A live-cell imaging system for visualizing the transport of Marburg virus nucleocapsid-like structures.

BACKGROUND: Live-cell imaging is a powerful tool for visualization of the spatio-temporal dynamics of moving signals in living cells. Although this technique can be utilized to visualize nucleocapsid transport in Marburg virus (MARV)- or Ebola virus-infected cells, the experiments require biosafety level-4 (BSL-4) laboratories, which are restricted to trained and authorized individuals. METHODS: To overcome this limitation, we developed a live-cell imaging system to visualize MARV nucleocapsid-like structures using fluorescence-conjugated viral proteins, which can be conducted outside BSL-4 laboratories. RESULTS: Our experiments revealed that nucleocapsid-like structures have similar transport characteristics to those of nucleocapsids observed in MARV-infected cells, both of which are mediated by actin polymerization. CONCLUSIONS: We developed a non-infectious live cell imaging system to visualize intracellular transport of MARV nucleocapsid-like structures. This system provides a safe platform to evaluate antiviral drugs that inhibit MARV nucleocapsid transport.

Rousette Bat Dendritic Cells Overcome Marburg Virus-Mediated Antiviral Responses by Upregulation of Interferon-Related Genes While Downregulating Proinflammatory Disease Mediators.

Dysregulated and maladaptive immune responses are at the forefront of human diseases caused by infection with zoonotic viral hemorrhagic fever viruses. Elucidating mechanisms of how the natural animal reservoirs of these viruses coexist with these agents without overt disease, while permitting sufficient replication to allow for transmission and maintenance in a population, is important for understanding the viral ecology and spillover to humans. The Egyptian rousette bat (ERB) has been identified as a reservoir for Marburg virus (MARV), a filovirus and the etiological agent of the highly lethal Marburg virus disease. Little is known regarding how these bats immunologically respond to MARV infection. In humans, macrophages and dendritic cells (DCs) are primary targets of infection, and their dysregulation is thought to play a central role in filovirus diseases, by disturbing their normal functions as innate sensors and adaptive immune response facilitators while serving as amplification and dissemination agents for the virus. The infection status and responses to MARV in bat myeloid-lineage cells are uncharacterized and likely represent an important modulator of the bat's immune response to MARV infection. Here, we generate DCs from the bone marrow of rousette bats. Infection with a bat isolate of MARV resulted in a low level of transcription in these cells and significantly downregulated DC maturation and adaptive immune-stimulatory pathways while simultaneously upregulating interferon-related pathogen-sensing pathways. This study provides a first insight into how the bat immune response is directed toward preventing aberrant inflammatory responses while mounting an antiviral response to defend against MARV infection.IMPORTANCE Marburg viruses (MARVs) cause severe human disease resulting from aberrant immune responses. Dendritic cells (DCs) are primary targets of infection and are dysregulated by MARV. Dysregulation of DCs facilitates MARV replication and virus dissemination and influences downstream immune responses that result in immunopathology. Egyptian rousette bats (ERBs) are natural reservoirs of MARV, and infection results in virus replication and shedding, with asymptomatic control of the virus within weeks. The mechanisms that bats employ to appropriately respond to infection while avoiding disease are unknown. Because DC infection and modulation are important early events in human disease, we measured the transcriptional responses of ERB DCs to MARV. The significance of this work is in identifying cell type-specific coevolved responses between ERBs and MARV, which gives insight into how bat reservoirs are able to harbor MARV and permit viral replication, allowing transmission and maintenance in the population while simultaneously preventing immunopathogenesis.

Marburg virus regulates the IRE1/XBP1-dependent unfolded protein response to ensure efficient viral replication.

