De nombreuses maladies infectieuses animales et zoonotiques font l’objet d’une surveillance chez l’animal. Dans certaines filières animales, plusieurs réseaux ou dispositifs sont mis en place sans qu’ils ne soient reliés ni coordonnés. De plus dans certains cas, plusieurs dispositifs surveillent les mêmes maladies mais de manière indépendante. Cette absence d’articulation peut conduire à un défaut d’efficacité des efforts de surveillance et à un coût global plus élevé. Notre travail s’est fixé pour objectif d’étudier en quoi l’interconnexion entre des dispositifs de surveillance déjà existants pourrait améliorer la surveillance des maladies infectieuses et comment mettre en œuvre une telle interconnexion.Pour tenter de répondre à cet objectif, nous avons pris comme support d’application trois systèmes de surveillance présents en France ayant pour objet des maladies infectieuses équines : l’anémie infectieuse des équidés, l’artérite virale équine et la métrite contagieuse équine. Nous avons adopté une démarche en trois étapes. Tout d’abord, une évaluation quantitative de la sensibilité de la surveillance a été menée à l’aide d’une méthode de capture-recapture. Deuxièmement nous avons évalué de manière semi-quantitative et comparative l’organisation et le fonctionnement général des trois systèmes de surveillance en identifiant les pistes d’interconnexion les plus pertinentes. Troisièmement, un atelier participatif réunissant une trentaine d’acteurs sanitaires et professionnels a été conduit afin d’évaluer et de hiérarchiser les solutions d’interconnexion. Ces travaux ont permis de fournir une vision claire et détaillée des qualités et des défauts des systèmes de surveillance. Ils ont aussi abouti à des recommandations d’interconnexion concrètes, ayant recueilli une adhésion large de la part des acteurs de la surveillance et de la filière, et pour lesquelles les bénéfices attendus et les niveaux de faisabilité, d’acceptabilité et de priorité ont été évalués. Ces travaux successifs ont également permis d’impliquer progressivement dans le processus un grand nombre d’acteurs et de bénéficiaires de la surveillance sanitaire équine. Afin d’enrichir cette démarche de préparation et d’accompagnement à la mise en œuvre d’une interconnexion, nous proposons d’y intégrer à l’avenir des travaux complémentaires éventuellement à conduire selon les besoins recensés, tels que des évaluations économiques, sociales ou multicritères. / Numerous animal and zoonotic infectious diseases are monitored in animals. In many animal industries, several epidemiological surveillance systems or components are implemented but neither connected nor coordinated. Furthermore, in some cases, several components monitor the same diseases in an independent way. This lack of coordination may impair the efficiency of the surveillance and increase the global costs. Our objective was to investigate how the interconnection between existing surveillance systems could improve the infectious diseases surveillance and how to implement such an interconnection.To address this question, we studied three French surveillance systems of equine infectious diseases: equine infectious anaemia, equine viral arteritis and equine contagious metritis. We used a three-step approach. First, we evaluated quantitatively the surveillance sensibility by using a capture-recapture method. Secondly, we assessed in a semi-quantitative and comparative way the organization and operation of the three systems and we identified the most relevant ways for interconnection. Thirdly, we organized a participative workshop gathering thirty stakeholders involved in equine healthcare and/or equine industry to estimate and prioritize the ways of interconnection. These studies have accurately underlined the strengths and weaknesses of the surveillance systems and they have resulted in practical recommendations of interconnection. The professionals from equine industry and the specialists in epidemiological surveillance involved in this work have approved and supported these recommendations. Feasibility, acceptability, priority and expected benefits of the recommendations were estimated. Our successive works have allowed to gradually involve numerous actors and beneficiaries of the surveillance in the interconnection process. In order to improve and enhance this approach -designed to help the preparation and the implementation of an interconnection- we propose to include optional complementary studies, such as economic, social and/or multicriteria evaluations.

Myeloid-derived suppressor cells (MDSC) expand during sepsis, suppress both innate and adaptive immunity, and promote chronic immunosuppression, which characterizes the late/chronic phase of sepsis. We previously reported that the transcription factors Stat3 and C/EBPb synergize to induces the expression of microRNA (miR)-21 and miR-181b to promote MDSC expansion in a mouse model of polymicrobial sepsis that progresses from an early/acute proinflammatory phase to a late/chronic immunosuppressive stage. We also showed that Gr1+CD11b+ cells, the precursors of MDSCs, from mice genetically deficient in the inflammatory protein S100A9 lack miR-21 or miR-181b in late sepsis, and are not immunosuppressive. In the present study, we show that S100A9 induces miR-21 and miR-181b during the late sepsis phase. We find that S100A9 associates with and stabilizes the Stat3-C/EBPb protein complex that activates the miRNA promoters. Reconstituting Gr1+CD11b+ cells from the S100A9 knockout mice with late sepsis with S100A9 protein restores the Stat3-C/EBPb protein complex and miRNA expressions, and switches the Gr1+CD11b+ cells into the immunosuppressive, MDSC phenotype. Importantly, we find that this process requires IL-10 mediated signaling, which induces S100A9 translocation from the cytosol to the nucleus. These results demonstrate that S100A9 promotes MDSC expansion and immunosuppression in late/chronic sepsis by inducing the expression of miR-21 and miR-181b.

