Lipid Selectivity of Membrane Action of the Fragments of Fusion Peptides of Marburg and Ebola Viruses.

The life cycle of Ebola and Marburg viruses includes a step of the virion envelope fusion with the cell membrane. Here, we analyzed whether the fusion of liposome membranes under the action of fragments of fusion peptides of Ebola and Marburg viruses depends on the composition of lipid vesicles. A fluorescence assay and electron microscopy were used to quantify the fusogenic activity of the virus fusion peptides and to identify the lipid determinants affecting membrane merging. Differential scanning calorimetry of lipid phase transitions revealed alterations in the physical properties of the lipid matrix produced by virus fusion peptides. Additionally, we found that plant polyphenols, quercetin, and myricetin inhibited vesicle fusion induced by the Marburg virus fusion peptide.

Longitudinal proteome-wide antibody profiling in Marburg virus survivors identifies wing domain immunogen for vaccine design.

Limited knowledge exists on the quality of polyclonal antibody responses generated following Marburg virus (MARV) infection and its evolution in survivors. In this study, we evaluate MARV proteome-wide antibody repertoire longitudinally in convalescent phase approximately every six months for five years following MARV infection in ten human survivors. Differential kinetics were observed for IgM vs IgG vs IgA epitope diversity, antibody binding, antibody affinity maturation and Fc-receptor interaction to MARV proteins. Durability of MARV-neutralizing antibodies is low in survivors. MARV infection induces a diverse epitope repertoire with predominance against GP, VP40, VP30 and VP24 that persisted up to 5 years post-exposure. However, the IgM and IgA repertoire declines over time. Within MARV-GP, IgG recognize antigenic sites predominantly in the amino-terminus, wing domain and GP2-heptad repeat. Interestingly, MARV infection generates robust durable FcɣRI, FcɣRIIA and FcɣRIIIA IgG-Fc receptor interactions. Immunization with immunodominant MARV epitopes reveals conserved wing region between GP1 and GP2, induces neutralizing antibodies against MARV. These findings demonstrate that MARV infection generates a diverse, long-lasting, non-neutralizing, IgG antibody repertoire that perturbs disease by FcɣR activity. This information, along with discovery of neutralizing immunogen in wing domain, could aid in development of effective therapeutics and vaccines against Marburg virus.

Epidemiological description of Marburg virus disease outbreak in Kagera region, Northwestern Tanzania.

INTRODUCTION: In March 2023, a Marburg Virus Disease (MVD) outbreak was declared in Kagera region, Northwestern Tanzania. This was the first MVD outbreak in the country. We describe the epidemiological characteristics of MVD cases and contacts. METHODS: The Ministry of Health activated an outbreak response team. Outbreak investigation methods were applied to cases identified through MVD standard case definitions and confirmed through reverse-transcriptase polymerase chain reaction (RT PCR). All identified case contacts were added into the contact listing form and followed up in-person daily for any signs or symptoms for 21 days. Data collected from various forms was managed and analyzed using Excel and QGIS software for mapping. RESULTS: A total of nine MVD cases were reported with eight laboratory-confirmed and one probable. Two of the reported cases were frontline healthcare workers and seven were family related members. Cases were children and adults between 1-59 years of age with a median age of 34 years. Six were males. Six cases died equivalent to a case fatality rate (CFR) of 66.7%. A total of 212 individuals were identified as contacts and two (2) became cases. The outbreak was localized in two geo-administrative wards (Maruku and Kanyangereko) of Bukoba District Council. CONCLUSION: Transmission during this outbreak occurred among family members and healthcare workers who provided care to the cases. The delay in detection aggravated the spread and possibly the consequent fatality but once confirmed the swift response stemmed further transmission containing the disease at the epicenter wards. The outbreak lasted for 72 days but as the origin is still unknown, further research is required to explore the source of this outbreak.

Modified oxymatrine as novel therapeutic inhibitors against Monkeypox and Marburg virus through computational drug design approaches.

