Plant viruses have developed a number of strategies that enable them to become obligate intracellular parasites of many agricultural crops. Potato virus X (PVX) belongs to a group of positive-sense, single-stranded plant RNA viruses that replicate on host membranes and form elaborate structures known as viral replication complexes (VRCs) that contain viral RNA (vRNA), proteins and host cellular components. VRCs are the principal sites of viral genome replication, virion assembly and packaging of vRNA for export into neighbouring cells. For many animal viruses, host membrane association is crucial for RNA export. For plant viruses, it is not yet known how vRNA is transported to and through plant plasmodesmata. PVX encodes genetic information required for its movement between cells; three viral triple gene block (TGB) movement proteins and a viral coat protein are essential for viral trafficking. This research project studies the relationship between PVX and its host plants, Nicotiana benthamina and Nicotiana tabacum. A particular focus of this project is exploration of the structural and functional significance of the PVX VRC and how the virus recruits cell host components for its replication and movement between cells. The role of specific viral proteins in establishing the VRC, and the ways in which these interact with host organelles, was investigated. A combination of different approaches was used, including RNA-binding dyes and a Pumilio-based bimolecular fluorescence complementation assay for detection of the vRNA, fluorescent reporters for virusencoded proteins, fluorescent reporters for host organelles involved in viral replication, and also transgenic tobacco plants expressing reporters for specific plant components (endoplasmic reticulum, Golgi, actin, microtubules and plasmodesmata). In addition, mutagenesis was used to study the functions of individual viral proteins in replication and movement. All of these approaches were combined to achieve live-cell imaging of the PVX infection process. The PVX VRC was shown to be a highly compartmentalised structure; (+)-stranded vRNA was concentrated around the viral TGB1 protein, which was localised in discrete circular compartments within the VRC while coat protein was localised to the external edges of the VRC. The vRNA was closely associated with host components (endoplasmic reticulum and actin) shown to be involved in the formation of the VRC. The TGB2/TGB3 viral proteins were shown to colocalise with the host endomembranes (ER) and to exit these compartments in the form of motile granules. vRNA, TGB1, TGB2 and CP localised to plasmodesmata of the infected cells. TGB1 was shown to move cell-to-cell and recruit ER, Golgi and actin in the absence of viral infection. In the presence of virus, TGB1 targeted the VRCs in several neighbouring cells. A model of PVX replication and movement is proposed in which TGB1 functions as a key component for recruitment of host components into the VRC to enable viral replication and spread.

Insects are host to a diverse range of vertically transmitted micro-organisms, but while their bacterial symbionts are well-studied, little is known about their vertically transmitted viruses. The sigma virus (DMelSV) is currently the only natural hostspecific pathogen to be described in Drosophila melanogaster. In this thesis I have examined; the diversity and evolution of sigma viruses in Drosophila, their transmission and population dynamics, and their ability to host shift. I have described six new rhabdoviruses in five Drosophila species — D. affinis, D. obscura, D. tristis, D. immigrans and D. ananassae — and one in a member of the Muscidae, Muscina stabulans (Chapters two and four). These viruses have been tentatively named as DAffSV, DObsSV, DTriSV, DImmSV, DAnaSV and MStaSV respectively. I sequenced the complete genomes of DObsSV and DMelSV, the L gene from DAffSV and partial L gene sequences from the other viruses. Using this new sequence data I created a phylogeny of the rhabdoviruses (Chapter two). The sigma viruses form a distinct clade which is closely related to the Dimarhabdovirus supergroup, and the high levels of divergence between these viruses suggest that they may deserve to be recognised as a new genus. Furthermore, this analysis produced the most robustly supported phylogeny of the Rhabdoviridae to date, allowing me to reconstruct the major transitions that have occurred during the evolution of the family. This data suggests that the bias towards research into plants and vertebrates means that much of the diversity of rhabdoviruses has been missed, and rhabdoviruses may be common pathogens of insects. In Chapter three I examined whether the new sigma viruses in Drosophila affinis and Drosophila obscura are both vertically transmitted. As is the case for DMelSV, both males and females can transmit these viruses to their offspring. Males transmit lower viral titres through sperm than females transmit through eggs, and a lower proportion of their offspring become infected. I