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Les systèmes de génétique inverse permettent de manipuler les génomes viraux et se sont révélés essentiels pour étudier les virus à ARN. Récemment, une méthode basée sur la PCR, la méthode ISA (Infectious Subgenomic Amplicons), a été développée. La première partie de cette thèse se concentre sur la simplification de la méthode ISA. La principale contrainte d'ISA est l'exigence de produire des fragments génomiques modifiés qui nécessite un promoteur de transcription à l’extrémité 5’ du premier fragment et un ribozyme du virus de l'hépatite delta, suivi du signal de polyadénylation du virus simien 40 (HDR / SV40pA) à l’extrémité 3’ du dernier fragment. Ici, nous proposons une nouvelle méthode simplifiée "Haiku", dans laquelle sont fournis ces deux séquences en tant qu'amplicons séparés. Cette technique améliorée a été appliquée avec succès à une large gamme de virus dans des cellules de moustiques et de mammifères. La deuxième partie de cette thèse est axée sur la caractérisation de la population virale issue de divers systèmes de génétique inverse en utilisant le HRV-B14 comme modèle.Nos résultats montrent que le choix de la méthode a influencé la diversité génétique des populations virales mais quelle que soit la méthode utilisée, la fitness réplicative était similaire. En outre, nos données ont révélé que le poly(A)25 est la longueur optimale pour récupérer le HRV-B14 avec une efficacité élevée. La dernière partie du présent travail a examiné le potentiel de la méthode «ISA» pour conserver le spectre mutant présent dans l'échantillon viral d'origine. Nous avons montré que cette méthode récapitule au moins partiellement les quasi-espèces de la population virale native. / Reverse genetics systems allow manipulating viral genomes and have proved to be essential for studying RNA viruses. Recently, a PCR-based method, named ISA (Infectious Subgenomic Amplicons), was developed to facilitate the study of single-stranded positive-sense RNA viruses. The first part of the present work focused on simplifying the ISA method. The main constraint of the canonic protocol of the ISA method is the requirement to produce modified genomic fragments encompassing the transcription promoter and the terminator. Here, we propose the ultimately simplified "Haiku" design in which the promoter and the terminator are provided as additional separate DNA amplicons. This improved procedure was successfully applied to the rescue of a wide range of viruses in mosquito and mammalian cells. The second part of this work assessed the viral population issued from different reverse genetics systems. Using HRV B-14 as a model, we compared the genetic diversity and the replicative fitness of viruses generated using the most commonly used reverse genetics methods. Our results showed that the choice of the method influenced the genetic diversity of viral populations but whatever the method used, the replicative fitness was similar. In addition, Our data revealed that poly(A)25 is the optimal length to recover HRV-B14 with high efficiency and could be used to recover polyadenylated RNA viruses other than HRV-B14. The last part of the present work investigated the potential of the “ISA” method to conserve the mutant spectrum present in the original viral sample. We have showed that this method recapitulate at least partially the quasispecies of the native viral population.

Réovirus, connu sous le nom REOLYSIN®, est présentement à l'étude à titre d'agent oncolytique. Or, la spécificité du virus pour les cellules cancéreuses pourrait être optimisée par une modification au niveau de la protéine d'attachement σ1. La présente étude vise à démontrer qu'une telle amélioration est possible par l'utilisation de la méthode nouvellement décrite de génétique inverse. Par cette technique, il est possible d'ajouter un polypeptide d'une longueur de quarante acides aminés à l'extrémité C-terminale de σ1. Il est aussi possible d'engendrer des virus mutés en leur site d'activité mucinolytique. Les virus nouvellement créés démontrent une efficacité de réplication diminuée, mais demeurent infectieux. Contrairement aux méthodes traditionnellement utilisées avec réovirus, la méthode de génétique inverse permet de conserver les mutations engendrées, par substitution ou addition, au cours des cycles de réplication. Une telle étude démontre qu'il serait possible de modifier le tropisme de réovirus. / Reovirus, also known under the tradename REOLYSIN®, is currently under clinical trial as an oncolytic agent. The specificity of the virus for transformed (cancerous) cells could be improved by modifications of the σ1 attachment protein. This study presents two ways to achieve this improvement by using a newly developed method of reverse genetics for double-stranded RNA viruses. As a proof of concept, we engineered the addition of an exogenous polypeptide of forty amino-acids to the C-terminal end of σ1. Subsequently, we created viruses with compromised mucinolytic activities by introducing amino-acids substitutions in the appropriate genetic sites. A decreased replicative efficacy was observed in all experimentally generated viruses, despite maintaining their infectious capacity. Reverse genetics allows the generation of viruses that retain their mutations throughout successive replicative cycles, which was impossible with traditionnal techniques. This project demonstrates the feasibility of tropism modification by using the reverse genetics method.

