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Open AccessEditorial

An Interagency Collaboration to Facilitate Development of Filovirus Medical Countermeasures

by Nicole Kilgore and Edwin O. Nuzum

Viruses 2012, 4(10), 2312-2316; https://doi.org/10.3390/v4102312 - 19 Oct 2012

Cited by 18 | Viewed by 7005

Abstract

The Filovirus Animal Non-Clinical Group (FANG) is a US interdepartmental and interagency group established to support and facilitate the advanced development of filovirus Medical Countermeasures (MCM), both vaccines and therapeutics. It is co-led by one representative from the Department of Defense (DoD), the [...] Read more.

The Filovirus Animal Non-Clinical Group (FANG) is a US interdepartmental and interagency group established to support and facilitate the advanced development of filovirus Medical Countermeasures (MCM), both vaccines and therapeutics. It is co-led by one representative from the Department of Defense (DoD), the first author, and one from the Department of Health and Human Services (HHS), the second author. The FANG membership includes operational level program staff and Subject Matter Experts (SME) from performing organizations as well as scientific staff and program managers from DoD and HHS funding and regulatory agencies. Focus areas include animal models, assays, reagents, product manufacture and characterization, and other interagency product development issues that will support Food and Drug Administration (FDA) licensure of safe and effective filovirus MCMs. The FANG continues to develop strategies to address broadly applicable and interagency product development challenges relevant to filovirus MCM development. This paper summarizes FANG structure and accomplishments and is meant to heighten community awareness of this government-led collaborative effort. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

Research

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Open AccessArticle

Standardization of the Filovirus Plaque Assay for Use in Preclinical Studies

by Amy C. Shurtleff, Julia E. Biggins, Ashley E. Keeney, Elizabeth E. Zumbrun, Holly A. Bloomfield, Ana Kuehne, Jennifer L. Audet, Kendra J. Alfson, Anthony Griffiths, Gene G. Olinger and Sina Bavari

Viruses 2012, 4(12), 3511-3530; https://doi.org/10.3390/v4123511 - 6 Dec 2012

Cited by 49 | Viewed by 16424

Abstract

The filovirus plaque assay serves as the assay of choice to measure infectious virus in a cell culture, blood, or homogenized tissue sample. It has been in use for more than 30 years and is the generally accepted assay used to titrate virus [...] Read more.

The filovirus plaque assay serves as the assay of choice to measure infectious virus in a cell culture, blood, or homogenized tissue sample. It has been in use for more than 30 years and is the generally accepted assay used to titrate virus in samples from animals treated with a potential antiviral therapeutic or vaccine. As these animal studies are required for the development of vaccines and therapeutics under the FDA Animal Rule, it is essential to have a standardized assay to compare their efficacies against the various filoviruses. Here, we present an evaluation of the conditions under which the filovirus plaque assay performs best for the Ebola virus Kikwit variant and the Angola variant of Marburg virus. The indicator cell type and source, inoculum volumes, length of incubation and general features of filovirus biology as visualized in the assay are addressed in terms of the impact on the sample viral titer calculations. These optimization studies have resulted in a plaque assay protocol which can be used for preclinical studies, and as a standardized protocol for use across institutions, to aid in data comparison. This protocol will be validated for use in GLP studies supporting advanced development of filovirus therapeutics and vaccines. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessArticle

Development of a Murine Model for Aerosolized Ebolavirus Infection Using a Panel of Recombinant Inbred Mice

by Elizabeth E. Zumbrun, Nourtan F. Abdeltawab, Holly A. Bloomfield, Taylor B. Chance, Donald K. Nichols, Paige E. Harrison, Malak Kotb and Aysegul Nalca

Viruses 2012, 4(12), 3468-3493; https://doi.org/10.3390/v4123468 - 3 Dec 2012

Cited by 34 | Viewed by 9570

Abstract

Countering aerosolized filovirus infection is a major priority of biodefense research. Aerosol models of filovirus infection have been developed in knock-out mice, guinea pigs and non-human primates; however, filovirus infection of immunocompetent mice by the aerosol route has not been reported. A murine [...] Read more.

