The Experts below are selected from a list of 1494 Experts worldwide ranked by ideXlab platform
Stefan Kunz - One of the best experts on this subject based on the ideXlab platform.
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Cleavage of the Glycoprotein of Arenaviruses
Activation of Viruses by Host Proteases, 2018Co-Authors: Antonella Pasquato, Laura Cendron, Stefan KunzAbstract:The Arenaviruses are a large family of emerging negative-stranded RNA viruses that include several severe human pathogens causing hemorrhagic fevers with high mortality. During the Arenavirus life cycle, processing of the viral envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is crucial for productive Infection. The ability of newly emerging Arenaviruses to hijack human SKI-1/S1P is a key factor for zoonotic transmission and human disease potential. Apart from being an essential host factor for Arenavirus Infection, SKI-1/S1P is involved in the regulation of important physiological processes and linked to major human diseases. This chapter provides an overview of the mechanisms of Arenavirus GPC processing by SKI-1/S1P including recent findings. We will highlight to what extent the molecular mechanisms of SKI-1/S1P cleavage of viral GPC differ from processing of SKI-1/S1P’s cellular substrates and discuss the implications for virus-host interaction and coevolution. Moreover, we will show how the use of the viral GPC as a “molecular probe” uncovered novel and unusual aspects of SKI-1/S1P biosynthesis and maturation. The crucial role of SKI-1/S1P in Arenavirus Infection and other major human diseases combined with its nature as an enzyme makes SKI-1/S1P further an attractive target for therapeutic intervention. In the last part, we will therefore cover past and present efforts to identify specific SKI-1/S1P inhibitors.
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Lassa virus cell entry via dystroglycan involves an unusual pathway of macropinocytosis
Journal of virology, 2016Co-Authors: Joel Oppliger, Giulia Torriani, Antonio Herrador, Stefan KunzAbstract:ABSTRACT The pathogenic Old World Arenavirus Lassa virus (LASV) causes a severe hemorrhagic fever with a high rate of mortality in humans. Several LASV receptors, including dystroglycan (DG), TAM receptor tyrosine kinases, and C-type lectins, have been identified, suggesting complex receptor use. Upon receptor binding, LASV enters the host cell via an unknown clathrin- and dynamin-independent pathway that delivers the virus to late endosomes, where fusion occurs. Here we investigated the mechanisms underlying LASV endocytosis in human cells in the context of productive Arenavirus Infection, using recombinant lymphocytic choriomeningitis virus (rLCMV) expressing the LASV glycoprotein (rLCMV-LASVGP). We found that rLCMV-LASVGP entered human epithelial cells via DG using a macropinocytosis-related pathway independently of alternative receptors. Dystroglycan-mediated entry of rLCMV-LASVGP required sodium hydrogen exchangers, actin, and the GTPase Cdc42 and its downstream targets, p21-activating kinase-1 (PAK1) and Wiskott-Aldrich syndrome protein (N-Wasp). Unlike other viruses that enter cells via macropinocytosis, rLCMV-LASVGP entry did not induce overt changes in cellular morphology and hardly affected actin dynamics or fluid uptake. Screening of kinase inhibitors identified protein kinase C, phosphoinositide 3-kinase, and the receptor tyrosine kinase human hepatocyte growth factor receptor (HGFR) to be regulators of rLCMV-LASVGP entry. The HGFR inhibitor EMD 1214063, a candidate anticancer drug, showed antiviral activity against rLCMV-LASVGP at the level of entry. When combined with ribavirin, which is currently used to treat human Arenavirus Infection, EMD 1214063 showed additive antiviral effects. In sum, our study reveals that DG can link LASV to an unusual pathway of macropinocytosis that causes only minimal perturbation of the host cell and identifies cellular kinases to be possible novel targets for therapeutic intervention. IMPORTANCE Lassa virus (LASV) causes several hundred thousand Infections per year in Western Africa, with the mortality rate among hospitalized patients being high. The current lack of a vaccine and the limited therapeutic options at hand make the development of new drugs against LASV a high priority. In the present study, we uncover that LASV entry into human cells via its major receptor, dystroglycan, involves an unusual pathway of macropinocytosis and define a set of cellular factors implicated in the regulation of LASV entry. A screen of kinase inhibitors revealed HGFR to be a possible candidate target for antiviral drugs against LASV. An HGFR candidate inhibitor currently being evaluated for cancer treatment showed potent antiviral activity and additive drug effects with ribavirin, which is used in the clinic to treat human LASV Infection. In sum, our study reveals novel fundamental aspects of the LASV-host cell interaction and highlights a possible candidate drug target for therapeutic intervention.
