Lethal infection of primates with variola virus as a model for human smallpox
P. B. Jahrling1, L. Hensley1, J. Huggins1, M. Martinez1, S. Ibrahim 1 , J. LeDuc2
United States Army Research Institute of Infectious Diseases, Frederick, MD, USA1 and Centers for Disease Control , Atlanta, USA2.
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Smallpox virus is acknowledged to pose a significant threat as a bioterrorist weapon because of its contagiousness and the absence of effective antiviral drugs. Development of an animal model for Variola virus infection is essential to development and licensure of effective countermeasures and improved vaccines. We are developing a model for human smallpox using cynomolgus monkeys infected with a variola strain isolated from India in 1964. Intravenous inoculation of high doses (10 9 PFU) resulted in a rapidly fatal disease course with end stage lesions resembling human smallpox. Detection of viral genomes in peripheral blood leukocytes and throat fluids by TaqMan PCR was achieved within 48 hours of exposure, suggesting the possibility of achieving a definitive diagnosis of smallpox during the prodrome. Lower viral doses are being evaluated with the objective of extending the mean time to death. The observation that certain strains of variola can produce fulminent disease in monkeys is a breakthrough in the quest for effective countermeasures for smallpox.
The disease course was evaluated in cynomolgus monkeys exposed to different strains of high-titer (108-109) smallpox virus by combinations of intravenous inoculation and aerosol exposure. The majority of monkeys died between 4 and 7 days post exposure. Clinically, these monkeys showed cutaneous erythema and hemorrhage as early as day 2. At necropsy, in addition to cutaneous hemorrhage, features of a coagulopathy included hemorrhagic effusions, and visceral and mucosal hemorrhage. By day 3, early development of vesicles and pustules, ballooning degeneration and necrosis of epidermal and mucosal epithelial cells were evident. Fibrin deposition and thrombi, and systemic lymphoid apoptosis involving secondary lymphoid organs were also present. By light and electron microscopy, immunohistochemistry, in situ hybridization, and detection of DNA strand breaks (TUNEL assay); those monkeys dying acutely showed lesions compatible with coagulopathy, lymphocyte apoptosis, virus-induced visceral degeneration and necrosis, and generalized, centrifugally distributed virus-induced exanthem and enanthem. Other monkeys died on days 6 or 13; one monkey survived. In addition to lesions due to coagulopathy, and the lymphoid and visceral lesions noted above, those monkeys dying on day 6 also showed progressive development of exanthem and enanthem. Both the monkey dying on day 13 and the survivor developed generalized centrifugally distributed exanthem and enanthem strikingly similar to classical smallpox (variola major). Like the acutely dying monkeys, the day 13 monkey had gross and microscopic changes compatible with disseminated intravascular coagulopathy; however, visceral degenerative changes were minimal. Lymphoid hyperplasia characterized the secondary lymphoid organs in both animals. The monkeys infected by both aerosol and intravenous routes developed minimal to mild bronchiolitis by the time of death (days 4 or 6). Marked leukocytosis, thrombocytopenia, and elevated serum creatinine, blood urea nitrogen, hepatic aspartate and alanine aminotransaminases values dominated the clinicopathologic profile.
The disease course clearly resembles hemorrhagic smallpox; however, for evaluation of antiviral drugs, a less severe disease resembling common smallpox is preferable. Thus, lower inoculum doses of the virulent strains (Harper or India 7124) will be tested, and quantifiable parameters of disease will be evaluated, including lesion counts, infectious virus in blood and secretions, clinical blood chemistries, and cytokine profiles.. As model refinement continues, concurrent advances in the identification of useful antiviral drugs are anticipated. These advancements, combined with further enhancements in diagnostic strategies, are reasonable milestones projected for this collaborative research program. Effective mitigation of an adversary’s most potent biological weapon (smallpox) must be an international priority.