Limited G-dependent residual infectivity of G gene-deleted vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) -ΔG vector particles are widely applied in vaccine development, gene delivery, and immunotherapy studies due to their high transduction efficiency and replication-defective design. However, G-pseudotyped VSV-ΔG preparations may exhibit residual multicycle infectivity associated with VSV-G-dependent entry. Therefore, in this study, we generated VSV-ΔG particles and corresponding M51R and ΔM51 derivatives to examine the relationship between G-dependent residual infectivity and post-entry innate immune regulation. Serial passage experiments were performed with removal of the inoculum and repeated PBS washing, followed by RT-PCR and Sanger sequencing of viral genomic regions. Detectable infectivity persisted transiently during early passages but progressively declined, while no evidence of restoration of the deleted VSV-G gene was identified. Neutralization with anti-VSV-G antibody markedly reduced infection, supporting the dependence of the observed residual infectivity on functional VSV-G-mediated entry. M51 mutations were associated with enhanced interferon-related signaling after infection. Together, these findings distinguish limited G-dependent residual infectivity from restoration of replication competence and highlight the need to consider such activity during the characterization and interpretation of G-pseudotyped VSV-ΔG systems.