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PubMed2025

Conserved Filovirus Proteins as Targets of Broad-Spectrum Antivirals.

Filoviruses are enveloped, non-segmented, negative-strand RNA viruses belonging to the Filoviridae family, which includes five genera: Ebolavirus, Marburgvirus, Cuevavirus, Striavirus, and Thamnovirus. Members of this family cause severe and, often, fatal hemorrhagic fevers in humans and non-human primates, with high mortality rates. To date, only two filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), are known to infect humans and are listed as priority pathogens by the World Health Organization due to their potential for re-emergence and the current lack of effective vaccines and antiviral treatments. In this study, we identify and characterize conserved binding sites within key filoviral proteins to support the development of broad-spectrum, direct-acting antiviral agents. We validated the significance of these conserved regions for drug discovery using existing experimental data. Our analysis revealed notably high sequence similarity among proteins from filoviruses capable of infecting humans (EBOV, TAFV, BDBV, SUDV, MARV, and RAVV) compared to those from non-zoonotic species, with the highest conservation observed in the L and VP40 proteins-both critical for viral genome transcription and replication. Furthermore, we compiled and analyzed available experimental data on known antiviral compounds targeting these proteins, identifying several agents with cross-filovirus activity, including Galidesivir, Remdesivir, and Favipiravir. The integrated approach described here-combining sequence and structural conservation analysis with chemical structure and antiviral activity data-demonstrates a strategy that could be extended to the development of broad-spectrum therapeutics across multiple viral families.

Broad Spectrum Antiviral↗
PubMed2025

Amplification of Filovirus Genomes from Clinical Samples for Next Generation Sequencing.

Viral genome sequencing has become a critical tool in outbreak mitigation. Due to their small size relative to the host genome, viral genomes comprise a small fraction of next generation sequencing reads in clinical samples when using unbiased sequencing approaches. Long-range polymerase chain reaction facilitates the amplification of viral genomes from clinical and environmental samples with minimal primer sites, allowing researchers to target regions of the genome that are conserved across available variants. Here, we describe the amplification and sequencing of the Ebola virus genome from tissue samples collected from infected nonhuman primates. This protocol facilitates full viral genome recovery from as low as 103 median tissue culture infectious doses per milliliter.

High-Throughput Nucleotide Sequencing↗
PubMed2026

The Re-Emergence of Bundibugyo Ebolavirus in Uganda and the Democratic Republic of Congo: Epidemiological Drivers, Response Strategies, and Implications for Global Health Security.

Bundibugyo ebolavirus (BDBV) is one of the least studied species within the genus Orthoebolavirus (family Filoviridae), despite its capacity to cause severe Ebola virus disease (EVD) with substantial mortality. First identified during a 2007-2008 outbreak in Bundibugyo District, western Uganda (149 reported cases, 37 deaths; case-fatality rate [CFR] approximately 25-36%), BDBV re-emerged in 2012 in Orientale Province, Democratic Republic of the Congo (DRC) (57-59 cases, 29-34 deaths; CFR 34-58%), before resurfacing in Ituri Province, DRC, in April-May 2026. By 11 August 2026, this third outbreak had grown to 4566 laboratory-confirmed cases and 2128 deaths (CFR ≈ 47%) across five DRC provinces and Uganda, becoming the largest, fastest-growing BDBV epidemic on record and the second-largest Ebola-family outbreak overall. This narrative review, not a systematic review or meta-analysis, summarizes peer-reviewed literature, preprints, and official situation reports from WHO, Africa CDC, US CDC, ECDC, and national health ministries, identified through PubMed, Scopus, Web of Science, Google Scholar, and Embase from inception to 12 August 2026, to examine BDBV historical evolution, virology and pathogenesis, drivers of re-emergence, surveillance and response, therapeutic and vaccine gaps, and global health security implications. The 2026 outbreak, unfolding amid conflict and mass displacement in eastern DRC, has been marked by an estimated basic reproduction number of 1.4-2.1 (central estimate 1.71), disproportionate infection among healthcare workers (7.2% of confirmed cases in DRC, 20% in Uganda), and the continued absence of licensed BDBV-specific vaccines or therapeutics. Findings underscore the need for sustained genomic and ecological surveillance, decentralized rapid diagnostics, broadly protective pan-filovirus vaccines, conflict-sensitive response strategies, and strengthened Uganda-DRC collaboration. Because the evidence base for the ongoing outbreak remains preliminary, findings should be interpreted cautiously and revisited as further peer-reviewed data emerge.

