Die Phosphorylierung des Ebolavirus VP30 reguliert die virale Transkription und Replikation

Das Ebolavirus bildet zusammen mit dem Marburgvirus die Familie der Filoviridae, die ein einzelsträngiges nichtsegmentiertes RNA Genom in negativer Orientierung besitzen. Filoviren verursachen schwere hämorrhagische Fieber in Menschen und Affen, weswegen sie als BSL4-Pathogene klassifiziert werden. Die Transkriptions- und Replikationseinheit des Virus bildet der Nukleokapsidkomplex, der sich aus dem RNA-Genom sowie den Nukleokapsidproteinen NP, VP30, VP35 und L zusammensetzt. Dabei agiert VP30 als essentieller Ebolavirus-spezifischer Transkriptionsfaktor, der für die Replikation nicht benötigt wird. Die Aktivität als Transkriptionsfaktor wird über die Phosphorylierung des Proteins reguliert. Nichtphosphoryliertes VP30 unterstützt die Synthese der viralen mRNAs in einem Minigenomsystem, während das phosphorylierte VP30 die Transkription nicht aktivieren kann. Im ersten Teil der vorliegenden Arbeit wurde der Einfluss der VP30 Phosphorylierung auf die Regulation des Übergangs von viraler Transkription zu Replikation untersucht. Dabei stand im Vordergrund der Einfluss der Phosphorylierung auf die Replikation. Mit Hilfe von phospho-mimetischen Mutanten des VP30 konnte gezeigt werden, dass phosphoryliertes VP30 die Replikation fördert, während dephosphoryliertes VP30 einen hemmenden Effekt auf die Replikation besaß. Weiterhin wurde die Interaktion von VP30 mit den anderen Komponenten des Nukleokapsidkomplexes untersucht. Dabei wurde eine bisher unbekannte Interaktion des VP30 mit dem Polymerase Co-Faktor VP35 beschrieben, die vom Phosphorylierungsstatus des VP30 beeinflusst wurde. Möglicherweise führt die phosphorylierungsabhängige Interaktion des VP30 mit VP35 zu einem Ausschluss von VP30 aus einem putativen Transkriptasekomplex und dadurch zur Hemmung der Transkription und Stimulierung der Replikation. Außerdem konnte in der vorliegenden Arbeit gezeigt werden, dass eine dynamische Phosphorylierung des VP30 essentiell für die initialen Schritte der Primären Transkription in frühen Stadien des viralen Lebenszyklus ist. Eine dynamische Phosphorylierung des VP30 an Serinrest 29 war ausreichend für die Generierung eines rekombinanten Ebolavirus mit wildtypischen Eigenschaften. Im Gegensatz dazu ließ sich ein stabiles rekombinantes Virus mit Serinrest 30 als einziger Phosphoakzeptorstelle nicht herstellen und resultierte im Auftreten von kompensatorischen Mutationen innerhalb der VP30 Phosphorylierungsdomäne. Die im Rahmen dieser Arbeit gewonnen Ergebnisse unterstreichen die Bedeutung der VP30 Phosphorylierung für den viralen Replikationszyklus: die dynamische Phosphorylierung des VP30 ist in frühen Stadien des Infektionszyklus für die Primäre Transkription essentiell und besitzt ebenfalls einen Einfluss auf den Übergang von viraler Transkription zu Replikation

From hybridomas to a robust microalgal-based production platform: molecular design of a diatom secreting monoclonal antibodies directed against the Marburg virus nucleoprotein

Background: The ideal protein expression system should provide recombinant proteins in high quality and quantity involving low production costs only. However, especially for complex therapeutic proteins like monoclonal antibodies many challenges remain to meet this goal and up to now production of monoclonal antibodies is very costly and delicate. Particularly, emerging disease outbreaks like Ebola virus in Western Africa in 2014–2016 make it necessary to reevaluate existing production platforms and develop robust and cheap alternatives that are easy to handle. Results: In this study, we engineered the microalga Phaeodactylum tricornutum to produce monoclonal IgG antibodies against the nucleoprotein of Marburg virus, a close relative of Ebola virus causing severe hemorrhagic fever with high fatality rates in humans. Sequences for both chains of a mouse IgG antibody were retrieved from a murine hybridoma cell line and implemented in the microalgal system. Fully assembled antibodies were shown to be secreted by the alga and antibodies were proven to be functional in western blot, ELISA as well as IFA studies just like the original hybridoma produced IgG. Furthermore, synthetic variants with constant regions of a rabbit IgG and human IgG with optimized codon usage were produced and characterized. Conclusions: This study highlights the potential of microalgae as robust and low cost expression platform for monoclonal antibodies secreting IgG antibodies directly into the culture medium. Microalgae possess rapid growth rates, need basically only water, air and sunlight for cultivation and are very easy to handle

Safety and immunogenicity of rVSVΔG-ZEBOV-GP Ebola vaccine in adults and children in Lambaréné, Gabon: A phase I randomised trial.

