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The filoviruses Marburg virus (MARV) and Ebola virus (EBOV) cause hemorrhagic fever in humans and nonhuman primates, with high case fatality rates. MARV VP30 is known to be phosphorylated and to interact with nucleoprotein (NP), but its role in regulation of viral transcription is disputed. Here, we analyzed phosphorylation of VP30 by mass spectrometry, which resulted in identification of multiple phosphorylated amino acids. Modeling the full-length three-dimensional structure of VP30 and mapping the identified phosphorylation sites showed that all sites lie in disordered regions, mostly in the N-terminal domain of the protein. Minigenome analysis of the identified phosphorylation sites demonstrated that phosphorylation of a cluster of amino acids at positions 46 through 53 inhibits transcription. To test the effect of VP30 phosphorylation on its interaction with other MARV proteins, coimmunoprecipitation analyses were performed. They demonstrated the involvement of VP30 phosphorylation in interaction with two other proteins of the MARV ribonucleoprotein complex, NP and VP35. To identify the role of protein phosphatase 1 (PP1) in the identified effects, a small molecule, 1E7-03, targeting a noncatalytic site of the enzyme that previously was shown to increase EBOV VP30 phosphorylation was used. Treatment of cells with 1E7-03 increased phosphorylation of VP30 at a cluster of phosphorylated amino acids from Ser-46 to Thr-53, reduced transcription of MARV minigenome, enhanced binding to NP and VP35, and dramatically reduced replication of infectious MARV particles. Thus, MARV VP30 phosphorylation can be targeted for development of future antivirals such as PP1-targeting compounds. IMPORTANCE The largest outbreak of MARV occurred in Angola in 2004 to 2005 and had a 90% case fatality rate. There are no approved treatments available for MARV. Development of antivirals as therapeutics requires a fundamental understanding of the viral life cycle. Because of the close similarity of MARV to another member of Filoviridae family, EBOV, it was assumed that the two viruses have similar mechanisms of regulation of transcription and replication. Here, characterization of the role of VP30 and its phosphorylation sites in transcription of the MARV genome demonstrated differences from those of EBOV. The identified phosphorylation sites appeared to inhibit transcription and appeared to be involved in interaction with both NP and VP35 ribonucleoproteins. A small molecule targeting PP1 inhibited transcription of the MARV genome, effectively suppressing replication of the viral particles. These data demonstrate the possibility developing antivirals based on compounds targeting PP1.
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Marburgvirus/crecimiento & desarrollo , Nucleoproteínas/metabolismo , Proteínas Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Replicación Viral/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Genoma Viral/genética , Células HEK293 , Humanos , Indoles/farmacología , Marburgvirus/genética , Espectrometría de Masas , Fosforilación , ARN Viral/genética , Transcripción Genética/genética , Urea/análogos & derivados , Urea/farmacología , Células Vero , Proteínas Virales/genéticaRESUMEN
The filovirus Ebola (EBOV) causes the most severe hemorrhagic fever known. The EBOV RNA-dependent polymerase complex includes a filovirus-specific VP30, which is critical for the transcriptional but not replication activity of EBOV polymerase; to support transcription, VP30 must be in a dephosphorylated form. Here we show that EBOV VP30 is phosphorylated not only at the N-terminal serine clusters identified previously but also at the threonine residues at positions 143 and 146. We also show that host cell protein phosphatase 1 (PP1) controls VP30 dephosphorylation because expression of a PP1-binding peptide cdNIPP1 increased VP30 phosphorylation. Moreover, targeting PP1 mRNA by shRNA resulted in the overexpression of SIPP1, a cytoplasm-shuttling regulatory subunit of PP1, and increased EBOV transcription, suggesting that cytoplasmic accumulation of PP1 induces EBOV transcription. Furthermore, we developed a small molecule compound, 1E7-03, that targeted a non-catalytic site of PP1 and increased VP30 dephosphorylation. The compound inhibited the transcription but increased replication of the viral genome and completely suppressed replication of EBOV in cultured cells. Finally, mutations of Thr(143) and Thr(146) of VP30 significantly inhibited EBOV transcription and strongly induced VP30 phosphorylation in the N-terminal Ser residues 29-46, suggesting a novel mechanism of regulation of VP30 phosphorylation. Our findings suggest that targeting PP1 with small molecules is a feasible approach to achieve dysregulation of the EBOV polymerase activity. This novel approach may be used for the development of antivirals against EBOV and other filovirus species.