then examined natural populations of D. obscura in the UK; 39% of flies were infected and the viral population shows clear evidence of a recent expansion, with extremely low genetic diversity and a large excess of rare polymorphisms. Using sequence data I estimate that the virus has swept across the UK within the last ~11 years, during which time the viral population size doubled approximately every 9 months. Using simulations based on lab estimates of transmission rates, I show that the biparental mode of transmission allows the virus to invade and rapidly spread through populations, at rates consistent with those measured in the field. Therefore, as predicted by the simulations, the virus has undergone an extremely rapid and recent increase in population size. In Chapter four I investigated for the first time whether vertically transmitted viruses undergo host shifts or cospeciate with their hosts. Using a phylogenetic approach I show that sigma viruses have switched between hosts during their evolutionary history. These results suggest that sigma virus infections may be short-lived in a given host lineage, so that their long-term persistence relies on rare horizontal transmission events between hosts. In Chapter five I examined the ability of three Drosophila sigma viruses to persist and replicate in 51 hosts sampled across the Drosophilidae phylogeny. I used a phylogenetic mixed model to account for the non-independence of host taxa due to common ancestry, which additionally allows integration over the uncertainty in the host phylogeny. In two out of the three viruses there was a negative correlation between viral titre and genetic distance from the natural host. Additionally the host phylogeny explains an extremely high proportion of the variation (after considering genetic distance from the natural host) in the ability of these viruses to replicate in novel hosts (>0.8 for all viruses). There were strong phylogenetic correlations between all the viruses (>0.65 for all pairs), suggesting a given species’ level of resistance to one virus is strongly correlated with its resistance to other viruses. This suggests the host phylogeny, and genetic distance from the natural host, may be important in determining viruses ability to host switch. This work has aimed to address fundamental questions relating to host-parasite coevolution and pathogen emergence. The data presented suggests that sigma viruses are likely to be widespread vertically transmitted insect viruses, which have dynamic interactions with their hosts. These viruses appear to have switched between hosts during their evolutionary history and it is likely the host phylogeny is a determinant of such host shifts.

RACK1 (Receptor for activated protein C kinase 1) est une protéine ribosomale associée à de nombreuses voies de signalisation. RACK1 est nécessaire à la traduction sélective de virus contenant des sites d’entrée interne du ribosome (IRES). En outre, l’expression de RACK1 est nécessaire au cours du développement, suggérant que ce facteur participe à la traduction de certains ARNm cellulaires. Dans le but de mieux comprendre la fonction de RACK1 chez la drosophile, j’ai au cours de ma thèse caractérisé l’interactome de RACK1 et un IRES viral régulé par ce facteur. J’ai également essayé d’établir un lien entre signalisation cellulaire et traduction, et montré que la région du knob est importante pour la fonction de RACK1 au ribosome. Enfin, j’ai établi que RACK1 est nécessaire à la réponse à des stress abiotiques, et identifié les gènes cellulaires régulés par RACK1 dans ce contexte. J’ai en particulier découvert que RACK1 était un régulateur négatif de l’expression de plusieurs gènes de l’immunité innée. Mes résultats suggèrent que RACK1 joue un rôle pivot au sein du ribosome, régulant la traduction de façon positive ou négative selon l’ARNm et le contexte cellulaire. / RACK1 (Receptor for activated protein C kinase 1) is a ribosomal protein associated to many signaling pathways. RACK1 is required for the selective translation of viruses containing internal ribosome entry sites (IRES). In addition, expression of RACK1 is necessary during development, suggesting that it regulates the translation of cellular mRNAs. In order to better understand the function of RACK1 in Drosophila, I have participated in the characterization of the RACK1 interactome and of a RACK1-dependent viral IRES. I have also attempted to establish a connection between the function of RACK1 in signaling and in translation, and I have shown that the knob domain of RACK1 is important for IRES-dependent translation. Finally, I have established that RACK1 is required for the response to abiotic stresses, and I have identified cellular genes regulated by RACK1 in this context. In particular, I discovered that RACK1 is a negative regulator of several innate immunity genes. My results suggest that RACK1 plays a pivotal role within the ribosome, regulating translation positively or negatively in an mRNA- and possibly context-specific manner.

