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Hemorrhagic smallpox, caused by variola virus (VARV), was a rare but nearly 100% lethal human disease manifestation. Hemorrhagic smallpox is frequently characterized by secondary bacterial infection, coagulopathy, and myocardial and subendocardial hemorrhages. Previous experiments have demonstrated that intravenous (IV) cowpox virus (CPXV) exposure of macaques mimics human hemorrhagic smallpox. The goal of this experiment was to further understand the onset, nature, and severity of cardiac pathology and how it may contribute to disease. The findings support an acute late-stage myocarditis with lymphohistiocytic infiltrates in the CPXV model of hemorrhagic smallpox.

To characterize T cell epitopes in monkeypox virus (MPXV) infected rhesus macaques, we utilized IFN γ Elispot assay to screen 400 predicted peptides from 20 MPXV proteins. Two peptides from the F8L protein, an analog of E9L protein in vaccinia, were found to elicit CD8+ T cell responses. Prediction and in vitro MHC binding analyses suggest that one is restricted by Mamu-A1 ⁎ 001 and another by Mamu-A1 ⁎ 002. The Mamu-A1 ⁎ 002 epitope is completely identical in all reported sequences for variola, vaccinia, cowpox and MPXV. The Mamu-A1 ⁎ 001 epitope is conserved in MPXV and vaccinia, and has one residue substitution (V6>I) in some cowpox sequences and all variola sequences. Given CD8+ T-cell epitopes from E9L were also identified in humans and mice, our data suggested that F8L/E9L may be a dominant pox viral protein for CD8+ T cell responses, and may be considered as a target when designing vaccines that target pox-specific T cell responses.

In the fall of 2014, an international news agency reported that patients suffering from Ebola virus disease (EVD) in Liberia were treated successfully with lamivudine, an antiviral drug used to treat human immunodeficiency virus-1 and hepatitis B virus infections. According to the report, 13 out of 15 patients treated with lamivudine survived and were declared free from Ebola virus disease. In this study, the anti-Ebola virus (EBOV) activity of lamivudine and another antiretroviral, zidovudine, were evaluated in a diverse set of cell lines against two variants of wild-type EBOV. Variable assay parameters were assessed to include different multiplicities of infection, lengths of inoculation times, and durations of dosing. At a multiplicity of infection of 1, lamivudine and zidovudine had no effect on EBOV propagation in Vero E6, Hep G2, or HeLa cells, or in primary human monocyte-derived macrophages. At a multiplicity of infection of 0.1, zidovudine demonstrated limited anti-EBOV activity in Huh 7 cells. Under certain conditions, lamivudine had low anti-EBOV activity at the maximum concentration tested (320 μM). However, lamivudine never achieved greater than 30% viral inhibition, and the activity was not consistently reproducible. Combination of lamivudine and zidovudine showed no synergistic antiviral activity. Independently, a set of in vitro experiments testing lamivudine and zidovudine for antiviral activity against an Ebola-enhanced green fluorescent protein reporter virus was performed at the Centers for Disease Control and Prevention. No antiviral activity was observed for either compound. A study evaluating the efficacy of lamivudine in a guinea pig model of EVD found no survival benefit. This lack of benefit was observed despite plasma lamivudine concentrations in guinea pig of about 4 μg/ml obtained in a separately conducted pharmacokinetics study. These studies found no evidence to support the therapeutic use of lamivudine for the treatment of EVD.

