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Monocytes and macrophages play pivotal roles in host defense against microbial and viral pathogens through production of inflammatory and cytotoxic mediators, phagocytic activity, and activation of T cells. In previous studies, we reported significant expansion of CD14+ cells following intravenous cowpox virus Brighton Red (CPXV) infection of cynomologus macaques accompanied by increased levels of pro-inflammatory cytokines in plasma. Here, monocytes were assayed for production of pro-inflammatory cytokines and chemotactic factors during CPXV disease progression at days 3, 4, 6, and 7 post-infection in a serial sacrifice study of cynomologus macaques intravenously inoculated with 5 x 106 PFU CPXV. We measured monocyte absolute cell numbers in blood, frequencies of monocytes and macrophages in blood, and compared normal and infected monocytes through production of pro-inflammatory cytokines IL-6, IL-8, MCP-1, and IFNγ. Our results demonstrated an early increase in frequency and absolute numbers of total monocytes in blood with a rapid decline in moribund animals. Cytokine and chemokine analysis demonstrated an increase in IL-8 and MCP-1 expression over the course of disease but little IFNγ or IL-6. Pox antigen positive monocytes also expressed IL-8, but were not observed to express MCP-1. This suggests pox infection may alter cytokine production profiles of monocytes and may play a role in disease progression.

Evaluation of countermeasures against variola virus, the causative agent of smallpox, must be performed through extrapolation from suitable animal models of human smallpox. In previous studies, the gross and histopathological findings of nonhuman primates intravenously (IV) infected with Cowpox Brighton Red (CPXV) included the presence of necrotic areas of bone marrow that were orthopox-viral antigen positive by immunohistochemistry (IHC). Here, we describe changes in bone marrow in regards to cellular composition, orthopox-viral antigen positivity, viral load, and histological findings during a serial disease progression study of cynomolgus macaques following IV inoculation of CPXV. Our results demonstrate infectious CPXV as early as day 3 post inoculation in the bone marrow. Flow cytometry and histological analysis indicate changes in bone marrow composition. Furthermore, flow cytometry analysis indicates many cell populations including monocytes, CD34+ stem cells, and neutrophils were orthopox-viral antigen positive, while mesenchymal stromal cells were not. We have previously shown immunopositivity in the bone marrow by IHC. Flow cytometry revealed no significant differences in early or late apoptosis between CPXV- and mock-infected NHPs. These data indicate an important role for bone marrow function during the progression of disease in the CPXV- nonhuman primate model of smallpox.

In the current study of MPXV infection in rhesus macaques, we analyzed the NK cell responses in absolute cell numbers, cell proliferation, chemokine receptor expression, and function. Our results showed that the absolute number of total NK cells in the blood increased in response to MPXV infection at a magnitude of 23-fold, manifested by similar increases in CD56+, CD16+ and CD16-CD56- double negative (DN) NK cells. Similarly, the frequency and NK cell number in the lymph nodes also largely increased with total NK cell number increasing nearly 50-fold. Ki67 staining showed that NK cells both in the blood and lymph nodes proliferated during acute infection. Chemokine analysis revealed an early reduction of CXCR3, CCR7 and CCR6 on NK cells, followed with an increased expression of CXCR3 and CCR5 at later time points (day 7). Furthermore, MPXV infection impaired NK cell degranulation and ablated secretion of IFN-g and TNF-a. Taken together, MPXV infection induced massive NK cell proliferation, but suppressed NK cell function.