RESUMEN
CD8(+) T cells and NK cells protect from viral infections by killing virally infected cells and secreting interferon-γ. Several inhibitory receptors limit the magnitude and duration of these anti-viral responses. NKG2A, which is encoded by Klrc1, is a lectin-like inhibitory receptor that is expressed as a heterodimer with CD94 on NK cells and activated CD8(+) T cells. Previous studies on the impact of CD94/NKG2A heterodimers on anti-viral responses have yielded contrasting results and the in vivo function of NKG2A remains unclear. Here, we generated Klrc1(-/-) mice and found that NKG2A is selectively required for resistance to ectromelia virus (ECTV). NKG2A functions intrinsically within ECTV-specific CD8(+) T cells to limit excessive activation, prevent apoptosis, and preserve the specific CD8(+) T cell response. Thus, although inhibitory receptors often cause T cell exhaustion and viral spreading during chronic viral infections, NKG2A optimizes CD8(+) T cell responses during an acute poxvirus infection.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Células Asesinas Naturales/inmunología , Subfamília C de Receptores Similares a Lectina de Células NK/inmunología , Infecciones por Poxviridae/inmunología , Animales , Citotoxicidad Inmunológica/inmunología , Modelos Animales de Enfermedad , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Double-stranded (ds) RNA, both synthetic and produced during virus replication, rapidly stimulates MAPK and NF-κB signaling that results in expression of the inflammatory genes inducible nitric oxide synthase, cyclooxygenase 2, and IL-1ß by macrophages. Using biochemical and genetic approaches, we have identified the chemokine ligand-binding C-C chemokine receptor type 5 (CCR5) as a cell surface signaling receptor required for macrophage expression of inflammatory genes in response to dsRNA. Activation of macrophages by synthetic dsRNA does not require known dsRNA receptors, as poly(inosinic:cytidylic) acid [poly(I:C)] activates signaling pathways leading to expression of inflammatory genes to similar levels in wild-type and Toll-like receptor 3- or melanoma differentiation antigen 5-deficient macrophages. In contrast, macrophage activation in response to poly(I:C) is attenuated in macrophages isolated from mice lacking CCR5. These findings support a role for CCR5 as a cell surface signaling receptor that participates in activation of inflammatory genes in macrophages in response to the viral dsRNA mimetic poly(inosinic:cytidylic) acid by pathways that are distinct from classical dsRNA receptor-mediated responses.
Asunto(s)
Inflamación/metabolismo , Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Poli I-C/farmacología , Receptores CCR5/agonistas , Transducción de Señal/efectos de los fármacos , Animales , Citocinas/genética , Citocinas/metabolismo , Regulación de la Expresión Génica , Inflamación/genética , Inflamación/inmunología , Helicasa Inducida por Interferón IFIH1/deficiencia , Helicasa Inducida por Interferón IFIH1/genética , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células RAW 264.7 , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismoRESUMEN
Exogenous cytokine therapy can induce systemic toxicity, which might be prevented by activating endogenously produced cytokines in local cell niches. Here we developed antibody-based activators of cytokine signaling (AcCS), which recognize cytokines only when they are bound to their cell surface receptors. AcCS were developed for type I interferons (IFNs), which induce cellular activities by binding to cell surface receptors IFNAR1 and IFNAR2. As a potential alternative to exogenous IFN therapy, AcCS were shown to potentiate the biological activities of natural IFNs by â¼100-fold. Biochemical and structural characterization demonstrates that the AcCS stabilize the IFN-IFNAR2 binary complex by recognizing an IFN-induced conformational change in IFNAR2. Using IFN mutants that disrupt IFNAR1 binding, AcCS were able to enhance IFN antiviral potency without activating antiproliferative responses. This suggests AcCS can be used to manipulate cytokine signaling for basic science and possibly for therapeutic applications.