Humans infected with yellow fever virus (YFV), a mosquito-borne flavivirus, can develop illness ranging from a mild febrile disease to hemorrhagic fever and death. The 17D vaccine strain of YFV was developed in the 1930s, has been used continuously since development and has proven very effective. Genetic differences between vaccine and wild-type viruses are few, yet viral or host mechanisms associated with protection or disease are not fully understood. Over the past 20 years, a number of cases of vaccine-associated disease have been identified following vaccination with 17D; these cases have been correlated with reduced immune status at the time of vaccination. Recently, several studies have evaluated T cell responses to vaccination in both humans and non-human primates, but none have evaluated the response to wild-type virus infection. In the studies described here, monocyte-derived macrophages (MDM) and dendritic cells (MoDC) from both humans and rhesus macaques were evaluated for their ability to support infection with either wild-type Asibi virus or the 17D vaccine strain and the host cytokine and chemokine response characterized. Human MoDC and MDM were also evaluated for their ability to stimulate CD4+ T cells. It was found that MoDC and MDM supported viral replication and that there were differential cytokine responses to infection with either wild-type or vaccine viruses. Additionally, MoDCs infected with live 17D virus were able to stimulate IFN-γ and IL-2 production in CD4+ T cells, while cells infected with Asibi virus were not. These data demonstrate that wild-type and vaccine YFV stimulate different responses in target antigen presenting cells and that wild-type YFV can inhibit MoDC activation of CD4+ T cells, a critical component in development of protective immunity. These data provide initial, but critical insight into regulatory capabilities of wild-type YFV in development of disease., Author Summary Yellow fever virus (YFV) is a mosquito-borne flavivirus that can cause lethal hemorrhagic fever in infected humans. An effective live-attenuated vaccine, 17D, was developed in 1937 and continues to be used today. Over the past several years, a number of cases of vaccine-associated disease have been identified and linked to a compromised immune status. In the studies presented here we evaluated the susceptibility of macrophages and dendritic cells (DCs) to YFV infection, their cytokine response to infection and their ability to interact with and stimulate CD4+ T cells. These studies found that macrophages and DCs derived from both humans and non-human primates were susceptible to infection with either a wild-type Asibi YFV or the 17D vaccine virus, that the two viruses stimulated different cytokine responses in these cells and that human DCs infected with the 17D virus were able to stimulate CD4+ T cells while those infected with Asibi virus did not. These data demonstrate clear differences between the wild-type and vaccine viruses that may be related to the success of the 17D vaccine. These data also suggest that wild-type YFV may be able to inhibit critical components of the immune response to allow virus propagation and dissemination.

Natural killer (NK) cells play critical roles in innate immunity and in bridging innate and adaptive immune responses against viral infection. However, the response of NK cells to monkeypox virus (MPXV) infection is not well characterized. In this intravenous challenge study of MPXV infection in rhesus macaques (Macaca mulatta), we analyzed blood and lymph node NK cell changes in absolute cell numbers, cell proliferation, chemokine receptor expression, and cellular functions. 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 increases in CD56+, CD16+, CD16-CD56- double negative, and CD16+CD56+ double positive NK cell subsets. Similarly, the frequency and NK cell numbers in the lymph nodes also largely increased with the total NK cell number increasing 46.1-fold. NK cells both in the blood and lymph nodes massively proliferated in response to MPXV infection as measured by Ki67 expression. Chemokine receptor analysis revealed reduced expression of CXCR3, CCR7, and CCR6 on NK cells at early time points (days 2 and 4 after virus inoculation), followed by an increased expression of CXCR3 and CCR5 at later time points (days 7-8) of infection. In addition, MPXV infection impaired NK cell degranulation and ablated secretion of interferon-γ and tumor necrosis factor-α. Our data suggest a dynamic model by which NK cells respond to MPXV infection of rhesus macaques. Upon virus infection, NK cells proliferated robustly, resulting in massive increases in NK cell numbers. However, the migrating capacity of NK cells to tissues at early time points might be reduced, and the functions of cytotoxicity and cytokine secretion were largely compromised. Collectively, the data may explain, at least partially, the pathogenesis of MPXV infection in rhesus macaques.

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.

Within the erythroid lineage, erythropoietin (EPO) responsiveness is manifested by cell division and growth as well as specific changes in heme and globin production that ultimately result in the production of erythrocytes. However, the nascent relationship between EPO-associated mitosis and globin gene regulation has not been fully defined. In this study, cultured adult human CD34+ cells from peripheral blood were used to investigate early cellular responses to erythropoietin in the context of mitosis. Matched cultures were performed in replicate using human cells from at least two healthy adults. To detect mitosis, one million cells were labeled with 2uM carboxyfluorescein diacetate succinamidyl ester (CFSE). The CFSE-labeled cells were then cultured in the presence [EPO(+)] or absence [EPO(−)] of 4U/mL EPO, and analyzed using flow cytometry. No cell divisions were detected in either condition during the first 24 hours in culture, and multiple cell divisions were noted on subsequent culture days only in the EPO(+) cultures. Remarkably, dual-staining with CFSE and CD71 revealed a small (90% of those cells. These novel results suggest that the frequency and levels of gamma-globin transcripts are quite low at the earliest stages of an EPO response among adult human CD34+ cells. However, it is clearly demonstrated that the cells can increase their capacity to import iron and transcribe beta-globin mRNA at very high levels prior to their first EPO-dependent cell division.