Global impact of viral diseases specially Monkeypox (mpox) and Marburg virus, emphasizing the urgent need for effective drug interventions. Oxymatrine is an alkaloid which has been selected and modified using various functional groups to enhance its efficacy. The modifications were evaluated using various computatioanal analysis such as pass prediction, molecular docking, ADMET, and molecular dynamic simulation. Mpox and Marburg virus were chosen as target diseases based on their maximum pass prediction spectrum against viral disease. After that, molecular docking, dynamic simulation, DFT, calculation and ADMET prediction were determined. The main objective of this study was to enhance the efficacy of oxymatrine derivatives through functional group modifications and computational analyses to develop effective drug candidates against mpox and Marburg viruses. The calculated binding affinities indicated strong interactions against both mpox virus and Marburg virus. After that, the molecular dynamic simulation was conducted at 100 ns, which confirmed the stability of the binding interactions between the modified oxymatrine derivatives and target proteins. Then, the modified oxymatrine derivatives conducted theoretical ADMET profiling, which demonstrated their potential for effective drug development. Moreover, HOMO-LUMO calculation was performed to understand the chemical reactivity and physicochemical properties of compounds. This computational analysis indicated that modified oxymatrine derivatives for the treatment of mpox and Marburg virus suggested effective drug candidates based on their binding affinity, drug-like properties, stability and chemical reactivity. However, further experimental validation is necessary to confirm their clinical value and efficacy as therapeutic candidates.

Rational design of self-amplifying virus-like vesicles with Ebola virus glycoprotein as vaccines.

As emerging and re-emerging pathogens, filoviruses, especially Ebola virus (EBOV), pose a great threat to public health and require sustained attention and ongoing surveillance. More vaccines and antiviral drugs are imperative to be developed and stockpiled to respond to unpredictable outbreaks. Virus-like vesicles, generated by alphavirus replicons expressing homogeneous or heterogeneous glycoproteins (GPs), have demonstrated the capacity of self-propagation and shown great potential in vaccine development. Here, we describe a novel class of EBOV-like vesicles (eVLVs) incorporating both EBOV GP and VP40. The eVLVs exhibited similar antigenicity as EBOV. In murine models, eVLVs were highly attenuated and elicited robust GP-specific antibodies with neutralizing activities. Importantly, a single dose of eVLVs conferred complete protection in a surrogate EBOV lethal mouse model. Furthermore, our VLVs strategy was also successfully applied to Marburg virus (MARV), the representative member of the genus Marburgvirus. Taken together, our findings indicate the feasibility of an alphavirus-derived VLVs strategy in combating infection of filoviruses represented by EBOV and MARV, which provides further evidence of the potential of this platform for universal live-attenuated vaccine development.

Inactivation Validation of Ebola, Marburg, and Lassa Viruses in AVL and Ethanol-Treated Viral Cultures.

High-consequence pathogens such as the Ebola, Marburg, and Lassa viruses are handled in maximum-containment biosafety level 4 (BSL-4) laboratories. Genetic material is often isolated from such viruses and subsequently removed from BSL-4 laboratories for a multitude of downstream analyses using readily accessible technologies and equipment available at lower-biosafety level laboratories. However, it is essential to ensure that these materials are free of viable viruses before removal from BSL-4 laboratories to guarantee sample safety. This study details the in-house procedure used for validating the inactivation of Ebola, Marburg, and Lassa virus cultures after incubation with AVL lysis buffer (Qiagen) and ethanol. This study's findings show that no viable virus was detectable when high-titer cultures of Ebola, Marburg, and Lassa viruses were incubated with AVL lysis buffer for 10 min, followed by an equal volume of 95% ethanol for 3 min, using a method with a sensitivity of ≤0.8 log10 TCID50 as the limit of detection.

Efficacy and Immunogenicity of a Recombinant Vesicular Stomatitis Virus-Vectored Marburg Vaccine in Cynomolgus Macaques.

Filoviruses, like the Marburg (MARV) and Ebola (EBOV) viruses, have caused outbreaks associated with significant hemorrhagic morbidity and high fatality rates. Vaccines offer one of the best countermeasures for fatal infection, but to date only the EBOV vaccine has received FDA licensure. Given the limited cross protection between the EBOV vaccine and Marburg hemorrhagic fever (MHF), we analyzed the protective efficacy of a similar vaccine, rVSV-MARV, in the lethal cynomolgus macaque model. NHPs vaccinated with a single dose (as little as 1.6 × 107 pfu) of rVSV-MARV seroconverted to MARV G-protein prior to challenge on day 42. Vaccinemia was measured in all vaccinated primates, self-resolved by day 14 post vaccination. Importantly, all vaccinated NHPs survived lethal MARV challenge, and showed no significant alterations in key markers of morbid disease, including clinical signs, and certain hematological and clinical chemistry parameters. Further, apart from one primate (from which tissues were not collected and no causal link was established), no pathology associated with Marburg disease was observed in vaccinated animals. Taken together, rVSV-MARV is a safe and efficacious vaccine against MHF in cynomolgus macaques.