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Ebolavirus/fisiología , Proteína Fosfatasa 1/metabolismo , ARN Viral/biosíntesis , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Proteínas Virales/metabolismo , Replicación Viral/fisiología , Animales , Antivirales/farmacología , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Humanos , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteína Fosfatasa 1/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Viral/genética , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacos , Células Vero , Proteínas Virales/genética , Replicación Viral/efectos de los fármacosRESUMEN
Machupo virus (MACV) is the etiological agent of Bolivian hemorrhagic fever (BHF), a reemerging and neglected tropical disease associated with high mortality. The prototypical strain of MACV, Carvallo, was isolated from a human patient in 1963, but minimal in vitro and in vivo characterization has been reported. To this end, we utilized reverse genetics to rescue a pathogenic MACV from cloned cDNAs. The recombinant MACV (rMACV) had in vitro growth properties similar to those of the parental MACV. Both viruses caused similar disease development in alpha/beta and gamma interferon receptor knockout mice, including neurological disease development and high mortality. In addition, we have identified a novel murine model with mortality and neurological disease similar to BHF disease reported in humans and nonhuman primates.
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Arenavirus del Nuevo Mundo/genética , ADN Complementario/genética , Modelos Animales de Enfermedad , Fiebre Hemorrágica Americana/genética , Análisis de Varianza , Animales , Secuencia de Bases , Línea Celular , Chlorocebus aethiops , Cricetinae , Cartilla de ADN/genética , Técnicas Histológicas , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Plásmidos/genética , Receptor de Interferón alfa y beta/deficiencia , Receptor de Interferón alfa y beta/genética , Receptores de Interferón/deficiencia , Receptores de Interferón/genética , Genética Inversa/métodos , Análisis de Secuencia de ARN , Células Vero , Receptor de Interferón gammaRESUMEN
Ebola virus (EBOV) infections are characterized by deficient T lymphocyte responses, T lymphocyte apoptosis, and lymphopenia in the absence of direct infection of T lymphocytes. In contrast, dendritic cells (DC) are infected but fail to mature appropriately, thereby impairing the T cell response. We investigated the contributions of EBOV proteins in modulating DC maturation by generating recombinant viruses expressing enhanced green fluorescent protein and carrying mutations affecting several potentially immunomodulating domains. They included envelope glycoprotein (GP) domains, as well as innate response antagonist domains (IRADs) previously identified in the VP24 and VP35 proteins. GP expressed by an unrelated vector, but not the wild-type EBOV, was found to strongly induce DC maturation, and infections with recombinant EBOV carrying mutations disabling GP functional domains did not restore DC maturation. In contrast, each of the viruses carrying mutations disabling any IRAD in VP35 induced a dramatic upregulation of DC maturation markers. This was dependent on infection, but not interaction with GP. Disabling of IRADs also resulted in up to a several hundredfold increase in secretion of cytokines and chemokines. Furthermore, these mutations induced formation of homotypic DC clusters, which represent close correlates of their maturation and presumably facilitate transfer of antigen from migratory DC to lymph node DC. Thus, an individual IRAD is insufficient to suppress DC maturation; rather, the suppression of DC maturation and the "immune paralysis" observed during EBOV infections results from a cooperative effect of two or more individual IRADs.