Οι εντεροϊοί έχουν ιδιαίτερη σημασία για τη δημόσια υγεία, δεδομένου ότι σχετίζονται με επιδημίες γαστρεντερίτιδας (μη βακτηριογενούς προέλευσης) από την κατανάλωση μολυσμένου νερού. Οι ιοί αυτοί μπορούν να απομονωθούν σε μεγάλες ποσόστητες από κόπρανα και ούρα ανθρώπινης προέλευσης, καθώς και από λύματα και μολυσμένα νερά. Επιπλέον, οι Αδενοϊοί είναι παθογόνοι για τον άνθρωπο και η παρουσία τους σε περιβαλλοντικά δείγματα δύναται να προκαλέσει σημαντικές μολύνσεις. Οι Αδενοϊοί είναι ιοί ανθρώπινης εντερικής προέλευσης που περιέχουν DNA και ορισμένοι ορότυποι τους είτε δεν καλλιεργούνται, είτε καλλιεργούνται δύσκολα στις συνήθεις κυτταρικές σειρές. Γι’ αυτόν το λόγο, η ανίχνευσή τους σε μολυσμένο νερό και ο ρόλος τους ως παραγόντων πρόκλησης γαστρεντερίτιδας έχουν υποτιμηθεί. Οι Ρότα ιοί είναι υπεύθυνοι για οξεία περιστατικά γαστρεντερίτιδας στον άνθρωπο και τα ζώα. Κατόπιν του διπλασιασμού του γενετικού τους υλικού στον γαστρεντερικό σωλήνα οι ιοί αυτοί εκκρίνονται και δύνανται να διασπαρούν στο περιβάλλον και το νερό. Γενικά, οι Ρότα ιοί έχουν εμπλακεί σε περιστατικά γαστρεντερίτιδας σε πολλές χώρες. Η σταθερότητα των ανθρωπίνων Ρότα ιών στο νερό και η ανθεκτικότητά τους σε φυσικοχημικές διαδικασίες εξυγίανσης κατά την επεξεργασία των λυμάτων συντείνουν στην εξάπλωσή τους. Στην παρούσα διατριβή ανιχνεύθηκαν και απομονώθηκαν εντεροϊοί, αδενοϊοί και ρότα ιοί από ακατέργαστα λύματα, τα οποία ελήφθησαν από την είσοδο τεσσάρων σταθμών βιολογικού καθαρισμού (δύο στην Αττική και δύο στην Αχαΐα). Συνολικά ελήφθησαν 118 δείγματα ακατέργαστων λυμάτων κατά την περίοδο Σεπτέμβριο 2000 – Σεπτέμβριο 2003. Η μεθοδολογία αφορούσε στην συμπύκνωση των ιών ακολουθούμενη από RT-nested PCR, προκειμένου να επιτευχθεί αύξηση της ευαισθησίας απομόνωσης των ιών. Μετά την απομόνωση γενετικού υλικού των ιών πραγματοποιήθηκε τυποποίησή τους εφαρμόζοντας nucleotide sequencing analysis. Οι Ρότα ιοί ανιχνεύθηκαν σε 17 δείγματα (14.2%). Τα αποτελέσματα της τυποποίησής τους ήταν rotavirus τύπος G1 (88.2%) και τύπος G2 (11.8%). Οι Αδενοϊοί βρέθηκαν σε 55 δείγματα (45.8%). Η Sequencing ανάλυση είχε ως αποτέλεσμα την παρουσία στα δείγματα αδενοϊών της ομάδας F [τύποι 40 (34.6%) και 41 (63.6%)] και της ομάδας C [τύπος 2 (1.8%)]. Οι Εντεροϊοί ανιχνεύθηκαν σε 30 δείγματα (40%) και η sequencing ανάλυση είχε ως αποτέλεσμα την παρουσία αρκετών τύπων όπως (α) coxsackievirus (τύποι A6 - 3.3%, A9 - 3.3%, A16 - 3.3%, B4 - 16.7%, B5 - 3.3%), (β) echovirus (τύποι 2 -6.7%, 6 - 13.3%, 30 -10%), (γ) εντεροϊοί τύποι 68 - 3.3%, 71 - 13.3% καθώς και porcine εντεροϊός (6.7%), poliovirus 1 (6.7%) και poliovirus 2 (10%). Η μικροβιολογική ποιότητα του νερού επομένως και η ανθρώπινη υγεία επηρεάζονται σημαντικά από την παρουσία μικροοργανισμών εντερικής προέλευσης, οι οποίοι προέρχονται από λύματα που καταλήγουν στο υδάτινο περιβάλλον. Πολλές επιδημίες από ιούς εντερικής προέλευσης έχουν κατά καιρούς συνδυαστεί με το νερό. Οι υδατογενείς επιδημίες γενικά εξαπλώνονται στον πληθυσμό από κατανάλωση μολυσμένου νερού, κολύμβηση σε ακατάλληλα νερά αναψυχής καθώς επίσης μεταδίδονται απο τη σωματική επαφή και την εισπνοή. Τα μη επεξεργασμένα λύματα της μελέτης περιέχουν πολλούς και διαφορετικούς τύπους ιών εντερικής προέλευσης οι οποίοι κατά κύριο λόγο προκαλούν γαστρεντερίτιδα. Επομένως καθίσταται αναγκαία η επξεργασία των λυμάτων στο μέγιστο δυνατό βαθμό στους σταθμούς βιολογικού καθαρισμού. Η Sequencing ανάλυση έδειξε την παρουσία ανθρώπινων Ρότα ιών A (τύποι G1 και G2), οι οποίοι προκαλούν παγκοσμίως διάρροια σε παιδιά καθώς επίσης και την παρουσία αδενοϊών τύπου 40 και 41, οι οποίοι είναι σημαντικοί αιτιολογικοί παράγοντες γαστρεντερίτιδας, κυρίως σε θερμά κλίματα. Από την άλλη πλευρά η ποικιλία των εντεροϊών που ανιχνεύθηκε στα ακατέργαστα λύματα ήταν μεγαλύτερη συγκρινόμενη με την αντίστοιχη των υπολοίπων ιών της μελέτης. Η παρούσα διατριβή αναδεικνύει την αποτελεσματικότητα της μεθόδου «nucleotide sequencing analysis», ως μέσου επιδημιολογικής μελέτης και