Le virus de l’hépatite B (VHB) utilise la machinerie transcriptionnelle nucléaire pour sa réplication. Le génome viral est transporté de la périphérie cellulaire à l’enveloppe nucléaire. Généralement, ce transport intracytoplasmique rétrograde est facilité par le réseau de Mt via l’utilisation du complexe moteur de la dynéine. Nous avons montré que le transport des capsides du VHB dépend des Mt, ce qui permet l’adressage des capsides aux complexes du pore nucléaire (NPC) ; lequel est requis pour l’étape de libération du génome de la capside dans le noyau.Dans cette étude, nous avons utilisé des capsides provenant de virus récupérés dans du surnageant de HepG2.2.15, qui contiennent le génome mature partiellement double brin (capsides matures), et des capsides exprimées chez E.coli. Ces dernières sont utilisées telles quelles, capsides E.coli contenant de l’ARN, ou bien sont utilisées pour préparer des capsides vides. Après microinjection dans des ovocytes de Xenopus laevis, nous avons observé que les capsides vides et les capsides matures sont transloquées aux NPC avec une cinétique similaire. Les capsides contenant de l’ARN ne sont pas identifiées aux NPCs ce qui implique que le transport des deux autres types de capsides est actif. Cela a été confirmé par la pré-injection d’anticorps anti tubuline qui neutralisent le transport assuré par les Mt.L’attachement spécifique des capsides matures et vides aux Mt a été confirmé en utilisant des Mt polymérisés in vitro, nous avons montré que cette interaction nécessitait des protéines cytosoliques. En utilisant des expériences de coïmmunoprécipitation et de cosédimentation nous avons identifié une chaîne légère de la dynéine (DynLL1 membre de la famille Lc8) comme partenaire des capsides. Dans les expériences de microinjection, la comicroinjection d’un excès de DynLL1 avec les capsides inhibe leur transport vers les NPCs, indiquant que DynLL1 est impliquée dans le transport actif des capsides.DynLL2 qui n’interagit pas avec les capsides diffère de DynLL1 de seulement six acides aminés. Par mutagénèse dirigée de DynLL1, nous avons montré l’implication de deux acides aminés dans l’interaction directe avec les capsides. Ces deux acides aminés sont présents à la surface du dimère de DynLL1 et absents dans le sillon résultant de la dimérisation de DynLL1, sillon impliqué dans l’interaction avec la DynIC. Nous avons partiellement reconstitué le complexe DynIC, DynLL1 et capsides vides qui doit en partie refléter la situation in vivo. / Hepatitis B virus (HBV) needs the nuclear transcription machinery for replication. The virus thus depends on the transport of its genome from the cell periphery to the nuclear envelope. In general this retrograde intracytoplasmic trafficking is facilitated along Mt (MT) using motor protein complexes of the dynein family. As we showed earlier HBV capsid transport also depends upon intact MT in order to allow their arrival at the nuclear pores, which in turn is required for genome liberation from the capsid.In the analysis we used virus-derived HBV capsids obtained from the supernatant of HepG2.2.15, which contain the mature partially double-stranded DNA genome (mature capsids) and capsids expressed in E. coli. The latter were applied in two forms: as unspecific E. coli RNA- containing capsids and as empty capsids. Upon microinjection into Xenopus laevis oocytes we observed that mature and empty capsids were translocated to the nuclear pores with a similar kinetic. RNA-containing capsids failed to arrive at the pores implying that transport of the two other capsid types was active. Active translocation was confirmed by pre-injecting anti tubulin antibodies which interfere with MT-mediated translocation.In vitro reconstitution assays confirmed the specific attachment of mature and empty capsids to MTs and showed the need of further cytosolic proteins. Using pull-down and co-sedimentation experiments we identified one dynein light chain (DYNLL1, member of the Lc8 family) as interaction partner of the capsids. Injecting an excess of recombinant DYNLL1 with empty capsids into Xenopus laevis oocytes inhibited capsid transport to the nuclear pores indicating that DYNLL1 was only functional interaction partner implied in active transport.DNYLL2 did not interact with the capsids although differing from DYNLL1 by just six amino acids. Site directed mutagenesis of DYNLL1 revealed that two amino acids were critical for a direct interaction with the capsids. Both localized at the exterior of the DYNLL1 dimer and not in the groove of DYNLL1, which interacts with the dynein intermediate chain. Accordingly we could reconstitute a complex consisting of empty capsids, DYNLL1 and dynein intermediate chain as it should be in the in vivo situation.