Countering aerosolized filovirus infection is a major priority of biodefense research. Aerosol models of filovirus infection have been developed in knock-out mice, guinea pigs and non-human primates; however, filovirus infection of immunocompetent mice by the aerosol route has not been reported. A murine model of aerosolized filovirus infection in mice should be useful for screening vaccine candidates and therapies. In this study, various strains of wild-type and immunocompromised mice were exposed to aerosolized wild-type (WT) or mouse-adapted (MA) Ebola virus (EBOV). Upon exposure to aerosolized WT-EBOV, BALB/c, C57BL/6 (B6), and DBA/2 (D2) mice were unaffected, but 100% of severe combined immunodeficiency (SCID) and 90% of signal transducers and activators of transcription (Stat1) knock-out (KO) mice became moribund between 7–9 days post-exposure (dpe). Exposure to MA-EBOV caused 15% body weight loss in BALB/c, but all mice recovered. In contrast, 10–30% lethality was observed in B6 and D2 mice exposed to aerosolized MA-EBOV, and 100% of SCID, Stat1 KO, interferon (IFN)-γ KO and Perforin KO mice became moribund between 7–14 dpe. In order to identify wild-type, inbred, mouse strains in which exposure to aerosolized MA-EBOV is uniformly lethal, 60 BXD (C57BL/6 crossed with DBA/2) recombinant inbred (RI) and advanced RI (ARI) mouse strains were exposed to aerosolized MA-EBOV, and monitored for disease severity. A complete spectrum of disease severity was observed. All BXD strains lost weight but many recovered. However, infection was uniformly lethal within 7 to 12 days post-exposure in five BXD strains. Aerosol exposure of these five BXD strains to 10-fold less MA-EBOV resulted in lethality ranging from 0% in two strains to 90–100% lethality in two strains. Analysis of post-mortem tissue from BXD strains that became moribund and were euthanized at the lower dose of MA-EBOV, showed liver damage in all mice as well as lung lesions in two of the three strains. The two BXD strains that exhibited 90–100% mortality, even at a low dose of airborne MA-EBOV will be useful mouse models for testing vaccines and therapies. Additionally, since disease susceptibility is affected by complex genetic traits, a systems genetics approach was used to identify preliminary gene loci modulating disease severity among the panel BXD strains. Preliminary quantitative trait loci (QTLs) were identified that are likely to harbor genes involved in modulating differential susceptibility to Ebola infection. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessArticle

Discovery and Early Development of AVI-7537 and AVI-7288 for the Treatment of Ebola Virus and Marburg Virus Infections

by Patrick L. Iversen, Travis K. Warren, Jay B. Wells, Nicole L. Garza, Dan V. Mourich, Lisa S. Welch, Rekha G. Panchal and Sina Bavari

Viruses 2012, 4(11), 2806-2830; https://doi.org/10.3390/v4112806 - 6 Nov 2012

Cited by 112 | Viewed by 22588

Abstract

There are no currently approved treatments for filovirus infections. In this study we report the discovery process which led to the development of antisense Phosphorodiamidate Morpholino Oligomers (PMOs) AVI-6002 (composed of AVI-7357 and AVI-7539) and AVI-6003 (composed of AVI-7287 and AVI-7288) targeting Ebola [...] Read more.

There are no currently approved treatments for filovirus infections. In this study we report the discovery process which led to the development of antisense Phosphorodiamidate Morpholino Oligomers (PMOs) AVI-6002 (composed of AVI-7357 and AVI-7539) and AVI-6003 (composed of AVI-7287 and AVI-7288) targeting Ebola virus and Marburg virus respectively. The discovery process involved identification of optimal transcript binding sites for PMO based RNA-therapeutics followed by screening for effective viral gene target in mouse and guinea pig models utilizing adapted viral isolates. An evolution of chemical modifications were tested, beginning with simple Phosphorodiamidate Morpholino Oligomers (PMO) transitioning to cell penetrating peptide conjugated PMOs (PPMO) and ending with PMOplus containing a limited number of positively charged linkages in the PMO structure. The initial lead compounds were combinations of two agents targeting separate genes. In the final analysis, a single agent for treatment of each virus was selected, AVI-7537 targeting the VP24 gene of Ebola virus and AVI-7288 targeting NP of Marburg virus, and are now progressing into late stage clinical development as the optimal therapeutic candidates. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessArticle

Niemann-Pick C1 (NPC1)/NPC1-like1 Chimeras Define Sequences Critical for NPC1’s Function as a Filovirus Entry Receptor

by Anuja Krishnan, Emily Happy Miller, Andrew S. Herbert, Melinda Ng, Esther Ndungo, Sean P. Whelan, John M. Dye and Kartik Chandran

Viruses 2012, 4(11), 2471-2484; https://doi.org/10.3390/v4112471 - 25 Oct 2012

Cited by 32 | Viewed by 11325

Abstract

We recently demonstrated that Niemann-Pick C1 (NPC1), a ubiquitous 13-pass cellular membrane protein involved in lysosomal cholesterol transport, is a critical entry receptor for filoviruses. Here we show that Niemann-Pick C1-like1 (NPC1L1), an NPC1 paralog and hepatitis C virus entry factor, lacks filovirus [...] Read more.

We recently demonstrated that Niemann-Pick C1 (NPC1), a ubiquitous 13-pass cellular membrane protein involved in lysosomal cholesterol transport, is a critical entry receptor for filoviruses. Here we show that Niemann-Pick C1-like1 (NPC1L1), an NPC1 paralog and hepatitis C virus entry factor, lacks filovirus receptor activity. We exploited the structural similarity between NPC1 and NPC1L1 to construct and analyze a panel of chimeras in which NPC1L1 sequences were replaced with cognate sequences from NPC1. Only one chimera, NPC1L1 containing the second luminal domain (C) of NPC1 in place of its own, bound to the viral glycoprotein, GP. This engineered protein mediated authentic filovirus infection nearly as well as wild-type NPC1, and more efficiently than did a minimal NPC1 domain C-based receptor recently described by us. A reciprocal chimera, NPC1 containing NPC1L1’s domain C, was completely inactive. Remarkably, an intra-domain NPC1L1-NPC1 chimera bearing only a ~130-amino acid N–terminal region of NPC1 domain C could confer substantial viral receptor activity on NPC1L1. Taken together, these findings account for the failure of NPC1L1 to serve as a filovirus receptor, highlight the central role of the luminal domain C of NPC1 in filovirus entry, and reveal the direct involvement of N–terminal domain C sequences in NPC1’s function as a filovirus receptor. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessArticle

