Treatment of Marburg and Ebola hemorrhagic fevers: A strategy for testing new drugs and vaccines under outbreak conditions.

The filoviruses, Marburg and Ebola, have the dubious distinction of being associated with some of the highest case-fatality rates of any known infectious disease-approaching 90% in many outbreaks. In recent years, laboratory research on the filoviruses has produced treatments and vaccines that are effective in laboratory animals and that could potentially drastically reduce case-fatality rates and curtail outbreaks in humans. However, there are significant challenges in clinical testing of these products and eventual delivery to populations in need. Most cases of filovirus infection are recognized only in the setting of large outbreaks, often in the most remote and resource-poor areas of sub-Saharan Africa, with little infrastructure and few personnel experienced in clinical research. Significant political, legal, and socio-cultural barriers also exist. Here, we review the present research priorities and environment for field study of the filovirus hemorrhagic fevers and outline a strategy for future prospective clinical research on treatment and vaccine prevention

Establishment of Fruit Bat Cells (Rousettus aegyptiacus) as a Model System for the Investigation of Filoviral Infection

Marburg virus and several species of Ebola virus are endemic in central Africa and cause sporadic outbreaks in this region with mortality rates of up to 90%. So far, there is no vaccination or therapy available to protect people at risk in these regions. Recently, different fruit bats have been identified as potential reservoirs. One of them is Rousettus aegyptiacus. It seems that within huge bat populations only relatively small numbers are positive for filovirus-specific antibodies or filoviral RNA, a phenomenon that is currently not understood. As a first step towards understanding the biology of filoviruses in bats, we sought to establish a model system to investigate filovirus replication in cells derived from their natural reservoir. Here, we provide the first insights into this topic by monitoring filovirus infection of a Rousettus aegyptiacus derived cell line, R06E. We were able to show that filoviruses propagate well in R06E cells, which can, therefore, be used to investigate replication and transcription of filovirus RNA and to very efficiently perform rescue of recombinant Marburg virus using reverse genetics. These results emphasize the suitability of the newly established bat cell line for filovirus research

Structure and Functional Analysis of the RNA- and Viral Phosphoprotein-Binding Domain of Respiratory Syncytial Virus M2-1 Protein

Respiratory syncytial virus (RSV) protein M2-1 functions as an essential transcriptional cofactor of the viral RNA-dependent RNA polymerase (RdRp) complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P, another component of the RdRp complex. These binding properties are related to the core region of M2-1 encompassing residues S58 to K177. Here we report the NMR structure of the RSV M2-158–177 core domain, which is structurally homologous to the C-terminal domain of Ebola virus VP30, a transcription co-factor sharing functional similarity with M2-1. The partial overlap of RNA and P interaction surfaces on M2-158–177, as determined by NMR, rationalizes the previously observed competitive behavior of RNA versus P. Using site-directed mutagenesis, we identified eight residues located on these surfaces that are critical for an efficient transcription activity of the RdRp complex. Single mutations of these residues disrupted specifically either P or RNA binding to M2-1 in vitro. M2-1 recruitment to cytoplasmic inclusion bodies, which are regarded as sites of viral RNA synthesis, was impaired by mutations affecting only binding to P, but not to RNA, suggesting that M2-1 is associated to the holonucleocapsid by interacting with P. These results reveal that RNA and P binding to M2-1 can be uncoupled and that both are critical for the transcriptional antitermination function of M2-1

Filovirus RefSeq Entries: Evaluation and Selection of Filovirus Type Variants, Type Sequences, and Names

Sequence determination of complete or coding-complete genomes of viruses is becoming common practice for supporting the work of epidemiologists, ecologists, virologists, and taxonomists. Sequencing duration and costs are rapidly decreasing, sequencing hardware is under modification for use by non-experts, and software is constantly being improved to simplify sequence data management and analysis. Thus, analysis of virus disease outbreaks on the molecular level is now feasible, including characterization of the evolution of individual virus populations in single patients over time. The increasing accumulation of sequencing data creates a management problem for the curators of commonly used sequence databases and an entry retrieval problem for end users. Therefore, utilizing the data to their fullest potential will require setting nomenclature and annotation standards for virus isolates and associated genomic sequences. The National Center for Biotechnology Information’s (NCBI’s) RefSeq is a non-redundant, curated database for reference (or type) nucleotide sequence records that supplies source data to numerous other databases. Building on recently proposed templates for filovirus variant naming [ ()////-], we report consensus decisions from a majority of past and currently active filovirus experts on the eight filovirus type variants and isolates to be represented in RefSeq, their final designations, and their associated sequences