Bundibugyo ebolavirus↗
PubMed2025

Transcription- and Replication-Competent Virus-like Particle Systems for Marburg Virus.

Here, we describe the transcription- and replication-competent virus-like particle (trVLP) system for Marburg virus (MARV), which recapitulates transcription and replication of the viral genome in addition to viral particle assembly, egress, and entry. This protocol includes instructions for transfections for producer and acceptor cells and the use of trVLPs for infection.

Marburgvirus↗
PubMed2025

Droplet-Based Single-Cell 3' mRNA Sequencing of Marburg Virus-Infected Samples.

Single-cell technologies are continually evolving with emerging methods that are gradually uncovering the central DNA-RNA-protein dogma. Single-cell RNA sequencing is one arm of a multi-omic approach that achieves an astounding level of granularity to reveal the complexity of virus-host interactions at the transcriptomic level. Cell tropism, virus replication, pathogenesis, and gene expression changes mediated by the virus and the host's immune response to infection are just some areas of study that are gaining better clarity due to the high-resolution analysis afforded by the technology.We describe a single-cell sequencing protocol for Marburg virus infection in vivo using nonhuman primate blood and the 10× Chromium Next GEM single-cell genomics methodology. Working with pathogens of high consequence is logistically complicated, requiring containment in biosafety level (BSL)-4 laboratories and harsh inactivation procedures before samples can safely be removed to lower biosafety conditions. We provide procedural insight into sample isolation and processing conducted in BSL-4 and describe the requirements for safe sample removal without jeopardizing quality for down-stream sequencing and analysis in BSL-2 conditions. Characterization of complicated biological processes mediated by high-containment pathogens, typically restricted to analogous model systems, e.g., minigenome, can be achieved using live virus.

Animals↗
PubMed2025

Proximity Proteomics to Profile Ebola Virus Protein Interactome in Its Functional Context.

Proximity labeling-based proteomics (proximity proteomics) has emerged as a popular and versatile approach to illuminate the molecular interactions between viruses and their hosts. In this approach, a proximity labeling enzyme tag is fused to a bait protein and labels neighboring proteins with a chemical handle such as biotin, allowing for downstream affinity purification. Compared to another widely used technique, affinity purification coupled mass spectrometry, proximity proteomics enables the detection of low affinity or transient interactors that might have important functions in the viral life cycle. Further, proximity proteomics can identify interactors of a labile bait protein, of which affinity purification is technically challenging. Here, we describe a proximity proteomic protocol to identify cellular interactors of the Ebola virus polymerase. A similar strategy is readily applicable to elucidate the virus-host interactions for Marburg virus.

Ebolavirus↗
PubMed2025

Marburg Virus Minigenome Assays.

This chapter describes minigenome systems for Marburg virus (MARV), which reconstitute the viral polymerase complex functions of gene expression and genome replication. Procedures covered herein include passage and seeding of cells, transfection, sample collection, and reporter gene assays.

Marburgvirus↗
PubMed2025

Ebola virus VP35 NNLNS motif modulates viral RNA synthesis and MIB2-mediated signaling.

Ebola virus (EBOV) is a nonsegmented, negative-sense virus (NNSV) with a single-stranded RNA genome. EBOV encodes for a limited number of proteins and thus depends on host factors to facilitate viral replication and pathogenesis. Of the virus-encoded proteins, multifunctional EBOV VP35 (eVP35) is necessary for host immune evasion and viral RNA synthesis. Previous proteomics studies identified an interaction between eVP35 and the host E3 ubiquitin ligase Mindbomb 2 (MIB2). Here, we show how an NNLNS (Asn-Asn-Leu-Asn-Ser) motif (residues 201 to 205) within eVP35 serves as a binding site for MIB2. This motif is critical for eVP35-dependent inhibition of MIB2-mediated interferon induction. It is also important for EBOV RNA synthesis as MIB2 binding to eVP35 inhibited EBOV minigenome activity. Altogether, these findings highlight the importance of the eVP35 protein and the role of host factors in EBOV infection.

Ebolavirus↗