Evolutionary Dynamics of Rift Valley Fever Virus in Saudi Arabia: Whole-Genome Analysis of the 2000–2001 Outbreak Strains

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen that causes severe disease in livestock and humans, with epidemic potential and a capacity for geographic expansion beyond its African endemic range. The 2000–2001 outbreak in Saudi Arabia represented the first confirmed introduction of RVFV outside Africa, yet comprehensive multi-segment genomic characterization of the circulating strains has remained incomplete. Here, we performed an integrated evolutionary and molecular analysis of the three complete-genome Saudi RVFV strains (Saudi 2000-10911, Saudi 200010901, and SA01-1322) using publicly available sequences. Whole-genome comparisons revealed substantial nucleotide and amino acid diversity, with the L segment exhibiting the highest mutation burden (182 nucleotide substitutions, 17 amino acid changes), while the NSs region of the S segment accumulated 52 nucleotide and 11 amino acid mutations compared to the highly conserved nucleocapsid domain. Maximum-likelihood phylogenetic analyses of all three segments consistently placed the Saudi strains within Lineage C (bootstrap = 100/100), clustering with East African strains from Kenya (2006–2007), Tanzania (2007), and South Africa (2008–2010), with congruent topologies across segments confirming the absence of reassortment. Bayesian phylogeographic analysis of 118 L-segment sequences estimated a mean substitution rate of 1.697 × 10−4 substitutions/site/year (95% HPD: 1.5–1.9 × 10−4) and a time to the most recent common ancestor of approximately 1857 (95% HPD: 1835–1876). Selection-pressure analyses using four complementary methods (MEME, FEL, FUBAR, and SLAC) demonstrated pervasive purifying selection across all three segments, with only sporadic episodic positive selection detected at a limited number of codons (six in L, seven in M, and one in S). In silico glycosylation prediction identified conserved N- and O-glycosylation sites on the M segment, with the Saudi strains displaying a Lineage C-typical profile lacking geographically restricted markers. However, the absence of canonical signal peptides in all sequences warrants cautious interpretation of these predictions. Collectively, our findings are consistent with a single introduction of Lineage C RVFV from East Africa among the sampled strains, with no subsequent reassortment, and that the virus has evolved under strong functional constraints with only rare, lineage-restricted episodes of positive selection. The identified Saudi-specific and lineage-defining mutations provide molecular markers for surveillance, while the high conservation of core proteins supports the feasibility of broadly protective vaccines.