| Canine diarrhea virus infections are persistent and fatal.The characteristics of long treatment cycles and low cure rate of puppies caused great psychological harm and economic losses to owners.Canine parvovirus(CPV),canine coronavirus(CCoV),canine astrovirus(CaAstV),and canine kobuviruses(CaKoV)are the main members of diarrhea virus.Their clinical symptoms are similar and the clinical differential diagnosis is extremely difficult.Therefore,rapid diagnostic method for the preparation of the four pathogens of diarrhea virus disease is crucial.Among them,CPV poses a particularly serious threat.CPV is an acute contagious infectious disease,which was widely spreading in dogs and causing high morbidity to canine,additionally,it often causes severe economic and spiritual losses to the owner.Therefore,a comprehensive epidemiological investigation and basic research can help to provide a basis for effective prevention and treatment.Compared with common real-time PCR detection methods,multiplex quantitative PCR can detect multiple pathogens in one reaction at the same time,which is more convenient and specific.In this experiment,we first designed a quadruple TaqMan multiplex real-time quantitative PCR detection method that can detect CPV,CCoV,CaAstV.and CaKoV at the same time.The optimal primer and probe concentrations in the quadruplex real-time PCR detection method were 0.2 μM and 0.05 μM,respectively,the most suitable annealing temperature was 56℃,and the detection sensitivity for the four target viruses mixed templates was 102 copies/uL.There was no cross-reaction with torque teno canine virus(TTCaV),canine influenza virus(H3N2 CIV),and canine distemper virus(CDV).The multiplex real-time PCR detection method has the advantages of strong specificity,high sensitivity and good repeatability,and can be effectively applied to clinical detection and large-scale epidemic situation analysis.Based on this detection method,this study collected 1004 samples of anal swabs or fecal material from sick dogs from six provinces to detect potential CPV,CCoV,CaAstV,and CaKoV.Among these,611 non-diarrhea and 393 diarrheal disease samples were included.The results showed that 228 were positive for CPV.The positive rates in non-diarrhea and diarrhea disease samples were 46/611(7.5%)and 182/393(46.3%),respectively.In addition,the co-infection of CPV with other diarrhea viruses were counted.The epidemiological investigation in this study provide the evidence that the high infection rate of CPV in dogs.Furthermore,the full length of the major VP2 gene sequence was amplified from the positive samples,and 127 sequences were finally obtained.Non-synonymous mutations within CPV-2 that have not previously been reported,including I219V and Q386K.It shows that the virus continues to mutate,which makes continuous epidemiological surveillance and real-time vaccine development particularly important.According to the previously reported amino acid corresponding to genotypes,it was found that the current epidemic genotype in 5 provinces in China were all CPV-2c,and further satistic analysis showed that the change of the epidemic genotype in China was also closely related to the time.In addition,genetic evolution analysis with sequences of reference strains in the world and strains in China,showed that these viruses may be easily transferred internationally.Overall,our research provides detailed information about the current prevalence of CPV in China,which indicates that the continued prevalence of CPV may pose new challenges for diagnosis and future control strategies.Furthermore,purifying the VP2 prokaryotic expression protein to immunize BALB/c mice,hybridoma cells were prepared form the mouse spleen cells and fuse SP2/0 cells,and perform hybridoma cell lines by indirect ELISA.For screening,the positive clones were subcloned 3 times.Finally,four hybridoma cell lines 2F5,3C2,3D7,and 4E7,which were capable of stably secreting specific monoclonal antibodies against the VP2 protein were obtained.The monoclonal antibody subtype analysis results showed that all four monoclonal antibody subtypes belong to IgG1κ.WB and IFA assay indicated that all the four monoclonal antibodies were able to fast react with the CPV-2a subtype strain in this experiment.In addition,using purified truncated proteins and immunoblotting,the range of B-cell epitopes recognized by the four monoclonal antibodies were determined to be 71-107aa.Overall,this study has potential applications in the diagnosis of CPV infection and the function and antigenicity of its VP2 protein. |
