Nanopore sequencing of recombinant VSV stock bearing Rs3367-CoV spike protein

SARS-like betacoronaviruses (sarbecoviruses) endemic in bats pose a significant zoonotic threat to humans. Genetic pathways associated with spillover of bat sarbecoviruses into humans are incompletely understood. Herein, we show that the rescue of a recombinant VSV surrogate virus bearing the Rs3367-CoV spike gene is associated with the selection of amino acid substitutions in the S1 SD2 subdomain and the S2 fusion-peptide proximal peptide region. Our findings suggest that these substitutions are adaptations required for spike opening, virus-receptor recognition, and cell entry. We propose that the "locked-down" nature of these spikes and their requirement for S1-S2 cleavage to engage ACE2 represent viral optimizations for a fecal-oral lifestyle and immune evasion in their natural hosts. These adaptations may be a broader property of bat sarbecoviruses than currently recognized.