Zaire Ebolavirus Research Articles

RVSVΔG-ZEBOV-GP (also designated V920) recombinant vesicular stomatitis virus pseudotyped with Ebola Zaire Glycoprotein: Standardized template with key considerations for a risk/benefit assessment

The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety and characteristics of live, recombinant viral vector vaccines. A recent publication by the V3SWG described live, attenuated, recombinant vesicular stomatitis virus (rVSV) as a chimeric virus vaccine for HIV-1 (Clarke et al., 2016). The rVSV vector system is being explored as a platform for development of multiple vaccines. This paper reviews the molecular and biological features of the rVSV vector system, followed by a template with details on the safety and characteristics of a rVSV vaccine against Zaire ebolavirus (ZEBOV). The rVSV-ZEBOV vaccine is a live, replication competent vector in which the VSV glycoprotein (G) gene is replaced with the glycoprotein (GP) gene of ZEBOV. Multiple copies of GP are expressed and assembled into the viral envelope responsible for inducing protective immunity. The vaccine (designated V920) was originally constructed by the National Microbiology Laboratory, Public Health Agency of Canada, further developed by NewLink Genetics Corp. and Merck & Co., and is now in final stages of registration by Merck. The vaccine is attenuated by deletion of the principal virulence factor of VSV (the G protein), which also removes the primary target for anti-vector immunity. The V920 vaccine caused no toxicities after intramuscular (IM) or intracranial injection of nonhuman primates and no reproductive or developmental toxicity in a rat model. In multiple studies, cynomolgus macaques immunized IM with a wide range of virus doses rapidly developed ZEBOV-specific antibodies measured in IgG ELISA and neutralization assays and were fully protected against lethal challenge with ZEBOV virus. Over 20,000 people have received the vaccine in clinical trials; the vaccine has proven to be safe and well tolerated. During the first few days after vaccination, many vaccinees experience a mild acute-phase reaction with fever, headache, myalgia, and arthralgia of short duration; this period is associated with a low-level viremia, activation of anti-viral genes, and increased levels of chemokines and cytokines. Oligoarthritis and rash appearing in the second week occur at a low incidence, and are typically mild-moderate in severity and self-limited. V920 vaccine was used in a Phase III efficacy trial during the West African Ebola epidemic in 2015, showing 100% protection against Ebola Virus Disease, and it has subsequently been deployed for emergency control of Ebola outbreaks in central Africa. The template provided here provides a comprehensive picture of the first rVSV vector to reach the final stage of development and to provide a solution to control of an alarming human disease.

Safety, tolerability, pharmacokinetics, and immunogenicity of the therapeutic monoclonal antibody mAb114 targeting Ebola virus glycoprotein (VRC 608): an open-label phase 1 study

mAb114 is a single monoclonal antibody that targets the receptor-binding domain of Ebola virus glycoprotein, which prevents mortality in rhesus macaques treated after lethal challenge with Zaire ebolavirus. Here we present expedited data from VRC 608, a phase 1 study to evaluate mAb114 safety, tolerability, pharmacokinetics, and immunogenicity. In this phase 1, dose-escalation study (VRC 608), conducted at the US National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA), healthy adults aged 18-60 years were sequentially enrolled into three mAb114 dose groups of 5 mg/kg, 25 mg/kg, and 50 mg/kg. The drug was given to participants intravenously over 30 min, and participants were followed for 24 weeks. Participants were only enrolled into increased dosing groups after interim safety assessments. Our primary endpoints were safety and tolerability, with pharmacokinetic and anti-drug antibody assessments as secondary endpoints. We assessed safety and tolerability in all participants who received study drug by monitoring clinical laboratory data and self-report and direct clinician assessment of prespecified infusion-site symptoms 3 days after infusion and systemic symptoms 7 days after infusion. Unsolicited adverse events were recorded for 28 days. Pharmacokinetic and anti-drug antibody assessments were completed in participants with at least 56 days of data. This trial is registered with ClinicalTrials.gov, number NCT03478891, and is active but no longer recruiting. Between May 16, and Sept 27, 2018, 19 eligible individuals were enrolled. One (5%) participant was not infused because intravenous access was not adequate. Of 18 (95%) remaining participants, three (17%) were assigned to the 5 mg/kg group, five (28%) to the 25 mg/kg group, and ten (55%) to the 50 mg/kg group, each of whom received a single infusion of mAb114 at their assigned dose. All infusions were well tolerated and completed over 30-37 min with no infusion reactions or rate adjustments. All participants who received the study drug completed the safety assessment of local and systemic reactogenicity. No participants reported infusion-site symptoms. Systemic symptoms were all mild and present only in four (22%) of 18 participants across all dosing groups. No unsolicited adverse events occurred related to mAb114 and one serious adverse event occurred that was unrelated to mAb114. mAb114 has linear pharmacokinetics and a half-life of 24·2 days (standard error of measurement 0·2) with no evidence of anti-drug antibody development. mAb114 was well tolerated, showed linear pharmacokinetics, and was easily and rapidly infused, making it an attractive and deployable option for treatment in outbreak settings. Vaccine Research Center, US National Institute of Allergy and Infectious Diseases, and NIH.