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a molecular sensor to characterize Arenavirus envelope glycoprotein cleavage by subtilisin kexin isozyme 1 site 1 protease
Journal of Virology, 2016Co-Authors: Joel Oppliger, Joel Ramos Da Palma, Dominique J Burri, Abdelmajid Khatib, Antonella Pasquato, Christina F. Spiropoulou, Eric Bergeron, Stefan KunzAbstract:Arenaviruses are emerging viruses including several causative agents of severe hemorrhagic fevers in humans. The advent of next-generation sequencing technology has greatly accelerated the discovery of novel Arenavirus species. However, for many of these viruses, only genetic information is available, and their zoonotic disease potential remains unknown. During the Arenavirus life cycle, processing of the viral envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) is crucial for productive Infection. The ability of newly emerging Arenaviruses to hijack human SKI-1/S1P appears, therefore, to be a requirement for efficient zoonotic transmission and human disease potential. Here we implement a newly developed cell-based molecular sensor for SKI-1/S1P to characterize the processing of Arenavirus GPC-derived target sequences by human SKI-1/S1P in a quantitative manner. We show that only nine amino acids flanking the putative cleavage site are necessary and sufficient to accurately recapitulate the efficiency and subcellular location of Arenavirus GPC processing. In a proof of concept, our sensor correctly predicts efficient processing of the GPC of the newly emergent pathogenic Lujo virus by human SKI-1/S1P and defines the exact cleavage site. Lastly, we employed our sensor to show efficient GPC processing of a panel of pathogenic and nonpathogenic New World Arenaviruses, suggesting that GPC cleavage represents no barrier for zoonotic transmission of these pathogens. Our SKI-1/S1P sensor thus represents a rapid and robust test system for assessment of the processing of putative cleavage sites derived from the GPCs of newly discovered Arenavirus by the SKI-1/S1P of humans or any other species, based solely on sequence information. IMPORTANCE Arenaviruses are important emerging human pathogens that can cause severe hemorrhagic fevers with high mortality in humans. A crucial step in productive Arenavirus Infection of human cells is the processing of the viral envelope glycoprotein by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P). In order to break the species barrier during zoonotic transmission and cause severe disease in humans, newly emerging Arenaviruses must be able to hijack human SKI-1/S1P efficiently. Here we implement a newly developed cell-based molecular sensor for human SKI-1/S1P to characterize the processing of Arenavirus glycoproteins in a quantitative manner. We further use our sensor to correctly predict efficient processing of the glycoprotein of the newly emergent pathogenic Lujo virus by human SKI-1/S1P. Our sensor thus represents a rapid and robust test system with which to assess whether the glycoprotein of any newly emerging Arenavirus can be efficiently processed by human SKI-1/S1P, based solely on sequence information.
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Plasmacytoid Dendritic Cells Are Productively Infected and Activated through TLR-7 Early after Arenavirus Infection
Cell host & microbe, 2012Co-Authors: Monica Macal, Stefan Kunz, Gavin M. Lewis, Richard A. Flavell, James A. Harker, Elina I. ZunigaAbstract:The antiviral response is largely mediated by dendritic cells (DCs), including conventional (c) DCs that function as antigen-presenting cells, and plasmacytoid (p) DCs that produce type I interferons, making them an attractive target for viruses. We find that the Old World Arenaviruses lymphocytic choriomeningitis virus clone 13 (LCMV Cl13) and Lassa virus bind pDCs to a greater extent than cDCs. Consistently, LCMV Cl13 targets pDCs early after in vivo Infection of its natural murine host and establishes a productive and robust replication cycle. pDCs coproduce type I interferons and proinflammatory cytokines, with the former being induced in both infected and uninfected pDCs, demonstrating a dissociation from intrinsic virus replication. TLR7 globally mediates pDC responses, limits pDC viral load, and promotes rapid innate and adaptive immune cell activation. These early events likely help dictate the outcome of Infections with Arenaviruses and other DC-replicating viruses and shed light on potential therapeutic targets.
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Current drug discovery strategies against Arenavirus Infections
Expert review of anti-infective therapy, 2012Co-Authors: Antonella Pasquato, Dominique J Burri, Stefan KunzAbstract:Arenaviruses are a large group of emerging viruses including several causative agents of severe hemorrhagic fevers with high mortality in man. Considering the number of people affected and the currently limited therapeutic options, novel efficacious therapeutics against Arenaviruses are urgently needed. Over the past decade, significant advances in knowledge about the basic virology of Arenaviruses have been accompanied by the development of novel therapeutics targeting different steps of the arenaviral life cycle. High-throughput, small-molecule screens identified potent and broadly active inhibitors of Arenavirus entry that were instrumental for the dissection of unique features of Arenavirus fusion. Novel inhibitors of Arenavirus replication have been successfully tested in animal models and hold promise for application in humans. Late in the Arenavirus life cycle, the proteolytic processing of the Arenavirus envelope glycoprotein precursor and cellular factors critically involved virion assembly and budding provide further promising 'druggable' targets for novel therapeutics to combat human Arenavirus Infection.
Brian B. Gowen - One of the best experts on this subject based on the ideXlab platform.
