Images in Clinical Medicine. Progression of the Lesion at the Site of Inoculation After Smallpox Vaccination

The New England Journal of Medicine. Jan, 2003  |  Pubmed ID: 12556544

Exploring the Potential of Variola Virus Infection of Cynomolgus Macaques As a Model for Human Smallpox

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2004  |  Pubmed ID: 15477589

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.

The Host Response to Smallpox: Analysis of the Gene Expression Program in Peripheral Blood Cells in a Nonhuman Primate Model

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2004  |  Pubmed ID: 15477590

Smallpox has played an unparalleled role in human history and remains a significant potential threat to public health. Despite the historical significance of this disease, we know little about the underlying pathophysiology or the virulence mechanisms of the causative agent, variola virus. To improve our understanding of variola pathogenesis and variola-host interactions, we examined the molecular and cellular features of hemorrhagic smallpox in cynomolgus macaques. We used cDNA microarrays to analyze host gene expression patterns in sequential blood samples from each of 22 infected animals. Variola infection elicited striking and temporally coordinated patterns of gene expression in peripheral blood. Of particular interest were features that appear to represent an IFN response, cell proliferation, immunoglobulin gene expression, viral dose-dependent gene expression patterns, and viral modulation of the host immune response. The virtual absence of a tumor necrosis factor alpha/NF-kappaB-activated transcriptional program in the face of an overwhelming systemic infection suggests that variola gene products may ablate this response. These results provide a detailed picture of the host transcriptional response during smallpox infection, and may help guide the development of diagnostic, therapeutic, and prophylactic strategies.

Early Days: Genomics and Human Responses to Infection

Current Opinion in Microbiology. Jun, 2006  |  Pubmed ID: 16679048

DNA microarray-based gene transcript-profiling of the responses of primates to infection has begun to yield new insights into host-pathogen interactions; this approach, however, remains plagued by challenges and complexities that have yet to be adequately addressed. The rapidly changing nature over time of acute infectious diseases in a host, and the genetic diversity of microbial pathogens present unique problems for the design and interpretation of functional-genomic studies in this field. In addition, there are the more common problems related to heterogeneity within clinical samples, the complex, non-standardized confounding variables associated with human subjects and the complexities posed by the analysis and validation of highly parallel data. Whereas various approaches have been developed to address each of these issues, there are significant limitations that remain to be overcome. The resolution of these problems should lead to a better understanding of the dialogue between the host and pathogen.

The Temporal Program of Peripheral Blood Gene Expression in the Response of Nonhuman Primates to Ebola Hemorrhagic Fever

Genome Biology. 2007  |  Pubmed ID: 17725815

Infection with Ebola virus (EBOV) causes a fulminant and often fatal hemorrhagic fever. In order to improve our understanding of EBOV pathogenesis and EBOV-host interactions, we examined the molecular features of EBOV infection in vivo.

Comparative Analysis of Viral Gene Expression Programs During Poxvirus Infection: a Transcriptional Map of the Vaccinia and Monkeypox Genomes

PloS One. 2008  |  Pubmed ID: 18612436

Poxviruses engage in a complex and intricate dialogue with host cells as part of their strategy for replication. However, relatively little molecular detail is available with which to understand the mechanisms behind this dialogue.

Interferon-alpha Initiates Type 1 Diabetes in Nonobese Diabetic Mice

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2008  |  Pubmed ID: 18716002

With the goal of identifying changes in gene expression in CD4(+) T cells during the development of diabetes in the nonobese diabetic (NOD) mouse, we used DNA microarrays to analyze gene expression in CD4(+) T cells from the pancreatic draining lymph nodes of NOD/BDC 2.5 T cell receptor transgenic and WT NOD mice at different ages. At 4 and 6 weeks of age, we found up-regulation of a number of genes that are known to be induced by IFN-alpha. IFN-alpha levels and IFN-alpha-producing plasmacytoid dendritic cells were increased in the PLNs of 3- to 4-week-old NOD mice. Moreover, blockade of IFN-alpha receptor 1 in NOD mice by a neutralizing antibody at 2-3 weeks of age significantly delayed the onset and decreased the incidence of type 1 diabetes, increased the relative number of immature dendritic cells in the PLNs, and enhanced the ability of spleen CD4(+) T cells to produce IL-4 and IL-10. These findings demonstrate that IFN-alpha in the PLNs is an essential initiator in the pathogenesis of type 1 diabetes in NOD mice.

