Dissertations / Theses on the topic 'Viruses – Reproduction'

This research project deals with the characterization of self-replicating Kirsten murine viral DNA. The replication of this viral DNA and tetracycline resistance conferred to bacteria by this viral DNA will be studied. The restriction endonuclease and Southern blot analysis revealed a fragment of pBR322 from the Hind III and Pst I site that is located in the 3' end of the MLV-K:E molecule. Single stranded sequencing of the two terminal ends of this fragment verified that the 3' end of MLV-K:E contains identical sequence homology to pBR322. The presence of this pBR322 fragment explains the unusual properties of the MLV-K:E molecule. However, tetracycline resistance is less in E. Coli containing MLV-K:E than E. coli containing pBR322 as determined by zone of inhibition assay. This may be due to alteration in the promoter region of the tetracycline gene.

This project has been concerned with the application of the Baylis-Hillman methodology to the synthesis of medicinally important diketo acid analogues (cinnamate ester-AZT conjugates and 3-hydroxy ester-AZT conjugates) as dual-action HIV-1 IN/RT inhibitors; and on exploratory studies in the preparation of 3-(amidomethyl)-(1H)-2-quinolones as PR inhibitors; and (1H)-2- quinolone-AZT conjugates as dual action IN/RT inhibitors. A series of Baylis-Hillman adducts has been prepared, typically in moderate to excellent yield, by reacting 2-nitrobenzaldehyde with methyl acrylate, ethyl acrylate and methyl vinyl ketone in the presence of 1,4- diazabicyclo[2.2.2]octane (DABCO). Subsequently, various transformations that include conjugate addition of primary and secondary amines to the α,ß-unsaturated moiety to obtain 2- (aminomethyl)-3-hydroxy-3-(2-nitrophenyl)propanoate derivatives, effective SN2´ substitution of the BH ß-hydroxy by a Vilsmeier-Haack in situ-generated chloride to afford Baylis-Hillman allyl chlorides, iron in acetic acid-catalyzed cyclisation to 3-acetoxymethyl-(1H)-2-quinolone derivatives were achieved. Thus, using the Baylis-Hillman methodology, two nuanced classes of diketo acid analogues were constructed. These involved conjugating appropriate propargylamine derivatives with AZT using the „click‟ reaction. In an exploratory study, the quinolone derivative, precisely 3-acetoxymethyl- (1H)-quinol-2-one, was transformed into 3-hydroxymethyl-(1H)-quinol-2-one using potassium carbonate in a mixture of methanol and water (1:1). Following successful hydrolysis, the resulting alcohol was transformed to the corresponding chloride, 3-chloromethyl-(1H)-quinol-2- one, using thionyl chloride. Subsequent nucleophilic substitution afforded 3-(aminomethyl)- (1H)-2-quinolone derivatives which were subsequently transformed to 3-(amidomethyl)-(1H)-2- quinolones; and 3-[(propargylamino)-methyl]-(1H)-quinol-2-one as precursors to quinolone- AZT derivatives. All compounds were characterized by NMR, IR, and where appropriate, high resolution MS

Tetraviruses are a family of small non-enveloped positive sense RNA viruses that exclusively infect members of the order Lepidoptera. Their replication biology is poorly studied because, with the exception of Providence virus (PrV), tetraviruses are unable to replicate in tissue culture cells. The overall aim of the research described in this thesis was to develop a fundamental understanding of the replication of tetraviruses, focussing on the site of replication within host cells and in particular, the subcellular localisation of the viral replicase. Helicoverpa armigera stunt virus (HaSV, Genus: Omegatetravirus) was chosen for this study because it is the only tetravirus for which the cDNAs have been shown to be infectious. In the absence of tissue culture cell lines susceptible to HaSV infection, the approach was to use confocal fluorescence microscopy to examine the subcellular localisation of the HaSV replicase fused to enhanced green fluorescent protein (EGFP) in mammalian and insect tissue culture cells. The replicase (with EGFP fused at its C-terminus) localised to punctate structures throughout the cytoplasm of transfected HeLa and Sf9 cells. These structures were then shown – using live cell imaging and time lapse photography – to behave similarly to cellular endocytic organelles and fluorescence partially overlapped with membranes containing the late endosomal marker protein CD63. Biochemical fractionation of Sf9 cells expressing the replicase via a recombinant baculovirus (as well as transfected HeLa and Sf9 cells expressing EGFP-replicase fusion proteins) demonstrated that the replicase was strongly associated with detergentresistant membranes (DRMs) in these cells. Deletion analysis of the replicase coding sequence revealed two regions involved in the generation of the punctuate structures. Firstly, the C-terminal half of the replicase RNAdependant RNA polymerase domain was found to be essential for targeting and the tight association with DRMs while the second region, within the Nterminal 44 amino acids, enhanced localisation through a combination of secondary structural elements and sequence-specific functions. A comparative immunofluorescence study on PrV, which replicates as a persistent infection in an insect midgut cell line, showed that the PrV replicase also localised to punctate structures in the cytoplasm. Biochemical fractionation showed that the replicase was also strongly associated with DRMs. This thesis describes the development of new experimental systems for the study of tetravirus replication biology and the data lead to the conclusion that the HaSV replicase associates with DRMs derived from alternate endocytic pathway organelles.

In this thesis we describe novel approaches to the formal description of systems which reproduce, and show that the resulting models have explanatory power and practical applications, particularly in the domain of computer virology. We start by generating a formal description of computer viruses based on formal methods and notations developed for software engineering. We then prove that our model can be used to detect metamorphic computer viruses, which are designed specifically to avoid well-established signature-based detection methods. Next, we move away from the specific case of reproducing programs, and consider formal models of reproducing things in general. We show that we can develop formal models of the ecology of a reproducer, based on a formalisation of Gibson's theory of affordances.

The aim of this study was to examine the mechanisms of transcription and replication of the paramyxovirus, simian virus type 5 (SV5). This was initially attempted using reverse genetics techniques and subsequently examining specific viral protein: protein interactions within the replication complex. A cDNA clone encoding a synthetic negative-sense RNA genome analogue was constructed. Reverse genetics techniques were used to attempt to characterise conditions which supported the transcription and replication of this genome analogue, with or without the use of wild-type helper virus but were unsuccessful. During the course of these studies, a number of mammalian cell lines inducibly expressing SV5 proteins were isolated. These cell lines were subsequently used to examine viral protein: protein interactions within the replication complex. When expressed alone, both P and V proteins exhibited diffuse cytoplasmic fluorescence and V was also found in the nucleus. However, when NP was expressed alone, it was seen as punctate and granular cytoplasmic fluorescence. The distribution patterns of the proteins changed when expressed in combination. Large cytoplasmic aggregates similar to those at late times in an SV5 infection were seen in cells which co-expressed NP and P. When NP was co-expressed with V, however, NP was partially redistributed to give diffuse cytoplasmic and nuclear fluorescence. This showed that both P and V proteins could interact with NP and suggested that V may play a role in keeping NP soluble prior to an ordered encapsidation process. Extracts from these cell lines were then used in a novel protein: protein capture assay and demonstrated that NP could interact with both P and V proteins. NP expressed by the cell line was shown to contained both soluble and polymeric forms of NP. P was shown to bind both forms of NP, while V could only bind soluble NP. Since P and V proteins are amino co-terminal, the site of interaction between P and polymeric NP was predicted to be in the P unique C-terminus. This was strengthened when a P-specific C- terminal mAb was found to block the binding of P with polymeric NP. Deletion mutant analysis in the C-terminus of the P protein showed that the mAb binding site was at the extreme C-terminus of the protein suggesting this is the point of interaction between P and polymeric NP. Possible roles for these protein: protein interactions and implications for the paramyxovirus replication complex are discussed.

 In Epstein-Barr virus (EBV)-associated malignancies, the virus is harbored in every tumor cell and persists in a tightly latent form (latency I, II or III) expressing a limited number of viral latent proteins. Induction of EBV lytic cycle, which triggers expression of a much larger number of viral proteins, may lead to therapeutic effects against EBV-associated cancers. We previously found that suberoylanilide hydroxamic acid (SAHA), a FDA-approved histone deacetylase inhibitor, induced EBV lytic cycle and mediated enhanced cell death in EBV-positive gastric carcinoma cells (latency II). In this thesis, we sought to investigate SAHA’s induction of EBV lytic cycle and its cellular consequences in EBV-associated epithelial malignancies, with particular focus on nasopharyngeal carcinoma (NPC) due to its strong association with EBV and high prevalence in southern Chinese populations. SAHA effected strong induction of EBV lytic cycle in EBV-positive epithelial malignancies, including gastric carcinoma and NPC, as evidenced by strong expression of EBV lytic proteins, replication of viral DNA and production of infectious viral particles. Immunofluorescent staining revealed that up to 70% EBV-positive epithelial cancers expressed EBV lytic proteins following treatment with micromolar concentrations of SAHA. However, SAHA could not induce EBV lytic cycle in NK lymphoma cells (both NPC and NK lymphoma express EBV latency II pattern), indicating preferential viral lytic induction in epithelial rather than lymphoid malignancies. EBV lytic cycle induction in NPC by SAHA required activation of protein kinase C-delta (PKC-) and acetylation of non-histone protein but required neither phosphatidylinositol 3’-kinase (PI3K), MAPK/ERK kinase (MEK), c-Jun aminoterminal kinase (JNK) nor p38 stress mitogen-activated protein kinase (MAPK) signaling pathway. Conflicting observations regarding the effect of EBV lytic cycle induction on apoptosis were reported. Thus, we investigated the relationship between EBV lytic cycle induction and apoptosis in NPC following treatment with SAHA. EBV-positive NPC showed a higher percentage of apoptosis and proteolytic cleavage of PARP, caspases-3, -7 and -9 over EBV-negative NPC and greater than 85% of NPC cells co-expressed EBV immediate-early (Zta), early (BMRF1) or late (gp350/220) lytic proteins and cleaved caspase-3. Tracking of expression of these lytic proteins over time demonstrated that NPC proceeded to apoptosis following EBV lytic cycle induction, contrary to the previously reported anti-apoptotic effect of EBV lytic proteins in Burkitt lymphoma. Analyses of cleaved caspase-3 expression upon RNAi knockdown and exogenous expression of Zta further supported that EBV lytic cycle directly led to apoptosis of EBV-positive NPC cells. Interestingly, inhibition of EBV DNA replication and late lytic protein expression by phosphonoformic acid did not impact on SAHA’s induced cell death in NPC, indicating that early rather than late phase of EBV lytic cycle contributed to the apoptotic effect. Finally, in vivo effects of SAHA on EBV lytic cycle induction and tumor growth suppression were observed in NPC tumors established in nude mice. In conclusion, activation of EBV lytic cycle from latent cycle in EBV-positive epithelial malignancies including NPC by SAHA effected apoptosis and tumor growth suppression of the cancer cells and provided experimental evidence for virus-targeted therapy against EBV-positive cancers.
published_or_final_version
Paediatrics and Adolescent Medicine
Doctoral
Doctor of Philosophy

Thesis (Ph. D.)--University of Adelaide, Dept. of Microbiology & Immunology, 1998.
Copy of author's previously published article on back end-paper. Includes bibliographical references (leaves 105-158).