Computational analysis in designing T cell epitopes enriched peptides of Ebola glycoprotein exhibiting strong binding interaction with HLA molecules

Computational approach has shown remarkable progress in epitope mapping, paving the way to finding vaccine candidates against different viruses. In the current study, prediction algorithms and molecular docking were applied to select peptides containing multiple Ebola glycoprotein epitopes showing interaction with different HLA molecules. Six peptides containing overlapping multiple HLA I (CD8+) and II (CD4+) restricted T cell epitopes were generated via consensus approach applying six different prediction tools. Four (P1, P2, P5 and P6) out of six peptides were selected after screening for absence of undesirable responses and presence of B cell epitopes. Peptide-HLA interaction analysis based on Autodock Vina and CABS-dock showed strong binding of these four peptides with eighteen HLA molecules. HLA coverage analysis from each prediction tool showed that these peptides were able to bind to diverse HLA-A, HLA-B, HLA-DP, HLA-DQ and HLA-DR alleles. Population coverage analysis of peptides for expected immune response in four different continents (Africa, America, Asia and Europe) have shown average population coverage viz, P1 (95%), P2 (96%), P5 (91%) and P6 (94%). Further, these peptides were found to be nearly 100% conserved in Zaire Ebola virus while LANETTQALQLF (P5) was found to be 100% conserved in Zaire, Sudan, Bundibugyo and Tai Forest species. Therefore, these peptides capable of inducing T and B cell response and being presented by a wide range of HLA molecules have a strong potential to be part of diagnostic and preventive tools against Ebola virus disease.

The efficacy of poly-ICLC against Ebola-Zaire virus (EBOV) infection in mice and cynomolgus monkeys

The potential protection of poly-ICLC (Hiltonol®) a double stranded RNA (dsRNA) against EBOV infection was assessed with prophylactic and therapeutic administration to wild type and TLR3-negative mice, and in non-human primates (NHPs) by measuring EBOL serum titers, survival extension, and serum liver and kidney function markers. Various doses of aqueous and liposomal poly-ICLC monotherapy provided robust protection in otherwise lethal murine EBOV challenge models, when treatment is started on the day 0 or one day after virus challenge. There was no advantage of liposomal vs. the aqueous poly-ICLC form. Protection appeared to be independent of TLR-3. NHPs treated with poly-ICLC and challenged with EBOV survived longer but eventually succumbed to Ebola infection. Nevertheless, the liver and kidney serum markers were markedly reduced in the infected and treated NHPs. In the two longest surviving poly-ICLC- treated NHPs, the day 10 serum EBOV titer was reduced 2.1 and 30 fold respectively.

Exploring the human-animal interface of Ebola virus disease outbreaks.

