Special Issue "Advances in Filovirus Research"

Guest Editor
Dr. Sina Bavari
Target Discovery and Experimental Microbiology, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, USA
E-Mail: sina.bavari@amedd.army.mil
Phone: +1 301 619 4258
Interests: viral biology; therapeutics; vaccines; viral immunology; genomics; drug discovery; target identification; host-based anti-microbial

Guest Editor
Dr. Jens H. Kuhn
Integrated Research Facility at Fort Detrick (IRF-Frederick), Office 3A110, NIH/NIAID/DCR, B-8200 Research Plaza, Fort Detrick, Frederick, MD 21702, USA
E-Mail: kuhnjens@mail.nih.gov
Phone: +1-301-631-7245
Fax: +1-301-619-5029

Submission

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Type of Paper: Article
Title: Some Observations on the Effect of in vitro Cell Passage on Virulence and Recognition of Ebolaviruses
Authors: Sophie Smither, Lin Eastaugh, Jackie Steward and Mark Stephen Lever
Affiliations: Biomedical Sciences Department, Defence Science and Technology Laboratory (Dstl), Porton Down, UK. E-mail sjsmither@dstl.gov.uk
Abstract: Three of the five ebolavirus species are pathogenic to humans, whilst all species have been isolated from primates, bats and, more recently, pigs.  In research laboratories, ebolaviruses are typically used after growth in cell culture. The effect of continuous growth in cell culture, however, has not been reported for ebolaviruses, although passage of other viruses is known to alter virulence and other characteristics. This paper describes the effect of cell passage on the virulence of Ebola virus and Reston virus. Whether recognition of Ebola virus, via antibodies or genetic sequence, is affected by cell passage is also discussed. Alongside conventional cell culture, bat and pig cells were used for growth of Ebola virus and Reston virus respectively, to mimic the suspected natural hosts of these species. Passage of Ebola virus and Reston virus in typical, monkey-derived cell line, did not alter the virulence or recognition by ELISA or PCR. Reston virus did not become virulent after passage in pig-derived cells. Ebola virus did not grow well in bat-derived cells, and may become avirulent after repeated passage. These findings suggest the role of bats in the ecology of Ebola virus warrants further investigation.
Keywords: Ebola virus; Reston virus; passage; cells; virulence; bat

Type of Paper: Article
Title: Efficacy of Disinfectants against Filoviruses
Authors: J.A. Steward, L. Eastaugh, S. Smither, A.J.Gates, T.J.Piercy and M.S. Lever
Affiliation: Defence Science and Technology Laboratory (Dstl), Porton Down, Salisbury, Wiltshire, UK. SP4 0JQ. Tel.: +44 1980 613169. Email: jasteward@dstl.gov.uk
Abstract: Effective disinfectants are critical for the safe laboratory handling of highly pathogenic viruses such Ebola virus or Marburg virus.  In this study we evaluated the virucidal efficacy of commercially available Viragri Plus VT49 (JohnsonDiversey, UK) and Desintex (Laboratories Rochex, France) disinfectants against the filoviruses. All manipulations were performed within a BSL4 laboratory at Dstl Porton Down, UK.  Viragri Plus VT49 is a blend of glutaraldehyde and quaternary ammonium compounds.  Desintex is a mixture of quaternary ammonium, biguanide and alkylamine. The inactivation of Ebola virus variant E718 (EBOV) and Marburg virus variant Popp (MARV) were evaluated against a final concentration of either 5% Viragri or 5% Desintex with a five minute contact time at room temperature.  The efficacies of working solutions of disinfectant were assessed over time. The test method was based on European Standard EN 14476.  Equal volumes of disinfectant and virus were thoroughly mixed and incubated for a 5 minute contact time.  An aliquot of this was then added to a T12.5 tissue culture flask containing Vero C1008 cells in Dulbecco’s Modified Eagle Medium (DMEM) and 2% Fetal Calf Serum (FCS).  After 24 hours incubation at 37°C in a 5% CO2 atmosphere the media was replaced with fresh DMEM and 2% FCS incubated for a further 6 days.  Following incubation, supernatants were decanted into new T25 flasks containing fresh Vero C1008 cells and media changed after 24 hours as described previously.  After a further incubation of 6 days this was repeated with a T75 flask and Vero C1008 cells.  The sequential passage into larger tissue culture flasks allowed for the detection of remaining viable virus. Following a contact time of five minutes, with each individual disinfectant applied at a working concentration of 5%, the titres of Ebola virus and Marburg virus were reduced by more than 6 log10. The disinfectant was effective, at these concentrations, for a period of 20 weeks.
Keywords: Ebola virus, Marburg virus, inactivation, disinfectants
© Crown Copyright. Dstl, 2012.

