ABSTRACT
Measures to control the COVID-19 pandemic such as antiviral therapy and vaccination have been challenged by ongoing virus evolution under antiviral and immune pressures. Understanding viral evolutionary dynamics is crucial for responding to SARS-CoV-2, and preparing for the next pandemic, by informing prediction of virus adaptation, public health strategies, and design of broadly effective therapies. Whole-genome sequencing (WGS) of SARS-CoV-2 during the pandemic enabled fine-grained studies of virus evolution in the human population. Serial passaging in vitro offers a controlled environment to investigate the emergence and persistence of genetic variants that may confer selective advantage. Nine virus lineages, including four "variants of concern" and three former "variants under investigation" as designated by the World Health Organisation, were chosen to investigate intra- and inter-lineage evolution through long-term serial passaging in Vero E6 cells. Viruses were sampled over at least 33 passages (range 33-100) and analysed using WGS to examine evolutionary dynamics and identify key mutations with implications for virus fitness, transmissibility, and immune evasion. All passages continued to replicate in culture, despite regular accumulation of mutations. There was evidence of convergent acquisition of mutations both across passage lines and compared with contemporaneous SARS CoV-2 clinical sequences from population studies. Some of these convergent mutations are hypothesised to be important in proliferation of SARS-CoV-2 lineages, such as by evading host immune responses (e.g. S:A67V, S:H655Y). Given these mutations arose in vitro, in the absence of a multicellular host immune response, this suggests virus genome mutation resulted from stochastic events, rather than immune-driven mutation. There was a regular gain and loss of low-frequency variants during serial passaging, but some became fixed in subsequent multiple passages, suggesting either a benefit of the mutation in vitro, or at least a lack of deleterious effect. Our findings reveal valuable insights into the evolution of SARS-CoV-2 by quantitatively investigating evolutionary dynamics of the virus over the greatest number of serial passages to date. Knowledge of these evolutionary trends will be useful for public health and the development of antiviral and vaccine measures to reduce the effects of SARS CoV-2 infection on the human population.