BACKGROUND: The rVSVΔG-ZEBOV-GP vaccine prevented Ebola virus disease when used at 2 × 107 plaque-forming units (PFU) in a trial in Guinea. This study provides further safety and immunogenicity data. METHODS AND FINDINGS: A randomised, open-label phase I trial in Lambaréné, Gabon, studied 5 single intramuscular vaccine doses of 3 × 103, 3 × 104, 3 × 105, 3 × 106, or 2 × 107 PFU in 115 adults and a dose of 2 × 107 PFU in 20 adolescents and 20 children. The primary objective was safety and tolerability 28 days post-injection. Immunogenicity, viraemia, and shedding post-vaccination were evaluated as secondary objectives. In adults, mild-to-moderate adverse events were frequent, but there were no serious or severe adverse events related to vaccination. Before vaccination, Zaire Ebola virus (ZEBOV)-glycoprotein (GP)-specific and ZEBOV antibodies were detected in 11% and 27% of adults, respectively. In adults, 74%-100% of individuals who received a dose 3 × 104, 3 × 105, 3 × 106, or 2 × 107 PFU had a ≥4.0-fold increase in geometric mean titres (GMTs) of ZEBOV-GP-specific antibodies at day 28, reaching GMTs of 489 (95% CI: 264-908), 556 (95% CI: 280-1,101), 1,245 (95% CI: 899-1,724), and 1,503 (95% CI: 931-2,426), respectively. Twenty-two percent of adults had a ≥4-fold increase of ZEBOV antibodies, with GMTs at day 28 of 1,015 (647-1,591), 1,887 (1,154-3,085), 1,445 (1,013-2,062), and 3,958 (2,249-6,967) for the same doses, respectively. These antibodies persisted up to day 180 for doses ≥3 × 105 PFU. Adults with antibodies before vaccination had higher GMTs throughout. Neutralising antibodies were detected in more than 50% of participants at doses ≥3 × 105 PFU. As in adults, no serious or severe adverse events related to vaccine occurred in adolescents or children. At day 2, vaccine RNA titres were higher for adolescents and children than adults. At day 7, 78% of adolescents and 35% of children had recombinant vesicular stomatitis virus RNA detectable in saliva. The vaccine induced high GMTs of ZEBOV-GP-specific antibodies at day 28 in adolescents, 1,428 (95% CI: 1,025-1,989), and children, 1,620 (95% CI: 806-3,259), and in both groups antibody titres increased up to day 180. The absence of a control group, lack of stratification for baseline antibody status, and imbalances in male/female ratio are the main limitations of this study. CONCLUSIONS: Our data confirm the acceptable safety and immunogenicity profile of the 2 × 107 PFU dose in adults and support consideration of lower doses for paediatric populations and those who request boosting. TRIAL REGISTRATION: Pan African Clinical Trials Registry PACTR201411000919191

Phase 1 Trials of rVSV Ebola Vaccine in Africa and Europe.

BACKGROUND: The replication-competent recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing a Zaire ebolavirus (ZEBOV) glycoprotein was selected for rapid safety and immunogenicity testing before its use in West Africa. METHODS: We performed three open-label, dose-escalation phase 1 trials and one randomized, double-blind, controlled phase 1 trial to assess the safety, side-effect profile, and immunogenicity of rVSV-ZEBOV at various doses in 158 healthy adults in Europe and Africa. All participants were injected with doses of vaccine ranging from 300,000 to 50 million plaque-forming units (PFU) or placebo. RESULTS: No serious vaccine-related adverse events were reported. Mild-to-moderate early-onset reactogenicity was frequent but transient (median, 1 day). Fever was observed in up to 30% of vaccinees. Vaccine viremia was detected within 3 days in 123 of the 130 participants (95%) receiving 3 million PFU or more; rVSV was not detected in saliva or urine. In the second week after injection, arthritis affecting one to four joints developed in 11 of 51 participants (22%) in Geneva, with pain lasting a median of 8 days (interquartile range, 4 to 87); 2 self-limited cases occurred in 60 participants (3%) in Hamburg, Germany, and Kilifi, Kenya. The virus was identified in one synovial-fluid aspirate and in skin vesicles of 2 other vaccinees, showing peripheral viral replication in the second week after immunization. ZEBOV-glycoprotein-specific antibody responses were detected in all the participants, with similar glycoprotein-binding antibody titers but significantly higher neutralizing antibody titers at higher doses. Glycoprotein-binding antibody titers were sustained through 180 days in all participants. CONCLUSIONS: In these studies, rVSV-ZEBOV was reactogenic but immunogenic after a single dose and warrants further evaluation for safety and efficacy. (Funded by the Wellcome Trust and others; ClinicalTrials.gov numbers, NCT02283099, NCT02287480, and NCT02296983; Pan African Clinical Trials Registry number, PACTR201411000919191.)