Norovirus (NoV) and Sapovirus (SaV) are major causes of outbreak gastroenteritis worldwide. NoV and SaV are highly infectious, have multiple transmission routes and have a short incubation period, thereby facilitating rapid intercontinental spread of new variants. Consequently, a treatment would be advantageous for controlling them. However, currently little is known about the replication cycle and evolution of human NoV or SaV as neither are culturable. NoV and SaV are RNA viruses of the Caliciviridae family and have great genetic diversity which is thought to facilitate irnmune evasion. Consequently variants of NoV GI1.4 arose in 1996, 2002, 2004 and in 2006 and resulted in pandemics. Therefore, in this study, the role of the two main mechanisms associated with generating viral diversity; recombination, and point mutation were investigated for NoV and SaV. Physiological and kinetic properties of three NoV RdRps (genotypes, Gll.b, Gll.4, Gll.7) and two SaV RdRps (genogroups GI, GII) were also investigated. RNA recombination is a significant driving force in viral evolution. Increased awareness of recombination within the Calicivirus genus Norovirus (NoV) has led to a rise in the identification of NoV recombinants and they are now reported at high frequency. Despite this no classification system exists for recombinant NoVs As a result, there is duplication in reporting novel recombinants and the precise number of novel NoV recombinant types is unknown. Therefore, in order to elucidate thero!e of recombination in NoV evolution, 121 NoV nucleotide sequences, compiled from the GenBank database and published literature, were analysed for recombination events. NoV recombinants and their recombination breakpoint were identified using three methods: phylogenetic analysis, Simplot analysis and the Maximum Chi-Squared method. In total 19 unique NoV recombinant types were identified in circulation across the globe and they had a common recombination point near the ORF1/2 overlap. Recombination at the ORF1/0RF2 overlap could have important implications in NoV evolution as it enables a virus to swap its antigenic determinates (capsid) and thereby avoid immune clearance in an analogous manner to antigenic shift in influenza virus. This study also examined the role of NoV and SaV replication in generating viral diversity by comparing the physiological, kinetic and biochemical properties of five genotypically distinct RdRps from two different genera of the Caliciviridae. Genetically diverse HuCV RdRps were expressed in Escherichia coli and characterised in an in vitro assay designed for this study. The results indicated that despite high sequence variation between the five enzymes (between 6% and 71% amino acid difference) they shared similar physiological properties. Though there was some variation in their template usage and kinetic properties. SaV was able to perform primer dependent replication on homopolymeric A RNA whereas the NoV RdRps were not. Additionally, NoV RdRps had a higher incorporation rate and were more kinetically efficient than the two SaV RdRps. The incorporation fidelity of the five enzymes was similar (between 2.2x10-5 to 8.9x10-4 ), although interestingly the most prevalent strain, Gll.4, had the lowest fidelity of the caliciviruses. Therefore, suggesting that RdRp fidelity has an important role in NoV evolution. Overall, this study illustrated that NoV and SaV generate genetic diversity in a similar fashion to other RNA viruses, that is, a delicate combination of recombination, point mutation and replication efficiency. Understanding the mechanisms involved in viral replication and genomic diversity of the calicivirus RdRps is essential if a successful control strategy for the human caliciviruses is going to be developed.

Picornavirus proteins VP1 to VP3 are exposed on the surface of the virus particle whereas VP4 is internal and modified at its amino terminus by the addition of myristic acid (Chow et al., 1987; Paul et al., 1987). Myristic acid occupies a position in the core of mature poliovirus particles; it has been suggested that it may be important for particle integrity or in the localization of the capsid protein precursor on the hydrophobic membranes during virion assembly (Chow et al., 1987). To determine the function of the amino-terminal myristylation of VP4 in picornaviruses, and to establish whether competition for the acylation site is a possible approach to antiviral chemotherapy, the effect of fatty acids on virus replication has been examined. Some fatty acids are able to enter picornavirus-infected cells and compete for the myristylation site on VP4. Unexpectedly, it was found that short chain fatty acids also inhibit an early event in the replication of bovine enterovirus (BEV) at concentrations which have no detectable effect on cellular macromolecular synthesis and cloning. These findings indicate that fatty acids inhibit cell-mediated uncoating. Short chain fatty acids inhibit the replication of bovine enterovirus but are almost ineffective against poliovirus type 1, coxsackievirus B5, encephalomyocarditis virus and human rhinovirus lB. Lauric acid binds to bovine enterovirus, thereby stabilizing the virus particle to heat degradation. Fatty acid-bound virions attach to susceptible cells but fail to undergo cell-mediated uncoating. The inhibitory effect is reversible with chloroform and may result from a hydrophobic interaction between the fatty acid and a specific site on the virus particie.

The influenza A virus non-structural (NS1) protein is multifunctional, and during virus-infection NS1 interacts with several factors in order to manipulate host-cell processes. This study reports that NS1 binds directly to p85β, a regulatory subunit of phosphoinositide 3-kinase (PI3K), but not to the related p85α. Expression of NS1 was sufficient to activate PI3K and cause the phosphorylation of a downstream mediator of PI3K signalling, Akt. However, in virus-infected MDCK cells, the kinetics of Akt phosphorylation did not correlate with NS1 expression, and suggested that negative regulation of this signalling pathway occurs subsequent to ~8h post-infection. Mapping studies showed that the NS1:p85β interaction is primarily mediated by the NS1 C-terminal domain and the p85β inter-SH2 (Src homology 2) domain. Additionally, the highly conserved tyrosine at residue 89 (Y89) of NS1 was found to be important for binding and activating PI3K in a phosphorylation-independent manner. The inter-SH2 domain of p85β is a coiled-coil structure that acts as a scaffold for the p110 catalytic subunit of PI3K. As NS1 does not displace p110 from the inter-SH2 domain, a model is proposed whereby NS1 forms an active heterotrimeric complex with PI3K, and disrupts the ability of p85β to control p110 function. Biological studies revealed that a mutant influenza A virus (Udorn/72) expressing NS1 with phenylalanine substituted for tyrosine-89 (Y89F) exhibited a small-plaque phenotype, and grew more slowly in MDCK cells than wild-type virus. Unexpectedly, another mutant influenza A virus strain (WSN/33) expressing NS1-Y89F was not attenuated in MDCK cells, yet appeared to be less pathogenic than wild-type in vivo. Overall, these data indicate a role for NS1-mediated PI3K activation in efficient influenza A virus replication. The potential application of this work to the design of novel anti-influenza drugs and vaccine production is discussed.

The picornavirus family includes significant human and animal viruses such as poliovirus (PV), human rhinovirus (HRV) and foot-and-mouth-disease virus (FMDV). Current disease treatment and control strategies are limited by an incomplete understanding of the interactions between the non-structural, replicative picornavirus proteins and host cell components. To investigate these interactions, Theiler's murine encephalomyelitis virus (TMEV) 2B, 2C, 2BC and 3A proteins were transiently expressed in BHK-21 cells and detected by indirect immunostaining and laser-scanning or epifluorescence microscopy. The signal of the 2B protein overlapped with that of the ER marker protein, ERp60, as well as that of the peripheral Golgi marker protein, β-COP. The 2C protein overlapped with ERp60 in a faint reticular stain, and localised to large punctate structures that partially overlapped with β-COP at higher levels of expression. The 2BC protein located to large perinuclear structures that overlapped exclusively with β-COP. The TMEV 3A protein signal overlapped with both ERp60 and β-COP stains, in addition in cells expressing the 3A protein the ER appeared swollen and bulbous while the Golgi was dispersed in some cells. 2C and 2BC proteins with C-terminal deletions localised in the same manner as the wild type proteins indicating that the localisation signals that determine subcellular localisation of the proteins are within the N-terminal 60 amino acids of the 2C protein. The significance of the high degree of conservation of the N-terminal domain of the 2C protein throughout the Picornaviridae was investigated through the introduction of amino acid substitution mutations at highly conserved residues in the N-terminal domain of 2C into the viral cDNA. Upon transfection of the viral RNA into BHK-21 cells, it was observed that substitution of amino acid residues 8, 18 and 29 abolished the ability of TMEV to induce cytopathic effect (CPE), while substitution of residues 4, 14 and 23 only attenuated the ability of TMEV to induce CPE. To determine whether amino acid substitution mutations would affect the localisation of the 2C protein, 2C proteins with substitution mutations at amino acids 4, 8, 14, 18, 23 and 29 were transiently expressed in BHK-21 cells and detected by indirect imrnunostaining and examination by laser-scanning confocal and epifluorescence microscopy. The 2C mutant 4, 8 and 29 proteins showed slightly altered localisation patterns compared to the wild type protein with a significant portion of the proteins localising in a perinuclear stain suggesting possible localisation to the nuclear envelop. The 2C mutant 14 and 18 proteins localised to a diffuse pattern in BHK-21 cells while the 2C mutant 23 protein located to small punctate structures that partially overlapped with the ERp60 stain but were completely separate from the β-COP stain. Finally, a hydrophilic, antigenic region of the 2C protein was expressed in frame with an N-terminal GST tag and was successfully purified on a pilot-scale and detected by Western analysis. This 2C178 peptide will be used to generate antibodies against the 2C and 2BC proteins for use in future studies. This study has furthered our knowledge of the localisation of the picornavirus 2B, 2C, 2BC and 3A proteins in host cells and identified a possible link between this localisation and an ability of TMEV to replicate in BHK-21 cells.

