Comparison of monkeypox viruses pathogenesis in mice by in vivo imaging.

Monkeypox viruses (MPXV) cause human monkeypox, a zoonotic smallpox-like disease endemic to Africa, and are of worldwide public health and biodefense concern. Using viruses from the Congo (MPXV-2003-Congo-358) and West African (MPXV-2003-USA-044) clades, we constructed recombinant viruses that express the luciferase gene (MPXV-Congo/Luc+and MPXV-USA-Luc+) and compared their viral infection in mice by biophotonic imaging. BALB/c mice became infected by both MPXV clades, but they recovered and cleared the infection within 10 days post-infection (PI). However, infection in severe combined immune deficient (SCID) BALB/c mice resulted in 100% lethality. Intraperitoneal (IP) injection of both MPXV-Congo and MPXV-Congo/Luc+resulted in a systemic clinical disease and the same mean time-to-death at 9 (+/-0) days post-infection. Likewise, IP injection of SCID-BALB/c mice with MPXV-USA or the MPXV-USA-Luc+, resulted in similar disease but longer (P<0.05) mean time-to-death (11+/-0 days) for both viruses compared to the Congo strains. Imaging studies in SCID mice showed luminescence in the abdomen within 24 hours PI with subsequent spread elsewhere. Animals infected with the MPXV-USA/Luc+had less intense luminescence in tissues than those inoculated with MPXV-Congo/Luc+, and systemic spread of the MPXV-USA/Luc+virus occurred approximately two days later than the MPXV-Congo/Luc+. The ovary was an important target for viral replication as evidenced by the high viral titers and immunohistochemistry. These studies demonstrate the suitability of a mouse model and biophotonic imaging to compare the disease progression and tissue tropism of MPX viruses

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

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

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

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

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

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

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