Virus nomenclature below the species level : a standardized nomenclature for filovirus strains and variants rescued from cDNA

Specific alterations (mutations, deletions, insertions) of virus genomes are crucial for the functional characterization of their regulatory elements and their expression products, as well as a prerequisite for the creation of attenuated viruses that could serve as vaccine candidates. Virus genome tailoring can be performed either by using traditionally cloned genomes as starting materials, followed by site-directed mutagenesis, or by de novo synthesis of modified virus genomes or parts thereof. A systematic nomenclature for such recombinant viruses is necessary to set them apart from wild-type and laboratoryadapted viruses, and to improve communication and collaborations among researchers who may want to use recombinant viruses or create novel viruses based on them. A large group of filovirus experts has recently proposed nomenclatures for natural and laboratory animal-adapted filoviruses that aim to simplify the retrieval of sequence data from electronic databases. Here, this work is extended to include nomenclature for filoviruses obtained in the laboratory via reverse genetics systems. The previously developed template for natural filovirus genetic variant naming,\virus name[(\strain[/)\isolation host-suffix[/ \country of sampling[/\year of sampling[/\genetic variant designation[-\isolate designation[, is retained, but we propose to adapt the type of information added to each field for cDNA clone-derived filoviruses. For instance, the full-length designation of an Ebola virus Kikwit variant rescued from a plasmid developed at the US Centers for Disease Control and Prevention could be akin to ‘‘Ebola virus H.sapiens-rec/COD/1995/Kikwit-abc1’’ (with the suffix ‘‘rec’’ identifying the recombinant nature of the virus and ‘‘abc1’’ being a placeholder for any meaningful isolate designator). Such a full-length designation should be used in databases and the methods section of publications. Shortened designations (such as ‘‘EBOV H.sap/COD/95/ Kik-abc1’’) and abbreviations (such as ‘‘EBOV/Kik-abc1’’) could be used in the remainder of the text, depending on how critical it is to convey information contained in the full-length name. ‘‘EBOV’’ would suffice if only one EBOV strain/variant/isolate is addressed.http://link.springer.com/journal/705hb201

Virus nomenclature below the species level : a standardized nomenclature for laboratory animal-adapted strains and variants of viruses assigned to the family Filoviridae

The International Committee on Taxonomy of Viruses (ICTV) organizes the classification of viruses into taxa, but is not responsible for the nomenclature for taxa members. International experts groups, such as the ICTV Study Groups, recommend the classification and naming of viruses and their strains, variants, and isolates. The ICTV Filoviridae Study Group has recently introduced an updated classification and nomenclature for filoviruses. Subsequently, and together with numerous other filovirus experts, a consistent nomenclature for their natural genetic variants and isolates was developed that aims at simplifying the retrieval of sequence data from electronic databases. This is a first important step toward a viral genome annotation standard as sought by the US National Center for Biotechnology Information (NCBI). Here, this work is extended to include filoviruses obtained in the laboratory by artificial selection through passage in laboratory hosts. The previously developed template for natural filovirus genetic variant naming ( //<year of sampling>/-) is retained, but it is proposed to adapt the type of information added to each field for laboratory animal-adapted variants. For instance, the full-length designation of an Ebola virus Mayinga variant adapted at the State Research Center for Virology and Biotechnology “Vector” to cause disease in guinea pigs after seven passages would be akin to “Ebola virus VECTOR/C.porcellus-lab/COD/1976/Mayinga- GPA-P7”. As was proposed for the names of natural filovirus variants, we suggest using the fulllength designation in databases, as well as in the method section of publications. Shortened designations (such as “EBOV VECTOR/C.por/COD/76/May-GPA-P7”) and abbreviations (such as “EBOV/May-GPA-P7”) could be used in the remainder of the text depending on how critical it is to convey information contained in the full-length name. “EBOV” would suffice if only one EBOV strain/variant/isolate is addressed.This work was funded in part by the Joint Science and Technology Office for Chem Bio Defense (proposal #TMTI0048_09_RD_T to SB).http://www.springerlink.com/content/0304-8608/hb2013ab201