ανάλυσης της συσχέτισης ιών που εμπλέκονται σε ανθρώπινες ασθένειες κι εκέινων που ανιχνεύονται σε ακατέργαστα λύματα. / Enteroviruses have been associated with outbreaks of waterborne non-bacterial gastroenteritis and are of important concern for public health. Significant numbers of viruses can be isolated from faeces and urine of humans as well as from sewage and polluted waters. Adenoviruses are also pathogenic to humans and their presence in environmental samples (polluted waters) may cause infections. Like rotaviruses, adenoviruses are causative agents of gastroenteritis, are the only human enteric viruses to contain DNA and many serotypes are difficult to culture in regular cell lines For this reason, and because adenoviruses are slow growing, their presence in polluted water and their role as originators of gastroenteritis have probably been underestimated. Rota viruses are responsible for severe gastroenteritis in humans and animals. After replicating in the gastrointestinal tract, these viruses are excreted and may be dispersed in environmental waters. Rota viruses have been implicated in waterborne gastroenteritis outbreaks in many countries. The stability of human rotaviruses in environmental water and their resistance to physicochemical treatment processes in sewage treatment plants may facilitate their transmission. In the present study, enteroviruses, adenoviruses and rota viruses were detected in raw sewage samples from inlets of four biological treatment plants in Greece (two in Athens, two in Patras}. Raw sewage samples (118) were analyzed for the presence of these viruses during the period September 2000 to September 2003. Our approach consisted of a simple concentration of viruses from raw sewage followed by RT-nested PCR in order to increase the sensitivity of virus detection. The viral sequences detected were then characterized by nucleotide sequencing analysis. Rota viruses were detected in 17 samples (14.2%). Sequencing analysis of the positive sewage samples revealed the presence of rotavirus type G1 (88.2%) and type G2 (11.8%). Adenoviruses were found in 55 samples (45.8%). Sequencing analysis of the positive sewage samples revealed the presence of adenovirus group F type 40 (34.6%), type 41 (63.6%) and group C type 2 (1.8%). Enteroviruses were detected in 30 samples (40%) and sequencing analysis of the positive sewage samples revealed the presence of several types such as (a) coxsackievirus types (A6 - 3.3%, A9 - 3.3%, A16 - 3.3%, B4 - 16.7%, B5 - 3.3%), (b) echovirus types (2 -6.7%, 6 - 13.3%, 30 -10%), (c) enterovirus types (68 - 3.3%, 71 - 13.3%) as well as porcine enterovirus (6.7%). poliovirus 1 (6.7%) and poliovirus 2 (10%). Water quality and, therefore human health, may be significantly affected by the presence of pathogenic enteric microorganisms derived from sewage discharged to the aquatic environment. Outbreaks of enteric virus disease have been linked to water at various times and to different causes. Waterborne disease may be transmitted by consumption of polluted drinking water, by immersion in recreational water or by contact with skin or inhalation. Raw sewage was found to be contaminated by different types of enteric viruses that mainly cause gastroenteritis; therefore, it is necessary to use the most efficient water treatment measures in sewage treatment plants. Sequencing analysis showed the presence of human rotavirus A type G1 and G2 which cause childhood diarrhea worldwide and enteric adenoviruses (types 40 and 41) which are important etiological agents of pediatric gastroenteritis, principally in temperate climates. On the other hand, the variety of enteroviruses identified in the raw sewage samples was more extensive compared to the other viruses of the study. The present study demonstrated the efficiency of the nucleotide sequencing analysis for studying epidemiological relationships between strains involved in human infections and those found in raw sewage.