Whereas the prevention and treatment of Ebola virus disease (EVD) have been well studied after the 2013-16 outbreak in West Africa, the emergence of human outbreaks and their mechanisms have yet to be explored in detail. In particular, it has yet to be clarified whether the emergence records offer any theoretical insight into the changing interface between humans and animal reservoirs. Here we explore the epidemiological record of emergence, investigating predominant causes of the introduction to the human population, their characteristics, and frequencies. We retrieved data of every outbreak that can be traced back to a single zoonotic spillover. Through statistical analysis, we have shown that (i) the leading cause of emergence was eating and hunting habits, (ii) primates act as the main source of zoonotic spillover, and (iii) Zaire ebolavirus is the most virulent type. Moreover, the trend of emergence was demonstrated not to be a Poisson process, indicating that some unknown, underlying, non-random mechanisms are likely to govern the spillover event. In the Democratic Republic of Congo, an increasing emergence trend was favored compared with a purely random emergence model. Outbreak event data and their causative viruses should be explored biologically and epidemiologically to possibly predict future outbreak events.

Cloning, Expression and Purification of Recombinant Forms of Full Length and Extracellular Domain EBOV Glycoprotein within Mammalian Cell-Lines

Background: Full length Ebolavirus glycoprotein (GP) intersperses the outer most lipid membrane to form spikes, where it mediates virus-host cell interaction. A secretory form of GP (sGP) is also produced by all 5 known Ebolavirus species. These attributes make GP an ideal target for research and development (R and D) of Ebolavirus and possibly pan-filovirus targeted Rapid Diagnostic Tests (RDTs), bio-therapeutics and vaccines. Prior cloning of recombinant Zaire ebolavirus (EBOV) GP has majorly used insect (baculovirus ) expression systems. We report the cloning, expression and purification of the full length and extracellular domain (ECD) forms of recombinant EBOV GP in mammalian cell-lines. Methods and results: 2034 and 1956 base-pair (bp) coding DNA sequences corresponding to the 669 and 643 amino acids (aa) residues of full length and ECD forms of EBOV GP were sub-cloned into the pTGE plasmids. Recombinant pTGE-plasmids were used to transfect 293-6E HEK mammalian cells grown in serum-free FreeStyleTM 293 Expression Medium. Cell lysates and or culture supernatants were used to obtain purified protein, followed by analysis on SDS-PAGE and Western blot. Purified full length GP was detected as membrane bound protein in cell lysates with estimated molecular weight of ~100 kDa (Cal.M.W.~71.67 kDa) on Western blot; and 0.02 mg GP (Concentration: 0.2 mg/mL, Purity: ~50%) derived. On the contrary, ECD GP was detected in supernatants of cell culture broth with estimated molecular weights of ~116 kDa based on SDS-PAGE and Western blot; and 1.6 mg (Concentration: 0.4 mg/ml, Purity: ~70%) of GP_ECD was obtained. Conclusion: Within mammalian cells, recombinant full length EBOV GP is predominantly expressed as transmembrane protein (tGP), while ECD GP is eluted into the culture medium. Both recombinant forms of GP are critical for the R and D of rapid diagnostic tests (RDTs).

In silico identified immunogenic Ebola nucleoprotein peptides elicit immune response

Immunoinformatics has dropped significantly to discovering strong antibody competitors against different microorganisms. In the momentum study, a blend of various T (CD4+ and CD8+) and B cell epitope expectation devices was applied to discover peptides containing numerous epitopes against Ebola nucleoprotein (NP) and the introduction of peptides to human leukocyte antigen (HLA) atoms was examined by forecast, docking and populace inclusion apparatuses. Further, ELISA was done to gauge IFN-γ in peptide animated fringe blood mononuclear cells disengaged from the blood of sound volunteers. Six peptides containing various T and B cell epitopes were acquired after expectation examines and dispensing with the peptides at risk to create immune system and unfavorably susceptible reaction. All peptides showed 100% conservancy in Zaire Ebola infection. Forecast devices, Auto dock Vina and CABS-dock results affirmed the capacity of anticipated peptides to tie with assorted HLA alleles. Populace inclusion investigation anticipated high inclusion (> 85%) for anticipated insusceptible reaction in four landmasses (Africa, America, Asia and Europe). Peptide animated cells showed upgraded IFN-γ emission when contrasted with unstimulated cells. Thusly, the recognized NP peptides can be considered as potential engineered antibody competitors against Ebola virus.

