Identification and Characterization of Neutralizing Epitopes on Spike Protein of Turkey Coronavirus
Abstract
Studies were carried out to identify and characterize neutralizing epitopes on the spike (S) protein of turkey coronavirus (TCoV). Turkey coronavirus S gene was arranged into 10 fragments for cloning. COS-7 cells transfected with DNA plasmid coding for 3'end of S1 gene and 5' end of S2 gene (pTriEx3-4F/4R, amino acid residue 482-678), 3' end of S1 gene (pTriEx3-Mod4F/Epi4R, acid residue 476-520), or 3' end of S2 gene (pTriEx3-6F/6R, acid residue 830-1071) were positively reacted with anti-His monoclonal antibody and anti-TCoV serum by immunofluorescent antibody assay. The ratio of virus neutralization (VN) titer between antiserum against expressed Mod4F/Epi4R protein, 4F/4R protein, or 6F/6R protein and a known positive anti-TCoV serum was 0.81, 0.93, or 0. The pathogenicity and immunogenicity of a high-passage TCoV (P3 TCoV 540), passaged serially in turkey embryonated eggs for 344 times (P344 TCoV 540), were studied by experimental infection. P344 TCoV did not cause enteritis-related clinical signs, gross, and microscopic lesions. The highest P344 TCoV 540 viral load was detected in the ileum by real-time reverse transcription polymerase chain reaction (RRT-PCR) at 3 days post-infection (dpi). Turkey poults inoculated with P344 TCoV 540 were protected against subsequent challenge by P344 TCoV 540 completely or P3 TCoV 540 partially. P344 TCoV 540 had 11 amino acid substitutions as compared to those of P3 TCoV in the epitope-containing fragments of S gene. Turkeys were vaccinated by priming with one or two doses of 100 or 750µg DNA (pTriEx3-4F/4R) and boosting with one dose of 200µg recombinant 4F/4R protein. Turkeys primed with two doses of 750µg DNA had the highest level of TCoV S protein-specific IgG and VN titers. Turkeys primed with two doses of 750µg DNA or one dose of 100µg DNA mixed with polyethyleneimine microparticle and one dose of 750µg DNA had 40% protection against infectious challenge at 3 and 10 dpi. In conclusion, C-terminal S1 region of TCoV possesses neutralizing epitope. An epitope-based DNA vaccine coding C-terminal S1 and N-terminal S2 protein of TCoV in DNA-prime protein-boost vaccination strategy elicits TCoV-specific humoral immune responses and confers protection of turkeys against TCoV infection.
Degree
Ph.D.
Advisors
Lin, Purdue University.
Subject Area
Virology|Veterinary services|Immunology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.