Filoviruses, which include ebola-, marburg-, and cuevaviruses, are notifiable animal disease agents. The natural hosts of these viruses are bats; however, livestock, and particularly pigs, have also been shown to harbor filoviruses. As zoonotic agents filoviruses can be transmitted from animals to humans, and then cause severe hemorrhagic fevers with extremely high case fatality rates. The unprecedented outbreak of ebolavirus hemorrhagic fever in Western Africa in 2014/2015 has demonstrated that these viruses constitute a major threat to public health.
Our focus is the molecular biology of filoviruses, with a particular emphasis on virus-host interactions and the identification of pathogenicity determinants. This aims at finding commonalities between filoviruses and other virus families, which can serve as targets for broad spectrum antivirals that are active not only against filoviruses, but also against other RNA viruses, and at gaining the ability to predict the pathogenic potential of novel filoviruses.
A methodological focus is the development and application of reverse genetics systems for filoviruses. This includes the generation of recombinant infectious ebolaviruses using full-length clone systems, which we work on in the BSL 4-lab for zoonoses of the FLI, as well as life-cycle-modelling systems, which allow work on filoviruses without the need for high containment laboratories.
- Hoenen T, Groseth A, Feldmann H. 2019. Therapeutic strategies to target the Ebola virus life cycle. Nature Reviews in Microbiology 17(10):593-606.
- Groseth A, Hoenen T. 2017. Forty years of ebolavirus molecular biology: Understanding a novel disease agent through the development and application of new technologies. Methods in Molecular Biology 1628:15-38.
- Hoenen T, Brandt J, Caì Y, Kuhn JH, Finch C. 2017. Reverse genetics of filoviruses. Current Topics in Microbiology and Immunology 411:421-445.
Development, optimization and application of life cycle modelling systems
Filoviruses are considered biosafety level 4 (BSL4) agents, which limits experimental work to a few maximum containing laboratories worldwide. In order to investigate the life cycle of filoviruses outside of such laboratories, we have developed life cycle modeling systems. These systems are based on minigenomes (miniature versions of the filoviral genome, from which essential virus genes have been removed, and which, therefore, can be used under BSL1 conditions) and model either individual aspects of the viral life cycle or its entirety. The most advanced of these systems, the so-called tetracistronic transcription and replication-competent virus-like particle (trVLP) system (Figure 1), allows us to model the entire life cycle over multiple infectious cycles using only filoviral components. Several variants of this system are being made available to scientific entities through the European Virus Archive, and can be used for multiple applications, including the identification and characterization of new antiviral drugs, as well as basic research on filoviruses. Our laboratory is constantly optimizing and further developing these systems, and applies them to study current questions in filovirus biology.
Links and further information about tetracistronic trVLP systems
- link to the European Virus Archive (EVAg): https://www.european-virus-archive.com/
- further information about the tetracistronic trVLP systems available through EVAg
- Wendt L, Bostedt L, Hoenen T, Groseth A. 2019. High-throughput screening for negative-stranded hemorrhagic fever viruses using reverse genetics. Antiviral Research 170:104569.
- Kämper L, Zierke L, Schmidt ML, Müller A, Wendt L, Brandt J, Hartmann E, Braun S, Holzerland J, Groseth A, Hoenen T. 2019. Assessment of the function and intergenus-compatibility of Ebola and Lloviu virus proteins. Journal of General Virology 100(5):760-772.
- Schmidt ML, Tews BA, Groseth A, Hoenen T. 2018. Generation and optimization of a GFP-expressing trVLP system for Ebola virus. Journal of Infectious Diseases 218 (suppl. 5):S360.
- Wendt L, Kämper L, Schmidt ML, Mettenleiter TC, Hoenen T. 2018. Analysis of a putative late domain using an Ebola virus transcription and replication-competent virus-like particle system. Journal of Infectious Diseases 218 (suppl. 5):S355.
- Schmidt ML, Hoenen T. 2017. Characterization of the catalytic center of the Ebola virus L polymerase. PLoS Neglected Tropical Diseases 11(10):e0005996.
- further literature
Study of the role of the host factors NXF1, DDX39B, and CAD for the filovirus life cycle
As intracellular parasites viruses rely on host cell proteins for many if not all aspects of their life cycle. Interactions of viral factors with host cell proteins represent interesting targets for therapeutics, since it is more difficult for viruses to develop resistance to such therapeutics than to therapeutics that are directly and exclusively acting against viral factors. In addition, various viruses often use identical interactions or host factors, so that targeted therapeutics often have a broader spectrum of action than classic, directly acting antiviral therapies. In a project funded by the German Research Foundation (Deutsche Forschungsgemeinschaft - DFG) we are working on three host factors (NXF1, DDX39B, and CAD) that play a role in viral genome replication and transcription (Figure 2). On the one hand we are characterizing interactions between these cellular proteins and viral factors on a biochemical level, and on the other hand we investigate the functional role of the host proteins in the viral life cycle. For this purpose, both life-cycle modeling systems and recombinant filoviruses are being used.
