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Ebola virus (EBOV) RNA has the potential to form hairpin structures at the transcription start sequence (TSS) and reinitiation sites of internal genes, both on the genomic and antigenomic/mRNA level. Hairpin formation involving the TSS and the spacer sequence between promotor elements (PE) 1 and 2 was suggested to regulate viral transcription. Here, we provide evidence that such RNA structures form during RNA synthesis by the viral polymerase and affect its activity. This was analysed using monocistronic minigenomes carrying hairpin structure variants in the TSS-spacer region that differ in length and stability. Transcription and replication were measured via reporter activity and by qRT-PCR quantification of the distinct viral RNA species. We demonstrate that viral RNA synthesis is remarkably tolerant to spacer extensions of up to ~54 nt, but declines beyond this length limit (~25% residual activity for a 66-nt extension). Minor incremental stabilizations of hairpin structures in the TSS-spacer region and on the mRNA/antigenomic level were found to rapidly abolish viral polymerase activity, which may be exploited for antisense strategies to inhibit viral RNA synthesis. Finally, balanced viral transcription and replication can still occur when any RNA structure formation potential at the TSS is eliminated, provided that hexamer phasing in the promoter region is maintained. Altogether, the findings deepen and refine our insight into structure and length constraints within the EBOV transcription and replication promoter and suggest a remarkable flexibility of the viral polymerase in recognition of PE1 and PE2.

Transcription of non-segmented negative sense (NNS) RNA viruses follows a stop-start mechanism and is thought to be initiated at the genome’s very 3’-end. The synthesis of short abortive leader transcripts (leaderRNAs) has been linked to transcription initiation for some NNS viruses. Here, we identified the synthesis of abortive leaderRNAs (as well as trailer RNAs) that are specifically initiated opposite to (anti)genome nt 2; leaderRNAs are predominantly terminated in the region of nt ~ 60–80. LeaderRNA synthesis requires hexamer phasing in the 3’-leader promoter. We determined a steady-state NP mRNA:leaderRNA ratio of ~10 to 30-fold at 48 h after Ebola virus (EBOV) infection, and this ratio was higher (70 to 190-fold) for minigenome-transfected cells. LeaderRNA initiation at nt 2 and the range of termination sites were not affected by structure and length variation between promoter elements 1 and 2, nor the presence or absence of VP30. Synthesis of leaderRNA is suppressed in the presence of VP30 and termination of leaderRNA is not mediated by cryptic gene end (GE) signals in the 3’-leader promoter. We further found different genomic 3’-end nucleotide requirements for transcription versus replication, suggesting that promoter recognition is different in the replication and transcription mode of the EBOV polymerase. We further provide evidence arguing against a potential role of EBOV leaderRNAs as effector molecules in innate immunity. Taken together, our findings are consistent with a model according to which leaderRNAs are abortive replicative RNAs whose synthesis is not linked to transcription initiation. Rather, replication and transcription complexes are proposed to independently initiate RNA synthesis at separate sites in the 3’-leader promoter, i.e., at the second nucleotide of the genome 3’-end and at the more internally positioned transcription start site preceding the first gene, respectively, as reported for Vesicular stomatitis virus., Author summary The RNA polymerase (RdRp) of Ebola virus (EBOV) initiates RNA synthesis at the 3’-leader promoter of its encapsidated, non-segmented negative sense (NNS) RNA genome, either at the penultimate 3’-end position of the genome in the replicative mode or more internally (position 56) at the transcription start site (TSS) in its transcription mode. Here we identified the synthesis of abortive replicative RNAs that are specifically initiated opposite to genome nt 2 (termed leaderRNAs) and predominantly terminated in the region of nt ~ 60–80 near the TSS. The functional role of abortive leaderRNA synthesis is still enigmatic; a role in interferon induction could be excluded. Our findings indirectly link leaderRNA termination to nucleoprotein (NP) availability for encapsidation of nascent replicative RNA or to NP removal from the template RNA. Our findings further argue against the model that leaderRNA synthesis is a prerequisite for each transcription initiation event at the TSS. Rather, our findings are in line with the existence of distinct replicase and transcriptase complexes of RdRp that interact differently with the 3’-leader promoter and intiate RNA synthesis independently at different sites (position 2 or 56 of the genome), mechanistically similar to another NNS virus, Vesicular stomatitis virus.

The nucleoprotein (NP) of Marburg virus (MARV), a close relative of Ebola virus (EBOV), encapsidates the single-stranded, negative-sense viral genomic RNA (vRNA) to form the helical NP–RNA complex. The NP–RNA complex constitutes the core structure for the assembly of the nucleocapsid that is responsible for viral RNA synthesis. Although appropriate interactions among NPs and RNA are required for the formation of nucleocapsid, the structural basis of the helical assembly remains largely elusive. Here, we show the structure of the MARV NP–RNA complex determined using cryo-electron microscopy at a resolution of 3.1 Å. The structures of the asymmetric unit, a complex of an NP and six RNA nucleotides, was very similar to that of EBOV, suggesting that both viruses share common mechanisms for the nucleocapsid formation. Structure-based mutational analysis of both MARV and EBOV NPs identified key residues for helical assembly and subsequent viral RNA synthesis. Importantly, most of the residues identified were conserved in both viruses. These findings provide a structural basis for understanding the nucleocapsid formation and contribute to the development of novel antivirals against MARV and EBOV., 致死的な出血熱を引き起こすマールブルグウイルスの増殖機構を解明 --エボラ・マールブルグウイルスの創薬に期待--. 京都大学プレスリリース. 2022-03-07., Viruses of a feather: Similar structures in Marburg and Ebola viruses provide clues for antivirals. 京都大学プレスリリース. 2022-07-25.

The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs that adopt non-canonical orientations, as compared to other proline-rich motifs. An affinity tag-purification mass spectrometry approach identified additional PPxPxY-containing host proteins hnRNP L, hnRNPUL1, and PEG10, as VP30 interactors. hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV infection, whereas hnRNPUL1 enhances. RBBP6 and hnRNP L modulate VP30 phosphorylation, increase viral transcription, and exert additive effects on viral RNA synthesis. PEG10 has more modest inhibitory effects on EBOV replication. hnRNPUL1 positively affects viral RNA synthesis but in a VP30-independent manner. Binding studies demonstrate variable capacity of the PPxPxY motifs from these proteins to bind VP30, define PxPPPPxY as an optimal binding motif, and identify the fifth proline and the tyrosine as most critical for interaction. Competition binding and hydrogen-deuterium exchange mass spectrometry studies demonstrate that each protein binds a similar interface on VP30. VP30 therefore presents a novel proline recognition domain that is targeted by multiple host proteins to modulate viral transcription.

The nucleoprotein (NP) of Marburg virus (MARV), a close relative of Ebola virus (EBOV), encapsidates the single-stranded, negative-sense viral genomic RNA (vRNA) to form the helical NP-RNA complex. The NP-RNA complex serves as a scaffold for the assembly of the nucleocapsid that is responsible for viral RNA synthesis. Although appropriate interactions among NPs and RNA are required for the formation of nucleocapsid, the structural basis of the helical assembly remains largely elusive. Here, we show the structure of the MARV NP-RNA complex determined using cryo-electron microscopy at a resolution of 3.1 Å. The structures of the asymmetric unit, a complex of an NP and six RNA nucleotides, was very similar to that of EBOV, suggesting that both viruses share common mechanisms for the nucleocapsid formation. Structure-based mutational analysis of both MARV and EBOV NPs identified key residues for the viral RNA synthesis as well as the helical assembly. Importantly, most of the residues identified were conserved in both viruses. These findings provide a structural basis for understanding the nucleocapsid formation and contribute to the development of novel antivirals against MARV and EBOV.

Background Ebola virus (EBOV) epidemics pose a major public health risk. There currently is no licensed human vaccine against EBOV. The safety and immunogenicity of a recombinant EBOV glycoprotein (GP) nanoparticle vaccine formulated with or without Matrix-M adjuvant were evaluated to support vaccine development. Methods A phase 1, placebo-controlled, dose-escalation trial was conducted in 230 healthy adults to evaluate 4 EBOV GP antigen doses as single- or 2-dose regimens with or without adjuvant. Safety and immunogenicity were assessed through 1-year postdosing. Results All EBOV GP vaccine formulations were well tolerated. Receipt of 2 doses of EBOV GP with adjuvant showed a rapid increase in anti-EBOV GP immunoglobulin G titers with peak titers observed on Day 35 representing 498- to 754-fold increases from baseline; no evidence of an antigen dose response was observed. Serum EBOV-neutralizing and binding antibodies using wild-type Zaire EBOV (ZEBOV) or pseudovirion assays were 3- to 9-fold higher among recipients of 2-dose EBOV GP with adjuvant, compared with placebo on Day 35, which persisted through 1 year. Conclusions Ebola virus GP vaccine with Matrix-M adjuvant is well tolerated and elicits a robust and persistent immune response. These data suggest that further development of this candidate vaccine for prevention of EBOV disease is warranted.

