Identification Of The Immunodominant Neutralizing Regions In The Spike Glycoprotein Of Porcine Deltacoronavirus

Porcine deltacoronavirus(PDCoV)belongs to the Coronavirus family and is a member of the delta coronavirus.It is a new enteric coronavirus that mainly infects piglets,causing severe enteritis and showing clinical symptoms of diarrhea,vomiting,and dehydration.PDCoV is widely popular among pigs,that poses a novel threat to swine husbandry worldwide.Crucial to halting PDCoV transmission and infection is the development of effective therapies and vaccines.During coronavirus infection,the spike(S)protein of coronavirus is the major target of host neutralizing antibodies.Therefore,this study intends to express PDCoV S protein in stages,prepare and identify its specific polyclonal antibody,and utilize virus neutralization assay(VN assay)and fluorescent focus neutralization assay(FFN)and Plaque-reduction neutralization tests(PRNT)detect the neutralizing activity of antibodies to identify the immunodominant regions of PDCoV S protein and lay the foundation for the design of subunit vaccines based on the immunodominant regions of PDCoV S protein.we aimed to identify the immunodominant region of PDCoV S protein,and its neutralizing epitopes.Based on previous structural data of PDCoV S protein,three truncated S proteins spanning the entire S domain were produced using an E.coli expression system.The constructs were designated S1-NTD(N-terminal domain of the S1 subunit,amino acids(aa)50-286),S1-CTD(C-terminal domain of the S1subunit(aa 278-616),and the S2 subunit(aa 601-1087).The purified protein was diluted to the same concentration,and the porcine anti-PDCoV positive serum was used as the primary antibody.The western blot was used to verify the truncated S protein reactogenicity.The results showed that the S1-CTD fusion protein was highly reactive with the PDCoV polyclonal antibody.Purified recombinant S1-NTD,S1-CTD and S2 proteins were injected into rabbits to produce specific antisera.All antisera were able to inhibit PDCoV infection in vitro,as determined by VN and FFN assays.Amongst the three antisera,S1-CTD-specific serum was the most effective.However,since the neutralizing antibody in these experiments was obtained from a single time point after immunization,the result is not conclusive.To verify the relative efficacies of the antisera,female BALB/c mice were inoculated with purified protein and the corresponding antibodies was evaluated several times after immunization.The inoculated mice showed a significant increase in anti-PDCoV antibody titer after receiving the second immunization.Anti-S1-CTD antiserum consistently showed the highest neutralizing antibody titers by VN assay across time points.At week 4,mice inoculated with S1-CTD showed a significant increase in neutralizing antibody titers to 1:545(± 81.7).Similarly,anti-S1-CTD antiserum collected at week 4 also had a markedly higher neutralizing titer(1:320)in FFN and PRNT assays.These results suggest that PDCoV S1-CTD has an important role in the viral infection process and can induce the body to produce strong neutralizing antibodies.