Reovirus Reassortants Reveal Context-Dependent Oncolytic Phenotypes Across Epithelial Cancer Cell Lines

Mammalian reoviruses are promising oncolytic agents, but most preclinical and clinical work has focused on the type 3 Dearing (T3D) prototype, potentially underestimating the therapeutic relevance of broader reovirus genetic diversity. Because reoviruses possess a segmented double-stranded RNA genome, reassortment can generate progeny with novel combinations of traits influencing infectivity, replication, and cytotoxicity. Here, we evaluated a panel of previously generated T1L × T3D reassortants and recombinant reoviruses across three epithelial tumor models: A549 lung adenocarcinoma and the oral squamous carcinoma cell lines OECM-1 and CAL-27. Across all three models, the tested viruses displayed marked cell line-dependent heterogeneity in both cytotoxicity and infectivity. Several reassortants reduced viability more effectively than the parental T1L and T3D strains in one or more cell lines, and DB62 emerged as the most broadly active candidate across the panel. Infectivity and cytotoxicity overlapped only partially, indicating that efficient infection alone does not fully predict oncolytic potency. Together, these findings show that reassortment can generate reoviruses with enhanced or selective activity across epithelial tumor contexts and support future studies examining how segment-dependent differences in interferon antagonism, entry, and cell death shape oncolytic potency.