La macroautophagie assure l'homéostasie cellulaire en recyclant du matériel cytosolique obsolète ou délétère et sa dérégulation est associée à plusieurs pathologies. Elle constitue aussi un mécanisme de défense car elle peut éliminer des pathogènes intracellulaires. L'étape cruciale de l'autophagie est la maturation lors de laquelle la vésicule renfermant des substrats cytosoliques, l'autophagosome, fusionne avec des lysosomes et la dégradation a lieu. Nous nous intéressons à la régulation de l'autophagie et aux conséquences de sa perturbation lors des infections, notamment par le virus de la rougeole (VR). Les données de l'équipe montrent qu'il induit et utilise toutes les étapes de l'autophagie, afin de se répliquer efficacement. Mes travaux montrent que des protéines du virus peuvent interagir avec au moins deux protéines cellulaires NDP52 et T6BP qui sont des récepteurs autophagiques (protéines cytosoliques ayant un domaine de liaison aux autophagosomes et un domaine de liaison au substrat à dégrader, par exemple des pathogènes). J'ai alors étudié le rôle des récepteurs autophagiques T6BP, NDP52 et Optineurine dans la réplication virale. J'ai aussi participé à une étude décrivant que NDP52 et Optineurine régulent en plus la maturation. Mes travaux de thèse démontrent un tel double rôle pour T6BP. Cependant, seuls T6BP et NDP52 sont nécessaires à la réplication du VR bien qu'elle requiert la maturation autophagique. Ainsi mes résultats suggèrent d'une part que les trois récepteurs puissent réguler la maturation d'autophagosomes distincts.D'autre part, le VR pourrait exploiter individuellement les autophagosomes dont la maturation dépend de T6BP et NDP52 pour se répliquer / Macroautophagy ensures cell homeostasis through the recycling of obsolete or deleterious cytosolic components and its deregulation is associated with several pathologies. It is also a defense mechanism as it allows the elimination of intracellular pathogens. The most important autophagic step is maturation, during which the cytosolic substrate-containing vesicle, the autophagosome, fuses with lysosomes and the degradation occurs. We study autophagy regulation and the consequences of its disruption during infections and in particular by measles virus (MeV). Our team has shown that MeV induces and exploits all steps of autophagy, to replicate more efficiently. My results indicate that viral proteins can interact with at least two cellular proteins, NDP52 and T6BP, which are autophagy receptors (cytosolic proteins that carry an autophagosome-binding domain and a domain binding substrates that would be degraded, such as intracellular pathogens). I then studied the role of autophagic receptors T6BP, NDP52 and OPTINEURIN in viral replication. I also took part in a study describing NDP52 and OPTINEURIN as autophagosome maturation regulators. My work depicts the same dual role for T6BP. However, only T6BP and NDP52 are necessary for MeV replication even though it requires autophagosome maturation. Thus, my results suggest that the three autophagy receptors might regulate distinct autophagosome maturation on one hand. On the other, MeV could individually exploit autophagosomes, the maturation of which is regulated by T6BP or NDP2 to replicate efficiently

Le syndrome d'immunodéficience acquise (SIDA) est une maladie provoquée chez l'homme par le virus de l'immunodéficience humaine (VIH), un lentivirus à ARN monocaténaire qui infecte les cellules humaines qui expriment les CD4 à leur surface. Depuis son apparition en 1982 chez l’homme, il y a eu environ 80 millions d'individus infectés dans le monde et près de la moitié d'entre eux sont déjà décédés. Aucun vaccin n'existe actuellement mais l'espérance de vie d’un grand nombre de patients est maintenant prolongée grâce au développement et la disponibilité d'un traitement antirétroviral hautement actif (HAART en anglais). En raison de la complexité des interactions hôte/pathogène liées à l'infection par le VIH-1 chez l'homme et les modèles primates non-humains actuels, le développement d’un modèle plus simple est nécessaire pour étudier et mieux comprendre les mécanismes sous-jacents de l'augmentation de la pathogenèse du VIH-1 chez l’humain. Dans ce but, un virus chimérique CAL-HIV-R1 a été construit dans notre laboratoire en échangeant les longues séquences répétées terminales (LTR) du VIH par celles du CAEV, un lentivirus caprin. Parce