Development And Preliminary Clinical Application Of Inactivated Vaccine Against Raccoon Dog Parvovirus

Raccoon dog parvovirus（RDPV）belongs to carnivorous parvovirus and is a member of Parvovirus family,parvovirus subfamily and Parvovirus genus.RDPV,along with Mink enteritis virus（MEV）,Feline parvovirus（FPV）,and Canine parvovirus（CPV）,all belong to the antigenic variant of carnivorous parvovirus..RDPV can cause severe enteritis in farm-raised raccoon dog.Clinical presentation includes diarrhea,dehydration,hematochezia and other clinical symptoms.It mostly occurs in groups of raccoon dogs and is easy to cause wide-spread death,especially in young raccoon dogs.At present,there is still no safe and effective vaccine to prevent the disease.Inactivated mink parvovirus enteritis vaccine or canine parvovirus vaccine are used in clinical emergency immunization.Although the three pathogens have partial cross-protection,the level of cross-protection is not complete.Therefore,the aim of this study includes multi-province sample collection,virus isolation and identification from 95 clinical disease samples viral stock generation,development of an inactivated vaccine and subsequent preliminary clinical trials.1.Isolation and identification of epidemic strains of raccoon dog parvovirus and establishment of pathogenesis modelSamples of suspected raccoon dog parvovirus enteritis were collected in major raccoon breeding areas in China.The samples were PCR amplified and sequenced,VP2 sequence was compared for amino acid sequence homology with VP2 genes of other major carnivorous parvoviruses.Amino acid mutations of target sequence were analyzed,and an evolutionary tree was constructed to analyze the evolutionary relationship between sequenced strains and isolated strains and reference sequence.Overall nucleaotide sequence similarity among the 23 sequenced strains was 99.5-100%,and the homology to the reference sequences in Gen Bank was 98.1%-99.5%.At the amino acid level,the similarity between the newly obtained sequencing strains was 99.5-100%,and the similarity with the reference sequence was 97.6-99.5%.Compared with the earlier published RDPV-VP2 protein sequence,mutations were found at residues 27,80,93,297 and 562 in the sequenced strains.Compared with CPV-b-VP2 protein sequence,amino acid residues 27,297,375 and 562 were mutated.RDPV were separated and identified by various methods from samples,and animal regression experiments were carried out to understand the biological characteristics of RPDV in China.The results showed that a 573bp specific band could be obtained by PCR with specific primers.The isolated virus can be cultured on F81 cells and produce significant CPE.Under electron microscope,microscopic virions of about 25nm were observed.The titer of the isolate fo porcine erythrocyte hemagglutination could reach 1:1024 by porcine erythrocyte agglutination（HA）test.The virus culture was used to infect healthy raccoon dogs.The results showed that the infected raccoon dogs showed typical clinical symptoms of parvovirus,while the normal control group did not.Finally,the raccoon dog parvovirus isolated from infected raccoon dog samples in this study was named RPDV-He B17.The data obtained in this study will provide reference for the genetic evolution analysis of the virus,vaccine and diagnostic research.Twenty raccoon dogs were selected and randomly divided into 5 groups.The virus(initial virus titer was 1×10^7.50TCID50/mL)was diluted into different poison values,and then the experimental raccoon dogs were given.A uninfected control group was set up.The test was set at a total dose of 5 mL per challenge.Raccoon dogs were kept separate.median infective dose(ID₅₀)was calculated according to the number of raccoon dogs in each group based on observation indicators including the mental state,appetite,fecal traits,fecal detoxification,leukocyte number and histopathological test results.,.The results showed that 4 to 9 days after attack,such as depression or depression,loss of appetite,diarrhea,blood stool,coma,death,swelling,congestion,bleeding,and thinning of intestinal wall.The pathological tissue sections showed extensive pathological changes in the mucosal epithelium and lamina propria,necrosis,and numerous connective tissue hyperplasia in the submucosa.The number of lymphocytes in the spleen was reduced compared to the control group.In addition,viral shedding in stool samples began 4 to 5days after the challenge,and the number of leukocytes decreased 2 to 3 days after challenge.The ID₅₀=1×10^-2.5/5 mL of raccoon dog parvovirus He B17 strain(virus titer of 1×10^7.50TCID₅₀/mL)was calculated according to the challenge results of different doses.It shows that He B17 virus can be used as the standard strain for evaluating the inactivated vaccine of raccoon dog parvovirus disease.Thus,a raccoon dog parvovirus disease model was successfully established in this study.2.Study on the genetic stability of Raccoon dog parvovirus and establishment the storage of the virus for efficacy test and vaccine preparationIn order to check the stability of the passage of raccoon dog parvovirus and ensure the stability of the efficacy test,continuous passage of raccoon dog parvovirus was carried out in compliance with the Good Manufacturing Principles（GMP）.,A database of basic and effective strains for production and testing was established and tested in detail.In this study,Raccoon dog parvovirus He B17 strain was continuously passaged to the 18th generation on F81 cells.The titer,virulence,purity and animal test of the 3rd,5rd,6rd and 8rd passaged