Asunto(s)
Citocinas/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Receptores de Citocinas/inmunología , Transducción de Señal , Antivirales/química , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Fragmentos de Inmunoglobulinas/farmacología , Interferón-alfa/farmacología , Cinética , Mutación/genética , Fosforilación , Conformación Proteica , Receptor de Interferón alfa y beta/química , Receptor de Interferón alfa y beta/metabolismo , Reproducibilidad de los Resultados , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Adenovirus infections of immunocompromised patients can develop into deadly multiorgan or systemic disease. The virus is especially threatening for pediatric allogeneic hematopoietic stem cell transplant recipients; according to some studies, 10% or more of these patients succumb to disease resulting from adenovirus infection. At present, there is no drug approved for the treatment or prevention of adenovirus infections. Compounds that are approved to treat other virus infections are used off-label to combat adenovirus, but only anecdotal evidence of the efficacy of these drugs exists. Ganciclovir, a drug approved for the treatment of herpesvirus infection, was previously reported to be effective against human adenoviruses in vitro. To model adenovirus infections in immunocompromised humans, we examined ganciclovir's efficacy in immunosuppressed Syrian hamsters intravenously infected with type 5 human adenovirus (Ad5). This animal model is permissive for Ad5 replication, and the animals develop symptoms similar to those seen in humans. We demonstrate that ganciclovir suppresses Ad5 replication in the liver of infected hamsters and that it mitigates the consequences of Ad5 infections in these animals when administered prophylactically or therapeutically. We show that ganciclovir inhibits Ad5 DNA synthesis and late gene expression. The mechanism of action for the drug is not clear; preliminary data suggest that it exerts its antiadenoviral effect by directly inhibiting the adenoviral DNA polymerase. While more extensive studies are required, we believe that ganciclovir is a promising drug candidate to treat adenovirus infections. Brincidofovir, a drug with proven activity against Ad5, was used as a positive control in the prophylactic experiment.
Asunto(s)
Infecciones por Adenoviridae/tratamiento farmacológico , Adenovirus Humanos/efectos de los fármacos , Antivirales/farmacología , Ganciclovir/farmacología , Huésped Inmunocomprometido , Proteínas Virales/antagonistas & inhibidores , Infecciones por Adenoviridae/inmunología , Infecciones por Adenoviridae/mortalidad , Infecciones por Adenoviridae/virología , Adenovirus Humanos/genética , Adenovirus Humanos/crecimiento & desarrollo , Adenovirus Humanos/patogenicidad , Animales , Peso Corporal/efectos de los fármacos , Línea Celular Tumoral , Citosina/análogos & derivados , Citosina/farmacología , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Femenino , Expresión Génica , Humanos , Masculino , Mesocricetus , Organofosfonatos/farmacología , Análisis de Supervivencia , Transaminasas/sangre , Carga Viral/efectos de los fármacos , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
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.
Asunto(s)
ARN Helicasas DEAD-box/inmunología , Interferón beta/inmunología , Membrana Mucosa/virología , Fiebre del Valle del Rift/enzimología , Fiebre del Valle del Rift/inmunología , Virus de la Fiebre del Valle del Rift/fisiología , Animales , Línea Celular , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , Células Dendríticas/inmunología , Células Dendríticas/virología , Femenino , Humanos , Interferón beta/genética , Macrófagos/inmunología , Macrófagos/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados/genética , Membrana Mucosa/inmunología , Fiebre del Valle del Rift/prevención & control , Fiebre del Valle del Rift/virología , Transducción de Señal , Receptores Toll-Like/genética , Receptores Toll-Like/inmunologíaRESUMEN
Poxviruses are dsDNA viruses with large genomes. Many genes in the genome remain uncharacterized, and recent studies have demonstrated that the poxvirus transcriptome includes numerous so-called anomalous transcripts not associated with open reading frames. Here, we characterize the expression and role of an apparently non-coding RNA in orthopoxviruses, which we call viral hairpin RNA (vhRNA). Using a bioinformatics approach, we predicted expression of a transcript not associated with an open reading frame that is likely to form a stem-loop structure due to the presence of a 21 nt palindromic sequence. Expression of the transcript as early as 2 h post-infection was confirmed by northern blot and analysis of publicly available vaccinia virus infected cell transcriptomes. The transcription start site was determined by RACE PCE and transcriptome analysis, and early and late promoter sequences were identified. Finally, to test the function of the transcript we generated an ectromelia virus knockout, which failed to form plaques in cell culture. The important role of the transcript in viral replication was further demonstrated using siRNA. Although the function of the transcript remains unknown, our work contributes to evidence of an increasingly complex poxvirus transcriptome, suggesting that transcripts such as vhRNA not associated with an annotated open reading frame can play an important role in viral replication.
