One-step growth curves for parental (MPXV-Congo, MPXV-USA-2003) and progeny recombinant (MPXV-Congo-Luc+, MPXV-USA-Luc+) viruses.

<p>Vero cell monolayers were infected at multiplicity of infection (MOI) of 0.1 with parental (MPXV-Congo, MPXV-USA-2003) or with progeny (MPXV-Congo-Luc+, MPXV-USA-Luc+) strains. After allowing for attachment (30 min), cells were washed twice with PBS to remove unattached virus. Then fresh medium as added and plates were incubated at 37°C 5% CO2. At various intervals thereafter, three wells per virus strain were harvested (media and cells) and placed at −70°C. After three cycles of freezing and thawing, the samples were sonicated and virus titers were determined by serial dilution and infection of Vero cell monolayers. Plaques were visualized by staining with 0.1% crystal violet in 20% ethanol and virus titers determined as described elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006592#pone.0006592-Carroll1" target="_blank">[39]</a>.