Viruses regulate cellular signalling pathways to ensure optimal viral replication. During Marburg virus (MARV) infection, large quantities of the viral glycoprotein GP are produced in the ER; this may result in the activation of the unfolded protein response (UPR). The most conserved pathway to trigger UPR is initiated by IRE1. Activation of IRE1 results in auto-phosphorylation, splicing of the XBP1 mRNA and translation of the XBP1s protein. XBP1s binds cis-acting UPR elements (UPRE) which leads to the enhanced expression of genes which should restore ER homeostasis. XBP1u protein is translated, if IRE1 is not activated. Here we show that ectopic expression of MARV GP activated the IRE1-XBP1 axis of UPR as monitored by UPRE luciferase assays. However, while at 24 h of infection with MARV IRE1 was phosphorylated, expression of XBP1s was only slightly enhanced and UPRE activity was not detected. The IRE1-XBP1 axis was not active at 48 h p.i. Co-expression studies of MARV proteins demonstrated that the MARV protein VP30 suppressed UPRE activation. Co-immunoprecipitation analyses revealed an RNA-dependent interaction of VP30 with XBP1u. Knock-out of IRE1 supported MARV infection at late time points. Taken together, these results suggest that efficient MARV propagation requires specific regulation of IRE1 activity.

Calu-3 cells are largely resistant to entry driven by filovirus glycoproteins and the entry defect can be rescued by directed expression of DC-SIGN or cathepsin L.

Priming of the viral glycoprotein (GP) by the cellular proteases cathepsin B and L (CatB, CatL) is believed to be essential for cell entry of filoviruses. However, pseudotyping systems that predominantly produce non-filamentous particles have frequently been used to prove this concept. Here, we report that GP-mediated entry of retroviral-, rhabdoviral and filoviral particles depends on CatB/CatL activity and that this effect is cell line-independent. Moreover, we show that the human cell line Calu-3, which expresses low amounts of CatL, is largely resistant to entry driven by diverse filovirus GPs. Finally, we demonstrate that Calu-3 cell entry mediated by certain filovirus GPs can be rescued upon directed expression of CatL or DC-SIGN. Our results identify Calu-3 cells as largely resistant to filovirus GP-driven entry and demonstrate that entry is limited at the stage of virion attachment and GP priming.

Marburg virus pathogenesis - differences and similarities in humans and animal models.

Marburg virus (MARV) is a highly pathogenic virus associated with severe disease and mortality rates as high as 90%. Outbreaks of MARV are sporadic, deadly, and often characterized by a lack of resources and facilities to diagnose and treat patients. There are currently no approved vaccines or treatments, and the chaotic and infrequent nature of outbreaks, among other factors, makes testing new countermeasures during outbreaks ethically and logistically challenging. Without field efficacy studies, researchers must rely on animal models of MARV infection to assess the efficacy of vaccines and treatments, with the limitations being the accuracy of the animal model in recapitulating human pathogenesis. This review will compare various animal models to the available descriptions of human pathogenesis and aims to evaluate their effectiveness in modeling important aspects of Marburg virus disease.

Phosphorylated VP30 of Marburg Virus Is a Repressor of Transcription

The filoviruses Marburg virus (MARV) and Ebola virus (EBOV) cause hemorrhagic fever in humans and nonhuman primates, with high case fatality rates. MARV VP30 is known to be phosphorylated and to interact with nucleoprotein (NP), but its role in regulation of viral transcription is disputed. Here, we analyzed phosphorylation of VP30 by mass spectrometry, which resulted in identification of multiple phosphorylated amino acids. Modeling the full-length three-dimensional structure of VP30 and mapping the identified phosphorylation sites showed that all sites lie in disordered regions, mostly in the N-terminal domain of the protein. Minigenome analysis of the identified phosphorylation sites demonstrated that phosphorylation of a cluster of amino acids at positions 46 through 53 inhibits transcription. To test the effect of VP30 phosphorylation on its interaction with other MARV proteins, coimmunoprecipitation analyses were performed. They demonstrated the involvement of VP30 phosphorylation in interaction with two other proteins of the MARV ribonucleoprotein complex, NP and VP35. To identify the role of protein phosphatase 1 (PP1) in the identified effects, a small molecule, 1E7-03, targeting a noncatalytic site of the enzyme that previously was shown to increase EBOV VP30 phosphorylation was used. Treatment of cells with 1E7-03 increased phosphorylation of VP30 at a cluster of phosphorylated amino acids from Ser-46 to Thr-53, reduced transcription of MARV minigenome, enhanced binding to NP and VP35, and dramatically reduced replication of infectious MARV particles. Thus, MARV VP30 phosphorylation can be targeted for development of future antivirals such as PP1-targeting compounds. IMPORTANCE The largest outbreak of MARV occurred in Angola in 2004 to 2005 and had a 90% case fatality rate. There are no approved treatments available for MARV. Development of antivirals as therapeutics requires a fundamental understanding of the viral life cycle. Because of the close similarity of MARV to another member of Filoviridae family, EBOV, it was assumed that the two viruses have similar mechanisms of regulation of transcription and replication. Here, characterization of the role of VP30 and its phosphorylation sites in transcription of the MARV genome demonstrated differences from those of EBOV. The identified phosphorylation sites appeared to inhibit transcription and appeared to be involved in interaction with both NP and VP35 ribonucleoproteins. A small molecule targeting PP1 inhibited transcription of the MARV genome, effectively suppressing replication of the viral particles. These data demonstrate the possibility developing antivirals based on compounds targeting PP1.