A Characterization of Aerosolized Sudan Virus Infection in African Green Monkeys, Cynomolgus Macaques, and Rhesus Macaques

by Elizabeth E. Zumbrun, Holly A. Bloomfield, John M. Dye, Ty C. Hunter, Paul A. Dabisch, Nicole L. Garza, Nicholas R. Bramel, Reese J. Baker, Roger D. Williams, Donald K. Nichols and Aysegul Nalca

Viruses 2012, 4(10), 2115-2136; https://doi.org/10.3390/v4102115 - 15 Oct 2012

Cited by 36 | Viewed by 11973

Abstract

Filoviruses are members of the genera Ebolavirus, Marburgvirus, and “Cuevavirus”. Because they cause human disease with high lethality and could potentially be used as a bioweapon, these viruses are classified as CDC Category A Bioterrorism Agents. Filoviruses are relatively stable [...] Read more.

Filoviruses are members of the genera Ebolavirus, Marburgvirus, and “Cuevavirus”. Because they cause human disease with high lethality and could potentially be used as a bioweapon, these viruses are classified as CDC Category A Bioterrorism Agents. Filoviruses are relatively stable in aerosols, retain virulence after lyophilization, and can be present on contaminated surfaces for extended periods of time. This study explores the characteristics of aerosolized Sudan virus (SUDV) Boniface in non-human primates (NHP) belonging to three different species. Groups of cynomolgus macaques (cyno), rhesus macaques (rhesus), and African green monkeys (AGM) were challenged with target doses of 50 or 500 plaque-forming units (pfu) of aerosolized SUDV. Exposure to either viral dose resulted in increased body temperatures in all three NHP species beginning on days 4–5 post-exposure. Other clinical findings for all three NHP species included leukocytosis, thrombocytopenia, anorexia, dehydration, and lymphadenopathy. Disease in all of the NHPs was severe beginning on day 6 post-exposure, and all animals except one surviving rhesus macaque were euthanized by day 14. Serum alanine transaminase (ALT) and aspartate transaminase (AST) concentrations were elevated during the course of disease in all three species; however, AGMs had significantly higher ALT and AST concentrations than cynos and rhesus. While all three species had detectable viral load by days 3-4 post exposure, Rhesus had lower average peak viral load than cynos or AGMs. Overall, the results indicate that the disease course after exposure to aerosolized SUDV is similar for all three species of NHP. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessArticle

High Content Image Based Analysis Identifies Cell Cycle Inhibitors as Regulators of Ebola Virus Infection

by Krishna P. Kota, Jacqueline G. Benko, Rajini Mudhasani, Cary Retterer, Julie P. Tran, Sina Bavari and Rekha G. Panchal

Viruses 2012, 4(10), 1865-1877; https://doi.org/10.3390/v4101865 - 25 Sep 2012

Cited by 14 | Viewed by 9079

Abstract

Viruses modulate a number of host biological responses including the cell cycle to favor their replication. In this study, we developed a high-content imaging (HCI) assay to measure DNA content and identify different phases of the cell cycle. We then investigated the potential [...] Read more.

Viruses modulate a number of host biological responses including the cell cycle to favor their replication. In this study, we developed a high-content imaging (HCI) assay to measure DNA content and identify different phases of the cell cycle. We then investigated the potential effects of cell cycle arrest on Ebola virus (EBOV) infection. Cells arrested in G1 phase by serum starvation or G1/S phase using aphidicolin or G2/M phase using nocodazole showed much reduced EBOV infection compared to the untreated control. Release of cells from serum starvation or aphidicolin block resulted in a time-dependent increase in the percentage of EBOV infected cells. The effect of EBOV infection on cell cycle progression was found to be cell-type dependent. Infection of asynchronous MCF-10A cells with EBOV resulted in a reduced number of cells in G2/M phase with concomitant increase of cells in G1 phase. However, these effects were not observed in HeLa or A549 cells. Together, our studies suggest that EBOV requires actively proliferating cells for efficient replication. Furthermore, multiplexing of HCI based assays to detect viral infection, cell cycle status and other phenotypic changes in a single cell population will provide useful information during screening campaigns using siRNA and small molecule therapeutics. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessArticle

Filovirus Research in Gabon and Equatorial Africa: The Experience of a Research Center in the Heart of Africa

by Eric Leroy and Jean Paul Gonzalez

Viruses 2012, 4(9), 1592-1604; https://doi.org/10.3390/v4091592 - 13 Sep 2012

Cited by 7 | Viewed by 9291

Abstract

Health research programs targeting the population of Gabon and Equatorial Africa at the International Center for Medical Research in Franceville (CIRMF), Gabon, have evolved during the years since its inception in 1979 in accordance with emerging diseases. Since the reemergence of Ebola virus [...] Read more.