Asunto(s)
Virus de la Ectromelia/crecimiento & desarrollo , Virus de la Ectromelia/genética , Regulación Viral de la Expresión Génica , Expresión Génica , ARN no Traducido/biosíntesis , Ensayo de Placa Viral , Animales , Northern Blotting , Línea Celular , Chlorocebus aethiops , Biología Computacional , Técnicas de Inactivación de Genes , Macaca mulatta , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , ARN no Traducido/genética , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
Protection of the human lung from infectious agents, allergens, and ultrafine particles is difficult with current technologies. High-efficiency particulate air (HEPA) filters remove airborne particles of >0.3 µm with 99.97% efficiency, but they are expensive to maintain. Electrostatic precipitation has been used as an inexpensive approach to remove large particles from airflows, but it has a collection efficiency minimum in the submicrometer size range, allowing for a penetration window for some allergens and ultrafine particles. Incorporating soft X-ray irradiation as an in situ component of the electrostatic precipitation process greatly improves capture efficiency of ultrafine particles. Here we demonstrate the removal and inactivation capabilities of soft-X-ray-enhanced electrostatic precipitation technology targeting infectious agents (Bacillus anthracis, Mycobacterium bovis BCG, and poxviruses), allergens, and ultrafine particles. Incorporation of in situ soft X-ray irradiation at low-intensity corona conditions resulted in (i) 2-fold to 9-fold increase in capture efficiency of 200- to 600-nm particles and (ii) a considerable delay in the mean day of death as well as lower overall mortality rates in ectromelia virus (ECTV) cohorts. At the high-intensity corona conditions, nearly complete protection from viral and bacterial respiratory infection was afforded to the murine models for all biological agents tested. When optimized for combined efficient particle removal with limited ozone production, this technology could be incorporated into stand-alone indoor air cleaners or scaled for installation in aircraft cabin, office, and residential heating, ventilating, and air-conditioning (HVAC) systems.
Asunto(s)
Microbiología del Aire , Alérgenos/efectos de la radiación , Bacterias/efectos de la radiación , Precipitación Química , Material Particulado/efectos de la radiación , Virus/efectos de la radiación , Rayos X , Alérgenos/química , Animales , Bacterias/química , Modelos Animales de Enfermedad , Ratones , Material Particulado/química , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/prevención & control , Infecciones del Sistema Respiratorio/virología , Electricidad Estática , Virus/químicaRESUMEN
Encephalomyocarditis virus (EMCV) is capable of stimulating inflammatory gene expression by macrophages as a result of interactions between EMCV capsid proteins and cell surface receptors. In this study, biochemical and genetic approaches identified a role for Ccr5, a chemokine receptor, in transducing the signals of EMCV infection that result in the expression of inflammatory genes in macrophages. Antibody neutralization and gene knockout strategies were used to show that the presence of Ccr5 is required for EMCV-stimulated mitogen-activated protein (MAP) kinase and nuclear factor-kappa B (NF-κB) activation, and the subsequent expression of the inflammatory gene-inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Ccr5 appears to participate in the early control of virus replication: EMCV mRNA accumulates to sevenfold higher levels in Ccr5-deficient mice when compared to wild-type controls. These findings support a regulatory role for Ccr5 in the antiviral response to EMCV in which this chemokine receptor participates in regulation of inflammatory gene expression in response to virus infection.