Deep-sequencing of Marburg virus genome during sequential mouse passaging and cell-culture adaptation reveals extensive changes over time.

Marburg virus (MARV) has caused outbreaks of filoviral hemorrhagic fever since its discovery in 1967. The largest and deadliest outbreak occurred in Angola in 2005, with 252 cases and 227 deaths. In 2014, we developed a mouse-adapted MARV, Angola variant through serial passaging in mice. The mouse-adapted MARV exhibits many of the hallmarks of MARV disease in humans. By applying deep-sequencing to every passage of the virus, we are able to study virus evolution in this host with surprising precision. We show that two regions go through substantial changes: the intergenic region between NP and VP35, as well as the first 100 amino acids of the VP40 protein. Our results also reveal that there were profound changes during the production of the final virus stock in cell culture. Overall, our results show that a handful of regions carry most of the mutations acquired during the adaptation of the virus to a new host and that many mutations become fixed very early during the adaptation process.

GS-5734 and its parent nucleoside analog inhibit Filo-, Pneumo-, and Paramyxoviruses.

GS-5734 is a monophosphate prodrug of an adenosine nucleoside analog that showed therapeutic efficacy in a non-human primate model of Ebola virus infection. It has been administered under compassionate use to two Ebola patients, both of whom survived, and is currently in Phase 2 clinical development for treatment of Ebola virus disease. Here we report the antiviral activities of GS-5734 and the parent nucleoside analog across multiple virus families, providing evidence to support new indications for this compound against human viruses of significant public health concern.

Ebola Virus and Marburg Virus in Human Milk Are Inactivated by Holder Pasteurization.

BACKGROUND: Potential donors of human milk are screened for Ebola virus (EBOV) using standard questions, but testing for EBOV and Marburg virus (MARV) is not part of routine serological testing performed by milk banks. Research aim: This study tested the hypothesis that EBOV would be inactivated in donor human milk (DHM) by standard pasteurization techniques (Holder) used in all North American nonprofit milk banks. METHODS: Milk samples were obtained from a nonprofit milk bank. They were inoculated with EBOV (Zaire strain) and MARV (Angola strain) and processed by standard Holder pasteurization technique. Plaque assays for EBOV and MARV were performed to detect the presence of virus after pasteurization. RESULTS: Neither EBOV nor MARV was detectable by viral plaque assay in DHM or culture media samples, which were pasteurized by the Holder process. CONCLUSION: EBOV and MARV are safely inactivated in human milk by standard Holder pasteurization technique. Screening for EBOV or MARV beyond questionnaire and self-deferral is not needed to ensure safety of DHM for high-risk infants.

Corning HYPERFlask® for viral amplification and production of diagnostic reagents.

Viral preparations are essential components in diagnostic research and development. The production of large quantities of virus traditionally is done by infecting numerous tissue culture flasks or roller bottles, which require large incubators and/or roller bottle racks. The Corning HYPERFlask® is a multilayer flask that uses a gas permeable film to provide gas exchange between the cells and culture medium and the atmospheric environment. This study evaluated the suitability of the HYPERFlask for production of Lassa, Ebola, Bundibugyo, Reston, and Marburg viruses and compared it to more traditional methods using tissue culture flasks and roller bottles. The HYPERFlask produced cultures were equivalent in virus titer and indistinguishable in immunodiagnostic assays. The use of the Corning HYPERFlask for viral production is a viable alternative to traditional tissue culture flasks and roller bottles. HYPERFlasks allow for large volumes of virus to be produced in a small space without specialized equipment.