Annual (2023) taxonomic update of RNA-directed RNA polymerase-encoding negative-sense RNA viruses (realm Riboviria: kingdom Orthornavirae: phylum Negarnaviricota)

55 Pág.In April 2023, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by one new family, 14 new genera, and 140 new species. Two genera and 538 species were renamed. One species was moved, and four were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.This work was supported in part through the Laulima Government Solutions, LLC, prime contract with the U.S. National Institute of Allergy and Infec tious Diseases (NIAID) under Contract No. HHSN272201800013C. J.H.K. performed this work as an employee of Tunnell Government Services (TGS), a subcontractor of Laulima Government Solutions, LLC, under Contract No. HHSN272201800013C. U.J.B. was supported by the Division of Intramural Resarch, NIAID. This work was also funded in part by Contract No. HSHQDC15-C-00064 awarded by DHS S and T for the management and operation of The National Biodefense Analysis and Countermeasures Centre, a federally funded research and development centre operated by the Battelle National Biodefense Institute (V.W.); and NIH contract HHSN272201000040I/HHSN27200004/D04 and grant R24AI120942 (N.V., R.B.T.). S.S. acknowl edges support from the Mississippi Agricultural and Forestry Experiment Station (MAFES), USDA-ARS project 58-6066-9-033 and the National Institute of Food and Agriculture, U.S. Department of Agriculture, Hatch Project, under Accession Number 1021494. The funders had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the U.S. Department of the Army, the U.S. Department of Defence, the U.S. Department of Health and Human Services, including the Centres for Disease Control and Prevention, the U.S. Department of Homeland Security (DHS) Science and Technology Directorate (S and T), or of the institutions and companies affiliated with the authors. In no event shall any of these entities have any responsibility or liability for any use, misuse, inability to use, or reliance upon the information contained herein. The U.S. departments do not endorse any products or commercial services mentioned in this publication. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S.Government retains a non-exclusive, paid up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for U.S. Government purposes.Peer reviewe

Die Phosphorylierung des Ebolavirus VP30 reguliert die virale Transkription und Replikation

Das Ebolavirus bildet zusammen mit dem Marburgvirus die Familie der Filoviridae, die ein einzelsträngiges nichtsegmentiertes RNA Genom in negativer Orientierung besitzen. Filoviren verursachen schwere hämorrhagische Fieber in Menschen und Affen, weswegen sie als BSL4-Pathogene klassifiziert werden. Die Transkriptions- und Replikationseinheit des Virus bildet der Nukleokapsidkomplex, der sich aus dem RNA-Genom sowie den Nukleokapsidproteinen NP, VP30, VP35 und L zusammensetzt. Dabei agiert VP30 als essentieller Ebolavirus-spezifischer Transkriptionsfaktor, der für die Replikation nicht benötigt wird. Die Aktivität als Transkriptionsfaktor wird über die Phosphorylierung des Proteins reguliert. Nichtphosphoryliertes VP30 unterstützt die Synthese der viralen mRNAs in einem Minigenomsystem, während das phosphorylierte VP30 die Transkription nicht aktivieren kann. Im ersten Teil der vorliegenden Arbeit wurde der Einfluss der VP30 Phosphorylierung auf die Regulation des Übergangs von viraler Transkription zu Replikation untersucht. Dabei stand im Vordergrund der Einfluss der Phosphorylierung auf die Replikation. Mit Hilfe von phospho-mimetischen Mutanten des VP30 konnte gezeigt werden, dass phosphoryliertes VP30 die Replikation fördert, während dephosphoryliertes VP30 einen hemmenden Effekt auf die Replikation besaß. Weiterhin wurde die Interaktion von VP30 mit den anderen Komponenten des Nukleokapsidkomplexes untersucht. Dabei wurde eine bisher unbekannte Interaktion des VP30 mit dem Polymerase Co-Faktor VP35 beschrieben, die vom Phosphorylierungsstatus des VP30 beeinflusst wurde. Möglicherweise führt die phosphorylierungsabhängige Interaktion des VP30 mit VP35 zu einem Ausschluss von VP30 aus einem putativen Transkriptasekomplex und dadurch zur Hemmung der Transkription und Stimulierung der Replikation. Außerdem konnte in der vorliegenden Arbeit gezeigt werden, dass eine dynamische Phosphorylierung des VP30 essentiell für die initialen Schritte der Primären Transkription in frühen Stadien des viralen Lebenszyklus ist. Eine dynamische Phosphorylierung des VP30 an Serinrest 29 war ausreichend für die Generierung eines rekombinanten Ebolavirus mit wildtypischen Eigenschaften. Im Gegensatz dazu ließ sich ein stabiles rekombinantes Virus mit Serinrest 30 als einziger Phosphoakzeptorstelle nicht herstellen und resultierte im Auftreten von kompensatorischen Mutationen innerhalb der VP30 Phosphorylierungsdomäne. Die im Rahmen dieser Arbeit gewonnen Ergebnisse unterstreichen die Bedeutung der VP30 Phosphorylierung für den viralen Replikationszyklus: die dynamische Phosphorylierung des VP30 ist in frühen Stadien des Infektionszyklus für die Primäre Transkription essentiell und besitzt ebenfalls einen Einfluss auf den Übergang von viraler Transkription zu Replikation