Root symbioses with arbuscular mycorrhizal fungi and rhizobial bacteria share a common signaling pathway in legumes. Among the common symbiosis genes are CASTOR and POLLUX, the twin homologous genes in Lotus japonicus that encode putative ion channel proteins. Orthologs of CASTOR and POLLUX are ubiquitously present in both legumes and non-legumes, but their function in non-legumes remains to be elucidated. Here, we use reverse genetic approaches to demonstrate that the rice (Oryza sativa) ortholog of POLLUX, namely Os-POLLUX, is indispensible for mycorrhizal symbiosis in rice. Furthermore, we show that Os-POLLUX can restore nodulation, but not rhizobial infection, to a M. truncatula dmi1 mutant.

Enteroviruses belong to the Picornaviridae family and are small icosahedral viruses with RNA genomes of positive polarity, containing a single open reading frame. They mostly cause mild or asymptomatic infections, but also a wide array of diseases including: poliomyelitis, encephalitis, gastroenteritis, aseptic meningitis, myocarditis, hand-foot-and-mouth disease, hepatitis and respiratory diseases, ranging from severe infections to the common cold. The projects described in this thesis have been carried out through reverse genetic studies of Enterovirus B and Rhinovirus C.                   In Papers I and II, a cassette vector was used to study recombination and translation of the RNA genome. It was found that the non-structural coding region could replicate when combined with the structural protein-coding region of other viruses of the same species. Furthermore, the genome could be translated and replicated without the presence of the structural protein-coding region. Moreover, it was found that when two additional nucleotides were introduced, shifting the reading frame, the virus could revert to the original reading frame, restoring efficient replication. In Paper III, a vector containing the genome of echovirus 5 was altered to produce an authentic 5’end of the in vitro transcribed RNA, which increased efficiency of replication initiation 20 times. This result is important, as it may lead to more efficient oncolytic virotherapy. An authentic 5’end was further used in Paper IV, where replication of Rhinovirus C in cell lines was attempted. Although passaging of the virus was unsuccessful, the genome was replicated and cytopathic effect induced after transfection. The restriction of efficient replication was therefore hypothesized to lie in the attachment and entry stages of the replication cycle. In Paper V, a cytolytic virus was found to have almost 10 times larger impact on gene expression of the host cell than a non-cytolytic variant. Furthermore, the lytic virus was found to build up inside the host cell, while the non-cytolytic virus was efficiently released.                   As a whole, this thesis has contributed to a deeper understanding of replication of enteroviruses, which may prove important in development of novel vaccines, antiviral agents and oncolytic virotherapies.

More than a decade after the completion of the human genome project, the function of a large number of genes remains to be elucidated. Forward and reverse genetic approaches have proven to be powerful tools to study gene function and have provided insights into fundamental biological processes. Furthermore, functional genetic screening can lead to a better understanding of the action of endogenous and exogenous stimuli such as hormones or drugs on biological systems. Thus far, systematic and unbiased studies have largely been limited to model organisms. However, complex disease-relevant genotypes and phenotypes cannot be studied in entirety in lower organisms creating a need for systematic approaches in human cells. This thesis describes a series of studies using forward and reverse genetic approaches combined with state-of-the-art technology in haploid human cells. The first chapter describes the development of a quantitative phenotypic read-out using a novel application of RNA-sequencing that allows the functional annotation of genes in signalling pathways. The presented data demonstrate that the employed shallow RNA-sequencing method is scalable and suitable as a read-out for reverse genetic screening. The second chapter focuses on the implementation of this method in a large reverse genetic study in human cells to functionally annotate tyrosine kinases in signalling pathways upon stimulation with a set of ten polypeptides and small molecules. The screens revealed known and unexpected interactions between different signalling molecules and pathways, validating the technical approach in a biological context. The third chapter presents a pilot study describing the set-up of a forward genetic technique for compound mode-of-action screening using a pooled human mutant cell line collection. The chemical genetic approach displayed sufficient sensitivity and allowed to monitor thousands of gene-drug interactions simultaneously. Together, this thesis combines elements to advance technological and biological aspects of functional genomics and chemical genetics.

