| Summary: | West Nile virus (\VNV) is a mosquito-borne flavivirus that has N-linked glycosylation sites on the premembrane (prM) envelope (E) and nonstructural 1 (NS1) proteins. NS1 contains three glycosylation (asparagine-X-threoine [NXT]) sites at NS1130, NS1175, and NSh07. The fIrst aim ofthe thesis was to attenuate WNV through the ablation of the NS1 glycosylation sites, individually and in all combinations, initially by replacing the asparagine (N) with alanine (A) in the glycosylation motifusing site-directed mutagenesis ofa WNV infectious clone. Two ofthe mutants were greatly attenuated, up to 50,000-fold, for lethal neuroinvasiveness in mice, had reduced viraemia at two and three days post-infection relative to the parental strain, but multiplied similarly to the parental strain in Vero cells. Although these viruses were highly attenuated, the most attenuated NS1 mutant virus reverted to virulence and reversion at the fIrst NS1 glycosylation site (NS1130) was seen in mice that succumbed to infection. The second aim was to diminish reversion. Thus, the fIrst NSI glycosylation site was mutated such that it contained two or three amino acid substitutions in the NS1130 glycosylation motif(NNT~SVT;NNT~QQA),while the second and third glycosylation sites remained a single N to A amino acid change. These viruses were highly attenuated for mouse neuroinvasiveness and neurovirulence, >1OO,OOO-foid and 3,000-fold, respectively, compared to the parental strain and did not revert to virulence. Multiplication kinetics ofthe attenuated viruses in mice showed a decrease in multiplication compared to the parental strain and a small plaque phenotype. The third aim was to examine a functional role for the NS1 protein using these mutant viruses. Electron microscopy studies ofthe most attenuated NS1 glycosylation mutant virus revealed changes in the virus-induced structures compared to the parent virusinfected Vero cells that may suggest involvement ofthe NSI protein in the fonnation ofthese structures. Cytokine expression experiments showed several down-regulated cytokines in the sera ofparent and attenuated NS1 mutant virus-infected mice compared to mock-infected serum suggesting that immune evasion by the suppression ofcytokines may contribute to the WNV virulence phenotype. Therefore, mutations in the NSI glycosylation sites were hypothesized to modify virus replication and the host response to infection leading to a mouse attenuated phenotype. Construction ofa virus with mutations ablating the glycosylation site in the E protein together with the ablation ofthe three NSI glycosylation sites completely attenuated this virus for mouse neuroinvasiveness with an ipLDso >100,000 PFU. It was hypothesized that the ablation ofthe prM glycosylation site would also attenuate WNV. Contrary to previous studies, however, the ablation ofthe prM glycosylation site did not attenuate the virus and multiplication kinetics in cell culture revealed that this mutant virus m~l~ipli.ed to higher titres at all time points when compared to the parental strain. UtIh~tlon ofthe mutant viruses generated in this thesis may have potential as effectIve components offuture vaccine candidates.
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