Identification and characterization of a neutralizing-epitope-containing spike protein fragment in turkey coronavirus

Abstract

Little is known about the neutralizing epitopes in turkey coronavirus (TCoV). The spike (S) protein gene of TCoV was divided into 10 fragments to identify the antigenic region containing neutralizing epitopes. The expression and antigenicity of S fragments was confirmed by immunofluorescence antibody (IFA) assay using an anti-histidine monoclonal antibody or anti-TCoV serum. Polyclonal antibodies raised against expressed S1 (amino acid position 1 to 573 from start codon of S protein), 4F/4R (482-678), 6F/6R (830-1071), or Mod4F/Epi4R (476-520) S fragment recognized native S1 protein and TCoV in the intestines of TCoV-infected turkey embryos. Anti-TCoV serum reacted with recombinant 4F/4R, 6F/6R, and Mod4F/Epi4R in a western blot. The results of a virus neutralization assay indicated that the carboxyl terminal region of the S1 protein (Mod4F/Epi4R) or the combined carboxyl terminal S1 and amino terminal S2 protein (4F/4R) possesses the neutralizing epitopes, while the S2 fragment (6F/6R) contains antigenic epitopes but not neutralizing epitopes.

Fig. 1

Positions of primers used to amplify fragments covering the whole spike (S) protein of turkey coronavirus (TCoV) isolate 540. The S protein of TCoV has 1203 amino acids and contains an N-terminal subunit (S1) and a C-terminal subunit (S2). The predicted cleavage site is located at amino acid 535. The amino acid (aa) position presented in the figure is from the start codon on the S protein gene of TCoV isolate 540 (GenBank accession number EU022525). The numbers above the names of the forward and reverse primers represent the position of the first or the last amino acid in the expressed S fragments

Fig. 2

Antigenicity of spike (S) protein fragments of turkey coronavirus (TCoV). IFA results with untransfected COS-7 cells as a negative control or cells transfected with various plasmids encoding fragments covering the whole S protein of TCoV are shown. The positive IFA result obtained with mouse anti-His monoclonal antibodies (Anti-His MAbs) indicates expression of recombinant protein fused with a His-tag from the transfected plasmid in COS-7 cells. The positive IFA result obtained with anti-TCoV serum indicates that the expressed S fragment from the transfected plasmid contained antigenic epitopes. (a) Summary of IFA results for anti-His MAbs or anti-TCoV serum reacting with COS-7 cells transfected with various plasmids encoding TCoV S fragments. (b) Photomicrography of untransfected COS-7 cells as negative acontrol or cells transfected with various plasmids encoding TCoV S fragments in an IFA with anti-His MAbs or anti-TCoV serum. Magnification, 400x

Fig. 3

Recombinant 4F/4R (amino acid position 482-678), 6F/6R (830-1071), and Mod4F/Epi4R (476-520) spike (S) proteins of turkey coronavirus (TCoV) expressed in E. coli and purified on His columns can be recognized by anti-TCoV serum in a western blot assay. (a) Recombinant 6F/6R and 4F/4R S proteins transferred from a 12.5% glycine SDS-PAGE gel to a nitrocellulose membrane were stained with 1:400-diluted anti-TCoV serum, followed by 1:400-diluted horseradish peroxidase (HRP)-conjugated goat anti-turkey IgG (H+L) secondary antibodies. The arrows point to 6F/6R S protein at 31 kD and 4F/4R S protein at 26 kD. (b) Recombinant Mod4F/Epi4R S protein transferred from a 16%/6 M urea Tricine SDS-PAGE gel to a nitrocellulose membrane was stained with 1:400-diluted anti-TCoV serum, followed by 1:400 HRP-conjugated goat anti-turkey IgG (H+L) secondary antibodies. The arrow points to a dimer of Mod4F/Epi4R S protein at 10 kD

Fig. 4

Results of deduced amino acid sequence alignment in the antigenic domain containing neutralizing epitopes (Mod4F/Epi4R S fragment) from TCoV isolates. TCoV 540, TCoV/TX-1038/98, TCoV/IN-517/94, and TCoV/VA-74/03 showed four different amino acid differences among four TCoV isolates and three differences among TCoV isolates excluding TCoV 540. The sequences of TCoV 540, TCoV/TX-1038/98, TCoV/IN-517/94, and TCoV/VA-74/03 are from GenBank accession numbers EU022525, GQ427176, GQ427175, and GQ4271731, respectively