Determina la relación entre el nivel de conocimientos sobre hepatitis B y el estado de vacunación de los estudiantes de medicina que formarán parte del programa internado médico 2019 del Hospital Nacional Dos de Mayo (HNDM). Realiza un estudio transversal, analítico, observacional y correlacional. La población está constituida por los 120 estudiantes de medicina que formarán parte del programa internado médico del 2019 del HNDM. Del total de estudiantes de medicina el 61% presentan un mal conocimiento sobre el virus de la hepatitis B (VHB); el 53% presentan estado de vacunación incompleto. Asimismo, se aprecia que del total de estudiantes que recibieron vacunación el 86,2% tienen edad de 22 a 27 años; el 66% son de sexo femenino; el 79.8% provienen de universidad privada; el 71.3% recibió tres dosis de la vacuna contra el VHB y el 88.3% no presentaron eventos supuestamente atribuidos a la vacunación e inmunización (ESAVI). De los estudiantes que tienen un mal conocimiento sobre el VHB el 54,1% tiene un esquema de vacunación incompleta, mientras que el 76% de los estudiantes con conocimiento regular poseen un esquema incompleto. Finalmente, del total de estudiantes con un buen conocimiento sobre el VHB el 92,9% presenta un esquema de vacunación completa. Concluye que el nivel de conocimientos sobre el VHB está relacionado con el estado de vacunación del estudiante de medicina ingresante al programa de internado médico 2019 del HNDM. / Tesis

Diese Arbeit beschäftigt sich mit Zusammenhängen kognitiver Fähigkeiten und der Befindlichkeit bei Patientinnen mit Hepatitis C aus der sogenannten „Anti-D-Kohorte“. Die Frauen (n=2867) dieser spezifischen Gruppe wurden 1978/79 im Rahmen einer Anti-D-Immunprophylaxe mit dem Hepatitis C (1b) Virus infiziert. Häufig werden von Patienten, welche mit Hepatitis C infiziert sind, Beschwer-den wie Abgeschlagenheit, Ermüdung und Depressivität beklagt. In Studien wurden zudem Einschränkungen in der Kognition, insbesondere der Aufmerk-samkeit und des Gedächtnis festgestellt. Dazu wurden im Rahmen eines größeren Forschungsprojektes (Herzig et al in prep) 58 Hepatitis C Patientinnen und 25 gesunde Kontrollprobandinnen aus-gewählt. In dieser Arbeit werden die Ergebnisse der psychometrischen Testun-gen von Kognition und der Befindlichkeit vorgestellt und diskutiert. In den Komponenten Aufmerksamkeit und kognitive Flexibilität sowie in der Befindlichkeit konnten signifikante Unterschiede zur Kontrollgruppe nachgewiesen werden. Zudem fiel ein tendenzieller Vorteil antiviral therapierter Patientinnen und Patientinnen ohne nachweisbare Viruslast auf, sodass selbst HCV negative Frauen nicht als „gesund“ angesehen werden können und die Empfehlung zur medikamentösen Therapie aller viruspositiven Patientinnen besteht.