Untersuchungen zur Replikation und Transkription von Marburg- und Ebolavirus

Filoviren, zu denen das Marburg- (MARV) und das Ebolavirus Zaire (EBOV-Z) gehören, zählen zu den tödlichsten Humanpathogenen, gegen die es weder ein Therapeutikum noch einen Impfstoff gibt. Innerhalb der Mononegavirales besitzen sie das längste Genom (etwa 19 kb) und als einzige Mitglieder neben den Pneumoviren ein viertes Nukleokapsidprotein, VP30. Dieses dient beim EBOV als Transkriptionsaktivator, die Funktion für das MARV ist aber ungeklärt. In der vorliegenden Arbeit wurden die cis-aktiven Signale für Replikation und Transkription des MARV, EBOV-Z und Ebolavirus Reston (EBOV-R) untersucht und gemeinsame Motive verglichen. Weiter wurde ein Volle-Länge-Rescue-System für das MARV etabliert, mithilfe dessen die Rolle des VP30 untersucht wurde. Als ein Kooperationsprojekt wurden die inhibitorischen Eigen-schaften synthetischer DNA-Analoga auf die Vermehrung von EBOV-Z in Zellkultur analysiert. Die Sekundärstruktur des Transkriptionsstart-Signal (TSS) des MARV wurde mittels chemischer Modifizierung ermittelt und unterschied sich gravierend von der des EBOV-TSS. Wurde die bei EBOV-Z gebildete Sekundärstruktur zerstört, so war kein VP30 mehr für die Transkription notwendig (Weik et al., 2002). Chimären der beiden Sekun-därstrukturen führten zum Verlust der Transkription und zu starker Reduzierung der Replikationsfähigkeit; VP30 hatte dabei keinen Einfluss auf die Transkription. Durch weitere Experimente konnte gezeigt werden, dass wahrscheinlich die Primärsequenz entscheidend für die Replikation und Transkription ist und nicht die Sekundärstruktur. Mithilfe des rekonstituierten Minigenomsystems für EBOV-Z wurde der genomische Replikationspromotor eingehend untersucht. Es war bereits bekannt, dass zwei Promotorelemente vorliegen, und die dazwischenliegende Sequenz unwichtig war und nur um 6 nts verlängert oder verkürzt werden konnte, ohne die Replikation zu beeinträchtigen (Schlenz, 2002, Weik, 2001). In dieser Arbeit konnte gezeigt werden, dass nur 3 von 8 vorkommenden UN5-Hexameren nötig sind, um Replikation zu unterstützen. Dieses Motiv wurde auch bei EBOV-R und MARV gefunden. Allerdings deuten die Daten darauf hin, dass der genomische Replikationspromotor des MARV nur aus einem Element besteht, in dem die Replikations- und Transkriptionssignale überlappen. Als ein sehr nützliches Hilfsmittel zur detaillierten Untersuchung des MARV wurde ein Volle-Länge-Rescue-System etabliert, in welchem rekombinantes MARV durch Transfektion des Antigenoms und Plasmiden der Nukleokapsidproteine VP30, VP35, NP und L erzeugt werden konnte. Somit war eine gezielte Manipulation des viralen Genoms möglich. Arbeiten mit rekombinanten Viren wurden in Kooperation mit Prof. Volchkov in Lyon durchgeführt. Mithilfe dieses Systems konnte gezeigt werden, dass VP30 eine wichtige Rolle für die effiziente Vermehrung des MARV in Zellkultur darstellt, obwohl eher eine strukturelle als katalytische Funktion angenommen wird. Bisher waren solche Untersuchungen nicht möglich, da VP30 im Minigenomsystem keine Funkti-on zukam. Zudem war es möglich, ein rekombinantes MARV zu erzeugen, welches 18 nts des Replikationspromotors des EBOV-Z inseriert hatte. Schließlich wurde eine Inhibition der Vermehrung des EBOV-Z in Zellkultur durch ein gegen die Translationsstart-Sequenz des VP35-Gens gerichtetes, peptidkonjugiertes Phosphorodiamidat-Morpholinooligomer (P-PMO) gezeigt. Die Daten zeigten eine sehr gute prophylaktische Nutzbarkeit, jedoch eingeschränkte Wirkung, wenn das P-PMO nach der Infektion appliziert wurde. Diese Ergebnisse wurden von der Kooperationsgruppe des USAMRIID in Mausexperimenten mit unkonjugierten PMOs bestätigt. Der Einsatz von (P)-PMOs zur Hemmung von VP35 ist ein vielversprechender prophylaktischer und evtl. sogar therapeutischer Ansatz für Filovirusinfektionen