Fine level epitope mapping and conservation analysis of two novel linear B-cell epitopes of the avian infectious bronchitis coronavirus nucleocapsid protein

Abstract

The nucleocapsid (N) protein of the infectious bronchitis virus (IBV) may play an essential role in the replication and translation of viral RNA. The N protein can also induce high titers of cross-reactive antibodies and cell-mediated immunity, which protects chickens from acute infection. In this study, we generated two monoclonal antibodies (mAbs), designated as 6D10 and 4F10, which were directed against the N protein of IBV using the whole viral particles as immunogens. Both of the mAbs do not cross react with Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV) and subtype H9 avian influenza virus (AIV). After screening a phage display peptide library and peptide scanning, we identified two linear B-cell epitopes that were recognized by the mAbs 6D10 and 4F10, which corresponded to the amino acid sequences (242)FGPRTK(247) and (195)DLIARAAKI(203), respectively, in the IBV N protein. Alignments of amino acid sequences from a large number of IBV isolates indicated that the two epitopes, especially (242)FGPRTK(247), were well conserved among IBV strains. This conclusion was further confirmed by the relationships of 18 heterologous sequences to the 2 mAbs. The novel mAbs and the epitopes identified will be useful for developing diagnostic assays for IBV infections.

Two novel neutralizing antigenic epitopes of the s1 subunit protein of a QX-like avian infectious bronchitis virus strain Sczy3 as revealed using a phage display peptide library

“…The amplified fragments were linked with pET-32a (+) to generate two recombinant plasmids pET-32a-Se1 and pET-32a-Se2, and the fusion proteins pET-se1 and pET-se2 were induced with 1 mM IPTG, and analyzed by SDS-PAGE. The fusion proteins were purified by HisTrap FF crude Columns, and used for further ELISA and Western-blot analysis, as previously described (Han et al, 2013).…”

“…Recently, RPPD libraries were used to map the antigenic site more elaborately. Han, (2013) identified two novel linear B-cell epitopes 242 FGPRTK 247 and 195 DLIARAAKI 203 of IBV nucleocapsid protein by using a Ph.D.12 TM Phage Display Peptide Library and two mAbs (Han et al, 2013). To update, there was no report of antigenic site mapping in IBV S1 subunit by RPPD library screening.…”

Altered pathogenicity of a tl/CH/LDT3/03 genotype infectious bronchitis coronavirus due to natural recombination in the 5′- 17 kb region of the genome

“…Gross lesions were also carefully examined and recorded for the dead chickens. The kidneys of the dead chickens infected with IBV strain tl/CH/LDT3/03 were subjected to immunohistochemistry (IHC) for the detection of IBV antigen using monoclonal antibody 6D10 (Han et al, 2013) directed against the nucleoprotein as previously described (de Wit et al, 2011a,b;Xu et al, 2015).…”

Recombinant duck enteritis viruses expressing major structural proteins of the infectious bronchitis virus provide protection against infectious bronchitis in chickens

“…In addition, the primer pair DF and DR, corresponding to the flanking sequence of the DEV US10 gene (Table 1), were used to differentiate wild-type (WT) DEV from the rDEVs. Western blotting was performed to detect the expression of IBV proteins from the rDEVs (Han et al, 2013). Rabbit anti-GFP IgG (SigmaeAldrich Corporation, St. Louis, MO, USA), mouse anti-IBV N protein monoclonal antibody (4F10) (Han et al, 2013), and chicken anti-IBV serum were used as primary antibodies for detection of EGFP, N, S, and S1 expressed from rDEV-EGFP, rDEV-N, rDEV-S, and rDEV-S1, respectively.…”