que ces LTR de CAEV ont un promoteur constitutif qui est indépendant du trans-activateur de la transcription, ce virus chimérique ne devrait pas subir de latence dans les cellules T CD4+ mémoire. Pour rendre son efficacité réplicative plus performante, cette chimère a subi plusieurs passages successifs sur des cellules humaines en culture. En plus de la présence de son récepteur primaire, la protéine CD4, le VIH doit interagir avec une seconde molécule co-réceptrice pour entrer dans la cellule hôte. Des clones moléculaires infectieux contenant des génomes proviraux complets de plusieurs isolats de VIH-1 ont été reçus de la banque de produits "NIH AIDS Reagent Program Repository". Trois d'entre eux, à savoir pNL4-3, p89.6 et WARO, ont été utilisés pour produire des stocks de virus après transfection des cellules de la lignée humaine HEK-293T et utilisés pour infecter d’autres lignées cellulaires telles que : 1) des cellules GHOST, utilisées pour examiner le tropisme des virus en fonction de leur utilisation des co-récepteurs et qui sont respectivement X4, X4/R5 et R5; 2) la lignée cellulaire M8166, utilisée comme cellules indicatrices du fait de ses propriétés fusogéniques, et qui sert à examiner les capacités de réplication et enfin, 3) la lignée cellulaire TZM-bl utilisée pour évaluer le titre infectieux des virus. Par ailleurs, un vaccin basé sur un vecteur ADN lentiviral chimérique, le CAL-SHIV-IN-, a été développé au laboratoire et testé chez des macaques. Dans le cadre de cette étude, un test de séro-neutralisation a été réalisé sur des échantillons de sérum des macaques vaccinés avec ce vecteur, et des animaux témoins, pour examiner la présence d'anticorps pouvant neutraliser le virus. Bien que des anticorps furent présents aucune capacité neutralisante n'a pu être détectée. / Acquired Immuno-Deficiency Syndrome (AIDS) is a disease caused by immunodeficiency viruses in human (HIV-1) and some animal species. The virus is a small enveloped particle that has a single-strand RNA genome and belongs to the lentivirus genus that belongs to the Retroviridae family. In human the virus infects and replicates mainly in cells that express the CD4 on their surface. Since its apparition in human in 1982 the virus has infected around 80 million individuals worldwide and caused the death of nearly half of them. No vaccine exists but life expectancy of near half of HIV-1-infected individuals has been now prolonged due to extensive highly active antiretroviral therapy (HAART). Because of the complexity of the host/pathogen interactions that are associated with HIV-1 infection in human and non-human primate models, a simple model system is strongly needed to ease the studies aiming at better understanding the underlying mechanisms of increased pathogenesis of HIV-1 in human. A chimeric virus CAL-HIV-R1 was created in our laboratory by exchanging the long terminal repeats (LTRs) of HIV with those of CAEV, a caprine lentivirus. Because these CAEV LTRs have a constitutive promoter, which is independent of the trans-activator of transcription, we expect that this chimeric virus should not undergo latency in memory CD4+ T cells. To increase the potency of this chimera, serial passages on cultured human cells were performed. Besides its primary receptor, CD4, HIV needs to interact with another molecule as a co-receptor. Several infectious molecular clones of HIV-1 isolates pDNAs containing the complete proviral genomes were received from the NIH AIDS Reagent Program Repository. Three of these, namely pNL4-3, p89.6 and WARO, were used to produce virus stocks following transfection in the human HEK-293T cell line and used to infect a variety of cell lines such as: 1) GHOST cells that were used to examine the tropism for the co-receptor that were X4, X4/R5 and R5 respectively; 2) M8166 a fusogenic indicator cell line to evaluate the replication competency, 3) TZM-bl to determine the infectivity titers of the viruses by scoring the blue cells enabled by infections. A vaccine based on a chimeric DNA vector, CAL-SHIV-IN-, has been developed in our laboratory and tested in macaques. A sero-neutralization assay was performed on sera of macaques, which had been vaccinated with this vector and challenged in parallel with control animals with a pathogenic virus. This assay was used to verify the presence of neutralizing antibodies, but, unfortunately none could be detected.

