Transcriptomics of Marburg virus-infected primary proximal tubular cells reveals negative correlation of immune response and energy metabolism.

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.

Coordinated inflammatory responses dictate Marburg virus control by reservoir bats.

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.

Contrasting effects of filamin A and B proteins in modulating filovirus entry.

Ebola (EBOV) and Marburg viruses (MARV) cause severe hemorrhagic fever associated with high mortality rates in humans. A better understanding of filovirus-host interactions that regulate the EBOV and MARV lifecycles can provide biological and mechanistic insight critical for therapeutic development. EBOV glycoprotein (eGP) and MARV glycoprotein (mGP) mediate entry into host cells primarily by actin-dependent macropinocytosis. Here, we identified actin-binding cytoskeletal crosslinking proteins filamin A (FLNa) and B (FLNb) as important regulators of both EBOV and MARV entry. We found that entry of pseudotype psVSV-RFP-eGP, infectious recombinant rVSV-eGP-mCherry, and live authentic EBOV and MARV was inhibited in filamin A knockdown (FLNaKD) cells, but was surprisingly enhanced in filamin B knockdown (FLNbKD) cells. Mechanistically, our findings suggest that differential regulation of macropinocytosis by FLNa and FLNb likely contributes to their specific effects on EBOV and MARV entry. This study is the first to identify the filamin family of proteins as regulators of EBOV and MARV entry. These findings may provide insight into the development of new countermeasures to prevent EBOV and MARV infections.

Transcriptional profiling of immune responses in NHPs after low-dose, VSV-based vaccination against Marburg virus.

Infection with Marburg virus (MARV), the causative agent of Marburg virus disease (MVD), results in haemorrhagic disease and high case fatality rates (>40%) in humans. Despite its public health relevance, there are no licensed vaccines or therapeutics to prevent or treat MVD. A vesicular stomatitis virus (VSV)-based vaccine expressing the MARV glycoprotein (VSV-MARV) is currently in clinical development. Previously, a single 10 million PFU dose of VSV-MARV administered 1-5 weeks before lethal MARV challenge conferred uniform protection in nonhuman primates (NHPs), demonstrating fast-acting potential. Additionally, our group recently demonstrated that even a low dose VSV-MARV (1000 PFU) protected NHPs when given 7 days before MARV challenge. In this study, we longitudinally profiled the transcriptional responses of NHPs vaccinated with this low dose of VSV-MARV either 14 or 7 days before lethal MARV challenge. NHPs vaccinated 14 days before challenge presented with transcriptional changes consistent with an antiviral response before challenge. Limited gene expression changes were observed in the group vaccinated 7 days before challenge. After challenge, genes related to lymphocyte-mediated immunity were only observed in the group vaccinated 14 days before challenge, indicating that the length of time between vaccination and challenge influenced gene expression. Our results indicate that a low dose VSV-MARV elicits distinct immune responses that correlate with protection against MVD. A low dose of VSV-MARV should be evaluated in clinical rails as it may be an option to deliver beneficial public health outcomes to more people in the event of future outbreaks.

VP24 matrix proteins of eight filoviruses downregulate innate immune response by inhibiting the interferon-induced pathway.

Filoviruses encode viral protein 24 (VP24) which effectively inhibit the innate immune responses in infected cells. Here we systematically analysed the effects of nine mammalian filovirus VP24 proteins on interferon (IFN)-induced immune response. We transiently expressed Ebola, Bombali, Bundibugyo, Reston, Sudan and Taï Forest ebolavirus (EBOV, BOMV, BDBV, RESTV, SUDV, TAFV, respectively), Lloviu virus (LLOV), Mengla dianlovirus (MLAV) and Marburgvirus (MARV) VP24 proteins and analysed their ability to inhibit IFN-α-induced activation of myxovirus resistance protein 1 (MxA) and interferon-induced transmembrane protein 3 (IFITM3) promoters. In addition, we analysed the expression of endogenous MxA protein in filovirus VP24-expressing cells. Eight filovirus VP24 proteins, including the VP24s of the recently discovered MLAV, BOMV and LLOV, inhibited IFN-induced MxA and IFITM3 promoter activation. MARV VP24 was the only protein with no inhibitory effect on the activation of either promoter. Endogenous MxA protein expression was impaired in cells transiently expressing VP24s with the exception of MARV VP24. We mutated nuclear localization signal (NLS) of two highly pathogenic filoviruses (EBOV and SUDV) and two putatively non-pathogenic filoviruses (BOMV and RESTV), and showed that the inhibitory effect on IFN-induced expression of MxA was dependent on functional cluster 3 of VP24 nuclear localization signal. Our findings suggest that filovirus VP24 proteins are both genetically and functionally conserved, and that VP24 proteins of most filovirus species are capable of inhibiting IFN-induced antiviral gene expression thereby efficiently downregulating the host innate immune responses.

