Multiple amino acid changes at the first glycosylation motif in NS1 protein of West Nile virus are necessary for complete attenuation for mouse neuroinvasiveness

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.