Molecular dynamics and combined docking studies for the identification of Zaire ebola virus inhibitors

Ebola virus (EBOV) is a lethal human pathogen with a risk of global spread of its zoonotic infections, and Ebolavirus Zaire specifically has the highest fatality rate amongst other species. There is a need for continuous effort towards having therapies, as a single licensed treatment to neutralize the EBOV is yet to come into reality. This present study virtually screened the MCULE database containing almost 36 million compounds against the structure of a Zaire Ebola viral protein (VP) 35 and a consensus scoring of both MCULE and CLCDDW docking programs remarked five compounds as potential hits. These compounds, with binding energies ranging from –7.9 to –8.9 kcal/mol, were assessed for predictions of their physicochemical and bioactivity properties, as well as absorption, distribution, metabolism, excretion, and toxicity (ADMET) criteria. The results of the 50 ns molecular dynamics simulations showed the presence of dynamic stability between ligand and protein complexes, and the structures remained significantly unchanged at the ligand-binding site throughout the simulation period. Both docking analysis and molecular dynamics simulation studies suggested strong binding affinity towards the receptor cavity and these selected compounds as potential inhibitors against the Zaire Ebola VP 35. With respect to inhibition constant values, bioavailability radar and other physicochemical properties, compound A (MCULE-1018045960-0-1) appeared to be the most promising hit compound. However, the ligand efficiency and ligand efficiency scale need improvement during optimization, and also validation via in vitro and in vivo studies are necessary to finally make a lead compound in treating Ebola virus diseases.Communicated by Ramaswamy H. Sarma

Serological Detection of Ebola Virus Exposures in Native Non-human Primates of Southern Nigeria

Ebola viruses (family: Filoviridae) are the cause of Ebola virus disease (EVD), a highly fatal illness characterised by haemorrhagic fever syndrome in both humans and non-human primates (NHPs). West Africa was the epicentre of the 2013–2015 EVD epidemic which caused the death of over 11,000 people, including eight casualties in southern Nigeria. Antibodies to filoviruses have been detected among NHPs in some countries, but there is no documented evidence of exposures to filoviruses among NHPs in Nigeria. From August 2015 to February 2017, a total of 142 serum samples were obtained from individual captive and wild animals, belonging to 11 NHP species, in southern Nigeria, and screened for species-specific antibodies to filoviruses belonging to the species; Zaire ebolavirus [Ebola virus (EBOV)], Sudan ebolavirus [Sudan virus (SUDV)], and Marburg marburgvirus [Ravn virus (RAVV)]–using a modifed filovirus species-specific ELISA technique. Of the sera tested, 2.1% (3/142) were positive for antibodies to EBOV. The entire 142 sera were negative for SUDV or RAVV. These findings point to the existence of natural exposures of NHPs in southern Nigeria to EBOV. There is need to discourage, the uncontrolled hunting of NHPs in Nigeria for public health safety.

Survey of Ebola Viruses in Frugivorous and Insectivorous Bats in Guinea, Cameroon, and the Democratic Republic of the Congo, 2015-2017.

To clarify the role of bats in the ecology of Ebola viruses, we assessed the prevalence of Ebola virus antibodies in a large-scale sample of bats collected during 2015–2017 from countries in Africa that have had previous Ebola outbreaks (Guinea, the Democratic Republic of the Congo) or are at high risk for outbreaks (Cameroon). We analyzed 4,022 blood samples of bats from >12 frugivorous and 27 insectivorous species; 2–37 (0.05%–0.92%) bats were seropositive for Zaire and 0–30 (0%–0.75%) bats for Sudan Ebola viruses. We observed Ebola virus antibodies in 1 insectivorous bat genus and 6 frugivorous bat species. Certain bat species widespread across Africa had serologic evidence of Zaire and Sudan Ebola viruses. No viral RNA was detected in the subset of samples tested (n = 665). Ongoing surveillance of bats and other potential animal reservoirs are required to predict and prepare for future outbreaks.

