https serendipbrynmawredu oneworld virus EBOLA VIRUS Ebola Virus Discovery 1976 Inject in mice Electron microscopy Transmission of EBOV and search for reservoir species Microbes and Infection 2005 ID: 1047283
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1. From Swiss Inst of Bioinformaticshttps://serendip.brynmawr.edu/oneworld/virusEBOLA VIRUS
2. Ebola Virus Discovery1976Inject in miceElectron microscopy
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4. Transmission of EBOV (and search for reservoir species)Microbes and Infection, 2005,The Natural History of Ebola Virus in AfricaSome infections occurFollowing bat sightings.Related viruses have bat reservoirsExperiments have failed toreproducibly infect a wide varietyof putative reservoir species with EBOV Susceptible to EBOVGorillaChimpanzeeDuiker(laboratory mice)
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6. Ghana 2008-2011 SurveyMethodology—surveyed fever patients Central/North Ghana—18 hospitals/285 patientsPCR to amplify EBOV L geneELISA (hepatitis) SequencingNo EbolaMajor illness Hepatitis B & C (adults), A (kids)
7. NE J. Med.4/27/14TEMContact tracing
8. Diagnosis –PCRRT-PCR to find high viral loadsGenome sequencingViral Growth cell culture/FL-anti Ebola AbEMphylogeny
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10. Eurosurveillance, Volume 19, Issue 36, 11 September 2014 Rapid communicationsEarly transmission dynamics of Ebola virus disease (EVD), West Africa, March to August 2014 H Nishiura , G ChowellMath modellingTransmission RatesRt need to be<1 to control epidemicRt= number of peopleInfected by each patient
11. Science Express, 28 Aug. 2014
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13. Pathogenesis of Ebola-Hartman et al, CDCFilovirusesHemorraghic fever/vascular dyfunctionEbola (Zaire & Sudan)MarburgReston & Ivory Coast (animals)4-10 day incubation period; 50-90% fatalNon-specific initial symptoms (fever, pains, nausea)Virus and antibodies detectable with symptoms
14. Ebola pathogenesisCoagulation connection—TF (tissue factor) levels increased Ebola macrophages engulfed in fibrin but Protein C level decreased (decreases coagulation)Animal fatality reduced by anti-coagulation factor Endothelial leakageshock, hemorrhage Glycoprotein GP1, GP2 connection not clear
15. Ebola pathogeneisFatal patients—bleeding, coagulation defects, high viral levels many organs, low antibody levelsTransmission body fluids, not aerosolEntry—mucous tissue, cuts, common receptor, replicate many cell typesImmunosuppression—enters dendritic (immature immune) cellslymph system. Dendritic cells coordinate innate/adaptive immune response—cytokine signalling (VP35), T-cells, interferonLymphocyte apoptosis
16. Ebola GoalsHistory and GeographyPathogenesisBiochemistry & Molecular Biology (and limitations & unknowns)Nucleic Acids7 ProteinsImplications for understanding biologyTherapeutic implications
17. Annual Rev Genetics, 1998, Conzelman
18. Replication/Transcription
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20. “universal” procedure for RT-PCRExtract RNA from 140 uL serum 20 min reverse transcriptase 50°C95°C 5 min10 “precycles” 95°C 5 s, 60-55°C 5s, 72°C 25 sec40 cycles 95°C (denature), 56°C 10 s (anneal), 72°C 10 s (elongate)$$$ equipment/hoursUnder development—10 min test for “field”
21. Advertising e-mail arrived Aug. 8, 2014
22. Comprehensive Functional Analysis of N-Linked Glycans on Ebola Virus GP1—Lenneman et al MBIO Aug. 1, 2014Endosome proteolytic processing
23. Why study GP1/GP2?Viral entry—RBD=receptor binding siteVaccine developmentUnderstand role of conserved glycosylation—Mutate conserved N, Y so GP1 will be expressed –sugarPseudovirus in tissue cultureResultsSugar removal allows protein production, increases viral entry (including macrophages), cell binding, protease susceptibility, cathespin B independence, but decreases binding NPC1 receptor Ca 2+ binds receptor lectins that bind glycans has some reduced binding when glycosylation is removedNear complete sugar removal allows greater anti-sera recognition for GP1 core onlyAuthors suggest GLYCAN SHIELD more important than entry reduction as reason evolutionary conservation.