Ebola virus is the causative agent of Ebola virus disease, a severe, often fatal illness in humans. So far, there are no US Food and Drug Administration (FDA)-approved therapeutics directed against Ebola virus. Here, we selected the host factor Niemann-Pick C1 (NPC1), which has been shown to be essential for Ebola virus entry into host cytoplasm, as a therapeutic target for suppression by locked nucleic acid-modified antisense oligonucleotides. Screening of antisense oligonucleotides in human and murine cell lines led to identification of candidates with up to 94% knockdown efficiency and 50% inhibitory concentration (IC50) values in the submicromolar range. Selected candidate oligonucleotides led to efficient NPC1 protein knockdown in vitro without alteration of cell viability. Furthermore, they did not have immune stimulatory activity in cell-based assays. Treatment of Ebola-virus-infected HeLa cells with the most promising candidates resulted in significant (>99%) virus titer reduction, indicating that antisense oligonucleotides against NPC1 are a promising therapeutic approach for treatment of Ebola virus infection. Keywords: Ebola virus, antisense oligonucleotide, NPC1, LNA

BackgroundThe 2013-16 Ebola virus disease epidemic in west Africa caused international alarm due to its rapid and extensive spread resulting in a significant death toll and social unrest within the affected region. The large number of cases provided an opportunity to study the long-term kinetics of Zaire ebolavirus-specific immune response of survivors in addition to known contacts of those infected with the virus.MethodsIn this observational cohort study, we worked with leaders of Ebola virus disease survivor associations in two regions of Guinea, Guéckédou and Coyah, to recruit survivors of Ebola virus disease, contacts from households of individuals known to have had Ebola virus disease, and individuals who were not knowingly associated with infected individuals or had not had Ebola virus disease symptoms to serve as negative controls. We did Zaire ebolavirus glycoprotein-specific T cell analysis on peripheral blood mononuclear cells (PBMCs) on location in Guinea and transported plasma and PBMCs back to Europe for antibody quantification by ELISA, functional neutralising antibody analysis using live Zaire ebolavirus, and T cell phenotype studies. We report on the longitudinal cellular and humoral response among Ebola virus disease survivors and highlight potentially paucisymptomatic infection.FindingsWe recruited 117 survivors of Ebola virus disease, 66 contacts, and 23 negative controls. The mean neutralising antibody titre among the Ebola virus disease survivors 3-14 months after infection was 1/174 (95% CI 1/136-1/223). Individual results varied greatly from 1/10 to more than 1/1000 but were on average ten times greater than that induced after 1 month by single dose Ebola virus vaccines. Following reactivation with glycoprotein peptide, the mean T cell responses among 116 Ebola virus disease survivors as measured by ELISpot was 305 spot-forming units (95% CI 257-353). The dominant CD8+ polyfunctional T cell phenotype, as measured among 53 Ebola virus disease survivors, was interferon γ+, tumour necrosis factor+, interleukin-2-, and the mean response was 0·046% of total CD8+ T cells (95% CI 0·021-0·071). Additionally, both neutralising antibody and T cell responses were detected in six (9%) of 66 Ebola virus disease contacts. We also noted that four (3%) of 117 individuals with Ebola virus disease infections did not have circulating Ebola virus-specific antibodies 3 months after infection.InterpretationThe continuous high titre of neutralising antibodies and increased T cell response might support the concept of long-term protective immunity in survivors. The existence of antibody and T cell responses in contacts of individuals with Ebola virus disease adds further evidence to the existence of sub-clinical Ebola virus infection.FundingUS Food & Drug Administration, Horizon 2020 EU EVIDENT, Wellcome, UK Department for International Development.TranslationFor the French translation of the abstract see Supplementary Materials section.

Summary Ebola virus (EBOV) is responsible for outbreaks with case fatality rates of up to 90% and for an epidemic in West Africa with more than ten thousand deaths. EBOV glycoprotein (EBOV-GP) is the only viral surface protein and is responsible for viral entry into cells. Here, by employing pseudotyped EBOV-GP viral particles, we uncover a critical role for sphingolipids in inhibiting viral entry. Sphingosine kinase 1 (SphK1) catalyzes the phosphorylation of sphingosine to sphingosine 1-phosphate (S1P). The administration of the SphK1 activator, K6PC-5, or S1P, or the overexpression of SphK1 consistently exhibited striking inhibitory effects in EBOV-GP-driven entry in diverse cell lines. Finally, K6PC-5 markedly reduced the EBOV titer in infected cells and the de novo production of viral proteins. These data present K6PC-5 as an efficient tool to inhibit EBOV infection in endothelial cells and suggest further studies to evaluate its systemic effects., Graphical Abstract, Highlights • K6PC-5, a sphingosine kinase 1 activator, inhibits Ebola virus infection • Sphingosine 1-phosphate, the product of SphK1, attenuates the viral entry • Inhibiton/activation of S1P receptors has no influence on Ebola virus entry • These data support the endogen effect of S1P in Ebola virus infection, Virology ; Molecular Microbiology ; Cell Biology

The severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as the infectious agent causing the pandemic coronavirus disease 2019 (COVID-19) with dramatic consequences for global human health and economics. Previously, we reached clinical evaluation with our vector vaccine based on vaccinia virus MVA against the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes an infection in humans similar to SARS and COVID-19. Here, we describe the construction and preclinical characterization of a recombinant MVA expressing full-length SARS-CoV-2 spike (S) protein (MVA-SARS-2-S). Genetic stability and growth characteristics of MVA-SARS-2-S, plus its robust synthesis of S antigen, make it a suitable candidate vaccine for industrial scale production. Vaccinated mice produced S antigen-specific CD8+ T cells and serum antibodies binding to S glycoprotein that neutralized SARS-CoV-2. Prime-boost vaccination with MVA-SARS-2-S protected mice sensitized with a human ACE2-expressing adenovirus from SARS-CoV-2 infection. MVA-SARS-2-S is currently being investigated in a phase I clinical trial as aspirant for developing a safe and efficacious vaccine against COVID-19.Significance StatementThe highly attenuated vaccinia virus MVA is licensed as smallpox vaccine, and as vector it is a component of the approved Adenovirus-MVA-based prime-boost vaccine against Ebola virus disease. Here we provide results from testing the COVID-19 candidate vaccine MVA-SARS-2-S, a poxvirus-based vector vaccine that proceeded to clinical evaluation. When administered by intramuscular inoculation, MVA-SARS-2-S expresses and safely delivers the full-length SARS-CoV-2 spike (S) protein, inducing balanced SARS-CoV-2-specific cellular and humoral immunity, and protective efficacy in vaccinated mice. Substantial clinical experience has already been gained with MVA vectors using homologous and heterologous prime-boost applications, including the immunization of children and immunocompromised individuals. Thus, MVA-SARS-2-S represents an important resource for developing further optimized COVID-19 vaccines.

Background In October 2015, 65 people came into direct contact with a healthcare worker presenting with a late reactivation of Ebola virus disease (EVD) in the United Kingdom. Vaccination was offered to 45 individuals with an initial assessment of high exposure risk. Methods Approval for rapid expanded access to the recombinant vesicular stomatitis virus–Zaire Ebola virus (rVSV-ZEBOV) vaccine as an unlicensed emergency medicine was obtained from the relevant authorities. An observational follow-up study was carried out for 1 year following vaccination. Results Twenty-six of 45 individuals elected to receive vaccination between 10 and 11 October 2015 following written informed consent. By day 14, 39% had seroconverted, increasing to 87% by day 28 and 100% by 3 months, although these responses were not always sustained. Neutralizing antibody responses were detectable in 36% by day 14 and 73% at 12 months. Common side effects included fatigue, myalgia, headache, arthralgia, and fever. These were positively associated with glycoprotein-specific T-cell but not immunoglobulin (Ig) M or IgG antibody responses. No severe vaccine-related adverse events were reported. No one exposed to the virus became infected. Conclusions This paper reports the use of the rVSV-ZEBOV vaccine given as an emergency intervention to individuals exposed to a patient presenting with a late reactivation of EVD. The vaccine was relatively well tolerated, but a high percentage developed a fever ≥37.5°C, necessitating urgent screening for Ebola virus, and a small number developed persistent arthralgia., The rVSV-ZEBOV vaccine was used as postexposure prophylaxis in individuals exposed to Ebola virus in the United Kingdom. It was rolled out rapidly and was generally well tolerated. Side effects correlated with the magnitude of CD8+ T-cell responses.

