Identification Of B-cell Epitopes And T-cell Epitopes In Glycoprotein 3 Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome?PRRS?is an infectious disease caused by porcine reproductive and respiratory syndrome virus?PRRSV?,which is characterized by reproductive failure in sows and respiratory diseases in all ages of pigs.PRRSV can infect different types of pigs and had a devastating ecomomic impact on the swine industry worldwide.Current commercial vaccines cannot provid complete protection to the vaccinated pigs mainly due to antibody dependent enhancement?ADE?,high variability and persistent infection.Thus,it is urgent to develop novel anti-PRRSV strategies to control this disease.Previous studies show that PRRSV can induce antibody responses and adaptive immunity.Glycoprotein 3 is one of the minor structural proteins of PRRSV,which plays an important role in the replication,assembly,mutation and pathogenicity of the virus.In this study,two B-cell epitopes and three T-cell epitopes of Glycoprotein 3 were identified by ELISA and ELISPOT,which may provide theoretical basis and data support for the development of vaccines against PRRSV.JXA1-R strain of PRRSV was used for vaccination in this study.Eight healthy5-weeks-old piglets were split in two groups?A and B?.Five animals in group A were vaccinated with 3 mL of JXA1-R and other three in group B were vaccinated with 3mL of supernatant of MARC-145 cells as controls.After vaccination,serum were collected every week and the peripheral blood mononuclear cells were collected at 12weeks post-infection.Serum were used to analyze the anti-N protein antibodies and viremia in vivo by ELISA and Real-Time PCR.PBMCs were used to ELISPOT assay.ELISA shows that anti-N protein antibodies appeared at 7 days post-vaccination and peaked between 14 and 21 days after infection.Real-Time PCR shows that viremia peaked between 7 and 14 days after infection and then declined,as excepted,viremia peaked again after the booster immunization,by 49 days post-infection,serum virus declined to undetectable levels in most pigs.A total of 24 peptides consisting of 18 amino acids with 9 amino acids overlaps were designed and synthesized based on the sequence of 226 amino acids of Glycoprotein3 extracellular domain of PRRSV JXA1-R strains.These synthetic peptides were used in ELISA assay and ELISPOT assay to define the location of epitopes recognized by B-lymphocytes and T-lymphocytes from PRRSV-infected gilts.ELISA shows that the immunodominant B-cell epitopes of Glycoprotein 3 were mainly distributed in the amino acids 46-99 of the N-terminus,of these immunodominant epitopes,peptide 4:P⁵⁵LCPTRQAAAEILEPGKS⁷² and peptide 7:C⁸²SENDHDELGFMVPPGLS⁹⁹ showed stronger immunoreactivity.ELISPOT showsthatthreepeptides(A¹⁰⁹WLAFLSFSYTAQFHPEI¹²⁶,Y¹³⁶VDIKHQFICAVHDGDNA¹⁵³and L²³⁵GMATRPLRRFAKVLSAA²⁵²)appeared to contain immunodominant T-cell epitopes.The amino acid sequence of identified B-cell epitopes and T-cell epitopes were aligned to the respective regions of Glycoprotein 3 of 21 PRRSV strains using DNASTAR.The results showed that the two B-cell epitopes identified in this study were 84%and 90%homologous to North American strains,respectively.The three T-cell epitopes?peptides No.10,No.13 and No.24?were 99.7%,78%,and 66%homologous to North American strains,respectively.