ABSTRACT
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.
Subject(s)
Macaca fascicularis , Marburg Virus Disease , Marburgvirus , Vesiculovirus , Viral Vaccines , Animals , Marburg Virus Disease/prevention & control , Marburg Virus Disease/immunology , Marburg Virus Disease/virology , Marburgvirus/immunology , Marburgvirus/genetics , Viral Vaccines/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/genetics , Vesiculovirus/genetics , Vesiculovirus/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Vaccines, Synthetic/immunology , Vaccines, Synthetic/administration & dosage , Disease Models, Animal , Vaccination , Male , Female , Vaccine Efficacy , Genetic Vectors , Immunogenicity, VaccineABSTRACT
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.
Subject(s)
Chiroptera , Marburg Virus Disease , Marburgvirus , Siphonaptera , Animals , Chiroptera/virology , Marburgvirus/genetics , Marburgvirus/physiology , Siphonaptera/virology , Marburg Virus Disease/virology , Marburg Virus Disease/transmission , Disease Reservoirs/virology , Viremia , Flea Infestations/veterinary , Flea Infestations/transmission , Flea Infestations/virology , RNA, Viral/genetics , EgyptABSTRACT
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.
Subject(s)
Antiviral Agents , Ebolavirus , Pyrroles , Virus Internalization , Pyrroles/pharmacology , Pyrroles/chemistry , Pyrroles/chemical synthesis , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/chemical synthesis , Humans , Structure-Activity Relationship , Ebolavirus/drug effects , Virus Internalization/drug effects , Heterocyclic Compounds/pharmacology , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/chemical synthesis , Filoviridae/drug effects , Marburgvirus/drug effectsABSTRACT
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.
Subject(s)
Marburg Virus Disease , Marburgvirus , Viral Vaccines , Animals , Cricetinae , Viral Vaccines/immunology , Marburgvirus/immunology , Marburg Virus Disease/prevention & control , Marburg Virus Disease/immunology , Disease Models, Animal , Adenoviridae/genetics , Adenoviridae/immunology , Vesiculovirus/immunology , Vesiculovirus/genetics , Antibodies, Viral/immunology , Vaccination/methodsABSTRACT
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.
Subject(s)
Endocytosis , Marburgvirus , Viral Matrix Proteins , Virion , rab GTP-Binding Proteins , rab GTP-Binding Proteins/metabolism , rab GTP-Binding Proteins/genetics , Marburgvirus/physiology , Marburgvirus/genetics , Marburgvirus/metabolism , Humans , Viral Matrix Proteins/metabolism , Viral Matrix Proteins/genetics , Animals , Virion/metabolism , Microtubules/metabolism , Microtubules/virology , Virus Release , Cell Line , HEK293 Cells , Virus ReplicationSubject(s)
Antiviral Agents , Disease Outbreaks , Marburg Virus Disease , Marburgvirus , Adult , Female , Humans , Male , Middle Aged , Equatorial Guinea/epidemiology , Equatorial Guinea/ethnology , Marburg Virus Disease/complications , Marburg Virus Disease/diagnosis , Marburg Virus Disease/drug therapy , Marburg Virus Disease/epidemiology , Marburgvirus/isolation & purification , Antiviral Agents/therapeutic use , Viral Load , Child, Preschool , AgedABSTRACT
Although next-generation sequencing (NGS) has been instrumental in determining the genomic sequences of emerging RNA viruses, de novo sequence determination often lacks sufficient coverage of the 5' and 3' ends of the viral genomes. Since the genome ends of RNA viruses contain the transcription and genome replication promoters that are essential for viral propagation, a lack of terminal sequence information hinders the efforts to study the replication and transcription mechanisms of emerging and re-emerging viruses. To circumvent this, we have developed a novel method termed ViBE-Seq (Viral Bona Fide End Sequencing) for the high-resolution sequencing of filoviral genome ends using a simple yet robust protocol with high fidelity. This technique allows for sequence determination of the 5' end of viral RNA genomes and mRNAs with as little as 50 ng of total RNA. Using the Ebola virus and Marburg virus as prototypes for highly pathogenic, re-emerging viruses, we show that ViBE-Seq is a reliable technique for rapid and accurate 5' end sequencing of filovirus RNA sourced from virions, infected cells, and tissue obtained from infected animals. We also show that ViBE-Seq can be used to determine whether distinct reverse transcriptases have terminal deoxynucleotidyl transferase activity. Overall, ViBE-Seq will facilitate the access to complete sequences of emerging viruses.
