A glycosylated peptide in the West Nile virus envelope protein is immunogenic during equine infection

1 School of Molecular and Microbial Sciences, The University of Queensland, St Lucia, Queensland 4072, Australia 2 AGEN Biomedical Limited, Acacia Ridge, Queensland, Australia 3 Australian Biosecurity CRC for Emerging Infectious Disease, St Lucia, Queensland, Australia 4 Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 5 CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, Victoria, Australia Correspondence Roy A. Hall roy.hall{at}uq.edu.au Using a monoclonal antibody directed to domain I of the West Nile virus (WNV) envelope (E) protein, we identified a continuous (linear) epitope that was immunogenic during WNV infection of horses. Using synthetic peptides, this epitope was mapped to a 19 aa sequence (WN19: E147–165) encompassing the WNV NY99 E protein glycosylation site at position 154. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N -linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognized the recombinant peptide. Failure of most WNV- and MVEV-positive horse sera to recognize the epitope as a deglycosylated fusion protein confirmed that the N -linked glycan was important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. Present address: The International Livestock Research Institute, PO Box 30709, Nairobi 00100, Kenya. Present address: Department of Pathology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA. Present address: Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA, USA. || Present address: Mosquito Control Laboratory, Queensland Institute of Medical Research, Herston, Queensland, Australia.