Update on the Phylodynamic and Genetic Variability of Marburg Virus.

The COVID-19 pandemic has not only strained healthcare systems in Africa but has also intensified the impact of emerging and re-emerging diseases. Specifically in Equatorial Guinea, mirroring the situation in other African countries, unique zoonotic outbreaks have occurred during this challenging period. One notable resurgence is Marburg virus disease (MVD), which has further burdened the already fragile healthcare system. The re-emergence of the Marburg virus amid the COVID-19 pandemic is believed to stem from a probable zoonotic spill-over, although the precise transmission routes remain uncertain. Given the gravity of the situation, addressing the existing challenges is paramount. Though the genome sequences from the current outbreak were not available for this study, we analyzed all the available whole genome sequences of this re-emerging pathogen to advocate for a shift towards active surveillance. This is essential to ensure the successful containment of any potential Marburg virus outbreak in Equatorial Guinea and the wider African context. This study, which presents an update on the phylodynamics and the genetic variability of MARV, further confirmed the existence of at least two distinct patterns of viral spread. One pattern demonstrates a slower but continuous and recurring virus circulation, while the other exhibits a faster yet limited and episodic spread. These results highlight the critical need to strengthen genomic surveillance in the region to effectively curb the pathogen's dissemination. Moreover, the study emphasizes the importance of prompt alert management, comprehensive case investigation and analysis, contact tracing, and active case searching. These steps are vital to support the healthcare system's response to this emerging health crisis. By implementing these strategies, we can better arm ourselves against the challenges posed by the resurgence of the Marburg virus and other infectious diseases.

Marburg virus in Egyptian Rousettus bats in Guinea: Investigation of Marburg virus outbreak origin in 2021.

In 2021, a patient died from Marburg virus (MARV) disease in Guinea and it was the first confirmed case in West Africa. The origin of the outbreak has not been identified. It was revealed that the patient didn't travel anywhere before the illness. Prior to outbreak, MARV had been found in bats in the neighboring Sierra Leone, but never in Guinea. Therefore, the origin of infection is unclear: was it an autochthonous case with spillover from a local population of bats or an imported case with spillover from fruit bats foraging/migrating from Sierra Leone? In this paper, we studied Rousettus aegyptiacus in Guinea as the possible source of MARV infection caused the patient death in 2021 in Guinea. We caught bats in 32 sites of Guéckédou prefecture, including seven caves and 25 locations of the flight path. A total of 501 fruit bats (Pteropodidae) were captured, including 66 R. aegyptiacus. The PCR screening showed three positive MARV R. aegyptiacus, roosting in two caves discovered in Guéckédou prefecture. After Sanger sequencing and phylogenetic analyses it was shown that found MARV belongs to the Angola-like lineage but it is not identical to the isolate obtained during the outbreak of 2021.

Pseudotyped Viruses for Marburgvirus and Ebolavirus.

Marburg virus (MARV) and Ebola virus (EBOV) of the Filoviridae family are the most lethal viruses in terms of mortality rate. However, the development of antiviral treatment is hampered by the requirement for biosafety level-4 (BSL-4) containment. The establishment of BSL-2 pseudotyped viruses can provide important tools for the study of filoviruses. This chapter summarizes general information on the filoviruses and then focuses on the construction of replication-deficient pseudotyped MARV and EBOV (e.g., lentivirus system and vesicular stomatitis virus system). It also details the potential applications of the pseudotyped viruses, including neutralization antibody detection, the study of infection mechanisms, the evaluation of antibody-dependent enhancement, virus entry inhibitor screening, and glycoprotein mutation analysis.

Marburg and Ebola Virus Infections Elicit a Complex, Muted Inflammatory State in Bats.

The Marburg and Ebola filoviruses cause a severe, often fatal, disease in humans and nonhuman primates but have only subclinical effects in bats, including Egyptian rousettes, which are a natural reservoir of Marburg virus. A fundamental question is why these viruses are highly pathogenic in humans but fail to cause disease in bats. To address this question, we infected one cohort of Egyptian rousette bats with Marburg virus and another cohort with Ebola virus and harvested multiple tissues for mRNA expression analysis. While virus transcripts were found primarily in the liver, principal component analysis (PCA) revealed coordinated changes across multiple tissues. Gene signatures in kidney and liver pointed at induction of vasodilation, reduction in coagulation, and changes in the regulation of iron metabolism. Signatures of immune response detected in spleen and liver indicated a robust anti-inflammatory state signified by macrophages in the M2 state and an active T cell response. The evolutionary divergence between bats and humans of many responsive genes might provide a framework for understanding the differing outcomes upon infection by filoviruses. In this study, we outline multiple interconnected pathways that respond to infection by MARV and EBOV, providing insights into the complexity of the mechanisms that enable bats to resist the disease caused by filoviral infections. The results have the potential to aid in the development of new strategies to effectively mitigate and treat the disease caused by these viruses in humans.

