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

The Ebola Virus Genomic Replication Promoter Is Bipartite and Follows the Rule of Six

In this work we investigated the cis-acting signals involved in replication of Ebola virus (EBOV) genomic RNA. A set of mingenomes with mutant 3′ ends were generated and used in a reconstituted replication and transcription system. Our results suggest that the EBOV genomic replication promoter is bipartite, consisting of a first element located within the leader region of the genome and a second, downstream element separated by a spacer region. While proper spacing of the two promoter elements is a prerequisite for replication, the nucleotide sequence of the spacer is not important. Replication activity was only observed when six or a multiple of six nucleotides were deleted or inserted, while all other changes in length abolished replication completely. These data indicate that the EBOV replication promoter obeys the rule of six, although the genome length is not divisible by six. The second promoter element is located in the 3′ nontranslated region of the first gene and consists of eight UN(5) hexamer repeats, where N is any nucleotide. However, three consecutive hexamers, which could be located anywhere within the promoter element, were sufficient to support replication as long as the hexameric phase was preserved. By using chemical modification assays, we could demonstrate that nucleotides 5 to 44 of the EBOV leader are involved in the formation of a stable secondary structure. Formation of the RNA stem-loop occurred independently of the presence of the trailer, indicating that a panhandle structure is not formed between the 3′ and 5′ ends

Rescue of Recombinant Marburg Virus from cDNA Is Dependent on Nucleocapsid Protein VP30

Here we report recovery of infectious Marburg virus (MARV) from a full-length cDNA clone. Compared to the wild-type virus, recombinant MARV showed no difference in terms of morphology of virus particles, intracellular distribution in infected cells, and growth kinetics. The nucleocapsid protein VP30 of MARV and Ebola virus (EBOV) contains a Zn-binding motif which is important for the function of VP30 as a transcriptional activator in EBOV, whereas its role for MARV is unclear. It has been reported previously that MARV VP30 is able to support transcription in an EBOV-specific minigenome system. When the Zn-binding motif was destroyed, MARV VP30 was shown to be inactive in the EBOV system. While it was not possible to rescue recombinant MARV when the VP30 plasmid was omitted from transfection, MARV VP30 with a destroyed Zn-binding motif and EBOV VP30 were able to mediate virus recovery. In contrast, rescue of recombinant EBOV was not supported by EBOV VP30 containing a mutated Zn-binding domain

The Iminosugar UV-4 is a Broad Inhibitor of Influenza A and B Viruses ex Vivo and in Mice

Iminosugars that are competitive inhibitors of endoplasmic reticulum (ER) α-glucosidases have been demonstrated to have antiviral activity against a diverse set of viruses. A novel iminosugar, UV-4B, has recently been shown to provide protection against lethal infections with dengue and influenza A (H1N1) viruses in mice. In the current study, the breadth of activity of UV-4B against influenza was examined ex vivo and in vivo. Efficacy of UV-4B against influenza A and B viruses was shown in primary human bronchial epithelial cells, a principal target tissue for influenza. Efficacy of UV-4B against influenza A (H1N1 and H3N2 subtypes) and influenza B was demonstrated using multiple lethal mouse models with readouts including mortality and weight loss. Clinical trials are ongoing to demonstrate safety of UV-4B and future studies to evaluate antiviral activity against influenza in humans are planned

Two key residues in ephrinB3 are critical for its use as an alternative receptor for Nipah virus

EphrinB2 was recently discovered as a functional receptor for Nipah virus (NiV), a lethal emerging paramyxovirus. Ephrins constitute a class of homologous ligands for the Eph class of receptor tyrosine kinases and exhibit overlapping expression patterns. Thus, we examined whether other ephrins might serve as alternative receptors for NiV. Here, we show that of all known ephrins (ephrinA1-A5 and ephrinB1-B3), only the soluble Fc-fusion proteins of ephrinB3, in addition to ephrinB2, bound to soluble NiV attachment protein G (NiV-G). Soluble NiV-G bound to cell surface ephrinB3 and B2 with subnanomolar affinities (Kd = 0.58 nM and 0.06 nM for ephrinB3 and B2, respectively). Surface plasmon resonance analysis indicated that the relatively lower affinity of NiV-G for ephrinB3 was largely due to a faster off-rate (K(off) = 1.94 x 10(-3) s(-1) versus 1.06 x 10(-4) s(-1) for ephrinB3 and B2, respectively). EphrinB3 was sufficient to allow for viral entry of both pseudotype and live NiV. Soluble ephrinB2 and B3 were able to compete for NiV-envelope-mediated viral entry on both ephrinB2- and B3-expressing cells, suggesting that NiV-G interacts with both ephrinB2 and B3 via an overlapping site. Mutational analysis indicated that the Leu-Trp residues in the solvent exposed G-H loop of ephrinB2 and B3 were critical determinants of NiV binding and entry. Indeed, replacement of the Tyr-Met residues in the homologous positions in ephrinB1 with Leu-Trp conferred NiV receptor activity to ephrinB1. Thus, ephrinB3 is a bona fide alternate receptor for NiV entry, and two residues in the G-H loop of the ephrin B-class ligands are critical determinants of NiV receptor activity

VP35 Knockdown Inhibits Ebola Virus Amplification and Protects against Lethal Infection in Mice

Phosphorodiamidate morpholino oligomers (PMO) are a class of uncharged single-stranded DNA analogs modified such that each subunit includes a phosphorodiamidate linkage and morpholine ring. PMO antisense agents have been reported to effectively interfere with the replication of several positive-strand RNA viruses in cell culture. The filoviruses, Marburg virus and Ebola virus (EBOV), are negative-strand RNA viruses that cause up to 90% lethality in human outbreaks. There is currently no commercially available vaccine or efficacious therapeutic for any filovirus. In this study, PMO conjugated to arginine-rich cell-penetrating peptide (P-PMO) and nonconjugated PMO were assayed for the ability to inhibit EBOV infection in cell culture and in a mouse model of lethal EBOV infection. A 22-mer P-PMO designed to base pair with the translation start site region of EBOV VP35 positive-sense RNA generated sequence-specific and time- and dose-dependent inhibition of EBOV amplification in cell culture. The same oligomer provided complete protection to mice when administered before or after an otherwise lethal infection of EBOV. A corresponding nonconjugated PMO, as well as nonconjugated truncated versions of 16 and 19 base residues, provided length-dependent protection to mice when administered prophylactically. Together, these data suggest that antisense PMO and P-PMO have the potential to control EBOV infection and are promising therapeutic candidates