Health research programs targeting the population of Gabon and Equatorial Africa at the International Center for Medical Research in Franceville (CIRMF), Gabon, have evolved during the years since its inception in 1979 in accordance with emerging diseases. Since the reemergence of Ebola virus in Central Africa, the CIRMF “Emerging Viral Disease Unit” developed diagnostic tools and epidemiologic strategies and transfers of such technology to support the response of the National Public Health System and the World Health Organization to epidemics of Ebola virus disease. The Unit carries out a unique investigation program on the natural history of the filoviruses, emergence of epidemics, and Ebola virus pathogenesis. In addition, academic training is provided at all levels to regional and international students covering emerging conditions (host factors, molecular biology, genetics) that favor the spread of viral diseases. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessArticle

Genetics-Based Classification of Filoviruses Calls for Expanded Sampling of Genomic Sequences

by Chris Lauber and Alexander E. Gorbalenya

Viruses 2012, 4(9), 1425-1437; https://doi.org/10.3390/v4091425 - 31 Aug 2012

Cited by 18 | Viewed by 9251

Abstract

We have recently developed a computational approach for hierarchical, genome-based classification of viruses of a family (DEmARC). In DEmARC, virus clusters are delimited objectively by devising a universal family-wide threshold on intra-cluster genetic divergence of viruses that is specific for each level of [...] Read more.

We have recently developed a computational approach for hierarchical, genome-based classification of viruses of a family (DEmARC). In DEmARC, virus clusters are delimited objectively by devising a universal family-wide threshold on intra-cluster genetic divergence of viruses that is specific for each level of the classification. Here, we apply DEmARC to a set of 56 filoviruses with complete genome sequences and compare the resulting classification to the ICTV taxonomy of the family Filoviridae. We find in total six candidate taxon levels two of which correspond to the species and genus ranks of the family. At these two levels, the six filovirus species and two genera officially recognized by ICTV, as well as a seventh tentative species for Lloviu virus and prototyping a third genus, are reproduced. DEmARC lends the highest possible support for these two as well as the four other levels, implying that the actual number of valid taxon levels remains uncertain and the choice of levels for filovirus species and genera is arbitrary. Based on our experience with other virus families, we conclude that the current sampling of filovirus genomic sequences needs to be considerably expanded in order to resolve these uncertainties in the framework of genetics-based classification. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessArticle

PAirwise Sequence Comparison (PASC) and Its Application in the Classification of Filoviruses

by Yiming Bao, Vyacheslav Chetvernin and Tatiana Tatusova

Viruses 2012, 4(8), 1318-1327; https://doi.org/10.3390/v4081318 - 20 Aug 2012

Cited by 34 | Viewed by 8710

Abstract

PAirwise Sequence Comparison (PASC) is a tool that uses genome sequence similarity to help with virus classification. The PASC tool at NCBI uses two methods: local alignment based on BLAST and global alignment based on Needleman-Wunsch algorithm. It works for complete genomes of [...] Read more.

PAirwise Sequence Comparison (PASC) is a tool that uses genome sequence similarity to help with virus classification. The PASC tool at NCBI uses two methods: local alignment based on BLAST and global alignment based on Needleman-Wunsch algorithm. It works for complete genomes of viruses of several families/groups, and for the family of Filoviridae, it currently includes 52 complete genomes available in GenBank. It has been shown that BLAST-based alignment approach works better for filoviruses, and therefore is recommended for establishing taxon demarcation criteria. When more genome sequences with high divergence become available, these demarcations will most likely become more precise. The tool can compare new genome sequences of filoviruses with the ones already in the database, and propose their taxonomic classification. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Graphical abstract

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Open AccessCommunication

A Limited Structural Modification Results in a Significantly More Efficacious Diazachrysene-Based Filovirus Inhibitor

by Života Selaković, Dejan Opsenica, Brett Eaton, Cary Retterer, Sina Bavari, James C. Burnett, Bogdan A. Šolaja and Rekha G. Panchal

Viruses 2012, 4(8), 1279-1288; https://doi.org/10.3390/v4081279 - 15 Aug 2012

Cited by 13 | Viewed by 6897

Abstract

Ebola (EBOV) and Marburg (MARV) filoviruses are highly infectious pathogens causing deadly hemorrhagic fever in humans and non-human primates. Promising vaccine candidates providing immunity against filoviruses have been reported. However, the sporadic nature and swift progression of filovirus disease underlines the need for [...] Read more.

Ebola (EBOV) and Marburg (MARV) filoviruses are highly infectious pathogens causing deadly hemorrhagic fever in humans and non-human primates. Promising vaccine candidates providing immunity against filoviruses have been reported. However, the sporadic nature and swift progression of filovirus disease underlines the need for the development of small molecule therapeutics providing immediate antiviral effects. Herein we describe a brief structural exploration of two previously reported diazachrysene (DAAC)-based EBOV inhibitors. Specifically, three analogs were prepared to examine how slight substituent modifications would affect inhibitory efficacy and inhibitor-mediated toxicity during not only EBOV, but also MARV cellular infection. Of the three analogs, one was highly efficacious, providing IC₅₀ values of 0.696 µM ± 0.13 µM and 2.76 µM ± 0.21 µM against EBOV and MARV infection, respectively, with little or no associated cellular toxicity. Overall, the structure-activity and structure-toxicity results from this study provide a framework for the future development of DAAC-based filovirus inhibitors that will be both active and non-toxic in vivo. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessArticle

Evaluation of the Operator Protection Factors Offered by Positive Pressure Air Suits against Airborne Microbiological Challenge

by Jackie A. Steward and Mark S. Lever

Viruses 2012, 4(8), 1202-1211; https://doi.org/10.3390/v4081202 - 7 Aug 2012

Cited by 8 | Viewed by 7364

Abstract

Laboratories throughout the world that perform work with Risk Group 4 Pathogens generally adopt one of two approaches within BSL-4 environments: either the use of positive pressure air-fed suits or using Class III microbiological safety cabinets and isolators for animal work. Within the [...] Read more.