Marburg Virus- A Threat During SARS-CoV-2 Era: A Review.

In the German towns of Marburg, Frankfurt, and Belgrade in 1967, this single negativestranded RNA virus was initially discovered. The importation of infected grivet monkeys from Uganda is what caused this virus-related sickness. As a result of the early link between viruses and non-human primates, this virus is frequently referred to as vervet monkey sickness. This virus causes Marburg hemorrhagic fever in humans and non-human primates. Human endothelial cells serve as the primary vehicle for replication. According to a 2009 report, the virus was being stored in Egyptian fruit bats (Rousettus aegyptiacus). Body fluids, unprotected sex, broken or injured skin, and other bodily fluids are the main routes of transmission. After the incubation period, symptoms like chills, headaches, myalgia, and stomach pain start to show up. There is no specific medication for such an infection, only hydration therapy and adequate oxygenation are followed. The following diagnostic techniques can be used to confirm the diagnosis: (i) an antibody-capture enzyme linked immunosorbent assay (ELISA); ii) an antigen capture ELISA test; iii) a serum neutralization test; iv) an RT PCR assay; v) electron microscopy; or vi) virus isolation by cell culture. Because MARV is a risk group 4 infection, laboratory staff must take strict precautions (RG-4).

Combination treatment of mannose and GalNAc conjugated small interfering RNA protects against lethal Marburg virus infection.

Marburg virus (MARV) infection results in severe viral hemorrhagic fever with mortalities up to 90%, and there is a pressing need for effective therapies. Here, we established a small interfering RNA (siRNA) conjugate platform that enabled successful subcutaneous delivery of siRNAs targeting the MARV nucleoprotein. We identified a hexavalent mannose ligand with high affinity to macrophages and dendritic cells, which are key cellular targets of MARV infection. This ligand enabled successful siRNA conjugate delivery to macrophages both in vitro and in vivo. The delivered hexa-mannose-siRNA conjugates rendered substantial target gene silencing in macrophages when supported by a mannose functionalized endosome release polymer. This hexa-mannose-siRNA conjugate was further evaluated alongside our hepatocyte-targeting GalNAc-siRNA conjugate, to expand targeting of infected liver cells. In MARV-Angola-infected guinea pigs, these platforms offered limited survival benefit when used as individual agents. However, in combination, they achieved up to 100% protection when dosed 24 h post infection. This novel approach, using two different ligands to simultaneously deliver siRNA to multiple cell types relevant to infection, provides a convenient subcutaneous route of administration for treating infection by these dangerous pathogens. The mannose conjugate platform has potential application to other diseases involving macrophages and dendritic cells.

A rabies virus-vectored vaccine expressing two copies of the Marburg virus glycoprotein gene induced neutralizing antibodies against Marburg virus in humanized mice.

Marburg virus disease (MVD) is a lethal viral haemorrhagic fever caused by Marburg virus (MARV) with a case fatality rate as high as 88%. There is currently no vaccine or antiviral therapy approved for MVD. Due to high variation among MARV isolates, vaccines developed against one strain fail to protect against other strains. Here we report that three recombinant rabies virus (RABV) vector vaccines encoding two copies of GPs covering both MARV lineages induced pseudovirus neutralizing antibodies in BALB/c mice. Furthermore, high-affinity human neutralizing antibodies were isolated from a humanized mouse model. The three vaccines produced a Th1-biased serological response similar to that of human patients. Adequate sequential immunization enhanced the production of neutralizing antibodies. Virtual docking suggested that neutralizing antibodies induced by the Angola strain seemed to be able to hydrogen bond to the receptor-binding site (RBS) in the GP of the Ravn strain through hypervariable regions 2 (CDR2) and CDR3 of the VH region. These findings demonstrate that three inactivated vaccines are promising candidates against different strains of MARV, and a novel fully humanized neutralizing antibody against MARV was isolated.

Landscape and age dynamics of immune cells in the Egyptian rousette bat.

Bats harbor high-impact zoonotic viruses often in the absence of disease manifestation. This restriction and disease tolerance possibly rely on specific immunological features. In-depth molecular characterization of cellular immunity and imprinting of age on leukocyte compartments remained unexplored in bats. We employ single-cell RNA sequencing (scRNA-seq) and establish immunostaining panels to characterize the immune cell landscape in juvenile, subadult, and adult Egyptian rousette bats (ERBs). Transcriptomic and flow cytometry data reveal conserved subsets and substantial enrichments of CD79a+ B cells and CD11b+ T cells in juvenile animals, whereas neutrophils, CD206+ myeloid cells, and CD3+ T cells dominate as bats reach adulthood. Despite differing frequencies, phagocytosis of circulating and tissue-resident myeloid cells and proliferation of peripheral and splenic lymphocytes are analogous in juvenile and adult ERBs. We provide a comprehensive map of the immune landscape in ERBs and show age-imprinted resilience progression and find that variability in cellular immunity only partly recapitulates mammalian archetypes.