Dual monoclonal antibody-based sandwich ELISA for detection of in vitro packaged Ebola virus

BackgroundRapid transmission and high mortality of Ebola virus disease (EVD) highlight a urgent need of large scale, convenient and effective measure for Ebola virus screening. Application of monoclonal antibodies (mAbs) are crucial for establishment of an enzyme-linked immunosorbent assay (ELISA) with high sensitivity and specificity.MethodsThe traditional cell fusion technique was used to generate a panel of hybridomas. Two mAbs were characterized by SDS-PAGE, Western blot, Indirect immunofluorescence assay (IFA). A sandwich ELISA was established using the two mAbs. The detection capability of the ELISA was evaluated.ResultsIn the current study, we produced two murine-derived mAbs (designated as 6E3 and 3F21) towards Zaire Ebola virus glycoprotein (GP), the major viral transmembrane spike protein associated with viral attachment. It was shown that 6E3 and 3F21 recognized GP1 and GP2 subunits of the GP respectively. Furthermore, 6E3 and 3F21 bound to corresponding epitopes on GP without reciprocal topographical interpretation. Subsequently, a sandwich ELISA based on the two mAbs were established and evaluated. The detection limit was 3.6 ng/ml, with a linear range of 3.6–100 ng/ml. More importantly, Ebola virus like particles (eVLPs) were able to be detected by this established virus detection measure.ConclusionsWe produced and characterized two murine-derived mAbs (designated as 6E3 and 3F21) towards Zaire Ebola virus glycoprotein (GP), and established a sandwich ELISA based on the mAbs. It was suggested that the sandwich ELISA provided an alternative method for specific and sensitive detection of Ebola virus in the field setting.

Standardized focus assay protocol for biosafety level four viruses

Working in accordance with biosafety level four practices is highly complex and time-consuming. Therefore, the respective laboratory protocols should be as uniform as possible, simple to perform and straightforward in readout. Here we describe the successful application of a standardized 24-well plate focus assay protocol for the titration of Zaire ebolavirus (two isolates), Marburg virus (three isolates), Lassa virus (two isolates), Crimean Congo hemorrhagic fever virus (one isolate), and tick-borne encephalitis virus (two isolates). Viral titers are determined based on a simple visual readout. The protocol exhibits high precision, with coefficients of variation for interassay variability ranging between 0.05 and 0.21 and those for intraassay variability between 0.08 and 0.23. All reagents required for the test, including primary and secondary antibodies, are commercially available, facilitating the establishment of the protocol in other laboratories.

Chaperones, Membrane Trafficking and Signal Transduction Proteins Regulate Zaire Ebola Virus trVLPs and Interact With trVLP Elements.

Ebolavirus (EBOV) life cycle involves interactions with numerous host factors, but it remains poorly understood, as does pathogenesis. Herein, we synthesized 65 siRNAs targeting host genes mostly connected with aspects of the negative-sense RNA virus life cycle (including viral entry, uncoating, fusion, replication, assembly, and budding). We produced EBOV transcription- and replication-competent virus-like particles (trVLPs) to mimic the EBOV life cycle. After screening host factors associated with the trVLP life cycle, we assessed interactions of host proteins with trVLP glycoprotein (GP), VP40, and RNA by co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP). The results demonstrate that RNAi silencing with 11 siRNAs (ANXA5, ARFGAP1, FLT4, GRP78, HSPA1A, HSP90AB1, HSPA8, MAPK11, MEK2, NTRK1, and YWHAZ) decreased the replication efficiency of trVLPs. Co-IP revealed nine candidate host proteins (FLT4, GRP78, HSPA1A, HSP90AB1, HSPA8, MAPK11, MEK2, NTRK1, and YWHAZ) potentially interacting with trVLP GP, and four (ANXA5, GRP78, HSPA1A, and HSP90AB1) potentially interacting with trVLP VP40. Ch-IP identified nine candidate host proteins (ANXA5, ARFGAP1, FLT4, GRP78, HSPA1A, HSP90AB1, MAPK11, MEK2, and NTRK1) interacting with trVLP RNA. This study was based on trVLP and could not replace live ebolavirus entirely; in particular, the interaction between trVLP RNA and host proteins cannot be assumed to be identical in live virus. However, the results provide valuable information for further studies and deepen our understanding of essential host factors involved in the EBOV life cycle.

