Isolation And Identification Of Porcine Epidemic Diarrhea Virus KB4 Strain And Development Of Its Inactivated Vaccine

Porcine epidemic diarrhea(PED),with the clinical symptoms of vomiting,diarrhea and dehydration,is an Acute,high contact mortality infectious disease caused by porcine epidemic diarrhea virus(PEDV).Morbidity of sows is different,the sows mainly manifest as the rapid recovery after anorexia,depression and diarrhea.Morbidity of piglets is close to 100%.Piglets within 1 week of age often die of dehydration after 3-4 days of persistent diarrhea,with high mortality.Growing pigs are mainly characterized by diarrhea,anorexia and depression,which can recover quickly without death.After recovery,the pigs will get strong immune response.PED is becoming prevalence and results in a huge effect on the pig industry in the world since 2010.Based on genetic analysis PEDV is divided into G1a,G1b,G2a and G2b subtypes.Currently,the subtypes G2a and G2b of PEDV are dominated,while the widely used vaccine CV777 is classified to the subtype Gla,which cannot provide full protection against the current prevalent strain.Therefore,it is urgent to develop new PED vaccine.In this study,we detected the suspected PED cases in the eastern China,isolated and identified PEDV and developed the inactivated vaccine against PEDV.1.Isolation and identification of PEDV and its pathogenicity220 suspected samples for porcine epidemic diarrhea were collected from 2015 to 2016 in eastern China and subjected to RT-PCR detection.Result show that the positive rate was 30.00%(66/220).The S genes of 15 PEDV positive samples were amplified and subjected to sequencing and analysis.Phylogenetic analysis of S genes of 15 PEDV strains indicated that all strains were classified as G2 genotypes,four strain of them were belonged to the G2a subtype,while 11 strains of them were belonged to the G2b subtype,which was different from vaccine strain CV777.After the continuous blind passage in Vero cell for 12 generations one of 15 strains could produce cytopathic effect in Vero cell.The isolated strain was identified as PEDV by RT-PCR and named as PEDV KB4.Its TCID50 was about 106.0/ml.The susceptible 5-day-old piglets were challenged orally with 2 ml of 105.5 TCID50/ml,104.5 TCID50/ml and 103.5 TCID50/ml,respectively.All challenged piglets showed diarrhea symptoms and viral shedding.The mortalities for the 105.5 TCID50/ml group,104.5 TCID50/ml group,and 103.5 TCID50/ml group were 3/3,3/3 and 1/3,respectively.Therefore,the isolated PEDV was highly pathogenic to piglets and belonged to G2b subtype.2.Screening of PEDV vaccine strainIn order to obtain PEDV vaccine candidate with high antigenic titer and good immunogenicity,PEDV KB4 strain was grown in the medium with gradually reduced trypsin,and this virus was purified by plaque clone method.Virus titer,pathogenicity and immunogenicity were determined for different generations of PEDV KB4 virus.The results showed that the morphology and size of plaque formed by PEDV(KB4 strains)was stable and uniform at the F22 generation.During the passage,the concentration of trypsin added in media was reduced from 2 ?g/ml to 0 ?g/ml at the F17 and F19 generations.We also found that the virus titer was increased with the adaptability to the cells,and the titer of F32 virus was more than 107.50TCID50/ml.The F22 generation of PEDV was highly pathogenic,which can cause 100%morbidity and fecal shedding in infected piglets.In contrast,the F32 generation of PEDV can cause 60%morbidity and fecal shedding in infected piglets.The results of immune protection showed that KB4-F22 strain can provide 60%protection with once immunization and were 100%protection with twice immunization,and KB4-F32 strain can provide 60-80%protection with once immunization and were 100%protection with twice immunization.Therefore,KB4-F32 was selected as the vaccine candidate strain.3.Screening of immune adjuvants for PEDV vaccineIn order to determine the best adjuvant for PEDV KB4-F32 inactivated vaccine.Three adjuvants,including oil-in-water adjuvant A,water-in-oil adjuvant B,bidirectional(water-in-oil-in water)adjuvant C,were selected to prepare the vaccine after the inactivation of PEDV and their safety and efficacy were determined.The results showed that the temperature,feeding,drinking of vaccinated 3-day-old piglets were all normal.No side effect was observed.After vaccination with 3 different vaccines,the adjuvant C group induced higher neutralization antibody against PEDV than the adjuvants A and B groups.The adjuvant C group provided a 100%protection against PEDV and no virus shedding in the feces,while adjuvant A and B groups had mild diarrhea and virus shedding in the feces.Based on the above results,the cebico adjuvant C was selected for production of the inactivated vaccine against porcine epidemic diarrhea.4.Preparation of PED inactivated vaccine(KB4)and evaluation of its immune efficacyThree batches of inactivated vaccines(KB4-F32)against PED were produced in this study.The safety test confirmed that the vaccines were well absorbed,the feeding,drinking and mental status of vaccinated piglets were normal,and there were no side effect such as increased body temperature,redness,swelling and lumps observed in the injection site.In order to verify the protection efficacy of the inactivated vaccine of PEDV,3-day-old piglets were immunized once and boosted at 14 days post vaccination with 1 ml vaccine.The sera were collected at 7 days after secondary immunization and the piglets were challenged with 2 ml PEDV KB(105.5TCID50/ml).The clinical symptoms of each group of pigs were observed for 10 days post challenge,and the anal swabs were collected daily for virus detection by RT-PCR.The results showed that the antibody titers induced by all the 3 batches vaccine were more than 1:32,and that of the control group was less than 1:4.In the challenge test,all of the 3 batches vaccine could provide a 100%protection against PEDV induced diarrhea,only a few piglets showed transient anorexia and depression in 2016001 and 2016003 vaccination groups and recovered within 2 days.All the pigs in the control group had diarrhea,and the mortality was 10%.Our studies suggested that the vaccine can provide high protection and no virus shedding in feces against PEDV.In conclusion,a PEDV KB4 strain was isolated and identified;its titer was more than 107.5 TCID50/ml after 32 generation passage.The preparation of inactivated vaccine can provide 100%protection against PEDV challenge after twice immunization,which is useful for prevention and control of PED.