CPV antigen variation to develop more effective vaccine.A rapid way of detecting CPV by PCR method with the minimum detected threshold value as 38.4 pg/μL DNA was established in the present study. The detection of CPV method by PCR had been successfully used in detecting 327 clinical samples random collected in and around Beijing (feces, urine, saliva, the muzzle secretions, serum etc).Among this samples, 51 CPV positive samples had been screened by PCR detection method. In comparison with two methods, PCR positive detection rate was 15.6%, whereas gold chromatography test had the positive detection rate 12.2% .It was clear that the detection of CPV method by PCR had higher specificity and sensitivity.As the main encoding gene of nuclear capsid protein, VP2 gene contains all the antigenic sites of CPV. We chose VP2 as the target gene for canine parvovirus infection molecular epidemiology investigation. By cloning and sequence analysis the VP2 gene , the homologous analysis of VP2 nuclear and amino acid sequences was 98.2% 100%, 97.7% 100% respectively between samples and references strains. The result showed that the mutation of VP2 was little from 2010 for the homologous analysis of VP2 nuclear and amino acid sequences was 99.0%～100% and 98.8～100% respectively among different samples. The major subtype in Beijing from 2010 to 2011 was CPV-2a accounting for 90.2%(46/51), while the percentage of CPV-2b was as low as 9.8% (5/51) and there was no CPV-2c or CPV-2. Comparing with classic CPV-2a/b strains, the mutation of VP2 in Beijing was sporadic point mutation, containing 555Ile→Val,324Tyr→Ile,267Phe→Tyr,440Thr→Ala,101Thr→Ile,87Leu→Met,139Val→Ile. There were also some individual mutations such as 187(Pro→Ile,Gln),188(Ala→Gln),308(Val→Ile). DNAStar analysis showed in addition to Ile 101, Ile 139, Ile187, Gln 187, Gln 188 and Tyr 267, all of the mutation Met 87, Ile 308, Ile 324, Ala 440 and Val 555 had the important biological significance because of their location, as they were in the potential antigen epitope area of VP2 protein. Phylogenies analysis of the VP2 gene sequences indicated that all the 51samples were closely related to China strains composing a big branch of CPV-2a/b but not the same branch with FPV. Samples shared the nearest phylogenetic relationship with Taiwan, Beijing, Wuhan and Nanjing isolate strains, followed by Brazil, France, Vietnam, the United States, Italy, Poland for the farther.According to the homologous analysis, 51 samples could be divided into six groups and each group with 100% homologous. Feces samples A1, B4, B6, B8, B25, E4 were taken as model for each group. Five strains of the virus were successfully isolated by feces infecting and passaging in F81 cells through synchronizing culture method. After a series of systematic identification, such as the virus morphological observation, PCR detection, IFA test, the TCID₅₀ test, the five strains of virus were confirmed to be CPV and named CPV-BJ-A1,CPV-BJ-B4,CPV-BJ-B8,CPV-BJ-B25,CPV-BJ-E4 respectively. VP2 complete gene sequence analysis showed that, the five strains were all CPV-2a. However, the vaccines widespread in the market were based on CPV-2, which could be one of the reasons why the vaccine provided low immunity.To sum up, the full epidemiology investigation of canine parvovirus infection and phylogenic analysis had laid a theoretical foundation for analysis of failure in immunity in clinical and pertinently developing CPV vaccines in pets.