Subject(s)
Ebolavirus , Filoviridae , Genome, Viral , High-Throughput Nucleotide Sequencing , RNA, Viral , Sequence Analysis, RNA , RNA, Viral/genetics , High-Throughput Nucleotide Sequencing/methods , Ebolavirus/genetics , Sequence Analysis, RNA/methods , Filoviridae/genetics , Marburgvirus/genetics , Humans , AnimalsABSTRACT
Quantitative immunoassays, such as the traditional enzyme-linked immunosorbent assay (ELISA), are used to determine concentrations of an antigen in a matrix of unknown antigen concentration. Magnetic immunoassays, such as the Luminex xMAP technology, allow for the simultaneous detection of multiple analytes and offer heightened sensitivity, specificity, low sample volume requirements, and high-throughput capabilities. Here, we describe a quantitative immunoassay using the Luminex MAGPIX® System to determine the antigen concentration from liquid samples with unknown concentrations. In detail, we describe a newly developed assay for determining production yields of Drosophila S2-produced Marburg virus (MARV) glycoprotein in insect-cell-culture-derived supernatant. The potential applications of this assay could extend to the quantification of viral antigens in fluids derived from both in vitro and in vivo models infected with live MARV, thereby providing additional applications for virological research.
Subject(s)
Antigens, Viral , Microspheres , Animals , Immunoassay/methods , Antigens, Viral/immunology , Antigens, Viral/analysis , Marburgvirus/immunology , Marburgvirus/isolation & purification , Drosophila , Cell Culture Techniques/methods , Cell Line , Enzyme-Linked Immunosorbent Assay/methodsABSTRACT
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.
Subject(s)
Computational Biology , Marburg Virus Disease , Marburgvirus , Viral Vaccines , Marburgvirus/immunology , Marburg Virus Disease/prevention & control , Marburg Virus Disease/immunology , Viral Vaccines/immunology , Computational Biology/methods , Animals , Humans , Epitopes, T-Lymphocyte/immunology , Epitopes, T-Lymphocyte/genetics , Epitopes/immunology , Epitopes/genetics , Epitopes/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , ImmunoinformaticsABSTRACT
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.
