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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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Construction of MPXV-Luc+viruses.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.
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pone-0006592-g008: Construction of MPXV-Luc+viruses.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.

Mentions: Plasmid pUC18 was digested with Pvu II and gel-purified to remove the LacZ and polycloning site sequences. This plasmid contributed the backbone for the MPX transfer vector. Then, two oligonucleotides (5′-GGCCGGCCGGACCGACACCCTAGGACTAGTCGATGCTAGCGCCAGGCGCGCCGGGCCC-3′ and 5′-GGGCCCGGCGCGCCTGGCGCTAGCATCGACTAGTCCTAGGGTGTCGGTCCGGCCGGCC-3′) were synthesized and annealed to form a double stranded molecule containing multiple cloning sites. For this purpose, 2 µg of each oligonucleotide were resuspended in 100 µl of 50 mM Tris pH 8.0, and incubated at 72°C for 10 min. The mixture was then allowed to slowly cool to room temperature. Ten microliters of the annealed mixture were employed in a blunt-end ligation reaction (room temperature, overnight) with the Pvu II-digested pUC18 plasmid. The resulting plasmid, designated pPCSII, was sequenced and then purified for further manipulation (Figure 8A).


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

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

Construction of MPXV-Luc+viruses.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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2719101&req=5

pone-0006592-g008: Construction of MPXV-Luc+viruses.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.
Mentions: Plasmid pUC18 was digested with Pvu II and gel-purified to remove the LacZ and polycloning site sequences. This plasmid contributed the backbone for the MPX transfer vector. Then, two oligonucleotides (5′-GGCCGGCCGGACCGACACCCTAGGACTAGTCGATGCTAGCGCCAGGCGCGCCGGGCCC-3′ and 5′-GGGCCCGGCGCGCCTGGCGCTAGCATCGACTAGTCCTAGGGTGTCGGTCCGGCCGGCC-3′) were synthesized and annealed to form a double stranded molecule containing multiple cloning sites. For this purpose, 2 µg of each oligonucleotide were resuspended in 100 µl of 50 mM Tris pH 8.0, and incubated at 72°C for 10 min. The mixture was then allowed to slowly cool to room temperature. Ten microliters of the annealed mixture were employed in a blunt-end ligation reaction (room temperature, overnight) with the Pvu II-digested pUC18 plasmid. The resulting plasmid, designated pPCSII, was sequenced and then purified for further manipulation (Figure 8A).

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