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Bakgrund: Ebola upptäcktes för första gången 1976 i ett dubbelt utbrott i Sudan och Zaire i Afrika. Det pågående utbrottet av ebola virus sjukdom (EVD) startade i Guinea 2013 och har sedan dess spridits vidare till Liberia, Sierra Leone, Nigeria, Mali och Senegal. T.o.m. 4 mars 2015 har nästan 24 000 smittats och 10 000 avlidit vilket är mer än 50 gånger fler än i det tidigare största utbrottet i Uganda 2000. Viruset består av fem arter: Bundibugyo, Reston, Sudan, Tai Forest och Zaire. Vektorn för viruset är fortfarande okänd men förmodas vara flygande hundar (fruit bats) som själva är resistenta. Symtomen innefattar bl.a. feber, trötthet, kräkning, diarré, ledsmärtor och mukosala blödningar. Behandlingen består vanligen av vätskeersättning. Det finns varken godkänt vaccin eller läkemedel i dagsläget men flertalet studier pågår för att utveckla bl.a. antikropparna ZMapp och antiviral behandling med Brincidofovir. Syfte: Redogöra för tidigare och pågående utbrott av ebola och undersöka vilka faktorer som bidragit till att epidemin 2013-2015 inträffat och fått stora proportioner i Västafrika. Metod: Till det aktuella ämnet har den senaste informationen inhämtats via WHO och CDCs respektive hemsidor mellan 23/2-9/3 2015. Resultat: En kombination av faktorer har bidragit till att det pågående utbrottet blivit större än tidigare. Länderna som drabbats har inte varit förberedda, virusarten ebola Zaire har hög dödlighet, sjukvård och infrastruktur är svag och kulturen är djupt grundad i ett samhälle där utbildningen är låg. Befolkningen behöver respekteras och utbildas för att de aktivt ska kunna vara delaktiga i att stoppa smittspridningen av viruset. Ju mindre geografisk yta viruset är fördelat på desto lättare blir det att kontrollera och bekämpa det. Diskussion: Guinea, Liberia och Sierra Leone har alla drabbats extra hårt av pågående EVD utbrott och behöver nu få hjälp med återuppbyggnad av ett fungerande sjukvårdssystem som befolkningen kan lita på och med personal som kan arbeta under säkra förhållanden. Stort fokus bör också läggas på att implementera kulturen i de skyddsåtgärder som behövs för att förhindra ytterligare spridning och framtida utbrott. Ytterligare forskning krävs för att kunna erbjuda bättre behandling och profylax.

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Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Ebolavirus is responsible for a deadly hemorrhagic fever that has claimed thousands of lives in Africa and could become a global health threat. Because of the danger of infection, novel Ebola research is restricted to BSL-4 laboratories; this slows progress due to both the cost and expertise required to operate these laboratories. The development of a safe surrogate would speed research and reduce risk to researchers. Two highly conserved Ebola gene segments—from the glycoprotein and nucleoprotein genes—were designed with modifications preventing expression while maintaining sequence integrity, spliced into high copy number plasmids, cloned into E.coli, and tested for stability, safety, and potential research applications. The surrogates were stable over 2-3 months, had a negligible mutation rate (<0.165% over the experiment), and were detectable in human blood down to 5.8E3-1.17E4 surrogates/mL. These protocols could be used to safely simulate other pathogens and promote infectious disease treatment and detection research. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

Ebolavirus (EBOV) is a single stranded RNA virus that causes haemorrhagic fever in humans and other mammals. The EBOV encodes 7 proteins, NP, L, VP30, VP35, VP40, GP and VP24. VP24 is believed to be one of the EBOV proteins that causes the extreme virulence of the pathogen. The protein blocks the interaction between PY-STAT1 and KPNA, a protein that is involved in the import of PY-STAT1 into the nucleus. The nuclear import of PYSTAT1 is therefore blocked. This leads to the inhibition of IFN signalling. The purpose of this study was to express and purify VP24 and KPNA5. The proteins recombinantly expressed as a fusion tag in E. coli in lysogeny broth. Purification of VP24 was done using immobilized metal ion affinity chromatography, size exclusion chromatography and ion exchange chromatography. Characterization of the protein was analysed using circular dichroism. The results obtained from this study showed that VP24 could be purified in pH 10 buffers with little loss of protein due to aggregation and the protein was folded with an alphahelical structure. The expression and purification of KPNA5 was more complicated and further evaluation is left for future studies. The established protocol for expression and purification of VP24 and the initial work on KPNA5 will go a long way to aiding future studies on the system, thus the answer to the question regarding the extreme virulence of EBOV will be closer.

