The Identification and Characterization of Two Novel Epitopes on the Nucleocapsid Protein of the Porcine Epidemic Diarrhea Virus

Abstract

Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that causes severe diarrhea and death, particularly in neonatal piglets. The nucleocapsid protein (N protein) of PEDV presents strong immunogenicity and contributes to the cross-reactivity between PEDV and TGEV. However, the characterization of epitopes on the PEDV N protein remains largely unknown. Here, two monoclonal antibodies (MAbs) specific to the N protein of a PEDV strain, FJzz1/2011, were generated and screened against a partially overlapping library of 24 GST-fusion N protein-truncated constructs. We confirmed that residues 18–133 (designated NEP-D4) and residues 252–262 (designated NEP-D6) were the epitopes targeted by MAbs PN-D4 and PN-D6, respectively. Sequence analysis revealed that these two epitopes were highly conserved among PEDV strains but were significantly different from other members of the Coronavirinae subfamily. Western blot analysis showed that they could be specifically recognized by PEDV antisera but could not be recognized by TGEV hyperimmune antisera. Indirect immunofluorescence (IFA) assays confirmed no cross-reaction between these two MAbs and TGEV. In addition, the freeze-thaw cycle and protease treatment results indicated that NEP-D4 was intrinsically disordered. All these results suggest that these two novel epitopes and their cognate MAbs could serve as the basis for the development of precise diagnostic assays for PEDV.

Molecular characterization of US-like and Asian non-S INDEL strains of porcine epidemic diarrhea virus (PEDV) that circulated in Japan during 2013–2016 and PEDVs collected from recurrent outbreaks

“…Compared to the vaccine strains, all the field strains in the present study had 9 aa substitutions (Additional file 2: Figure S1B); the substitution K123 N resulted in forming a predicted N-glycosylation site on the N protein from the field strains, while the aa substitutions A142T and N255S observed in field strains formed two predicted phosphorylation sites. Of note, the substitutions K123 N and N255S belong to the reported antigenic epitopes NEP-D4 (aa 18-133) and NEP-D6 (aa 252-262), respectively [18].…”

“…The N protein of coronavirus interacts with viral genomic RNA, serving as the critical basis for the helical nucleocapsid during the viral assembly [17]. Two antigenic epitopes (NEP-D4 and NEP-D6) have been identified in the N protein of PEDV [18].…”

Identification of a Novel Linear B-Cell Epitope on the Nucleocapsid Protein of Porcine Deltacoronavirus

“…N protein also owns multiple functions in pathogenesis, viral replication, and immune system interference [17]. These characteristics make the N protein an ideal target for development of serological methods based on purified protein [19] or antigenic epitopes [20].…”

Cellular hnRNP A1 Interacts with Nucleocapsid Protein of Porcine Epidemic Diarrhea Virus and Impairs Viral Replication

“…Firstly, as a structural protein, N Protein along with the genomic RNA forms the nucleocapsid of PEDV. Furthermore, N protein plays an important role in PEDV RNA synthesis and enhancing the PEDV transcription and virion assembly [ 8 , 9 , 10 ]. However, the effects of N protein on PEDV infection usually depend on its interaction with some host factors.…”