New Perspectives on the Biogenesis of Viral Inclusion Bodies in Negative-Sense RNA Virus Infections

Infections by negative strand RNA viruses (NSVs) induce the formation of viral inclusion bodies (IBs) in the host cell that segregate viral as well as cellular proteins to enable efficient viral replication. The induction of those membrane-less viral compartments leads inevitably to structural remodeling of the cellular architecture. Recent studies suggested that viral IBs have properties of biomolecular condensates (or liquid organelles), as have previously been shown for other membrane-less cellular compartments like stress granules or P-bodies. Biomolecular condensates are highly dynamic structures formed by liquid-liquid phase separation (LLPS). Key drivers for LLPS in cells are multivalent protein:protein and protein:RNA interactions leading to specialized areas in the cell that recruit molecules with similar properties, while other non-similar molecules are excluded. These typical features of cellular biomolecular condensates are also a common characteristic in the biogenesis of viral inclusion bodies. Viral IBs are predominantly induced by the expression of the viral nucleoprotein (N, NP) and phosphoprotein (P); both are characterized by a special protein architecture containing multiple disordered regions and RNA-binding domains that contribute to different protein functions. P keeps N soluble after expression to allow a concerted binding of N to the viral RNA. This results in the encapsidation of the viral genome by N, while P acts additionally as a cofactor for the viral polymerase, enabling viral transcription and replication. Here, we will review the formation and function of those viral inclusion bodies upon infection with NSVs with respect to their nature as biomolecular condensates

Risk communication and crisis communication in infectious disease outbreaks in Germany: what is being done, and what needs to be done

Objective: Risk communication plays a central role in the management of infectious disease. The World Health Organization's 2005 International Health Regulations have highlighted the need for countries to strengthen their capacities in this area to ensure effective responses to public health emergencies. We surveyed laboratories, hospitals, and public health institutions in Germany to detail the current situation regarding risk communication and crisis management and to identify which areas require further development. Methods: A mixed methods approach was adopted. An initial questionnaire was distributed to relevant persons in laboratories and hospitals, and semistructured interviews were conducted with selected participants. Representatives from state public health authorities, federal agencies, and media also were interviewed to add additional contextual information to the questionnaire responses. Results: Based on the responses received, the universal sense among key stakeholders was that risk communication and crisis communication measures must be improved. Collaborative working was a consistent theme, with participants suggesting that a partnering strategy could help to improve performance. This approach could be achieved through better coordination between groups, for example, through a knowledge-sharing policy. Conclusions: More research is needed on how such collaboration might be implemented, along with a general conceptual framework for risk communication to underpin the overall strategy. (Disaster Med Public Health Preparedness. 2014;0:1-6)

Marburg biosafety and biosecurity scale (MBBS): a framework for risk assessment and risk communication

Current risk assessment and risk communication of biosafety and biosecurity concerns lack a convenient metric and conceptual framework. The absence of such a systematic tool makes communication more difficult and can lead to ambiguous public perception of and response to laboratory biosafety incidents and biosecurity threats. A new 7-category scoring scale is proposed for incidents and situations in laboratories related to the handling of human and animal pathogens. The scale aims to help clarify risk categories, facilitate coordination and communication, and improve public understanding of risk related to biosafety and biosecurity