Zaire ebolavirus is a long filamentous single-stranded RNA virus belonging to the family Filoviridae. Due to the virus’ high mortality rate, lack of an approved vaccine, and potential use as a bioterrorism weapon, research on this topic has been of high demand. To address this issue, several vector platforms have been investigated as vaccine candidates. DNA and adenovirus vaccine platforms are known to elicit robust cellmediated immune responses, while adeno-associated virus and vesicular stomatitis virus vaccines are recognized for strong humoral responses. The leading hypothesis of the present project was to determine whether these four vaccination platforms, in a heterologous prime-boost regimen, increase survival and the breadth of the immune response. To test this hypothesis, the main objectives were to evaluate the cell-mediated and humoral immune responses, as well as correlate the induced immunity to protection against MA-EBOV. The heterologous pairings were strategically designed to induce both arms of the immune response. An optimized Zaire ebolavirus glycoprotein was inserted into each of the vaccine platforms and evaluated against mouse-adapted Zaire ebolavirus. Serum obtained from vaccinated mice was analyzed by a neutralizing antibody assay and IgG ELISA to determine the humoral response. The cell-mediated immune response was monitored via ELISPOT. Collectively, the data indicates that regardless of whether homologous or heterologous, a more robust immune response was observed in prime-boost strategies compared to an individual vaccination alone. In addition, the cell-mediated and humoral data show that heterologous combinations induce higher IgG specific titers in comparison to homologous regimens. As expected and consequent with immune responses, survival studies demonstrate that prime-boost III vaccinations, heterologous or homologous, are superior to vaccination regimens involving only one strategy. This data supports further evaluation of the prime-boost strategies to develop an optimal immunization strategy that can be applied to other disease models.

Zaire ebolavirus is a long filamentous single-stranded RNA virus belonging to the family Filoviridae. Due to the virus’ high mortality rate, lack of an approved vaccine, and potential use as a bioterrorism weapon, research on this topic has been of high demand. To address this issue, several vector platforms have been investigated as vaccine candidates. DNA and adenovirus vaccine platforms are known to elicit robust cellmediated immune responses, while adeno-associated virus and vesicular stomatitis virus vaccines are recognized for strong humoral responses. The leading hypothesis of the present project was to determine whether these four vaccination platforms, in a heterologous prime-boost regimen, increase survival and the breadth of the immune response. To test this hypothesis, the main objectives were to evaluate the cell-mediated and humoral immune responses, as well as correlate the induced immunity to protection against MA-EBOV. The heterologous pairings were strategically designed to induce both arms of the immune response. An optimized Zaire ebolavirus glycoprotein was inserted into each of the vaccine platforms and evaluated against mouse-adapted Zaire ebolavirus. Serum obtained from vaccinated mice was analyzed by a neutralizing antibody assay and IgG ELISA to determine the humoral response. The cell-mediated immune response was monitored via ELISPOT. Collectively, the data indicates that regardless of whether homologous or heterologous, a more robust immune response was observed in prime-boost strategies compared to an individual vaccination alone. In addition, the cell-mediated and humoral data show that heterologous combinations induce higher IgG specific titers in comparison to homologous regimens. As expected and consequent with immune responses, survival studies demonstrate that prime-boost III vaccinations, heterologous or homologous, are superior to vaccination regimens involving only one strategy. This data supports further evaluation of the prime-boost strategies to develop an optimal immunization strategy that can be applied to other disease models.

Indiana University-Purdue University Indianapolis (IUPUI) / The purpose of this paper is to examine the extent to which elements of culture (values, beliefs, and behaviors) have contributed to the construction of health meaning in regards to 2014 Ebola outbreak in Guinea. I conducted 14 interviews with people who lived in Guinea during the 2014 Ebola outbreak about their own experiences of the crisis and how health related messages were received by the general population. All the participants in this study were between 25 and 56 with an average age of 41. All participants agreed that culture played a crucial role in how people perceived the disease. It has also impacted the way people responded the prevention plans. When the ones did not believe in the existence of the disease, others did believe but because of certain customs, they were unable to follow public health safety recommendation.