L’infection par HIV-1 représente un des problèmes de santé publique majeurs de notre société actuelle. Le traitement HAART (Highly Active AntiRetroviral Therapy) inhibe le cycle réplicatif viral mais ne permet pas l’éradication du HIV-1. La principale cause de cet échec thérapeutique est la persistance de réservoirs cellulaires infectés de manière latente par HIV-1, qui, lors de l’arrêt du traitement HAART, sont à l’origine d’un rebond de la charge plasmatique virale. Le défi actuel est donc de découvrir de nouvelles méthodes d’élimination des cellules réservoirs. Une des stratégies envisagées est de forcer l’expression virale dans les cellules infectées de manière latente afin d’entraîner leur destruction suite à leur détection par le système immunitaire ou suite aux effets cytopathiques viraux. Parallèlement, le traitement HAART serait maintenu afin de limiter la propagation des virions néo-synthétisés. Plusieurs éléments sont impliqués dans la répression transcriptionnelle associée à la latence post-intégrationnelle du virus HIV-1 :la nature du site d’intégration ;l’absence de facteurs cellulaires inductibles tels que NF-κB ;la structure chromatinienne du provirus et les modifications post-traductionnelles des histones ;l’absence de niveaux suffisants de la protéine trans-activatrice Tat. De plus, notre laboratoire a précédemment mis en évidence un lien entre deux de ces éléments, en démontrant, dans une lignée modèle de latence post-intégrationnelle, que la cytokine pro-inflammatoire TNFα, un activateur de la voie de signalisation NF-κB, permet une réactivation synergique de l’expression virale combinée à l’inhibiteur d’histone-désacétylases (HDACI) TSA. Cependant, l’utilisation thérapeutique du TNFα et de la TSA est inenvisageable en raison de leurs toxicités.

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Doctorat en Sciences
info:eu-repo/semantics/nonPublished

The influenza A virus genome codes for up to 12 proteins. Segment 2 encodes three proteins, the polymerase subunit PB1, a small protein PB1-F2 and an N-terminally truncated version of PB1 called N40. Different functions have been reported for PB1-F2 such as induction of apoptosis, regulation of the viral polymerase activity, enhancement of secondary bacterial infections and modulation of the innate immune system. So far, no function has been ascribed to N40. To study PB1-F2 in more detail, its coding sequence was deleted from its original position and inserted downstream of the PB1 (segment 2), NA (segment 6) or M (segment 7) open reading frames (ORF) employing different strategies, including the use of an overlapping Stop-Start cassette, a duplicated promoter sequence and the self-cleaving 2A peptide derived from foot-and-mouth disease virus. Viruses with bicistronic segments were rescued and tested for their ability to express PB1-F2. Whereas no expression of PB1-F2 was detected from bicistronic segments 2 and 7, expression of PB1-F2 from segment 6 was observed in high levels. However, the phenotype of all these viruses was similar to that of viruses lacking PB1-F2 which made mutational analysis of PB1-F2 not worthwhile. Previously, the function of PB1-F2 was mainly studied using a virus deficient in PB1-F2 production but showing increased N40 expression. In the present study, recombinant WSN viruses lacking either PB1-F2 or N40, or both proteins were engineered and the effects of these mutations on the viral life cycle were examined. Viruses deficient for PB1-F2 that overexpressed N40 showed the most attenuated phenotype, whereas the loss of PB1-F2 alone did not obviously affect virus replication. Reduced viral polymerase activity was observed for viruses lacking N40, however attenuation in vivo was only seen in combination with the loss of PB1-F2. Neither the loss of PB1-F2 nor N40 alone had a great impact, but changes in the expression level of both proteins were disadvantageous for the virus. Increased levels of N40 shifted the polymerase activity towards replication, suggesting a new function for N40. Thus, it was shown that the segment 2 gene products and their expression level influence viral replication and pathogenicity, and a careful design of mutant recombinant viruses is vital for determining the experimental outcome.

La vitesse de réplication du HIV-1(Human Immunodeficiency Virus type 1), qui semble corrélée de manière directe à la vitesse de progression de la maladie vers le stade SIDA, est essentiellement contrôlée au niveau transcriptionnel. La transcription du HIV-1 est régulée par la structure chromatinienne, des éléments agissant en cis localisés dans les LTRs, des facteurs de transcription agissant en trans et par la protéine virale trans-activatrice Tat (revu dans Quivy et al. 2007, Bisgrove et al. 2005, Rohr et al. 2003, Rabson and Graves 1997). En plus de l’enhancer localisé dans le LTR5’ du HIV-1, un enhancer intragénique, localisé dans le gène pol du HIV-1, inductible par le phorbol 12-myristate 13-acétate (PMA) a été identifié. La localisation progressive de l’activité enhancer a permis de définir deux domaines distincts et indépendants dans cet enhancer intragénique :les fragments 5103 et 5105 localisés respectivement dans la partie centrale du gène pol et dans une région couvrant la fin du gène pol, le gène vif, le gène vpr et le premier exon codant des gènes tat et rev (Verdin et al. 1990). Les fragments 5103 et 5105 se comportent tous deux comme des enhancers inductibles par le PMA lorsqu’ils sont clonés en amont du promoteur de la thymidine kinase dans un vecteur rapporteur en cellules HeLa. Notre laboratoire a précédemment identifié trois sites de liaison pour les facteurs de transcription AP-1 dans le fragment 5103 (Van Lint et al. 1991).

Au cours de notre thèse, nous avons poursuivi la caractérisation de ces sites de liaison AP-1 et avons montré que les facteurs c Fos, JunB et JunD interagissent in vitro avec ces motifs. Pour chaque site, nous avons identifié des mutations qui abolissent la liaison des facteurs AP-1 sans altérer la séquence en acides aminés sous-jacente de la transcriptase inverse. Par des expériences de transfection transitoire, nous avons démontré que les sites AP 1 intragéniques sont entièrement responsables de l’activité enhancer PMA-dépendante du fragment 5103. De plus, l’activité PMA-inductible du fragment 5103 est inhibée par le mutant dominant négatif A-Fos à condition que les sites ne soient pas mutés. A l’inverse, l’expression ectopique de dimères forcés AP-1 affecte positivement l’activité enhancer du fragment 5103. Enfin, nous avons étudié le rôle biologique des sites AP-1 intragéniques dans la réplication virale et avons montré que ces sites contribuent positivement à l’infectivité du virus.

Durant la seconde partie de notre thèse, nous avons entamé la caractérisation physique et fonctionnelle du fragment 5105. Nos résultats de transfection transitoire montrent que l’activité PMA inductible du fragment 5105 est localisée dans le dernier tiers de ce dernier :le sous fragment 5105.3. L’analyse bioinformatique de cette région a permis de mettre en évidence un site de liaison pour les facteurs AP-1 in vitro. Des mutations ponctuelles permettent d’abolir la liaison des facteurs à leur site mais altèrent la séquence en acides aminés sous-jacente codant pour les protéines Tat et Rev. Nous avons montré que ce site est impliqué dans l’activité transcriptionnelle de ce fragment. L’expression ectopique du mutant dominant négatif A-Fos inhibe l’activité transcriptionnelle PMA-inductible du fragment 5105. Une analyse bioinformatique plus large nous a ensuite permis d’identifier in vitro, par retard de migration sur gel, 5 sites de liaison pour le facteur YY1 et 2 sites de liaison pour le facteur PU.1 dont les implications pour le virus restent encore à déterminer.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Comme pour la plupart des virus à ARN+, le VHC induit des remaniements membranaires appelés membranous web. Les protéines non structurales virales formant le complexe de réplication du virus sont associées à ces membranes néosynthétisées. La compréhension de la mise en place de ces membranes cellulaire est encore actuellement mal connue. Afin d’étudier ce phénomène, nous avons dans un premier temps sélectionné des clones cellulaires Huh7.5 hébergeants un réplicon sous-génomiquedu virus. Nous avons ainsi pu mettre en évidence la présence d’un réseau multivésiculaire semblant provenir de l’induction de mécanismes d’autophagie. Plus récemment l’utilisation du modèle de propagation du virus complet nous a permis de mieux caractériser ce réseau multivésiculaire en déterminant trois sous réseaux vésiculaires structuralement différents. L’analyse de cette étude est effectuée principalement par microscopie électronique avec des techniques innovantes tels que la reconstruction tridimensionnelle et des immunogolds
As other RNA viruses, HCV induces membrane alterations termed membranous web and its nonstructural proteins forming the viral replication complex are associated to these neo-synthesized membranes. The mechanism underlying these host cell membranes alterations is still currently unknown. To investigate this mechanism, we initially selected Huh7.5 cells clones harbouring a HCV subgenomic replicon. We were able to demonstrate the presence of a multivesicular network apparently linked to the autophagy induction mechanisms. More recently, using the cell culture-adapted HCVsystem, we better characterized this network by determining three multivesiculars vesicles structurally different subnets. This study was carried out mainly by performing electron microscopy observations,with using innovative techniques such as three-dimensional reconstruction and immunogold

Parmi les rétrovirus tumorigènes, les rétrovirus appelés « complexes » dont font partie HTLV-1 chez l’homme et BLV chez le mouton, sont responsables de leucémies lymphoïdes chroniques caractérisées par des pathogenèses similaires. Bien que la contribution essentielle de la protéine Tax dans la leucémogenèse soit bien établie pour ces deux virus, il existe toujours une contreverse quant à l’expression des protéines virales -dont l’oncoprotéine Tax- dans les cellules transformées. Dans le cas de HTLV-1 et BLV, le virus est latent. Une hypothèse attrayante suggère que l’extinction complète du provirus accompagnée du blocage transcriptionnel de l’expression de Tax dans la cellule leucémique serait un élément indispensable au développement de la tumeur en réduisant son immunogénicité. Le silencing viral permettrait à la cellule infectée d’échapper à la reconnaissance par le système immunitaire de l’hôte.