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Development of a New Tacaribe Arenavirus Infection Model and Its Use to Explore Antiviral Activity of a Novel Aristeromycin Analog
2013Co-Authors: Brian B. Gowen, Eric J Sefing, Donald F Smee, Min-hui Wong, Deanna Larson, Kie-hoon Jung, John D. Morrey, Stewart W SchnellerAbstract:Background: A growing number of Arenaviruses can cause a devastating viral hemorrhagic fever (VHF) syndrome. They pose a public health threat as emerging viruses and because of their potential use as bioterror agents. All of the highly pathogenic New World Arenaviruses (NWA) phylogenetically segregate into clade B and require maximum biosafety containment facilities for their study. Tacaribe virus (TCRV) is a nonpathogenic member of clade B that is closely related to the VHF Arenaviruses at the amino acid level. Despite this relatedness, TCRV lacks the ability to antagonize the host interferon (IFN) response, which likely contributes to its inability to cause disease in animals other than newborn mice. Methodology/Principal Findings: Here we describe a new mouse model based on TCRV challenge of AG129 IFN-a/b and-c receptor-deficient mice. Titration of the virus by intraperitoneal (i.p.) challenge of AG129 mice resulted in an LD 50 of,100 fifty percent cell culture infectious doses. Virus replication was evident in the serum, liver, lung, spleen, and brain 4–8 days after inoculation. MY-24, an aristeromycin derivative active against TCRV in cell culture at 0.9 mM, administered i.p. once daily for 7 days, offered highly significant (P,0.001) protection against mortality in the AG129 mouse TCRV Infection model, without appreciably reducing viral burden. In contrast, in a hamster model of arenaviral hemorrhagic fever based on challenge with clade A Pichinde Arenavirus, MY-24 did not offer significant protection against mortality. Conclusions/Significance: MY-24 is believed to act as an inhibitor of S-adenosyl-L-homocysteine hydrolase, but our finding
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Open Access Assessing changes in vascular permeability in a hamster model of viral hemorrhagic fever
2013Co-Authors: Brian B. Gowen, Min-hui Wong, Deanna Larson, John D. Morrey, Justin G Jul, Nyall R London, Mike BrayAbstract:Background: A number of RNA viruses cause viral hemorrhagic fever (VHF), in which proinflammatory mediators released from infected cells induce increased permeability of the endothelial lining of blood vessels, leading to loss of plasma volume, hypotension, multi-organ failure, shock and death. The optimal treatment of VHF should therefore include both the use of antiviral drugs to inhibit viral replication and measures to prevent or correct changes in vascular function. Although rodent models have been used to evaluate treatments for increased vascular permeability (VP) in bacterial sepsis, such studies have not been performed for VHF. Results: Here, we use an established model of Pichinde virus Infection of hamsters to demonstrate how changes in VP can be detected by intravenous infusion of Evans blue dye (EBD), and compare those measurements to changes in hematocrit, serum albumin concentration and serum levels of proinflammatory mediators. We show that EBD injected into sick animals in the late stage of Infection is rapidly sequestered in the viscera, while in healthy animals it remains within the plasma, causing the skin to turn a marked blue color. This test could be used in live animals to detect increased VP and to assess the ability of antiviral drugs and vasoactive compounds to prevent its onset. Finally, we describe a multiplexed assay to measure levels of serum factors during the course of Pichinde Arenavirus Infection and demonstrate that viremia and subsequent increase in white blood cell count
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Vascular leak ensues a vigorous proinflammatory cytokine response to Tacaribe Arenavirus Infection in AG129 mice
Virology Journal, 2013Co-Authors: Eric J Sefing, Min-hui Wong, Deanna P Larson, Brett L Hurst, Arnaud J Van Wettere, Stewart W Schneller, Brian B. GowenAbstract:Background Tacaribe virus (TCRV) is a less biohazardous relative of the highly pathogenic clade B New World Arenaviruses that cause viral hemorrhagic fever syndromes and require handling in maximum containment facilities not readily available to most researchers. AG129 type I and II interferon receptor knockout mice have been shown to be susceptible to TCRV Infection, but the pathogenic mechanisms contributing to the lethal disease are unclear. Methods To gain insights into the pathogenesis of TCRV Infection in AG129 mice, we assessed hematologic and cytokine responses during the course of Infection, as well as changes in the permeability of the vascular endothelium. We also treated TCRV-challenged mice with MY-24, a compound that prevents mortality without affecting viral loads during the acute Infection, and measured serum and tissue viral titers out to 40 days post-Infection to determine whether the virus is ultimately cleared in recovering mice. Results We found that the development of viremia and splenomegaly precedes an elevation in white blood cells and the detection of high levels of proinflammatory mediators known to destabilize the endothelial barrier, which likely contributes to the increased vascular permeability and weight loss that was observed several days prior to when the mice generally succumb to TCRV challenge. In surviving mice treated with MY-24, viremia and liver virus titers were not cleared until 2–3 weeks post-Infection, after which the mice began to recover lost weight. Remarkably, substantial viral loads were still present in the lung, spleen, brain and kidney tissues at the conclusion of the study. Conclusions Our findings suggest that vascular leak may be a contributing factor in the demise of TCRV-infected mice, as histopathologic findings are generally mild to moderate in nature, and as evidenced with MY-24 treatment, animals can survive in the face of high viral loads.
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Vascular leak ensues a vigorous proinflammatory cytokine response to Tacaribe Arenavirus Infection in AG129 mice.
Virology journal, 2013Co-Authors: Eric J Sefing, Min-hui Wong, Brett L Hurst, Arnaud J Van Wettere, Stewart W Schneller, Deanna Larson, Brian B. GowenAbstract:Background Tacaribe virus (TCRV) is a less biohazardous relative of the highly pathogenic clade B New World Arenaviruses that cause viral hemorrhagic fever syndromes and require handling in maximum containment facilities not readily available to most researchers. AG129 type I and II interferon receptor knockout mice have been shown to be susceptible to TCRV Infection, but the pathogenic mechanisms contributing to the lethal disease are unclear.
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Use of recombinant adenovirus vectored consensus IFN-α to avert severe Arenavirus Infection.