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Diferenciación Celular/inmunología , Células Dendríticas/virología , Ebolavirus/genética , Proteínas del Envoltorio Viral/inmunología , Animales , Biomarcadores/metabolismo , Western Blotting , Chlorocebus aethiops , Citocinas/metabolismo , Células Dendríticas/citología , Ebolavirus/metabolismo , Citometría de Flujo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Mutación/genética , Células Vero , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
INTRODUCTION: The purpose of this review is to examine African Ebola outbreaks from their first discovery to the present, to determine how the medical and public health response has changed and identify the causes for those changes. We sought to describe what is now known about the epidemiology and spread of Ebola virus disease (EVD) from the significant outbreaks that have occurred and outbreak control methods applied under often challenging circumstances. Given the substantial role that the U.S. Government and the U.S. DoD have played in the 2014 to 2016 West African Ebola outbreak, the role of the DoD and the U.S. Africa Command in controlling EVD is described. MATERIALS AND METHODS: A descriptive method design was used to collect and analyze all available Ebola outbreak literature using the PubMed database. An initial literature search was conducted by searching for, obtaining, and reading original source articles on all major global Ebola outbreaks. To conduct a focused search, we used initial search terms "Ebola outbreak," "Ebola virus disease," "Ebola response," "Ebola countermeasures," and also included each country's name where Ebola cases are known to have occurred. From the 4,673 unique articles obtained from this search and subsequent article title review, 307 articles were identified for potential inclusion. Following abstract and article review, 45 original source articles were used to compile the history of significant Ebola outbreaks. From this compilation, articles focused on each respective subsection of this review to delineate and describe the history of EVD and response, identifying fundamental changes, were obtained and incorporated. RESULTS: We present known Ebola virus and disease attributes, including a general description, seasonality and location, transmission capacity, clinical symptoms, surveillance, virology, historical EVD outbreaks and response, international support for Ebola outbreak response, U.S. DoD support, medical countermeasures supporting outbreak response, remaining gaps to include policy limitations, regional instability, climate change, migration, and urbanization, public health education and infrastructure, and virus persistence and public awareness. CONCLUSIONS: The health and societal impacts of EVD on Africa has been far-reaching, with about 35,000 cases and over 15,000 deaths, with small numbers of cases spreading globally. However, the history of combatting EVD reveals that there is considerable hope for African nations to quickly and successfully respond to Ebola outbreaks, through use of endemic resources including Africa CDC and African Partner Outbreak Response Alliance and the U.S. Africa Command with greater DoD reachback. Although there remains much to be learned about the Ebola virus and EVD including whether the potential for novel strains to become deadly emerging infections, invaluable vaccines, antivirals, and public health measures are now part of the resources that can be used to combat this disease.
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Brotes de Enfermedades , Fiebre Hemorrágica Ebola , Humanos , Brotes de Enfermedades/prevención & control , Ebolavirus , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/prevención & control , Salud Pública/métodos , Salud Pública/tendencias , Estados UnidosRESUMEN
Rift Valley fever is a mosquito-borne zoonotic disease endemic to sub-Saharan Africa. Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) causes high rates of abortion and fetal malformation in pregnant ruminants, and haemorrhagic fever, neurological disorders or blindness in humans. The MP-12 strain is a highly efficacious and safe live-attenuated vaccine candidate for both humans and ruminants. However, MP-12 lacks a marker to differentiate infected from vaccinated animals. In this study, we originally aimed to characterize the efficacy of a recombinant RVFV MP-12 strain encoding Toscana virus (TOSV) NSs gene in place of MP-12 NSs (rMP12-TOSNSs). TOSV NSs promotes the degradation of dsRNA-dependent protein kinase (PKR) and inhibits interferon-ß gene up-regulation without suppressing host general transcription. Unexpectedly, rMP12-TOSNSs increased death in vaccinated outbred mice and inbred BALB/c or C57BL/6 mice. Immunohistochemistry showed diffusely positive viral antigens in the thalamus, hypothalamus and brainstem, including the medulla. No viral antigens were detected in spleen or liver, which is similar to the antigen distribution of moribund mice infected with MP-12. These results suggest that rMP12-TOSNSs retains neuroinvasiveness in mice. Our findings demonstrate that rMP12-TOSNSs causes neuroinvasion without any hepatic disease and will be useful for studying the neuroinvasion mechanism of RVFV and TOSV.
