In vivo evidence of expanded single-cell viral bottlenecks and quasispecies heterogeneity
This study investigates the diversity and single-cell bottlenecks of viral quasispecies using a synthetic mutant library of Pepper mild mottle virus (PMMoV), a tobamovirus expressing GFP. A highly diverse 10N mutant pool was generated by inserting a 14-base degenerate sequence upstream of the RNA-dependent RNA polymerase (RdRp) start codon, outside subgenomic regions, to avoid disrupting downstream gene expression. The pool was introduced into Nicotiana benthamiana plants via Agrobacterium-mediated delivery. At 11 days post-infection, single infected mesophyll cells were isolated based on GFP fluorescence, and viral minus-strand RNA was amplified and deep sequenced to capture the sequence diversity of replicating viral genomes. The dataset includes 29 single-cell and 3 bulk leaf samples, providing in vivo resolution of viral sequence heterogeneity at the cellular level. These data reveal a higher-than-expected single-cell multiplicity of infection and shed light on the early dynamics of quasispecies formation and propagation.