PloS one, 2011Co-Authors: Brian B. Gowen, Eric J Sefing, Jane Ennis, Andrew Russell, Min-hui Wong, Jeffrey W. TurnerAbstract:Several Arenaviruses can cause viral hemorrhagic fever, a severe disease with case-fatality rates in hospitalized individuals ranging from 15-30%. Because of limited prophylaxis and treatment options, new medical countermeasures are needed for these viruses classified by the National Institutes of Allergy and Infectious Diseases (NIAID) as top priority biodefense Category A pathogens. Recombinant consensus interferon alpha (cIFN-α) is a licensed protein with broad clinical appeal. However, while cIFN-α has great therapeutic value, its utility for biodefense applications is hindered by its short in vivo half-life, mode and frequency of administration, and costly production. To address these limitations, we describe the use of DEF201, a replication-deficient adenovirus vector that drives the expression of cIFN-α, for pre- and post-exposure prophylaxis of acute arenaviral Infection modeled in hamsters. Intranasal administration of DEF201 24 h prior to challenge with Pichinde virus (PICV) was highly effective at protecting animals from mortality and preventing viral replication and liver-associated disease. A significant protective effect was still observed with a single dosing of DEF201 given two weeks prior to PICV challenge. DEF201 was also efficacious when administered as a treatment 24 to 48 h post-virus exposure. The protective effect of DEF201 was largely attributed to the expression of cIFN-α, as dosing with a control empty vector adenovirus did not protect hamsters from lethal PICV challenge. Effective countermeasures that are highly stable, easily administered, and elicit long lasting protective immunity are much needed for arena and other viral Infections. The DEF201 technology has the potential to address all of these issues and may serve as a broad-spectrum antiviral to enhance host defense against a number of viral pathogens.
Min-hui Wong - One of the best experts on this subject based on the ideXlab platform.
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Development of a New Tacaribe Arenavirus Infection Model and Its Use to Explore Antiviral Activity of a Novel Aristeromycin Analog
2013Co-Authors: Brian B. Gowen, Eric J Sefing, Donald F Smee, Min-hui Wong, Deanna Larson, Kie-hoon Jung, John D. Morrey, Stewart W SchnellerAbstract:Background: A growing number of Arenaviruses can cause a devastating viral hemorrhagic fever (VHF) syndrome. They pose a public health threat as emerging viruses and because of their potential use as bioterror agents. All of the highly pathogenic New World Arenaviruses (NWA) phylogenetically segregate into clade B and require maximum biosafety containment facilities for their study. Tacaribe virus (TCRV) is a nonpathogenic member of clade B that is closely related to the VHF Arenaviruses at the amino acid level. Despite this relatedness, TCRV lacks the ability to antagonize the host interferon (IFN) response, which likely contributes to its inability to cause disease in animals other than newborn mice. Methodology/Principal Findings: Here we describe a new mouse model based on TCRV challenge of AG129 IFN-a/b and-c receptor-deficient mice. Titration of the virus by intraperitoneal (i.p.) challenge of AG129 mice resulted in an LD 50 of,100 fifty percent cell culture infectious doses. Virus replication was evident in the serum, liver, lung, spleen, and brain 4–8 days after inoculation. MY-24, an aristeromycin derivative active against TCRV in cell culture at 0.9 mM, administered i.p. once daily for 7 days, offered highly significant (P,0.001) protection against mortality in the AG129 mouse TCRV Infection model, without appreciably reducing viral burden. In contrast, in a hamster model of arenaviral hemorrhagic fever based on challenge with clade A Pichinde Arenavirus, MY-24 did not offer significant protection against mortality. Conclusions/Significance: MY-24 is believed to act as an inhibitor of S-adenosyl-L-homocysteine hydrolase, but our finding
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Open Access Assessing changes in vascular permeability in a hamster model of viral hemorrhagic fever
2013Co-Authors: Brian B. Gowen, Min-hui Wong, Deanna Larson, John D. Morrey, Justin G Jul, Nyall R London, Mike BrayAbstract:Background: A number of RNA viruses cause viral hemorrhagic fever (VHF), in which proinflammatory mediators released from infected cells induce increased permeability of the endothelial lining of blood vessels, leading to loss of plasma volume, hypotension, multi-organ failure, shock and death. The optimal treatment of VHF should therefore include both the use of antiviral drugs to inhibit viral replication and measures to prevent or correct changes in vascular function. Although rodent models have been used to evaluate treatments for increased vascular permeability (VP) in bacterial sepsis, such studies have not been performed for VHF. Results: Here, we use an established model of Pichinde virus Infection of hamsters to demonstrate how changes in VP can be detected by intravenous infusion of Evans blue dye (EBD), and compare those measurements to changes in hematocrit, serum albumin concentration and serum levels of proinflammatory mediators. We show that EBD injected into sick animals in the late stage of Infection is rapidly sequestered in the viscera, while in healthy animals it remains within the plasma, causing the skin to turn a marked blue color. This test could be used in live animals to detect increased VP and to assess the ability of antiviral drugs and vasoactive compounds to prevent its onset. Finally, we describe a multiplexed assay to measure levels of serum factors during the course of Pichinde Arenavirus Infection and demonstrate that viremia and subsequent increase in white blood cell count
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Vascular leak ensues a vigorous proinflammatory cytokine response to Tacaribe Arenavirus Infection in AG129 mice
Virology Journal, 2013Co-Authors: Eric J Sefing, Min-hui Wong, Deanna P Larson, Brett L Hurst, Arnaud J Van Wettere, Stewart W Schneller, Brian B. GowenAbstract:Background Tacaribe virus (TCRV) is a less biohazardous relative of the highly pathogenic clade B New World Arenaviruses that cause viral hemorrhagic fever syndromes and require handling in maximum containment facilities not readily available to most researchers. AG129 type I and II interferon receptor knockout mice have been shown to be susceptible to TCRV Infection, but the pathogenic mechanisms contributing to the lethal disease are unclear. Methods To gain insights into the pathogenesis of TCRV Infection in AG129 mice, we assessed hematologic and cytokine responses during the course of Infection, as well as changes in the permeability of the vascular endothelium. We also treated TCRV-challenged mice with MY-24, a compound that prevents mortality without affecting viral loads during the acute Infection, and measured serum and tissue viral titers out to 40 days post-Infection to determine whether the virus is ultimately cleared in recovering mice. Results We found that the development of viremia and splenomegaly precedes an elevation in white blood cells and the detection of high levels of proinflammatory mediators known to destabilize the endothelial barrier, which likely contributes to the increased vascular permeability and weight loss that was observed several days prior to when the mice generally succumb to TCRV challenge. In surviving mice treated with MY-24, viremia and liver virus titers were not cleared until 2–3 weeks post-Infection, after which the mice began to recover lost weight. Remarkably, substantial viral loads were still present in the lung, spleen, brain and kidney tissues at the conclusion of the study. Conclusions Our findings suggest that vascular leak may be a contributing factor in the demise of TCRV-infected mice, as histopathologic findings are generally mild to moderate in nature, and as evidenced with MY-24 treatment, animals can survive in the face of high viral loads.