Marburg virus inclusions: A virus-induced microcompartment and interface to multivesicular bodies and the late endosomal compartment.

Filovirus infection of target cells leads to the formation of virally induced cytoplasmic inclusions that contain viral nucleocapsids at different stages of maturation. While the role of the inclusions has been unclear since the identification of Marburg and Ebola viruses, it recently became clear that the inclusions are the sites of viral replication, nucleocapsid formation and maturation. Live cell imaging analyses revealed that mature nucleocapsids are transported from inclusions to the filopodia, which represent the major budding sites. Moreover, inclusions recruit cellular proteins that have been shown to support the transport of nucleocapsids. For example, the tumor susceptibility gene 101 protein (Tsg101) interacts with a late domain motif in the nucleocapsid protein NP and recruits the actin-nucleation factor IQGAP1. Complexes of nucleocapsids together with Tsg101 and IQGAP1 are then co-transported along actin filaments. We detected additional proteins (Alix, Nedd4 and the AAA-type ATPase VPS4) of the endosomal sorting complex required for transport (ESCRT) that are recruited into inclusions. Together, the results suggest that nucleocapsids recruit the machinery that enhances viral budding at the plasma membrane. Furthermore, we identified Lamp1 as a marker of the late endosomal compartment in inclusions, while ER, Golgi, TGN and early endosomal markers were absent. In addition, we observed that LC3, a marker of autophagosomal membranes, was present in inclusions. The 3D structures of inclusions show an intricate structure that seems to accommodate an intimate cooperation between cellular and viral components with the intention to support viral transport and budding.

[Characterization of Marburg virus morphology].

Ebola virus (EBOV) and Marburg virus (MARV) belong to the family Filoviridae. Filoviruses cause severe filovirus hemorrhagic fever (FHF) in humans, with high case fatality rates, and represent potential agents for bioterrorism and biological weapons. It is necessary to keep surveillance of filoviruses, even though there is no report of their isolation and patients in China so far. To characterize MARV morphology, the Lake Victoria marburgvirus--Leiden was stained negatively and observed under a transmission electron microscope which is one of important detection methods for filoviruses in emergencies and bioterrorism. MARV showed pleomorphism, with filamentous, rod-shaped, cobra-like, spherical, and branch-shaped particles of uniform diameter but different lengths. Pleomorphism of negatively stained MARV is summarized in this article, so as to provide useful information for possible electron microscopic identification of filoviruses in China.

Standardization of the filovirus plaque assay for use in preclinical studies.

The filovirus plaque assay serves as the assay of choice to measure infectious virus in a cell culture, blood, or homogenized tissue sample. It has been in use for more than 30 years and is the generally accepted assay used to titrate virus in samples from animals treated with a potential antiviral therapeutic or vaccine. As these animal studies are required for the development of vaccines and therapeutics under the FDA Animal Rule, it is essential to have a standardized assay to compare their efficacies against the various filoviruses. Here, we present an evaluation of the conditions under which the filovirus plaque assay performs best for the Ebola virus Kikwit variant and the Angola variant of Marburg virus. The indicator cell type and source, inoculum volumes, length of incubation and general features of filovirus biology as visualized in the assay are addressed in terms of the impact on the sample viral titer calculations. These optimization studies have resulted in a plaque assay protocol which can be used for preclinical studies, and as a standardized protocol for use across institutions, to aid in data comparison. This protocol will be validated for use in GLP studies supporting advanced development of filovirus therapeutics and vaccines.

Establishment of fruit bat cells (Rousettus aegyptiacus) as a model system for the investigation of filoviral infection.