Mycobacterium tuberculosis (Mtb) causes human tuberculosis, and more people die of it than of any other pathogen in the world. Immunodominant antigens elicit the large majority of T cells during an infection, making them logical vaccine candidates. Yet, it is still unknown whether these immunodominant antigen-specific T cells recognize Mtb-infected cells. Two immunodominant antigens, TB10.4 and Ag85b, have been incorporated into vaccine strategies. Surprisingly, mice vaccinated with TB10.4 generate TB10.4-specific memory CD8+ T cells but do not lead to additional protection compared to unvaccinated mice during TB. Ag85b-specific CD4+ T cells are also generated during vaccination, but the literature on whether these cells recognize Mtb-infected cells is also inconsistent. We demonstrate that TB10.4-specific CD8+ T cells do not recognize Mtb-infected cells. However, under the same conditions, Ag85b-specific CD4+ T cells recognize Mtb-infected macrophages and inhibit bacterial growth. In contrast, polyclonal CD4+ and CD8+ T cells from the lungs of infected mice can specifically recognize Mtb-infected macrophages, suggesting macrophages present antigens other than the immunodominant TB10.4. The antigen location may also be critical for presentation to CD8+ T cells, and live Mtb may inhibit antigen presentation of TB10.4. Finally, we propose that TB10.4 is a decoy antigen as it elicits a robust CD8+ T cell response that poorly recognizes Mtb-infected macrophages, allowing Mtb to evade host immunity.

The rapid development of DNA sequencing technologies has opened up new avenues of research, including the investigation of population structure within infectious diseases (both within patient and between populations). In order to take advantage of these advances in technologies and the generation of new types of data, novel bioinformatics tools are needed that won’t succumb to artifacts introduced by the data generation, and thus provide accurate and precise results. To achieve this goal I have create several tools. First, SeekDeep, a pipeline for analyzing targeted amplicon sequencing datasets from various technologies, is able to achieve 1-base resolution even at low frequencies and read depths allowing for accurate comparison between samples and the detection of important SNPs. Next, PathWeaver, a local haplotype assembler designed for complex infections and highly variable genomic regions with poor reference mapping. PathWeaver is able to create highly accurate haplotypes without generating chimeric assemblies. PathWeaver was used on the key protein in pregnancy-associated malaria Plasmodium falciparum VAR2CSA which revealed population sub-structuring within the key binding domain of the protein observed to be present globally along with confirming copy number variation. Finally, the program Carmen is able to utilize PathWeaver to augment the results from targeted amplicon approaches by reporting where and when local haplotypes have been found previously. These rigorously tested tools allow the analysis of local haplotype data from various technologies and approaches to provide accurate, precise and easily accessible results.

Abstract Background Measles is a serious infectious disease that contributes significantly to the burden of disease in many developing countries. In most developed nations, such as Canada, endemic transmission of measles has been declared eliminated thanks to rigorous vaccination programs, but isolated outbreaks of the disease continue to happen. Therefore, a thorough understanding of the factors contributing to these outbreaks is necessary. Objectives There were two main objectives of this thesis. The first objective was to assess the geospatial distribution of reported measles cases in Ontario with a goal of identifying clusters of reported measles. For this objective, the main hypothesis was that measles cases would not be randomly distributed across Ontario and instead would cluster in certain regions. The second objective was to explore some of the factors that may be associated with measles clusters. For this objective, the main hypothesis was that the proportion of immigrants, population density, low-income prevalence and education level would be associated with measles clusters. Methods The first objective was achieved through a thorough geospatial analysis using SaTScan and R. Individual forward sortation areas were used as the spatial unit of analysis. The analysis leveraged data from multiple sources: 2016 Census data, Ontario measles cases data from iPHIS from 2008 to 2019, a shapefile of all forward sortation areas in Canada from Statistics Canada and centroid coordinates of forward sortation areas that were obtained using web scrapping techniques on the geolocation service of Natural Resources Canada. The maximal window size of the geospatial analysis was chosen using the maximum clustering heterogeneous set-proportion technique. The geospatial analysis was run with 99,999 Monte Carlo repetitions under a Poisson distribution using the purely spatial analysis. The Ontario population from the 2016 Census was used as the population at risk. Any cluster with a p ≤ 0.05 was deemed statistically significant. The second objective was achieved through a case-control study: Forward sortation areas that were within statistically significant measles clusters were considered as cases and the rest of the forward sortation areas were considered as controls. Demographic data necessary to assess the factors of interest were extracted from the 2016 Census. A univariable logistic regression model was run to compute the odds ratio and test the association between the factors of interest and measles clusters. 95% confidence intervals were computed for each odds ratio. Data-curation techniques and data analysis were performed in R 4.0.4. Results From 2008 through 2019, 178 measles cases were identified. 82% of cases lacked necessary vaccination or vaccination records against measles, 35% of cases were linked to traveling outside of Ontario, 20% of cases reported being in contact with a known case, and 72% of cases were less than 5 years old or older than 21. Ten measles clusters were identified of which six were deemed statistically significant. These six significant clusters represented 7% of the population at risk but contained nearly 40% of all reported measles cases between 2008 and 2019. Measles clusters had a strong association with the proportion of immigrants living within them, population density and prevalence of low-income. No association was found between education level and measles clusters. Conclusion The results indicate that most measles cases in Ontario are unvaccinated or lack proof of vaccination; arise through secondary transmission within the province; arise from undetected transmission; and are adults or infants. Additionally, it is possible to see that the risk of reported measles cases is not randomly distributed across the province, but instead measles cases tend to cluster in certain regions. Such clusters tend to be characterized by specific population-level factors that may be contributing to the risk of reported measles. Targeted and equitable interventions are needed as we continue on the path to eradication.

