Simple SummarySerial passaging of oncolytic viruses in a new host system can increase their infectivity and anti-cancer efficacy by the directed evolution of naturally occurring variants. RNA viruses have a high nucleotide substitution and proof-reading error rate due to the low fidelity of viral RNA-dependent RNA polymerase. The directed evolution of oncolytic RNA viruses is a practical strategy to select for virus variants with an increased therapeutic efficacy. The Seneca Valley virus (SVV) is a promising oncolytic virotherapy candidate for a range of human cancers. Here, we used deep genome sequencing to analyse viral genome changes during the serial passaging of the SVV in two cell culture models of small-cell lung cancer, i.e., monolayer cells and tumorsphere. We observed the improved infectivity of the SVV in tumorspheres over time associated with the accumulation of several mutations across the genome, possibly involved in the optimization of infectiousness in tumors.AbstractThe Seneca Valley virus (SVV) is an oncolytic virus from the picornavirus family, characterized by a 7.3-kilobase RNA genome encoding for all the structural and functional viral proteins. Directed evolution by serial passaging has been employed for oncolytic virus adaptation to increase the killing efficacy towards certain types of tumors. We propagated the SVV in a small-cell lung cancer model under two culture conditions: conventional cell monolayer and tumorspheres, with the latter resembling more closely the cellular structure of the tumor of origin. We observed an increase of the virus-killing efficacy after ten passages in the tumorspheres. Deep sequencing analyses showed genomic changes in two SVV populations comprising 150 single nucleotides variants and 72 amino acid substitutions. Major differences observed in the tumorsphere-passaged virus population, compared to the cell monolayer, were identified in the conserved structural protein VP2 and in the highly variable P2 region, suggesting that the increase in the ability of the SVV to kill cells over time in the tumorspheres is acquired by capsid conservation and positively selecting mutations to counter the host innate immune responses.