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Encéfalo/virología , Enfermedades del Sistema Nervioso/virología , Fiebre del Valle del Rift/prevención & control , Virus de Nápoles de la Fiebre de la Mosca de los Arenales/genética , Virus de Nápoles de la Fiebre de la Mosca de los Arenales/patogenicidad , Vacunas Atenuadas/efectos adversos , Proteínas no Estructurales Virales/metabolismo , Vacunas Virales/efectos adversos , Animales , Línea Celular , Chlorocebus aethiops , Femenino , Humanos , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fiebre del Valle del Rift/inmunología , Virus de la Fiebre del Valle del Rift/inmunología , Virus de Nápoles de la Fiebre de la Mosca de los Arenales/inmunología , Vacunación , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Células Vero , Proteínas no Estructurales Virales/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunologíaRESUMEN
The Ebola virus (EBOV) transcriptional regulation involves host protein phosphatases PP1 and PP2A, which dephosphorylate the transcriptional cofactor of EBOV polymerase VP30. The 1E7-03 compound, which targets PP1, induces VP30 phosphorylation and inhibits EBOV infection. This study aimed to investigate the role of PP1 in EBOV replication. When EBOV-infected cells were continuously treated with 1E7-03, the NP E619K mutation was selected. This mutation moderately reduced EBOV minigenome transcription, which was restored by the treatment with 1E7-03. Formation of EBOV capsids, when NP was co-expressed with VP24 and VP35, was impaired with NPE 619K. Treatment with 1E7-03 restored capsid formation by NP E619K mutation, but inhibited capsids formed by WT NP. The dimerization of NP E619K, tested in a split NanoBiT assay, was significantly decreased (~ 15-fold) compared to WT NP. NP E619K bound more efficiently to PP1 (~ 3-fold) but not B56 subunit of PP2A or VP30. Cross-linking and co-immunoprecipitation experiments showed fewer monomers and dimers for NP E619K which were increased with 1E7-03 treatment. NP E619K showed increased co-localization with PP1α compared to WT NP. Mutations of potential PP1 binding sites and NP deletions disrupted its interaction with PP1. Collectively, our findings suggest that PP1 binding to the NP regulates NP dimerization and capsid formation, and that NP E619K mutation, which has the enhanced PP1 binding, disrupts these processes. Our results point to a new role for PP1 in EBOV replication in which NP binding to PP1 may facilitate viral transcription by delaying capsid formation and EBOV replication.
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Members of the Ebolavirus genus demonstrate a marked differences in pathogenicity in humans with Ebola (EBOV) being the most pathogenic, Bundibugyo (BDBV) less pathogenic, and Reston (RESTV) is not known to cause a disease in humans. The VP24 protein encoded by members of the Ebolavirus genus blocks type I interferon (IFN-I) signaling through interaction with host karyopherin alpha nuclear transporters, potentially contributing to virulence. Previously, we demonstrated that BDBV VP24 (bVP24) binds with lower affinities to karyopherin alpha proteins relative to EBOV VP24 (eVP24), and this correlated with a reduced inhibition in IFN-I signaling. We hypothesized that modification of eVP24-karyopherin alpha interface to make it similar to bVP24 would attenuate the ability to antagonize IFN-I response. We generated a panel of recombinant EBOVs containing single or combinations of point mutations in the eVP24-karyopherin alpha interface. Most of the viruses appeared to be attenuated in both IFN-I-competent 769-P and IFN-I-deficient Vero-E6 cells in the presence of IFNs. However, the R140A mutant grew at reduced levels even in the absence of IFNs in both cell lines, as well as in U3A STAT1 knockout cells. Both the R140A mutation and its combination with the N135A mutation greatly reduced the amounts of viral genomic RNA and mRNA suggesting that these mutations attenuate the virus in an IFN-I-independent attenuation. Additionally, we found that unlike eVP24, bVP24 does not inhibit interferon lambda 1 (IFN-λ1), interferon beta (IFN-ß), and ISG15, which potentially explains the lower pathogenicity of BDBV relative to EBOV. Thus, the VP24 residues binding karyopherin alpha attenuates the virus by IFN-I-dependent and independent mechanisms.