Title: The Impact of Regulations, Safety Considerations and Physical Limitations on Research Progress at High Biocontainment
Authors: Amy C. Shurtleff, Nicole Garza, et al.
Affiliation: Integrated Toxicology Division, USAMRIID, 1425 Porter St, Frederick, MD 21702; Email:  amy.c.shurtleff.ctr@us.army.mil
Abstract: The purpose of this article is to describe some of the limitations imposed upon research at high biocontainment (BSL-4), with regard to biosecurity regulations, safety considerations, research space limitations and physical inabilities in executing experimental procedures. These limitations impact the number and size of research collaborations on projects to investigate many agents of biodefense concern. Several governmental institutions regulate biocontainmentresearch and handling of select agents (BSAT), and procedures for personnel approval for BSAT access, inventory of agent stocks and documentation requirements are all impacted by these regulations. Safety considerations govern the process for safely handling and inactivating samples for removal from the BSL-4 laboratory for further processing, the types of tools and items available for use within the laboratories, as well as the workflow and time required for performing experiments. Wearing the fully encapsulating, positive-pressure suit imposes physical limitations on the researcher which are not experienced by laboratory personnel working in lower biocontainment environments. Mitigating these constraints takes extra time, effort, good communication practices between personnel and creative solutions. Biodefense research funding has necessitated increasing the number of high containment laboratories in existence over the last 10 years, but space for performing in vitro and in vivo experiments is still limited. Advanced development GLP studies supporting approval of novel vaccines and therapeutics under the FDA Animal Rule of Efficacy must be prioritized, but they frequently share the same physical space as important ongoing basic research studies.  Scheduling of studies to be executed at BSL-4 is challenging, especially when many collaborating researchers at an institute have large portfolios of funded projects with similar priorities and deadlines. A description of these and other BSL-4 research considerations will be provided to educate the reader about the possibilities and limitations in BSL-4 research.

Type of Paper: Review Article
Title: Potential Vaccines, Therapeutics, and Drug Targets for Filovirus Infections
Authors: Friedrich, B.M. ^1§, Trefry, J.C.^1§, Biggins, J. E. ¹, Honko, A.N. 1, Smith, D.R. ¹, and Olinger, G.G.^1*
Affiliations: ¹ United States Army Medical Research Institute of Infectious Diseases, Division of Virology, 1425 Porter Street, Frederick, Maryland. ^§ Authors contributed equally. *Author to whom correspondence should be addressed; E-mail: gene.olinger@us.army.mil
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 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 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.

Type of Paper: Article
Title: An Interagency Collaboration to Facilitate Development of Filovirus Medical Countermeasures
Authors: Nicole Kilgore ¹ and Edwin O. Nuzum ^2*
Affiliations: ¹ Chemical Biological Medical Systems Joint Vaccine Acquisition Program, 1564 Freedman Dr, Fort Detrick, MD 21702, USA; E-mail: Nicole.kilgore@us.army.mil
² Office of Biodefense Research Affairs, DMID, NIAID, NIH, 6610 Rockledge, Bethesda, MD 20892, USA; E-Mail: enuzum@niaid.nih.gov.
*Author to whom correspondence should be addressed; E-mail: enuzum@niaid.nih.gov
Abstract: The Filovirus Animal Non-Clinical Group (FANG) is an 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) and one from the Department of Health and Human Services (DHHS). The FANG membership includes operational level program staff and subject matter experts from performing organizations as well as scientific staff and program managers from DoD and DHHS funding and regulatory agencies. Focus areas include animal models, assays, reagents, product manufacture and characterization, and other interagency product development issues relevant to Food and Drug Administration (FDA) approval of filovirus MCMs. The FANG continues to develop strategies to address broadly applicable and interagency product development challenges relevant to filovirus MCM development. This paper will discuss FANG structure and accomplishments as well as increase community awareness of this government-led collaborative effort.
Keywords: Filovirus; Collaboration; Government; Product Development; Medical Countermeasures