Laboratories throughout the world that perform work with Risk Group 4 Pathogens generally adopt one of two approaches within BSL-4 environments: either the use of positive pressure air-fed suits or using Class III microbiological safety cabinets and isolators for animal work. Within the UK at present, all laboratories working with Risk Group 4 agents adopt the use of Class III microbiological safety cabinet lines and isolators. Operator protection factors for the use of microbiological safety cabinets and isolators are available however; there is limited published data on the operator protection factors afforded by the use of positive pressure suits. This study evaluated the operator protection factors provided by positive pressure air suits against a realistic airborne microbiological challenge. The suits were tested, both intact and with their integrity compromised, on an animated mannequin within a stainless steel exposure chamber. The suits gave operator protection in all tests with an intact suit and with a cut in the leg. When compromised by a cut in the glove, a very small ingress of the challenge was seen as far as the wrist. This is likely to be due to the low airflow in the gloves of the suit. In all cases no microbiological penetration of the respiratory tract was observed. These data provide evidence on which to base safety protocols for use of positive pressure suits within high containment laboratories. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessArticle

Authentication of the R06E Fruit Bat Cell Line

by Ingo Jordan, Vincent J. Munster and Volker Sandig

Viruses 2012, 4(5), 889-900; https://doi.org/10.3390/v4050889 - 23 May 2012

Cited by 11 | Viewed by 8775

Abstract

Fruit bats and insectivorous bats are believed to provide a natural reservoir for a wide variety of infectious diseases. Several lines of evidence, including the successful isolation of infectious viruses, indicate that Marburg virus and Ravn virus have found a major reservoir in [...] Read more.

Fruit bats and insectivorous bats are believed to provide a natural reservoir for a wide variety of infectious diseases. Several lines of evidence, including the successful isolation of infectious viruses, indicate that Marburg virus and Ravn virus have found a major reservoir in colonies of the Egyptian rousette (Rousettus aegyptiacus). To facilitate molecular studies on virus-reservoir host interactions and isolation of viruses from environmental samples, we established cell lines from primary cells of this animal. The cell lines were given to several laboratories until we realized that a contamination with Vero cells in one of the cultures had occurred. Here we describe a general diagnostic procedure for identification of cross-species contamination with the focus on Vero and Rousettus cell lines, and summarize newly discovered properties of the cell lines that may pertain to pathogen discovery. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessArticle

Structural Basis for Differential Neutralization of Ebolaviruses

by Shridhar Bale, Joao M. Dias, Marnie L. Fusco, Takao Hashiguchi, Anthony C. Wong, Tong Liu, Ana I. Keuhne, Sheng Li, Virgil L. Woods, Jr., Kartik Chandran, John M. Dye and Erica Ollmann Saphire

Viruses 2012, 4(4), 447-470; https://doi.org/10.3390/v4040447 - 5 Apr 2012

Cited by 58 | Viewed by 17649

Abstract

There are five antigenically distinct ebolaviruses that cause hemorrhagic fever in humans or non-human primates (Ebola virus, Sudan virus, Reston virus, Taï Forest virus, and Bundibugyo virus). The small handful of antibodies known to neutralize the ebolaviruses bind to the surface glycoprotein termed [...] Read more.

There are five antigenically distinct ebolaviruses that cause hemorrhagic fever in humans or non-human primates (Ebola virus, Sudan virus, Reston virus, Taï Forest virus, and Bundibugyo virus). The small handful of antibodies known to neutralize the ebolaviruses bind to the surface glycoprotein termed GP_1,2. Curiously, some antibodies against them are known to neutralize in vitro but not protect in vivo, whereas other antibodies are known to protect animal models in vivo, but not neutralize in vitro. A detailed understanding of what constitutes a neutralizing and/or protective antibody response is critical for development of novel therapeutic strategies. Here, we show that paradoxically, a lower affinity antibody with restricted access to its epitope confers better neutralization than a higher affinity antibody against a similar epitope, suggesting that either subtle differences in epitope, or different characteristics of the GP_1,2 molecules themselves, confer differential neutralization susceptibility. Here, we also report the crystal structure of trimeric, prefusion GP_1,2 from the original 1976 Boniface variant of Sudan virus complexed with 16F6, the first antibody known to neutralize Sudan virus, and compare the structure to that of Sudan virus, variant Gulu. We discuss new structural details of the GP₁-GP₂ clamp, thermal motion of various regions in GP_1,2 across the two viruses visualized, details of differential interaction of the crystallized neutralizing antibodies, and their relevance for virus neutralization. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Review