Dissecting Interferon-induced Transcriptional Programs in Human Peripheral Blood Cells

PloS One. 2010  |  Pubmed ID: 20339534

Interferons are key modulators of the immune system, and are central to the control of many diseases. The response of immune cells to stimuli in complex populations is the product of direct and indirect effects, and of homotypic and heterotypic cell interactions. Dissecting the global transcriptional profiles of immune cell populations may provide insights into this regulatory interplay. The host transcriptional response may also be useful in discriminating between disease states, and in understanding pathophysiology. The transcriptional programs of cell populations in health therefore provide a paradigm for deconvoluting disease-associated gene expression profiles.We used human cDNA microarrays to (1) compare the gene expression programs in human peripheral blood mononuclear cells (PBMCs) elicited by 6 major mediators of the immune response: interferons alpha, beta, omega and gamma, IL12 and TNFalpha; and (2) characterize the transcriptional responses of purified immune cell populations (CD4+ and CD8+ T cells, B cells, NK cells and monocytes) to IFNgamma stimulation. We defined a highly stereotyped response to type I interferons, while responses to IFNgamma and IL12 were largely restricted to a subset of type I interferon-inducible genes. TNFalpha stimulation resulted in a distinct pattern of gene expression. Cell type-specific transcriptional programs were identified, highlighting the pronounced response of monocytes to IFNgamma, and emergent properties associated with IFN-mediated activation of mixed cell populations. This information provides a detailed view of cellular activation by immune mediators, and contributes an interpretive framework for the definition of host immune responses in a variety of disease settings.

Stunned Silence: Gene Expression Programs in Human Cells Infected with Monkeypox or Vaccinia Virus

PloS One. 2011  |  Pubmed ID: 21267444

Poxviruses use an arsenal of molecular weapons to evade detection and disarm host immune responses. We used DNA microarrays to investigate the gene expression responses to infection by monkeypox virus (MPV), an emerging human pathogen, and Vaccinia virus (VAC), a widely used model and vaccine organism, in primary human macrophages, primary human fibroblasts and HeLa cells. Even as the overwhelmingly infected cells approached their demise, with extensive cytopathic changes, their gene expression programs appeared almost oblivious to poxvirus infection. Although killed (gamma-irradiated) MPV potently induced a transcriptional program characteristic of the interferon response, no such response was observed during infection with either live MPV or VAC. Moreover, while the gene expression response of infected cells to stimulation with ionomycin plus phorbol 12-myristate 13-acetate (PMA), or poly (I-C) was largely unimpaired by infection with MPV, a cluster of pro-inflammatory genes were a notable exception. Poly(I-C) induction of genes involved in alerting the innate immune system to the infectious threat, including TNF-alpha, IL-1 alpha and beta, CCL5 and IL-6, were suppressed by infection with live MPV. Thus, MPV selectively inhibits expression of genes with critical roles in cell-signaling pathways that activate innate immune responses, as part of its strategy for stealthy infection.

Development of Vaccinia Reporter Viruses for Rapid, High Content Analysis of Viral Function at All Stages of Gene Expression

Antiviral Research. Jul, 2011  |  Pubmed ID: 21569797

Vaccinia virus is the prototypical orthopoxvirus of Poxviridae, a family of viruses that includes the human pathogens Variola (smallpox) and Monkeypox. Core viral functions are conserved among orthopoxviruses, and consequently Vaccinia is routinely used to study poxvirus biology and screen for novel antiviral compounds. Here we describe the development of a series of fluorescent protein-based reporter Vaccinia viruses that provide unprecedented resolution for tracking viral function. The reporter viruses are divided into two sets: (1) single reporter viruses that utilize temporally regulated early, intermediate, or late viral promoters; and (2) multi-reporter viruses that utilize multiple temporally regulated promoters. Promoter and reporter combinations were chosen that yielded high signal-to-background for stage-specific viral outputs. We provide examples for how these viruses can be used in the rapid and accurate monitoring of Vaccinia function and drug action.