Dans la première partie de notre travail, nous avons pu montrer, grâce à l’observation et le suivi de deux moutons infectés par BLV, que l’extinction virale était associée au développement tumoral. Alors que la phase asymptomatique est caractérisée par l’existence de différents clones cellulaires infectés et exprimant le virus, la phase tumorale est caractérisée par l’existence d’un seul clone cellulaire dans lequel l’expression virale est éteinte. Dans le cas du mouton S2531, nous avons mis en évidence que le silencing observé dans la phase tumorale est d’origine génétique. L’extinction de l’expression virale et la domination du clone muté au niveau de la protéine Tax (K303) sont associées à l’émergence de la leucémie et constituent une caractéristique de la phase tumorale. Le deuxième mouton étudié, S267, est caractérisé par la présence de cellules infectées non transformées et transformées au même moment. Par ailleurs, le mouton S267 est caractérisé par une absence de mutations ou délétions au niveau du provirus intégré dans les cellules tumorales. Dans la deuxième partie de notre travail, nous avons pu, grâce à l’établissement de la lignée L267 dans laquelle le silencing viral n’est pas lié à une défection dans la structure génomique du provirus, élucider les mécanismes épigénétiques responsables du silencing. Nous montrons que ce provirus silencieux peut être réactivé in vitro 1) lorsque la protéine Tax sauvage est introduite par transfert rétroviral, 2) après traitement des cellules par la trichostatine A (TSA), un inhibiteur des histones désacétylases, 3) par traitement des cellules à la 5’azacytidine, un agent inhibiteur de DNA méthyltransférases. La réactivation du provirus silencieux lui confère la capacité d’infecter des moutons sains, suggérant que le provirus est complet et exempt de mutation qui pourrait altérer son fonctionnement. Les complexes de désacétylation des histones msin3A et HDAC-1 jouent un rôle important dans l’établissement du silencing viral via la condensation de la chromatine, et ce changement de la structure chromatinienne est lié a un ensemble de modifications ciblant les acides aminés des queues N-terminales des histones H3 et H4 établissant ainsi un code histone pour l’extinction et l’expression du provirus. Enfin, l’étude comparative des profils d’expression génique de cellules B tumorales dans lesquelles le provirus BLV est soit silencieux, soit ré-activé suite à l’expression exogène de Tax a montré que les mécanismes épigénétiques de silencing se superposent même dans le modèle génétique de silencing. De plus le rétablissement de l’expression virale par Tax est associé à une élimination des complexes de désacétylation des histones au niveau du promoteur viral.

Nos résultats ouvrent le champ vers l’utilisation de modèle de leucémie ovine dans des essais thérapeutiques basés sur la combinaison du traitement par des inhibiteurs des HDACs et des DNMTs et une immunothérapie ciblant des antigènes tumoraux d’origine virale ou cellulaire. De telles expériences in vivo constitueront un modèle utile pour le traitement l’ATLL et de pathologies prolifératives touchant les cellules B chez l’homme.

Doctorat en sciences biomédicales
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L’infection par le cytomégalovirus est le plus souvent asymptomatique chez les sujets immunocompétents mais entraine une morbidité et une mortalité importantes chez les patients immunocompromis et en cas d’infection congénitale.

Après l’infection primaire, le virus persiste tout au long de la vie à l’état latent mais peut se réactiver de manière intermittente. Ceci est associé à l’expansion de lymphocytes T CD4+ fortement différenciés ayant des fonctions auxiliaires et cytolytiques. L’infection primaire est, par contre, caractérisée par une réplication virale intense qui dure plusieurs mois. Il a été montré que l’exposition prolongée à des concentrations élevées d’antigènes entraine une perte progressive de fonction par les lymphocytes T appelée épuisement et caractérisée par l’expression de récepteurs inhibiteurs. L’impact de la réplication virale intense observée au cours de l’infection primaire par le CMV sur la fonction des lymphocytes T CD4+ n’est pas bien connu.

La fonctionnalité des lymphocytes T CD4+ a été explorée chez l’humain et le singe rhésus au cours de l’infection primaire et comparée à celle de sujets porteurs chroniques du virus.

Les résultats montrent que l’infection primaire par le CMV est associée à la détection de lymphocytes T CD4+ circulants ayant une faible capacité de prolifération et de production de cytokines et d’IL-2 en particulier.

L’impact de la différenciation sur la fonction des lymphocytes a été exploré en détail chez l’humain. Il a été observé qu’un degré de différenciation plus élevé des lymphocytes T CD4+ spécifiques du CMV joue un rôle dans la production réduite d’IL-2. Toutefois, la fraction moins différenciée (exprimant la molécule CD28) présente également une sécrétion d’IL-2 moindre au cours de l’infection primaire. Ceci fait partie d’une diminution globale de la production de cytokines au cours de l’infection primaire qui affecte également la sécrétion d’IFNγ et TNFα, entraine une polyfonctionnalité réduite et est indépendante de la différenciation. L’épuisement des lymphocytes T CD4+ spécifiques du CMV contribue à leur fonctionnalité moindre comme l’indique l’expression accrue du récepteur inhibiteur PD-1 et l’augmentation des réponses prolifératives en présence d’anticorps bloquant PD-1.

Le lien entre excrétion virale et fonction lymphocytaire a été étudié chez le macaque rhésus. L’infection par le CMV est observée chez les singes juvéniles et adultes mais pas chez les nourrissons. L’excrétion urinaire et salivaire est significativement plus fréquente et intense chez les singes juvéniles par rapport aux adultes. Comme chez l’humain au cours de l’infection primaire, les lymphocytes T CD4+ spécifiques du virus sont moins

polyfonctionnels et prolifèrent moins efficacement chez les singes juvéniles par rapport aux singes adultes. Ceci est associé à l’expression accrue du récepteur inhibiteur PD-1 chez les singes juvéniles. La réponse proliférative des lymphocytes T CD4+ est accrue en présence d’anticorps bloquant PD-1 ou d’IL-2 exogène. Enfin, une association inverse entre fonction lymphocytaire et excrétion urinaire a été mise en évidence chez les macaques adultes.

Ces résultats indiquent que l’infection par le CMV présente des caractéristiques semblables chez l’humain et le singe rhésus. L’infection primaire est associée à la détection de lymphocytes T CD4+ ayant une fonctionnalité moindre qu’au cours de l’infection chronique. L’expression du récepteur inhibiteur PD-1 typique des cellules épuisées est l’un des mécanismes impliqués et pourrait être la cible de stratégies immunomodulatrices visant à améliorer les fonctions lymphocytaires et le contrôle de la réplication virale. Les résultats présentés indiquent que l’infection naturelle chez le singe rhésus constitue un modèle potentiellement utile à l’étude de la réponse immune au CMV humain et à l’évaluation de stratégies immunomodulatrices.

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Cytomegalovirus infection is mostly asymptomatic in immunocompetent hosts but leads to severe morbidity and mortality in immunocompromised subjects and foetuses.

After primary infection, CMV establishes lifelong persistence but can reactivate intermittently. This is associated with the expansion of highly differentiated CD4+ T lymphocytes exhibiting helper functions and cytolytic activity.

Primary infection is characterised by an intense viral replication lasting several months. It has been shown that prolonged exposure to elevated antigen concentrations induces a progressive loss of function by T lymphocytes called exhaustion. This state of functional impairment is associated to the expression of inhibitory receptors. The consequence of the intense viral replication seen in primary CMV infection on CD4+ T cell function is unknown.

CD4+ T cell function has been studied in human and rhesus macaque during primary CMV infection. Chronic CMV carriers have been used as controls.

The results show that primary CMV infection is associated to the detection of circulating CD4+ T lymphocytes exhibiting weak proliferative capacities and reduced cytokine production affecting IL-2 in particular.

The impact of differentiation on lymphocyte function has been explored in detail in human. An increased proportion of terminally differentiated CD4+ T cells (CD28-) is observed during primary infection. These lymphocytes are unable to secrete IL-2 in response to CMV antigens. Interestingly, CD28+ CMV-specific CD4+ T cells also exhibit reduced IL-2 production during primary infection. This is part of a global reduction of cytokine production affecting IFNγ and TNFα as well. The impaired cytokine production is associated to reduced polyfunctionality and is independent of differentiation. Exhaustion of CMV-specific CD4+ T lymphocytes contributes to the reduced functionality as shown by an increased expression of the inhibitory receptor PD-1 and improved proliferative responses in the presence of PD-1 blocking antibodies.

The relationship between viral replication and lymphocyte function has been explored in rhesus macaques. CMV infection is observed in juvenile and adult monkeys but not in newborns. Excretion in urine and saliva is significantly more frequent and intense in juvenile monkeys than adults. As in primary infection in human, CMV-specific CD4+ T lymphocytes are less polyfunctional and have lower proliferative capacities in juveniles as compared to adults. This is associated with an increased expression of PD-1 in juvenile monkeys. CD4+ T cell proliferative responses are increased when PD-1 blocking antibodies or exogenous IL-2 are added to the culture medium. Finally, an inverse association between lymphocyte function and urinary excretion has been observed in adult macaques.