Flexible docking-based molecular dynamics simulation of natural product compounds and Ebola virus Nucleocapsid (EBOV NP): a computational approach to discover new drug for combating Ebola

BackgroundEbola still remains as one of the most problematic infectious diseases in Africa with a high rate of mortality. Although this disease has been known for an almost half-century, there are no vaccines and drugs available in the market to treat Ebola. Zaire ebolavirus (EBOV), a single-stranded RNA virus which belongs to Filoviridae family and Mononegavirales order, is one of the virus causing Ebola. As one of seven proteins that EBOV encodes, Ebola virus nucleoprotein (EBOV NP) plays an imperative role in EBOV proliferation cycle. Therefore, the development of a new Ebola treatment can be targeted towards EBOV NP.ResultsIn this work, we screened about 190,084 natural product compounds from ZINC15 database through in silico virtual screening and flexible docking simulation. Furthermore, the bioavailability and toxicity prediction have been conducted as well. Two best ligands according to the simulation and prediction tests were progressed into the molecular dynamics simulation.ConclusionIn the end, we found that our proposed ligands, namely α-lipomycin (ZINC56874155) and 3-(((S)-1-amino-1,2,3,4-tetrahydroisoquinolin-5-yl)methyl)-5-((5-((5R,7S)-5,7-dihydroxy-3-oxodecyl)-2-hydroxyphenoxy) methyl)pyrrolo[3,4-b]pyrrol-5-ium (ZINC85628951), showed the promising results to be developed as a lead compounds for treating Ebola. Therefore, an experimental study is required to validate their inhibition activities against EBOV NP.

Chiroptera as a Potential Reservoir of Dangerous for Humans Viruses in the territory of the Republic of Guinea. Part 1

Chiroptera is one of the largest in numbers orders of mammals which can take on the role of natural host and vector of various viruses, bacteria, and pathogenic fungi. Over the period of 2007–2013, 248 new viruses pertaining to 24 families were detected in chiropterans. Lately, a range of novel infections that provoked severe diseases in humans were associated with bats-carriers. Presently they are viewed as a potential reservoir of Ebola, Marburg, and Hendra viruses, severe acute respiratory syndrome agents, as well as many other lethal diseases. Chiropterans started drawing particular attention after establishment of the fact that they are involved in circulation of ebolavirus Zaire and possibly participate in the formation of natural foci of this infection. Currently chiropterans are considered to be natural reservoirs of ebolavirus Zaire and some other filoviruses (Filoviridae). Accept from filoviruses, other significant for public healthcare viruses were isolated from chiropterans on the African continent – lyssaviruses (Rhabdoviridae, Lyssavirus), henipaviruses (Paramixoviridae, Henipavirus), and coronaviruses (Coronaviridae, Alphacoronavirus, Betacoronavirus) that can also circulate among chiropterans in West Africa. The data available from literature sources on dissemination of chiropterans in the territory of the African continent, their species diversity, peculiarities of ecology and behavior are analyzed in this paper. The information on the infections for which chiropterans can serve as potential reservoirs is also provided. Specifics of interaction between the population residing on African continent and bats of different species are described too. The review is devoted to the assessment of results of studies on population, ecological, and epidemiological factors contributing to maintenance of circulation of a number of dangerous for humans viruses (filoviruses, lyssaviruses, henipaviruses, and coronaviruses) among African chiropterans. The need for further investigation of the mentioned factors affecting chiropteran populations in the Republic of Guinea is substantiated.

Identity and validity of conserved B cell epitopes of filovirus glycoprotein: towards rapid diagnostic testing for Ebola and possibly Marburg virus disease