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Ebolavirus , Fiebre Hemorrágica Ebola , Humanos , Interferones/metabolismo , Ebolavirus/fisiología , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , Proteínas Virales/metabolismo , Interferón beta/genética , Interferón beta/metabolismoRESUMEN
INTRODUCTION: Cholera remains a significant public health threat for many countries, and the severity largely varies by the population and local conditions that drive disease spread, especially in endemic areas prone to natural disasters and flooding. Epidemiological models can provide useful information to military planners for understanding disease spread within populations and the effectiveness of response options for preventing the transmission among deployed and stationed personnel. This study demonstrates the use of epidemiological modeling to understand the dynamics of cholera transmission to inform emergency planning and military preparedness in areas with highly communicable diseases. MATERIALS AND METHODS: Areas with higher probability for a potential cholera outbreak in Haiti followed by a natural disaster were identified. The hotspots were then used to seed an extended compartmental model, EpiGrid, to simulate notional spread scenarios of cholera originating in three distinct areas in Haiti. Disease parameters were derived from the 2010 cholera outbreak in Haiti, and disease spread was simulated over a 12-week period under uncontrolled and controlled spread. RESULTS: For each model location, scenarios of mitigated (intervention with 30% transmission reduction via international aid) and unmitigated (without intervention) are simulated. The results depict the geographical spread and estimate the cumulative cholera infection for each notional scenario over the course of 3 months. Disease transmission differs considerably across origin site with an outbreak originating in the department of Nippes spanning the largest geographic area and resulting in the largest number of cumulative cases after 12 weeks under unmitigated (79,518 cases) and mitigated (35,667 cases) spread scenarios. CONCLUSIONS: We modeled the notional re-emergence and spread of cholera following the August 2021 earthquake in Haiti while in the midst of the global COVID-19 pandemic. This information can help guide military and emergency response decision-making during an infectious disease outbreak and considerations for protecting military personnel in the midst of a humanitarian response. Military planners should consider the use of epidemiological models to assess the health risk posed to deployed and stationed personnel in high-risk areas.
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INTRODUCTION: Throughout the Coronavirus Disease 2019 (COVID-19) pandemic, military commanders have been challenged with providing appropriate travel guidance for their military and civilian personnel and dependents. This guidance, where promulgated, lacks uniformity. Travel aids and computer applications similarly differ and are not updated as often as jurisdictional travel health guidance is changed. Given the ever-evolving Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants with differing degrees of infectivity, COVID-19 travel guidance will remain relevant for military travelers during the transition from pandemic to endemic phases and for the foreseeable future. MATERIALS AND METHODS: We reviewed all germane travel guidance promulgated by the U.S, Department of Defense; the U.S. Centers for Disease Control and Prevention; and other federal, state, and international agencies. From these materials, we identified and delineated applicable universal components for COVID-19 travel risk and created a universal Travel Risk Assessment Questionnaire (TRAQ). RESULTS: We present a universal TRAQ that identifies and allows for a graded most-appropriate response to known travel risk assessment factors including travel restrictions, travel mode, travel time, travel party size, trip duration, COVID-19 incidence rate at travel destination, lodging, planned activities, personal interaction level, vaccination coverage at destination, travel location, traveler's vaccination status, previous COVID-19 infection, mask wear compliance, mask type, and work environment, along with additional considerations and post-travel COVID-19 questions. We provide examples of the use of this questionnaire that describe low, medium, and high risk to the traveler for contracting COVID-19. CONCLUSION: Our TRAQ provides an easy-to-use format that can enable military, business, or personal travelers to more completely assess their likelihood of COVID-19 exposure and help them to reduce their potential for contracting COVID-19 during travel and subsequently transmitting it to others upon return. It should help commanders and traveling personnel to better assess COVID-19 travel risks through application of known travel risk factors.