These results indicate that CMV infection shares common features in human and rhesus macaque. Primary infection is associated to the detection of CD4+ T lymphocyte displaying lower functional capacities as compared to chronic infection. Exhaustion contributes to the functional impairment and the inhibitory receptor PD-1 could be targeted by immunomodulatory strategies aiming at improving lymphocyte functions and controlling viral replication. Natural CMV infection in rhesus macaque might be useful as a model to evaluate the efficacy and safety of immunomodulatory approaches.
Doctorat en Sciences médicales
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Les virus font partie des entités les plus abondantes sur Terre, mais la diversité des virus est très peu connue puisque biaisée en faveur d’hôtes animaux d’intérêts sociétal, agronomique et économique. L’apport des nouvelles techniques de séquençage permet actuellement d’obtenir des informations qui étaient tout simplement inaccessibles. Le but de mon travail de thèse a été l’étude de la diversité virale présente au sein d’un grand nombre d’animaux non-modèles. Pour répondre à cette problématique il m’a fallu mettre en place une méthodologie analytique innovante de découverte de nouveaux virus par une approche de méta-transcriptomique. Ce travail i) montre que la méthodologie bioinformatique mise en place est pertinente, ii) permet de découvrir de nouveaux virus ayant des caractéristiques génomiques particulières relevant de nouveaux genres ou familles de virus, iii) révèle de nouveaux hôtes pour des virus appartenant à des familles virales très étudiées et iv) montre que la gamme d’hôte de virus connus peut être plus étendue qu’attendu grâce à un focus sur la diversité des virus d’hyménoptères. D’une manière plus globale, mon travail permet de combler quelques lacunes existantes dans les connaissances liées à l’étude de la diversité virale et met en évidence l’importance de l’étude des animaux non-modèles
Viruses are among the most abundant entities on Earth, but the viral diversity remains mostly unknown as currently biased in favour of animals of social, agronomic and economic interest. Next Generation Sequencing technologies provide access to so far inaccessible information. The aim of my PhD thesis was the study of the viral diversity within a large range of non-model animals. To address this question I set up an innovative analytical framework to discover new viruses based on a meta-transcriptomic approach. This work i) shows that this bioinformatics method is efficient and powerful, ii) allows the discovery of new viruses with particular genomic organisations suggesting they belong to new virus genera of families, iii) uncovered new viruses from new hosts in well-known viral families and iv) shows wider viral host range than previously expected based on a particular focus on hymenopteran viral diversity. Overall, my work allows to fill some gaps in the knowledge of viral diversity and shows the importance of studying non-model animal species in virology

La cuantificación de la transmisión del Virus del síndrome reproductivo y respiratorio porcino (VSRRP) es crucial para estimar el potencial de la vacunación como medio de control de la infección a escala poblacional. El objetivo de la presente tesis fue determinar la tasa de reproducción (R) del VSRRP en condiciones de campo y experimentalmente, usando como modelo cerdos destetados y de engorde. El primer estudio tuvo como objetivo estimar de forma preliminar de la R del VSRRP en dos granjas de ciclo cerrado (F1 y F2) infectadas de forma endémica. En ambos casos, un lote entero de lechones fue monitoreado desde destete hasta matadero. Basándose en los resultados serológicos, el tiempo necesario para alcanzar el 50% de animales infectados fue de 7 y 3,5 semanas en la F1 y F2, respectivamente. El valor de R para la transmisión del VSRRP fue estimado en 3,53 (CI95%: 2,89-4.18) y 7,11 (CI95%: 3,55-10,68) para F1 y F2, respectivamente. El segundo estudio pretendía determinar la eficacia de la vacunación en un modelo experimental de transmisión por contacto en grupos. Lechones de 3 semanas de edad (n=98) fueron divididos en dos grupos: 1) cerdos V, vacunados con una vacuna comercial atenuada del genotipo 1, y 2) cerdos NV, sin tratamiento. Cinco semanas más tarde, 14 NV (a partir de ahora animales “semillas” (S)) fueron inoculados por vía intranasal (IN) con la cepa 3267 del VSRRP (genotipo 1). Dos días después, un S fue introducido en corrales de 5 V o 5 NV (8 grupos de 1S:5V y 6 grupos de 1S:5NV). Tras 21 días de contacto con el S, todos los NV desarrollaron viremia mientras que solo el 52.5% de V se infectó. El tiempo medio de supervivencia fue de 7 y 21 días para los NV y V, respectivamente (p<0,05). El valor de R fue 2,78 (CI95%: 2,13-3,43) y 0,53 (CI95%: 0,19-0,76) para los NV y V, respectivamente (p<0,05). Además, la vacunación redujo significativamente la duración de la viremia y del periodo de excreción del virus por las vías nasal y fecal. En el tercer estudio, la transmisión del virus y la eficacia vacunal fueron evaluadas en modelo experimental de transmisión por contacto de tipo uno a uno. Como en el estudio anterior, 44 lechones de 3 semanas de vida fueron divididos en dos grupos: V y NV. Cuatro semanas más tarde, 18 NV fueron separados e inoculados IN con el mismo aislado usado en el estudio 2 (animales S). Dos días después, cada S fue reagrupado con un V o un NV formando 12 réplicas de 1S:1V y seis de 1S:1NV. Los animales se siguieron por 21 días. Los V infectados desde el S (Vinf) venían trasladados (en menos de 24 horas) a un nuevo corral donde venían dejados en contacto con un nuevo V no infectado y reservado desde el inicio (Vc ). Todos los NV y V se infectaron tras contacto con el S pero la duración de la viremia se redujo desde 12,5±2,7 días en los NV a 5,5±4,3 días en los V (p<0,005). Además, el tiempo necesario para infectarse fue de 13.6±3 y 5,4±2,7 días en los NV y V, respectivamente. La transmisión desde Vinf a Vc tuvo lugar en 7/8 animales (87.5%), pero la duración de la viremia y el área bajo la curva (AUC) de la carga vírica en suero se redujeron significativamente en los Vc (AUC: 0.98, 0.87, 0.79 para NV, Vinf y Vc, respectivamente, p<0.05). In conclusión, los resultados de la presente tesis indican que para el VSRRP de genotipo 1 y subtipo 1, la vacunación masiva de los animales puede ser una herramienta efectiva para parar o disminuir significativamente la transmisión.
The quantification of Porcine reproductive and respiratory syndrome virus (PRRSV) transmission is crucial to foresee the potential of vaccination as a mean of control of the infection at a population level. The aim of the present thesis was to determine the reproduction rate (R) of PRRSV under field and experimental conditions using weaners/growers as a model. Study 1 was directed to preliminary assess R of PRRSV in two endemically infected farrow-to-finish farms (F1 and F2). In both cases, a whole batch of weaners was followed serologically from weaning to slaughtering age. Based on serological data, the average time needed to reach 50% of infected pigs was 7 and 3.5 weeks in F1 and F2, respectively. R value for PRRSV transmission was estimated to be 3.53 (CI95%: 2.89-4.18) and 7.11 (CI95%: 3.55-10.68) for F1 and F2, respectively. The second study was directed to assess the efficacy of the vaccination in an experimental study of transmission by contact in groups. Ninety-eight 3-week-old piglets were divided in two groups: 1) V pigs, vaccinated with a commercial attenuated PRRSV genotype 1 vaccine, and 2) NV piglets, kept as unvaccinated controls. Five weeks later, 14 NV pigs (from now on seeders (S)) were inoculated intranasally (IN) with PRRSV genotype 1 isolate 3267. Two days later, one S was mingled with 5 V or 5 NV pigs (8 groups of 1S:5V and 6 replicas of 1S:5NV). After 21 days of contact with the S, all NV developed viremia while only 52.5% of V pigs were become viremic. The average 50% survival period was 7 and 21 days for NV and V pigs, respectively (p<0.05). The R value was 2.78 (CI95%: 2.13-3.43) and 0.53 (CI95%: 0.19-0.76) for NV and for V pigs, respectively (p<0.05). Moreover, vaccination significantly reduced the biological parameters related to transmission, such as the duration of viremia and shedding of virus by the nasal and fecal routes. In the third study, the transmission of PRRSV and vaccine efficacy were assessed using one-to-one experiments, a scenario where the probability of transmission between animals was the highest possible. Forty-four 3-week-old piglets were divided in 2 groups as above: V and NV. Four weeks later, 18 NV were inoculated IN with the same isolate as in study 2 (S pigs). Two days later, S pigs were mixed in a 1:1 basis with V and NV pigs, namely 1S:1V (n=12) and 1S:1NV (n=6) and were then followed for 21 days. Once a V pig was detected as viremic as a result of the contact with a S, the infected V (Vinf) was transferred (in less than 24 h) to a new pen where it was left in contact with a new V (from now Vc) for at least 14 days. All NV and V pigs became viremic after contacting S pigs; however, the average duration of viremia was reduced from 12.5±2.7 to 5.5±4.3 days comparing V pigs to NV (p<0.05). Also, V animals needed on average 13.6±3 days in order to become infected from the S compared to NV (5.4±2.7 days, p< 0.05). Transmission from Vinf to Vc pigs occurred in 7/8 animals (87.5%) but the mean length of viremia as well as the area under the curve (AUC) for viral load in sera of Vc pigs were also significantly reduced (AUC: 0.98, 0.87 and 0.79 for NV, Vinf and Vc, respectively, p<0.05). In conclusion, the data shown in the present thesis indicated that for genotype 1 subtype 1 PRRSV, mass vaccination of pigs can be a tool effective to stop or to significantly reduce the transmission of the virus.