Subject(s)
Antiviral Agents , Ebolavirus , Marburgvirus , Molecular Docking Simulation , Ebolavirus/drug effects , Ebolavirus/metabolism , Marburgvirus/drug effects , Marburgvirus/metabolism , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Viral Matrix Proteins/metabolism , Viral Matrix Proteins/antagonists & inhibitors , Viral Matrix Proteins/chemistry , Hemorrhagic Fever, Ebola/drug therapy , Hemorrhagic Fever, Ebola/virology , Humans , Computer Simulation , Molecular Dynamics Simulation , Viral Regulatory and Accessory ProteinsABSTRACT
Marburg virus infection in humans is associated with case fatality rates that can reach up to 90%, but to date, there are no approved vaccines or monoclonal antibody (mAb) countermeasures. Here, we immunized Rhesus macaques with multivalent combinations of filovirus glycoprotein (GP) antigens belonging to Marburg, Sudan, and Ebola viruses to generate monospecific and cross-reactive antibody responses against them. From the animal that developed the highest titers of Marburg virus GP-specific neutralizing antibodies, we sorted single memory B cells using a heterologous Ravn virus GP probe and cloned and characterized a panel of 34 mAbs belonging to 28 unique lineages. Antibody specificities were assessed by overlapping pepscan and binding competition analyses, revealing that roughly a third of the lineages mapped to the conserved receptor binding region, including potent neutralizing lineages that were confirmed by negative stain electron microscopy to target this region. Additional lineages targeted a protective region on GP2, while others were found to possess cross-filovirus reactivity. Our study advances the understanding of orthomarburgvirus glycoprotein antigenicity and furthers efforts to develop candidate antibody countermeasures against these lethal viruses. IMPORTANCE: Marburg viruses were the first filoviruses characterized to emerge in humans in 1967 and cause severe hemorrhagic fever with average case fatality rates of ~50%. Although mAb countermeasures have been approved for clinical use against the related Ebola viruses, there are currently no approved countermeasures against Marburg viruses. We successfully isolated a panel of orthomarburgvirus GP-specific mAbs from a macaque immunized with a multivalent combination of filovirus antigens. Our analyses revealed that roughly half of the antibodies in the panel mapped to regions on the glycoprotein shown to protect from infection, including the host cell receptor binding domain and a protective region on the membrane-anchoring subunit. Other antibodies in the panel exhibited broad filovirus GP recognition. Our study describes the discovery of a diverse panel of cross-reactive macaque antibodies targeting orthomarburgvirus and other filovirus GPs and provides candidate immunotherapeutics for further study and development.
Subject(s)
Antibodies, Monoclonal , Antibodies, Neutralizing , Antibodies, Viral , Cross Reactions , Macaca mulatta , Marburg Virus Disease , Marburgvirus , Animals , Marburgvirus/immunology , Antibodies, Viral/immunology , Antibodies, Neutralizing/immunology , Antibodies, Monoclonal/immunology , Marburg Virus Disease/immunology , Marburg Virus Disease/prevention & control , Cross Reactions/immunology , Glycoproteins/immunology , Viral Envelope Proteins/immunology , Immunization , Humans , Ebolavirus/immunology , Antigens, Viral/immunologyABSTRACT
Viral haemorrhagic fevers (VHF) pose a significant threat to human health. In recent years, VHF outbreaks caused by Ebola, Marburg and Lassa viruses have caused substantial morbidity and mortality in West and Central Africa. In 2022, an Ebola disease outbreak in Uganda caused by Sudan virus resulted in 164 cases with 55 deaths. In 2023, a Marburg disease outbreak was confirmed in Equatorial Guinea and Tanzania resulting in over 49 confirmed or suspected cases; 41 of which were fatal. There are no clearly defined correlates of protection against these VHF, impeding targeted vaccine development. Any vaccine developed should therefore induce strong and preferably long-lasting humoral and cellular immunity against these viruses. Ideally this immunity should also cross-protect against viral variants, which are known to circulate in animal reservoirs and cause human disease. We have utilized two viral vectored vaccine platforms, an adenovirus (ChAdOx1) and Modified Vaccinia Ankara (MVA), to develop a multi-pathogen vaccine regime against three filoviruses (Ebola virus, Sudan virus, Marburg virus) and an arenavirus (Lassa virus). These platform technologies have consistently demonstrated the capability to induce robust cellular and humoral antigen-specific immunity in humans, most recently in the rollout of the licensed ChAdOx1-nCoV19/AZD1222. Here, we show that our multi-pathogen vaccines elicit strong cellular and humoral immunity, induce a diverse range of chemokines and cytokines, and most importantly, confers protection after lethal Ebola virus, Sudan virus and Marburg virus challenges in a small animal model.