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The objective of this study was to evaluate the feasibility of using clinical parameters to demonstrate disease progression and differentiate between Omsk hemorrhagic fever virus (OHFV) and Russian spring-summer encephalitis virus (RSSEV) infection in the mouse model. Adult C57BL/6 and balb/c mice were infected with either OHFV or RSSEV by footpad inoculation and their temperature, body weight, clinical signs complete blood count, and blood chemistries were evaluated for up to 15 days post-infection (dpi). Clinical evaluation showed that OHFV infection seriously affects balb/c mice, which had shorter average survival times (ASTs) than other groups. On the contrary, RSSEV infection of C57BL/6 mice was more severe than in balb/c mice. During these studies, the development of fever was not observed and the body weight of OHFV infected balb/c and C57BL/6 mice began to decline sharply starting from day 7 and 8, respectively, which correlated with disease onset. Peak increase of globulin and neutrophils was demonstrated after 9 dpi in OHFV infected mice; however, the lymphocyte number was not affected. Viremia was undetectable in these animals with either virus infection, but virus was found in most organs tested. These results indicate marked differences in the clinical signs, pathology, and immune response of mice infected with either OHFV or RSSEV and further validate the use of this mouse model system to evaluate human disease.
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Infecciones por Flavivirus/patología , Infecciones por Flavivirus/fisiopatología , Flavivirus/patogenicidad , Estructuras Animales/virología , Animales , Recuento de Células Sanguíneas , Temperatura Corporal , Peso Corporal , Diagnóstico Diferencial , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Índice de Severidad de la Enfermedad , Análisis de Supervivencia , ViremiaRESUMEN
Ebola virus (EBOV) protein VP35 inhibits production of interferon alpha/beta (IFN) by blocking RIG-I-like receptor signaling pathways, thereby promoting virus replication and pathogenesis. A high-throughput screening assay, developed to identify compounds that either inhibit or bypass VP35 IFN-antagonist function, identified five DNA intercalators as reproducible hits from a library of bioactive compounds. Four, including doxorubicin and daunorubicin, are anthracycline antibiotics that inhibit topoisomerase II and are used clinically as chemotherapeutic drugs. These compounds were demonstrated to induce IFN responses in an ATM kinase-dependent manner and to also trigger the DNA-sensing cGAS-STING pathway of IFN induction. These compounds also suppress EBOV replication in vitro and induce IFN in the presence of IFN-antagonist proteins from multiple negative-sense RNA viruses. These findings provide new insights into signaling pathways activated by important chemotherapy drugs and identify a novel therapeutic approach for IFN induction that may be exploited to inhibit RNA virus replication.IMPORTANCE Ebola virus and other emerging RNA viruses are significant but unpredictable public health threats. Therapeutic approaches with broad-spectrum activity could provide an attractive response to such infections. We describe a novel assay that can identify small molecules that overcome Ebola virus-encoded innate immune evasion mechanisms. This assay identified as hits cancer chemotherapeutic drugs, including doxorubicin. Follow-up studies provide new insight into how doxorubicin induces interferon (IFN) responses, revealing activation of both the DNA damage response kinase ATM and the DNA sensor cGAS and its partner signaling protein STING. The studies further demonstrate that the ATM and cGAS-STING pathways of IFN induction are a point of vulnerability not only for Ebola virus but for other RNA viruses as well, because viral innate immune antagonists consistently fail to block these signals. These studies thereby define a novel avenue for therapeutic intervention against emerging RNA viruses.
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Antivirales/farmacología , Daño del ADN/inmunología , Ebolavirus/fisiología , Evasión Inmune/efectos de los fármacos , Interferones/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Replicación Viral/efectos de los fármacos , Línea Celular , Ebolavirus/inmunología , HumanosRESUMEN
High-throughput screening (HTS) has been integrated into the drug discovery process, and multiple assay formats have been widely used in many different disease areas but with limited focus on infectious agents. In recent years, there has been an increase in the number of HTS campaigns using infectious wild-type pathogens rather than surrogates or biochemical pathogen-derived targets. Concurrently, enhanced emerging pathogen surveillance and increased human mobility have resulted in an increase in the emergence and dissemination of infectious human pathogens with serious public health, economic, and social implications at global levels. Adapting the HTS drug discovery process to biocontainment laboratories to develop new drugs for these previously uncharacterized and highly pathogenic agents is now feasible, but HTS at higher biosafety levels (BSL) presents a number of unique challenges. HTS has been conducted with multiple bacterial and viral pathogens at both BSL-2 and BSL-3, and pilot screens have recently been extended to BSL-4 environments for both Nipah and Ebola viruses. These recent successful efforts demonstrate that HTS can be safely conducted at the highest levels of biological containment. This review outlines the specific issues that must be considered in the execution of an HTS drug discovery program for high-containment pathogens. We present an overview of the requirements for HTS in high-level biocontainment laboratories.
