The mutational dynamics of the SARS-CoV-2 virus in serial passages in vitro

Abstract

Since its outbreak in 2019, Severe Acute Respiratory Syndrome Coronavirus 2 keeps surprising the medical community by evolving diverse immune escape mutations in a rapid and effective manner. To gain deeper insight into mutation frequency and dynamics, we isolated ten ancestral strains of SARS-CoV-2 and performed consecutive serial incubation in ten replications in a suitable and common cell line and subsequently analysed them using RT-qPCR and whole genome sequencing. Along those lines we hoped to gain fundamental insights into the evolutionary capacity of SARS-CoV-2 in vitro . Our results identified a series of adaptive genetic changes, ranging from unique convergent substitutional mutations and hitherto undescribed insertions. The region coding for spike proved to be a mutational hotspot, evolving a number of mutational changes including the already known substitutions at positions S:484 and S:501. We discuss the evolution of all specific adaptations as well as possible reasons for the seemingly inhomogeneous potential of SARS-CoV-2 in the adaptation to cell culture. The combination of serial passage in vitro with whole genome sequencing uncovers the immense mutational potential of some SARS-CoV-2 strains. The observed genetic changes of SARS-CoV-2 in vitro could not be explained solely by selectively neutral mutations but possibly resulted from the action of directional selection accumulating favourable genetic changes in the evolving variants, along the path of increasing potency of the strain. Competition among a high number of quasi-species in the SARS-CoV-2 in vitro population gene pool may reinforce directional selection and boost the speed of evolutionary change.

Phylogenetic landscape of Monkeypox Virus (MPV) during the early outbreak in New York City, 2022

“…This observation is consistent with findings from previous studies, in which mutational adaptive changes are usually not recorded until after at least four passages in vitro. This phenomenon has been described for RNA viruses [ 18 , 19 ], as well as some DNA viruses, including poxviruses, such as Fowlpox virus [ 20 ]…”

“…On the other hand, several studies have assessed the genomic behaviour of viruses maintained in cultures, especially RNA viruses. Some of these studies have revealed adaptive changes after serial viral passages in vitro, but none earlier than four passages [ 18 , 19 ]. Others have entirely failed to document differences at a genomic level when comparing viruses from directly collected samples and in-vitro grown viruses [ 20 ].…”

Magnipore: Prediction of differential single nucleotide changes in the Oxford Nanopore Technologies sequencing signal of SARS-CoV-2 samples

“…The RNAs we sequenced originate mainly from cell lysates but may also contain portions of extracellular virions. Another aspect is the occurrence of subpopulations in vivo and in vitro , so-called quasi-species [53], which, as explained in the previous section, can influence the results. In addition, the RNA was prepared after the occurrence of a cytopathic effect detectable by light microscopy.…”

Analysis of spike protein variants evolved in a novel mouse model of persistent SARS-CoV-2 infection

“…Previously, mutations involved in SARS-CoV-2 entry into cells were evolved by serial passage in human cell lines without ACE2 or cellular proteases 22, 29 . Furthermore, several mutations reported in natural variants were recapitulated by serial passaging in several human cell lines, with some differences between colon epithelial cells and lung epithelial cells 30 .…”