Development of SARS-CoV-2 replicons for the ancestral virus and variant of concern Delta for antiviral screening. (preprint)

SARS-CoV-2 is the aetiologic agent of COVID-19 and the associated ongoing pandemic. As the pandemic has progressed, Variants of Concern (VOC) have emerged with lineage defining mutations. Using a SARS-CoV-2 reverse genetic system, based on transformation associated recombination in yeast, a series of replicons were produced for the ancestral Wuhan virus and the SARS-CoV-2 VOC Delta in which different combinations of the Spike, membrane, ORF6 and ORF7a coding sequences were replaced with sequences encoding the selectable marker puromycin N-acetyl transferase and reporter proteins (Renilla luciferase, mNeonGreen and mScarlet). Replicon RNAs were replication competent in African green monkey kidney (Vero E6) derived cells and a range of human cell lines, with a Vero E6 cell line expressing ACE2 and TMPRSS2 showing much higher transfection efficiency and overall levels of Renilla luciferase activity. The replicons could be used for transient gene expression studies, but cell populations that stably maintained the replicons could not be propagated. Replication of the transiently expressed replicon RNA genomes was sensitive to remedesivir, providing a system to dissect the mechanism of action of antiviral compounds.

Rapid selection of P323L in the SARS-CoV-2 polymerase (NSP12) in humans and non-human primate models and confers a large plaque phenotype (preprint)

The mutational landscape of SARS-CoV-2 varies at both the dominant viral genome sequence and minor genomic variant population. An early change associated with transmissibility was the D614G substitution in the spike protein. This appeared to be accompanied by a P323L substitution in the viral polymerase (NSP12), but this latter change was not under strong selective pressure. Investigation of P323L/D614G changes in the human population showed rapid emergence during the containment phase and early surge phase of wave 1 in the UK. This rapid substitution was from minor genomic variants to become part of the dominant viral genome sequence. A rapid emergence of 323L but not 614G was observed in a non-human primate model of COVID-19 using a starting virus with P323 and D614 in the dominant genome sequence and 323L and 614G in the minor variant population. In cell culture, a recombinant virus with 323L in NSP12 had a larger plaque size than the same recombinant virus with P323. These data suggest that it may be possible to predict the emergence of a new variant based on tracking the distribution and frequency of minor variant genomes at a population level, rather than just focusing on providing information on the dominant viral genome sequence e.g., consensus level reporting. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.