Asunto(s)
Virus de la Encefalomiocarditis/fisiología , Interferón Tipo I/biosíntesis , Macrófagos/virología , Receptores CCR5/fisiología , Transducción de Señal/fisiología , Animales , Anticuerpos Neutralizantes/farmacología , Células Cultivadas , Ciclooxigenasa 2/metabolismo , ARN Helicasas DEAD-box/fisiología , Expresión Génica , Helicasa Inducida por Interferón IFIH1 , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Receptores CCR5/deficiencia , Receptor Toll-Like 3/fisiologíaRESUMEN
Poxviruses produce complement regulatory proteins to subvert the host's immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host's immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement's role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE's regulatory capacity. These results suggest that EMICE's role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.
Asunto(s)
Inactivadores del Complemento/inmunología , Proteínas del Sistema Complemento/inmunología , Virus de la Ectromelia/inmunología , Evasión Inmune , Proteínas Virales/inmunología , Factores de Virulencia/inmunología , Animales , Línea Celular , Chlorocebus aethiops , Cricetinae , Cricetulus , Virus de la Ectromelia/patogenicidad , Ratones , Ratones Endogámicos C57BL , Pruebas de NeutralizaciónRESUMEN
Adenoviruses (Ads) cause a wide array of end-organ and disseminated diseases in severely immunosuppressed patients. For example, approximately 20% of pediatric allogeneic hematopoietic stem cell transplant recipients develop disseminated Ad infection, and the disease proves fatal in as many as 50-80% of these patients. Ad infections are a serious problem for solid-organ transplant recipients and AIDS patients as well. Unfortunately, there are no antiviral drugs approved specifically to treat these infections. A suitable animal model that is permissive for Ad replication would help in the discovery process. Here we identify an animal model to study Ad pathogenesis and the efficacy of antiviral compounds. We show that human serotype 5 Ad (Ad5) causes severe systemic disease in immunosuppressed Syrian hamsters that is similar to that seen in immunocompromised patients. We also demonstrate that hexadecyloxypropyl-cidofovir (CMX001) rescues the hamsters from a lethal challenge with Ad5. The antiviral drug provided protection both prophylactically and when given up to 2 days after i.v. exposure to Ad5. CMX001 acts by reducing Ad replication in key target organs. Thus, the immunosuppressed Syrian hamster is a powerful model to evaluate anti-Ad drugs, and its use can facilitate the entry of drugs such as CMX001 into clinical trials.
Asunto(s)
Citosina/análogos & derivados , Inmunosupresores/uso terapéutico , Organofosfonatos/farmacología , Adenoviridae/metabolismo , Infecciones por Adenoviridae/metabolismo , Infecciones por Adenoviridae/terapia , Animales , Antivirales/farmacología , Cricetinae , Citosina/farmacología , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Células Madre Hematopoyéticas/citología , Humanos , Hígado/metabolismo , Mesocricetus , Modelos BiológicosRESUMEN
Src family kinases (SFKs) are non-receptor tyrosine kinases that have been implicated as regulators of the inflammatory response. In this study, the role of SFK activation in the inflammatory response of macrophages to encephalomyocarditis virus (EMCV) infection was examined. Virus infection of macrophages stimulates the expression of cyclooxygenase-2 (COX-2), interleukin (IL)-1beta and inducible nitric oxide synthase (iNOS). Inhibition of SFK attenuates EMCV-induced COX-2 expression and prostaglandin E(2) production, iNOS expression and subsequent nitric oxide production, and IL-1beta expression. EMCV-induced COX-2 expression requires the activation of nuclear factor-kappaB and the mitogen-activated protein kinase p38. Consistent with these previous findings, inhibition of SFKs attenuated the phosphorylation of p38 in response to EMCV infection, suggesting that SFKs may act upstream of p38. These findings provide evidence that SFK activation plays an active role in the regulation of inflammatory gene expression by virus-infected macrophages.
