Differences Between Immunogeicity Of S Proteins Of Porcine Epidemic Diarrhea Virus Variant Strain

Since October 2010, porcine epidemic diarrhea has been outbreaking in China and other countries. In this thesis, sequences of spike genes of 42 PEDV strains which include 24 PEDV Chinese strains isolated after 2010, 7 US strains, 6 Korean strains and 5 reference strains were compared. The results showed that mutations in S genes of PEDV isolates after 2010 mainly located between 22 aa and 380 aa region which was designed as SA. The 2010 epidemic strains showed closer genetic relationship and falled in same branch; however the CV777 vaccine strains and earlier isolates were grouped in another branch, and they exhibited closer relationship with high homology of 95%-99.1%. The 2010 PEDV strains showed 92.3%- 94.1% homology with earlier isolates, and only 93.3% homology with vaccine strain CV777. The predication of antigenic epitopes and N-glycosylation sites of SA region indicated that there were differences in antigenic epitopes numbers and locations in 22aa-380 aa region, especially in 66aa-280 aa between 2010 epidemic strains and earlier strains. There was an extra N-glycosylation site at 62aa-65 aa of SA in all 2010 epidemic strains. The above results suggested that PEDV isolates after 2010 had developed mutation compared with classical strains. Based on these information, the SA genes of PEDV variant CH-ZMDZY-11 and vaccine strain CV777 were cloned and expressed in E.coli. The expressed SA proteins showed good immunogenicity and immune reactivity. The successful expression laid a foundation for development of an ELISA for detecting anti-PEDV antibody in clinical samples; analysis of difference in immune reactivity between S proteins of PEDV epidemic strains and vaccine strains.By using purified recombinant PEDV SA protein as coating antigen, an indirect ELISA was established for detecting anti-PEDV antibody. The optimum assay conditions were as follow: optimal antigen coating amount of 1ug / well, the coated plates were placed at 37 ℃ for 1h and then at 4 ℃ overnight; serum working dilution at 1:100, incubation time was 1.5h; a secondary antibody HRP-labeled rabbit anti-pig Ig G at a dilution of 1: 4000, TMB reaction time was 15 min. sample with S / P value greater than or equal 0.058 was identified as positive, less than 0.045 as negative. The developed ELISA showed good specificity and reproducibility. For 50 suspected porcine epidemic diarrhea serum samples tested, the results showed high compliance rate with the results of pathogen detection, which indicated that the established ELISA in this study could be used to detect anti-PEDV variant antibodies and for disease monitoring.To analyze the effects of defferences of S genes between PEDV variant and the vaccine strains on vaccine efficacy, SA genes from PEDV variant and vaccine strain were expressed, and anti-SA proteins polyclonal antivodies were prepared by immunized mouse and rabbits with purified recombinant SA proteins respectively. Both Western blotting assay and ELISA showed that there were significant differences in immune reactivity between S proteins of PEDV variant and vaccine strain. Reactivity between vaccine strain SA protein(YSA) and anti-YSA serum was much higher than that of between PEDV variant SA protein(LSA) and anti-YSA serum. And reactivity between PEDV variant SA protein(LSA) and anti-LSA serum was much higher than that of between vaccine strain SA protein(YSA) and anti-YSA serum. The neutralizing titer of anti-YSA serum against vaccine strain CV777 was much higher that that of anti-LSA against CV777.In summary, our results indicated that there were differences in immune reactivities between S proteins of PEDV variant and vaccine strains. And differences in antigenic epitopes numbers and locations in 22aa-380 aa region could lead to vaccination failure of CV777-based PED vaccines against PEDV variant infection.