Climate change has already led to an increase in the frequency and intensity of storm events in many parts of the world. With further increases predicted, and growing evidence of the link between extreme precipitation and waterborne disease, it is important to elucidate the role of sediments in pathogen transmission. Intense rainfall can trigger discharges from combined sewer overflows, increase surface and subsurface faecal inputs and can re-suspend microorganisms already present in sediments. Previous research has shown that sediments can act as environmental reservoirs of numerous waterborne pathogens, including enteric viruses and antibiotic resistant bacteria. Sediments are therefore a growing public health concern and are an increasing focus of human health protection strategies. However, further research is needed in order to elucidate the behaviour of microorganisms within these matrices. Routine monitoring for pathogens within sediments has not to date been considered feasible in many parts of the world and although low-cost microbial tools to detect faecal pollution of human origin, such as those that detect phages infecting Bacteroides fragilis (GB-124), have shown promise in many situations, they have seldom been applied to sediment matrices. Sediments might offer a more sensitive and longer-term assessment of contamination sources and hazards to health, compared with analysis of overlying waters from the same location. This study therefore sought to determine an effective elution method to extract GB-124 phages from sediments and to use it in an intensive six month investigation of the River (Ouse) catchment in Southeast England. The results (ANOVA with post-hoc Tukey) revealed that a low-cost elution method, involving 10% beef extract, provided the most effective means of recovering GB-124 phage from a range of river sediments (66% recovery). GB124 phages were subsequently enumerated in sediment and overlying water collected from 25 sites across the study catchment, along with somatic coliphage (SC), faecal coliforms (FC), and intestinal enterococci (IE). Physicochemical data were also collected. Analyses revealed evidence of faecal contamination at all sites, and human contamination at 13 of the 25 sites. Whilst levels of microorganism in the water and sediment were significantly correlated: GB-124 (p= 0.015); SC (p= 0.000); FC (p= 0.018); and IE (p= 0.038), importantly GB-124, SC, FC and IE (p= 0.00) were detected at significantly higher levels (Mann-Whitney) in the sediment samples. Significant correlations (p= < 0.01) were also observed between levels of FC, IE and SC and sediment temperature, but not between water temperature and any of the parameters in the water column. Interestingly, GB-124 phage showed no significant correlation with the non human-specific parameters (SC, FC, and IE) in the sediment matrices, which were found to co-correlate with one another (p= 0.00). The findings suggest that the application of low-cost monitoring approaches to analyse river sediments may not only provide a better assessment of dominant pollution sources than grab samples of overlying water (due to the higher levels and incidence of phages in sediments), but that they may also provide a better indication of potential risks to health from human enteric viruses. As such, the findings of this study add significantly to the body of extant knowledge relating to the behaviour of GB-124 phages in the environment and further support their use for microbial source tracking (MST) and as a potential component of quantitative microbial risk assessment (QMRA) studies.

Le virus de l'hépatite C (VHC) est détecté dans les sérums de patients infectés sous forme de particules infectieuses lipidées de très faibles densités. Le VHC est un virus enveloppé dont l'assemblage de particules virales se produit à la membrane du réticulum endoplasmique consécutivement au clivage séquentiel de sa polyprotéine et à sa maturation en protéines structurales et non structurales. Dans ce travail, nous avons cherché à mieux comprendre les mécanismes d'assemblage, d'enveloppement et de sécrétion des particules infectieuses. Dans une première étude, nous avons montré la connexion fonctionnelle entre les complexes de réplication et les sites d'assemblage. Dans une seconde étude, nous avons montré que p7 ralentissait de manière dose-dépendante le trafic ER-Golgi, conduisant à une rétention intracellulaire de la glycoprotéine virale E2. En outre, nous avons montré que le clivage du précurseur protéique E2p7 contrôle l'expression intracellulaire E2 et les niveaux de sécrétion des particules subvirales et des virions infectieux. Enfin, nous avons également mis en évidence que l'extrémité N-terminale de p7 gouverne l'infectivité spécifique des particules en coordonnant la rencontre des composants de la nucléocapside avec les glycoprotéines, mais aussi l'enveloppement de la nucléocapside. Dans une troisième étude, nous avons découvert des fonctions et des facteurs spécifiques du sérum, des cellules productrices et des séquences du VHC qui modulent la lipidation des particules virales au cours de leur assemblage et de leur sécrétion. Au total, ces différents travaux ont contribué à mieux comprendre les étapes de l'assemblage du VHC et les mécanismes modulant i) le transfert des ARN viraux des complexes de réplication vers les sites d’assemblage, ii) la rencontre des nucléocapsides et des glycoprotéines, et enfin, iii) l'acquisition de lipides par des particules virales / Hepatitis C virus (HCV) is detected in the sera of infected patients as lipidated infectious particles of very-low density. HCV is an enveloped virus whose assembly of viral particles occurs at the endoplasmic reticulum membrane following sequential cleavage of its polyprotein and its maturation as structural and non-structural viral proteins. In this work, we aimed at better understanding the mechanisms of assembly, envelopment and secretion