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

Osorio JE, Iams KP, Meteyer CU, Rocke TE - PLoS ONE (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, USA. osorio@svm.vetmed.wisc.edu

ABSTRACT
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.

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One-step growth curves for parental (MPXV-Congo, MPXV-USA-2003) and progeny recombinant (MPXV-Congo-Luc+, MPXV-USA-Luc+) viruses.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 [39].
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pone-0006592-g001: One-step growth curves for parental (MPXV-Congo, MPXV-USA-2003) and progeny recombinant (MPXV-Congo-Luc+, MPXV-USA-Luc+) viruses.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 [39].

Mentions: Sequencing and PCR analysis showed that recombinant MPXV-USA-Luc+and MPXV-Congo-Luc+contained the luciferase gene inserted into the 176–177 intergenic regions. To determine whether this insertion adversely affected the overall growth characteristics of MPX viruses, we carried out one-step growth experiments in Vero cell monolayers. Total virus production, expressed as PFU/ml, was determined for samples collected at various times after infection. For both wt and Luc+viruses, the lag and rise period of exponential growth were of similar duration, and gave comparable yields (Figure 1). Thus, within experimental limitations, we concluded that the insertion of the luciferase gene in our engineered viruses did not limit growth in Vero cells.


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

Osorio JE, Iams KP, Meteyer CU, Rocke TE - PLoS ONE (2009)

One-step growth curves for parental (MPXV-Congo, MPXV-USA-2003) and progeny recombinant (MPXV-Congo-Luc+, MPXV-USA-Luc+) viruses.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 [39].
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2719101&req=5

pone-0006592-g001: One-step growth curves for parental (MPXV-Congo, MPXV-USA-2003) and progeny recombinant (MPXV-Congo-Luc+, MPXV-USA-Luc+) viruses.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 [39].
Mentions: Sequencing and PCR analysis showed that recombinant MPXV-USA-Luc+and MPXV-Congo-Luc+contained the luciferase gene inserted into the 176–177 intergenic regions. To determine whether this insertion adversely affected the overall growth characteristics of MPX viruses, we carried out one-step growth experiments in Vero cell monolayers. Total virus production, expressed as PFU/ml, was determined for samples collected at various times after infection. For both wt and Luc+viruses, the lag and rise period of exponential growth were of similar duration, and gave comparable yields (Figure 1). Thus, within experimental limitations, we concluded that the insertion of the luciferase gene in our engineered viruses did not limit growth in Vero cells.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, USA. osorio@svm.vetmed.wisc.edu

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus