West Nile virus (WNV), like all members of the Japanese encephalitis (JE) serogroup except JE virus, contains three N-linked glycosylation (N-X-S/T) sites in the NS1 protein at asparagine residues NS1 130, NS1 175 and NS1 207. Previously we showed that the ablation of these glycosylation sites in WNV, by substitution of asparagine for alanine, attenuated mouse neuroinvasiveness; however, full attenuation was not achieved and the virus retained a neurovirulence phenotype. Sequence of viral RNA extracted from mouse brains revealed a reversion at the NS1 130 site in some mice that succumbed to the attenuated NS1 130A/175A/207A strain. Here, we further attenuated WNV by mutating the asparagine to serine or glutamine in addition to mutating other residues in the NS1 130-132 glycosylation motif. These mutants proved to further attenuate WNV for both neuroinvasiveness and neurovirulence in mice. NS1 130-132QQA/175A/207A, the most attenuated mutant virus, showed modest changes in infectivity titers versus the parental strain, was not temperature sensitive, and did not show reversion in mice. Mutant virus was completely attenuated for neuroinvasiveness after intraperitoneal inoculation with >1,000,000 PFU, and mice were protected against lethal challenge. Overall, we showed that changing the asparagine of the NS1 130 glycosylation motif to a serine or glutamine attenuated WNV further than the asparagine to alanine substitution. Further, mutating all three of the amino acids of the NS1 130-132 glycosylation motif (NTT-QQA) along with NS1 175 and NS1 207 asparagine to alanine mutations gave the most stable and attenuated strain.
- West Nile virus
ASJC Scopus subject areas
- Molecular Medicine
- Immunology and Microbiology(all)
- Public Health, Environmental and Occupational Health
- Infectious Diseases