Le virus West Nile (VWN) est un virus neurotrope principalement transmis par piqûre de moustique et dont le réservoir est constitué par la faune aviaire sauvage. Les souches circulant en Europe appartiennent à 4 lignages génétiques différents à l’origine de nombreuses épidémies d’ampleur modérée à faible et limitées géographiquement, contrairement à ce qui a été observé en Amérique du Nord. En 1998 en Israël, une importante épidémie a a été associée pour la première fois à une forte mortalité de la faune aviaire sauvage. Le virus (souche IS-98-ST1, lignage 1a) a été isolé du cerveau d’une cigogne moribonde. L’objet de cette thèse a été de construire un clone infectieux de la souche IS-98-ST1 afin d’en explorer les propriétés de neuroinvasion et de pouvoir mettre en évidence les déterminants moléculaires de sa virulence.Le virus obtenu à partir de la construction clone infectieux s’est révélé posséder les mêmes propriétés biologiques que le virus parental, que ce soit in vitro sur cellules Vero ou in vivo sur souris sensibles ou résistantes ou encore sur l’embryon de poulet. L’embryon de poulet est présenté ici comme un nouveau modèle d’évaluation de la virulence du VWN. Un modèle cellulaire neuronale (lignée de neuroblastomes humains, SK-N-SH) est aussi évalué dans ce manuscrit. En conclusion, un nouvel outil de génétique inverse a été obtenu pour le VWN. Cet outil permettra de travailler sur l’impact de mutations ponctuelles, ou de modifications plus importantes touchant un ou plusieurs gènes viraux sur la virulence du VWN, spécifiquement dans le contexte européen. / West Nile virus (WNV) is a neurotropic virus mainly transmitted through mosquito bites. Wild birds represent the main reservoir hosts. Strains circulating in Europe belong to four lineages and have caused numerous but limited epidemics over the last few years. In 1998, an important outbreak associated to huge bird fatalities caused by a highly neuroinvasive strain (IS-98-ST1) took place in Israel. We aimed at producing a new infectious clone, based on the lineage 1a IS-98-ST1 WNV strain, for the characterization of its neuroinvasion properties as well as the molecular determinants of European WNV virulence. The growth kinetics of recombinant and parental WNV were similar in Vero cells. Moreover, the phenotypes of recombinant and parental WNV were indistinguishable in terms of viremia, viral load in the brain and mortality in susceptible and resistant mice. Finally, the pathobiology of the infectious clone was examined in embryonated chicken eggs, proposed as a new model for the evaluation of WNV virulence. The potential of human neuroblastoma cells (SK-N-SH) to discriminate between highly and mildly virulent WNV strains was assayed. In conclusion: a new molecular tool that is useful for the study of molecular determinants of WNV virulence has been generated. We take advantage of the high genetic stability of our one-piece infectious WNV cDNA clone to produce mutant viruses through the insertion of point mutations or the exchange of genetic fragments between WNV strains into the backbone of the IS-98-ST1 infectious clone.

Gonorrhea is caused by the human pathogen Neisseria gonorrhoeae. More than 106 million new cases of N. gonorrhoeae infections occur each year worldwide. There is no vaccine available against gonococcal infections and treatment of gonorrhea with antibiotics is the only way to eradicate infection. The high prevalence of antibiotic resistance (AMR) in this microorganism makes the effective treatment of gonococcal infections increasingly problematic. The emergence of AMR, especially to extended spectrum cephalosporins (i.e. cefixime and ceftriaxone) which are the last possibilities for single dose treatment options for gonococcal infections, is a serious concern. Gonorrhea may become an untreatable infection in the near future. Saskatchewan (SK) has one of the highest rates of gonorrhea in Canada. In order to better characterize the gonorrhea epidemic in SK, the objectives of the present research were to determine the prevalence and trends of AMR and emerging AMR mechanisms in N. gonorrhoeae isolates. AMR mechanisms were ascertained for the first time in SK in order to identify genetic causes of resistance. This was completed by determining and analyzing the DNA sequences of various genes - penA, mtrR, porB ponA, gyrA, parC mtrR, 23S rRNA alleles and erm –implicated in gonococcal AMR. The population dynamics of the N. gonorrhoeae isolates in SK was investigated by DNA based molecular methods to determine strain distribution, evolution of AMR phenotypes, and association between strain types (STs) and AMR genotypes and phenotypes. N. gonorrhoeae isolates (n=427) from Saskatchewan (2003-2011) were susceptible to antibiotics now recommended for treatment - cefixime, ceftriaxone and spectinomycin. Over 95% of the isolates tested were also susceptible to penicillin (96%) and ciprofloxacin (95.5%), antibiotics no longer recommended for treatment, and azithromycin (99.4%). Tetracycline resistance was also high (50.1%). N. gonorrhoeae isolates that were resistant to the antibiotics tested and also those isolates with MICs ≥0.003 mg/L to cefixime and ceftriaxone were analyzed (n=146) to determine their resistance mechanisms. This analysis revealed that reduced susceptibility to ceftriaxone and cefixime and resistance to penicillin is mediated by specific mutations in penicillin binding protein 2 (PBP2), in the promoter and dimerization domains of MtrR and porin protein (PorB). Novel mutations and combinations of mutations were noted. Ciprofloxacin resistant N. gonorrhoeae isolates carried double mutations in GyrA (S91F and D95G/N) and a S87R or S88P substitution in ParC. Isolates