BackgroundEbolavirus and Marburgvirus are genera of the virus family Filoviridae. Filoviruses cause rare but fatal viral hemorrhagic fevers (VHFs) in remote villages of equatorial Africa with potential for regional and international spread. Point-of-care (POC) rapid diagnostic tests (RDTs) are critical for early epidemic detection, reponse and control. There are 2 RDTs for Zaire ebolavirus (EBOV), but not other Ebolavirus spp. or Marburg marburgvirus (MARV). We validate 3 conserved B cell epitopes of filovirus glycoprotein (GP) using ebola virus diseases (EVD) survivor samples, towards devising pan-filovirus RDTs.MethodsIn-silico Immuno-informatics:- (a) multiple and basic local alignments of amino-acid sequences of filovirus (4 Ebolavirus spp. & MARV) Gp1, 2 and epitope prediction and conservation analyses within context of ClusterW, BLAST-P and the immune epitope database analysis resource (IEDB-AR); alongside (b) in-vitro enzyme immuno-assays (EIAs) for SUDV Gp1, 2 antigen and host-specific antibodies (IgM and IgG) among 94 gamma irradiated EVD survivor serum and 9 negative controls.ResultsLinear B cell epitopes were present across the entire length of all Gp1, 2, most lying in the region between amino acids positioned 350 and 500. Three seperate epitopes 97/80_GAFFLYDRLAST, 39_YEAGEWAENCY and 500_CGLRQLANETTQALQLFLRATTELR (designated UG-Filo-Peptide− 1, 2 and 3 respectively) were conserved within all studied filovirus species Gp1, 2. Gp1, 2 host specific IgM levels were comparably low (av. ODs < 0.04 [95% CI: 0.02837 to 0.04033]) among the 9 negative controls and 57 survivor samples analyzed. Host specific IgG levels, on the other hand, were elevated (av. ODs > 1.7525 [95% CI: 0.3010 to 3.1352]) among the 92 survivor samples relative to the 9 negative controls (av. ODs < 0.2.321 [95% CI: -0.7596 to 0.5372]). Filovirus Gp1, 2 antigen was not detected [av. ODs < 0.20] within EVD survivor serum relative to recombinant protein positive controls [av. ODs = 0.50].ConclusionsThese conserved B cell epitopes of filovirus Gp1, 2 and their derivative antibodies are promising for research and development of RDTs for EVD, with potential for extension to detect MVD.

Safety of the rVSV ZEBOV vaccine against Ebola Zaire among frontline workers in Guinea

BackgroundAs part of the ring vaccination trial in Guinea, Front Line Workers were invited to participate in a sub-study to provide additional information on the immunogenicity and safety of rVSVΔG/ZEBOV-GP. Here we summarize the information on the safety follow-up. MethodsAn open-label, non-randomized, immunogenicity evaluation of one dose of rVSVΔG/ZEBOV-GP was conducted in Conakry, Guinea between March 2015 and July 2016. Front-line workers refusing vaccination were invited to participate as a control group. Participants were followed for 3 months with a subset followed-up for 6 months after vaccination. Women becoming pregnant during the follow-up were followed until pregnancy outcome. Solicited and unsolicited adverse events were monitored at each contact with participants using standardized study forms. Results2016 vaccinated participants and 99 controls were included in the safety cohort. On the 3 days post-vaccination visit adverse events were very common, with over 70% of participants reporting at least one adverse event. The most frequently reported symptoms were headache, fatigue, arthralgia, subjective fever and myalgia. Among participants that completed fever diaries (n = 887), post-vaccination fever was reported by 15.22%. Comparing to the unvaccinated group, local reaction, fatigue, headache, arthralgia, myalgia and subjective fever occurring within the first 3 days post-vaccination were statistically significantly different in the vaccinated group compared to the unvaccinated. A total of 8 Serious Adverse Events were identified during follow-up. 2 SAEs were related to pregnancy. ConclusionsResults confirm that adverse events 3 days after vaccination with the rVSV candidate vaccine are common. The occurrence of fever is of particular concern in the context of ongoing Ebola transmission. Additional studies should address important data gaps regarding the use of the vaccine in pregnancy and other vulnerable populations.

Growth-Adaptive Mutations in the Ebola Virus Makona Glycoprotein Alter Different Steps in the Virus Entry Pathway.