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Vascular leak ensues a vigorous proinflammatory cytokine response to Tacaribe Arenavirus Infection in AG129 mice.
Virology journal, 2013Co-Authors: Eric J Sefing, Min-hui Wong, Brett L Hurst, Arnaud J Van Wettere, Stewart W Schneller, Deanna Larson, Brian B. GowenAbstract:Background Tacaribe virus (TCRV) is a less biohazardous relative of the highly pathogenic clade B New World Arenaviruses that cause viral hemorrhagic fever syndromes and require handling in maximum containment facilities not readily available to most researchers. AG129 type I and II interferon receptor knockout mice have been shown to be susceptible to TCRV Infection, but the pathogenic mechanisms contributing to the lethal disease are unclear.
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Use of recombinant adenovirus vectored consensus IFN-α to avert severe Arenavirus Infection.
PloS one, 2011Co-Authors: Brian B. Gowen, Eric J Sefing, Jane Ennis, Andrew Russell, Min-hui Wong, Jeffrey W. TurnerAbstract:Several Arenaviruses can cause viral hemorrhagic fever, a severe disease with case-fatality rates in hospitalized individuals ranging from 15-30%. Because of limited prophylaxis and treatment options, new medical countermeasures are needed for these viruses classified by the National Institutes of Allergy and Infectious Diseases (NIAID) as top priority biodefense Category A pathogens. Recombinant consensus interferon alpha (cIFN-α) is a licensed protein with broad clinical appeal. However, while cIFN-α has great therapeutic value, its utility for biodefense applications is hindered by its short in vivo half-life, mode and frequency of administration, and costly production. To address these limitations, we describe the use of DEF201, a replication-deficient adenovirus vector that drives the expression of cIFN-α, for pre- and post-exposure prophylaxis of acute arenaviral Infection modeled in hamsters. Intranasal administration of DEF201 24 h prior to challenge with Pichinde virus (PICV) was highly effective at protecting animals from mortality and preventing viral replication and liver-associated disease. A significant protective effect was still observed with a single dosing of DEF201 given two weeks prior to PICV challenge. DEF201 was also efficacious when administered as a treatment 24 to 48 h post-virus exposure. The protective effect of DEF201 was largely attributed to the expression of cIFN-α, as dosing with a control empty vector adenovirus did not protect hamsters from lethal PICV challenge. Effective countermeasures that are highly stable, easily administered, and elicit long lasting protective immunity are much needed for arena and other viral Infections. The DEF201 technology has the potential to address all of these issues and may serve as a broad-spectrum antiviral to enhance host defense against a number of viral pathogens.
Eric J Sefing - One of the best experts on this subject based on the ideXlab platform.
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alterations in favipiravir t 705 pharmacokinetics and biodistribution in a hamster model of viral hemorrhagic fever
Antiviral Research, 2015Co-Authors: Ia Gowe, Yousuke Furuta, Eric J Sefing, Jonna Westove, Donald F Smee, Joseph Hagloch, Jeffery O HallAbstract:Favipiravir (T-705) is a new anti-influenza drug approved for human use in Japan and progressing through Phase 3 clinical trials in the U.S. In addition to its potent inhibitory effects against influenza virus Infection, the compound has been shown to be broadly active against RNA viruses from 9 different families, including the Arenaviridae. Several members of the Arenaviridae family of viruses are significant human pathogens that cause viral hemorrhagic fever, a severe systemic syndrome where vascular leak is a cardinal feature. Because arenaviral Infections are unlikely to be diagnosed and treated until the illness has progressed to a more advanced state, it is important to understand the effects of the disease state on favipiravir pharmacokinetics (PK) and biodistribution to help guide therapeutic strategy. During acute Arenavirus Infection in hamsters, we found reduced plasma favipiravir concentrations and altered kinetics of absorption, elimination and time to maximum drug concentration. In addition, the amounts of the favipiravir M1 primary metabolite were higher in the infected animals, suggesting that favipiravir metabolism may favor the formation of this inactive metabolite during viral Infection. We also discovered differences in favipiravir and M1 PK parameters associated with Arenavirus Infection in a number of hamster tissues. Finally, analysis at the individual animal level demonstrated a correlation between reduced plasma favipiravir concentration with increased disease burden as reflected by weight loss and viral load. Our study is the first to show the impact of active viral Infection and disease on favipiravir PK and biodistribution, highlighting the need to consider alterations in these parameters when treating individuals with viral hemorrhagic fever of Arenavirus or other etiology.