resistant to azithromycin had specific mutations in all the four alleles of 23S rRNA as well as in the DNA binding domain of MtrR. Most resistance was chromosomally mediated while plasmid-mediated resistance to penicillin (0.93% of penicillin resistant isolates) and tetracycline (3.3%) was low. DNA based strain typing methods such as porB-DNA sequencing, N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and multilocus sequence typing (MLST) showed that the gonococcal population in SK differs appreciably from both other Canadian provinces and from strains reported internationally. MLST analysis, which ascertains the evolution of isolates over time, demonstrated that penicillin and tetracycline resistant isolates in SK evolved through spontaneous mutations in established lineages. Ciprofloxacin and azithromycin resistant N. gonorrhoeae isolates, on the other hand, were introduced into SK from outside the province. Significant associations between particular mutation pattern combinations in resistance determining genes and specific NG-MAST STs were identified e.g. NG-MAST ST 25 was associated with specific combined mutation patterns in PBP2, MtrR and PorB and antibiotic susceptibility; and, NG-MAST ST 3654 was associated with another PBP2/MtrR/PorB mutation pattern, chromosomal resistance to penicillin and tetracycline and elevated MICs to cefixime. This research shows the importance of regional antimicrobial susceptibility monitoring. In the context of SK, this means that local surveillance of gonococcal AMR may be used to develop policies for regional treatment guidelines which promote the prudent use of antimicrobials for treatment, including those antibiotics which may no longer be used in other regions due to higher AMR rates. Further, the significant association between particular AMR mutation pattern combinations and specific STs indicates that AMR might be predicted. These results should assist in the development of non-culture-based tests for the diagnosis of gonococcal AMR similar to nucleic acid amplification tests used to diagnose N. gonorrhoeae infections.

Le cancer colorectal (CCR) représente un problème majeur de Santé publique. Des facteurs de risque environnementaux, ainsi que l'apparition séquentielle d'altérations génétiques et épigénétiques corrélant avec la progression tumorale ont été extensivement décrites. Cependant, les variations épigénétiques préexistant dans le tissu sain, potentiellement responsables de différences notoires de susceptibilité au CCR, sont restées élusives jusqu'à ce jour. Afin de répondre à cette problématique, la lignée murine Apcd14 (porteuse d'une mutation hétérozygote constitutive au niveau du gène Adenomatous polyposis coli) a servi de modèle au cours de ce projet. Les individus de cette lignée comportent une expressivité très variable, au sens qu'ils développent spontanément un nombre plus ou moins important de tumeurs intestinales, impactant sur leur survie. Cette hétérogénéité est observée en dépit de conditions d'élevage et de génomes considérés identiques. L'analyse exhaustive de cette lignée a conduit à y caractériser deux groupes de souris, présentant une gravité différente de phénotype. La variation d'expression génétique dans le tissu sain (i.e en amont de la tumorigenèse) a ensuite été analysée dans le but de comprendre l'établissement de cette hétérogénéité. Ceci a mené à la découverte d'une signature de gènes différemment exprimés entre les deux groupes, permettant de corréler de façon parfaite données moléculaires et phénotype. La potentielle héritabilité de cette signature a par la suite conduit à remettre en cause le statut considéré syngénique de la lignée. Les approches expérimentales effectuées ont déjà permis de cibler une région chromosomique, ce qui mènera à court terme à la caractérisation d'un nouvel acteur impliqué dans la tumorigenèse intestinale. De manière plus générale, les expériences développées durant ce projet ouvrent la voie à la notion de susceptibilité individuelle face au cancer dépendante de la variation d'expression génétique. / Colorectal cancer (CRC) is a major public health concern. Environmental clues, as well as sequential genetic and epigenetic alterations correlating with tumor progression have been extensively described. Meanwhile, pre-existing epigenetic variations in the healthy intestinal mucosa, potentially leading to differences in CRC suceptibility, have generally been overlooked. In order to answer to this question, we have made use of the Apcd14 mouse model (carrying a heterozygous mutation in the Adenomatous polyposis coli gene). Although all Apcd14 mice apparently share the same genome and are raised in the same environmental conditions, they exhibit a huge phenotypic variability at the individual level, spontaneously developping a few or numerous intestinal tumors, that ultimately results in differences in survival rates. Through detailed analysis of this strain, two groups have been characterized, exhibiting several phenotypic specificities. Gene expression analysis in the healthy intestinal tissue was then performed in order to understand the differences already existing, prior to tumorigenesis. This led to the identification of a group-specific gene expression signature, allowing a correlation between macroscopic phenotype and molecular data. Consideration of the hereditary potential of this signature led to reconsider the syngeneic status of the Apcd14 strain. Several experimental data already targeted a specific genomic region, and this will allow to identify the genetic alteration involved. More generally, this project opens the way to discover a link between individual susceptibility to intestinal tumorigenesis and gene expression variability.