Type of Paper: Article
Title: Role of Cell Cycle in Ebola virus Infection
Authors: Kota K. P. et al.
Affiliation: United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, Frederick, MD 21702, USA; Email: Rekha.Panchal@us.army.mil
Abstract: Many viruses subvert the host cell cycle to favor their own replication. The progression from G1/S to G2/M phases of cell cycle is highly regulated and is governed by activities of kinase complexes made up of cyclins bound to cyclin dependent kinases (CDK).The molecular mechanisms by which viruses perturb the regulatory cell cycle machinery is not well understood. In this study we apply high content imaging (HCI) to generate distinct phenotypic finger prints for each phase of the cell cycle and use these finger prints to demonstrate that cells infected with Ebola virus (EBOV) are arrested in G0/G1 phase of the cell cycle. Furthermore, cells synchronized in G0/G1 phase favored EBOV replication compared to unsynchronized cells or cells blocked in other phases of cell cycle. Finally, siRNA screening with a focused library targeting 131 human cell cycle regulating proteins revealed that genes that arrest cells in the G0/G1 phase enhance EBOV replication. Collectively, our data suggests that EBOV infection induces cell cycle arrest in the G0/G1 phase which in turn favors EBOV replication.

Type of Paper: Review
Title: Shedding Light on Viral Diseases with High-Content Imaging
Authors: Pegoraro G. et al.
Affiliation: United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, Frederick, MD 21702, USA; Email: Rekha.Panchal@us.army.mil
Abstract: Microscopy has been instrumental for 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 single-cell levels. Here we a present a comprehensive overview of the most significant applications of High-Content Imaging (HCI) in the context of the cell biology of viral infection and disease. HCI has been used to elucidate host-virus interactions in genome-wide reverse genetic screens and discover novel cellular genes implicated in the binding, entry, replication and egress of several pathogenic viruses. HCI is also increasingly becoming instrumental in the screening of small-molecules for the identification and characterization of compounds with therapeutic, antiviral properties. A unique and powerful application of HCI is to study the effect of subpopulation variations in the metabolic state of host-cells and their effect on viral infection dynamics at the single cell level. Finally, we discuss possible novel applications of HCI in the context of filovirus research with particular emphasis on the identification of cellular biomarkers of virus infection.

Type of Paper: Article
Title: An Evaluation of the Operator Protection Factors Offered by Positive Pressure Air Suits Against Airborne Microbiological Challenge.
Authors: J.A. Steward and M.S. Lever
Affiliation: Defence Science and Technology Laboratories (Dstl), Porton Down, Salisbury, Wiltshire, UK. SP4 0JQ. Tel.: +44 1980 613169. Email: jasteward@dstl.gov.uk
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 (PPS) or primary containment using 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 primary containment. Operator Protection Factors (OPF) for the use of microbiological safety cabinets and isolators are available; however, there are no published data on the OPF afforded by the use of positive suits.
This study evaluated the OPF 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 PPS within high containment laboratories.

Type of Paper: Review
Title: Review of Filovirus Work and Facilities at Porton Down
Authors: Mark Stephen Lever and Sophie Smither
Affliation: Dstl Porton Down, Salisbury, Wiltshire, SP4 0JQ email:mslever@dstl.gov.uk
Abstract: Porton Down houses two separate sites capable of conducting high containment research on ACDP 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 focussed 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.

Type of Paper: Review
Title: Evaluation of the Syrian Hamster as a New Model of Ebola Virus Disease and Comparison to Existing Rodent Models
Authors: Victoria Wahl-Jensen ^{1, 2}, Hideki Ebihara ³, David Safronetz ^3
1 Integrated Research Facility at Fort Detrick, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), National Interagency Biodefense Campus, B-8200 Research Plaza, Fort Detrick, Frederick, Maryland 21702, USA. ² Tunnell Government Services, Inc., 6701 Democracy Blvd., Suite 515, Bethesda, MD 20817, USA. ³ Integrated Research Facility at Rocky Mountain Labs, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), 903 South 4^th Street, Hamilton, Montana 59840, USA.
* Author to whom correspondence should be addressed: victoria.jensen@nih.gov, Tel.: +1-301-631-7248; Fax: +1-301-631-7389
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 model for the study of EVD. In this review the history of the hamster as a research laboratory animal, as well as current benefits and challenges of this model will be discussed and compared directly with mouse and guinea pig models.

