The West Nile virus (WNV) subtype Kunjin virus (WNVKUN) is endemic to Australia. Here, we characterized the classical WNVKUN strain, OR393. The original OR393 strain contained two types of viruses: small plaque-forming virus (SP) and large plaque-forming virus (LP). The amino acid residues at positions 156 and 332 in the E protein (E156 and E332) of SP were Ser and Lys (E156S/332K), respectively, whereas those in LP were Phe and Thr (E156F/332T). SP grew slightly faster than LP in vitro. The E protein of SP was N-glycosylated, whereas that of LP was not. Analysis using two recombinant single-mutant LP viruses, rKUNV-LP-EF156S and rKUNV-LP-ET332K, indicated that E156S enlarged plaques formed by LP, but E332K potently reduced them, regardless of the amino acid at E156. rKUNV-LP-EF156S showed significantly higher neuroinvasive ability than LP, SP, and rKUNV-LP-ET332K. Our results indicate that the low-pathogenic classical WNVKUN can easily change its pathogenicity through only a few amino acid substitutions in the E protein. It was also found that Phe at E156 of the rKUNV-LP-ET332K was easily changed to Ser during replication in vitro and in vivo, suggesting that E156S is advantageous for the propagation of WNVKUN in mammalian cells.
Keywords: E protein; Kunjin virus; West Nile virus; glycosylation; pathogenicity; reverse genetics.