Gonorrhea, caused by the human pathogen Neisseria gonorrhoeae, is a severe public health problem worldwide with more than 82 million new infections each year. N. gonorrhoeae is transmitted by sexual contact and primarily causes urogenital mucosal infections in men and women. Left untreated, this infection may cause severe complications, especially in females. Eye infections of the newborn can occur. Gonorrhea infections enhance HIV transmission. The highly prevalent antibiotic resistance and the emergence of new drug resistances render treatment of the infections increasingly difficult. Close monitoring of antimicrobial susceptibility of this pathogen is crucial, and enhanced knowledge of molecular mechanisms of gonococcal antimicrobial resistance is urgently needed. There are no vaccines available against N. gonorrhoeae. Control of gonorrhea relies on comprehensive strategies which can be better formulated by understanding, at molecular levels, how N. gonorrhoeae is transmitted in communities. My research aimed to illustrate the severe burden of antimicrobial resistance in N. gonorrhoeae temporally and geographically in China and to reveal the molecular mechanisms of antibiotic resistance particularly the development of reduced susceptibility to ceftriaxone in N. gonorrhoeae isolates. To determine specific strain distributions, N. gonorrhoeae isolates were characterized using molecular typing methods such as a modified porB-based typing scheme and the N. gonorrhoeae Multi-Antigen Typing (NG-MAST) method, compared to traditional epidemiological approaches. The ultimate goal was to provide information for better formulating disease control strategies for gonorrhea. In this research, male patients with gonorrhea and their sex partners were recruited in Shanghai (2005 and 2008) and in Urumchi (2007-2008), China. Epidemiological information pertaining to sexual contacts was collected. N. gonorrhoeae isolates were investigated for their antimicrobial susceptibility. Molecular mechanisms of antimicrobial resistance were explored by analysis of potential resistant determinants (gyrA, parC, porB, mtrR, ponA and penA). The molecular data were combined with bioinformatic analysis and traditional epidemiological data. High percentages of N. gonorrhoeae isolates (11% - 19% in Shanghai, 4.5% in Urumchi) exhibited reduced susceptibility to ceftriaxone (MICs = 0.125-0.25 mg/L), the first line drug recommended for the treatment of gonorrhea in China. The majority of isolates (>98%) were susceptible to spectinomycin, an alternative regimen for gonorrhea treatment; however, the proportion of isolates having intermediate levels of susceptibility increased from 1.9% in 2005 to 9.9% in 2008. The majority of isolates tested were resistant to penicillin (80% - 93%), tetracycline (56% - 65%) and ciprofloxacin (98% - 100%). Plasmid-mediated resistance in N. gonorrhoeae isolates were highly prevalent (51% - 79%) in Shanghai and Urumchi. Analysis of 60 clinical isolates revealed that reduced susceptibility to ceftriaxone is mediated by porB1b allele and is associated with specific mutations in penicillin binding protein 2 and in the DNA binding and dimerization domains of MtrR. Penicillin binding protein 1 is not involved in reduced susceptibility to ceftriaxone. Although mutation patterns in quinolone resistant determinant regions (QRDRs) varied, the majority of ciprofloxacin resistant isolates had double mutations in GyrA (S91F and D95G/A/N) and most isolates also carried a S87R/N mutation in ParC. The presence of mutations in the QRDR of ParC is correlated with elevated ciprofloxacin MICs. A modified porB-based molecular typing scheme was developed and involved ~82% of the DNA sequence of gonococcal porB. This typing method proved to have high discriminatory ability (index of discrimination = 0.93 0.96), and was cost effective and easy to perform as compared to the NG-MAST analysis. Using the modified porB-based typing method, N. gonorrhoeae isolates were reliably differentiated, and transmission clusters were identified. Molecular epidemiology using the porB-based method confirmed direct sexual connections and identified sexual networks otherwise unrevealed by the patient self-reporting or traditional case-tracing methods.