Ebolavirus (EboV) is a highly pathogenic enveloped virus that causes outbreaks of zoonotic infection in Africa. The clinical symptoms are manifestations of the massive production of pro-inflammatory cytokines in response to infection and in many outbreaks, case fatality rate exceeds 75%. The unpredictable onset, ease of transmission, rapid progression of disease, high mortality and lack of effective vaccine or therapy have created a high level of public concern about EboV. Here we report the properties of a benzylpiperazine adamantane diamide-derived compound identified in a screen for inhibitors of EboV infection. We found that the inhibitor is specific, reversible, and that the target(s) for inhibition are present in cells and not in virus particles. The compound is not an inhibitor of acid pH-dependent endosome protease activity, which is required for EboV infection. Treatment of cells with this compound causes accumulation of cholesterol in late endosomes and lysosomes (LE/LY), suggesting it inhibits one or more proteins involved in regulation of cholesterol uptake into cells. Using mutant cell lines and informative derivatives of the inhibitor, we found the inhibitor target is the endosomal membrane protein Niemann-Pick C1 (NPC1). NPC1 is a polytopic LE/LY membrane protein that mediates uptake of lipoprotein-derived cholesterol into cells. We find that NPC1 is essential for EboV infection, that NPC1 binds to the protease-cleaved GP1 subunit of the EboV glycoprotein, and that the anti-viral compound inhibits infection by targeting NPC1 and interfering with binding to GP1. Furthermore, analysis of viral variants resistant to the anti-viral compound revealed that the residues which confer resistance are located on the surface of the receptor binding domain of GP1. Combined with the results of previous studies of GP structure and function, our findings support a model of EboV infection in which cleavage of the GP1 subunit by endosomal cathepsin proteases removes heavily glycosylated domains to expose the N-terminal domain, which is a ligand for NPC1 and regulates membrane fusion by the GP2 subunit. Thus, NPC1 is essential for EboV entry and a target for anti-viral therapy.

Introduction: Since the first outbreaks of Ebola virus disease 1976 in the Democratic Republic of the Congo (DRC) and Sudan there has been recurrent epidemics. The biggest outbreak so far hit West Africa between 2013-2016 when at least 28 600 people were estimated to have been infected and approximately 11 300 died in the most severely affected countries Guinea, Sierra Leone and Liberia. Ebola hemorrhagic fever (EHF) or Ebola virus disease (EVD) is caused by a virus that together with the Marburg virus belongs to the Filovirus family. The Ebola virus can cause hemorrhagic bleeding, has a rapid disease progression rate and a high mortality (25-90%). The symptoms are initially headache, fever, sore throat and muscle pain but during the latter part of the disease the characteristic bleedings from all bodily orifices occurs. Many internal organs do damage and survivors often have persistent sequels and long convalescense. EVD is a zoonosis and might infect antelopes, monkeys, porcupines among others. The natural reservoir is believed to be different species of bats. In august 2018, another outbreak of Ebola was reported in DRC. This was the tenth time the disease hit the country. However, for the first time there was candidate vaccine in clinical trial that could be used during an emergency. Experts within the WHO recommended that vaccination was initiated using Merck`s Ebolavaccine rVSV-∆G-ZEBOV-GP that was ready for a phase III clinical trial. This vaccine had already been used during the West African outbreak and had been known to provide a good safety profile as well as immunogenicity. The second vaccine to be used was a two-dose heterologous prime-boost vaccine regimen called Ad26.ZEBOV/MVA-BN-Filo which was