Porcine Reproductive and Respiratory Syndrome virus (PRRSV) causes the disease - Porcine Reproductive and Respiratory Syndrome (PRRS) which is one of the most economically important diseases for pig farmers. Since it was discovered in the United States and Europe, it has quickly affected the swine industry all over the world. Studying and controlling PRRSV has become an important issue in swine industry and scientific community, and has raised the concerns of governments like US and China. By using different bioinformatics and phylogenetics tools, scientists could understand the epidemiology and evolution of PRRSV from genomic data. However, a well-designed database for PRRSV sequence and relevant meta-information are generally required for the tools to produce insightful results. Therefore, I would like to introduce an easily accessible web platform for PRRSV analysis - PRRSV-Webtool. The core component of PRRSV-Webtool is phylogenetic reconstruction. Instead of using traditional phylogenetic reconstruction, a new method of reconstruction was introduced - Reconstruction by Addition of Taxon (RAT). RAT could build a phylogenetic tree from known existing phylogeny. Simulation tests were performed to evaluate the accuracies of RAT using PRRSV dataset. The percentages of correct branch reconstruction are 73.81% for type 1 PRRSV dataset and 80.68% for type 2 PRRSV dataset. Another important function of PRRSV-Webtool is genotyping. RAT could correctly identify the genotype of all sequences in the testing datasets. PRRSV-Webtool combined three main components: database, phylogenetic tool and World Wide Web. By using PRRSV-Webtool, the users can study their own PRRSV genome data easily via the web browser. Tools in PRRSV-Webtool can allow users to know more about their PRRSV isolates related to other field samples. With our PRRSV-Webtool, scientists and veterinaries can help to improve their understanding of PRRSV and help to control the virus by accelerating the process of virus surveillance and field sampling.
published_or_final_version
Biological Sciences
Master
Master of Philosophy

Les Archaea ont été identifiées en 1978 par Carl Woese, séparant le domaine du vivant en trois domaines distinct. Ces organismes ont principalement été isolés à partir d'environnements extrêmes (haute température, haute salinité. . . ). A ce jour, une cinquantaine de virus d'archaea ont été identifiés, et ils présentent une grande variété de morphotypes. Les Fusellovirus, qui infectent les archaea thermoacidophile du genre Sulfolobus, sont parmi les plus étudiés. Les génomes de neuf Fusellovirus ont été séquencés jusqu'à présent, et treize CDS sont communes à l'ensemble de ces virus. Il a été prédit sur la base d'analyses in silico que l'une de ces treize CDS, B251, code un initiateur de réplication de type bactérien. Nous avons montré que B251 possède les caractéristiques d'un initiateur de réplication de type bactérien. Nous avons également identifié une protéine pouvant être impliquée dans l'initiation de la réplication des Fusellovirus. B129 est une protéine à doigt de zinc qui se fixe à la fois sur l'origine de réplication et à proximité du site de recombinaison attP, propriété similaire à celles d'IHF d'E. Coli. En parallèle de ces analyses in vitro, nous avons étudié in silico l'organisation des génomes des Fusellovirus afin de localiser leur origine de réplication. Ces analyses nous ont permis de séparer les Fusellovirus en deux groupes se répliquant potentiellement selon des mécanismes différents. L'ensemble des résultats obtenus participe à une meilleure compréhension du cycle viral des Fusellovirus
Ln 1978, Carl Woese identified a third domain of life, the Archaea. These organisms were first identified in extreme environmental conditions, such as high temperature and high salt concentration. There are about fifty archaeal viruses currently identified, and they have a large variety of morphotypes. Among these viruses, the Fuselloviruses are the best characterized. They infect members of the Sulfolobus genus, isolated from sulfurous hot springs. There are currently nine Fuselloviruses genomes sequenced, with thirteen CDS common to all these viruses. Ln silico analysis predicted that one of these thirteen CDS, B251, could encode a bacterial-like replication initiator. We found that B251 have the characteristics of a bacterial-like replication initiator. We also identified a protein, which could be involved in replication initiation. B 129 is a zinc finger protein wich binds both to the origin of replication and close to the attP site, reminiscent of IHF in E. Coli. We performed in silico analyses of the nine Fuselloviruses genomes to identify their replication origin. Using GC skew analyses, we found that the Fuselloviruses could be divided in two subgroups, each of which replicate their DNA in a different way. All these results help to understand how the Fuselloviruses replicate their DNA and will provide a better understanding of their life cycle

Etude de la regulation negative liee a l'activateur b des polyomavirus. Cette regulation a ete etudiee in vitro avec des lignees lymphocytaires t etablies en culture. Le tropisme du virus dans le systeme lymphocytaire t pourrait etre correle a l'expression de l'antigene thy-1. 2. D'autre part, le tropisme d'infection du virus a ete suivi in vitro chez des souris athymiques ou non

La protéine EED (Embryonic Ectoderm Development) appartient à la famille des Polycomb Group agissant comme répresseurs épigénétiques. EED interagit avec la matrice du VIH (MA) et Nef. Nous avons mis en évidence qu'EED se lie aussi à l'intégrase (IN), et forme un complexe ternaire EED-MAIN. Ce ‘ménage-à-trois' est retrouvé aux pores nucléaires (NPC) et dans le noyau des cellules MT4 infectées par le VIH entre 1. 5 et 6h après l'infection. A la phase tardive du cycle viral, EED provoque un effet negatif important sur la production de VIH (~ 2 log). Nef restaure la production virale, et joue le rôle d'antagoniste d'EED. EED et Nef se relocalisent dans une fraction cellulaire insoluble, différente des microdomaines membranaires (ou ‘lipid rafts'). L'imagerie cellulaire révèle qu'EED induit la localisation aberrante de NPC dans le cytoplasme des cellules 293T. Ces NPC ectopiques pourraient séquestrer ou/et perturber le trafic cellulaire des génomes viraux, et donc l'assemblage des virions
Human protein EED (Embryonic Ectoderm Development) belongs to the Polycomb Group family, which act as gene silencers. EED interacts with the matrix protein of HIV-1 (MA) and Nef. We found that EED also binds to integrase (IN), and forms a ternary complex with MA and IN. This ‘menage-a-trois’ EEDIN- MA was found at the nuclar pore complexes (NPC) and in the nucleus of HIV-infected MT4 cells at early times post-infection (1. 5-6h pi). Overexpression of EED in transfected 293T cells resulted in a strong negative effect on HIV-1 yields (~ 2 log) at late times pi. This late negative effect was antagonized by Nef (and its G2A mutant), and was associated with a relocation of EED and Nef to a non-raft, pelletable cellular fraction. Cellular imaging showed that EED induced an aberrant location of clusters of NPC in the cytoplasm of 293T cells. These ectopic NPC might sequester the viral genomic RNA (gRNA), provoke a mistrafficking of gRNA, and result in a lower efficiency of virion assembly

L'étude du cycle du virus de l'hépatite C ( VHC) et la découverte de nouveaux traitements antiviraux ont été entravés par l'absence de systèmes cellulaires permettant la multiplication efficace du virus. Ce constat a motivé la première partie de nos travaux. Nous avons évalué l'adsorption et la multiplication du VHC dans cinq lignées cellulaires. Les cellules Vero (rein de singe) et AP61 (moustique) ont permis la détection de l'ARN viral pendant 28 jours et la réalisation de 4 passages viraux successifs. Le système cellulaire Vero , associé à une technique originale de quantification des ARN viraux par RT-PCR en temps réel a permis l'étude de l'adsorption de CHC. Ce travail a révélé le rôle des glycosaminoglycanes cellulaires dans l'adsorption du VHC mais aussi de deux autres Flaviviridae, les virus de la dengue et de la fièvre jaune utilisés, en parallèle, comme modèle et étudiés à l'aide d'une techique de titrage viral en culture de cellules. Le rôle important du récepteur des lipoproéines de faible densité (LDL) dans l'adsorption du VHC a été confirmé (inhibition de la fixation virale par les LDL et des anticorps anti-recepteurs des LDL). Les techniques développées dans cette étude pourraient être utilisées pour le criblage de molécules inhibant l'adsorption cellulaire des Flaviviridae
The study of Hepatitis C virus ( HCV) cycle and the discovery of new therapy have been hampered by the lack efficient virus culture systems. We assessed adsorption and replication of HCV on five cell lines. Monkey Vero cells and mosquito AP61 cells were selected for their ability to blind and replicate HCV and to support 4 virus passages. HCV adsorption was studies using Vero cells and quantification of virus RNA by a real time RT-PCR method. This work showed that cellular glycosaminoglycans were involved in HCV adsorption. These molecules were shown to have an important role in the binding of tow other Flaviviridea , Dengue virus and Yellow fever virus which were studies in parralel, as models, by virus titration in cell culture. The important role of low density lipoprotein (LDL) receptor in HCV adsporption was confirmed ( the viral binding was inhibited by LDL and anti-LDL receptor antibody). The methods descripted in this study might be screening of molecules inhibiting Flaviviridae cell-adsorption

La réplication des éléments extrachromosomales des Crenarchaeota est un sujet qui reste inconnu. Plus de 96% des ORFs virales n’ont pas des homologues dans des basses des données publiques et donc leurs fonctions ne peuvent pas être prédites par similarité de séquences. L’objectif de cet étude a été de comprendre les mécanismes de réplication des éléments extrachromosomales chez les Crenarchaea dans l’exemple du génome linaire du virus filamenteux d’Acidianus 1 (AFV1) qu’infecte Acidianus hospitalis, et celle du plasmide pRN1 de Sulfolobus islandicus. En utilisant l’analyse in silico de la séquence génétique, l’électrophorèse en deux dimensions des gel d’agarose (2DAGE) et la microscopie électronique en champ noir on postule que le mécanisme de réplication du AFV1 c’est un mécanisme dépendante de la recombinassions. On a trouvé une protéine terminal attache aux extrémités 5’ du génome viral. La formation des structures tertiaires sont favorises par l’interaction de cette protéine avec des régions internes du génome. La technique de 2DAGE a été utilisée pour étudier les caractéristiques des intermédiaires de réplication du plasmide pRN1. La présence des intermédiaires du même taille, mais différent structure secondaire support l’hypothèse d’un boucle stem à l’intérieur du réplicon minimal du plasmide. L’origine de réplication par 2DAGE se trouve au même site. La caractérisation biochimique de la protéine AFV1-ORF157 est incluse aussi dans cet manuscrit. Une activité de nucléase en ADN double brin est prouvée d’être présent dans l’ORF-157. Cette propriété a inspiré l’hypothèse que cette protéine peut être implique dans la maturation du génome avant son encapsidation
Replication of extrachromosomal elements of the Crenarchaeota remains to be poorly understood. More than 96% of viral ORFs do not have homologs in public databases, and thus their functions cannot be predicted based on sequence similarity. The aim of the present study was to understand the mechanisms of replication of extrachromosomal elements in Crenarchaeota in the frame of the genome of the Acidianus filamentous virus 1 (AFV1) of Acidianus hospitalis, and of the plasmid pRN1 of Sulfolobus islandicus. By applying in silico genome sequence analysis, two dimensional agarose gel electrophoresis (2DAGE) and dark field electron microscopy we postulated that the replication mechanism exploited by AFV1 is recombination-dependent replication. A terminal protein was found to be strongly attached to the 5’- ends of the encapsidated viral genome. Tertiary structures were promoted by the interaction of this protein with internal regions of the genome. Characterization of the replicative intermediates of pRN1 was performed using 2DAGE. The existence of intermediates of the same size, but different secondary structure supported the hypothesis of a stem-loop that was experimentally found to be inside the minimal replicon of the plasmid. A replication origin was found in the 2DAGE at this same site. The biochemical characterization of AFV1-ORF157 is also included in this dissertation. A nuclease activity on linear double-stranded DNA was shown to be present in ORF-157, giving rise to the hypothesis that this protein might be involved in edition of mature genomes for encapsidation