Gonorrhea, caused by the human pathogen Neisseria gonorrhoeae, is a severe public health problem worldwide with more than 82 million new infections each year. N. gonorrhoeae is transmitted by sexual contact and primarily causes urogenital mucosal infections in men and women. Left untreated, this infection may cause severe complications, especially in females. Eye infections of the newborn can occur. Gonorrhea infections enhance HIV transmission. The highly prevalent antibiotic resistance and the emergence of new drug resistances render treatment of the infections increasingly difficult. Close monitoring of antimicrobial susceptibility of this pathogen is crucial, and enhanced knowledge of molecular mechanisms of gonococcal antimicrobial resistance is urgently needed. There are no vaccines available against N. gonorrhoeae. Control of gonorrhea relies on comprehensive strategies which can be better formulated by understanding, at molecular levels, how N. gonorrhoeae is transmitted in communities. My research aimed to illustrate the severe burden of antimicrobial resistance in N. gonorrhoeae temporally and geographically in China and to reveal the molecular mechanisms of antibiotic resistance particularly the development of reduced susceptibility to ceftriaxone in N. gonorrhoeae isolates. To determine specific strain distributions, N. gonorrhoeae isolates were characterized using molecular typing methods such as a modified porB-based typing scheme and the N. gonorrhoeae Multi-Antigen Typing (NG-MAST) method, compared to traditional epidemiological approaches. The ultimate goal was to provide information for better formulating disease control strategies for gonorrhea. In this research, male patients with gonorrhea and their sex partners were recruited in Shanghai (2005 and 2008) and in Urumchi (2007-2008), China. Epidemiological information pertaining to sexual contacts was collected. N. gonorrhoeae isolates were investigated for their antimicrobial susceptibility. Molecular mechanisms of antimicrobial resistance were explored by analysis of potential resistant determinants (gyrA, parC, porB, mtrR, ponA and penA). The molecular data were combined with bioinformatic analysis and traditional epidemiological data. High percentages of N. gonorrhoeae isolates (11% - 19% in Shanghai, 4.5% in Urumchi) exhibited reduced susceptibility to ceftriaxone (MICs = 0.125-0.25 mg/L), the first line drug recommended for the treatment of gonorrhea in China. The majority of isolates (>98%) were susceptible to spectinomycin, an alternative regimen for gonorrhea treatment; however, the proportion of isolates having intermediate levels of susceptibility increased from 1.9% in 2005 to 9.9% in 2008. The majority of isolates tested were resistant to penicillin (80% - 93%), tetracycline (56% - 65%) and ciprofloxacin (98% - 100%). Plasmid-mediated resistance in N. gonorrhoeae isolates were highly prevalent (51% - 79%) in Shanghai and Urumchi. Analysis of 60 clinical isolates revealed that reduced susceptibility to ceftriaxone is mediated by porB1b allele and is associated with specific mutations in penicillin binding protein 2 and in the DNA binding and dimerization domains of MtrR. Penicillin binding protein 1 is not involved in reduced susceptibility to ceftriaxone. Although mutation patterns in quinolone resistant determinant regions (QRDRs) varied, the majority of ciprofloxacin resistant isolates had double mutations in GyrA (S91F and D95G/A/N) and most isolates also carried a S87R/N mutation in ParC. The presence of mutations in the QRDR of ParC is correlated with elevated ciprofloxacin MICs. A modified porB-based molecular typing scheme was developed and involved ~82% of the DNA sequence of gonococcal porB. This typing method proved to have high discriminatory ability (index of discrimination = 0.93 0.96), and was cost effective and easy to perform as compared to the NG-MAST analysis. Using the modified porB-based typing method, N. gonorrhoeae isolates were reliably differentiated, and transmission clusters were identified. Molecular epidemiology using the porB-based method confirmed direct sexual connections and identified sexual networks otherwise unrevealed by the patient self-reporting or traditional case-tracing methods.

No description available.