Epitope mapping and characterization of a novel Nsp10-specific monoclonal antibody that differentiates genotype 2 PRRSV from genotype 1 PRRSV

Zhibang Zhang,Jianguo Dong,Hanchun Yang,Lei Zhou,Xuexia Wen,Xinna Ge,Xin Guo

doi:10.1186/s12985-017-0782-9

Abstract

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of PRRS, has two distinct and highly diverse genotypes (genotype 1 and genotype 2) in the field. Accurate diagnosis and differentiation of the two genotypes of PRRSV are critical to the effective prevention and control of PRRS. The non-structural protein 10 (Nsp10) plays a vital role in viral replication and is one of the most conserved proteins of PRRSV, thus constituting a good candidate for PRRSV diagnosis.ResultsIn this study, we generated a monoclonal antibody (mAb) 4D9 against Nsp10 by immunizing BALB/c mice with purified recombinant Nsp10 expressed by an Escherichia coli system. Through fine epitope mapping of mAb 4D9 using a panel of eukaryotic expressed polypeptides with GFP-tags, we identified the motif 286AIQPDYRDKL295 as the minimal unit of the linear B-cell epitope recognized by mAb 4D9. Protein sequence alignment indicated that 286AIQPDYRDKL295 was highly conserved in genotype 2 PRRSV strains, whereas genotype 1 PRRSV strains had variable amino acids in this motif. Furthermore, a mutant of the motif carrying two constant amino acids of genotype 1 PRRSV, Cys290 and Glu293, failed to react with mAb 4D9. More importantly, the mAb 4D9 could differentiate genotype 2 PRRSV strains from genotype 1 PRRSV strains using Western blotting and immunofluorescence analysis.ConclusionOur findings suggest that Nsp10-specific mAb generated in this study could be a useful tool for basic research and may facilitate the establishment of diagnostic methods to discriminate between genotype 1 and genotype 2 PRRSV infection.

Highlights

Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of PRRS, has two distinct and highly diverse genotypes in the field
The results indicated that the purified recombinant His × 6-non-structural protein 10 (Nsp10) had good reactivity and was suitable for immunization
Production and characterization of Nsp10-specific monoclonal antibody (mAb) Hybridomas were screened by testing the supernatants with PRRSV Nsp10-specific indirect enzyme-linked immunosorbent assay (ELISA)

Summary

Introduction

Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of PRRS, has two distinct and highly diverse genotypes (genotype 1 and genotype 2) in the field. Porcine reproductive and respiratory syndrome (PRRS) is one of the most important viral diseases of pigs worldwide, causing annual economic losses of about US $664 million to the US swine industry [1]. The causative agent, porcine reproductive and respiratory syndrome virus (PRRSV), is an enveloped, single-stranded RNA (+). PRRSV Nsp lies in ORF1b region and encodes helicase [10], which possesses ATPase activity and can unwind dsRNA [11]. We generated a PRRSV Nsp10-specific mAb, fine mapped its epitope and demonstrated that it can differentiate the Nsp of the genotype 2 from that of genotype 1

Methods

Results

Conclusion