The intracellular transport of nucleocapsids of the highly pathogenic Marburg, as well as Ebola virus (MARV, EBOV), represents a critical step during the viral life cycle. Intriguingly, a population of these nucleocapsids is distributed over long distances in a directed and polar fashion. Recently, it has been demonstrated that the intracellular transport of filoviral nucleocapsids depends on actin polymerization. While it was shown that EBOV requires Arp2/3-dependent actin dynamics, the details of how the virus exploits host actin signaling during intracellular transport are largely unknown. Here, we apply a minimalistic transfection system to follow the nucleocapsid-like structures (NCLS) in living cells, which can be used to robustly quantify NCLS transport in live cell imaging experiments. Furthermore, in cells co-expressing LifeAct, a marker for actin dynamics, NCLS transport is accompanied by pulsative actin tails appearing on the rear end of NCLS. These actin tails can also be preserved in fixed cells, and can be visualized via high resolution imaging using STORM in transfected, as well as EBOV infected, cells. The application of inhibitory drugs and siRNA depletion against actin regulators indicated that EBOV NCLS utilize the canonical Arp2/3-Wave1-Rac1 pathway for long-distance transport in cells. These findings highlight the relevance of the regulation of actin polymerization during directed EBOV nucleocapsid transport in human cells.

The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs. In these interactions the largely alpha-helical carboxy-terminal domain of the EBOV VP30 engages with the motif such that the prolines adopt non-canonical orientations, as compared to other proline-rich motifs. Affinity tag-purification mass spectrometry identified additional PPxPxY-containing host proteins, including hnRNP L, hnRNPUL1 and PEG10, as VP30 interactors. Of these, hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV replication, whereas hnRNPUL1 enhances. Further, double knockdown studies support additive effects of RBBP6 and hnRNP L. Binding studies demonstrate variable capacity of PPxPxY motifs to bind VP30 and the extended motif PxPPPPxY is demonstrated to confer optimal binding and to inhibit RNA synthesis, with the fifth proline and the tyrosine being most critical. Competition binding and hydrogen-deuterium exchange studies demonstrate that each protein binds a similar interface on VP30 and impacts VP30 phosphorylation. VP30 therefore represents a novel proline recognition domain that allows multiple host proteins to target a single viral protein-protein interface to modulate viral transcription.

While it is well appreciated that late domains in the viral matrix proteins are crucial to mediate efficient virus budding, little is known about roles of late domains in the viral nucleocapsid proteins. Here, we characterized the functional relevance of a YxxL motif with potential late-domain function in the Ebola virus nucleocapsid protein VP24. Mutations in the YxxL motif had two opposing effects on the functions of VP24. On the one hand, the mutation affected the regulatory function of VP24 in viral RNA transcription and replication, which correlated with an increased incorporation of minigenomes into released transcription- and replication-competent virus-like particles (trVLPs). Consequently, cells infected with those trVLPs showed higher levels of viral transcription. On the other hand, mutations of the YxxL motif greatly impaired the intracellular transport of nucleocapsid-like structures (NCLSs) composed of the viral proteins NP, VP35, and VP24 and the length of released trVLPs. Attempts to rescue recombinant Ebola virus expressing YxxL-deficient VP24 failed, underlining the importance of this motif for the viral life cycle. IMPORTANCE Ebola virus (EBOV) causes a severe fever with high case fatality rates and, so far, no available specific therapy. Understanding the interplay between viral and host proteins is important to identify new therapeutic approaches. VP24 is one of the essential nucleocapsid components and is necessary to regulate viral RNA synthesis and condense viral nucleocapsids before their transport to the plasma membrane. Our functional analyses of the YxxL motif in VP24 suggested that it serves as an interface between nucleocapsid-like structures (NCLSs) and cellular proteins, promoting intracellular transport of NCLSs in an Alix-independent manner. Moreover, the YxxL motif is necessary for the inhibitory function of VP24 in viral RNA synthesis. A failure to rescue EBOV encoding VP24 with a mutated YxxL motif indicated that the integrity of the YxxL motif is essential for EBOV growth. Thus, this motif might represent a potential target for antiviral interference.

The intracytoplasmic movement of nucleocapsids is a crucial step in the life cycle of enveloped viruses. Determination of the viral components necessary for viral nucleocapsid transport competency is complicated by the dynamic and complex nature of nucleocapsid assembly and the lack of appropriate model systems. Here, we established a live-cell imaging system based on the ectopic expression of fluorescent Ebola virus (EBOV) fusion proteins, allowing the visualization and analysis of the movement of EBOV nucleocapsid-like structures with different protein compositions. Only three of the five EBOV nucleocapsid proteins-nucleoprotein, VP35, and VP24-were necessary and sufficient to form transport-competent nucleocapsid-like structures. The transport of these structures was found to be dependent on actin polymerization and to have dynamics that were undistinguishable from those of nucleocapsids in EBOV-infected cells. The intracytoplasmic movement of nucleocapsid-like structures was completely independent of the viral matrix protein VP40 and the viral surface glycoprotein GP. However, VP40 greatly enhanced the efficiency of nucleocapsid recruitment into filopodia, the sites of EBOV budding.

Zaire Ebolavirus (EBOV) causes a severe feverish disease with high case fatality rates. Transcription of EBOV is dependent on the activity of the nucleocapsid protein VP30 which represents an essential viral transcription factor. Activity of VP30 is regulated via phosphorylation at six N-terminal serine residues. Recent data demonstrated that dynamic phosphorylation and dephosphorylation of serine residue 29 is essential for transcriptional support activity of VP30. To analyze the spatio/temporal dynamics of VP30 phosphorylation, we generated a peptide antibody recognizing specifically VP30 phosphorylated at serine 29. Using this antibody we could demonstrate that (i) the majority of VP30 molecules in EBOV-infected cells is dephosphorylated at the crucial position serine 29, (ii) both, VP30 phosphorylation and dephosphorylation take place in viral inclusion bodies that are induced by the nucleoprotein NP and (iii) NP influences the phosphorylation state of VP30.

Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause haemorrhagic fever. Filoviruses are within the order Mononegavirales, which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein and other viral proteins to form a helical nucleocapsid. The nucleocapsid acts as a scaffold for virus assembly and as a template for genome transcription and replication. Insights into nucleoprotein-nucleoprotein interactions have been derived from structural studies of oligomerized, RNA-encapsidating nucleoprotein, and cryo-electron microscopy of nucleocapsid or nucleocapsid-like structures. There have been no high-resolution reconstructions of complete mononegavirus nucleocapsids. Here we apply cryo-electron tomography and subtomogram averaging to determine the structure of Ebola virus nucleocapsid within intact viruses and recombinant nucleocapsid-like assemblies. These structures reveal the identity and arrangement of the nucleocapsid components, and suggest that the formation of an extended α-helix from the disordered carboxy-terminal region of nucleoprotein-core links nucleoprotein oligomerization, nucleocapsid condensation, RNA encapsidation, and accessory protein recruitment.