Jump to: Editorial, Research

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Open AccessReview

The Impact of Regulations, Safety Considerations and Physical Limitations on Research Progress at Maximum Biocontainment

by Amy C. Shurtleff, Nicole Garza, Matthew Lackemeyer, Ricardo Jr. Carrion, Anthony Griffiths, Jean Patterson, Samuel S. Edwin and Sina Bavari

Viruses 2012, 4(12), 3932-3951; https://doi.org/10.3390/v4123932 - 19 Dec 2012

Cited by 20 | Viewed by 8532

Abstract

We describe herein, limitations on research at biosafety level 4 (BSL-4) containment laboratories, with regard to biosecurity regulations, safety considerations, research space limitations, and physical constraints in executing experimental procedures. These limitations can severely impact the number of collaborations and size of research [...] Read more.

We describe herein, limitations on research at biosafety level 4 (BSL-4) containment laboratories, with regard to biosecurity regulations, safety considerations, research space limitations, and physical constraints in executing experimental procedures. These limitations can severely impact the number of collaborations and size of research projects investigating microbial pathogens of biodefense concern. Acquisition, use, storage, and transfer of biological select agents and toxins (BSAT) are highly regulated due to their potential to pose a severe threat to public health and safety. All federal, state, city, and local regulations must be followed to obtain and maintain registration for the institution to conduct research involving BSAT. These include initial screening and continuous monitoring of personnel, controlled access to containment laboratories, accurate and current BSAT inventory records. Safety considerations are paramount in BSL-4 containment laboratories while considering the types of research tools, workflow and time required for conducting both in vivo and in vitro experiments in limited space. Required use of a positive-pressure encapsulating suit imposes tremendous physical limitations on the researcher. Successful mitigation of these constraints requires additional time, effort, good communication, and creative solutions. Test and evaluation of novel vaccines and therapeutics conducted under good laboratory practice (GLP) conditions for FDA approval are prioritized and frequently share the same physical space with important ongoing basic research studies. The possibilities and limitations of biomedical research involving microbial pathogens of biodefense concern in BSL-4 containment laboratories are explored in this review. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessReview

Use of the Syrian Hamster as a New Model of Ebola Virus Disease and Other Viral Hemorrhagic Fevers

by Victoria Wahl-Jensen, Laura Bollinger, David Safronetz, Fabian De Kok-Mercado, Dana P. Scott and Hideki Ebihara

Viruses 2012, 4(12), 3754-3784; https://doi.org/10.3390/v4123754 - 14 Dec 2012

Cited by 53 | Viewed by 11367

Abstract

Historically, mice and guinea pigs have been the rodent models of choice for therapeutic and prophylactic countermeasure testing against Ebola virus disease (EVD). Recently, hamsters have emerged as a novel animal model for the in vivo study of EVD. In this review, we [...] Read more.

Historically, mice and guinea pigs have been the rodent models of choice for therapeutic and prophylactic countermeasure testing against Ebola virus disease (EVD). Recently, hamsters have emerged as a novel animal model for the in vivo study of EVD. In this review, we discuss the history of the hamster as a research laboratory animal, as well as current benefits and challenges of this model. Availability of immunological reagents is addressed. Salient features of EVD in hamsters, including relevant pathology and coagulation parameters, are compared directly with the mouse, guinea pig and nonhuman primate models. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessReview

Endocytic Pathways Involved in Filovirus Entry: Advances, Implications and Future Directions

by Suchita Bhattacharyya, Nirupama Mulherkar and Kartik Chandran

Viruses 2012, 4(12), 3647-3664; https://doi.org/10.3390/v4123647 - 11 Dec 2012

Cited by 15 | Viewed by 12727

Abstract

Detailed knowledge of the host-virus interactions that accompany filovirus entry into cells is expected to identify determinants of viral virulence and host range, and to yield targets for the development of antiviral therapeutics. While it is generally agreed that filovirus entry into the [...] Read more.

Detailed knowledge of the host-virus interactions that accompany filovirus entry into cells is expected to identify determinants of viral virulence and host range, and to yield targets for the development of antiviral therapeutics. While it is generally agreed that filovirus entry into the host cytoplasm requires viral internalization into acidic endosomal compartments and proteolytic cleavage of the envelope glycoprotein by endo/lysosomal cysteine proteases, our understanding of the specific endocytic pathways co-opted by filoviruses remains limited. This review addresses the current knowledge on cellular endocytic pathways implicated in filovirus entry, highlights the consensus as well as controversies, and discusses important remaining questions. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessReview

Host Cell Factors in Filovirus Entry: Novel Players, New Insights

by Heike Hofmann-Winkler, Franziska Kaup and Stefan Pöhlmann

Viruses 2012, 4(12), 3336-3362; https://doi.org/10.3390/v4123336 - 26 Nov 2012

Cited by 33 | Viewed by 13888

Abstract

Filoviruses cause severe hemorrhagic fever in humans with high case-fatality rates. The cellular factors exploited by filoviruses for their spread constitute potential targets for intervention, but are incompletely defined. The viral glycoprotein (GP) mediates filovirus entry into host cells. Recent studies revealed important [...] Read more.