Type of Paper: Article
Title: Development of a Murine Model for Aerosolized Filovirus Infection Using a Panel of BXD Recombinant Inbred Mice
Author: Elizabeth E. Zumbrun, Holly A. Bloomfield, Taylor B. Chance, Donald K. Nichols, Nourtan Abdeltawab, Malak Kotb, and Aysegul Nalca
Affiliation: Center for Aerobiologial Sciences, USAMRIID, 1425 Porter St, Frederick, MD 21702; Division of Pathology, USAMRIID, 1425 Porter St, Frederick, MD 21702; Department of Molecular Genetics, Biochemistry and Molecular Biology, University of Cincinnati; E-Mail: Elizabeth.Zumbrun@us.army.mil
Abstract: Countering aerosolized filovirus infections is a major priority of biodefense research.  Aerosol models of filovirus infection have been developed in guinea pig and non-human primates; however, filovirus infection of wild-type mice by the aerosol route has not been reported.  A murine model of aerosolized filovirus infection in mice will 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, C57Bl6, and DBA mice were unaffected, but 100% of SCID and 90% of Stat1 knock-out (KO) mice succumbed to infection between 7-9 days post-exposure (dpe).  Balb/C mice lost approximately 15% of their body weight upon exposure to MA-EBOV, but all mice recovered.  In contrast, 10-30% lethality was observed in C57Bl6 and DBA mice exposed to aerosolized MA-EBOV, and 100% of  SCID, Stat1 KO, Ifnγ -/- and perforin -/- mice succumbed to infection between 7-14 dpe. To identify one or more immunocompetent mouse strains in which exposure to aerosolized MA-EBOV is uniformly lethal, 62 different BXD recombinant inbred (RI) mouse strains were tested for susceptibility or resistance to infection.  Aerosol exposure to a high dose of MA-EBOV produced a spectrum of disease severity, with most strains losing weight but recovering, and a uniformly lethal disease within 7 to 12 dpe in five strains.  Aerosol exposure of these five BXD strains to 10-fold less MA-EBOV resulted in lethality ranging from 0% in two strains to 100% lethality in only one strain.  In the three BXD strains with lethality at the lower dose of MA-EBOV, analysis of post-mortem tissue showed liver damage as well as lung lesions in two of these strains, but no lung lesions in the third strain.  A computational genetic analysis of the RI strains on the basis of outcome to aerosolized filovirus infection may allow for identification of gene loci involved in susceptibility or resistance to infection. Furthermore, the BXD strain for which even a low dose exposure to MA-EBOV is uniformly lethal may be useful for testing vaccines and therapies.

Type of Paper: Article
Title: PASC and its Application in the Classification of the Family Filoviridae
Author: Yiming Bao, Vyacheslav Chetvernin
Affiliation: National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD 20892; Email:  bao@mail.nih.gov and chetvern@ncbi.nlm.nih.gov
Abstract: Pairwise sequence comparison (PASC) is a sequence-based virus classification method. Here, we describe the application of PASC in filovirus classification using the web tool created by the National Center for Biotechnology Information, and compare it with the classification criteria established by the ICTV Filoviridae Study Group.