The Zaire ebolavirus (EBOV) glycoprotein (GP) is cleaved into two subunits (GP1 and GP2) that are both required for virus attachment and entry into cells. Sequence changes in the GP have been proposed to increase pathogenesis and to alter virus growth properties. Mutations in GP acquired during EBOV tissue culture passage have also been reported to change virus growth properties. Here, we report the isolation of six amino acid mutations in EBOV GP that spontaneously appeared during recovery and passage of an EBOV-Makona GP-pseudotyped vesicular stomatitis virus (VSV), two of which also occur during passage of EBOV clinical isolates in tissue culture. Each of the six mutations resulted in increased virus growth in monkey and human cell lines. All mutations are located in the GP2 fusion subunit and increase entry kinetics of EBOV virus-like particles (VLPs). The gain-of-entry function mapped to two mechanistic phenotypes. Mutations in heptad repeat 1 (HR1) decreased the requirement for cathepsin B activity for viral infection. Mutations directly within the fusion loop increased entry kinetics without altering the cathepsin B dependence. Several mutations in the fusion loop were substitutions of residues present in other ebolavirus glycoproteins, illustrating the evolutionary paths for maintaining an optimally functioning fusion loop under selection pressure.IMPORTANCEZaire ebolavirus (EBOV) is the causative agent of the highly lethal Ebola virus disease and poses a significant threat to the global health community. Approved antivirals against EBOV are lacking; however, promising therapies targeting the EBOV glycoprotein are being developed. Efficacy testing of these candidate therapeutics relies on EBOV laboratory stocks, which when grown in tissue culture may acquire mutations in the glycoprotein. These mutations can produce inaccurate results in therapeutic testing. Until recently, distinguishing between tissue culture mutations and naturally occurring polymorphisms in EBOV GP was difficult in the absence of consensus clinical GP sequences. Here, we utilize recombinant VSV (rVSV) pseudotyped with the consensus clinical EBOV Makona GP to identify several mutations that have emerged or have potential to emerge in EBOV GP during tissue culture passage. Identifying these mutations informs the EBOV research community as to which mutations may arise during preparation of laboratory virus stocks.

Acetylation of lysine residues in the recombinant nucleoprotein and VP40 matrix protein of Zaire Ebolavirus by eukaryotic histone acetyltransferases

Acetylation of histones and other proteins plays crucial roles in transcriptional regulation, chromatin organization, and other biological processes. It has been recently reported that the nucleoprotein (NP) of influenza virus is acetylated in infected cells, and this modification contributes to the RNA polymerization activity of the virus. As the influenza virus, the Ebolavirus contains single-stranded negative-sense RNA as its viral genome, which interacts with NP and other viral proteins. In this study, we performed a series of biochemical experiments and revealed that the recombinant Ebolavirus NP and the viral matrix protein VP40, which binds with NP, were acetylated by eukaryotic histone acetyltransferases, such as P300/CREB-binding protein (P300/CBP) and P300/CBP-associated factor (PCAF), in vitro. Mass spectrometry was used to identify the lysine residues that were potential acetylation targets in NP and VP40. The identified lysine residues in NP were located in the RNA-binding cleft and the VP35-binding domain. Potentially acetylated lysine targets in VP40 were identified in the basic patch, which is necessary for constructing oligomers. These results suggest that the acetylation of these lysine residues is involved in the interactions between viral proteins.

SEARCHING OF FLAVONOID COMPOUNDS AS A NEW ANTIVIRAL FOR SUDAN EBOLAVIRUS GLYCOPROTEIN USING IN SILICO METHODS

Ebola hemorrhagic fever is a viral disease from Ebolavirus genus and lethal to primates, including humans. The case fatality rate is 30%-90%. Until now, no vaccines nor drugs that could effectively combat Ebola hemorrhagic fever. Sudan ebolavirus (SEBOV) is the second deadliest species after Zaire ebolavirus, with afatality rate of 50-70%. In Ebola life cycle, glycoprotein (GP) is crucial for mediating Ebolavirus entry into the host cell. Thus, molecules that could inhibit GP activity has a potential to become an ideal therapeutic compoundof Ebola hemorrhagic fever. Flavonoid compounds are potential because of its antiviral properties. In this research, the in silico method was utilized to investigate the potency of flavonoid compounds as an inhibitor of SEBOV GP through molecular docking and computational ADMET test. Moreover, the oral bioavailability and toxicity prediction of the flavonoid compounds were performed as well to get the best flavonoid compounds. In this research, about 1358 flavonoid compounds and 3D structure of SEBOV GP were retrieved from ChEBI database and RCSB PDB, respectively. Moreover, MOE 2014.09 software was used as the primary software. Furthermore, the Osiris DataWarrior and SwissADME were used as the software for conducting computational ADMET test. In the end, cyanidin-3-(p-coumaroyl)-rutinoside-5-glucoside, myricitrin V, and 7-O-(6-feruoylglucosy) were selected as the potential inhibitor of SEBOV GP because they have the best binding affinity and low toxicity risk.