Asunto(s)
Ciclooxigenasa 2/biosíntesis , Virus de la Encefalomiocarditis/patogenicidad , Macrófagos/metabolismo , Macrófagos/virología , Familia-src Quinasas/metabolismo , Animales , Secuencia de Bases , Línea Celular , Ciclooxigenasa 2/genética , Cartilla de ADN/genética , Dinoprostona/biosíntesis , Activación Enzimática , Interacciones Huésped-Patógeno , Proteínas I-kappa B/metabolismo , Mediadores de Inflamación/metabolismo , Interleucina-1beta/biosíntesis , Interleucina-1beta/genética , Ratones , Modelos Biológicos , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Activación Transcripcional , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
Poxviruses subvert the host immune response by producing immunomodulatory proteins, including a complement regulatory protein. Ectromelia virus provides a mouse model for smallpox where the virus and the host's immune response have co-evolved. Using this model, our study investigated the role of the complement system during a poxvirus infection. By multiple inoculation routes, ectromelia virus caused increased mortality by 7 to 10 days post-infection in C57BL/6 mice that lack C3, the central component of the complement cascade. In C3(-/-) mice, ectromelia virus disseminated earlier to target organs and generated higher peak titers compared to the congenic controls. Also, increased hepatic inflammation and necrosis correlated with these higher tissue titers and likely contributed to the morbidity in the C3(-/-) mice. In vitro, the complement system in naïve C57BL/6 mouse sera neutralized ectromelia virus, primarily through the recognition of the virion by natural antibody and activation of the classical and alternative pathways. Sera deficient in classical or alternative pathway components or antibody had reduced ability to neutralize viral particles, which likely contributed to increased viral dissemination and disease severity in vivo. The increased mortality of C4(-/-) or Factor B(-/-) mice also indicates that these two pathways of complement activation are required for survival. In summary, the complement system acts in the first few minutes, hours, and days to control this poxviral infection until the adaptive immune response can react, and loss of this system results in lethal infection.
Asunto(s)
Proteínas del Sistema Complemento/fisiología , Ectromelia Infecciosa/inmunología , Ectromelia Infecciosa/mortalidad , Animales , Complemento C3/genética , Complemento C4/genética , Factor B del Complemento/genética , Proteínas del Sistema Complemento/genética , Ectromelia Infecciosa/genética , Ectromelia Infecciosa/patología , Hepatitis Viral Animal/genética , Hepatitis Viral Animal/patología , Hepatitis Viral Animal/virología , Inmunidad Innata/genética , Hígado/patología , Hígado/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Necrosis/genética , Necrosis/patología , Necrosis/veterinaria , Necrosis/virología , Viremia/mortalidad , Replicación Viral/genéticaRESUMEN
The Poxvirus Bioinformatics Resource Center (PBRC) has been established to provide informational and analytical resources to the scientific community to aid research directed at providing a better understanding of the Poxviridae family of viruses. The PBRC was specifically established as the result of the concern that variola virus, the causative agent of smallpox, as well as related viruses, might be utilized as biological weapons. In addition, the PBRC supports research on poxviruses that might be considered new and emerging infectious agents such as monkeypox virus. The PBRC consists of a relational database and web application that supports the data storage, annotation, analysis and information exchange goals of the project. The current release consists of over 35 complete genomic sequences of various genera, species and strains of viruses from the Poxviridae family. Sequence and annotation information for these viruses has been obtained from sequences publicly available from GenBank as well as sequences not yet deposited in GenBank that have been obtained from ongoing sequencing projects. In addition to sequence data, the PBRC provides comprehensive annotation and curation of virus genes; analytical tools to aid in the understanding of the available sequence data, including tools for the comparative analysis of different virus isolates; and visualization tools to help better display the results of various analyses. The PBRC represents the initial development of what will become a more comprehensive Viral Bioinformatics Resource Center for Biodefense that will be one of the National Institute of Allergy and Infectious Diseases' 'Bioinformatics Resource Centers for Biodefense and Emerging or Re-Emerging Infectious Diseases'. The PBRC website is available at http://www.poxvirus.org.