Marburg Virus VP30 Is Required for Transcription Initiation at the Glycoprotein Gene.

Marburg virus (MARV) is an enveloped, negative-sense RNA virus from the filovirus family that causes outbreaks of severe, frequently fatal illness in humans. Of the seven MARV proteins, the VP30 protein stands out because it is essential for viral growth but lacks a definitive function. Here, we used model MARV genome RNAs for one or two reporter genes and the MARV VP40, glycoprotein (GP), and VP24 genes to demonstrate that VP30 is dispensable for the transcription of some genes but critical for transcription reinitiation at the GP gene. This results in the loss of the expression of GP and downstream genes and the impaired production of infectious particles when VP30 is absent. Bicistronic minigenome assays demonstrate that the VP40 gene end/GP gene start junction specifically confers VP30 dependence. A region at the GP gene start site predicted to form a stem-loop contributes to VP30 dependence because the replacement of the GP stem-loop with corresponding sequences from the MARV VP35 gene relieves VP30 dependence. Finally, a Cys3-His zinc binding motif characteristic of filovirus VP30 proteins was demonstrated to be critical for reinitiation at GP. These findings address a long-standing gap in our understanding of MARV biology by defining a critical role for VP30 in MARV transcription. IMPORTANCE Marburg virus and Ebola virus encode VP30 proteins. While the role of VP30 in Ebola virus transcription has been well studied, the role of VP30 in the Marburg virus life cycle is not well understood. The work here demonstrates that different gene start sites within the Marburg viral genome have variable levels of dependence on Marburg virus VP30, with its expression being critical for transcription reinitiation at the GP gene start site. These findings address a long-standing question regarding Marburg virus VP30 function and further our understanding of how Marburg virus gene expression is regulated.

Immunoinformatic screening of Marburgvirus epitopes and computational investigations of epitope-allele complexes.

Marburgvirus (MARV), a member of the Filovirus family, causes severe hemorrhagic fever in humans. Currently, there are no approved vaccines or post exposure treatment methods available against MARV. With the aim of identifying vaccine candidates against MARV, we employ different sequence-based computational methods to predict the MHC-I and MHC-II T-cell epitopes as well as B-cell epitopes for the complete MARV genome. We analyzed the variations in the predicted epitopes among four MARV variants, the Lake Victoria, Angola, Musoke, and Ravn. We used a consensus approach to identify several epitopes, including novel epitopes, and narrowed down the selection based on different parameters such as antigenicity and IC50 values. The selected epitopes can be used in various vaccine constructs that give effective antibody responses. The MHC-I epitope-allele complexes for GP and NP with favorably low IC50 values were investigated using molecular dynamics computations to determine the molecular details of the epitope-allele complexes. This study provides information for further experimental validation of the potential epitopes and the design and development of MARV vaccines.

Assembly and transport of filovirus nucleocapsids.

Filovirus-infected cells are characterized by typical cytoplasmic inclusion bodies (IBs) located in the perinuclear region. The formation of these IBs is induced mainly by the accumulation of the filoviral nucleoprotein NP, which recruits the other nucleocapsid proteins, the polymerase co-factor VP35, the polymerase L, the transcription factor VP30 and VP24 via direct or indirect protein-protein interactions. Replication of the negative-strand RNA genomes by the viral polymerase L and VP35 occurs in the IBs, resulting in the synthesis of positive-strand genomes, which are encapsidated by NP, thus forming ribonucleoprotein complexes (antigenomic RNPs). These newly formed antigenomic RNPs in turn serve as templates for the synthesis of negative-strand RNA genomes that are also encapsidated by NP (genomic RNPs). Still in the IBs, genomic RNPs mature into tightly packed transport-competent nucleocapsids (NCs) by the recruitment of the viral protein VP24. NCs are tightly coiled left-handed helices whose structure is mainly determined by the multimerization of NP at its N-terminus, and these helices form the inner layer of the NCs. The RNA genome is fixed by 2 lobes of the NP N-terminus and is thus guided by individual NP molecules along the turns of the helix. Direct interaction of the NP C-terminus with the VP35 and VP24 molecules forms the outer layer of the NCs. Once formed, NCs that are located at the border of the IBs recruit actin polymerization machinery to one of their ends to drive their transport to budding sites for their envelopment and final release. Here, we review the current knowledge on the structure, assembly, and transport of filovirus NCs.