Subject(s)
Ebolavirus , Hemorrhagic Fever, Ebola , Lassa Fever , Lassa virus , Marburg Virus Disease , Marburgvirus , Animals , Mice , Ebolavirus/immunology , Lassa virus/immunology , Marburgvirus/immunology , Hemorrhagic Fever, Ebola/prevention & control , Hemorrhagic Fever, Ebola/immunology , Lassa Fever/immunology , Lassa Fever/prevention & control , Marburg Virus Disease/immunology , Marburg Virus Disease/prevention & control , Viral Vaccines/immunology , Humans , Vaccination , Female , Antibodies, Viral/immunology , Immunogenicity, Vaccine , Ebola Vaccines/immunologyABSTRACT
The increasing frequency of filovirus outbreaks in African countries has led to a pressing need for the development of effective antifilovirus agents. In continuation of our previous research on the antifilovirus activity of monoterpenoid derivatives, we synthesized a series of (+)-fenchol and (-)-isopinocampheol derivatives by varying the type of heterocycle and linker length. Derivatives with an N-alkylpiperazine cycle proved to be the most potent antiviral compounds, with half-maximal inhibitory concentration (IC50) 1.4-20 µÐ against Lenti-EboV-GP infection and 11.3-47 µÐ against Lenti-MarV-GP infection. Mechanism-of-action experiments revealed that the compounds may exert their action by binding to surface glycoproteins (GPs). It was demonstrated that the binding of the synthesized compounds to the Marburg virus GP is less efficient as compared to the Ebola virus GP. Furthermore, it was shown that the compounds possess lysosomotropic properties. Thus, the antiviral activity may be due to dual effects. This study offers new antiviral agents that are worthy of further exploration.
Subject(s)
Antiviral Agents , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/chemical synthesis , Humans , Virus Internalization/drug effects , Structure-Activity Relationship , Ebolavirus/drug effects , Molecular Structure , Dose-Response Relationship, Drug , Animals , Microbial Sensitivity Tests , Chlorocebus aethiops , Marburgvirus/drug effectsABSTRACT
Marburg virus disease (MVD) is a dreadful but exceptional disease. Formerly mainly identified in Uganda, Angola and the Democratic Republic of Congo, it has recently appeared in the Republic of Guinea, Ghana, Equatorial Guinea and Tanzania, adding West Africa to the affected regions. Humans become infected through exposure to bats Roussettus aegyptiacus or during unprotected care of infected people. Five cases are linked to travellers, the last one dates to 2008 and involved a visit to caves colonized by bats. At present, there is no specific treatment or vaccine. Despite its rarity, adventurous travelers should be aware of the risks of exposure and avoid entering places inhabited by bats.
La maladie à virus Marburg est une maladie redoutable mais exceptionnelle. Autrefois identifiée en Ouganda, Angola et République démocratique du Congo, elle a récemment fait son apparition en République de Guinée, au Ghana, en Guinée équatoriale et en Tanzanie, ajoutant l'Afrique de l'Ouest aux régions touchées. Les humains s'infectent lors d'une exposition avec les chauves-souris roussettes d'Égypte ou lors de la prise en charge sans protection de personnes infectées. Cinq cas sont liés à des voyageurs, le dernier remonte à 2008 et était associé à la visite de grottes colonisées par des roussettes d'Égypte. Actuellement, il n'existe aucun traitement spécifique ni vaccin. Malgré sa rareté, les voyageurs aventureux doivent être informés des risques d'exposition et éviter de pénétrer dans des lieux habités par des chauves-souris.