Background The recent West African Ebola epidemic led to accelerated efforts to test Ebola vaccine candidates. As part of the World Health Organisation-led VSV Ebola Consortium (VEBCON), we performed a phase I clinical trial investigating rVSV-ZEBOV (a recombinant vesicular stomatitis virus-vectored Ebola vaccine), which has recently demonstrated protection from Ebola virus disease (EVD) in phase III clinical trials and is currently in advanced stages of licensing. So far, correlates of immune protection are incompletely understood and the role of cell-mediated immune responses has not been comprehensively investigated to date. Methods: We recruited 30 healthy subjects aged 18–55 into an open-label, dose-escalation phase I trial testing three doses of rVSV-ZEBOV (3 × 105 plaque-forming units (PFU), 3 × 106 PFU, 2 × 107 PFU) (ClinicalTrials.gov; NCT02283099). Main study objectives were safety and immunogenicity, while exploratory objectives included lymphocyte dynamics, cell-mediated immunity and cytokine networks, which were assessed using flow cytometry, ELISpot and LUMINEX assay. Findings: Immunization with rVSV-ZEBOV was well tolerated without serious vaccine-related adverse events. Ebola virus-specific neutralizing antibodies were induced in nearly all individuals. Additionally, vaccinees, particularly within the highest dose cohort, generated Ebola glycoprotein (GP)-specific T cells and initiated a cascade of signaling molecules following stimulation of peripheral blood mononuclear cells with Ebola GP peptides. Interpretation: In addition to a benign safety and robust humoral immunogenicity profile, subjects immunized with 2 × 107 PFU elicited higher cellular immune responses and stronger interlocked cytokine networks compared to lower dose groups. To our knowledge these data represent the first detailed cell-mediated immuneprofile of a clinical trial testing rVSV-ZEBOV, which is of particular interest in light of its potential upcoming licensure as the first Ebola vaccine. VEBCON trial Hamburg, Germany (NCT02283099)., Highlights • A phase I clinical trial was conducted to investigate the live-attenuated Ebola vaccine rVSV-ZEBOV. • Ebola-specific humoral and cell-mediated immune responses show a favorable profile for subjects immunized with 2 × 107 PFU of rVSV-ZEBOV. • The highest dose cohort induced stronger antigen-specific CTL-responses and interlocked cytokine networks compared to lower dose groups. rVSV-ZEBOV is the first Ebola vaccine with human efficacy data, currently undergoing an accelerated licensing process. Nevertheless, to date no human immunological correlate of protection has been identified and mechanisms of immune responses elicited by rVSV-ZEBOV remain incompletely understood. We conducted a phase I trial to test rVSV-ZEBOV in 30 healthy subjects using three dosage levels. We here present a comprehensive evaluation of humoral and cell-mediated responses with an in-depth analysis of signaling molecules following ex vivo stimulation with Ebola GP peptides. Our data suggest a favorable immune response profile for subjects immunized with 2 × 107 PFU. These data address critical knowledge gaps with respect to mechanisms of immuneprotection in the context of Ebola vaccines and may provide additional evidence to support the current dosage used in later stage clinical trials., Graphical Abstract Image 1

The largest Ebola virus (EBOV) epidemic in West Africa ever caused more than 28,000 cases and 11,000 deaths, and the current EBOV epidemic in the Democratic Republic of the Congo continues, with more than 3,000 cases to date. Therefore, it is essential to develop antivirals against EBOV. Recently, an inhibitor of the cellular phosphatase PP2A-mediated dephosphorylation of the EBOV transcription factor VP30 has been shown to suppress the spread of Ebola virus. Here, we identified the protein kinase SRPK1 as a VP30-specific kinase that phosphorylates serine 29, the same residue that is dephosphorylated by PP2A. SRPK1-mediated phosphorylation of serine 29 enabled primary viral transcription. Mutation of the SRPK1 recognition motif in VP30 resulted in significant growth inhibition of EBOV. Similarly, elevation of the phosphorylation status of serine 29 by overexpression of SRPK1 inhibited EBOV growth, highlighting the importance of reversible phosphorylation of VP30 as a potential therapeutic target., Ebola virus (EBOV) causes a severe and often fatal disease for which no approved vaccines or antivirals are currently available. EBOV VP30 has been described as a viral phosphoprotein, and nonphosphorylated VP30 is essential and sufficient to support secondary transcription in an EBOV-specific minigenome system; however, phosphorylatable serine residues near the N terminus of VP30 are required to support primary viral transcription as well as the reinitiation of VP30-mediated transcription at internal EBOV genes. While the dephosphorylation of VP30 by the cellular phosphatase PP2A was found to be mediated by nucleoprotein, the VP30-specific kinases and the role of phosphorylation remain unknown. Here, we report that serine-arginine protein kinase 1 (SRPK1) and SRPK2 phosphorylate serine 29 of VP30, which is located in an N-terminal R26xxS29 motif. Interaction with VP30 via the R26xxS29 motif recruits SRPK1 into EBOV-induced inclusion bodies, the sites of viral RNA synthesis, and an inhibitor of SRPK1/SRPK2 downregulates primary viral transcription. When the SRPK1 recognition motif of VP30 was mutated in a recombinant EBOV, virus replication was severely impaired. It is presumed that the interplay between SRPK1 and PP2A in the EBOV inclusions provides a comprehensive regulatory circuit to ensure the activity of VP30 in EBOV transcription. Thus, the identification of SRPK1 is an important mosaic stone that completes our picture of the players involved in Ebola virus transcription regulation.

Recombinant vesicular stomatitis virus–Zaire Ebola virus (rVSV-ZEBOV) is the most advanced Ebola virus vaccine candidate and is currently being used to combat the outbreak of Ebola virus disease (EVD) in the Democratic Republic of the Congo (DRC). Here we examine the humoral immune response in a subset of human volunteers enrolled in a phase 1 rVSV-ZEBOV vaccination trial by performing comprehensive single B cell and electron microscopy structure analyses. Four studied vaccinees show polyclonal, yet reproducible and convergent B cell responses with shared sequence characteristics. EBOV-targeting antibodies cross-react with other Ebolavirus species, and detailed epitope mapping revealed overlapping target epitopes with antibodies isolated from EVD survivors. Moreover, in all vaccinees, we detected highly potent EBOV-neutralizing antibodies with activities comparable or superior to the monoclonal antibodies currently used in clinical trials. These include antibodies combining the IGHV3–15/IGLV1–40 immunoglobulin gene segments that were identified in all investigated individuals. Our findings will help to evaluate and direct current and future vaccination strategies and offer opportunities for novel EVD therapies. Analysis of monoclonal antibody responses to the Ebola virus vaccine rVSV-ZEBOV in human volunteers.

Ebola virus is the causative agent of a severe fever with high fatality rates in humans and nonhuman primates. The regulation of Ebola virus transcription and replication currently is not well understood. An important factor regulating viral transcription is VP30, an Ebola virus-specific transcription factor associated with the viral nucleocapsid. Previous studies revealed that the phosphorylation status of VP30 impacts viral transcription. Together with NP, L, and the polymerase cofactor VP35, nonphosphorylated VP30 supports viral transcription. Upon VP30 phosphorylation, viral transcription ceases. Phosphorylation weakens the interaction between VP30 and the polymerase cofactor VP35 and/or the viral RNA. VP30 thereby is excluded from the viral transcription complex, simultaneously leading to increased viral replication which is supported by NP, L, and VP35 alone. Here, we use an infectious virus-like particle assay and recombinant viruses to show that the dynamic phosphorylation of VP30 is critical for the cotransport of VP30 with nucleocapsids to the sites of viral RNA synthesis, where VP30 is required to initiate primary viral transcription. We further demonstrate that a single serine residue at amino acid position 29 was sufficient to render VP30 active in primary transcription and to generate a recombinant virus with characteristics comparable to those of wild-type virus. In contrast, the rescue of a recombinant virus with a single serine at position 30 in VP30 was unsuccessful. Our results indicate critical roles for phosphorylated and dephosphorylated VP30 during the viral life cycle. IMPORTANCE The current Ebola virus outbreak in West Africa has caused more than 28,000 cases and 11,000 fatalities. Very little is known regarding the molecular mechanisms of how the Ebola virus transcribes and replicates its genome. Previous investigations showed that the transcriptional support activity of VP30 is activated upon VP30 dephosphorylation. The current study reveals that the situation is more complex and that primary transcription as well as the rescue of recombinant Ebola virus also requires the transient phosphorylation of VP30. VP30 encodes six N-proximal serine residues that serve as phosphorylation acceptor sites. The present study shows that the dynamic phosphorylation of serine at position 29 alone is sufficient to activate primary viral transcription. Our results indicate a series of phosphorylation/dephosphorylation events that trigger binding to and release from the nucleocapsid and transcription complex to be essential for the full activity of VP30.