Attempted Transmission of Marburg Virus by Bat-Associated Fleas Thaumapsylla breviceps breviceps (Ischnopsyllidae: Thaumapsyllinae) to the Egyptian Rousette Bat (Rousettus aegyptiacus).

Egyptian rousette bats (ERBs) are implicated as reservoir hosts for Marburg virus (MARV), but natural mechanisms involved in maintenance of MARV in ERB populations remain undefined. A number of hematophagous ectoparasites, including fleas, parasitize bats. Subcutaneous (SC) inoculation of ERBs with MARV consistently results in viremia, suggesting that infectious MARV could be ingested by blood-sucking ectoparasites during feeding. In our study, MARV RNA was detected in fleas that took a blood meal during feeding on viremic bats on days 3, 7, and 11 after SC inoculation. Virus concentration in individual ectoparasites was consistent with detectable levels of viremia in the blood of infected host bats. There was neither seroconversion nor viremia in control bats kept in close contact with MARV-infected bats infested with fleas for up to 40 days post-exposure. In fleas inoculated intracoelomically, MARV was detected up to 14 days after intracoelomic (IC) inoculation, but the virus concentration was lower than that delivered in the inoculum. All bats that had been infested with inoculated, viremic fleas remained virologically and serologically negative up to 38 days after infestation. Of 493 fleas collected from a wild ERB colony in Matlapitsi Cave, South Africa, where the enzootic transmission of MARV occurs, all tested negative for MARV RNA. While our findings seem to demonstrate that bat fleas lack vectorial capacity to transmit MARV biologically, their role in mechanical transmission should not be discounted. Regular blood-feeds, intra- and interhost mobility, direct feeding on blood vessels resulting in venous damage, and roosting behaviour of ERBs provide a potential physical bridge for MARV dissemination in densely populated cave-dwelling bats by fleas. The virus transfer might take place through inoculation of skin, mucosal membranes, and wounds when contaminated fleas are squashed during auto- and allogrooming, eating, biting, or fighting.

The cellular protein phosphatase 2A is a crucial host factor for Marburg virus transcription.

Little is known regarding the molecular mechanisms that highly pathogenic Marburg virus (MARV) utilizes to transcribe and replicate its genome. Previous studies assumed that dephosphorylation of the filoviral transcription factor VP30 supports transcription, while phosphorylated VP30 reduces transcription. Here, we focused on the role of the host protein phosphatase 2A (PP2A) for VP30 dephosphorylation and promotion of viral transcription. We could show that MARV NP interacts with the subunit B56 of PP2A, as previously shown for the Ebola virus, and that this interaction is important for MARV transcription activity. Inhibition of the interaction between PP2A and NP either by mutating the B56 binding motif encoded on NP, or the use of a PP2A inhibitor, induced VP30 hyperphosphorylation, and as a consequence a decrease of MARV transcription as well as viral growth. These results suggest that NP plays a key role in the dephosphorylation of VP30 by recruiting PP2A. Generation of recombinant (rec) MARV lacking the PP2A-B56 interaction motif on NP was not possible suggesting an essential role of PP2A-mediated VP30 dephosphorylation for the MARV replication cycle. Likewise, we were not able to generate recMARV containing VP30 phosphomimetic mutants indicating that dynamic cycles of VP30 de- and rephosphorylation are a prerequisite for an efficient viral life cycle. As the specific binding motifs of PP2A-B56 and VP30 within NP are highly conserved among the filoviral family, our data suggest a conserved mechanism for filovirus VP30 dephosphorylation by PP2A, revealing the host factor PP2A as a promising target for pan-filoviral therapies. IMPORTANCE: Our study elucidates the crucial role of host protein phosphatase 2A (PP2A) in Marburg virus (MARV) transcription. The regulatory subunit B56 of PP2A facilitates VP30 dephosphorylation, and hence transcription activation, via binding to NP. Our results, together with previous data, reveal a conserved mechanism of filovirus VP30 dephosphorylation by host factor PP2A at the NP interface and provide novel insights into potential pan-filovirus therapies.

Vaccine Platform Comparison: Protective Efficacy against Lethal Marburg Virus Challenge in the Hamster Model.

