Targeted Epstein–Barr virus capture sequencing identifies BBLF4-L322M as an independent prognostic variant in nasopharyngeal carcinoma

Epstein-Barr virus (EBV) is etiologically linked to nasopharyngeal carcinoma (NPC) and shows geographic heterogeneity. To determine whether viral genotype affects prognosis, we performed capture-based whole-genome sequencing of EBV from 114 Guangxi NPC biopsies (mean breadth ~93%, depth ~979×) with Sanger confirmation at BNRF1-G696R. We detected 5,683 high-confidence variants with a mean of 1,112 per genome enriched in latent genes (LMP1, LMP2A/B, and the EBNA family). Variants with minor allele frequency ≥0.15 and missingness ≤0.05 were tested for association with 5-year overall survival. A total of 46 loci were significant in the univariate Cox models, and 19 missense variants underwent unsupervised clustering by genotype correlation, yielding two blocks. From these, BZLF1-A205S and BBLF4-L322M were selected as the index variants. In multivariable Cox models adjusted for age and dichotomized American Joint Committee on Cancer (AJCC) stage, BBLF4-L322M remained independently associated with poorer overall survival (OS), whereas BZLF1-A205S was not significant. A 720-genome phylogeny integrating our sequences with public data showed several Chinese sublineages with partial regional structuring. Guangxi strains were closely related to, yet partly distinct from, Guangdong and Hong Kong. These results support the incorporation of the EBV genotype, particularly BBLF4-L322M, into prognostic models and motivate functional studies to define mechanisms and validate their clinical utility. IMPORTANCE: The prognostic Epstein-Barr virus (EBV) variants in nasopharyngeal carcinoma (NPC) remain largely unknown. This study presents the first genome-wide survey of EBV sequence diversity in patients with NPC in Guangxi, China. The identified mutations, including BZLF1-A205S and BBLF4-L322M, are candidate viral biomarkers linked to patient survival. The abundance of nonsynonymous variants in latent phase genes implies a role for the viral genotype in the disease course. These insights have laid the groundwork for EBV-based prognostic biomarkers and precise medical strategies tailored to endemic NPC populations.