The Ebola virus (EBOV) can cause severe infections in humans, leading to a fatal outcome in a high percentage of cases. Neutralizing antibodies against the EBOV surface glycoprotein (GP) can prevent infections, demonstrating a straightforward way for an efficient vaccination strategy. Meanwhile, many different anti-EBOV antibodies have been identified, whereas the exact binding epitopes are often unknown. Here, we present the analysis of serum samples from an EBOV vaccine trial with the viral vector vaccine rVSV-ZEBOV and an Ebola virus disease (EVD) survivor, using high-density peptide arrays. In this proof-of-principle study, we detected distinct IgG and IgM antibodies binding to different epitopes of EBOV GP: By mapping the whole GP as overlapping peptide fragments, we found new epitopes and confirmed epitopes from the literature. Furthermore, we could validate the highly selective binding epitope of a neutralizing monoclonal anti-EBOV GP antibody. This shows that peptide arrays can be a valuable tool to study the humoral immune response to vaccines in patients and to support Ebola vaccine development. This article is protected by copyright. All rights reserved

The recent Ebola virus outbreak has highlighted the therapeutic potential of antisera and renewed interest in this treatment approach. While human convalescent sera may not be readily available in the early stages of an outbreak, antisera of animal origin can be produced in a short time frame. Here, we compared adjuvanted virus-like particles (VLP) with recombinant modified vaccinia virus Ankara and vesicular stomatitis virus (VSV), both expressing the Ebola virus antigens. The neutralizing antibody titers of rabbits immunized with adjuvanted VLPs were similar to those immunized with the replication-competent VSV, indicating that presentation of the antigen in its native conformation rather than de novo antigen expression is essential for production of functional antibodies. This approach also yielded high-titer antisera against Nipah virus glycoproteins, illustrating that it is transferable to other virus families. Multiple-step immunoglobulin G purification using a two-step 20–40% ammonium sulfate precipitation followed by protein A affinity chromatography resulted in 90% recovery of functionality and sustained in vivo stability. Adjuvanted VLP-based immunization strategies are thus a promising approach for the rapid generation of therapeutic antisera against emerging infections., Anti-serum: Rapid generation for viral outbreaks Passive immunity through the transfer of anti-serum represents the earliest clinical application of antibodies and is still widely used to this day in the form of anti-venoms. Veronika von Messling and colleagues at the Paul Ehrlich Institute investigate the potential of generating neutralizing anti-serum to the emerging viruses Ebola and Nipah. The authors compare different vaccination platforms in mice and rabbits and find that following multiple vaccine challenges, neutralizing antibody titers equivalent to that seen in convalescent patients could be obtained. Purification of the IgG fraction and processing into F(ab’)2 fragments has the potential to significantly reduce xeno-responses yet the authors find that neutralizing capacity is largely retained albeit at the cost of a shorter in vivo half-life. These findings offer the hope of rapidly generating large quantities of neutralizing anti-serum that could be used in a viral outbreak scenario.

Ebola (EBOV) and Zika viruses (ZIKV) are responsible for recent global health threats. As no preventive vaccines or antiviral drugs against these two re-emerging pathogens are available, we evaluated whether the molecular tweezer CLR01 may inhibit EBOV and ZIKV infection. This small molecule has previously been shown to inactivate HIV-1 and herpes viruses through a selective interaction with lipid-raft-rich regions in the viral envelope, which results in membrane disruption and loss of infectivity. We found that CLR01 indeed blocked infection of EBOV and ZIKV in a dose-dependent manner. The tweezer inhibited infection of epidemic ZIKV strains in cells derived from the anogenital tract and the central nervous system, and remained antivirally active in the presence of semen, saliva, urine and cerebrospinal fluid. Our findings show that CLR01 is a broad-spectrum inhibitor of enveloped viruses with prospects as a preventative microbicide or antiviral agent., Highlights • The molecular tweezer CLR01 inhibits EBOV and ZIKV infection. • ZIKV infection of cells derived from the anogenital tract and the central nervous system is blocked. • Anti-ZIKV activity is retained in semen, urine, saliva, and CSF. • CLR01 represents a broad-spectrum inhibitor of enveloped viruses with prospects as a microbicide or antiviral agent.

Ebola virus (EBOV) and Nipah virus (NiV) infection of humans can cause fatal disease and constitutes a public health threat. In contrast, EBOV and NiV infection of fruit bats, the putative (EBOV) or proven (NiV) natural reservoir, is not associated with disease, and it is currently unknown how these animals control the virus. The human interferon (IFN)-stimulated antiviral effector protein tetherin (CD317, BST-2) blocks release of EBOV- and NiV-like particles from cells and is counteracted by the EBOV glycoprotein (GP). In contrast, it is unknown whether fruit bat tetherin restricts virus infection and is susceptible to GP-driven antagonism. Here, we report the sequence of fruit bat tetherin and show that its expression is IFN stimulated and associated with strong antiviral activity. Moreover, we demonstrate that EBOV-GP antagonizes tetherin orthologues of diverse species but fails to efficiently counteract fruit bat tetherin in virus-like particle (VLP) release assays. However, unexpectedly, tetherin was dispensable for robust IFN-mediated inhibition of EBOV spread in fruit bat cells. Thus, the VLP-based model systems mimicking tetherin-mediated inhibition of EBOV release and its counteraction by GP seem not to adequately reflect all aspects of EBOV release from IFN-stimulated fruit bat cells, potentially due to differences in tetherin expression levels that could not be resolved by the present study. In contrast, tetherin expression was essential for IFN-dependent inhibition of NiV infection, demonstrating that IFN-induced fruit bat tetherin exerts antiviral activity and may critically contribute to control of NiV and potentially other highly virulent viruses in infected animals. IMPORTANCE Ebola virus and Nipah virus (EBOV and NiV) can cause fatal disease in humans. In contrast, infected fruit bats do not develop symptoms but can transmit the virus to humans. Why fruit bats but not humans control infection is largely unknown. Tetherin is an antiviral host cell protein and is counteracted by the EBOV glycoprotein in human cells. Here, employing model systems, we show that tetherin of fruit bats displays higher antiviral activity than human tetherin and is largely resistant against counteraction by the Ebola virus glycoprotein. Moreover, we demonstrate that induction of tetherin expression is critical for interferon-mediated inhibition of NiV but, for at present unknown reasons, not EBOV spread in fruit bat cells. Collectively, our findings identify tetherin as an antiviral effector of innate immune responses in fruit bats, which might allow these animals to control infection with NiV and potentially other viruses that cause severe disease in humans.

The journal of infectious diseases 212(suppl 2), S247-S257 (2015). doi:10.1093/infdis/jiv140, Published by Oxford Univ. Press, Oxford [u.a.]

Ebola- and marburgviruses belong to the family of filoviruses and cause severe haemorrhagic fevers in humans and nonhuman primates. Since their discovery in 1967, during outbreaks in Germany and former Yugoslavia originating with nonhuman primates imported from Uganda, they have been responsible for numerous disease outbreaks in Africa, including that of unprecedented size, which is currently ongoing in West Africa. Filoviruses have also been implicated in massive die-offs of great apes, and in recent years there have been several cases of imported infections into Europe and North America. Filoviral haemorrhagic fevers are severe diseases with case fatality rates of up to 90%. Currently, there is neither a licensed vaccine nor a specific therapy available, although experimental vaccines and treatments have shown promise in nonhuman primates, and are now being vigorously pursued in human clinical trials. Key Concepts Human pathogenic filoviruses (Ebola virus (EBOV), Sudan virus (SUDV), Tai Forest virus (TAFV), Bundibugyo virus (BDBV) and Marburg virus (MARV)) are found in Africa, primarily in bats and nonhuman primates. In contrast, Reston virus (RESTV), which is nonpathogenic in humans, originates in the Philippines, where it is found in both nonhuman primates and pigs. Imported nonhuman primates have been the cause of outbreaks of filoviral haemorrhagic fever in Europe and North America, both among humans and nonhuman primates. Tourism and the return of medical aid workers have also resulted in several imported infections during the last years. Bats represent the most likely reservoir for filoviruses. The recent outbreak in West Africa has been responsible for over 27 000 cases (as of 27 May 2015) and is still ongoing; highlighting the threat that filovirus infection continues to pose to human health. While the absolute number of filoviral haemorrhagic fever cases remain low compared to other diseases, the severe disease picture and high case fatality rates have led to a high public profile of filoviral haemorrhagic fevers. The pathophysiology of filoviral haemorrhagic fevers involves vascular dysfunction, impairment of the immune system and massive dysregulation of cytokine production. Death is caused by multiple organ failure as result of a syndrome resembling septic shock. Currently, the only measures to combat filovirus infections are supportive therapy and patient isolation. However, basic public health measures are very effective at controlling outbreaks, when consistently and rigorously implemented. Experimental treatments and vaccines in nonhuman primates exist, but are not yet licensed for use in humans. However, public interest in the current West African EBOV outbreak has accelerated efforts to see several of the more advanced therapeutic and vaccination approaches licenced. Keywords: Ebola; Marburg; Filovirus; haemorrhagic fever

