Genotype-Specific Amino Acid Signatures in the Gn/Gc Glycoprotein of Severe Fever with Thrombocytopenia Syndrome Virus Associated with Neurological Symptoms

Severe fever with thrombocytopenia syndrome (SFTS) is a highly fatal tick-borne disease caused by SFTS virus (SFTSV). The Gn/Gc glycoprotein plays a critical role in viral entry, but whether its amino acid polymorphisms directly determine neurological symptoms remains unclear. We sequenced the M segment from 99 SFTS patients (31 with neurological symptoms, 68 without) in Shandong Province, China. After removing recombinant sequences, 91 non-recombinant sequences were analyzed and genotyped based on M segment phylogeny. Systematic signature screening, phylogenetic reconstruction, genotype-stratified analysis, public database validation, cross-genotype comparison, and selection pressure inference were applied to distinguish functional determinants from genotype-specific markers. M segment-based phylogeny classified the sequences into four genotypes (D, E, F, A), with D accounting for 79.3% of NS cases. Although 10 candidate sites initially distinguished NS from Non-NS, their associations disappeared upon genotype-stratified analysis; all were low-frequency variants (<15%) in public databases. Cross-genotype comparison revealed D-specific (“GERHVGIIFL”) and E/F-specific (“DGKQMRVTSF”) patterns, none of which overlapped with positively selected sites. These findings demonstrate that the apparent NS association reflected genotype confounding rather than functional determinants, identifying the 10 sites as potential genotype D-specific markers with high discriminatory potential, warranting further validation in independent datasets. This study provides a cautionary example for virus–phenotype association studies, emphasizing that genotype stratification is essential before interpreting amino acid differences as functional.