Marburg virus (MARV), a filovirus, was first identified in 1967 in Marburg, Germany, and Belgrade, former Yugoslavia. Since then, MARV has caused sporadic outbreaks of human disease with high case fatality rates in parts of Africa, with the largest outbreak occurring in 2004/05 in Angola. From 2021 to 2023, MARV outbreaks occurred in Guinea, Ghana, New Guinea, and Tanzania, emphasizing the expansion of its endemic area into new geographical regions. There are currently no approved vaccines or therapeutics targeting MARV, but several vaccine candidates have shown promise in preclinical studies. We compared three vaccine platforms simultaneously by vaccinating hamsters with either a single dose of an adenovirus-based (ChAdOx-1 MARV) vaccine, an alphavirus replicon-based RNA (LION-MARV) vaccine, or a recombinant vesicular stomatitis virus-based (VSV-MARV) vaccine, all expressing the MARV glycoprotein as the antigen. Lethal challenge with hamster-adapted MARV 4 weeks after vaccination resulted in uniform protection of the VSV-MARV and LION-MARV groups and 83% of the ChAdOx-1 MARV group. Assessment of the antigen-specific humoral response and its functionality revealed vaccine-platform-dependent differences, particularly in the Fc effector functions.

Cheminformatics-based analysis identified (Z)-2-(2,5-dimethoxy benzylidene)-6-(2-(4-methoxyphenyl)-2-oxoethoxy) benzofuran-3(2H)-one as an inhibitor of Marburg replication by interacting with NP.

The highly pathogenic Marburg virus (MARV) is a member of the Filoviridae family, a non-segmented negative-strand RNA virus. This article represents the computer-aided drug design (CADD) approach for identifying drug-like compounds that prevent the MARV virus disease by inhibiting nucleoprotein, which is responsible for their replication. This study used a wide range of in silico drug design techniques to identify potential drugs. Out of 368 natural compounds, 202 compounds passed ADMET, and molecular docking identified the top two molecules (CID: 1804018 and 5280520) with a high binding affinity of -6.77 and -6.672 kcal/mol, respectively. Both compounds showed interactions with the common amino acid residues SER_216, ARG_215, TYR_135, CYS_195, and ILE_108, which indicates that lead compounds and control ligands interact in the common active site/catalytic site of the protein. The negative binding free energies of CID: 1804018 and 5280520 were -66.01 and -31.29 kcal/mol, respectively. Two lead compounds were re-evaluated using MD modeling techniques, which confirmed CID: 1804018 as the most stable when complexed with the target protein. PC3 of the (Z)-2-(2,5-dimethoxybenzylidene)-6-(2-(4-methoxyphenyl)-2-oxoethoxy) benzofuran-3(2H)-one (CID: 1804018) was 8.74 %, whereas PC3 of the 2'-Hydroxydaidzein (CID: 5280520) was 11.25 %. In this study, (Z)-2-(2,5-dimethoxybenzylidene)-6-(2-(4-methoxyphenyl)-2-oxoethoxy) benzofuran-3(2H)-one (CID: 1804018) unveiled the significant stability of the proteins' binding site in ADMET, Molecular docking, MM-GBSA and MD simulation analysis studies, which also showed a high negative binding free energy value, confirming as the best drug candidate which is found in Angelica archangelica which may potentially inhibit the replication of MARV nucleoprotein.

N-Substituted Pyrrole-Based Heterocycles as Broad-Spectrum Filoviral Entry Inhibitors.

Since the largest and most fatal Ebola virus epidemic during 2014-2016, there have been several consecutive filoviral outbreaks in recent years, including those in 2021, 2022, and 2023. Ongoing outbreak prevalence and limited FDA-approved filoviral therapeutics emphasize the need for novel small molecule treatments. Here, we showcase the structure-activity relationship development of N-substituted pyrrole-based heterocycles and their potent, submicromolar entry inhibition against diverse filoviruses in a target-based pseudovirus assay. Inhibitor antiviral activity was validated using replication-competent Ebola, Sudan, and Marburg viruses. Mutational analysis was used to map the targeted region within the Ebola virus glycoprotein. Antiviral counter-screen and phospholipidosis assays were performed to demonstrate the reduced off-target activity of these filoviral entry inhibitors. Favorable antiviral potency, selectivity, and drug-like properties of the N-substituted pyrrole-based heterocycles support their potential as broad-spectrum antifiloviral treatments.

Marburg virus exploits the Rab11-mediated endocytic pathway in viral-particle production.