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Bioensayo/instrumentación , Contención de Riesgos Biológicos/instrumentación , Evaluación Preclínica de Medicamentos/instrumentación , Ensayos Analíticos de Alto Rendimiento/instrumentación , Laboratorios , Tecnología Farmacéutica/instrumentación , Diseño de Fármacos , Diseño de Equipo , Análisis de Falla de Equipo , Robótica/instrumentación , Manejo de Especímenes/instrumentaciónRESUMEN
Nipah virus is a biosafety level 4 (BSL-4) pathogen that causes severe respiratory illness and encephalitis in humans. To identify novel small molecules that target Nipah virus replication as potential therapeutics, Southern Research Institute and Galveston National Laboratory jointly developed an automated high-throughput screening platform that is capable of testing 10,000 compounds per day within BSL-4 biocontainment. Using this platform, we screened a 10,080-compound library using a cell-based, high-throughput screen for compounds that inhibited the virus-induced cytopathic effect. From this pilot effort, 23 compounds were identified with EC50 values ranging from 3.9 to 20.0 µM and selectivities >10. Three sulfonamide compounds with EC50 values <12 µM were further characterized for their point of intervention in the viral replication cycle and for broad antiviral efficacy. Development of HTS capability under BSL-4 containment changes the paradigm for drug discovery for highly pathogenic agents because this platform can be readily modified to identify prophylactic and postexposure therapeutic candidates against other BSL-4 pathogens, particularly Ebola, Marburg, and Lassa viruses.
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Antivirales/administración & dosificación , Antivirales/química , Evaluación Preclínica de Medicamentos/instrumentación , Ensayos Analíticos de Alto Rendimiento/instrumentación , Virus Nipah/efectos de los fármacos , Virus Nipah/fisiología , Sulfonamidas/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Animales , Chlorocebus aethiops , Contención de Riesgos Biológicos/instrumentación , Relación Dosis-Respuesta a Droga , Diseño de Equipo , Análisis de Falla de Equipo , Robótica/instrumentación , Células Vero , Replicación Viral/fisiologíaRESUMEN
BACKGROUND: Junín virus (JUNV), the etiologic agent of Argentine hemorrhagic fever (AHF), is classified by the NIAID and CDC as a Category A priority pathogen. Presently, antiviral therapy for AHF is limited to immune plasma, which is readily available only in the endemic regions of Argentina. T-705 (favipiravir) is a broadly active small molecule RNA-dependent RNA polymerase inhibitor presently in clinical evaluation for the treatment of influenza. We have previously reported on the in vitro activity of favipiravir against several strains of JUNV and other pathogenic New World arenaviruses. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the efficacy of favipiravir in vivo, guinea pigs were challenged with the pathogenic Romero strain of JUNV, and then treated twice daily for two weeks with oral or intraperitoneal (i.p.) favipiravir (300 mg/kg/day) starting 1-2 days post-infection. Although only 20% of animals treated orally with favipiravir survived the lethal challenge dose, those that succumbed survived considerably longer than guinea pigs treated with placebo. Consistent with pharmacokinetic analysis that showed greater plasma levels of favipiravir in animals dosed by i.p. injection, i.p. treatment resulted in a substantially higher level of protection (78% survival). Survival in guinea pigs treated with ribavirin was in the range of 33-40%. Favipiravir treatment resulted in undetectable levels of serum and tissue viral titers and prevented the prominent thrombocytopenia and leucopenia observed in placebo-treated animals during the acute phase of infection. CONCLUSIONS/SIGNIFICANCE: The remarkable protection afforded by i.p. favipiravir intervention beginning 2 days after challenge is the highest ever reported for a small molecule antiviral in the difficult to treat guinea pig JUNV challenge model. These findings support the continued development of favipiravir as a promising antiviral against JUNV and other related arenaviruses.