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Hepatitis B virus (HBV) is a Hepadnaviridae virus infecting mammals. Its infection can result in an acute or chronic infection. Chronic infection can result in hepatocellular carcinoma and liver cirrhosis, potentially leading to death of the patient. HBV is a small 42 nm virus with a genome length of 3.2 kb encoding seven viral proteins. HBV Core protein (HBc) is a capsid forming protein which is pleiotropic in function. We have identified two ubiquitin ligases which could interact with this protein: F-box only protein 3 (FBXO3; E3 ubiquitin ligase) and Ubiquitin conjugating enzyme E2 O (UBE2O; E2/E3 ubiquitin ligase). By employing multiple methods we have confirmed these interactions. Co- immunoprecipitation and further western blot analysis unveiled multiple new insights into the ligases′ impact on HBc: FBXO3-mediated HBc polyubiquitination stimulation and UBE2O-mediated HBc monoubiquitination promotion. FBXO3's and UBE2O's role in HBV life cycle was investigated as well. By silencing the expression of FBXO3 and UBE2O respectively, we have observed changes in HBV replication levels: FBXO3 serves as an inhibitor of HBV replication, while UBE2O stimulates the course of HBV life cycle. Further investigation of these newly-discovered understandings may lead to a whole new HBV - host interplay...

Orientador: José Paes de Ameida Nogueira Pinto / Coorientador: / Banca: Vera Lúcia Mores Rall / Banca: Luciano dos Santos Bersot / Resumo: O vírus da hepatite E (HEV) é um importante problema para a saúde pública e uma das principais causas de hepatite entérica em todo o mundo, podendo ser transmitido pelo consumo de carne suína contaminada. O trabalho teve por objetivos avaliar a prevalência do HEV em suínos de criações intensivas e tecnificadas, abatidos em matadouro-frigorífico sob Inspeção Sanitária. Foram coletadas 140 amostras de fezes de suínos de 14 propriedades distintas dos estados de São Paulo e Santa Catarina. As técnicas de diagnósticos moleculares nas amostras de fezes foram Nested RT-PCR e sequenciamento de Sanger para identificação da presença do vírus da hepatite E e o genótipo para elucidar os dados epidemiológicos. Os suínos não apresentaram doenças ou sinais clínicos no exame ante mortem e no exame post mortem. Os fígados não apresentaram lesões macroscópicas e contaminações gastrointestinais visíveis, portanto, as vísceras e carcaças foram destinadas ao consumo humano. Foram identificados 1,4% de suínos infectados pelo HEV genótipo 3. O vírus da Hepatite E, genótipos 3 e 4 tem caráter zoonótico, podendo gerar grande impacto na Saúde Pública. Diante disso, deve-se haver cuidados na manipulação de produtos possivelmente contaminados pelo HEV, evitando assim a contaminação cruzada, e, também, tratando termicamente a carne e derivados de suínos, a fim de garantir a inocuidade alimentar. / Abstract: Hepatitis E virus (HEV) is a major public health problem and one of the leading causes of enteric hepatitis worldwide and can be transmitted through the consumption of contaminated pork. The objective of this study was to evaluate the prevalence of HEV in pigs from intensive and technified farms slaughtered at a slaughterhouse under Sanitary Inspection. A total of 140 swine feces samples were collected from 14 different properties in the states of São Paulo and Santa Catarina. Molecular diagnostic techniques in faecal samples were Nested RT-PCR and Sanger sequencing to identify the presence of hepatitis E virus and the genotype to elucidate epidemiological data. The pigs had no disease or clinical signs on antemortem and post-mortem examination. The livers did not present macroscopic lesions and visible gastrointestinal contaminations, therefore the viscera and carcasses were destined for human consumption. A total of 1.4% of HEV genotype 3 infected pigs were identified. The hepatitis E virus, genotypes 3 and 4, is zoonotic in nature and can have a great impact on public health. In view of this, care must be taken in the handling of products possibly contaminated by HEV, thus avoiding cross-contamination, and also by thermally treating meat and pork products in order to guarantee food safety. / Mestre