marketed by Johnson&amp;Johnson. Ad26.ZEBOV was given as a first dose to prime the immune system and then MVA-BN-Filo was administered as a second dose approximately 8 weeks later as a booster. This vaccine regimen had also been showing promising results in phase I and II clinical trials. Objective: This degree project was made in attempt to answer the issue: How effective and safe are the new Ebola vaccines against EVD? Methods: To answer this question, a literature study was conducted. The outbreak in DRC was ongoing by the time of this degree project and there were no phase III trials conducted for the Ad26.ZEBOV/MVA-BN-Filo vaccine regimen. A lot of information was therefore retrieved from WHO, Doctors without borders, and so on. Merck`s vaccine had been prequalified and received the generic name Ervebo in the fall of 2018. Randomised clinical trials (RCT) and clinical trials regarding Ervebo was downloaded from the internet using the search engine PubMed that`s accessing different databases. Results: Both Ervebo and the 2-dose vaccine regimen Ad26.ZEBOV/MVA-BN-Filo initiated an immune response against ebolavirus that was almost 100%. The most frequent solicited local and systemic adverse events were injection site pain and headache, myalgia and fever. All adverse events were reported to be mild and resolved in a short period of time. No severe adverse events were reported. Discussion: The biggest challenge in the future will concern how to initiate vaccination strategies in countries with a lack of financial resources and infrastructure as well as ongoing armed conflicts (mainly DRC). Conclusion: The new vaccines against Ebola virus infection are all effective with a good safety profile. / Bakgrund: Sedan de första utbrotten av ebolafeber 1976 i Demokratiska republiken Kongo (DRK) och Sudan har epidemier blossat upp med jämna mellanrum. Det hittills största utbrottet drabbade Västafrika 2013-2016 då minst 28 600 beräknades ha smittats och minst 11 300 avled i de hårdast drabbade länderna Guinea, Sierra Leone och Liberia. Ebolafeber, eller ebola hemorragisk feber, orsakas av ett virus som tillsammans med Marburgviruset hör till familjen Filovirus. Ebolaviruset kan orsaka hemorragiska blödningar, har snabbt sjukdomsförlopp och hög dödlighet (25-90%). Symptomen är inledningsvis influensaliknande men under den senare delen av sjukdomsförloppet förekommer de karaktäristiska blödningarna från alla kroppsöppningar. Många inre organ tar skada och överlevare har ofta bestående men och lång konvalescens. Ebolafeber är en zoonos och kan drabba antiloper, olika sorters apor m fl. Den naturliga reservoaren tros vara olika arter av fladdermöss. I augusti 2018 rapporterades om ännu ett utbrott av ebolafeber i DRK, det 10:e i ordningen. En avgörande skillnad vid detta utbrott var att det nu fanns kandidatvaccin i kliniska studier att tillgå. En expertgrupp inom WHO rekommenderade att vaccination inledddes med rVSV-∆G-ZEBOV, ett ebolavaccin tillverkat av Merck redo för kliniska studier fas III. Detta vaccin hade använts redan under utbrottet i Västafrika och visat goda resultat vad gällde immunogenicitet och säkerhet. Det andra vaccinet att sättas in var Ad26.ZEBOV/MVA-BN-Filo, tillhandahållet av Johnson&amp;Johnson. Detta bestod av två olika doser som gavs med ca 8 veckors mellanrum, ofta kallat ”prime/boost”-vaccin. Även för Ad26.ZEBOV/MVA-BN-Filo fanns dokumenterade positiva resultat. Syfte: Detta examensarbetet gjordes med syftet att svara på frågeställningen: Hur effektiva och säkra är de nya vaccinen mot ebolafeber? Metod: För att svara på frågan gjordes en litteraturstudie. När detta examensarbete författades pågick fortfarande utbrottet i DRK och fas III studierna för Ad26.ZEBOV/MVA-BN-Filo var inte avslutade. Mycket information fick hämtas från olika hemsidor, bl a WHO och Läkare utan gränser. Mercks vaccin prekvalificerades hösten 2019 och gavs handelsnamnet Ervebo. För detta vaccin