Type of Paper: Article
Title: Comparison of Sudan virus Yambio and Sudan virus Gulu in Non-human Primates to Support Non-clinical Testing of Filovirus Medical Countermeasures
Authors: Anna N. Honko, Joshua C. Johnson, Eric M. Mucker, Miriam A. Botto, Heather L. Esham, John C. Trefry, Christopher R. Reed, LTC Bridget S. Lewis, and Lisa E. Hensley
Affiliation: USAMRIID, Fort Detrick, MD  21701 and FDA, Silver Spring, MD 20993
Email: anna.honko@us.army.mil
Abstract: Because Sudan virus variants may differ in respect to disease severity and outcome in both humans and nonhuman primate models, test system development should include evaluation of multiple appropriate variants.  Previous non-human primate models have used the Boniface variant of Sudan virus.  Although the disease presentation and pathology in these models may be similar to with what is known about human disease, there are concerns relating to the isolation of the Boniface seed from guinea pigs for use in future comparisons. Therefore, two variants (Yambio and Gulu) were selected for comparison that had no history of previous animal passages and minimal cell culture expansions, and that have had a human case fatality rate similar to Boniface (41%, 53%, and 53% and respectively).  Survival data, serum chemistry and hematology analyses, immunological results, pathological findings and telemetry results are presented demonstrating similar disease profiles for each of these variants in cynomolgus macaques.

Type of Paper: Article
Title: Filovirus Plaque Assay: a Flexible, Robust and Validatable Assay to Support Advanced Development Studies
Author: Amy C. Shurtleff, Ashley Keeney, Jennifer Audet, Travis Warren, Jay Wells, Sina Bavari
Affiliation: Integrated Toxicology Division, USAMRIID, 1425 Porter St, Frederick, MD 21702; E-Mail: amy.c.shurtleff.ctr@us.army.mil
Abstract: The filovirus plaque assay serves as the assay of choice to measure live infectious virus in a cell culture, blood, or homogenized tissue sample. It has been in use for more than 30 years and is generally the assay used to describe the virus titer in samples from animals treated with a potential antiviral therapeutic vaccine. These animal studies are essential to the development of vaccines and therapeutics under the FDA Animal Rule of Efficacy. Sometimes feeling like more of an art form in performance, the assay is quite flexible and can be performed using a variety of conditions, all resulting in the same measurable virus titer. Here we will present various approaches to evaluate the conditions under which the assay performs best, and whether any of these conditions appreciably alters the expected viral titer in a sample. The indicator cell type and source, incubation times, inoculum volumes, length of incubation and general features of the biology of ebolavirus as visualized in the assay will be presented. The culmination of these optimization studies will result in an approach for validation of this assay for use in SOP-driven GLP studies supporting advanced development of filovirus therapeutics.

Type of Paper: Review
Title: The Baboon (Papio spp.) as a Model of Human Filovirus Infection
Authors: ¹ Donna L. Perry & ² Gary L. White
Affiliation: ¹ IRF Pathology Core (Contractor), Supporting the Integrated Research Facility, Division of Clinical Research, NIAID, NIH, Frederick, MD 21702; E-Mail: perrydl@niaid.nih.gov
² Director, Comparative Medicine, Professor, Dept. of Pathology, Principal Investigator, Director, University of Oklahoma Baboon Research Resource, Director, University of Oklahoma Ft. Reno Science Park; E-Mail: gary-white@ouhsc.edu
Abstract: Despite being susceptible to natural filovirus infection and sharing over 95% genetic homology with humans, baboons have been rarely utilized as experimental models of human infection or to evaluate the efficacy of filovirus prophylactics and therapeutics. This review will summarize what is known about the pathogenesis of filovirus infection in baboons, how well the baboon model reflects human filovirus infection, the use of the baboon for vaccine development, and discuss possible reasons underlying the scarce utilization of baboons in filovirus research.

Type of Paper: Article
Title: Mouse Models of Filovirus Infection
Author: Steven B. Bradfute
Affiliation: Department of Molecular Genetics and Microbiology, University of NewMexico, Albuquerque, New Mexico  87131; Email:  sbradfute@salud.unm.edu
Abstract: The study of filovirus infection has benefited from a range of animal models.  The mouse has served as a useful, and in some cases, irreplaceable model for the study of immune responses, therapeutics,vaccines, and basic research in filovirus infection.  This review covers the history of mouse model development in filovirus research, as well as its use in analysis of immunology and therapeutics in filovirus pathogenesis. Future directions for research, as well as the benefits and limitations of the various mouse models, are also discussed.

