Rabbitpox virus and vaccinia virus infection of rabbits as a model for human smallpox

Abstract

The threat of smallpox release and use as a bioweapon has encouraged the search for new vaccines and antiviral drugs, as well as development of new small-animal models in which their efficacy can be determined. Here, we reinvestigate a rabbit model in which the intradermal infection of rabbits with very low doses of either rabbitpox virus (RPV) or vaccinia virus Western Reserve (VV-WR) recapitulates many of the clinical features of human smallpox. Following intradermal inoculation with RPV, rabbits develop systemic disease characterized by extensive viremia, numerous secondary lesions on the skin and mucocutaneous tissues, severe respiratory disease, death by 9 days postinfection, and, importantly, natural aerosol transmission between animals. Contrary to previous reports, intradermal infection with VV-WR also resulted in a very similar lethal systemic disease in rabbits, again with natural aerosol transmission between animals. When sentinel and index animals were cohoused, transmission rates approached 100% with either virus, with sentinel animals exhibiting a similar, severe disease. Lower rates of transmission were observed when index and sentinel animals were housed in separate cages. Sentinel animals infected with RPV with one exception succumbed to the disease. However, the majority of VV-WR-infected sentinel animals, while becoming seriously ill, survived. Finally, we tested the efficacy of the drug 1-O-hexadecyloxypropyl-cidofovir in the RPV/rabbit model and found that an oral dose of 5 mg/kg twice a day for 5 days beginning 1 day before infection was able to completely protect rabbits from lethal disease.

FIG. 1.

Pathology of disease in 9-week-old rabbits infected intradermally with 1,000 PFU of RPV. (A) Average temperatures of animals. (B) Average weights of animals normalized against uninfected and age-matched controls. (C) Survival curves. (D) Time line description of the first appearance of key clinical signs of disease. The boxes indicate the range of days in which these symptoms were first observed. ▪, mock- infected rabbits (n = 9); ▴, RPV-infected rabbits (n = 25). The error bars indicate standard errors of the mean. Morbidity dictated that all animals be euthanized by 9 days p.i.

FIG. 2.

Images of mock-infected rabbits (A to C), RPV-infected rabbits (D to F), VV-WR_C-infected rabbits (G to I), and sentinel rabbits infected by contact exposure to RPV-infected (J to L) or VV-WR_C-infected (M to O) rabbits. (A to C) A mock-infected rabbit 7 days after intradermal injection with PBS showing the primary inoculation site (A), nose and mouth (B), and ear pinnae (C). (D to F) A 9-week-old rabbit 7 days after intradermal infection with 1,000 PFU of RPV showing the primary inoculation site (D), mucopurulent discharge from the nostrils (E), and secondary lesions on the ear pinnae (F, arrows). (G to I) A 9-week-old rabbit, 8 days after intradermal infection with 1,000 PFU of VV-WR_C showing the primary inoculation site (G), profuse mucopurulent discharge from the nostrils (H), and secondary lesions on the ear pinnae (I, arrows). (J to L) An RPV-infected sentinel animal 13 days after infection of the index animal showing hemorrhagic lungs (J), profound swelling of the face and neck with nasal discharge (K), and secondary lesions in the ear pinnae (L, arrows). (M to O) A VV-WR_C-infected sentinel animal 15 days after infection of the index animal showing secondary lesions on the eyelids (M, arrows), mucopurulent nasal discharge (N), and confluent secondary lesions in the ear pinnae (O). The bars on the images are 1 cm in length.

FIG. 3.

Pathology of disease in 9-week-old rabbits infected intradermally with 1,000 PFU of VV-WR_C. (A) Average temperatures of animals. (B) Average weights of animals normalized against uninfected and age-matched controls. (C) Survival curves. (D) Time line description of the first appearance of key clinical signs of disease. The boxes indicate the range of days in which these symptoms were first observed. ▪, mock-infected rabbits (n = 9); ▴, VV-WR_C-infected rabbits (n = 24). The error bars indicate standard errors of the mean.

FIG. 4.

Comparative pathology of disease in 6-month-old rabbits infected intradermally with 1,000 PFU of RPV or VV-WR_C. (A) Average temperatures of animals. (B) Average weights of animals normalized against uninfected and age-matched controls. (C) Survival curves. (D) Time of the first appearance of key clinical signs of disease. The boxes indicate the range of days in which these symptoms were first observed. ▪, mock-infected rabbits (n = 2); ▴, RPV-infected rabbits (n = 5); ×, VV-WR_C-infected rabbits (n = 5). The error bars indicate standard errors of the mean.

FIG. 5.

Comparative pathology of disease in 9-week-old sentinel rabbits infected by exposure to rabbits intradermally infected with 1,000 PFU of RPV. (A) Average temperatures of infected sentinel animals. (B) Average weights of infected sentinel animals normalized against uninfected and age-matched controls. (C) Survival curves of infected animals. (D) Time line description of the first appearance of key clinical signs of disease. The boxes indicate the range of days in which these symptoms were first observed in infected sentinel animals. ▪, uninfected sentinel rabbits (n = 11); ▴, RPV-infected by direct contact (n = 11); ⧫, RPV infected without contact (n = 5). The error bars indicate standard errors of the mean.

FIG. 6.

Comparative pathology of disease in 9-week-old sentinel rabbits infected by exposure to rabbits intradermally infected with 1,000 PFU of VV-WR_C. (A) Average temperatures of infected sentinel animals. (B) Average weights of infected sentinel animals normalized against uninfected and age-matched controls. (C) Survival curves of infected animals. (D) Time line description of the first appearance of key clinical signs of disease in infected sentinel animals. The boxes indicate the range of days in which these symptoms were first observed. ▪, uninfected sentinel rabbits (n = 4); ▴, VV-WR_C-infected by direct contact (n = 8); ⋄, VV-WR_C-infected without contact (n = 4). The error bars indicate standard errors of the mean.

FIG. 7.

Comparative pathology of disease in 9-week-old rabbits treated with HDP-CDV and infected with 500 PFU of RPV. (A) Average temperatures of infected and control animals. (B) Average weights of infected animals normalized against uninfected and age-matched controls. (C) Survival curves of infected animals. (D to F) Images of the primary inoculation site of a rabbit treated with 5 mg/kg of HDP-CDV from −1 to 3 days p.i. at 4 days p.i. (D), 8 days p.i. (E), and 12 days p.i. (F). ▪, placebo-treated rabbits (n = 4); ▴, 5 mg/kg HDP-CDV-treated rabbits (n = 4); ⧫, 1 mg/kg HDP-CDV-treated rabbits (n = 4); •, uninfected control animals (n = 4). The bars on the images are 1 cm in length. The primary lesion areas are demarcated by the circles. The error bars indicate standard errors of the mean.