Asunto(s)
Amidas/uso terapéutico , Antivirales/uso terapéutico , Fiebre Hemorrágica Americana/tratamiento farmacológico , Virus Junin/efectos de los fármacos , Pirazinas/uso terapéutico , Administración Oral , Amidas/farmacocinética , Animales , Antivirales/farmacocinética , Modelos Animales de Enfermedad , Cobayas , Fiebre Hemorrágica Americana/virología , Inyecciones Intraperitoneales , Masculino , Plasma/química , Pirazinas/farmacocinética , Análisis de Supervivencia , Viremia/prevención & controlRESUMEN
Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever, neurological disorders or blindness in humans, and a high rate of abortion in ruminants. MP-12 strain, a live-attenuated candidate vaccine, is attenuated in the M- and L-segments, but the S-segment retains the virulent phenotype. MP-12 was manufactured as an Investigational New Drug vaccine by using MRC-5 cells and encodes a functional NSs gene, the major virulence factor of RVFV which 1) induces a shutoff of the host transcription, 2) inhibits interferon (IFN)-ß promoter activation, and 3) promotes the degradation of dsRNA-dependent protein kinase (PKR). MP-12 lacks a marker for differentiation of infected from vaccinated animals (DIVA). Although MP-12 lacking NSs works for DIVA, it does not replicate efficiently in type-I IFN-competent MRC-5 cells, while the use of type-I IFN-incompetent cells may negatively affect its genetic stability. To generate modified MP-12 vaccine candidates encoding a DIVA marker, while still replicating efficiently in MRC-5 cells, we generated recombinant MP-12 encoding Punta Toro virus Adames strain NSs (rMP12-PTNSs) or Sandfly fever Sicilian virus NSs (rMP12-SFSNSs) in place of MP-12 NSs. We have demonstrated that those recombinant MP-12 viruses inhibit IFN-ß mRNA synthesis, yet do not promote the degradation of PKR. The rMP12-PTNSs, but not rMP12-SFSNSs, replicated more efficiently than recombinant MP-12 lacking NSs in MRC-5 cells. Mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs induced neutralizing antibodies at a level equivalent to those vaccinated with MP-12, and were efficiently protected from wild-type RVFV challenge. The rMP12-PTNSs and rMP12-SFSNSs did not induce antibodies cross-reactive to anti-RVFV NSs antibody and are therefore applicable to DIVA. Thus, rMP12-PTNSs is highly efficacious, replicates efficiently in MRC-5 cells, and encodes a DIVA marker, all of which are important for vaccine development for Rift Valley fever.
Asunto(s)
Phlebovirus/metabolismo , Virus de la Fiebre del Valle del Rift/metabolismo , Proteínas no Estructurales Virales/metabolismo , Línea Celular , Humanos , Phlebovirus/genética , Virus de la Fiebre del Valle del Rift/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
Omsk hemorrhagic fever virus (OHFV) and Russian spring-summer encephalitis virus (RSSEV) are tick-borne flaviviruses that have close homology but different pathology and disease outcomes. Previously, we reported that C57BL/6 and BALB/c mice were excellent models to study the pathology and clinical signs of human RSSEV and OHFV infection. In the study described here, we found that RSSEV infection induced robust release of proinflammatory cytokines (IL-1α, IL-1ß, IL-6 and TNF-α) and chemokines (MCP-1, MIP-1ß, RANTES and KC) in the brain at 9 and 11dpi, together with moderate to low Th1 and Th2 cytokines. In contrast, OHFV infection stimulated an early and prominent induction of IL-1α, TNF-α, IL-12p70, MCP-1, MIP-1α and MIP-1ß in the spleen of infected mice. Collectively our data suggest that a differential host response to infection may lead to the alternate disease outcomes seen following OHFV or RSSEV infection.