The severe Ebola virus disease epidemic occurring in West Africa stems from a single zoonotic transmission event to a 2-year-old boy in Meliandou, Guinea. We investigated the zoonotic origins of the epidemic using wildlife surveys, interviews, and molecular analyses of bat and environmental samples. We found no evidence for a concurrent outbreak in larger wildlife. Exposure to fruit bats is common in the region, but the index case may have been infected by playing in a hollow tree housing a colony of insectivorous free-tailed bats (Mops condylurus). Bats in this family have previously been discussed as potential sources for Ebola virus outbreaks, and experimental data have shown that this species can survive experimental infection. These analyses expand the range of possible Ebola virus sources to include insectivorous bats and reiterate the importance of broader sampling efforts for understanding Ebola virus ecology.

Background The rVSVΔG-ZEBOV-GP vaccine prevented Ebola virus disease when used at 2 × 107 plaque-forming units (PFU) in a trial in Guinea. This study provides further safety and immunogenicity data. Methods and findings A randomised, open-label phase I trial in Lambaréné, Gabon, studied 5 single intramuscular vaccine doses of 3 × 103, 3 × 104, 3 × 105, 3 × 106, or 2 × 107 PFU in 115 adults and a dose of 2 × 107 PFU in 20 adolescents and 20 children. The primary objective was safety and tolerability 28 days post-injection. Immunogenicity, viraemia, and shedding post-vaccination were evaluated as secondary objectives. In adults, mild-to-moderate adverse events were frequent, but there were no serious or severe adverse events related to vaccination. Before vaccination, Zaire Ebola virus (ZEBOV)–glycoprotein (GP)–specific and ZEBOV antibodies were detected in 11% and 27% of adults, respectively. In adults, 74%–100% of individuals who received a dose 3 × 104, 3 × 105, 3 × 106, or 2 × 107 PFU had a ≥4.0-fold increase in geometric mean titres (GMTs) of ZEBOV-GP-specific antibodies at day 28, reaching GMTs of 489 (95% CI: 264–908), 556 (95% CI: 280–1,101), 1,245 (95% CI: 899–1,724), and 1,503 (95% CI: 931–2,426), respectively. Twenty-two percent of adults had a ≥4-fold increase of ZEBOV antibodies, with GMTs at day 28 of 1,015 (647–1,591), 1,887 (1,154–3,085), 1,445 (1,013–2,062), and 3,958 (2,249–6,967) for the same doses, respectively. These antibodies persisted up to day 180 for doses ≥3 × 105 PFU. Adults with antibodies before vaccination had higher GMTs throughout. Neutralising antibodies were detected in more than 50% of participants at doses ≥3 × 105 PFU. As in adults, no serious or severe adverse events related to vaccine occurred in adolescents or children. At day 2, vaccine RNA titres were higher for adolescents and children than adults. At day 7, 78% of adolescents and 35% of children had recombinant vesicular stomatitis virus RNA detectable in saliva. The vaccine induced high GMTs of ZEBOV-GP-specific antibodies at day 28 in adolescents, 1,428 (95% CI: 1,025–1,989), and children, 1,620 (95% CI: 806–3,259), and in both groups antibody titres increased up to day 180. The absence of a control group, lack of stratification for baseline antibody status, and imbalances in male/female ratio are the main limitations of this study. Conclusions Our data confirm the acceptable safety and immunogenicity profile of the 2 × 107 PFU dose in adults and support consideration of lower doses for paediatric populations and those who request boosting. Trial registration Pan African Clinical Trials Registry PACTR201411000919191, Sanjeev Krishna and colleagues present a phase I randomised trial for safety and immunogenicity of an Ebola vaccine in both adults and children in Gabon., Author summary Why was this study done? The worst Ebola outbreak in history ended in 2016 after killing about 11,323 individuals and infecting 28,650 individuals worldwide. This public health emergency accelerated efforts to develop a vaccine as part of the strategy to contain the outbreak. Two vaccine candidates with preclinical safety and efficacy data obtained from non-human primates entered human trials. The one used in our study is the rVSVΔG-ZEBOV-GP vaccine, containing a non-infectious portion of a gene from the Zaire Ebola virus introduced into a recombinant vesicular stomatitis virus (rVSV), which itself is unlikely to cause disease in humans. To generate data for deployment of the vaccine, several dose-ranging phase I trials were initiated across centres in the United States, Europe, and Africa. What did the researchers do and find? We allocated 115 adults aged 18–50 years to receive 1 of the 5 doses used in the trial. A single intramuscular dose ranging from 3 × 103 to 2 × 107 plaque-forming units (PFU) was given, and participants were followed up until 6 months post-injection for safety and immunogenicity. Preliminary results led to the selection of the 2 × 107 PFU dose for further development. We also included 20 adolescents (13–17 years) and 20 children (6–12 years), who received the 2 × 107 PFU dose and were followed-up in a similar way as the adults. No vaccine-related serious or severe adverse event was reported by any participant. A high proportion of our population—even though residing in an area with no history of Ebola outbreak—had pre-vaccination antibodies specific to the Zaire Ebola virus. In adults, antibodies persisted up to 6 months post-injection at doses of 3 × 105 to 2 × 107 PFU. In participants with baseline antibodies, a dose as low as 3 × 104 PFU could induce high antibody titres up to day 56 post-injection. Higher vaccine replication, leading to shedding of the vaccine in saliva and urine, occurred in children and adolescents. What do these findings mean? Our results and other findings show that this vaccine is safe and immunogenic. Lower vaccine doses may be needed in paediatric populations as well as for boosting after primary vaccination or naturally acquired immunity.

During the recent Ebola outbreak in West Africa several international mobile laboratories were deployed to the mainly affected countries Guinea, Sierra Leone and Liberia to provide ebolavirus diagnostic capacity. Additionally, imported cases and small outbreaks in other countries required global preparedness for Ebola diagnostics. Detection of viral RNA by reverse transcription polymerase chain reaction has proven effective for diagnosis of ebolavirus disease and several assays are available. However, reliability of these assays is largely unknown and requires serious evaluation. Therefore, a proficiency test panel of 11 samples was generated and distributed on a global scale. Panels were analyzed by 83 expert laboratories and 106 data sets were returned. From these 78 results were rated optimal and 3 acceptable, 25 indicated need for improvement. While performance of the laboratories deployed to West Africa was superior to the overall performance there was no significant difference between the different assays applied., Author summary For the highly infectious and deadly ebolavirus disease (EVD) to date neither specific treatment nor vaccines are available. Rapid and adequate isolation of patients is the only option to contain and to combat spreading of the disease. Reliable and sensitive diagnosis that allows efficient identification of infected individuals is a pre-requisite for outbreak management. External Quality Assurance (EQA) studies are a vital tool to assess individual diagnostic laboratory performance particularly important during the outbreak of novel emerging infections. Therefore, a panel of inactivated ebolavirus samples was generated in order to perform an EQA for ebolavirus diagnostic during the recent outbreak in West Africa to assess performance of mobile laboratories sent to the outbreak countries from different parts of the world. Further, the panel was provided to laboratories in other parts of the world to improve global preparedness in case EVD would spread through international travel or evacuation of infected international staff members deployed to West Africa to fight the disease. While 73.6% of all results reported during this study were rated optimal the performance of the laboratories from the outbreak countries was even better with 82.1% of the results rated optimal.