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Development of a New Tacaribe Arenavirus Infection Model and Its Use to Explore Antiviral Activity of a Novel Aristeromycin Analog
2013Co-Authors: Brian B. Gowen, Eric J Sefing, Donald F Smee, Min-hui Wong, Deanna Larson, Kie-hoon Jung, John D. Morrey, Stewart W SchnellerAbstract:Background: A growing number of Arenaviruses can cause a devastating viral hemorrhagic fever (VHF) syndrome. They pose a public health threat as emerging viruses and because of their potential use as bioterror agents. All of the highly pathogenic New World Arenaviruses (NWA) phylogenetically segregate into clade B and require maximum biosafety containment facilities for their study. Tacaribe virus (TCRV) is a nonpathogenic member of clade B that is closely related to the VHF Arenaviruses at the amino acid level. Despite this relatedness, TCRV lacks the ability to antagonize the host interferon (IFN) response, which likely contributes to its inability to cause disease in animals other than newborn mice. Methodology/Principal Findings: Here we describe a new mouse model based on TCRV challenge of AG129 IFN-a/b and-c receptor-deficient mice. Titration of the virus by intraperitoneal (i.p.) challenge of AG129 mice resulted in an LD 50 of,100 fifty percent cell culture infectious doses. Virus replication was evident in the serum, liver, lung, spleen, and brain 4–8 days after inoculation. MY-24, an aristeromycin derivative active against TCRV in cell culture at 0.9 mM, administered i.p. once daily for 7 days, offered highly significant (P,0.001) protection against mortality in the AG129 mouse TCRV Infection model, without appreciably reducing viral burden. In contrast, in a hamster model of arenaviral hemorrhagic fever based on challenge with clade A Pichinde Arenavirus, MY-24 did not offer significant protection against mortality. Conclusions/Significance: MY-24 is believed to act as an inhibitor of S-adenosyl-L-homocysteine hydrolase, but our finding
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Vascular leak ensues a vigorous proinflammatory cytokine response to Tacaribe Arenavirus Infection in AG129 mice
Virology Journal, 2013Co-Authors: Eric J Sefing, Min-hui Wong, Deanna P Larson, Brett L Hurst, Arnaud J Van Wettere, Stewart W Schneller, Brian B. GowenAbstract:Background Tacaribe virus (TCRV) is a less biohazardous relative of the highly pathogenic clade B New World Arenaviruses that cause viral hemorrhagic fever syndromes and require handling in maximum containment facilities not readily available to most researchers. AG129 type I and II interferon receptor knockout mice have been shown to be susceptible to TCRV Infection, but the pathogenic mechanisms contributing to the lethal disease are unclear. Methods To gain insights into the pathogenesis of TCRV Infection in AG129 mice, we assessed hematologic and cytokine responses during the course of Infection, as well as changes in the permeability of the vascular endothelium. We also treated TCRV-challenged mice with MY-24, a compound that prevents mortality without affecting viral loads during the acute Infection, and measured serum and tissue viral titers out to 40 days post-Infection to determine whether the virus is ultimately cleared in recovering mice. Results We found that the development of viremia and splenomegaly precedes an elevation in white blood cells and the detection of high levels of proinflammatory mediators known to destabilize the endothelial barrier, which likely contributes to the increased vascular permeability and weight loss that was observed several days prior to when the mice generally succumb to TCRV challenge. In surviving mice treated with MY-24, viremia and liver virus titers were not cleared until 2–3 weeks post-Infection, after which the mice began to recover lost weight. Remarkably, substantial viral loads were still present in the lung, spleen, brain and kidney tissues at the conclusion of the study. Conclusions Our findings suggest that vascular leak may be a contributing factor in the demise of TCRV-infected mice, as histopathologic findings are generally mild to moderate in nature, and as evidenced with MY-24 treatment, animals can survive in the face of high viral loads.
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Vascular leak ensues a vigorous proinflammatory cytokine response to Tacaribe Arenavirus Infection in AG129 mice.
Virology journal, 2013Co-Authors: Eric J Sefing, Min-hui Wong, Brett L Hurst, Arnaud J Van Wettere, Stewart W Schneller, Deanna Larson, Brian B. GowenAbstract:Background Tacaribe virus (TCRV) is a less biohazardous relative of the highly pathogenic clade B New World Arenaviruses that cause viral hemorrhagic fever syndromes and require handling in maximum containment facilities not readily available to most researchers. AG129 type I and II interferon receptor knockout mice have been shown to be susceptible to TCRV Infection, but the pathogenic mechanisms contributing to the lethal disease are unclear.
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Use of recombinant adenovirus vectored consensus IFN-α to avert severe Arenavirus Infection.