Porcine reproductive and respiratory syndrome virus (PRRSV) is a rapidly evolving virus that has significant economic and welfare implications for the pig industry. Vaccination strategies have proved largely ineffective in controlling PRRSV, in some cases even reverting to virulence. An increasing body of evidence suggests a host genetic basis for PRRSV resistance so there is a need to examine the role of host genetics in a biologically relevant in vitro cell culture system. However, PRRSV research is inhibited by the current scarcity of suitable in vitro culture systems. With the aim of developing a convenient in vitro model, porcine bone marrow-derived macrophages (BMDM) were evaluated as a PRRSV cell culture system. BMDM were found to be highly permissive to Type I PRRSV and amenable to genetic manipulation. BMDM proved to be excellent cells for virus production, producing significantly higher titres of PRRSV than commonly used alternative cell types. Surprisingly, PRRSV entry into BMDM was found to be independent of both the prototypic PRRSV receptors, CD163 and CD169, providing further evidence for the existence of alternate PRRSV entry mechanisms in primary cell types. To explore the genetics of pig susceptibility to PRRSV, network-based analysis of host transcriptional datasets, following PRRSV challenge, revealed important differences in co-regulated gene pathways between samples from pigs with different PRRSV-permissiveness. These pathways included genes with important, recently characterised, anti-pathogen activities. The incorporation of network-based transcriptional analysis and published genetic variation data led to the identification of a member of the guanlyate binding protein family, GBP-1, as a candidate host gene involved in controlling PRRSV replication. Overexpression of GBP-1 in BMDM revealed a significant anti-PRRSV function for this protein. Further investigation of published genetic variation in GBP-1 suggested a potential role of this gene in PRRSV tolerance. The results presented in this thesis provide evidence for an alternate PRRSV entry pathway in a biologically relevant cell type. The discovery of a highly PRRSV-infectable cell type with potential for genetic manipulation adds a useful new tool to the area of PRRSV research. The identification of GBP-1 as a novel anti-viral protein with a significant inhibitory effect on PRRSV infection, together with genetic variation in this gene, prompts further research into the genetic basis for PRRSV resistance.

Introduction International guidelines endorse the screening of patients for human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and Chlamydia trachomatis before assisted reproductive techniques (ART). At present no such guidelines exists in South Africa. At the Reproductive and Endocrine Unit (referred to as “the Unit”) of Steve Biko Academic Hospital, all patients with unknown HIV status are counselled and a blood sample is collected during the initial visit for automated laboratory based HIV screening. These HIV results are not available before semen samples are processed. Furthermore, patients are not screened for HBV, HCV and Chlamydia trachomatis. Couples attending the Unit are of a low to middle socio-economic status and experience financial constraints. Moreover, automated laboratory based assays are expensive to perform. Rapid testing is a cost effective and practical method from screening patients, with a 20–30 minute result turnover time. Until screening at the Unit is improved, the possible identification of semen characteristics that could indicate HIV infection would be a useful tool. Materials and Methods The following rapid point-of-care assays were evaluated: Determine® HIV-1/2 combo test (n=100), Determine® HBsAg test (n=100), DIAQUICK HCV kit (n=74), and the DIAQUICK Chlamydia trachomatis kit (n=30). For profiling, parameters from a basic semen analysis of HIV-positive males (n=60) were compared with HIV-negative males (n=60). Information pertaining to CD4 count, antiretroviral treatment and plasma viral load of HIV-positive males were analysed. Results From all patients included in the study, 8% tested positive for HIV. The risk of a female being HIV-positive was 3.73 times higher than for males. In the pilot study to explore rapid testing for HBV and HCV, 1% and 1.4% of patients tested positive respectively. When testing for Chlamydia trachomatis 31.3% of females, but no males tested positive. Comparing semen profiles, no significant differences were found between samples from HIV positive and negative males or between HIV positive males categorised by CD4 cell count (p>0.05). For the HIV-positive group with a detectable plasma HIV viral load (>40 copies/ml), a significant difference was observed in the semen viscosity (p=0.0460). Significant differences were noted in the sperm motility (immotile sperm p=0.0456, progressive sperm p=0.0192) of patients receiving antiretroviral (ARV) therapy. Discussion and Conclusion The use of rapid testing is an acceptable and feasible option for improving current screening protocols at the Unit. The absence of definite alterations in the semen characteristics of HIV-positive men further motivates the need for a simpler, point-of-care screening protocol. The prevalence of HBV was lower than that reported in the general population of South Africa and further investigation is needed. Although the sample size was small, HCV prevalence was similar to that of the general population. One third of females tested positive for Chlamydia trachomatis. The methodology used was possibly not appropriate for males. This study highlighted the need for guidelines that address the specialised needs of ART clinics in resource-limited and developing countries with a high HIV prevalence.
Dissertation (MSc)--University of Pretoria, 2013.
gm2014
Obstetrics and Gynaecology
unrestricted

Le virus de l'hépatite C (VHC) affecte 170 millions de personnes dans le monde. Quatre-vingt pour cent des individus atteints vont développer une hépatite chronique voire une cirrhose puis un cancer du foie pour certains d'entre eux. Il n'existe actuellement qu'un seul traitement contre le virus, mais il n'est efficace que pour la moitié des patients. Notre projet consiste à mieux comprendre les mécanismes de réplication du virus dans le but de développer de nouveaux inhibiteurs. Au cours de ma thèse j'ai participé au développement d'un nouveau modèle de la réplication du VHC. Ce modèle cellulaire permet une étude dissociée des deux principales activités virales, ce qui est actuellement impossible avec les autres modèles utilisés. Pour cela, les protéines virales nécessaires à la réplication sont constamment produites dans des cellules. Elles s'associent en un complexe qui va répliquer une molécule d'acide nucléique contenant un gène rapporteur encadré par les séquences de reconnaissance virale. Cette molécule perpétuellement dupliquée à chaque cycle, révèle un mécanisme de réplication proche de celui utilisé par le virus. Ce modèle de la réplication du VHC a déjà permis de mettre en évidence une diminution de l'activité réplicative chez les virus de génotype 3. Sept variations nucléotidiques situées dans une région du génome viral impliquée dans la traduction et la réplication du virus en sont responsables. Cette spécificité de séquence pourrait ainsi expliquer la meilleure réponse au traitement par l'interféron observée chez les patients infectés par les virus de génotype 3. D'autre part, nous avons montré que les molécules répliquées dans ce système sont dépourvues d'une structure réputée indispensable à la synthèse du génome viral. Cette structure appelée 5BSL3. 2 est néanmoins indispensable à la prolifération du virus. La deuxième partie de mon projet de thèse a donc consisté à caractériser la 5BSL3. 2 et plus particulièrement à analyser son rôle dans la traduction des protéines virales
The hepatitis C virus (HCV) affects around 170 million people worldwide and 3-4 million persons are infected each year. This infection will lead to death in 5-7 % of patients infected with HCV as a consequence of liver disease. The virus was first identified by Choo et al. (1989) but until recently development of new treatment for this infection has been hampered by the lack of an efficient cellular system. We established a new model to study HCV replication. In this system, the genes coding for the HCV non structural proteins are introduced in Huh7 cells (human hepatoma cell line) in order to constitutively express the HCV complex. Its activity is analysed by transfection of non-coding RNA (RNA minigenome) in the modified Huh7 cells. Those RNAs include EGFP and hygromycine genes surrounded by 5'UTR HCV non coding sequences. Those regions are included in order to be recognised by the HCV complex. The actvity of the HCV replication complex was determined by flow cytometry. Only cells able to support RNA minigenome replication could express the EGFP gene. RNA minigenome replication was detected in cells and could be maintained under hygromycine selection. I used this model to analyze the differences in replication activities between HCV genotype 1 and 3. We have identified 7 non contiguous nucleotides specific of genotype 3 in the 5'UTR, and those nucleotides are only present in this genotype, I showed these changes could be responsible for the reduced efficiency of RNA replication in the genotype 3. I also used this model to study the role of a cis-acting replication element, previously shown to be critical for the virus' replication. We have shown that this sequence is not required for the replication of our minigenome. I designed experiments to understand and explain the differences observed