Asunto(s)
Biología Computacional , Bases de Datos de Ácidos Nucleicos , Poxviridae/genética , Análisis de Secuencia de ADN , ADN Viral/química , Genoma Viral , Genómica , Internet , Programas Informáticos , Interfaz Usuario-ComputadorRESUMEN
BACKGROUND: Vaccinia virus (VACV)-DUKE was isolated from a lesion on a 54 year old female who presented to a doctor at the Duke University Medical Center. She was diagnosed with progressive vaccinia and treated with vaccinia immune globulin. The availability of the VACV-DUKE genome sequence permits a first time genomic comparison of a VACV isolate associated with a smallpox vaccine complication with the sequence of culture-derived clonal isolates of the Dryvax vaccine. RESULTS: This study showed that VACV-DUKE is most similar to VACV-ACAM2000 and CLONE3, two VACV clones isolated from the Dryvax vaccine stock confirming VACV-DUKE as an isolate from Dryvax. However, VACV-DUKE is unique because it is, to date, the only Dryvax clone isolated from a patient experiencing a vaccine-associated complication. The 199,960 bp VACV-DUKE genome encodes 225 open reading frames, including 178 intact genes and 47 gene fragments. Between VACV-DUKE and the other Dryvax isolates, the major genomic differences are in fragmentation of the ankyrin-like, and kelch-like genes, presence of a full-length Interferon-alpha/beta receptor gene, and the absence of a duplication of 12 ORFs in the inverted terminal repeat. Excluding this region, the DNA sequence of VACV-DUKE differs from the other two Dryvax isolates by less than 0.4%. DNA sequencing also indicated that there was little heterogeneity in the sample, supporting the hypothesis that virus from an individual lesion is clonal in origin despite the fact that the vaccine is a mixed population. CONCLUSION: Virus in lesions that result from progressive vaccinia following vaccination with Dryvax are likely clonal in origin. The genomic sequence of VACV-DUKE is overall very similar to that of Dryvax cell culture-derived clonal isolates. Furthermore, with the sequences of multiple clones from Dryvax we can begin to appreciate the diversity of the viral population in the smallpox vaccine.
Asunto(s)
Genoma Viral , Vacuna contra Viruela/efectos adversos , Virus Vaccinia/genética , Vaccinia/virología , Femenino , Genes Virales , Humanos , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Filogenia , Análisis de Secuencia de ADN , Virus Vaccinia/clasificación , Virus Vaccinia/aislamiento & purificaciónRESUMEN
Cytokines are pivotal to a balanced innate or cell-mediated immune response, can be indicative of disease progression and/or resolution, and are being evaluated as therapeutics. There is a need to purify and/or to measure key cytokines rapidly with accuracy, precision, and sensitivity. The current assay technologies, which are based on RT-PCR, immunoassays, or bioassays, are limited in their use in the clinic, in particular because of the long time (1-3 h) required to carry out the assays. An alternative approach explored here is the use of pathogen-encoded cytokine-binding proteins, which have Kd in the nanomolar range. It is anticipated that pathogens have evolved binding proteins, antagonists, and/or specific neutralizing phenotypes directed against key signaling and effector molecules involved in the multifaceted host defense system. Thus, by screening the genomes of a wide range of microbial agents, we would expect to find coding sequences for binding proteins for the most important cytokines. Consistent with this view is the identification of poxvirus genes encoding binding activities for TNF type I and type II interferons, interleukin (IL)-1beta, IL-18, and beta-chemokines. These high-affinity receptors have the potential to act as surrogate antibodies in a number of applications in cytokine quantification and purification and could be potentially useful reagents to complement the existing panel of anti-cytokine, monoclonal, polyclonal, or engineered antibodies that are currently available.