PloS one, 2011Co-Authors: Brian B. Gowen, Eric J Sefing, Jane Ennis, Andrew Russell, Min-hui Wong, Jeffrey W. TurnerAbstract:Several Arenaviruses can cause viral hemorrhagic fever, a severe disease with case-fatality rates in hospitalized individuals ranging from 15-30%. Because of limited prophylaxis and treatment options, new medical countermeasures are needed for these viruses classified by the National Institutes of Allergy and Infectious Diseases (NIAID) as top priority biodefense Category A pathogens. Recombinant consensus interferon alpha (cIFN-α) is a licensed protein with broad clinical appeal. However, while cIFN-α has great therapeutic value, its utility for biodefense applications is hindered by its short in vivo half-life, mode and frequency of administration, and costly production. To address these limitations, we describe the use of DEF201, a replication-deficient adenovirus vector that drives the expression of cIFN-α, for pre- and post-exposure prophylaxis of acute arenaviral Infection modeled in hamsters. Intranasal administration of DEF201 24 h prior to challenge with Pichinde virus (PICV) was highly effective at protecting animals from mortality and preventing viral replication and liver-associated disease. A significant protective effect was still observed with a single dosing of DEF201 given two weeks prior to PICV challenge. DEF201 was also efficacious when administered as a treatment 24 to 48 h post-virus exposure. The protective effect of DEF201 was largely attributed to the expression of cIFN-α, as dosing with a control empty vector adenovirus did not protect hamsters from lethal PICV challenge. Effective countermeasures that are highly stable, easily administered, and elicit long lasting protective immunity are much needed for arena and other viral Infections. The DEF201 technology has the potential to address all of these issues and may serve as a broad-spectrum antiviral to enhance host defense against a number of viral pathogens.
Jack H. Nunberg - One of the best experts on this subject based on the ideXlab platform.
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2006. Role of the stable signal peptide of the Junín Arenavirus envelope glycoprotein in pH-dependent membrane fusion
2015Co-Authors: Joanne York, Jack H. NunbergAbstract:The envelope glycoprotein of the Arenaviruses (GP-C) is unusual in that the mature complex retains the cleaved, 58-amino-acid signal peptide. Association of this stable signal peptide (SSP) has been shown to be essential for intracellular trafficking and proteolytic maturation of the GP-C complex. We identify here a specific and previously unrecognized role of SSP in pH-dependent membrane fusion. Amino acid substitutions that alter the positive charge at lysine K33 in SSP affect the ability of GP-C to mediate cell-cell fusion and the threshold pH at which membrane fusion is triggered. Based on the presumed location of K33 at or near the luminal domain of SSP, we postulate that SSP interacts with the membrane-proximal or transmembrane regions of the G2 fusion protein. This unique organization of the GP-C complex may suggest novel strategies for intervention in Arenavirus Infection. Arenaviruses are endemic in rodent populations worldwide (43) and can be transmitted to humans to cause severe acute hemorrhagic fevers (35, 41). Phylogenetic analysis indicates that Arenavirus species have diversified, along with their rodent hosts, to form closely related clades of Old World and New World viruses (13). In Africa, up to 300,000 Infections by Lass
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Arenavirus Infection Induces Discrete Cytosolic Structures for RNA Replication
Journal of virology, 2012Co-Authors: Nicholas L. Baird, Joanne York, Jack H. NunbergAbstract:Arenaviruses are responsible for acute hemorrhagic fevers with high mortality and pose significant threats to public health and biodefense. These enveloped negative-sense RNA viruses replicate in the cell cytoplasm and express four proteins. To better understand how these proteins insinuate themselves into cellular processes to orchestrate productive viral replication, we have identified and characterized novel cytosolic structures involved in Arenavirus replication and transcription. In cells infected with the nonpathogenic Tacaribe virus or the attenuated Candid#1 strain of Junin virus, wefind that newly synthesized viral RNAs localize to cytosolic puncta containing the nucleoprotein (N) of the virus. Density gradient centrifugation studies reveal that these replication-transcription complexes (RTCs) are associated with cellular membranes and contain full-length genomic- and antigenomic-sense RNAs. Viral mRNAs segregate at a higher buoyant density and are likewise scant in immunopurified RTCs, consistent with their translation on bulk cellular ribosomes. In addition, confocal microscopy analysis reveals that RTCs contain the lipid phosphatidylinositol-4-phosphate and proteins involved in cellular mRNA metabolism, including the large and small ribosomal subunit proteins L10a and S6, the stress granule protein G3BP1, and a subset of translation initiation factors. Elucidating the structure and function of RTCs will enhance our understanding of virus-cell interactions that promote Arenavirus replication and mitigate against host cell immunity. This knowledge may lead to novel intervention strategies to limit viral virulence and pathogenesis.
