Exploring the potential of variola virus infection of cynomolgus macaques as a model for human smallpox

Abstract

Smallpox virus (variola) poses a significant threat as an agent of bioterrorism. To mitigate this risk, antiviral drugs and an improved vaccine are urgently needed. Satisfactory demonstration of protective efficacy against authentic variola will require development of an animal model in which variola produces a disease course with features consistent with human smallpox. Toward this end, cynomolgus macaques were exposed to several variola strains through aerosol and/or i.v. routes. Two strains, Harper and India 7124, produced uniform acute lethality when inoculated i.v. in high doses (10(9) plaque-forming units). Lower doses resulted in less fulminant, systemic disease and lower mortality. Animals that died had profound leukocytosis, thrombocytopenia, and elevated serum creatinine levels. After inoculation, variola was disseminated by means of a monocytic cell-associated viremia. Distribution of viral antigens by immunohistochemistry correlated with the presence of replicating viral particles demonstrated by electron microscopy and pathology in the lymphoid tissues, skin, oral mucosa, gastrointestinal tract, reproductive system, and liver. These particles resembled those seen in human smallpox. High viral burdens in target tissues were associated with organ dysfunction and multisystem failure. Evidence of coagulation cascade activation (D dimers) corroborated histologic evidence of hemorrhagic diathesis. Depletion of T cell-dependent areas of lymphoid tissues occurred, probably as a consequence of bystander apoptotic mechanisms initiated by infected macrophages. Elaboration of cytokines, including IL-6 and IFN-gamma, contribute to a cytokine storm formerly known as "toxemia." A more precise understanding of disease pathogenesis should provide targets for therapeutic intervention, to be used alone or in combination with inhibitors of variola virus replication.

Fig. 1.

Total white blood counts in monkeys infected with Harper and India strains of smallpox (a), platelet counts in plasma from infected monkeys (b), infectious viral titers recovered from throat swabs of infected monkeys (c), and skin lesions scores as a function of viral dose (India strain) (d). Lesion scores: 3, >500 lesions; 2, 50-500 lesions; 1, 1-50 lesions.

Fig. 2.

Gross lesions associated with smallpox virus infection of monkeys. (a) Hemorrhage on the serosal surface of the distal colon, monkey I-7, 6 days after exposure. Note the diffuse petechial hemorrhages and hemorrhagic, colic lymph nodes. (b) Mucosal surface of the distal colon described in a. Note the severe congestion, hemorrhage, and hemorrhagic colic lymph nodes. (c) Medial surface of the right arm of monkey I-4, 9 days after exposure. Smallpox pustules are predominantly discrete with occasional coalescence. Pock lesions developed synchronously and were more numerous distally, which is consistent with centrifugal distribution. (d). Palmar surface of the left hand of a monkey 11 days after exposure. Discrete and coalescing pustules retained integrity due to heavily keratinized palmar and digital epidermis. (e) Upper lip and nostrils from a cynomolgus monkey 11 days after exposure. Note synchronous development of pustules, some of which have umbilicated. Pustules that formed on the lightly keratinized mucosal surfaces of the nostrils have already ulcerated, resulting in dried exudates.

Fig. 3.

Microscopic localization of smallpox virus in tissues of infected monkeys. (a) Immunohistochemical localization of variola antigen in cutaneous epidermis in association with hydropic degeneration and microvesicles, day 3. (b) Variola localization in hepatocytes and Kupffer (arrow) cells of liver, day 6. (c) Variola antigen in association with proximal and distal tubules of kidney, day 6. (d) Electron micrograph of histiocytic cells in spleen, with evidence of replicating virus in association with lamellar membranous bodies and intercellular fibrin.

Fig. 4.

Immunofluorescence examination of tissues from nonhuman primates infected with variola. (a) Staining for monocytes/macrophages (green) and viral antigen (red) indicated the presence of infected monocytes/macrophages (gold) in the lymphoid tissues (lymph node shown) and in circulation. (b) In addition to monocytes/macrophages, virus-infected endothelial cells (green) were readily observed. (c) Staining for monocytes/macrophages (green) and apoptosis (red) revealed the presence of numerous tingible body macrophages in lymphoid tissues. The majority of apoptotic cells were not monocytes/macrophages but instead were lymphocytes. A rare apoptotic monocyte/macrophage (arrow) is shown. (d) A monocyte/macrophage with a clearly stained nonapoptotic nucleus (blue) is shown engulfing two separate apototic bodies.

Fig. 5.

Concentrations of D dimers, cytokines, and chemokines sequentially obtained from variola-infected monkeys. (a) D dimers (μg/ml) in sera of monkeys that received graded doses of variola strain India 1724 i.v. (b) IL-8 concentrations (pg/ml). (c) Monocyte chemoattractant protein 1 concentrations (pg/ml). (d) Macrophage inflammatory protein 1β concentrations (pg/ml). (e) IL-6 concentrations (pg/ml) in the sera of three monkeys infected i.v. with Harper strain (10⁹ pfu). (f) Mean (± 1 SD) IFN-γ concentrations (pg/ml) in sera of monkeys infected i.v. with India 7124 and Harper strains (10⁹ pfu).