Asunto(s)
Anticuerpos/metabolismo , Citocinas/metabolismo , Inmunoensayo/métodos , Proteínas/metabolismo , Virus/metabolismo , Citocinas/genéticaRESUMEN
Concern regarding the use of variola and monkeypox viruses as bioterrorist agents has led to an increased study of orthopoxviruses to understand the molecular and cellular basis of pathogenesis and develop safe and effective antivirals and vaccines against smallpox. Crucial to these efforts is the availability of animal models, which are inexpensive, genetically homogeneous, and recapitulate the human disease. The popular small-animal orthopoxvirus models employ the inbred mouse as the host, the respiratory tract as the site of virus inoculation, and orthopoxviruses-vaccinia, cowpox, and ectromelia viruses-as surrogates for variola virus. Ectromelia virus is likely the best surrogate for variola virus in a mouse model, as it is infectious at very low doses of virus, and the mousepox disease is associated with high mortality in the susceptible A, BALB/c, and DBA/2 stains of mice, but causes an unapparent infection in the C57BL/6 mouse strain. This chapter describes an ectromelia virus respiratory infection model in the mouse.
Asunto(s)
Modelos Animales de Enfermedad , Orthopoxvirus/inmunología , Infecciones por Poxviridae/inmunología , Infecciones del Sistema Respiratorio/virología , Animales , Orthopoxvirus/genética , Orthopoxvirus/fisiología , Infecciones por Poxviridae/prevención & control , Infecciones por Poxviridae/transmisión , Infecciones del Sistema Respiratorio/inmunologíaRESUMEN
There are no drugs approved specifically to treat disseminated adenovirus (Ad) infections in humans. Cidofovir is active against Ad in cell culture, and it is used frequently in the clinic with disseminated infection in pediatric transplant patients; however, controlled clinical studies have not been conducted to prove the anti-Ad efficacy of cidofovir. Brincidofovir, a lipid-linked derivative of cidofovir, which has strong activity against Ad in cell culture and in animal models, is a promising new drug currently in clinical trials. Ribavirin, which has modest activity against some Ad types in cell culture, has been used in the clinic against disseminated Ad, but the efficacy of ribavirin is unknown. In the current study, we have examined the activity of cidofovir, brincidofovir, and ribavirin against disseminated Ad5 infection in the immunosuppressed Syrian hamster model. Hamsters are immunosuppressed by treatment with cyclophosphamide, then infected intravenously with Ad5, leading to disseminated Ad5 infection, especially in the liver. We found that cidofovir and brincidofovir have excellent activity against Ad5 pathology and replication in the liver, even when administered therapeutically starting at 3 days post-challenge with Ad5. Ribavirin did not have anti-Ad5 activity in our model. Our data support the use of cidofovir and brincidofovir in humans for the treatment of disseminated Ad infections in humans.
Asunto(s)
Infecciones por Adenoviridae/tratamiento farmacológico , Infecciones por Adenoviridae/patología , Adenovirus Humanos/crecimiento & desarrollo , Citosina/análogos & derivados , Organofosfonatos/uso terapéutico , Ribavirina/uso terapéutico , Infecciones por Adenoviridae/virología , Alanina Transaminasa/sangre , Animales , Peso Corporal , Línea Celular , Cidofovir , Citosina/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Humanos , Huésped Inmunocomprometido , Hígado/patología , Hígado/virología , Mesocricetus , Análisis de Supervivencia , Resultado del Tratamiento , Carga ViralRESUMEN
Autoimmune destruction of insulin producing pancreatic ß-cells is the hallmark of type I diabetes. One of the key molecules implicated in the disease onset is the immunoproteasome, a protease with multiple proteolytic sites that collaborates with the constitutive 19S and the inducible 11S (PA28) activators to produce immunogenic peptides for presentation by MHC class I molecules. Despite its importance, little is known about the function and regulation of the immunoproteasome in pancreatic ß-cells. Of special interest to immunoproteasome activation in ß-cells are the effects of IFNß, a type I IFN secreted by virus-infected cells and implicated in type I diabetes onset, compared to IFNγ, the classic immunoproteasome inducer secreted by cells of the immune system. By qPCR analysis, we show that mouse insulinoma MIN6 cells and mouse islets accumulate the immune proteolytic ß1(i), ß2(i) and ß5(i), and 11S mRNAs upon exposure to IFNß or IFNγ. Higher concentrations of IFNß than IFNγ are needed for similar expression, but in each case the expression is transient, with maximal mRNA accumulation in 12 hours, and depends primarily on Interferon Regulatory Factor 1. IFNs do not alter expression of regular proteasome genes, and in the time frame of IFNß-mediated response, the immune and regular proteolytic subunits co-exist in the 20S particles. In cell extracts with ATP, these particles have normal peptidase activities and degrade polyubiquitinated proteins with rates typical of the regular proteasome, implicating normal regulation by the 19S activator. However, ATP depletion rapidly stimulates the catalytic rates in a manner consistent with levels of the 11S activator. These findings suggest that stochastic combination of regular and immune proteolytic subunits may increase the probability with which unique immunogenic peptides are produced in pancreatic ß-cells exposed to IFNß, but primarily in cells with reduced ATP levels that stimulate the 11S participation in immunoproteasome function.
