<i>In vitro</i> expression of CCP.

<p>BSC-40 cells were infected with the parental monkeypox virus (ROC 2003 or USA 2003), the recombinant monkeypox virus (ROCΔCCP or USA+CCP), or vaccinia WR. At 24 hpi, supernatants (A) and cell lysates (B) were harvested and probed with polyclonal α-VCP antibody. Complement control protein homologues in monkeypox virus (∼24 kDa) and vaccinia WR (∼35 kDa) were detected. Human transferrin (85 kDa) was used as the load control for each well. Numerical values below each lane in blots A and B represent the intensity value of the band using the Image Lab 3.0 band density tool. Each lane was loaded with (3 µg/ml) concentrated supernatant or cell lysate. BSC-40 cells were infected with either the ROC 2003 strain of monkeypox virus or vaccinia WR and supernatant was harvested and replaced with fresh media at the indicated times (C). The harvested supernatants were analyzed using a polyclonal α-VCP antibody. VCP expression was first detected in cell supernatants at 10 hpi and monkeypox CCP expression at 24 hpi. BSC-40 cells were infected with the parental monkeypox virus (ROC 2003 or USA 2003), the recombinant monkeypox virus (ROCΔCCP or USA+CCP), or vaccinia WR in the presence (+) or absence (−) of Ara-C. At 24 hpi, supernatants (D) were harvested and probed with polyclonal α-VCP antibody. Expression of CCP in the presence/absence of Ara-C in the West African recombinant monkeypox strain was similar to the Congo Basin strain.</p

Anti-orthopoxvirus antibody production.

<p>Orthopoxvirus specific ELISA to detect total IgG. The average value per infection group is shown here. The reported OD-COV (cutoff value) corresponds to the value above the average signal of the negative control wells plus two standard deviations. None of the average OD-COVs were significantly different between groups within a sample day (p>0.05). Dark bar borders indicate n<4 for the mean generated on that day.</p

Comparison of disease signs in the prairie dog challenge study.

*<p>- Prairie dog PD2 (underlined) died post-anesthesia on day 10.</p>†<p>- Prairie dogs were observed every other day.</p

Monkeypox growth curve.

<p>Single step growth curves of the parental (ROC 2003, USA 2003) and recombinant (ROCΔCCP, USA+CCP) monkeypox virus strains. No statistically significant differences were identified between the strains (p>0.05).</p

C4b binding activity of monkeypox virus CCP.

<p>Assay measuring the binding of MPXV complement control proteins from the media of infected cells to human C4b. Solid triangles and squares show binding activity of USA+CCP and ROC 2003, respectively, to C4b (solid lines) or to control wells with BSA alone (dashed lines). Open triangles and squares show binding activity of USA and ROCΔCCP, respectively, to C4b. Also graphed is a known concentration of rVCP (star symbol with dashed line) from which we estimated the amount of MPXV CCP in the media of infected cells. The starting amount of rVCP was 2.5 ng (1.75 nM) and was then similarly diluted 2-fold. The error bars in this figure were smaller than the symbols.</p

Viable virus in animal necropsy tissue samples in plaque-forming unit per gram of tissue.

*<p>Death due to anesthesia.</p

Constructs used for recombinant virus formation.

<p>Plasmid maps of pABgpt and pCDgpt illustrate the recombination sites within ROC 2003 and USA 2003 genomes. For both recombinant strains, gpt expression was controlled by a synthetic early-late promoter sequence. In the recombinant USA+CCP, the <i>ccp</i> gene was under its own native promoter from ROC 2003, and the insert was located in the non-coding region between open reading frames 177 and 176 of USA 2003.</p

Monkeypox disease progression timeline.

<p>Depiction of the comparative disease progression of the four challenge monkeypox strains in the prairie dog animal model. This diagram highlights monkeypox disease as observed lesions progressed from a local to systemic infection and eventual resolution. Note the slower progression of disease in the ROCΔCCP-infected group when compared to its ROC 2003 parental group. The USA+CCP-infected prairie dogs displayed earlier subjective signs of infection when compared to the USA 2003 group.</p

Viable monkeypox virus loads.

<p>Mean live virus titers for the oral (A), ocular (B), and anal (C) swabs taken from each animal in a given infection group in the prairie dog challenge study. Note that all animals in the ROC 2003 group were dead by day 16. Also, by day 16 there were only two remaining animals in the USA 2003 group and starting on day 20, there were only two remaining animals in the USA+CCP group. Dark bar borders indicate n<4 for the mean generated on that day. The mean viral titer for each infection group was graphed with standard deviations. Unless indicated by an asterisk, group comparisons within sampling days were not significantly different (p>0.05).</p