of infectious particles. In a first study, we highlighted the functional connection between replication complexes and assembly sites. In a second study, we showed that p7 dose-dependently slows down the ER-to-Golgi traffic, leading to intracellular retention of E2 viral glycoprotein. In addition, we showed that cleavage of an E2p7 precursor protein controls E2 intracellular expression and secretion levels of subviral particles and infectious virions. Finally, we also highlighted that p7 N-terminal extremity governs the specific infectivity of the infectious particles by coordinating the encountering of the nucleocapsid components with the glycoproteins and the envelopment of the nucleocapsids. In a third study, we discovered specific functions and factors from serum, producer cells, and HCV sequences that modulate lipidation of viral particles during their assembly and secretion. Altogether, these different works contributed at better understanding the steps of HCV assembly and the mechanisms modulating i) the transfer of viral RNAs from replication complexes to assembly sites, ii) the encountering of the nucleocapsids and glycoproteins followed by virion envelopment, and finally, iii) the acquisition of lipids by viral particles

L’épissage alternatif et l’export nucléaire de l’ARN pré-génomique 35S du virus de la mosaïque du chou-fleur (CaMV) ont fait l’objet de peu d’études. Quatre isoformes épissées de l’ARN 35S ont déjà été décrites. Nos résultats montrent que l’épissage alternatif est plus complexe vu que plus d’une douzaine d’isoformes d’ARN 35S sont générées par ce mécanisme. Ce processus ne semble pas avoir pour fonction d’augmenter la complexité du protéome viral. Le rôle de l’épissage alternatif apparait encore difficile à appréhender puisque l’inactivation des sites d’épissage est systématiquement contrecarrée par l’utilisation de sites cryptiques. Pour maintenir l’intégrité du génome viral, des molécules d’ARN 35S préservées de l’épissage sont exportées vers le cytoplasme. L’essentiel de nos travaux sur ce sujet a porté sur le développement d’approches destinées à l’étude de la voie d’export nucléaire de l’ARN 35S, et des séquences et protéines virales potentiellement impliquées dans ce processus. / Alternative splicing and nuclear export of the pregenomic 35S RNA are two steps of the infectious cycle of Cauliflower mosaic virus (CaMV) that are poorly understood. Four 35S RNA spliced isoforms were described in previous reports. Our results show that at least twelve spliced 35S RNA isoforms are generated upon infection. Alternative splicing is important for CaMV infectivity but apparently, it does not expand CaMV proteome. Its role is difficult to assess because inactivation of splice donor or acceptor sites is constantly rescued by the use of cryptic sites. In order to maintain CaMV genomic integrity, unspliced 35S RNA must be exported to the cytoplasm. Our work has been mainly focused on developing experimental tools dedicated to study the 35S RNA nuclear export pathway as well as viral sequences and proteins potentially involved in this process.

Les virus sont les entités les plus abondantes dans les océans et ces parasitoïdes interagissent avec chaque composante de la biosphère marine, soit par la sélection des communautés d'hôte, soit en influençant les cycles biogéochimiques. Cependant, l'impact des conditions environnementales sur ces assemblages viraux complexes est encore mal compris. L'objectif de ce travail est de comprendre comment les assemblages viraux sont influencés par les facteurs environnementaux. Durant un suivi mensuel des communautés de mars 2013 à avril 2014, nous nous sommes intéressés à un système hôte-virus abondant et largement répandu, les communautés de microalgues Mamiellophyceae et leurs Prasinovirus. Durant ces études, des approches de PCR quantitative et de séquençage massif ont été développées afin de décrire les populations environnementales présentes au sein du Golfe du Lion. Les populations de Mamiellophyceae dominent le compartiment des picoeucaryotes photosynthétiques, avec des dynamiques fortes au cours de l'année. Les communautés de Prasinovirus reflètent les dynamiques de leurs hôtes, elles-mêmes en lien avec les facteurs environnementaux. En revanche, la dispersion ainsi que le maintien des virions semblent directement impacté par les conditions environnementales, comme la température et l'hydrodynamisme. Certains groupes viraux, comme les virus d'Ostreococcus, sont inféodés aux lagunes et aux zones côtières, suggérant ainsi que la dispersion des populations virales est limitées par la présence des hôtes. / Viruses are the most abundant biological entities in oceans and they every member of the marine biosphere is affected by them, they influence the composition of communities and influence biogeochemical cycles. However, the influence of environmental conditions on complex viral populations is still poorly understood. The aim of this study is to understand how environmental factors influence viral communities. We followed viral communities monthly from March 2013 to April 2014 and investigated the host-virus system of Mamiellophyceae communities and their prasinoviruses, a model sytem that is abundant and widespread. Prasinovirus-host populations from the Gulf of Lion were quantified by PCR amplification and analysis of genetic marker genes and high throughput sequencing. Photosynthetic populations of picoeukaryotes were dominated by populations of Mamiellophyceae showing high levels of quantitative and qualitative annual variations that were related to environmental factors. Prasinovirus communities mimed host dynamics, but viral dispersion and persistence appeared to be impacted directly by environmental conditions such as temperature and hydrodynamics. Several viral groups, such as Ostreococcus viruses, were specific to lagoonal and coastal areas, suggesting that their dispersal is limited by host occurrence.