BACKGROUND: The fruit bat species Rousettus aegyptiacus was identified as a potential reservoir for the highly pathogenic filovirus Marburg virus. To establish a basis for a molecular understanding of the biology of filoviruses in the reservoir host, we have adapted a set of molecular tools for investigation of filovirus replication in a recently developed cell line, R06E, derived from the species Rousettus aegyptiacus. METHODOLOGY/PRINCIPAL FINDINGS: Upon infection with Ebola or Marburg viruses, R06E cells produced viral titers comparable to VeroE6 cells, as shown by TCID(50) analysis. Electron microscopic analysis of infected cells revealed morphological signs of filovirus infection as described for human- and monkey-derived cell lines. Using R06E cells, we detected an unusually high amount of intracellular viral proteins, which correlated with the accumulation of high numbers of filoviral nucleocapsids in the cytoplasm. We established protocols to produce Marburg infectious virus-like particles from R06E cells, which were then used to infect naïve target cells to investigate primary transcription. This was not possible with other cell lines previously tested. Moreover, we established protocols to reliably rescue recombinant Marburg viruses from R06E cells. CONCLUSION/SIGNIFICANCE: These data indicated that R06E cells are highly suitable to investigate the biology of filoviruses in cells derived from their presumed reservoir.

Basolateral budding of Marburg virus: VP40 retargets viral glycoprotein GP to the basolateral surface.

Virus budding from the basolateral domain of infected polarized cells could be one of the mechanisms underlying the quick systemic infection induced by Marburg virus (MARV). We found that MARV buds from the basolateral pole of hepatocytes and bile epithelial cells in infected guinea pigs, which leads to the release of infectious virus into the vascular system. Basolateral budding might be orchestrated by the basolaterally located MARV matrix protein VP40, which induces a partial relocalization of MARV glycoprotein from the apical to the basolateral plasma membrane. This redistribution is a prerequisite for budding of infectious virions from the basolateral domain.

Budding of Marburgvirus is associated with filopodia.

Viruses exploit the cytoskeleton of host cells to transport their components and spread to neighbouring cells. Here we show that the actin cytoskeleton is involved in the release of Marburgvirus (MARV) particles. We found that peripherally located nucleocapsids and envelope precursors of MARV are located either at the tip or at the side of filopodial actin bundles. Importantly, viral budding was almost exclusively detected at filopodia. Inhibiting actin polymerization in MARV-infected cells significantly diminished the amount of viral particles released into the medium. This suggested that dynamic polymerization of actin in filopodia is essential for efficient release of MARV. The viral matrix protein VP40 plays a key role in the release of MARV particles and we found that the intracellular localization of recombinant VP40 and its release in form of virus-like particles were strongly influenced by overexpression or inhibition of myosin 10 and Cdc42, proteins important in filopodia formation and function. We suggest that VP40, which is capable of interacting with viral nucleocapsids, provides an interface of MARV subviral particles and filopodia. As filopodia are in close contact with neighbouring cells, usurpation of these structures may facilitate spread of MARV to adjacent cells.

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.

Outbreak of Marburg hemorrhagic fever in Angola: a review of the history of the disease and its biological aspects.

Transmission of a dangerous infectious disease threatens not merely a local population but the world at large as the result of immigration and increased and faster travel. Any outbreak elicits considerable concern and demands that various precautionary methods be instituted and that the disease be contained as quickly as possible. Recently, an old disease, one that may have been present for centuries and was identified decades ago, reared its ugly head, killing more than 200 people before it was contained. Fortunately, the disease, Marburg hemorrhagic fever, was limited to a small geographic area, but the devastation of lives was much greater than that of many epidemics and was a warning of the numerous factors, including fear, lack of understanding, and deception, that can exacerbate the spread of disease and hinder implementation of restraints. This article reviews the history of the disease caused by Marburg virus and its biological components.

Organisation of health care during an outbreak of Marburg haemorrhagic fever in the Democratic Republic of Congo, 1999.

Organising health care was one of the tasks of the International Scientific and Technical Committee during the 1998-1999 outbreak in Durba/Watsa, in the north-eastern province (Province Orientale), Democratic Republic of Congo. With the logistical support of Médecins sans Frontières (MSF), two isolation units were created: one at the Durba Reference Health Centre and the other at the Okimo Hospital in Watsa. Between May 6th, the day the isolation unit was installed and May 19th, 15 patients were admitted to the Durba Health Centre. In only four of them were the diagnosis of Marburg haemorrhagic fever (MHF) confirmed by laboratory examination. Protective equipment was distributed to health care workers and family members caring for patients. Information about MHF, modes of transmission and the use of barrier nursing techniques was provided to health care workers and sterilisation procedures were reviewed. In contrast to Ebola outbreaks, there was little panic among health care workers and the general public in Durba and all health services remained operational.