Histological sections from mice infected with MPXV-USA-2003 stained with hematoxylin and eosin.

<p>A) Necrosis of ovarian follicles (arrow) with subacute inflammation infiltrating surrounding connective tissue and peri-ovarian fat. B) Skin of foot with intradermal bulla containing edema and cell debris (arrow). Surrounding epidermis is undergoing ballooning degeneration.</p

In vivo imaging of MPXV-Congo-Luc+ (left panel) and MPXV-USA-Luc+ (right panel) in BALB/c mice (Intraperitoneal inoculation).

<p>Groups of four, 4-week-old BALB/c mice were inoculated by the IP route with 105 PFU of either MPXV-Congo-Luc+or MPXV-USA-Luc+viruses. At indicated times post-infection, mice were injected IP with 1.5 mg luciferin in 100 µl of DPBS (Promega, Madison, WI) and imaged (ventral view) in an IVIS 200 imager (Caliper Life Sciences, Alameda, CA). Exposures for 30 sec (F8, medium binning) were taken at approximately 12 minutes post-luciferin injection following anaesthetization with isoflurane. An uninfected negative control animal (first animal on left side) was also injected with luciferin and imaged on the same times as infected animals. Images were analyzed with Living Image 3.0 software (Caliper Life Sciences, Alameda, CA). A) 24 h; B) 96 h; C)168 h; D) 240 h.</p

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>.</p

In vivo imaging of MPXV-Congo-Luc+ (left panel) and MPXV-USA-Luc+ (right panel) in SCID- BALB/c mice (Intraperitoneal inoculation).

<p>A group of four, 4-week-old SCID BALB/c mice was inoculated by the IP route with 105 PFU of MPXV-USA-Luc+virus and imaged as described as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006592#s4" target="_blank">materials and methods</a>. An uninfected negative control animal (on left) was also injected with luciferin and imaged on the same times as infected animals. Ventral view.</p

Immuno-histochemical staining in tissues of mice infected with MPXV.

<p>Tissues from 4-wk-old SCID/BALB/c mice infected IP with MPXV-USA-2003-Luc+and uninfected SCID/BALB/c mice were stained by using vaccinia mouse hyperimmune mouse and horse-radish peroxidase as a detection label. MPXV antigen was identified in the intestine, ovary and skin of the feet (7B, D, and F respectively). Poxviral inclusions were seen in the skin of a foot (7F arrows). MPXV antigen was also found in the nasal turbinate of IN infected mice (7H). None of the IHC-stained tissues of the control mice including intestine, ovary, and skin (7A, C, and E respectively) had viral antigen staining.</p

Virus titers from selected tissues and correlation with luminescence.

<p>Tissues samples from kidney, liver, lung and ovaries were aseptically harvested to compare viral titers between the parental MPXV-USA-2003 and recombinant progeny MPXV-USA-Luc+strains. Tissue homogenates were centrifuged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006592#s4" target="_blank">materials and methods</a> section. Viral titers were calculated per gram of tissue.</p

Construction of MPXV-Luc+viruses.

<p>A) Two synthetic DNA fragments containing the p7.5 promoter from vaccinia were annealed and cloned into the pUC18 plasmid, resulting into the pPCSII, plasmid. B) The GPT gene was PCR amplified and cloned into the pTK/Sel2 plasmid resulting in the pTK-GPT plasmid. C) The SE/L-GPT fragment was removed from the pTK-GPT plasmid and ligated into the pPCSII plasmid generating the pGPT/PCSII plasmid. D) The luciferase gene was cloned into the plasmid pTK/Sel2/luc plasmid. E) The SE/L-luciferase fragment was cloned into the pGPT/PCSII plasmid to generate pGPT/luc/PCSII construct. Then, MPXV regions, sequences for the left (176L) and right (176R) flanking sequences of the intergenic region 176–177 were cloned into the pGPT/luc/PCSII. The resulting plasmid was then used to generate recombinant MPXV.</p

Correlation between viral titers with luminescence levels.

<p>The luminescence of kidney, liver, and lung tissue lysates was measured with the IVIS imager. A calibration curve was then generated using inverse regression analysis and plotting virus titer (PFU/g) against luminescence (photons/sec). MPXV-USA-2003-Luc+(•). MPXV-Congo-Luc+(○).</p