No description available.

Oropouche virus (OROV) is a medically important orthobunyavirus, which causes frequent outbreaks of a febrile illness in the northern parts of Brazil. However, despite being the cause of an estimated half a million human infections since its first isolation in Trinidad in 1955, details of the molecular biology of this tripartite, negative-sense RNA virus remain limited. The work presented in this thesis has re-determined the nucleotide sequences of OROV strain BeAn19991 (GenBank accession numbers: L, KP052850; M, KP052851 and S, KP052852), and demonstrates that the S segment is significantly longer than the published sequence with an additional 204 nucleotides at the 3' end. Data analysis revealed that there is a critical nucleotide mismatch at position 9 within the base-paired terminal panhandle structure of each genomic segment. Using a combination of deep sequencing and Sanger sequencing the complete genome sequences of 10 field isolates of OROV were also determined for the first time, and led to the identification of a novel OROV reassortant virus. Phylogenetic analysis of these sequences and of published sequences showed that there are two genotypes of OROV, rather than the four genotypes previously proposed. Further work led to the development of a T7-RNA polymerase-driven minigenome and virus-like particle (VLP) production systems for OROV; the information from these was subsequently used to develop a reverse genetics system for OROV. Using reverse genetics, OROV mutants that lack either the non-structural proteins NSm or NSs were generated. In vitro growth properties of the OROV mutant lacking NSm were indistinguishable from the wild-type virus, but the NSs mutant was attenuated in growth, particularly in interferon (IFN) competent cells. Further work demonstrated NSs as a viral IFN antagonist and that it's C-terminus is required for this activity. Interestingly, OROV is more resistant to IFN-α treatment than Bunyamwera virus, but this is not related to its NSs protein. The development of a reverse genetics system for OROV, which is the main human pathogen within the Simbu serogroup of orthobunyaviruses, will prove invaluable for future studies designed to further investigate the molecular pathogenesis of this virus and in the development of attenuated vaccine strains.

Japanese encephalitis virus (JEV) is the causative agent of Japanese encephalitis (JE), the leading cause of vaccine-preventable neurological disease. JEV is a flavivirus that is primarily transmitted through the bite of infected mosquitoes, similar to dengue virus (DENV), St. Louis encephalitis virus (SLEV), West Nile virus (WNV), and Zika virus (ZIKV). The two viral characteristics that dictate virulence are (1) neuroinvasiveness, the ability of the virus to invade the central nervous system(CNS), and (2) neurovirulence, the capacity of the virus to kill resident cells in the CNS. The clinically proven live-attenuated JEV vaccine, SA14-14-2, lacks both pathogenic characteristics unlike its virulent parental virus, SA14. Previous work has revealed the viral E gene as the main determinant of these two pathogenic properties, though the molecular mechanisms behind their attenuation remain unclear. The E gene encodes for the viral envelope glycoprotein that is involved in viral entry into susceptible host cells. The E protein of SA14-14-2 differs from SA14 by nine amino acids. To investigate the role of these mutations in JEV virulence, we created a series of SA14E mutants using infectious cDNA technology. Here, we report the independent function of domains I (DI) and II (DII) of the viral E protein in JEV neurotropism. We reveal that an individual mutation in DI, E138K,and synergism between two mutations in DII, E244G and K279M,are independently sufficient for the attenuation of JEV neuroinvasion. Also, we report that multiple E mutations are required for full attenuation of JEV neurovirulence. Overall, our findings show the direct relationship between genetic factors and JEV neuroinvasion. These results provide a solid foundational base for the logical development of other, currently non-existing, live-attenuated neurotropic flavivirus vaccines and antivirals.