- Brandt J, Wendt L, Bodmer B, Mettenleiter TC, Hoenen T. 2020. The cellular protein CAD is recruited into Ebola virus inclusion bodies by the nucleoprotein NP to facilitate genome replication and transcription. Cells 9(5):1126. https://www.ncbi.nlm.nih.gov/pubmed/32370067
- Wendt L, Brandt J, Bodmer B, Reiche S, Schmidt ML, Traeger S, Hoenen T. 2020. The Ebola virus nucleoprotein recruits the nuclear RNA export factor NXF1 into inclusion bodies to facilitate viral protein expression. Cells 9(1):187. https://www.ncbi.nlm.nih.gov/pubmed/31940815
- Martin S, Chiramel AI, Schmidt ML, Chen Y, Whitt N, Watt A, Dunham EC, Shifflett K, Traeger S, Leske A, Buehler E, Martellaro C, Brandt J, Wendt L, Müller A, Peitsch S, Best SM, Stech J, Finke S, Römer-Oberdörfer A, Groseth A, Feldmann H, Hoenen T. 2018. A genome-wide siRNA screen identifies a druggable host pathway essential for the Ebola virus life cycle. Genome Medicine 10(1):58.
Study of the biology of filoviral inclusion bodies
During infection, filovirus proteins accumulate to form structures called inclusion bodies. These inclusion bodies play essential functions in viral genome replication and transcription of mRNAs, as well as in the assembly of nucleocapsids. However, the mechanism underlying the generation and organization of inclusion bodies are unknown. For a number of other negative-sense RNA viruses similar inclusion bodies have now been recognized as being liquid organelles, i.e. microenvironments that are not defined by delineating membranes, but are rather held together by liquid-liquid phase separation. In a project funded by the German Research Foundation (Deutsche Forschungsgemeinschaft - DFG) we are investigating whether filoviral inclusion bodies also are liquid organelles, and what mechanisms underlie their formation. Further, we want to understand whether phase separation plays a role in nucleocapsid export from inclusion bodies. To facilitate these studies, we are combining state-of-the-art live cell microscopy technology under BSL4 conditions with recombinant, reporter-expressing filoviruses. Ultimately, these studies are aimed at identifying commonalities between filoviruses and other negative-sense RNA viruses, and to provide information regarding possible molecular targets for antivirals targeting an essential aspect of the life cycle of these viruses.
Comparison of highly pathogenic and apathogenic filoviruses as part of the VISION research consortium
While filoviruses are best known for their highly pathogenic representatives such as Ebola virus (case fatality rate of up to 60%) and Marburg virus (case fatality rate of up to 80%), also viruses that are most likely apathogenic for humans such as Reston virus exist. However, the factors that determine these differences in pathogenic potential are currently unknown. Therefore, it is currently impossible to provide an evidence-based risk assessment for novel filoviruses such as Lloviu virus, which was recently discovered in Spain and Hungary. As part of the research consortium VISION (illuminating VIrus-hoSt interactIONs of high-consequence zoonotic viruses in vitro and in vivo) we compared specific parameters of the life cycle of Ebola virus and Reston virus (i.e. efficiency and kinetics of virus entry into host cells, efficiency and kinetics of genome replication, efficiency and kinetics of budding) to better understand a potential contribution of differences in these processes to pathogenic potential. Our studies revealed that Ebola virus and Reston virus differ in their efficiency and kinetics of genome replication, but not of entry or budding of virus particles. In parallel, we explored novel strategies to tag these viruses with fluorescent proteins in order to become able to study their spread and pathogenesis in infected host organisms, and were able to rescue a number of recombinant, reporter-expressing viruses that are now being used for further studies.
- Bodmer B, Greßler J, Schmidt ML, Holzerland J, Brandt J, Braun S, Groseth A, Hoenen T. 2020. Differences in viral RNA synthesis but not budding or entry contribute to the In vitro attenuation of Reston virus compared to Ebola virus. Microorganisms 8(8):1215. https://www.ncbi.nlm.nih.gov/pubmed/32796523
Investigations of the role of livestock animals for filovirus biology in West Africa
The 2013-2016 Ebola virus epidemic in West Africa has demonstrated that filoviruses are not only a problem for African countries, but that outbreaks of this kind can have worldwide consequences. At the same time, it has highlighted gaps in our knowledge of the biology and epidemiology of filoviruses. One of these gaps is the potential role of livestock as relevant hosts. While it is known that pigs can become infected with Asian filoviruses, and also seem capable of transmitting these viruses to humans, the importance of pigs or other farm animals for African filoviruses is poorly understood. In a project funded by the Federal Ministry of Food and Agriculture based on a decision of the German Bundestag through the Federal Agency for Food and Agriculture, the role of livestock for the biology of filoviruses in West Africa was examined in more detail. The focus of the Laboratory for Molecular Biology of Filoviruses was to investigate the ability of the cellular receptor NPC1 from African animal species to allow entry of filoviruses into the host cell, and to develop novel detection methods for previously unknown filoviruses. Furthermore, the laboratory participated in the training of African partners in molecular detection techniques for filoviruses and in sequencing using Nanopore technology.
- Mueller A, Fischer K, Suluku R, Hoenen T. 2019. Sequencing of mRNA from Whole Blood using Nanopore Sequencing. Journal of Visualized Experiments 148.
- Fischer K, Jabaty J, Suluku R, Strecker T, Groseth A, Fehling SK, Balkema-Buschmann A, Koroma B, Schmidt KM, Ataherstone C, Weingartl HM, Mettenleiter TC, Groschup MH, Hoenen T, Diederich S. 2018. Serological evidence for the circulation of Ebolaviruses in pigs from Sierra Leone. Journal of Infectious Diseases 218(suppl_5):S360-S364.
- Hoenen T. 2016. Sequencing of Ebola Virus Genomes Using Nanopore Technology. Bioprotocols 6(21):e1998.