Subject(s)
Marburgvirus , Travel , Female , Humans , Male , Marburg Virus Disease/epidemiology , Marburg Virus Disease/transmission , Marburg Virus Disease/virology , Marburgvirus/isolation & purification , Viral Zoonoses/epidemiology , Viral Zoonoses/transmission , Viral Zoonoses/virology , Chiroptera/virologyABSTRACT
Ebola virus (EBOV) and Marburg virus (MARV), members of the Filoviridae family, are highly pathogenic and can cause hemorrhagic fevers, significantly impacting human society. Bats are considered reservoirs of these viruses because related filoviruses have been discovered in bats. However, due to the requirement for maximum containment laboratories when studying infectious viruses, the characterization of bat filoviruses often relies on pseudoviruses and minigenome systems. In this study, we used RACE technology to sequence the 3'-leader and 5'-trailer of Menglà virus (MLAV) and constructed a minigenome. Similar to MARV, the transcription activities of the MLAV minigenome are independent of VP30. We further assessed the effects of polymorphisms at the 5' end on MLAV minigenome activity and identified certain mutations that decrease minigenome reporter efficiency, probably due to alterations in the RNA secondary structure. The reporter activity upon recombination of the 3'-leaders and 5'-trailers of MLAV, MARV, and EBOV with those of the homologous or heterologous minigenomes was compared and it was found that the polymerase complex and leader and trailer sequences exhibit intrinsic specificities. Additionally, we investigated whether the polymerase complex proteins from EBOV and MARV support MLAV minigenome RNA synthesis and found that the homologous system is more efficient than the heterologous system. Remdesivir efficiently inhibited MLAV as well as EBOV replication. In summary, this study provides new information on bat filoviruses and the minigenome will be a useful tool for high-throughput antiviral drug screening.
Subject(s)
Ebolavirus , Genome, Viral , Marburgvirus , Animals , Genome, Viral/genetics , Ebolavirus/genetics , Humans , Marburgvirus/genetics , Mengovirus/genetics , Virus Replication , RNA, Viral/genetics , Alanine/analogs & derivatives , Alanine/pharmacology , Chiroptera/virology , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Adenosine Monophosphate/metabolism , Filoviridae/genetics , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
Marburg virus (MARV) is one of the filovirus species that cause deadly hemorrhagic fever in humans, with mortality rates up to 90%. Neutralizing antibodies represent ideal candidates to prevent or treat virus disease. However, no antibody has been approved for MARV treatment to date. In this study, we identified a novel human antibody named AF-03 that targeted MARV glycoprotein (GP). AF-03 possessed a high binding affinity to MARV GP and showed neutralizing and protective activities against the pseudotyped MARV in vitro and in vivo. Epitope identification, including molecular docking and experiment-based analysis of mutated species, revealed that AF-03 recognized the Niemann-Pick C1 (NPC1) binding domain within GP1. Interestingly, we found the neutralizing activity of AF-03 to pseudotyped Ebola viruses (EBOV, SUDV, and BDBV) harboring cleaved GP instead of full-length GP. Furthermore, NPC2-fused AF-03 exhibited neutralizing activity to several filovirus species and EBOV mutants via binding to CI-MPR. In conclusion, this work demonstrates that AF-03 represents a promising therapeutic cargo for filovirus-caused disease.
Subject(s)
Ebolavirus , Hemorrhagic Fever, Ebola , Marburgvirus , Humans , Antibodies, Viral , Molecular Docking Simulation , Glycoproteins , Hemorrhagic Fever, Ebola/prevention & control , Ebolavirus/chemistryABSTRACT
Bats are increasingly recognized as reservoirs of emerging zoonotic pathogens. Egyptian rousette bats (ERBs) are the known reservoir of Marburg virus (MARV), a filovirus that causes deadly Marburg virus disease (MVD) in humans. However, ERBs harbor MARV asymptomatically, likely due to a coadapted and specific host immunity-pathogen relationship. Recently, we measured transcriptional responses in MARV-infected ERB whole tissues, showing that these bats possess a disease tolerant strategy that limits pro-inflammatory gene induction, presumably averting MVD-linked immunopathology. However, the host resistant strategy by which ERBs actively limit MARV burden remains elusive, which we hypothesize requires localized inflammatory responses unresolvable at bulk-tissue scale. Here, we use dexamethasone to attenuate ERB pro-inflammatory responses and assess MARV replication, shedding and disease. We show that MARV-infected ERBs naturally mount coordinated pro-inflammatory responses at liver foci of infection, comprised of recruited mononuclear phagocytes and T cells, the latter of which proliferate with likely MARV-specificity. When pro-inflammatory responses are diminished, ERBs display heightened MARV replication, oral/rectal shedding and severe MVD-like liver pathology, demonstrating that ERBs balance immunoprotective tolerance with discreet MARV-resistant pro-inflammatory responses. These data further suggest that natural ERB immunomodulatory stressors like food scarcity and habitat disruption may potentiate viral shedding, transmission and therefore outbreak risk.