fanns fler RCT-studier tillgängliga, vilka uteslutande valdes m h a sökmotorn PubMed som är länkad till olika databaser. Resultat: Både singeldosvaccinet Ervebo och dubbeldosvaccinen Ad26.ZEBOV/MVA-BN-Filo konstaterades ge ett nästan 100%-igt immunsvar mot ebolavirus. I den mån biverkningar förekom var de milda och övergående. Den mest rapporterade lokala biverkningen var ömhet vid injektionsstället och de vanligaste systemiska biverkningarna var huvudvärk, myalgi och feber. Diskussion: Den största utmaningen i framtiden blir att inleda vaccinationsprogram i länder med bristande finansiella resurser och infrastruktur samt pågående väpnade konflikter (främst DRK). Slutsatsen blev att de nya vaccinen mot ebolafeber är effektiva och säkra vid behandling mot ebolavirusinfektion.

<div>Ebolavirus, a genus of filoviruses, are responsible for outbreaks that cause up to 90% fatality, including the recent outbreak in West Africa that has resulted in over 28,603 reported cases and 11,301 deaths according to the WHO. Inhibitors of Vacuolar-ATPase (V-ATPase), a key protein complex that is responsible for endosomal acidification and represents a unique method to block this common viral pathway. V-ATPase inhibitors have previously been explored as therapies for many diseases but have failed due to high toxicity. Diphyllin is a natural, arylnaphthalene lignan that represents a novel structural class of V-ATPase inhibitors with a greater selectivity index than previous V-ATPase inhibitors. Diphyllin has shown promising anti-tumor and anti-osteoclast activity, as well as strong anti-viral activity against Influenza and Dengue viruses. </div><div>Herein, novel modifications of the lactone and phenol functional groups of diphyllin were explored for the ability to enhance the potency or therapeutic selectivity of the diphyllin core. Four initial sets of derivatives were synthesized and assayed for activity against ebolavirus infection, inhibition of cellular endosomal acidification, cytotoxicity and biochemical inhibition of isolated V-ATPase. Modification of diphyllin’s lactone functional group reduced both activity and selectivity, while alkylation of the phenol groups significantly enhanced activity. The incorporation of basic heterocycles to the alkyl group created an alkylamino series of derivatives that exhibited significantly improved therapeutic selectivity compared to diphyllin. Further investigation of the alkylamino class indicated that they retained activity against Marburgvirus infection, a filovirus related to Ebolavirus. Alkylamino derivatives inhibited ebolavirus infection of human macrophages at low micromolar levels with no apparent cytotoxicity.</div><div>Further investigation of the alkylamino class of diphyllin derivatives was conducted to determine if potency and/or therapeutic selectivity could be optimized. The addition of a 1-methylpiperazine moiety to the end of the alkyl chain improved potency 1260-fold over diphyllin, though therapeutic selectivity was not improved. The modification of the alkylamino linker to an acetamide eliminated cytotoxicity but decreased derivative activity against V-ATPase activity. To evaluate if the cytotoxicity evidenced by the alkylamino derivatives was evidenced in organisms, the derivative toxicity was assessed in zebrafish and mouse models. Derivatives displayed toxicity in a zebrafish developmental model but were all at least 10-fold less toxic than the known V-ATPase inhibitor bafilomycin A1. Three derivatives were well tolerated in CD-1 mice when administered at therapeutically relevant concentrations and caused no abnormal changes in their blood chemistry. Overall, these results demonstrate that the alkylamino and acetamide diphyllin phenol derivatives should be further studied as therapies for ebolavirus infection in addition to other V-ATPase mediated diseases.</div><div><br></div>

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