| Porcine epidemic diarrhea virus(PEDV) is an acute and contagious infectious disease that infects cells lining the small intestine of swine, resulting in vomiting, diarrhea, and dehydration. In China, the incidence of PEDV outbreaks has rapidly increased since 2010. The mortality due to PEDV infection can reach 80%–100% in piglets. Although the use of inactivated and attenuated vaccines may have helped to control the prevalence of disease, PEDV has continually emerged, causing tremendous losses to the swine industry in China.The amino acid sequences of the spike(S) protein, which can induce neutralizing antibody, has multiple insertions, deletions and mutations between the two PEDV genotypes, G1 and G2. To evaluate if these mutations lead to changes in antigenicity, we first optimized the codons of typical S genes of the CV777 vaccine strain(G1 subtype) and LNCT2 strain(G2 subtype) and expressed the recombinant full-length sequence of the S protein in a eukaryotic expression system. The IgG antibody levels of serum from mice immunized with purified S protein were markedly high. Comparison of antigenicity was based on polyclonal antibodies(PAbs) against the virus using enzyme-linked immunosorbent assay(ELISA), indirect immunofluorescence assay(IFA), Western blot and serum cross-neutralization(SN) assay. ELISA results indicated that the titers of anti-S PAbs reacting with same subtype S protein reached 1:204800, while the titers reacting with different subtype S protein reached 1:102400.IFA indicated that the highest titer of anti-S PAbs reacting with PEDV within subtypes(observing green fluorescence) reached 1:51200, while it was 1:25600 across subtypes. The average titer of anti-SCV777 PAb neutralizing CV777 was 1:336 while the titer cross-neutralizing LNCT2 was 1:150. The titer of anti-SLNCT2 PAbs neutralizing LNCT2 reached 1:387, while neutralizing CV777 was 1:230. Although reactivity with the PAbs revealed significant cross reactivity between the two PEDV subtypes, the results suggested that there was antigenic variation between PEDV subtypes G1 and G2.In order to detect the pivotal region of S protein that lead to the antigenic variation, we identified five monoclonal antibodies(MAbs) against S protein. All of MAbs could identify S proteins in natural viruses. L5F7 MAb could react with LNCT2 strian but not CV777 strain,so that it could be used to identify G1 and G2 subtype strains of PEDV. The conformational epitope was primarily located on 1 to 234 amino acids(aa) of N terminal of S protein by truncating S protein. According to squences alignment we found that high variation among S proteins was located on 21-402 aa. We expressed S121-402 aa proteins of CV777 and LNCT2 by Bac-to-Bac system and prepared polyclonal antibodies. There was no obvious difference in the antigenic responses based on PAb titers of ELISA and IFA. The results indicated that the mutation of S121-402 aa protein was not obviously related to antigenic variationFocused on PEDV S protein, we demonstrated that there were molecular and antigenic variations between G1 and G2 subtypes and preliminarily detected the variable antigenic regions. The analysis helps to understand the variation of PEDV in China and provide basic information on evaluating current vaccines, selecting and producing new vaccines. |