Bakgrund: Blodburen smitta sprids genom blod, blodtillblandade kroppsvätskor eller via blodprodukter. De mest framträdande blodburna smittorna är hiv, hepatit B och C och dessa klassas som allmänfarliga sjukdomar. Miljontals människor lever med hiv, hepatit B och C världen över. Inom vården är stick- och skärskador samt återanvänt engångsmaterial de som utgör de vanligaste smittoriskerna. Bland vårdpersonal råder det delade meningar om hur de upplever att det är att vårda patienter med blodburen smitta. Syfte: Att beskriva hur patienter med blodburen smitta (hiv, hepatit B och C) upplever bemötandet inom vården. Metod: En litteraturstudie med deskriptiv design baserad på tio vetenskapliga artiklar med kvalitativ ansats och en artikel med mixad ansats. Samtliga artiklar söktes i sökmotorn PubMed via databasen Medline. Huvudresultat: Resultaten från artiklarna visade att vårdpersonalens bemötande hade bidragit till att deltagarna känt sig stigmatiserade och stämplad i mötet med vården. Deltagarna hade upplevt vårdpersonalens rädsla för att själva bli smittad, vilket påverkade bemötandet negativt. Resultatet påvisade att känslig information hade avslöjats för obehöriga personer och deltagarna upplevde bemötandet som respektlöst då sekretessen inte upprätthölls. Okunskap om blodsmittor, bristande information, dålig respons på deltagarnas frågor samt att diagnoser hade överlämnats på fel sätt framkom även i resultatet. Av resultatet uppenbarade sig också att en del deltagarna i studierna hade känt sig jämlik behandlad av vårdpersonalens goda bemötande. Slutsats: Det har visat sig att deltagarna upplever stigmatiserande attityder, bristande kunskap samt att vårdpersonalen i deras bemötande uppvisar en rädsla för att själva bli smittad. Rädsla kan uppstå till följd av okunskap. Genom att förbättra vårdpersonalens kunskap om blodburna smittor skulle detta kunna bidra till att vårdpersonal blir mer säkra i vårdandet av patienter med blodsmitta. Detta skulle kunna främja att patienterna får en bättre upplevelse av bemötandet inom vården och att omvårdnaden skulle kunna bli bättre. / Background: Blood-borne infection is spread through blood, blood-mixed body fluids or through blood products. The most prominent blood-borne infections are HIV, hepatitis B and C and these are classified as general dangerous diseases. Millions of people live with HIV, hepatitis B and C worldwide. In healthcare, sharps injuries and recycled disposable materials are the most common risks of infection. There are divided opinions among healthcare professionals about how they feel about caring for patients with blood-borne infections. Aim: To describe how patients with bloodborne pathogens (HIV, hepatitis B and C) experience the treatment in healthcare. Method: A literature study with descriptive design based on ten scientific articles with a qualitative approach and one article with mixed methods. All articles were searched in the search engine PubMed through the database Medline. Results: The results from the articles showed that the healthcare personnel attitudes in treatment of the participants contributed to feeling stigmatized and stamped in the meeting with care. The participants had experienced the healthcare personnel fear of themselves being infected, which affected the treatment negatively. The results showed that sensitive information had been revealed to unauthorized persons and the participants experienced the treatment as disrespectful because confidentiality was not maintained. Ignorance about blood infections, shortage of information, poor response to the participants' questions and that diagnoses had been submitted incorrectly also appeared in the results. The result appeared that some participants in the studies had felt equal treatment from health professionals with a good attitude. Conclusion: It has been shown that participants feel stigmatizing attitudes, lack of knowledge and the caregivers in their treatment have a fear of becoming infected themselves. Fear can arise as a result of ignorance. By improving the caregivers' knowledge of blood-borne infections, this could contribute to healthcare professionals becoming more secure in the care of patients with bloodborne infections. This could promote that the patients get a better experience of the treatment in the care and that the nursing could be better.