Filoviruses cause severe hemorrhagic fever in humans with high case-fatality rates. The cellular factors exploited by filoviruses for their spread constitute potential targets for intervention, but are incompletely defined. The viral glycoprotein (GP) mediates filovirus entry into host cells. Recent studies revealed important insights into the host cell molecules engaged by GP for cellular entry. The binding of GP to cellular lectins was found to concentrate virions onto susceptible cells and might contribute to the early and sustained infection of macrophages and dendritic cells, important viral targets. Tyrosine kinase receptors were shown to promote macropinocytic uptake of filoviruses into a subset of susceptible cells without binding to GP, while interactions between GP and human T cell Ig mucin 1 (TIM-1) might contribute to filovirus infection of mucosal epithelial cells. Moreover, GP engagement of the cholesterol transporter Niemann-Pick C1 was demonstrated to be essential for GP-mediated fusion of the viral envelope with a host cell membrane. Finally, mutagenic and structural analyses defined GP domains which interact with these host cell factors. Here, we will review the recent progress in elucidating the molecular interactions underlying filovirus entry and discuss their implications for our understanding of the viral cell tropism. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessReview

The Baboon (Papio spp.) as a Model of Human Ebola Virus Infection

by Donna L. Perry, Laura Bollinger and Gary L.White

Viruses 2012, 4(10), 2400-2416; https://doi.org/10.3390/v4102400 - 23 Oct 2012

Cited by 27 | Viewed by 8225

Abstract

Baboons are susceptible to natural Ebola virus (EBOV) infection and share 96% genetic homology with humans. Despite these characteristics, baboons have rarely been utilized as experimental models of human EBOV infection to evaluate the efficacy of prophylactics and therapeutics in the United States. [...] Read more.

Baboons are susceptible to natural Ebola virus (EBOV) infection and share 96% genetic homology with humans. Despite these characteristics, baboons have rarely been utilized as experimental models of human EBOV infection to evaluate the efficacy of prophylactics and therapeutics in the United States. This review will summarize what is known about the pathogenesis of EBOV infection in baboons compared to EBOV infection in humans and other Old World nonhuman primates. In addition, we will discuss how closely the baboon model recapitulates human EBOV infection. We will also review some of the housing requirements and behavioral attributes of baboons compared to other Old World nonhuman primates. Due to the lack of data available on the pathogenesis of Marburg virus (MARV) infection in baboons, discussion of the pathogenesis of MARV infection in baboons will be limited. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessReview

Forty-Five Years of Marburg Virus Research

by Kristina Brauburger, Adam J. Hume, Elke Mühlberger and Judith Olejnik

Viruses 2012, 4(10), 1878-1927; https://doi.org/10.3390/v4101878 - 1 Oct 2012

Cited by 152 | Viewed by 50133

Abstract

In 1967, the first reported filovirus hemorrhagic fever outbreak took place in Germany and the former Yugoslavia. The causative agent that was identified during this outbreak, Marburg virus, is one of the most deadly human pathogens. This article provides a comprehensive overview of [...] Read more.

In 1967, the first reported filovirus hemorrhagic fever outbreak took place in Germany and the former Yugoslavia. The causative agent that was identified during this outbreak, Marburg virus, is one of the most deadly human pathogens. This article provides a comprehensive overview of our current knowledge about Marburg virus disease ranging from ecology to pathogenesis and molecular biology. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessReview

Clinical Management of Filovirus-Infected Patients

by Danielle V. Clark, Peter B. Jahrling and James V. Lawler

Viruses 2012, 4(9), 1668-1686; https://doi.org/10.3390/v4091668 - 20 Sep 2012

Cited by 52 | Viewed by 15098

Abstract

Filovirus infection presents many unique challenges to patient management. Currently no approved treatments are available, and the recommendations for supportive care are not evidence based. The austere clinical settings in which patients often present and the sporadic and at times explosive nature of [...] Read more.

Filovirus infection presents many unique challenges to patient management. Currently no approved treatments are available, and the recommendations for supportive care are not evidence based. The austere clinical settings in which patients often present and the sporadic and at times explosive nature of filovirus outbreaks have effectively limited the information available to evaluate potential management strategies. This review will summarize the management approaches used in filovirus outbreaks and provide recommendations for collecting the information necessary for evaluating and potentially improving patient outcomes in the future. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessReview

Potential Vaccines and Post-Exposure Treatments for Filovirus Infections

by Brian M. Friedrich, John C. Trefry, Julia E. Biggins, Lisa E. Hensley, Anna N. Honko, Darci R. Smith and Gene G. Olinger

Viruses 2012, 4(9), 1619-1650; https://doi.org/10.3390/v4091619 - 20 Sep 2012

Cited by 44 | Viewed by 12556

Abstract

Viruses of the family Filoviridae represent significant health risks as emerging infectious diseases as well as potentially engineered biothreats. While many research efforts have been published offering possibilities toward the mitigation of filoviral infection, there remain no sanctioned therapeutic or vaccine strategies. Current [...] Read more.