Orientadores: Eliana Amaral, Maria Yolanda Makuch
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Objetivo: Identificar quais Serviços de Reprodução Assistida (SRA) e Serviços de Assistência Especializada em HIV e Aids (SAE) do Sistema de Saúde Pública do Brasil, que oferecem atendimento a pessoas vivendo com HIV com desejo reprodutivo e descrever as vivências, informações adquiridas e barreiras encontradas pelos gestores de programas, profissionais de saúde e usuários, relacionados a essa demanda. Metodologia: Estudo descritivo, de corte transversal através de entrevistas telefônicas com 69 gestores dos programas de saúde da mulher (PSM) e 69 de DST/Aids, estaduais e municipais associado a estudo de casos através de entrevistas semi-estruturadas com profissionais e usuários soropositivos de um serviço de reprodução assistida (SRA) e um serviço de atenção especializada em HIV/Aids (SAE) por região geográfica do país. Foi realizada análise descritiva dos dados quantitativos e análise temática do conteúdo para os dados qualitativos. Resultados: Foram realizadas 64 entrevistas com gestores dos PSM, sendo identificado apenas um SRA universitário que atendia casais soropositivos. Nas 63 entrevistas realizadas com gestores dos Programas de DST/Aids, constatou-se que 64% dos SAE estaduais e 73% dos municipais ofereciam orientação reprodutiva. As dificuldades relatadas pelos gestores para não oferecimento de apoio à reprodução incluíram falta de decisão política, de recursos humanos e financeiros. Nas entrevistas com os profissionais dos seis SAE visitados, foi observado que o foco dos atendimentos era na prevenção, principalmente através do uso do preservativo. A falta de encaminhamentos apropriados e a desatualização do conhecimento científico foram frequentes nos relatos dos profissionais dos serviços. A dificuldade em falar sobre o desejo reprodutivo nas consultas foi observada nas falas dos profissionais e também dos usuários. Para os últimos, isso esteve associado ao medo da discriminação e do preconceito. Entretanto, através da 47 entrevistas realizadas com usuários, o desejo de ter filhos foi vivenciado de maneira natural e expresso independentemente de se ter ou não parceiro fixo, mas, para aqueles que possuem parceiros fixos, o fato de não ter filhos da atual união pareceu aumentar a intenção reprodutiva. Conclusão: Os resultados sugerem a existência de demanda reprimida para reprodução de casais vivendo com HIV, a falta de aconselhamento reprodutivo nos SAE e de investimento em SRA que atenda a essa população, havendo um único SRA universitário no país que oferece esse tipo de atendimento. A falta de integração entre os vários setores sugere a ausência de políticas públicas voltadas para o aconselhamento reprodutivo e a necessidade de diretrizes nacionais específicas voltadas para a redução da transmissão do HIV durante todo o contexto reprodutivo
Abstract: Objective: To identify assisted reproductive services(ARS) and specialized HIV/AIDS services within the Brazilian public health system that provide care to people living with HIV who desire a child and describe the experience, the information, and the barriers encountered by program managers, healthcare professionals and users with respect to this demand. Methods: A descriptive, cross-sectional study in which 69 women's healthcare program managers and 69 STD/AIDS program managers at both state and municipal level were interviewed by telephone, in association with a case study conducted through semistructured interviews with professionals and users of one ARS service and one HIV/AIDS service in each geographical region of the country. Descriptive analysis of the quantitative data and thematic content analysis of the qualitative data were performed. Results: Sixty-four interviews were conducted with women's healthcare program managers. Only one university ARS provided care to seropositive couples. Of the 63 interviews carried out with STD/AIDS program managers, 64% of the state and 73% of the municipal HIV/AIDS services were found to offer reproductive counseling. The difficulties offered by managers as reasons for not providing reproductive support included a lack of political decision and of human and financial resources. At the six HIV/AIDS services the professionals revealed that the focus of consultations was on the prevention, lack of appropriate referrals and outdated scientific knowledge were frequently reported. Difficulty in discussing reproductive issues was perceived in the interviews with the professionals and also with the users. In the latter case, this was associated with a fear of discrimination and prejudice. Nevertheless, as shown in the 47 interviews conducted with users, the desire to have a child was experienced as a natural part of life and was expressed irrespective of whether the individual had a steady partner or not; however, in the former case, the fact of not having a child with the individual's current partner appeared to increase the desire for a child. Conclusion: These findings suggest the existence of a repressed demand for reproduction of PLWHA and lack of reproductive counseling was observed at all HIV/Aids specialized services, as well as investment in ART services to be provided to HIV-positive couples, based on the finding that only one university ART service in the country offers this type of care. Lack of integration between the various sectors suggests an absence of public policies on reproductive counseling and a need for specific national guidelines aimed at reducing HIV transmission within the whole context of reproduction
Doutorado
Ciencias Biomedicas
Doutor em Tocoginecologia

Porcine reproductive and respiratory syndrome (PRRS) is the most significant infectious disease currently affecting the swine industry worldwide. Several inactivated and modified live vaccines (MLV) have been developed to curb PRRSV infections. The unsatisfactory efficacy and safety of these vaccines, drives for the development of new generation PRRS universal vaccines. Virus like particles (VLPs) based vaccines are gaining increasing acceptance compared to subunit vaccines, as they present the antigens in more veritable conformation and are even readily recognized by the immune system. Hepatitis B virus (HBV) core antigen (HBcAg) is very well studied and has been successfully used as a carrier for more than 100 other viral sequences. In this study, hybrid HBcAg VLPs are generated by fusion of the conserved protective epitopes of PRRSV and expressed in E. coli. An optimized purification protocol that overcomes issues from ultracentrifugation is developed to obtain hybrid HBcAg VLP protein from the inclusion bodies. This hybrid HBcAg VLP protein self assembled to 23nm VLPs that were shown to block virus infection of susceptible cells when tested on MARC 145 cells. Therefore, the safety of non-infectious and non-replicable VLPs and production through low-cost E. coli fermentation may make this vaccine competitive against current vaccines on both efficacy and cost.
Master of Science

L'infection de l'homme par le virus de l'hepatite b (vhb) est un probleme de sante mondial. Plus de 300 millions d'individus sont actuellement porteurs chroniques de ce virus. Cette chronicite augmente de 100 fois leur risque de developper un carcinome hepatocellulaire. Si il existe des vaccins pour lutter de maniere preventive contre la propagation de ce virus, les traitements proposes aux porteurs chroniques n'ont qu'une efficacite limitee. La recherche de nouvelles molecules antivirales ainsi que de nouvelles approches therapeutiques reste donc d'actualite. Ces recherches sont facilitees par les differents modeles animaux du vhb (dhbv ou whv). Ainsi l'objectif de notre travail a ete de poursuivre la mise au point de modeles de culture in vitro du dhbv et du whv. Ces modeles de cultures primaires d'hepatocytes ont ensuite ete utilises pour le criblage in vitro de nouvelles molecules antivirales. Parmi ces molecules, des bloqueurs du cycle cellulaire ont montre un impact important sur la replication du dhbv. La place de ce resultat dans la comprehension des relations entre les hepadnavirus et le cycle cellulaire est discutee

Le Cytomégalovirus humain (CMVH) et l'Herpèsvirus humain de type 6 (HHV6) sont deux ?- Herpesvirinae présentant un tropisme majeur pour le système hématopoi͏̈étique. Ce travail a visé à évaluer leurs capacités de réplication au cours de la différenciation hématopoi͏̈étique. Des outils méthodologiques autorisant la mise en évidence de la réplication virale ont été développés, permettant la détection de transcrits viraux tardifs par RT-PCR. Parmi les marqueurs développés pour le CMVH, l'ARNm tardif UL22, issu d'un épissage, a montré son intérêt, en particulier dans le cadre du diagnostic de l'infection active/maladie à CMVH. Pour l'HHV6, la détection du transcrit tardif multi-épissé de la gp 82-105 (U100) et la mise en évidence en cytométrie de flux d'antigènes nucléaires tardifs ont été développés à partir de cultures de souches virales de variant B. Ces essais ont permis de préciser que la forme épissée du gène U100 est exprimée en phase tardive du cycle de réplication, alors que la forme non épissée est un marqueur de la phase précoce. La possibilité de survenue d'une réplication des deux virus au cours de l'expansion ex vivo de 10 prélèvements de cellules souches hématopoi͏̈étiques CD34+ périphériques, a ensuite été envisagée. Ces essais ont été réalisés dans le cadre d'une nouvelle approche thérapeutique, qui vise à réduire la durée d'aplasie, chez des sujets traités par hautes doses de chimiothérapie, grâce à l'injection de cellules souches différenciées in vitro. Les cultures ont été menées en milieu liquide, en présence de cytokines permettant d'engager la différenciation vers les lignées myéloi͏̈des et monocytaires. Parmi les quatre cultures de cellules provenant de patients séropositifs pour le CMVH, aucun marqueur de réactivation virale n'a été détecté. A l'inverse, dans 5/10 cultures issues de prélèvements effectués chez les patients séropositifs pour l'HHV6, le transcrit U100 a été amplifié. Ces données montrent pour la première fois et sans avoir recours à une infection préalable par une souche virale, une réplication de l'HHV6 due à une réactivation au cours de la différenciation des progéniteurs hématopoi͏̈étiques. Ces résultats originaux soulèvent le problème de la sécurité virale lors des réinjections chez des patients immunodéprimés de cellules supportant une réplication de l'HHV6
Cytomegalovirus (CMV) and Human Herpesvirus 6 (HHV6) were two ? -Herpesvirus with a major tropism for hematopoietic system. Since their interactions with hematopoiesis are still poorly understood, we evaluated their replication capacity during differenciation of hematopoietic stem cells. We first developed and evaluated tools to follow viral replication, by detection of late viral transcripts, which assess occurrence of a complete replication cycle. Among the CMV transcripts amplified by reverse transcription-PCR, the late spliced UL22 mRNA was of particular interest, notably to predict the occurrence CMV disease during active infection. For HHV6, a one-step RT-PCR amplifying the late alternatively U100 gene encoding the gp 82-105 glycoprotein and a flow cytometry method to analyse nuclear late antigens were developed from reference strains cultures. Evaluation of these methods was then realized during a sequential culture of HST strain on MT4 cells. This allows to specify that the U100 gene splicing starts at a late stage of multiplication whereas unspliced forms were detectable earlier in the cycle. We then evaluated the occurrence of viral replication during ex vivo expansion of 10 peripheral hematopoietic stem cells (CD34+) samples. Cultures were maintained in a serum free liquid medium supplemented with cytokines of myeloid-monocyte lineage. Neither CMV DNA nor CMV UL22 or UL86 mRNA were detected among the 4 cultures from CMV seropositive patients. Conversely, the late alternatively spliced U100 mRNA (HHV6) was detected at day 14 or 21 in 5/10 cultures from HHV6 seropositive patients. Our data demonstrated for the first time that HHV6 could enter in a replicative cycle during hematopoietic differenciation in the absence of in vitro infection of cells. This also raised the question of the viral safety of the infusion of ex-vivo expanded CD34-positive PBPCs