As a result of transcriptional editing, Ebola virus (EBOV) produces multiple soluble products from its glycoprotein gene, the primary product of which is the secreted glycoprotein (sGP), in addition to the membrane-bound viral spike protein GP1,2. A lack of leukocyte infiltration is observed during EBOV infection, which is thought to allow virus replication to proceed unchecked and thus represents a significant role in the immunopathology of the disease. Currently the only know function of sGP is that it has an anti-inflammatory effect on endothelial cells treated with TNF-α, an effect that has been hypothesized to interfere with recruitment or extravasation of leukocytes. To better characterize this anti-inflammatory function, a link between sGP structure and function was sought. Mass spectrometry (MS) analysis of recombinant sGP demonstrated that it is a parallel-orientated disulphide-linked homodimer that contains Cys53-C53’ and Cys306-C306’ intermolecular disulphide bonds. In addition to being glycosylated with complex N-glycans, sGP also contained a novel post-translation modification, termed C-mannosylation. C-mannosylation was not required for the anti-inflammatory function of sGP; however, glycine mutations at amino acids 53 and 306 resulted in the complete loss of the anti-inflammatory effect on TNF-α treated endothelial cells. Thus, a specific structure mediated by intermolecular disulphide bonds is required for the proposed anti-inflammatory function of sGP, suggesting that this effect is the result of a specific interaction. The spike protein GP1,2, also contains C-mannosylation motifs. MS analysis of GP1,2 indicated that GP1 was C-mannosylated, while two adjacent motifs in the membrane proximal region (MPER) of GP2 were not. The infectious virus-like particle (iVLP) assay, a system for investigating virus particle assembly and entry, was utilized to determine the functional importance of these conserved tryptophans. Elimination of the C-mannosylation motif, which resides in an external loop region of GP1, increased reporter activity, suggesting that particle entry is enhanced and this region may interact with the cell surface despite being outside of the receptor binding site. Decreased reporter activity was observed for all MPER mutants, with multiple MPER tryptophan mutations resulting in decreased GP1,2 incorporation. These data place the MPER tryptophan residues in an important role for glycoprotein incorporation and particle entry. Given the tryptophan content and location is similar to the MPER of HIV gp41, where these residues are required for glycoprotein incorporation and fusion, the MPER of EBOV GP2 may function similarly.

Ebola virus (EBOV) is an enveloped, negative-sense single-stranded RNA virus that causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes multiple transcripts due to RNA editing at a conserved editing site (ES) (a hepta-uridine stretche). The majority of GP gene transcript is unedited and encodes for a soluble glycoprotein (sGP); a defined function has not been assigned for sGP. In contrast, the transmembrane glycoprotein (GP1,2) dictates viral tropism and is expressed through RNA editing by insertion of a nontemplate adenosine (A) residue. Hypothetically, the insertion/deletion of a different number of A residues through RNA editing would result in another yet unidentified GP gene product, the small soluble glycoprotein (ssGP). I have shown that ssGP specific transcripts were indeed produced during EBOV infection. Detection of ssGP during infection was challenging due to the abundance of sGP over ssGP and the absence of distinguishing antibodies for ssGP. Optimized two- dimensional (2-D) gel electrophoresis verified the expression of ssGP during infection. Biophysical characterization revealed ssGP is a disulfide-linked homodimer that is exclusively N-glycosylated. Although ssGP appears to share similar structural properties with sGP, it does not have the same anti-inflammatory function. Using a new rapid transcript quantification assay (RTQA), I was able to demonstrate that RNA editing is an inherent feature of the genus Ebolavirus and all species of EBOV produce multiple GP gene products. A newly developed dual-reporter minigenome system was utilized to characterize EBOV RNA editing and determined the conserved ES sequence and cis-acting sequences as primary and secondary requirements for RNA editing, respectively. Viral protein (VP) 30, a transcription activator, was identified as a contributing factor of RNA editing— a proposed novel function for this largely uncharacterized viral protein. Finally, I could show that EBOV RNA editing is GP gene-specific because a similar sequence located in L gene did not serve as an ES, most likely due to the lack of the necessary cis-acting sequences. In conclusion, I identified a novel soluble protein of EBOV whose function needs further characterization. I also shed light into the mechanism of EBOV RNA editing, a potential novel target for intervention.

As a result of transcriptional editing, Ebola virus (EBOV) produces multiple soluble products from its glycoprotein gene, the primary product of which is the secreted glycoprotein (sGP), in addition to the membrane-bound viral spike protein GP1,2. A lack of leukocyte infiltration is observed during EBOV infection, which is thought to allow virus replication to proceed unchecked and thus represents a significant role in the immunopathology of the disease. Currently the only know function of sGP is that it has an anti-inflammatory effect on endothelial cells treated with TNF-α, an effect that has been hypothesized to interfere with recruitment or extravasation of leukocytes. To better characterize this anti-inflammatory function, a link between sGP structure and function was sought. Mass spectrometry (MS) analysis of recombinant sGP demonstrated that it is a parallel-orientated disulphide-linked homodimer that contains Cys53-C53’ and Cys306-C306’ intermolecular disulphide bonds. In addition to being glycosylated with complex N-glycans, sGP also contained a novel post-translation modification, termed C-mannosylation. C-mannosylation was not required for the anti-inflammatory function of sGP; however, glycine mutations at amino acids 53 and 306 resulted in the complete loss of the anti-inflammatory effect on TNF-α treated endothelial cells. Thus, a specific structure mediated by intermolecular disulphide bonds is required for the proposed anti-inflammatory function of sGP, suggesting that this effect is the result of a specific interaction. The spike protein GP1,2, also contains C-mannosylation motifs. MS analysis of GP1,2 indicated that GP1 was C-mannosylated, while two adjacent motifs in the membrane proximal region (MPER) of GP2 were not. The infectious virus-like particle (iVLP) assay, a system for investigating virus particle assembly and entry, was utilized to determine the functional importance of these conserved tryptophans. Elimination of the C-mannosylation motif, which resides in an external loop region of GP1, increased reporter activity, suggesting that particle entry is enhanced and this region may interact with the cell surface despite being outside of the receptor binding site. Decreased reporter activity was observed for all MPER mutants, with multiple MPER tryptophan mutations resulting in decreased GP1,2 incorporation. These data place the MPER tryptophan residues in an important role for glycoprotein incorporation and particle entry. Given the tryptophan content and location is similar to the MPER of HIV gp41, where these residues are required for glycoprotein incorporation and fusion, the MPER of EBOV GP2 may function similarly.