Type of Paper: Article
Title: Clinical Pathology Parameters for Objective Euthanasia Assessment in Ebola Virus Infected Nonhuman Primates
Authors: Travis K. Warren et al.
Affiliations: USAMRIID, Fort Detrick, MD  21701;  email: travis.warren@amedd.army.mil
Abstract: Although no vaccine and therapeutic products have yet been licensed for use against filovirus infections, multiple products – including both vaccine and therapeutic candidates – are currently in development.   In the US, demonstrations of efficacy for these products will likely occur using any or a number of nonhuman primate models of filovirus infection, to satisfy requirements for licensure under the Animal Rule.  In most instances, the critical endpoint for efficacy assessment will be survival following experimental infection; however, as yet there exists no standardized approach for assessing the health status or euthanasia criteria for filovirus-exposed nonhuman primates.  Consideration of objective criteria during these assessment processes is important to a) ensure that unprotected or infection-control subjects are humanely euthanized without unnecessary suffering, b) enhance the likelihood that animals exhibiting mild or moderate signs of disease, but which may ultimately clear the infection and survive, are not prematurely euthanized, c) minimize the occurrence of spontaneous deaths, for which acquisition of important end-stage samples may be foregone, d) enhance the reproducibility of experimental results conducted by different research teams, and e) provide a defensible rationale for euthanasia decisions that withstands scrutiny by regulatory agencies.  To determine whether clinical pathology parameters routinely monitored during nonhuman primate efficacy studies were predictive of survival status, historic records were compiled for greater than 50 surviving and nonsurviving rhesus monkeys infected by the intramuscular route with Ebola virus at the US Army Medical Research Institute for Infectious Diseases.  These parameters were statistically analyzed and those exhibiting predicative value for survival outcome are reported.  These findings may be useful for standardization of objective animal-health and euthanasia assessments for the rhesus-monkey Ebola-virus infection model and may serve as a useful approach to guide similar standardization strategies for other test systems or viral hemorrhagic fever disease models.

Type of Paper: Review
Title: Treatment of Filovirus Patients
Authors: Danielle V. Clark, Peter B. Jahrling, and James V. Lawler
Affiliations: NIH/NIAID Integrated Research Facility at Fort Detrick, Fort Detrick, Frederick, Maryland, USA. Tel: +1-301-631-7313; E-mail: danielle.clark@nih.gov
Abstract: Filovirus infection presents many unique challenges to patient management. Currently there are no approved treatments available, and the recommendations for supportive care are not evidence based. The austere clinical settings in which patients often present, as well as 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.

Type of Paper: Review
Title: Filoviral Entry: Advances, Implications and Future Directions
Authors: Suchita Bhattacharyya ¹ and Kartik Chandran ²
Affiliations: ¹ Nomis Center for Immunobiology and Microbial Pathogenesis, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, California, USA. ² Department of Microbiology & Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA; E-mail: Suchita Bhattacharyya: suchita@salk.edu; Kartik Chandran: kartik.chandran@einstein.yu.edu
Abstract: There are no specific drugs or licensed vaccines currently available against filoviruses and hence it is imperative to understand the viral replication cycle in order to design effective anti-viral therapeutics. Since entry is the first step in any viral replication cycle, understanding filoviral entry mechanisms is critical in order to devise strategies to prevent these viruses from entering their target cells. Traditionally, our understanding of filoviral entry mechanisms has been impeded by several studies implicating or refuting the roles of various cellular endocytic pathways and host factors involved in this process. It is widely agreed upon that filoviruses enter via pH-dependent endocytosis and require proteolytic cleavage of their envelope glycoprotein by lysosomal cysteine proteases called cathepsins. Both macropinocytosis as well as clathrin endocytic pathways have been implicated in filoviral entry, while the role of caveolae has been supported and refuted by different groups. Also several cellular proteins have been implicated in filoviral entry such as folate receptor alpha, lectins, beta 1 integrins, TAM receptors and Niemann-Pick C1.  Filoviruses have been suggested to bind to a common receptor and a recent study has proposed T-cell immunoglobulin and mucin domain 1 (TIM-1) as the putative receptor for filoviruses. This review summarizes published research work in the field of filoviral entry highlighting the consensus as well as controversies, and also discusses important questions, the answers to which could significantly enhance our understanding of this field.

