Establishment Of Duplex PCR For Detecting Novel Raccoon Dogs Parvovirus And Development Of Inactivated Vaccine

Raccoon dog parvovirus(RDPV)infection causes raccoon dog viral enteritis,which has a high incidence rate and mortality in raccoon dog population,causing huge economic losses to raccoon dog breeding industry and seriously restricting the healthy development of raccoon dog breeding industry.RDPV is closely related to Feline parvovirus(FPV),Mink enteritis virus(MEV),Canine parvovirus(CPV)and other parvoviruses.In recent years,epidemiological investigations in our laboratory have shown that a novel RDPV is popular in raccoon dogs in China.The key amino acid sites determining typing on its main structural protein VP2 are consistent with the corresponding amino acids on VP2 of CPV type 2(CPV-2),indicating that the novel RDPV is most closely related to CPV-2,but four amino acid sites of VP2 protein of the novel RDPV have been mutated,S27T,S297A,N375D and V562L,of which S27T is the first discovery.At present,there is no specific detection method for novel RDPV,and there is no commercial RDPV vaccine for raccoon dogs in China.Clinically,raccoon dogs are generally immunized with CPV inactivated vaccine or MEV inactivated vaccine.Although it has a certain immune protection effect,immune failure occurs from time to time,which brings challenges to the detection and prevention and control of novel RDPV.Therefore,a novel RDPV duplex PCR detection method was established,and an inactivated vaccine was prepared at the same time.Referring to the structural protein VP2 gene sequences of parvovirus such as RDPV,CPV,FPV and MEV published on Gen Bank,this study analyzed and compared the relevant sequences with Snap Gene software,designed a pair of general primers P1-F/P1-R for the conserved region of VP2 protein gene of parvovirus such as RDPV,CPV,FPV and MEV,and a pair of specific primers P2-F/P2-R for novel RDPV,and established a duplex PCR detection method.The target fragment size of general primer P1-F/P1-R was 261 bp and that of specific primer P2-F/P2-R was 648 bp.The target bands amplified by single PCR reaction were recovered and purified,connected with pMD18-T vector and transformed into DH5?,The plasmids were extracted and identified by enzyme digestion,and the positive recombinants were screened for sequencing.The results showed that the designed two pairs of PCR primers had strong specificity.The PCR reaction procedures such as primer concentration,annealing temperature and cycle times were explored by matrix experiment.Finally,the optimal conditions of duplex PCR reaction were determined,and the specificity test,sensitivity test and repeatability test were carried out.The results showed that the established duplex PCR method had strong specificity and good repeatability.The universal primer P1-F/P1-R could detect 1.83×10~2copies/?L of virus DNA at the lowest level;The specific primer P2-F/P2-R could detect 1.64×10 copies/?L of viral DNA at least.The established duplex PCR method was further used to detect 50 fecal and intestinal samples of suspected raccoon dog viral enteritis sent from 20 raccoon dog farms in Zhucheng,Weifang and Dongping,Shandong.The results showed that 35 samples were positive by duplex PCR amplification,and the coincidence rate with the results of virus isolation and culture was 100%.This study shows that the established duplex PCR method is suitable for the rapid detection of novel RDPV.A novel RDPV Dongping isolate(RDPV-DP1)was selected as seed virus to develop an inactivated vaccine.Firstly,RDPV-DP1 was expanded and cultured on CRFK cells.The virus solution was determined by HA and TCID₅₀test.The titer of virus HA was 2~7and 10^6.4TCID₅₀/mL.Inactivating agents were added to the virus solution in the proportions of 1:3,000,1:4,000,1:5,000 and 1:6,000 respectively?-Propionolactone was treated at 4? for 6,12,18and 24 h,and then inactivated by water bath at 37? for 2 h?-Propionolactone decomposition.Take 500?L antigen solutions under different inactivation conditions,CRFK cells were inoculated respectively and passed through 3 generations blindly.If there was no cytopathy(CPE),it was determined that it was completely inactivated.Final determination of inactivator?-Propionolactone was added at 1:4,000 and inactivated at 4? for 24 h.The inactivated virus solution was mixed with aluminum hydroxide adjuvant in the ratio of 2:1 to prepare the inactivated vaccine.Sterility test,appearance test and inactivation test of the vaccine during storage.The results showed that the inactivated vaccine had normal appearance,no precipitated impurities and microbial pollution,and was stored at 4? for at least 6 months.In the safety test,20 KM mice were randomly divided into 4 groups,with 5mice in each group,including routine dose vaccination group,over dose vaccination group,adjuvant vaccination group and normal saline vaccination group.Each mouse in the routine dose vaccination group was inoculated with 0.3 mL inactivated vaccine,each mouse in the over dose vaccination group was inoculated with 0.6 mL inactivated vaccine,each mouse in the adjuvant vaccination group was inoculated with 0.3 mL aluminum hydroxide adjuvant,and each mouse in the normal saline vaccination group was inoculated with 0.3 mL normal saline.Clinical observation and body temperature measurement were carried out on all experimental animals.The results showed that the clinical manifestations of experimental animals in each group were normal,and there were no pathological changes in autopsy,indicating that the prepared inactivated vaccine had good safety.Then the immunogenicity of inactivated vaccine was tested.72 KM mouse were randomly divided into 3 groups with 24mice in each group.At the beginning of the experiment,each mouse in group 1 and group 2was inoculated with 0.3 mL of inactivated vaccine subcutaneously,and the mice in group 2were immunized for the second time in the same way 14 days after the first immunization.On the 7,14,21 and 28th days after the first vaccination,the blood and serum of experimental animals were collected,and the titer of anti RDPV antibody was detected by HI test.The results showed that on the 14th day,the HI antibody titers of group 1 and group 2 mice were above 2~4,but on the 21st day,the HI antibody titers of group 1 mice were above 2~5,but the HI antibody titers of group 2 mice increased,with an average of 2~6.On the 28th day,the HI antibody titers of group 1 mice did not increase significantly,but the HI antibody titers of group 2 mice increased,approximately 2~7.The results showed that the inactivated vaccine could induce the body to produce good antibody level and had good immunogenicity.The effect of twice immunization was better than that of once immunization.It provided a potential choice for the prevention and control of raccoon dog parvovirus enteritis.