Ebola virus (EBOV) is an enveloped, negative-sense single-stranded RNA virus that causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes multiple transcripts due to RNA editing at a conserved editing site (ES) (a hepta-uridine stretche). The majority of GP gene transcript is unedited and encodes for a soluble glycoprotein (sGP); a defined function has not been assigned for sGP. In contrast, the transmembrane glycoprotein (GP1,2) dictates viral tropism and is expressed through RNA editing by insertion of a nontemplate adenosine (A) residue. Hypothetically, the insertion/deletion of a different number of A residues through RNA editing would result in another yet unidentified GP gene product, the small soluble glycoprotein (ssGP). I have shown that ssGP specific transcripts were indeed produced during EBOV infection. Detection of ssGP during infection was challenging due to the abundance of sGP over ssGP and the absence of distinguishing antibodies for ssGP. Optimized two- dimensional (2-D) gel electrophoresis verified the expression of ssGP during infection. Biophysical characterization revealed ssGP is a disulfide-linked homodimer that is exclusively N-glycosylated. Although ssGP appears to share similar structural properties with sGP, it does not have the same anti-inflammatory function. Using a new rapid transcript quantification assay (RTQA), I was able to demonstrate that RNA editing is an inherent feature of the genus Ebolavirus and all species of EBOV produce multiple GP gene products. A newly developed dual-reporter minigenome system was utilized to characterize EBOV RNA editing and determined the conserved ES sequence and cis-acting sequences as primary and secondary requirements for RNA editing, respectively. Viral protein (VP) 30, a transcription activator, was identified as a contributing factor of RNA editing— a proposed novel function for this largely uncharacterized viral protein. Finally, I could show that EBOV RNA editing is GP gene-specific because a similar sequence located in L gene did not serve as an ES, most likely due to the lack of the necessary cis-acting sequences. In conclusion, I identified a novel soluble protein of EBOV whose function needs further characterization. I also shed light into the mechanism of EBOV RNA editing, a potential novel target for intervention.

archives@tulane.edu / During the 2013-2016 West African outbreak, severe gastrointestinal symptoms were common in Ebola patients and associated with poor outcome. The efficient spread of Ebola virus (EBOV) via vomitus and diarrheal fluids, which contain high concentrations of virus, likely contributed to the scale of the outbreak. The delta-peptide is a conserved product of post-translational processing of the abundant EBOV soluble glycoprotein (sGP). Here, the murine ligated ileal loop model, which is well-established for the study of diarrheal disease, was used to demonstrate that delta-peptide is a potent enterotoxin. Dramatic intestinal fluid accumulation peaked at 9-12 hours following injection of biologically relevant amounts of delta-peptide into ileal loops, along with gross destruction of the villous architecture, loss of goblet cell polysaccharides, and secretion of pro-inflammatory cytokines. Transcriptomic analyses showed that delta-peptide triggers immune and cell survival responses. Delta-peptide may contribute greatly to EBOV-induced gastrointestinal pathology in humans. These findings demonstrate that the EBOV delta-peptide is an enterotoxic viroporin and may be categorized as a novel virulence factor. We then hypothesized that the delta-peptide may also be a druggable target to explore a new avenue for novel EBOV therapeutics, which are direly needed. An unconventional coupling strategy was employed to conjugate a modified version of the 23-residue delta-peptide to a carrier protein Keyhole Limpet Hemocyanin (KLH) in order to immunize rabbits so that they may generate high-affinity binding antibodies against the delta-peptide. Antisera was collected from the rabbits after regular immunizations and the IgG fraction of the antisera demonstrated binding and recognition against several delta-peptide variants confirmed by Western blotting and ELISAs. We then used this knowledge to determine therapeutic index in vitro by testing the antibody against the delta-peptide in the context of synthetic PC-PG vesicles and CHO cells. The purified IgG was then tested against the peptide in vivo to determine therapeutic efficacy by returning to the mouse model of diarrheal pathology mentioned previously. In a small pilot experiment, we were able to successfully block the enterotoxic activity of the delta-peptide and did not observe gastrointestinal distress in the mice that were treated with the peptide and antibody together versus just the peptide alone, signifying that the delta-peptide is a druggable target and may reveal a new therapeutic avenue against EBOV pathogenesis. / 1 / Shantanu Guha

