Ectromelia Virus Infections of Mice as a Model to Support the Licensure of Anti-Orthopoxvirus Therapeutics

The absence of herd immunity to orthopoxviruses and the concern that variola or monkeypox viruses could be used for bioterroristic activities has stimulated the development of therapeutics and safer prophylactics. One major limitation in this process is the lack of accessible human orthopoxvirus infections for clinical efficacy trials; however, drug licensure can be based on orthopoxvirus animal challenge models as described in the “Animal Efficacy Rule”. One such challenge model uses ectromelia virus, an orthopoxvirus, whose natural host is the mouse and is the etiological agent of mousepox. The genetic similarity of ectromelia virus to variola and monkeypox viruses, the common features of the resulting disease, and the convenience of the mouse as a laboratory animal underscores its utility in the study of orthopoxvirus pathogenesis and in the development of therapeutics and prophylactics. In this review we outline how mousepox has been used as a model for smallpox. We also discuss mousepox in the context of mouse strain, route of infection, infectious dose, disease progression, and recovery from infection

VennVax, a DNA-prime, peptide-boost multi-T-cell epitope poxvirus vaccine, induces protective immunity against vaccinia infection by T cell response alone

The potential for smallpox to be disseminated in a bioterror attack has prompted development of new, safer smallpox vaccination strategies. We designed and evaluated immunogenicity and efficacy of a T-cell epitope vaccine based on conserved and antigenic vaccinia/variola sequences, identified using bioinformatics and immunological methods. Vaccination in HLA transgenic mice using a DNA-prime/peptide-boost strategy elicited significant T cell responses to multiple epitopes. No antibody response pre-challenge was observed, neither against whole vaccinia antigens nor vaccine epitope peptides. Remarkably, 100% of vaccinated mice survived lethal vaccinia challenge, demonstrating that protective immunity to vaccinia does not require B cell priming

RNA helicase signaling is critical for type I interferon production and protection against rift valley fever virus during mucosal challenge

Rift Valley fever virus (RVFV) is an emerging RNA virus with devastating economic and social consequences. Clinically, RVFV induces a gamut of symptoms ranging from febrile illness to retinitis, hepatic necrosis, hemorrhagic fever, and death. It is known that type I interferon (IFN) responses can be protective against severe pathology; however, it is unknown which innate immune receptor pathways are crucial for mounting this response. Using both in vitro assays and in vivo mucosal mouse challenge, we demonstrate here that RNA helicases are critical for IFN production by immune cells and that signaling through the helicase adaptor molecule MAVS (mitochondrial antiviral signaling) is protective against mortality and more subtle pathology during RVFV infection. In addition, we demonstrate that Toll-like-receptor-mediated signaling is not involved in IFN production, further emphasizing the importance of the RNA cellular helicases in type I IFN responses to RVFV

Multiple Phosphatidylinositol 3-Kinases Regulate Vaccinia Virus Morphogenesis

Poxvirus morphogenesis is a complex process that involves the successive wrapping of the virus in host cell membranes. We screened by plaque assay a focused library of kinase inhibitors for those that caused a reduction in viral growth and identified several compounds that selectively inhibit phosphatidylinositol 3-kinase (PI3K). Previous studies demonstrated that PI3Ks mediate poxviral entry. Using growth curves and electron microscopy in conjunction with inhibitors, we show that that PI3Ks additionally regulate morphogenesis at two distinct steps: immature to mature virion (IMV) transition, and IMV envelopment to form intracellular enveloped virions (IEV). Cells derived from animals lacking the p85 regulatory subunit of Type I PI3Ks (p85α−/−β−/−) presented phenotypes similar to those observed with PI3K inhibitors. In addition, VV appear to redundantly use PI3Ks, as PI3K inhibitors further reduce plaque size and number in p85α−/−β−/− cells. Together, these data provide evidence for a novel regulatory mechanism for virion morphogenesis involving phosphatidylinositol dynamics and may represent a new therapeutic target to contain poxviruses

Severe Acute Respiratory Syndrome Coronavirus Gene 7 Products Contribute to Virus-Induced Apoptosis▿

