Isolation,Identification And Pathogenicity Analysis Of Emerging Porcine Epidemic Diarrhea Virus And Prokaryotic Expression Of N Protein

The pathogen of porcine epidemic diarrhea?PED?is porcine epidemic diarrhea virus?PEDV?.It mainly causes intestinal disease in pigs and symptoms are characterized by vomiting,diarrhea,anorexia,dehydration,and significant day age dependent.Especially for sucking piglets within 7 days of age,mortality can be as high as 100%for lack of effective maternal antibody.In recent years,outbreak of PED indicated that the traditional PEDV commercial vaccines have been lack of effective resistance to the epidemic PEDV.Thus,the study of the epidemic PEDV strains and the analysis its molecular epidemiology can provide indispensable theory for revealing the pathogenic molecular mechanism of novel emerged PEDV infection and the development of specificity diagnosis.In order to confirm the two strains PED virus isolated from intestines of diarrhea with highly pathogenic,we observed syncytium formations induced by the virus by inoculating the clinical samples on the vero cell which was cultured with trypsin.Moreover,the viruses were confirmed to be PEDV by multiplex reverse transcriptase-polymerase chain reaction?RT-PCR?,designatedas"C H/GDZH01/1311"?"ZH01"forabbre.?and"C H/GDZH02/1401"?"ZH02"for abbre.?.Meanwhile,their titers were measured on cells and showed 1×10^5.50 TCID₅₀/mL and 1×10^6.25 TCID₅₀/mL respectively.According to the identification of RT-PCR special for wild/attenuated-type PEDV and animal regression experiment,these two isolates were confirmed to be wild-type PEDV which are highly virulent.C loning and sequencing their N gene showed that these two isolates belong to the G2-1 subgroup,which has closed relationship with most lately epidemic isolates.However,they show low homology with the classical virulent strain CV777,which belongs to G1subgroup.In this research,ZH02 had been conducted to cell serial passage for 65generations for attenuation.It showed a titer of 1×10^6.75 TCID₅₀/mL which is similar to parental strain,but resulted in a later clinical symptom and death in neonatal piglets than parental strain.In the process of passaging the virus in vitro,The ZH02 strains of generation 65 had been sequenced.It showed that there were two nucleotides deletion in S gene and six nucleotides deletion compared with its parental strain,respectively.We initially found ZH02-P65 contains a 2-nt deletion in the cytoplasmic domain of the S gene causing early termination of translation.As reported in other coronaviruses,the lack of cytoplasm domain can cause the overexpression S gene,which induced a stronger CPE of fusogenic activity.As the S protein plays an importanr role in inducing the neutralizing antibody,its overexpression will help improve the immunogenicity of the virus,and suggesting it may become a better candidate vaccine strain.In the study,we successfully induced prokaryotic expression of N gene.We found the conditions of 37?,with Transetta?DE3?expression host cell and inducing by IPTG concentration of 1.0 mmol/L for 5 h can get the best quantity of protein expression.The result of SDS-PAGE electrophoresisshowed that the object protein had a size of 54 KDa,which was similar with the predicted one by software.The Western Blot result verified this recombinant protein were PEDV N protein.The specificity of the recombinant protein were confirmed by the PEDV N protein antigen detection kits.