virus were tested.The results showed that P3,P5,P6 and P8 strains were not contaminated by bacteria,molds,mycoplasma and exogenous viruses,and could be neutralized by the positive serum of parvovirus.The virus titers were 10^7.20TCID₅₀/ml,10^7.33TCID₅₀/ml,10^7.50TCID₅₀/ml and 10^7.50TCID₅₀/ml,respectively.A series of typical symptoms of parvovirus disease,such as stool viral shedding,watery stool,mental and appetite abnormalities,were induced after the challenge,and the disease occurred in5/5 of the raccoon dogs.Therefore,the P3 of He B17 strain of Raccoon dog parvovirus was used as the seed strain,and the P4-P6 was used as the strong strain for detection.P6,P8,P10,P12,P15and P18 viruses were selected for viral titration,hemagglutination value,immunogenicity,specificity and purity of the viruses.Finally,the production strain of He B17 was determined.In this study,the primary strains of P6,the basic strains of P7～P10,and the production strains of P11～P15 were established.The maximum production strains of He B17 were determined to be P15.3.Study on manufacturing technology of inactivated vaccine of Raccoon dog parvovirusIn this study,the manufacturing process of raccoon dog parvovirus inactivated vaccine was studied,and the main technological parameters were determined.The vaccine was produced in rollingg bottle culture of F81 cells.F81 cells were cultured in MEM medium containing 8%fetal bovine serum at 37℃at a speed of 8～10 RPM.Infected cells were collected at 3～4 days post virus inoculation and frozen and thawed once at-20℃to harvest the virus.The titers of He B17strains were not lower than 10^7.00TCID₅₀/ml.Then the virus was inactivated for testing.Virus inactivation was conducted using 2m M BEI at 30℃for 24 hours,and then 2m M sodium thiosulfate was added to neutralize BEI.Aluminum hydroxide was used as vaccine adjuvant,at theoptimal concentration of 5%.4.Study on safety and efficacy of inactivated vaccine against Raccoon dog parvovirusIn order to study the safety of the vaccine,three batches of inactivated vaccines were used for safety evaluation in raccoon dog parvovirus model.The results showed that single dose injection,single dose repeated injection and one over-dose injection were safe for healthy young raccoon dogs（50～70 days of age）and healthy adult raccoon dogs（6～18 months）before breeding.Anatomical and histological examination of some immune raccoon dogs showed no visible pathological changes.The results indicated that the inactivated vaccine of raccoon dog parvovirus disease was safe.The empty count,litter number and survival number of raccoon dogs were calculated to evaluate the effects of the vaccine on the reproductive performance of the breeding animals.The results showed that the empty count of the experimental group and the control group were all 0,and the litter number were 156,157,155 and 152,respectively.The survival numbers were 138,136,136 and 136,respectively.The farrowing rate and empty pregnant were 100%,0%respectively.The survival rates were 88.5%,86.6%,87.7%,86.2%,respectively.The results showed that the injection of inactivated parvovirus disease vaccine had no adverse effects on reproductive performance of female raccoon dogs.The effects of vaccine on the growth and fur quality of raccoon dogs were studied by evaluating the performance of raccoon dogs during growing period.There were no significant differences in fur quality indexes between immune group and control group（P>0.05）.The results showed that the inactivated vaccine had no significant effect on the production performance of raccoon dogs.The efficacy of inactivated vaccine against racoon dog parvovirus disease was evaluated through the study of the minimum immune dose,the correlation between serum antibody titer and challenge protection,the duration of vaccine immunity,the vaccine immunization program and the immune challenge test.The results showed that the minimum immune dose of inactivated vaccine for raccoon dog parvovirus disease was 0.5ml each,and the recommended immune dose was1.0ml each.There was a good parallel relationship between HI antibody titer and challenge protection.When the titer of HI antibody in serum was no less than 1:32,100%protection against parvovirus could be obtained.Serum HI titer peaked at 30 days after immunization,and began to decline at 60 days after immunization.By 210 days after immunization,the HI titer of immunized raccoon dogs was no less than 1:32.No cases occurred after immunization,and the protection rate was no less than 80%,which reached the standard of protective efficacy against raccoon dog parvovirus.According to the antibody level after immunization and the result of challenge,the vaccine still had immune protection against raccoon dog parvovirus 210 days after immunization.In order to ensure the sufficient level of immune protection,the immune duration of inactivated vaccine against parvovirus disease of raccoon dogs was set for 6 months based on breeding characteristics of raccoon dogs.By monitoring the serum HI antibody titer of vaccinated raccoon dogs at 21,30,60,90,120,150,180 and 210 days before and after immunization,the young and adult raccoon dogs aged 50 to 70 days after birth were vaccinated with one dose of vaccine,or two doses of vaccine,respectively.The results showed that a single dose of vaccine resulted in sufficient level of protective response in both young and adult raccoon dogs of 50 to 70 days of age.The protection rate of test animals was 100%（5/5）,and the incidence rate of control group was 100%（5/5）,indicating that the vaccine had good immunity protection.