Asunto(s)
Adenosina Trifosfato/metabolismo , Células Secretoras de Insulina/inmunología , Interferón beta/metabolismo , Proteínas Musculares/metabolismo , Complejo de la Endopetidasa Proteasomal/inmunología , Animales , Western Blotting , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Cartilla de ADN/genética , Inmunoprecipitación , Células Secretoras de Insulina/virología , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Octoxinol , Reacción en Cadena de la Polimerasa , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Type 1 diabetes results from autoimmune destruction of the insulin producing pancreatic ß-cells. The immunoproteasome, a version of the proteasome that collaborates with the 11S/PA28 activator to generate immunogenic peptides for presentation by MHC class I molecules, has long been implicated in the onset of the disease, but little is known about immunoproteasome function and regulation in pancreatic ß-cells. Interesting insight into these issues comes from a recent analysis of the immunoproteasome expressed in pancreatic ß-cells during early antiviral defenses mediated by interferon ß (IFNß), a type I IFN implicated in the induction of the diabetic state in human and animal models. Using mouse islets and the MIN6 insulinoma cell line, Freudenburg et al. found that IFNß stimulates expression of the immunoproteasome and the 11S/PA28 activator in a manner fundamentally similar to the classic immuno-inducer IFNγ, with similar timing of mRNA accumulation and decline; similar transcriptional activation mediated primarily by the IRF1 and similar mRNA and protein levels. Furthermore, neither IFNß nor IFNγ altered the expression of regular proteolytic subunits or prevented their incorporation into proteolytic cores. As a result, immunoproteasomes had stochastic combinations of immune and regular proteolytic sites, an arrangement that would likely increase the probability with which unique immunogenic peptides are produced. However, immunoproteasomes were activated by the 11S/PA28 only under conditions of ATP depletion. A mechanism that prevents the activation of immunoproteasome at high ATP levels has not been reported before and could have a major regulatory significance, as it could suppress the generation of immunogenic peptides as cell accumulate immunoproteasome and 11S/PA28, and activate antigen processing only when ATP levels drop. We discuss implications of these new findings on the link between early antiviral response and the onset of type 1 diabetes.
RESUMEN
In 2001, Jackson et al. reported that murine IL-4 expression by a recombinant ectromelia virus caused enhanced morbidity and lethality in resistant C57BL/6 mice as well as overcame protective immune memory responses. To achieve a more thorough understanding of this phenomenon and to assess a variety of countermeasures, we constructed a series of ECTV recombinants encoding murine IL-4 under the control of promoters of different strengths and temporal regulation. We showed that the ECTV-IL-4 recombinant expressing the highest level of IL-4 was uniformly lethal for C57BL/6 mice even when previously immunized. The lethality of the ECTV-IL-4 recombinants resulted from virus-expressed IL-4 signaling through the IL-4 receptor but was not due to IL-4 toxicity. A number of treatment approaches were evaluated against the most virulent IL-4 encoding virus. The most efficacious therapy was a combination of two antiviral drugs (CMX001(®) and ST-246(®)) that have different mechanisms of action.