Therapeutics of Ebola Hemorrhagic Fever: Whole-genome Transcriptional Analysis of Successful Disease Mitigation

The Journal of Infectious Diseases. Nov, 2011  |  Pubmed ID: 21987740

The mechanisms of Ebola (EBOV) pathogenesis are only partially understood, but the dysregulation of normal host immune responses (including destruction of lymphocytes, increases in circulating cytokine levels, and development of coagulation abnormalities) is thought to play a major role. Accumulating evidence suggests that much of the observed pathology is not the direct result of virus-induced structural damage but rather is due to the release of soluble immune mediators from EBOV-infected cells. It is therefore essential to understand how the candidate therapeutic may be interrupting the disease process and/or targeting the infectious agent. To identify genetic signatures that are correlates of protection, we used a DNA microarray-based approach to compare the host genome-wide responses of EBOV-infected nonhuman primates (NHPs) responding to candidate therapeutics. We observed that, although the overall circulating immune response was similar in the presence and absence of coagulation inhibitors, surviving NHPs clustered together. Noticeable differences in coagulation-associated genes appeared to correlate with survival, which revealed a subset of distinctly differentially expressed genes, including chemokine ligand 8 (CCL8/MCP-2), that may provide possible targets for early-stage diagnostics or future therapeutics. These analyses will assist us in understanding the pathogenic mechanisms of EBOV infection and in identifying improved therapeutic strategies.

Progression of Pathogenic Events in Cynomolgus Macaques Infected with Variola Virus

PloS One. 2011  |  Pubmed ID: 21998632

Smallpox, caused by variola virus (VARV), is a devastating human disease that affected millions worldwide until the virus was eradicated in the 1970 s. Subsequent cessation of vaccination has resulted in an immunologically naive human population that would be at risk should VARV be used as an agent of bioterrorism. The development of antivirals and improved vaccines to counter this threat would be facilitated by the development of animal models using authentic VARV. Towards this end, cynomolgus macaques were identified as adequate hosts for VARV, developing ordinary or hemorrhagic smallpox in a dose-dependent fashion. To further refine this model, we performed a serial sampling study on macaques exposed to doses of VARV strain Harper calibrated to induce ordinary or hemorrhagic disease. Several key differences were noted between these models. In the ordinary smallpox model, lymphoid and myeloid hyperplasias were consistently found whereas lymphocytolysis and hematopoietic necrosis developed in hemorrhagic smallpox. Viral antigen accumulation, as assessed immunohistochemically, was mild and transient in the ordinary smallpox model. In contrast, in the hemorrhagic model antigen distribution was widespread and included tissues and cells not involved in the ordinary model. Hemorrhagic smallpox developed only in the presence of secondary bacterial infections - an observation also commonly noted in historical reports of human smallpox. Together, our results support the macaque model as an excellent surrogate for human smallpox in terms of disease onset, acute disease course, and gross and histopathological lesions.

Identification of a Pyridopyrimidinone Inhibitor of Orthopoxviruses from a Diversity-oriented Synthesis Library

Journal of Virology. Mar, 2012  |  Pubmed ID: 22205744

Orthopoxviruses include the prototypical vaccinia virus, the emerging infectious agent monkeypox virus, and the potential biothreat variola virus (the causative agent of smallpox). There is currently no FDA-approved drug for humans infected with orthopoxviruses. We screened a diversity-oriented synthesis library for new scaffolds with activity against vaccinia virus. This screen identified a nonnucleoside analog that blocked postreplicative intermediate and late gene expression. Viral genome replication was unaffected, and inhibition could be elicited late in infection and persisted upon drug removal. Sequencing of drug-resistant viruses revealed mutations predicted to be on the periphery of the highly conserved viral RNA polymerase large subunit. Consistent with this, the compound had broad-spectrum activity against orthopoxviruses in vitro. These findings indicate that novel chemical synthesis approaches are a potential source for new infectious disease therapeutics and identify a potentially promising candidate for development to treat orthopoxvirus-infected individuals.