Subject(s)
Chiroptera , Filoviridae , Marburg Virus Disease , Marburgvirus , Animals , Humans , Marburgvirus/genetics , ImmunityABSTRACT
Marburg virus, a member of the Filoviridae, is the causative agent of Marburg virus disease (MVD), a hemorrhagic fever with a case fatality rate of up to 90 %. Acute kidney injury is common in MVD and is associated with increased mortality, but its pathogenesis in MVD remains poorly understood. Interestingly, autopsies show the presence of viral proteins in different parts of the nephron, particularly in proximal tubular cells (PTC). These findings suggest a potential role for the virus in the development of MVD-related kidney injury. To shed light on this effect, we infected primary human PTC with Lake Victoria Marburg virus and conducted transcriptomic analysis at multiple time points. Unexpectedly, infection did not induce marked cytopathic effects in primary tubular cells at 20 and 40 h post infection. However, gene expression analysis revealed robust renal viral replication and dysregulation of genes essential for different cellular functions. The gene sets mainly downregulated in PTC were associated with the targets of the transcription factors MYC and E2F, DNA repair, the G2M checkpoint, as well as oxidative phosphorylation. Importantly, the downregulated factors comprise PGC-1α, a well-known factor in acute and chronic kidney injury. By contrast, the most highly upregulated gene sets were those related to the inflammatory response and cholesterol homeostasis. In conclusion, Marburg virus infects and replicates in human primary PTC and induces downregulation of processes known to be relevant for acute kidney injury as well as a strong inflammatory response.
Subject(s)
Acute Kidney Injury , Marburgvirus , Humans , Animals , Marburgvirus/genetics , Energy Metabolism , Gene Expression Profiling , ImmunityABSTRACT
Filoviridae is a family of negative-sense RNA viruses with genomes of about 13.1-20.9 kb that infect fish, mammals and reptiles. The filovirid genome is a linear, non-segmented RNA with five canonical open reading frames (ORFs) that encode a nucleoprotein (NP), a polymerase cofactor (VP35), a glycoprotein (GP1,2), a transcriptional activator (VP30) and a large protein (L) containing an RNA-directed RNA polymerase (RdRP) domain. All filovirid genomes encode additional proteins that vary among genera. Several filovirids (e.g., Ebola virus, Marburg virus) are pathogenic for humans and highly virulent. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Filoviridae, which is available at www.ictv.global/report/filoviridae.
Subject(s)
Ebolavirus , Marburgvirus , Rhabdoviridae , Animals , Humans , Ebolavirus/genetics , Rhabdoviridae/genetics , Phylogeny , Genome, Viral , Virus Replication , Mammals/geneticsABSTRACT
Marburg virus, the first filovirus discovered and a close cousin to the Ebola virus, is carried by the Egyptian rousette bat, a common cave-dwelling fruit bat endemic to sub-Saharan Africa whose populations can exceed 50 000 individuals. Community outbreaks of Marburg virus can result in high morbidity rates. In eastern Africa, favorite habitats of these bats include rural subterranean gold mines-sometimes worked illegally-that create environments conducive to zoonotic virus transmission. This commentary on a case describes how outbreaks of Marburg virus disease among people exposed to sub-Saharan African caves and mines containing these bats cause tensions among miners, companies, public health officials, and conservationists.