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

Abstract

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.

Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyltransferase and polymerase domains

“…S4, available in JGV Online). This indicates that the conformation of KUN-NS5-47R-13 is compromised after adsorbance to the solid phase in ELISA, while the extra amino acids in the longer peptide (KUN-NS5-47R-19) preserve the antigenic structure after attachment to the plastic (Hobson-Peters et al, 2008). In summary, the peptide binding data described above indicate that a 13 aa stretch (encompassing positions 41-53 of NS5) contains the core residues of the 5H1 epitope.…”

“…Peptides were solubilized in water, except for the control peptide which was solubilized in acetic acid, and peptide NS5-25I-19, which was solubilized in dimethylformamide. Following solubilization, peptides were tested by ELISA and microsphere immunoassay (Luminex) as described previously (Hobson-Peters et al, 2008).…”

Detection of Antibodies to West Nile Virus in Horses, Costa Rica, 2004

“…In addition to assessment by PRNT and blocking ELISA, this subset of sera were further assessed by Western blot using a peptide from domain I of the WNV NY99 envelope protein (WN19) that was previously shown to have diagnostic potential in WNV-specific serological assays (Hobson-Peters et al 2008). The sera from 4 of the 5 Costa Rican horses that had antibodies to WNV by blocking ELISA and PRNT (Table 2, samples A9, A11, B5, and B10) reacted to peptide WN19 by Western blot.…”

“…Samples that returned a WNV PRNT 90 titer that was at least fourfold greater than the corresponding SLEV PRNT 90 titer were considered to have antibodies to WNV. Sera were further assessed by Western blot using a peptide from domain I of the WNV NY99 envelope protein (WN19), using previously described procedures (Hobson-Peters et al 2008).…”

A Novel Bacterium-Free Method for Generation of Flavivirus Infectious DNA by Circular Polymerase Extension Reaction Allows Accurate Recapitulation of Viral Heterogeneity

“…The locations of mutations were chosen so that the phenotype of mutants could be easily distinguished from that of the wild type: i.e., removal of glycosylation motif in E (E S156F) and change in the reactivity of the NS5 protein to the 5H1 monoclonal antibody (NS5 R47G and NS5 V49I) (22)(23)(24)(25). To generate the E S156F mutant, two overlapping PCR fragments were generated using the BmgBI-BstEII fragment as the template (Fig.…”