Type of Paper: Review
Title: Filovirus Antibodies: What is Available?
Authors: John M. Dye and Kelly L. Warfield
Affiliations: USAMRIID, Fort Detrick, MD  21701 and Integrated Biotherapeutics, Inc., 20358 Seneca Meadows Pkwy, Germantown, MD  20876; email: john.m.dye1@us.army.mil and kelly@integratedbiotherapeutics.com
Abstract: Filoviruses cause severe and often fatal hemorrhagic fever in nonhuman primates and humans with no available treatments or vaccines.  The filoviruses are classified for use in BSL-4 laboratories, which means there are limited laboratories worldwide that can work with the live viruses.  Partially due to this constraint, there is a paucity of reagents including antibodies available for diagnostics or assay development.  Additionally, even when reagents exist, they are not readily available for broad use and many that may be available are unreported in the public domain.  This review article will summarize the status of filovirus-specific antibodies currently being used in the field to educate researchers about potentially available products.

Type of Paper: Review
Title: Advances in Treatments of Filovirus Disease
Authors: John M. Dye, Steven B. Bradfute, and William D. Pratt
Affiliations: USAMRIID, Fort Detrick, MD  21701 and Department of Molecular Genetics and Microbiology, 1 University of New Mexico, Albuquerque, NM 87131-0001; email: john.m.dye1@us.army.mil , SBradfute@salud.unm.edu  and williamd.pratt@us.army.mil
Abstract: Filoviruses cause sporadic outbreaks resulting severe and often fatal hemorrhagic fever in nonhuman primates and humans.  Currently, there are no clinically proven treatments for filovirus disease; however, great strides have recently been made in the development of efficacious therapeutics.  In this review article, we will summarize the literature regarding this progress and discuss the future for treatment of filovirus infection.

Type of Paper: Review
Title: Receptors Engaged by Filoviruses for Host Cell Entry: New Insights
Authors: Heike Hofmann, Franziska Kaup and Stefan Pöhlmann
Affiliations: Deutsches Primatenzentrum GmbH, Leibniz-Institut für Primatenforschung, Kellnerweg 4, 37077 Göttingen, Tel: 0551 3851-0
E-mail: S.Poehlmann@dpz.eu
Abstract: Filoviruses cause severe hemorrhagic fever in humans with high case-fatality rates. The cellular factors exploited by filoviruses for spread and pathogenesis constitute potential targets for intervention but are incompletely defined. The viral glycoprotein, in concert with cellular factors, mediates filovirus entry into host cells. Recent studies revealed important insights into the host cell receptors engaged by GP for cellular entry. The binding of GP to host cell lectins was found to concentrate virions onto susceptible cells and might contribute to the early and sustained infection of macrophages and dendritic cells. Tyro-3 kinases were shown to promote macropinocytic uptake of filoviruses into a subset of susceptible cells without binding to GP, while interactions between GP and 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 receptors. Here, we will review the recent progress in elucidating the molecular interactions underlying filovirus entry into host cells and discuss its implications for our understanding of the viral tropism, pathogenesis and targets for antiviral intervention.

Type of Paper: Review
Title: Filovirus Aerosol Experimentation
Authors: Matthew G. Lackemeyer and Peter B. Jahrling
Affiliations: NIH/NIAID Integrated Research Facility at Fort Detrick, Fort Detrick, Frederick, Maryland, USA. Tel: +1-301-631-7258; E-mail: matthew.lackemeyer@nih.gov
Abstract: Filoviruses are considered to be significant public health threats due to their extreme virulence and the absence of licensed medical countermeasures. In nature, transmission is usually associated with close personal contact through body fluids.  However, filoviruses have the potential to be used in a bioterrorism attack since they remain stable while maintaining virulence within experimental aerosols. To address this potential, limited progress has been achieved in the development of animal models using aerosol exposures. However, the data are still very limited and lack standardization. Due to the threat potential of these viruses in aerosol form, all aerosol work must be performed within Class III biosafety cabinets inside a biosafety level 4 laboratory (BSL-4). This review will discuss the basic methodology of filovirus aerosolization, including the challenges and difficulties of performing aerosols within the constraints of BSL-4 biocontainment. In addition, the review will compare filovirus aerosol pathogenesis with conventional routes of exposure to inform the design of critical challenge experiments for countermeasure development.