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t 705 favipiravir inhibition of Arenavirus replication in cell culture
Antimicrobial Agents and Chemotherapy, 2011Co-Authors: Michelle Mendenhall, Yousuke Furuta, Donald F Smee, Juan-carlos De La Torre, Andrew Russell, Terry L Juelich, Emily Messina, Alexande N Freiberg, Michael R Holbrook, Jack H. NunbergAbstract:A number of New World Arenaviruses (Junin [JUNV], Machupo [MACV], and Guanarito [GTOV] viruses) can cause human disease ranging from mild febrile illness to a severe and often fatal hemorrhagic fever syndrome. These highly pathogenic viruses and the Old World Lassa fever virus pose a significant threat to public health and national security. The only licensed antiviral agent with activity against these viruses, ribavirin, has had mixed success in treating severe arenaviral disease and is associated with significant toxicities. A novel pyrazine derivative currently in clinical trials for the treatment of influenza virus Infections, T-705 (favipiravir), has demonstrated broad-spectrum activity against a number of RNA viruses, including Arenaviruses. T-705 has also been shown to be effective against Pichinde Arenavirus Infection in a hamster model. Here, we demonstrate the robust antiviral activity of T-705 against authentic highly pathogenic Arenaviruses in cell culture. We show that T-705 disrupts an early or intermediate stage in viral replication, distinct from absorption or release, and that its antiviral activity in cell culture is reversed by the addition of purine bases and nucleosides, but not with pyrimidines. Specific inhibition of viral replication/transcription by T-705 was demonstrated using a lymphocytic choriomeningitis Arenavirus replicon system. Our findings indicate that T-705 acts to inhibit Arenavirus replication/transcription and may directly target the viral RNA-dependent RNA polymerase. Several New World Arenaviruses, including Junin (JUNV), Machupo (MACV), and Guanarito (GTOV) viruses, as well as the related Old World Lassa virus, are among a phylogenetically diverse group of negative-sense RNA viruses that cause severe viral hemorrhagic fevers (VHFs) in regions of the world where they are endemic (9). The National Institutes of Health has classified these viruses as category A agents because of the threat they pose to the U.S. population (20). Despite the biodefense and public health risks associated with these highly pathogenic viruses, there are no FDA-licensed Arenavirus vaccines and current antiarenaviral therapy is limited to an offlabel use of ribavirin (1--D-ribofuranosyl-1,2,4-triazole-3-carboxamide), which has had only mixed success in the treatment of severe Infections and is associated with significant toxicity in humans (4, 15, 27). Therefore, it is important to develop novel and effective antiviral drugs to combat arenaviral hemorrhagic fevers. T-705 (favipiravir; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) is a pyrazine derivative with broad antiviral activity against RNA viruses, including influenza viruses (6, 16, 24, 25), flaviviruses (13, 19), bunyaviruses, and several nonpathogenic Arenaviruses (10–12). Moreover, studies employing the hamster Pichinde virus (PICV) Infection model of acute arenaviral disease have demonstrated that T-705 can be used effectively to treat advanced Infections in animals (10). However, T-705 has not yet been tested against highly pathogenic human Arenaviruses. Evidence indicates that T-705 is ribosylated and phosphorylated to the active T-705-4-ribofuranosyl-5-triphosphate form (T-705RTP) that inhibits influenza virus Infection by interfering with viral RNA replication and transcription through inhibition of the virus RNA-dependent RNA polymerase (RdRp) (7). The broad activity of T-705 against a number of RNA viruses suggests that this inhibitor may target a conserved functional element in the viral polymerase. The ability of T-705 to specifically target the viral replication machinery may minimize the possibility of in vivo toxicity. In contrast, ribavirin also inhibits cellular IMP dehydrogenase (IMPDH), a key enzyme in guanosine biosynthesis, and thereby perturbs cellular nucleotide pools. In the present study, we explored the mechanism of action of T-705 in cell culture and assessed the in vitro activity of T-705 against three highly pathogenic Arenaviruses.
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Unique Small Molecule Entry Inhibitors of Hemorrhagic Fever Arenaviruses
Journal of Biological Chemistry, 2008Co-Authors: Andrew M. Lee, Christina F. Spiropoulou, Jillian M. Rojek, Anette Gundersen, Wei Jin, Alex Shaginian, Joanne York, Jack H. Nunberg, Dale L. Boger, Michael B. A. OldstoneAbstract:Viral hemorrhagic fevers caused by the Arenaviruses Lassa virus in Africa and Machupo, Guanarito, Junin, and Sabia virus in South America are among the most devastating emerging human diseases with fatality rates of 15–35% and a limited antiviral therapeutic repertoire available. Here we used high throughput screening of synthetic combinatorial small molecule libraries to identify inhibitors of Arenavirus Infection using pseudotyped virion particles bearing the glycoproteins (GPs) of highly pathogenic Arenaviruses. Our screening efforts resulted in the discovery of a series of novel small molecule inhibitors of viral entry that are highly active against both Old World and New World hemorrhagic Arenaviruses. We observed potent inhibition of Infection of human and primate cells with live hemorrhagic Arenaviruses (IC50 = 500–800 nm). Investigations of the mechanism of action revealed that the candidate compounds efficiently block pH-dependent fusion by the Arenavirus GPs (IC50 of 200–350 nm). Although our lead compounds were potent against phylogenetically distant Arenaviruses, they did not show activity against other enveloped viruses with class I viral fusion proteins, indicating specificity for Arenavirus GP-mediated membrane fusion.
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Role of the Stable Signal Peptide of Junín Arenavirus Envelope Glycoprotein in pH-Dependent Membrane Fusion
Journal of virology, 2006Co-Authors: Joanne York, Jack H. NunbergAbstract:The envelope glycoprotein of the Arenaviruses (GP-C) is unusual in that the mature complex retains the cleaved, 58-amino-acid signal peptide. Association of this stable signal peptide (SSP) has been shown to be essential for intracellular trafficking and proteolytic maturation of the GP-C complex. We identify here a specific and previously unrecognized role of SSP in pH-dependent membrane fusion. Amino acid substitutions that alter the positive charge at lysine K33 in SSP affect the ability of GP-C to mediate cell-cell fusion and the threshold pH at which membrane fusion is triggered. Based on the presumed location of K33 at or near the luminal domain of SSP, we postulate that SSP interacts with the membrane-proximal or transmembrane regions of the G2 fusion protein. This unique organization of the GP-C complex may suggest novel strategies for intervention in Arenavirus Infection.