The large scale of the Ebola virus disease (EVD) outbreak in West Africa in 2013-2016 raised the question whether the host cell interactions of the responsible Ebola virus (EBOV) strain differed from those of other ebolaviruses. We previously reported that the glycoprotein (GP) of the virus circulating in West Africa in 2014 (EBOV2014) exhibited reduced ability to mediate entry into two nonhuman primate (NHP)-derived cell lines relative to the GP of EBOV1976. Here, we investigated the molecular determinants underlying the differential entry efficiency. We found that EBOV2014-GP-driven entry into diverse NHP-derived cell lines, as well as human monocyte-derived macrophages and dendritic cells, was reduced compared to EBOV1976-GP, although entry into most human- and all bat-derived cell lines tested was comparable. Moreover, EBOV2014 replication in NHP but not human cells was diminished relative to EBOV1976, suggesting that reduced cell entry translated into reduced viral spread. Mutagenic analysis of EBOV2014-GP and EBOV1976-GP revealed that an amino acid polymorphism in the receptor-binding domain, A82V, modulated entry efficiency in a cell line-independent manner and did not account for the reduced EBOV2014-GP-driven entry into NHP cells. In contrast, polymorphism T544I, located in the internal fusion loop in the GP2 subunit, was found to be responsible for the entry phenotype. These results suggest that position 544 is an important determinant of EBOV infectivity for both NHP and certain human target cells. IMPORTANCE The Ebola virus disease outbreak in West Africa in 2013 entailed more than 10,000 deaths. The scale of the outbreak and its dramatic impact on human health raised the question whether the responsible virus was particularly adept at infecting human cells. Our study shows that an amino acid exchange, A82V, that was acquired during the epidemic and that was not observed in previously circulating viruses, increases viral entry into diverse target cells. In contrast, the epidemic virus showed a reduced ability to enter cells of nonhuman primates compared to the virus circulating in 1976, and a single amino acid exchange in the internal fusion loop of the viral glycoprotein was found to account for this phenotype.

Background The ideal protein expression system should provide recombinant proteins in high quality and quantity involving low production costs only. However, especially for complex therapeutic proteins like monoclonal antibodies many challenges remain to meet this goal and up to now production of monoclonal antibodies is very costly and delicate. Particularly, emerging disease outbreaks like Ebola virus in Western Africa in 2014–2016 make it necessary to reevaluate existing production platforms and develop robust and cheap alternatives that are easy to handle. Results In this study, we engineered the microalga Phaeodactylum tricornutum to produce monoclonal IgG antibodies against the nucleoprotein of Marburg virus, a close relative of Ebola virus causing severe hemorrhagic fever with high fatality rates in humans. Sequences for both chains of a mouse IgG antibody were retrieved from a murine hybridoma cell line and implemented in the microalgal system. Fully assembled antibodies were shown to be secreted by the alga and antibodies were proven to be functional in western blot, ELISA as well as IFA studies just like the original hybridoma produced IgG. Furthermore, synthetic variants with constant regions of a rabbit IgG and human IgG with optimized codon usage were produced and characterized. Conclusions This study highlights the potential of microalgae as robust and low cost expression platform for monoclonal antibodies secreting IgG antibodies directly into the culture medium. Microalgae possess rapid growth rates, need basically only water, air and sunlight for cultivation and are very easy to handle. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0745-2) contains supplementary material, which is available to authorized users.

Sequence determination of complete or coding-complete genomes of viruses is becoming common practice for supporting the work of epidemiologists, ecologists, virologists, and taxonomists. Sequencing duration and costs are rapidly decreasing, sequencing hardware is under modification for use by non-experts, and software is constantly being improved to simplify sequence data management and analysis. Thus, analysis of virus disease outbreaks on the molecular level is now feasible, including characterization of the evolution of individual virus populations in single patients over time. The increasing accumulation of sequencing data creates a management problem for the curators of commonly used sequence databases and an entry retrieval problem for end users. Therefore, utilizing the data to their fullest potential will require setting nomenclature and annotation standards for virus isolates and associated genomic sequences. The National Center for Biotechnology Information’s (NCBI’s) RefSeq is a non-redundant, curated database for reference (or type) nucleotide sequence records that supplies source data to numerous other databases. Building on recently proposed templates for filovirus variant naming [ ()////-], we report consensus decisions from a majority of past and currently active filovirus experts on the eight filovirus type variants and isolates to be represented in RefSeq, their final designations, and their associated sequences.

The host cell protein tetherin can restrict the release of enveloped viruses from infected cells. The HIV-1 protein Vpu counteracts tetherin by removing it from the site of viral budding, the plasma membrane, and this process depends on specific interactions between the transmembrane domains of Vpu and tetherin. In contrast, the glycoproteins (GPs) of two filoviruses, Ebola and Marburg virus, antagonize tetherin without reducing surface expression, and the domains in GP required for tetherin counteraction are unknown. Here, we show that filovirus GPs depend on the presence of their authentic transmembrane domains for virus-cell fusion and tetherin antagonism. However, conserved residues within the transmembrane domain were dispensable for membrane fusion and tetherin counteraction. Moreover, the insertion of the transmembrane domain into a heterologous viral GP, Lassa virus GPC, was not sufficient to confer tetherin antagonism to the recipient. Finally, mutation of conserved residues within the fusion peptide of Ebola virus GP inhibited virus-cell fusion but did not ablate tetherin counteraction, indicating that the fusion peptide and the ability of GP to drive host cell entry are not required for tetherin counteraction. These results suggest that the transmembrane domains of filoviral GPs contribute to tetherin antagonism but are not the sole determinants.

Therapeutic options for Ebola virus disease (EVD) are currently limited to (1) best supportive care, and (2) evolving virus-specific therapies, resulting from decades of analyzing one of the world's deadliest diseases. Supportive care ranges from oral or intravenous rehydration therapy and anti-emetics in developing countries to much more extensive life-support interventions in resource-rich countries. Current EVD-specific therapies attempt to either interfere with the earliest steps of viral replication or to elicit a strong immune response against the virus. An entirely new approach is the extracorporeal elimination of viruses and viral glycoproteins by lectin affinity plasmapheresis. Herein, we report for the first time the successful and safe use of lectin affinity plasmapheresis in a patient with severe Ebola virus disease.

The World Health Organization (WHO) published 2 alcohol-based formulations to be used in healthcare settings and for outbreak-associated infections, but inactivation efficacies of these products have not been determined against (re-)emerging viruses. In this study, we evaluated the virucidal activity of these WHO products in a comparative analysis. Zika virus (ZIKV), Ebola virus (EBOV), severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) as (re-)emerging viral pathogens and other enveloped viruses could be efficiently inactivated by both WHO formulations, implicating their use in healthcare systems and viral outbreak situations.

Ebola virus is a non-segmented negative-sense RNA virus causing severe hemorrhagic fever with high fatality rates in humans and nonhuman primates. For transcription of the viral genome four viral proteins are essential: the nucleoprotein NP, the polymerase L, the polymerase cofactor VP35, and VP30. VP30 represents an essential Ebola virus-specific transcription factor whose activity is regulated via its phosphorylation state. In contrast to viral transcription, VP30 is not required for viral replication. Using a minigenome assay, we show that phosphorylation of VP30 inhibits viral transcription while viral replication is increased. Concurrently, phosphorylation of VP30 reciprocally regulates a newly described interaction of VP30 with VP35, and strengthens the interaction with NP. Our results indicate a critical role of VP30 phosphorylation for viral transcription and replication, suggesting a mechanism by which VP30 phosphorylation modulates the composition of the viral polymerase complex presumably forming a transcriptase in the presence of non-phosphorylated VP30 or a replicase in the presence of phosphorylated VP30.

We report here a complete genome sequence of Ebola virus Makona from a nonfatal patient sample that originated in Sierra Leone during the last Ebola virus outbreak in West Africa (species Zaire ebolavirus ) using a highly accurate circle sequencing (Cir-seq) method.

The Ebola virus (EBOV) outbreak in West Africa started in December 2013, claimed more than 11,000 lives, threatened to destabilize a whole region, and showed how easily health crises can turn into humanitarian disasters. EBOV genomic sequences of the West African outbreak revealed nonsynonymous mutations, which induced considerable public attention, but their role in virus spread and disease remains obscure. In this study, we investigated the functional significance of three nonsynonymous mutations that emerged early during the West African EBOV outbreak. Almost 90% of more than 1,000 EBOV genomes sequenced during the outbreak carried the signature of three mutations: a D759G substitution in the active center of the L polymerase, an A82V substitution in the receptor binding domain of surface glycoprotein GP, and an R111C substitution in the self-assembly domain of RNA-encapsidating nucleoprotein NP. Using a newly developed virus-like particle system and reverse genetics, we found that the mutations have an impact on the functions of the respective viral proteins and on the growth of recombinant EBOVs. The mutation in L increased viral transcription and replication, whereas the mutation in NP decreased viral transcription and replication. The mutation in the receptor binding domain of the glycoprotein GP improved the efficiency of GP-mediated viral entry into target cells. Recombinant EBOVs with combinations of the three mutations showed a growth advantage over the prototype isolate Makona C7 lacking the mutations. This study showed that virus variants with improved fitness emerged early during the West African EBOV outbreak. IMPORTANCE The dimension of the Ebola virus outbreak in West Africa was unprecedented. Amino acid substitutions in the viral L polymerase, surface glycoprotein GP, and nucleocapsid protein NP emerged, were fixed early in the outbreak, and were found in almost 90% of the sequences. Here we showed that these mutations affected the functional activity of viral proteins and improved viral growth in cell culture. Our results demonstrate emergence of adaptive changes in the Ebola virus genome during virus circulation in humans and prompt further studies on the potential role of these changes in virus transmissibility and pathogenicity.