24. Ebola Vaccine DevelopmentWant anti-GP antibodies (acute infection)Want longer term protection (CD8/T cell/cytokine)Adenovirus can deliver GP, but many are immune to human vectorsChimp adenovirusUse non-replicating form of virusBooster with MVA protects 100% Macaques
25. Nature Medicine, 7 Sept. 2014
26. 10 month challenge with lethal Ebola Dose
27. Ebola Polymerase L associates withTopoisomerase I
28. Topoisomerase IKnown to be important in viral replication/transcriptionStrand breaking is importantInhibition of Top I results in less Ebola replicationEbola changes localization of TopITherapeutic implications??J. Virology Aug. 2013, Takahashi et al
29. L gene—RdRpRNA dependent RNA polymerase2000 amino acidsTwo domainsPolymeraseTranscription factorTemplate is RNPHumans don’t have this enzymeGood therapeutic targetLike HIV RT???Why so little research?
30. Co-factor
31. Potential PPIs for VP35(based on VP35 biochemistry/structure)
32. NMR—solution experimentVerifies that drugs bind.Perturbs chemical shift.Binding pocket mutants haveno chemical shift change
33. Do drugs disrupt NP/VP35-IID interaction? Stop polymerase?Pull down assay withamylose beads.If VP35/IID binds NP,an NP-His band is visible.2 drugs inhibit bindingEbola polymerase complexIncludesEBOV L, VP30, VP35, NPSeveral drugs show doseDependent inhibition
34. Do drugs stop virus?Some drugs reduce viralinfectivityandviral release
35. Ebola Virus Modulates Transforming Growth Factor Signaling andCellular Markers of Mesenchyme-Like Transition in HepatocytesProteomics/Kinomics ApproachHow does EBOV affect global signalling?How are phosphorylation patterns changed?Therapeutic targets?Kindrachuk et al J. Virology September, 2014
36. Kinome Array300 peptide targets in arrayexpose to active kinases in lysateP1P300PEPTIDE TARGET + cognate kinase PEPTIDE TARGET—PPattern of target phosphorylation kinase IDWhat pathways are activated 1 hr, 6 hr, 24 hr?
37. ResultsTGF-b pathway up-regulated (secretion TGF-b and VEGF confirmed by ELISA)Use inhibitors of pathway TGF-b, P13K/AKT, MAPK/EFK, raf, JNK, PKCEarly use of some inhibitorsReduced mouse fatalities
38. EMT (epithelial to mesenchyme transition)TGF-b normal roles—wound healing, cell growth/differentiation, migration, immune responseWhat is happening to EBOV-infected cells?Epithelial tissues first infected—adherens/tight junctions disassembled. Gene expression pattern changes to reduce epithelial and increase mesenchyme cytoskeletal expression
39. Western blots/phosphorylationEMT protein TGF-b pathway P antibodiesArticle error—figure legend switched 8&9
40. EMT—local plus systemic effects
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42. From Medical Microbiology 4th ededited by Baron (1996)
43. Interferon protects uninfected cellsby changing gene expression
44. Ebola Virus VP24 Targets a Unique NLS Binding Siteon Karyopherin Alpha 5 to Selectively Competewith Nuclear Import of Phosphorylated STAT1 Xu et al Cell Host & Microbe, 2014
45. Proposed ModelSTAT1 must enter nucleus for interferon responseSTAT1 binds to KPNA for nuclear transporteVP24 binds strongly to KPNAeVP24 competes with STAT1 for same KPNA binding siteeVP24 and STAT1 binding sites overlap and are distinct from binding sites for normal transported moleculeseVP24 prevents normal interferon response by blocking STAT1 transport to nucleus, but does allow entry other molecules
46. Evidence for modelCrystallography structure—shows hydrophobic and H-bondingat interface very strong bindingeVP24/KPNAKPNA can be severely truncatedand still bind eVP24
47. More evidence for modelImmunoprecipitation using anti-FLAG antibodiesTo pull down eVP24 plus “normal” cargo or STAT1WT eVP24 prevents STAT1 but not DMC1 from binding
48. Last evidence for modelAssay tests whether interferoncan induce promoterWT eVP24 reducesExpression by >90%Marburg Virus mVP24has different sequencein key positions anddoes not block STAT1nuclear entry or interferonresponse.