VP2 is an outer capsid protein of African horsesickness virus (AHSV) and is recognized by serotype-discriminatory neutralizing antibodies. With the objective of locating its antigenic regions, a filamentous phage library was constructed that displayed peptides derived from the fragmentation of a cDNA copy of the gene encoding VP2. Peptides ranging in size from approximately 30 to 100 amino acids were fused with pIII, the attachment protein of the display vector, fUSE2. To ensure maximum diversity, the final library consisted of three sub-libraries. The first utilized enzymatically fragmented DNA encoding only the VP2 gene, the second included plasmid sequences, while the third included a PCR step designed to allow different peptide-encoding sequences to recombine before ligation into the vector. The resulting composite library was subjected to immunoaffinity selection with AHSV-specific polyclonal chicken IgY, polyclonal horse immunoglobulins and a monoclonal antibody (MAb) known to neutralize AHSV. Antigenic peptides were located by sequencing the DNA of phages bound by the antibodies. Most antigenic determinants capable of being mapped by this method were located in the N-terminal half of VP2. Important binding areas were mapped with high resolution by identifying the minimum overlapping areas of the selected peptides. The MAb was also used to screen a random 17-mer epitope library. Sequences that may be part of a discontinuous neutralization epitope were identified. The amino acid sequences of the antigenic regions on VP2 of serotype 3 were compared with corresponding regions on three other serotypes, revealing regions with the potential to discriminate AHSV serotypes serologically.


Article metrics loading...

Loading full text...

Full text loading...



