Long-term serial passaging of SARS-CoV-2 reveals signatures of convergent evolution

Understanding viral evolutionary dynamics is crucial to pandemic responses, informing prediction of virus adaptation over time, virus adaptation to antiviral and immune pressures, design of effective therapies, and virus surveillance for public health strategies. Whole-genome sequencing (WGS) of SARS-CoV-2 has enabled fine-grained studies of virus evolution in the human population. Serial passaging in vitro offers a complementary controlled environment to investigate the emergence and persistence of genetic variants that may confer selective advantage. In this study, nine virus lineages, including four "variants of concern" and three former "variants under investigation", were sampled over ≥33 serial passages (range 33-100) in Vero E6 cells. WGS was used to examine virus evolutionary dynamics and identify key mutations with implications for fitness and/or transmissibility. Mutations accumulated regularly during serial passaging. Many low-frequency variants were lost but others became fixed, suggesting either in vitro benefits, or at least a lack of deleterious effect. Mutations arose convergently both across passage lines and compared with contemporaneous SARS-CoV-2 clinical sequences, including some hypothesised to drive lineage success through host immune evasion (e.g. S:A67V, S:H655Y). The appearance of these mutations in vitro suggested key mutations can arise convergently even in the absence of a multicellular host immune response through mechanisms other than immune-driven mutation. Such mutations may provide other benefits to the viruses in vitro , or arise stochastically. Our quantitative investigation into SARS-CoV-2 evolutionary dynamics spans the greatest number of serial passages to date, and will inform measures to reduce the effects of SARS-CoV-2 infection on the human population.