Hepatitis B Virus (HBV) is a DNA virus and belongs to the genus Orthohepadnavirus of the Hepadnaviridae family which represents one of two animal viruses with a DNA genome which replicates by reverse transcription of a viral RNA intermediate. Nucleotide variation led to further sub-classification into 8 genotypes (A to H). The reverse transcription step within its life cycle is prone to the introduction of errors and recombination when dually infected. This leads to a viral quasispecies which forms during the course of infection with many minor population variants; such variants can however only be detected by means of ultra-deep sequencing. A recent study in the Department of Medical Virology (UP) by Mayaphi et al. identified a number of the specimens that partitioned away from the typical subgenotype A1 clades with high bootstrap values and longer branch lengths. Thus, the main objective of the current study was to characterize the full genome of all variants for the outliers observed in the aforementioned study, inclusive of potential recombination, dual infection and minor populations. Twenty samples were selected from a previous cohort for purposes of the present study. The viral DNA was extracted and amplified by PCR according to the methods described by Günther et al. with modified primer sets. Nineteen of the samples were successfully amplified and 15 of these were sequenced. Specimens were sequenced by NGS on the Illumina MiSeq™ sequencer and sequence data used to reconstruct the viral quasispecies of each specimen. Further analyses of the reconstructed variants included molecular characterization as well as phylogenetic analysis and screening for recombination and drug resistance mutations. Full genome coverage was obtained for twelve of the fifteen samples and full genome variants reconstructed, generating nearly 40 full genomes. Phylogenetic analysis showed that the majority of the samples are of genotype A, more specifically of subgenotype A1, differing by less than 4% from known sequences. The phylogenetic analysis revealed a similar clade of outliers, where four samples clustered together with significant bootstrap support (75%) and a fifth sample partitioned separate from, yet close to, this clade, away from the typical African A1 clade. This clade was assigned to genogroup III. Three samples were of the Asian A1 clade (genogroup I) with remaining specimens grouping within genotype D and E. The variants showed low diversity within each specimen with some differing at but a few positions across the genome while even the most diverse quasispecies differed by less than a percentage (32 positions). Several unique and atypical positional variations were observed amongst study samples of which some were present in but one of the variants for that sample. Twenty-six lead to shared amino acid changes. Some observed changes, such as A1762T/G1764A and G1896A, could explain the serological patterns such as HBeAg negativity while others, such as C2002T, were previously implicated in disease progression and severity. Sample N199 presented a longer branch length and revealed short regions within the genome that display evidence of recombination between HBV/A1 and HBV/A2. The results illustrate the utility of NGS technology in characterizing viral variants. / Dissertation (MSc)--University of Pretoria, 2014. / lk2014 / Medical Virology / MSc / Unrestricted

Background: CD100, also known as Sema4D, is an immune semaphorin constitutively expressed on natural killer (NK) cells and T cells. As an immune activation molecule, CD100 has important immunoregulatory effects on NK functions by enhancing the interactions between NK cells and target cells. The aim of this study was to investigate whether hepatitis C virus (HCV) infection affects CD100 expression, and whether interferon-α treatment enhances NK killing activity to facilitate HCV clearance via CD100. Methods: Expression of CD100 on NK cells was evaluated by flow cytometry in patients with chronic HCV infection, with or without pegylated interferon-α-based therapy. NK cell cytotoxicity and interferon (IFN)-γ production were measured by flow cytometry upon culturing the NK cells with K562 and Huh7.5 or HCV JFH-1-infected Huh7.5 cells. Results: The frequency of CD100+ NK cells in HCV-infected individuals was slightly suppressed compared to healthy subjects. IFN-α treatment could significantly upregulate CD100 expression, which was confirmed by in vitro studies using peripheral blood mononuclear cells cocultured with HCV-expressing Huh7.5 cells or IFN-α. Importantly, the expression of CD100 on NK cells from HCV patients was inversely associated with the HCV-RNA levels in the early phase of IFN-α therapy, and the IFN-α upregulated CD100 led to an enhanced NK killing activity through ligations with its receptors plexin-B1/B2 on target cells. Conclusion: These results implied a novel mechanism by which IFN-α enhanced CD100/Plexin-B1/B2 interaction plays an important role in promoting NK functions in patients with chronic hepatitis C.

Combination antiviral drug therapy improves the survival rates of patients chronically infected with hepatitis B virus by controlling viral replication and enhancing immune responses. Some of these drugs have side effects that make them unsuitable for long-term administration. To address the trade-off between the positive and negative effects of the combination therapy, we investigated an optimal control problem for a delay differential equation model of immune responses to hepatitis virus B infection. Our optimal control problem investigates the interplay between virological and immunomodulatory effects of therapy, the control of viremia and the administration of the minimal dosage over a short period of time. Our numerical results show that the high drug levels that induce immune modulation rather than suppression of virological factors are essential for the clearance of hepatitis B virus.