  1. Atassi, M. Z., Dolimbek, B. Z., Hayakari, M., Middlebrook, J. L., Whitney, B. & Oshima, M. (1996). Mapping of the antibody-binding regions on botulinum neurotoxin H-chain domain 855–1296 with antitoxin antibodies from three host species. Journal of Protein Chemistry 15, 691-700.[CrossRef] [Google Scholar]
  2. Barlow, D. J., Edwards, M. S. & Thornton, J. M. (1986). Continuous and discontinuous protein antigenic determinants. Nature 322, 747-748.[CrossRef] [Google Scholar]
  3. Barnard, B. J. H. (1993). Circulation of African horsesickness virus in zebra (Equus burchelli) in the Kruger National Park, South Africa, as measured by the prevalence of type specific antibodies. Onderstepoort Journal of Veterinary Research 60, 111-117. [Google Scholar]
  4. Bonnycastle, L. L. C., Mehroke, J. S., Rashed, M., Gong, X. & Scott, J. K. (1996). Probing the basis of antibody reactivity with a panel of constrained peptide libraries displayed by filamentous phage. Journal of Molecular Biology 258, 747-762.[CrossRef] [Google Scholar]
  5. Burrage, T. G., Trevejo, R., Stone-Marschat, M. & Laegreid, W. W. (1993). Neutralizing epitopes of African horsesickness virus serotype 4 are located on VP2. Virology 196, 799-803.[CrossRef] [Google Scholar]
  6. Clark, M. F. & Adams, A. N. (1977). Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Journal of General Virology 34, 475-483.[CrossRef] [Google Scholar]
  7. Clarke, L. & Carbon, J. (1976). A colony bank containing synthetic Col E1 hybrid plasmids representative of the entire E. coli genome. Cell 9, 91-99.[CrossRef] [Google Scholar]
  8. Cwirla, S. E., Peters, E. A., Barrett, R. W. & Dower, W. J. (1990). Peptides on phage: a vast library of peptides for identifying ligands. Proceedings of the National Academy of Sciences, USA 87, 6378-6382.[CrossRef] [Google Scholar]
  9. Devlin, J. J., Panganiban, L. C. & Devlin, P. E. (1990). Random peptide libraries: a source of specific protein binding molecules. Science 249, 404-406.[CrossRef] [Google Scholar]
  10. Du Plessis, D. H. & Jordaan, F. (1996). Phage libraries displaying random peptides derived from a target sequence. In Phage Display of Peptides and Proteins, pp. 141-150. Edited by B. K. Kay, J. Winter & J. McCafferty. San Diego: Academic Press.
  11. Du Plessis, D. H., Wang, L. F., Jordaan, F. A. & Eaton, B. T. (1994). Fine mapping of a continuous epitope on VP7 of bluetongue virus using overlapping synthetic peptides and a random epitope library. Virology 198, 346-349.[CrossRef] [Google Scholar]
  12. Du Plessis, D. H., Romito, M. & Jordaan, F. (1995). Identification of an antigenic peptide specific for bluetongue virus using phage display expression of NS1 sequences. Immunotechnology 1, 221-230.[CrossRef] [Google Scholar]
  13. Fack, F., Hügle-Dörr, B., Song, D., Queitsch, I., Petersen, G. & Bautz, E. K. F. (1997). Epitope mapping by phage display: random versus gene-fragment libraries. Journal of Immunological Methods 206, 43-52.[CrossRef] [Google Scholar]
  14. Fehrsen, J. & Du Plessis, D. H. (1999). Cross-reactive epitope mimics in a fragmented-genome phage display library derived from the rickettsia, Cowdria ruminantium. Immunotechnology 4, 175-184.[CrossRef] [Google Scholar]
  15. Felici, F., Castagnoli, L., Musacchio, A., Jappelli, R. & Cesareni, G. (1991). Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector. Journal of Molecular Biology 222, 301-310.[CrossRef] [Google Scholar]
  16. Howell, P. G. (1962). The isolation and identification of further antigenic types of African horsesickness virus. Onderstepoort Journal of Veterinary Research 29, 139-149. [Google Scholar]
  17. Huismans, H. & Van Dijk, A. A. (1990). Bluetongue virus structural components. Current Topics in Microbiology and Immunology 162, 21-41. [Google Scholar]
  18. Huismans, H., van der Walt, N. T., Cloete, M. & Erasmus, B. J. (1987). Isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep. Virology 157, 172-179.[CrossRef] [Google Scholar]
  19. Ilyichev, A. A., Minenkova, O. O., Kishchenko, G. P., Tat’kov, S. I., Karpishev, N. N., Eroshkin, A. M., Ofitzerov, V. I., Akimenko, Z. A., Petrenko, V. A. & Sandakhchiev, L. S. (1992). Inserting foreign peptides into the major coat protein of bacteriophage M13. FEBS Letters 301, 322-324.[CrossRef] [Google Scholar]
  20. Iwata, H., Yamagawa, M. & Roy, P. (1992). Evolutionary relationships among the gnat-transmitted orbiviruses that cause African horse sickness, bluetongue, and epizootic hemorrhagic disease as evidenced by their capsid protein sequences. Virology 191, 251-261.[CrossRef] [Google Scholar]
  21. Kay, B. K., Adey, N. B., He, Y. S., Manfredi, J. P., Mataragnon, A. H. & Fowlkes, D. M. (1993). An M13 phage library displaying random 38-amino-acid peptides as a source of novel sequences with affinity to selected targets. Gene 128, 59-65.[CrossRef] [Google Scholar]
  22. Martı́nez-Torrecuadrada, J. L. & Casal, J. I. (1995). Identification of a linear neutralization domain in the protein VP2 of African horse sickness virus. Virology 210, 391-399.[CrossRef] [Google Scholar]
  23. Martı́nez-Torrecuadrada, J. L., Langeveld, J. P. M., Venteo, A., Sanz, A., Dalsgaard, K., Hamilton, W. D. O., Meloen, R. H. & Casal, J. I. (1999). Antigenic profile of African horse sickness virus serotype 4 VP5 and identification of a neutralizing epitope shared with bluetongue virus and epizootic hemorrhagic disease virus. Virology 257, 449-459.[CrossRef] [Google Scholar]
  24. Nagesha, H. S., Yu, M. & Wang, L. F. (1996). Application of linker-ligation-PCR for construction of phage display epitope libraries. Journal of Virological Methods 60, 147-154.[CrossRef] [Google Scholar]
  25. Parmley, S. F. & Smith, G. P. (1988). Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene 73, 305-318.[CrossRef] [Google Scholar]
  26. Petersen, G., Song, D., Hügle-Dörr, B., Oldenburg, I. & Bautz, E. K. F. (1995). Mapping of linear epitopes recognized by monoclonal antibodies with gene-fragment phage display libraries. Molecular and General Genetics 249, 425-431. [Google Scholar]
  27. Polson, A., Coetzer, T., Kruger, J., von Maltzahn, E. & van der Merwe, K. J. (1985). Improvements in the isolation of IgY from the yolks of eggs laid by immunized hens. Immunological Investigations 14, 323-327.[CrossRef] [Google Scholar]
  28. Roy, P. (1992). Bluetongue virus proteins. Journal of General Virology 73, 3051-3064.[CrossRef] [Google Scholar]
  29. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989).Molecular Cloning. A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  30. Scott, J. K. & Smith, G. P. (1990). Searching for peptide ligands with an epitope library. Science 249, 386-390.[CrossRef] [Google Scholar]
  31. Smith, G. P. (1985). Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315-1317.[CrossRef] [Google Scholar]
  32. Smith, G. P. & Scott, J. K. (1993). Libraries of peptides and proteins displayed on filamentous phage. Methods in Enzymology 217, 228-257. [Google Scholar]
  33. Specthrie, L., Bullitt, E., Horiuchi, K., Model, P., Russel, M. & Makowski, L. (1992). Construction of a microphage variant of filamentous bacteriophage. Journal of Molecular Biology 228, 720-724.[CrossRef] [Google Scholar]
  34. Van Regenmortel, M. H. V. (1990). The structure of viral epitopes. In Immunochemistry of Viruses: the Basis for Serodiagnosis and Vaccines, pp. 1-24. Edited by M. H. V. Van Regenmortel & A. R. Neurath. Amsterdam: Elsevier.
  35. Van Regenmortel, M. H. V. & de Marcillac, G. D. (1988). An assessment of prediction methods for locating continuous epitopes in proteins. Immunology Letters 17, 95-107.[CrossRef] [Google Scholar]
  36. Van Wyngaardt, W., Du Plessis, D. H., Van Wyngaardt, S. & Verschoor, J. A. (1992). Production and properties of monoclonal antibodies against African horsesickness virus, serotype 3. Onderstepoort Journal of Veterinary Research 59, 129-133. [Google Scholar]
  37. Venter, M. (1999).The characterisation of truncated fragments of the major neutralisation specific antigen of African horsesickness virus serotype nine. MSc thesis, University of Pretoria, South Africa.
  38. Vreede, F. T. & Huismans, H. (1994). Cloning, characterization and expression of the gene that encodes the major neutralization-specific antigen of African horsesickness virus serotype 3. Journal of General Virology 75, 3629-3633.[CrossRef] [Google Scholar]
  39. Wang, L. F., Du Plessis, D. H., White, J. R., Hyatt, A. D. & Eaton, B. T. (1995). Use of a gene-targeted phage display random epitope library to map an antigenic determinant on the bluetongue virus outer capsid protein VP5. Journal of Immunological Methods 178, 1-12.[CrossRef] [Google Scholar]
  40. Williams, C. F., Inoue, T., Lucus, A. M., Zanotto, P. M. & Roy, P. (1998). The complete sequence of four major structural proteins of African horse sickness virus serotype 6: evolutionary relationships within and between the orbiviruses. Virus Research 53, 53-73.[CrossRef] [Google Scholar]

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error