The proteins encoded by gene 7 of the severe acute respiratory syndrome coronavirus (SARS-CoV) have been demonstrated to have proapoptotic activity when expressed from cDNA but appear to be dispensable for virus replication. Recombinant SARS-CoVs bearing deletions in gene 7 were used to assess the contribution of gene 7 to virus replication and apoptosis in several transformed cell lines, as well as to replication and pathogenesis in golden Syrian hamsters. Deletion of gene 7 had no effect on SARS-CoV replication in transformed cell lines, nor did it alter the induction of early apoptosis markers such as annexin V binding and activation of caspase 3. However, viruses with gene 7 disruptions were not as efficient as wild-type virus in inducing DNA fragmentation, as judged by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, indicating that the gene 7 products do contribute to virus-induced apoptosis. Disruption of gene 7 did not affect virus replication or morbidity in golden Syrian hamsters, suggesting that the gene 7 products are not required for acute infection in vivo. The data indicate that open reading frames 7a and 7b contribute to but are not solely responsible for the apoptosis seen in SARS-CoV-infected cells

EVM005: An Ectromelia-Encoded Protein with Dual Roles in NF-κB Inhibition and Virulence

<div><p>Poxviruses contain large dsDNA genomes encoding numerous open reading frames that manipulate cellular signalling pathways and interfere with the host immune response. The NF-κB signalling cascade is an important mediator of innate immunity and inflammation, and is tightly regulated by ubiquitination at several key points. A critical step in NF-κB activation is the ubiquitination and degradation of the inhibitor of kappaB (IκBα), by the cellular SCF^β-TRCP ubiquitin ligase complex. We show here that upon stimulation with TNFα or IL-1β, <i>Orthopoxvirus</i>-infected cells displayed an accumulation of phosphorylated IκBα, indicating that NF-κB activation was inhibited during poxvirus infection. Ectromelia virus is the causative agent of lethal mousepox, a natural disease that is fatal in mice. Previously, we identified a family of four ectromelia virus genes (EVM002, EVM005, EVM154 and EVM165) that contain N-terminal ankyrin repeats and C-terminal F-box domains that interact with the cellular SCF ubiquitin ligase complex. Since degradation of IκBα is catalyzed by the SCF^β-TRCP ubiquitin ligase, we investigated the role of the ectromelia virus ankyrin/F-box protein, EVM005, in the regulation of NF-κB. Expression of Flag-EVM005 inhibited both TNFα- and IL-1β-stimulated IκBα degradation and p65 nuclear translocation. Inhibition of the NF-κB pathway by EVM005 was dependent on the F-box domain, and interaction with the SCF complex. Additionally, ectromelia virus devoid of EVM005 was shown to inhibit NF-κB activation, despite lacking the EVM005 open reading frame. Finally, ectromelia virus devoid of EVM005 was attenuated in both A/NCR and C57BL/6 mouse models, indicating that EVM005 is required for virulence and immune regulation <i>in vivo</i>.</p></div

ECTV-Δ005, ECTV-Δ002 and ECTV-Δ002-005 inhibit TNFα induced IκBα degradation.

<p>(A) HeLa cells were mock-infected or infected with ECTV, ECTV-Δ005 or ECTV-005-rev at a MOI of 5. (C) Alternatively, HeLa cells were mock-infected or infected with ECTV, ECTV-Δ002, ECTV-Δ005 or ECTV-Δ002-005 at a MOI of 5. (A and C) At 12 hours post hours post-infection, cells were stimulated with 10 µM MG132 for 1 hour and hour and/or 10 ng ng/ml TNFα for 20 minutes. Cells were harvested, fixed and permeablized, followed by staining with anti minutes. Cells were harvested, fixed and permeablized, followed by staining with anti-IκBα and anti-I3L. Samples were subjected to flow cytometry, IκBα (A panels a–d, and C panels i–m) or I3L (A panels e–h and C panels n–r) are measured along the x-axis. (B and D) The percentage of cells expressing IκBα were measured and plotted as the average of three independent experiments with SEM.</p

EVM005 is required for virulence during ECTV infection of C57BL/6 and A/NCR mice.

<p>(A and B) Female C57BL/6 mice were mock-infected or infected with 10,000 pfu of ECTV, ECTV pfu of ECTV, ECTV-Δ005, ECTV-005-rev or ECTV-005(1-593)-rev via intranasal inoculation. Mice were monitored daily for mortality, day of death (A), and body weight (B). (C and D) Alternatively, female A/NCR mice were mock-infected or infected with 10,000 pfu of ECTV, ECTV pfu of ECTV, ECTV-Δ005, ECTV-005-rev or ECTV-005(1-593)-rev via footpad injection. Mice were monitored daily for day of death and mortality (C) as well as body weight (D). Morality curves were statistically analyzed using a Log-rank (Mantel-Cox) test.</p