Bakgrund: Ebolaviruset är en smitta med hög dödlighet som sprids via kroppsvätskor vilket ger influensaliknande symtom och blödningar. Sjuksköterskan ansvarar för att kontinuerligt utveckla vården och vårda patienter utifrån ett helhetsperspektiv. Flertalet brister i vården utgjorde hinder för omvårdnaden. Syfte: Syftet var att belysa sjuksköterskors upplevelser av att vårda ebolasmittade patienter. Metod: Allmän litteraturstudie med induktiv ansats där sju kvalitativa artiklar och en artikel med mixad metod analyserades baserat på fem steg för innehållsanalys. Det resulterade i tre kategorier. Resultat: Sjuksköterskor beskrev ett behov av fungerande struktur i sitt arbete med god utbildning, riktlinjer, säkerhet och ett fungerande samarbete. Sjuksköterskorna upplevde rädsla för smittspridning. Smittan var stigmatiserad vilket påverkade sjuksköterskor negativt. Arbetet ledde till stress och ohälsa. Bortsett från utmaningarna upplevdes en meningsfullhet i arbetet. Konklusion: Sjuksköterskans upplevelse av att vårda ebolasmittade patienter var att brist på kunskap och material hindrar adekvat omvårdnad men samarbete och rutiner främjar den. Stress och rädsla omgav arbetet med viruset, utöver att det också var stigmatiserat. Trots lidande och brister i omvårdnaden upplevdes meningsfullhet. / Background: The Ebola virus is an infectious disease with a high mortality rate. It spreads through body fluids and causes flu-like symptoms and bleeding. The nurse is responsible for continual care development and caring for patients holistically. Numerous shortcomings posed obstacles to nursing. Aim: To illustrate nurses experiences working with patients infected with the Ebola virus. Method: A general literature review with an inductive approach, where seven qualitative articles and one mixed method article were studied, based on five steps for content analysis. It resulted in three categories. Results: Nurses described a need for structure in their work with a functional and favourable education, guidelines, safety, and teamwork. Nurses experienced fear of the spread of Ebola infection. Ebola was stigmatised, affecting the nurses. Working led to stress and ill health. The nurses believed their work was meaningful. Conclusion: The nurse’s experiences of caring for Ebola-infected patients was that a lack of knowledge and materials prevents adequate nursing, but teamwork and routines promote it. Stress and fear surrounded handling the virus, in addition the work was stigmatised. Despite suffering and shortcomings in nursing, it was a meaningful experience.

Genom kritiska diskursanalyser ämnar studien att undersöka nyhetsrapporteringen av ebola i de svenska tidningarna Dagens Nyheter och Aftonbladet. Delvis för att få förståelse för hur mediepanik skapas, men även hur relationen mellan nyhetsläsaren och den lidande konstrueras. Undersökningsperioden för studien är oktober 2014. Den tidigare forskningen som belyses i denna undersökning är studier om nyhetsrapporteringar kring svininfluensan, SARS-smittan och salmonellabakterien. Dessa visar på att de intensiva perioderna i nyhetsrapporteringen ofta innefattar användningen av visuella och lingvistiska medel som bidrar till att skapa en mediepanik. De teoretiska ramarna som denna uppsats utgår ifrån är Simon Cottles teorier om hur medier konstruerar mediepaniker i rapporterandet om globala kriser, Roger Fowlers teorier om nyheternas språk vid rapporteringar om kriser och Lilie Chouliarakis teorier om lidandets spektakel. Resultatet visar således att nyhetsmedierna talar om ebolan som hot mot Europa och USA, om regional spridning, katastrofhjälp, geopolitik och sjukvård. De röster som främst får en talan i dessa artiklar är Världshälsoorganisationen, myndigheter, politiker och experter som uttalar sig om olika situationer. Patienter är de som inte får komma till tals även om de nämns i artiklarna. Resultatet av de ingående analyserna i denna studie visar att avståndet till nyhetshändelserna påverkar hur nyheter om ebola skildras i medierapporteringen. I artiklar som behandlade nyhetshändelser om ebola i Afrika, betonades det att ebolasmittan fanns på en avlägsen plats. Samtidigt upptäcktes det att innehållet såsom språket blev intensivare när medierapporteringen skildrade nyheter om ebola i Europa. Därmed skapades även en känsla av kris och risk.