Filoviruses produce viral particles with characteristic filamentous morphology. The major viral matrix protein, VP40, is trafficked to the plasma membrane and promotes viral particle formation and subsequent viral egress. In the present study, we assessed the role of the small GTPase Rab11-mediated endocytic pathway in Marburg virus (MARV) particle formation and budding. Although Rab11 was predominantly localized in the perinuclear region, it exhibited a more diffuse distribution in the cytoplasm of cells transiently expressing MARV VP40. Rab11 was incorporated into MARV-like particles. Expression of the dominant-negative form of Rab11 and knockdown of Rab11 decreased the amount of VP40 fractions in the cell periphery. Moreover, downregulation of Rab11 moderately reduced the release of MARV-like particles and authentic MARV. We further demonstrated that VP40 induces the distribution of the microtubule network toward the cell periphery, which was partly associated with Rab11. Depolymerization of microtubules reduced the accumulation of VP40 in the cell periphery along with viral particle formation. VP40 physically interacted with α-tubulin, a major component of microtubules, but not with Rab11. Taken together, these results suggested that VP40 partly interacts with microtubules and facilitates their distribution toward the cell periphery, leading to the trafficking of transiently tethering Rab11-positive vesicles toward the cell surface. As we previously demonstrated the role of Rab11 in the formation of Ebola virus particles, the results here suggest that filoviruses in general exploit the vesicle-trafficking machinery for proper virus-particle formation and subsequent egress. These pathways may be a potential target for the development of pan-filovirus therapeutics.IMPORTANCEFiloviruses, including Marburg and Ebola viruses, produce distinct filamentous viral particles. Although it is well known that the major viral matrix protein of these viruses, VP40, is trafficked to the cell surface and promotes viral particle production, details regarding the associated molecular mechanisms remain unclear. To address this knowledge gap, we investigated the role of the small GTPase Rab11-mediated endocytic pathway in this process. Our findings revealed that Marburg virus exploits the Rab11-mediated vesicle-trafficking pathway for the release of virus-like particles and authentic virions in a microtubule network-dependent manner. Previous findings demonstrated that Rab11 is also involved in Ebola virus-particle production. Taken together, these data suggest that filoviruses, in general, may hijack the microtubule-dependent vesicle-trafficking machinery for productive replication. Therefore, this pathway presents as a potential target for the development of pan-filovirus therapeutics.

Design of a novel multi-epitope vaccine against Marburg virus using immunoinformatics studies.

Marburg virus (MARV) is a highly contagious and virulent agent belonging to Filoviridae family. MARV causes severe hemorrhagic fever in humans and non-human primates. Owing to its highly virulent nature, preventive approaches are promising for its control. There is currently no approved drug or vaccine against MARV, and management mainly involves supportive care to treat symptoms and prevent complications. Our aim was to design a novel multi-epitope vaccine (MEV) against MARV using immunoinformatics studies. In this study, various proteins (VP35, VP40 and glycoprotein precursor) were used and potential epitopes were selected. CTL and HTL epitopes covered 79.44% and 70.55% of the global population, respectively. The designed MEV construct was stable and expressed in Escherichia coli (E. coli) host. The physicochemical properties were also acceptable. MARV MEV candidate could predict comprehensive immune responses such as those of humoral and cellular in silico. Additionally, efficient interaction to toll-like receptor 3 (TLR3) and its agonist (ß-defensin) was predicted. There is a need for validation of these results using further in vitro and in vivo studies.

In silico exploration of deep-sea fungal metabolites as inhibitor of Ebola and Marburg VP35 and VP40.

VP30 and VP40 proteins of Ebola and Marburg viruses have been recognized as potential targets for antiviral drug development due to their essential roles in the viral lifecycle. Targeting these proteins could disrupt key stages of the viral replication process, inhibiting the viruses' ability to propagate and cause disease. The current study aims to perform molecular docking and virtual screening on deep-sea fungal metabolites targeting Marburg virus VP40 Dimer, matrix protein VP40 from Ebola virus Sudan, Ebola VP35 Interferon Inhibitory Domain, and VP35 from Marburg virus. The top ten compounds for each protein target were chosen using the glide score. All the compounds obtained indicate a positive binding interaction. Furthermore, AdmetSAR was utilized to investigate the pharmacokinetics of the inhibitors chosen. Gliotoxin was used as a ligand with Marburg virus VP40 Dimer, Austinol with matrix protein VP40 from Ebola virus Sudan, Ozazino-cyclo-(2,3-dihydroxyl-trp-tyr) with Ebola VP35 Interferon Inhibitory Domain, and Dehydroaustinol with VP35 from Marburg virus. MD modeling and MMPBSA studies were used to provide a better understanding of binding behaviors. Pre-clinical experiments can assist validate our in-silico studies and assess whether the molecule can be employed as an anti-viral drug.