Viruses of the family Filoviridae represent significant health risks as emerging infectious diseases as well as potentially engineered biothreats. While many research efforts have been published offering possibilities toward the mitigation of filoviral infection, there remain no sanctioned therapeutic or vaccine strategies. Current progress in the development of filovirus therapeutics and vaccines is outlined herein with respect to their current level of testing, evaluation, and proximity toward human implementation, specifically with regard to human clinical trials, nonhuman primate studies, small animal studies, and in vitro development. Contemporary methods of supportive care and previous treatment approaches for human patients are also discussed. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessReview

Mouse Models for Filovirus Infections

by Steven B. Bradfute, Kelly L. Warfield and Mike Bray

Viruses 2012, 4(9), 1477-1508; https://doi.org/10.3390/v4091477 - 7 Sep 2012

Cited by 56 | Viewed by 9241

Abstract

The filoviruses marburg- and ebolaviruses can cause severe hemorrhagic fever (HF) in humans and nonhuman primates. Because many cases have occurred in geographical areas lacking a medical research infrastructure, most studies of the pathogenesis of filoviral HF, and all efforts to develop drugs [...] Read more.

The filoviruses marburg- and ebolaviruses can cause severe hemorrhagic fever (HF) in humans and nonhuman primates. Because many cases have occurred in geographical areas lacking a medical research infrastructure, most studies of the pathogenesis of filoviral HF, and all efforts to develop drugs and vaccines, have been carried out in biocontainment laboratories in non-endemic countries, using nonhuman primates (NHPs), guinea pigs and mice as animal models. NHPs appear to closely mirror filoviral HF in humans (based on limited clinical data), but only small numbers may be used in carefully regulated experiments; much research is therefore done in rodents. Because of their availability in large numbers and the existence of a wealth of reagents for biochemical and immunological testing, mice have become the preferred small animal model for filovirus research. Since the first experiments following the initial 1967 marburgvirus outbreak, wild-type or mouse-adapted viruses have been tested in immunocompetent or immunodeficient mice. In this paper, we review how these types of studies have been used to investigate the pathogenesis of filoviral disease, identify immune responses to infection and evaluate antiviral drugs and vaccines. We also discuss the strengths and weaknesses of murine models for filovirus research, and identify important questions for further study. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessReview

Shedding Light on Filovirus Infection with High-Content Imaging

by Gianluca Pegoraro, Sina Bavari and Rekha G. Panchal

Viruses 2012, 4(8), 1354-1371; https://doi.org/10.3390/v4081354 - 23 Aug 2012

Cited by 14 | Viewed by 7614

Abstract

Microscopy has been instrumental in the discovery and characterization of microorganisms. Major advances in high-throughput fluorescence microscopy and automated, high-content image analysis tools are paving the way to the systematic and quantitative study of the molecular properties of cellular systems, both at the [...] Read more.

Microscopy has been instrumental in the discovery and characterization of microorganisms. Major advances in high-throughput fluorescence microscopy and automated, high-content image analysis tools are paving the way to the systematic and quantitative study of the molecular properties of cellular systems, both at the population and at the single-cell level. High-Content Imaging (HCI) has been used to characterize host-virus interactions in genome-wide reverse genetic screens and to identify novel cellular factors implicated in the binding, entry, replication and egress of several pathogenic viruses. Here we present an overview of the most significant applications of HCI in the context of the cell biology of filovirus infection. HCI assays have been recently implemented to quantitatively study filoviruses in cell culture, employing either infectious viruses in a BSL-4 environment or surrogate genetic systems in a BSL-2 environment. These assays are becoming instrumental for small molecule and siRNA screens aimed at the discovery of both cellular therapeutic targets and of compounds with anti-viral properties. We discuss the current practical constraints limiting the implementation of high-throughput biology in a BSL-4 environment, and propose possible solutions to safely perform high-content, high-throughput filovirus infection assays. Finally, we discuss possible novel applications of HCI in the context of filovirus research with particular emphasis on the identification of possible cellular biomarkers of virus infection. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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Open AccessReview

A Review of Filovirus Work and Facilities at The Defence Science and Technology Laboratory Porton Down

by Sophie J. Smither and Mark S. Lever

Viruses 2012, 4(8), 1305-1317; https://doi.org/10.3390/v4081305 - 17 Aug 2012

Cited by 7 | Viewed by 10196

Abstract

Porton Down houses two separate sites capable of conducting high containment research on ACDP (Advisory Committee on Dangerous Pathogens) Hazard Group 4 agents: the Defence Science and Technology Laboratory (Dstl) and the Health Protection Agency (HPA), and filovirus research has been performed at [...] Read more.

Porton Down houses two separate sites capable of conducting high containment research on ACDP (Advisory Committee on Dangerous Pathogens) Hazard Group 4 agents: the Defence Science and Technology Laboratory (Dstl) and the Health Protection Agency (HPA), and filovirus research has been performed at Porton Down since the first Marburg virus disease outbreak in 1967. All work is conducted within primary containment either within cabinet lines (for in vitro work) or large rigid half-suit isolators (for in vivo work). There are extensive aerobiological facilities at high containment and the use of these facilities will be reported. Research at Dstl is primarily focused on assessing and quantifying the hazard, and testing the efficacy of medical countermeasures against filoviruses. Fundamental research directed to the study and understanding of the infectious and pathogenic nature of the filoviruses, particularly in aerosols, will be reported. Full article

(This article belongs to the Special Issue Advances in Filovirus Research 2012)

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