Smallpox lesion characterization in placebo-treated and tecovirimat-treated macaques using traditional and novel methods

Smallpox was the most rampant infectious disease killer of the 20th century, yet much remains unknown about the pathogenesis of the variola virus. Using archived tissue from a study conducted at the Centers for Disease Control and Prevention we characterized pathology in 18 cynomolgus macaques intravenously infected with the Harper strain of variola virus. Six macaques were placebo-treated controls, six were tecovirimat-treated beginning at 2 days post-infection, and six were tecovirimat-treated beginning at 4 days post-infection. All macaques were treated daily until day 17. Archived tissues were interrogated using immunohistochemistry, in situ hybridization, immunofluorescence, and electron microscopy. Gross lesions in three placebo-treated animals that succumbed to infection primarily consisted of cutaneous vesicles, pustules, or crusts with lymphadenopathy. The only gross lesions noted at the conclusion of the study in the three surviving placebo-treated and the Day 4 treated animals consisted of resolving cutaneous pox lesions. No gross lesions attributable to poxviral infection were present in the Day 2 treated macaques. Histologic lesions in three placebo-treated macaques that succumbed to infection consisted of proliferative and necrotizing dermatitis with intracytoplasmic inclusion bodies and lymphoid depletion. The only notable histologic lesion in the Day 4 treated macaques was resolving dermatitis; no notable lesions were seen in the Day 2 treated macaques. Variola virus was detected in all three placebo-treated animals that succumbed to infection prior to the study's conclusion by all utilized methods (IHC, ISH, IFA, EM). None of the three placebo-treated animals that survived to the end of the study nor the animals in the two tecovirimat treatment groups showed evidence of variola virus by these methods. Our findings further characterize variola lesions in the macaque model and describe new molecular methods for variola detection. The worldwide 2022 mpox outbreak renewed research interest in defining and countering threats posed by orthopoxviruses, including smallpox. In the absence of optimized prevention and treatment strategies, natural re-emergence of smallpox or its use as a bioweapon released into unvaccinated populations could result in avoidable illnesses and deaths. Here we applied recently developed techniques to study the relationship between smallpox lesion development and the fate of virus in three groups of animals exposed to smallpox virus: those treated with the FDA-approved antiviral tecovirimat 2 or 4 days after infection and a placebo-treated group. We were able to characterize the development of lesions at gross and microscopic levels and describe new molecular methods for studying the distribution and persistence of variola virus (VARV) in tissue samples that were collected from the three treatment groups. These findings can be used to fine-tune the timing of administering tecovirimat, other antivirals, and vaccines, as well as help characterize the performance of in-development orthopoxvirus countermeasures.