The DEAD-box RNA helicase eIF4A, which is part of the heterotrimeric translation initiation complex in eukaryotes, is an important novel drug target in cancer research because its helicase activity is required to unwind extended and highly structured 5′-UTRs of several proto-oncogenes. Silvestrol, a natural compound isolated from the plant Aglaia foveolata , is a highly efficient, non-toxic and specific inhibitor of eIF4A. Importantly, 5′-capped viral mRNAs often contain structured 5′-UTRs as well, which may suggest a dependence on eIF4A for their translation by the host protein synthesis machinery. In view of the recent Ebola virus (EBOV) outbreak in West Africa, the identification of potent antiviral compounds is urgently required. Since Ebola mRNAs are 5′-capped and harbor RNA secondary structures in their extended 5′-UTRs, we initiated a BSL4 study to analyze silvestrol in EBOV-infected Huh-7 cells and in primary human macrophages for its antiviral activity. We observed that silvestrol inhibits EBOV infection at low nanomolar concentrations, as inferred from large reductions of viral titers. This correlated with an almost complete disappearance of EBOV proteins, comparable in effect to the translational shutdown of expression of the proto-oncoprotein PIM1, a cellular kinase known to be affected by silvestrol. Effective silvestrol concentrations were non-toxic in the tested cell systems. Thus, silvestrol appears to be a promising first-line drug for the treatment of acute EBOV and possibly other viral infections.

The West African Ebola virus disease (EVD) outbreak was the largest EVD outbreak in history. However, data on lymphocyte dynamics and the antigen specificity of T cells in Ebola survivors are scarce, and our understanding of EVD pathophysiology is limited. A case of EVD survival in which the patient cleared Ebola virus (EBOV) infection without experimental drugs allowed for the detailed examination of lymphocyte dynamics. We demonstrate the persistence of T-cell activation well beyond viral clearance and detect EBOV-specific T cells. Our study provides significant insights into lymphocyte specificity during the recovery phase of EVD and may inform novel strategies to treat EVD.

Background Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes high fever, rash, and recurrent arthritis in humans. It has efficiently adapted to Aedes albopictus, which also inhabits temperate regions and currently causes large outbreaks in the Caribbean and Latin America. Ebola virus (EBOV) is a member of the filovirus family. It causes the Ebola virus disease (EDV), formerly known as Ebola hemorrhagic fever in humans and has a mortality rate of up to 70 %. The last outbreak in Western Africa was the largest in history and has caused approximately 25,000 cases and 10,000 deaths. For both viral infections no specific treatment or licensed vaccine is currently available. The bis-hexasulfonated naphthylurea, suramin, is used as a treatment for trypanosome-caused African river blindness. As a competitive inhibitor of heparin, suramin has been described to have anti-viral activity. Methods We tested the activity of suramin during CHIKV or Ebola virus infection, using CHIKV and Ebola envelope glycoprotein pseudotyped lentiviral vectors and wild-type CHIKV and Ebola virus. Results Suramin efficiently inhibited CHIKV and Ebola envelope-mediated gene transfer while vesicular stomatitis virus G protein pseudotyped vectors were only marginally affected. In addition, suramin was able to inhibit wild-type CHIKV and Ebola virus replication in vitro. Inhibition occurred at early time points during CHIKV infection. Conclusion Suramin, also known as Germanin or Bayer-205, is a market-authorized drug, however shows significant side effects, which probably prevents its use as a CHIKV drug, but due to the high lethality of Ebola virus infections, suramin might be valuable against Ebola infections.

The template for Ebola virus (EBOV) transcription and replication is the helical viral nucleocapsid composed of the viral negative-sense (−) RNA genome, which is complexed by the nucleoprotein (NP), VP35, polymerase L, VP24, and VP30. While viral replication is exerted by polymerase L and its cofactor VP35, EBOV mRNA synthesis is regulated by the viral nucleocapsid protein VP30, an essential EBOV-specific transcription factor. VP30 is a homohexameric phosphoprotein containing a nonconventional zinc finger. The transcriptional support activity of VP30 is strongly influenced by its phosphorylation state. We studied here how RNA binding contributed to VP30's function in transcriptional activation. Using a novel mobility shift assay and the 3′-terminal 154 nucleotides of the EBOV genome as a standard RNA substrate, we detected that RNA binding of VP30 was severely impaired by VP30 mutations that (i) destroy the protein's capability to form homohexamers, (ii) disrupt the integrity of its zinc finger domain, (iii) mimic its fully phosphorylated state, or (iv) alter the putative RNA binding region. RNA binding of the mutant VP30 proteins correlated strongly with their transcriptional support activity. Furthermore, we showed that the interaction between VP30 and the polymerase cofactor VP35 is RNA dependent, while formation of VP30 homohexamers and VP35 homotetramers is not. Our data indicate that RNA binding of VP30 is essential for its transcriptional support activity and stabilizes complexes of VP35/L polymerase with the (−) RNA template to favor productive transcriptional initiation in the presence of termination-active RNA secondary structures. IMPORTANCE Ebola virus causes severe fevers with unusually high case fatality rates. The recent outbreak of Ebola virus in West Africa claimed more than 11,000 lives and threatened to destabilize a whole region because of its dramatic effects on the public health systems. It is currently not completely understood how Ebola virus manages to balance viral transcription and replication in the infected cells. This study shows that transcriptional support activity of the Ebola virus transcription factor VP30 is highly correlated with its ability to bind viral RNA. The interaction between VP30 and VP35, the Ebola virus polymerase cofactor, is dependent on the presence of RNA as well. Our data contribute to the understanding of the dynamic interplay between nucleocapsid proteins and the viral RNA template in order to promote viral RNA synthesis.

BACKGROUND\ud The West African outbreak of Ebola virus disease that peaked in 2014 has caused more than 11,000 deaths. The development of an effective Ebola vaccine is a priority for control of a future outbreak.\ud \ud METHODS\ud In this phase 1 study, we administered a single dose of the chimpanzee adenovirus 3 (ChAd3) vaccine encoding the surface glycoprotein of Zaire ebolavirus (ZEBOV) to 60 healthy adult volunteers in Oxford, United Kingdom. The vaccine was administered in three dose levels — 1×1010 viral particles, 2.5×1010 viral particles, and 5×1010 viral particles — with 20 participants in each group. We then assessed the effect of adding a booster dose of a modified vaccinia Ankara (MVA) strain, encoding the same Ebola virus glycoprotein, in 30 of the 60 participants and evaluated a reduced prime–boost interval in another 16 participants. We also compared antibody responses to inactivated whole Ebola virus virions and neutralizing antibody activity with those observed in phase 1 studies of a recombinant vesicular stomatitis virus–based vaccine expressing a ZEBOV glycoprotein (rVSV-ZEBOV) to determine relative potency and assess durability.\ud \ud RESULTS\ud No safety concerns were identified at any of the dose levels studied. Four weeks after immunization with the ChAd3 vaccine, ZEBOV-specific antibody responses were similar to those induced by rVSV-ZEBOV vaccination, with a geometric mean titer of 752 and 921, respectively. ZEBOV neutralization activity was also similar with the two vaccines (geometric mean titer, 14.9 and 22.2, respectively). Boosting with the MVA vector increased virus-specific antibodies by a factor of 12 (geometric mean titer, 9007) and increased glycoprotein-specific CD8+ T cells by a factor of 5. Significant increases in neutralizing antibodies were seen after boosting in all 30 participants (geometric mean titer, 139; P