A conserved E1 stem motif functions as a virulence switch in the emerging Getah virus.

Getah virus (GETV) is an emerging arthropod-borne zoonotic alphavirus that poses a growing threat to animal and public health, yet its virulence determinants remain poorly understood. Here, we report the first identification of a natural GETV variant isolated from the brain of diseased piglets. This variant harbors a 9-nucleotide deletion in the E1 glycoprotein stem region, resulting in the deletion of glutamine 397 (Q397) and valine 398 (V398), along with an asparagine‑to‑isoleucine substitution at position 396 (N396I) and the loss of phenylalanine 399 (F399). It also carries a threonine-to-methionine substitution at position 49 (T49M) in nonstructural protein 2 (NSP2). The NQVF motif (residues 396-399) in E1 protein is highly conserved among GETV strains and related alphaviruses, suggesting a shared functional role. Using reverse genetics, we demonstrated that the E1 deletion-but not the NSP2 mutation-drastically attenuates viral replication in vitro and completely abrogates lethality in neonatal mice by restricting systemic dissemination. Stepwise mutagenesis further revealed that the four‑residue motif functions synergistically as a virulence switch: single deletions partially reduce pathogenicity, while the quadruple deletion confers full attenuation, limiting tissue tropism and histopathology. Although both wild‑type and mutant viruses crossed the placental barrier in pregnant mice, the mutant exhibited reduced vertical transmission and maternal tissue replication. Our findings identify a highly conserved E1 stem motif as a critical regulator of GETV virulence, offering a strategic target for developing attenuated vaccines against GETV and other alphaviruses.