Les réactivations à Polyomavirus, BK-virus (BKv) et JC-virus (JCv), sont des complications majeures en transplantation rénale, responsables de néphropathie à BKv (Nx BKv) et de leuco-encéphalopathie multifocale progressive (LEMP). Sans thérapeutique antivirale spécifique, ces infections virales menent à la perte du rein transplanté ou au décès du patient. Notre groupe a conduit une étude observationnelle incluant 100 patients transplantés rénaux avec différents niveaux de réactivation BKv (Etude MelTyK). Nous avons mis en évidence une altération progressive de la fonctionnalité des lymphocytes T spécifiques du BKv, associée à une corrélation inverse entre la polyfonctionnalité lymphocytaire ou le nombre d’incompatibilités HLA et la charge virale BKv plasmatique. Cette altération de la fonctionnalité suggérait un état d’épuisement des lymphocytes T spécifiques du BKv en fonction du niveau de réactivation BKv. Ces données nous ont conduit à élaborer une méthode biologique non-invasive d’évaluation du risque individuel de Nx BKv (brevet FR1855342). Cette méthode a pour objectif d’aider au diagnostic de Nx BKv sans avoir recours à la biopsie du greffon rénal et de stratifier le risque de développer cette complication. Par ailleurs, nous avons décrit un cas fatal de LEMP associé à un état d’anergie des lymphocytes T spécifiques du JCv. L’étude de la fonctionnalité des lymphocytes T spécifiques des Polyomavirus pourrait ouvrir de futures pistes diagnostiques et/ou thérapeutiques. Elle pourrait permettre de dépister les patients à risques de Nx BKv et pourrait contribuer au développement d’immunothérapies innovantes. La restauration de la fonctionalité des lymphocytes T spécifiques des Polyomavirus pourrait ainsi fournir une piste thérapeutique prometteuse afin de contrôler ces réactivations virales sans majorer le risque de rejet allogénique. / Polyomavirus reactivations, BK-virus (BKv) and JC-virus (JCv), are major complications in kidney transplantation, responsable of BKv associated nephropathy (BKvAN) and progressive multifocal leukoencephalopathy (PML). Without antiviral treatment, these viral reactivations lead to kidney transplant loss or patient death. Our group has headed an observational study including 100 kidney transplant recipients with different BKv reactivation levels (the MelTyK study). We were able to highlight a gradual loss of functional BKv-specific T cells, associated with an inverse correlation between lymphocyte functionality or HLA mismatches and plasmatic BKv viral load. This functional impairment suggested an exhaustion of BKv-specific T cells according to BKv reactivation levels. These data have led us to develop a non-invasive biological method to assess the individual BKvAN risk (patent FR1855342). This method is intended to help the BKvAN diagnosis, without renal graft biopsy and to stratify the risk to develop this complication. Moreover, we have described a fatal case of PML associated with a anergy state of the JCv-specific T cells. Functional assessment of Polyomavirus-specific T cells could help to propose new diagnostic assays and immunotherapy approaches. Functional restauration of Polyomavirus-specific T cells could provide a promising therapeutic approache to control viral reactivations without increase of allogenic rejection risk.

Les bactéries pathogènes intracellulaires manipulent les fonctions de la cellule hôte en sécrétant des facteurs de virulence (qu'on appelle effecteurs) dans le cytoplasme de la cellule infectée. Ce processus permet au pathogène de proliférer dans un environnement autrement hostile. L'identification et la caractérisation des effecteurs spécifiques des divers agents pathogènes est donc d'une importance cruciale pour contrer les infections bactériennes. Coxiella burnetii est un agent pathogène à Gram négatif de classe 3, responsable de la Fièvre Q, une zoonose qui entraîne des épidémies majeures, avec un fort impact sur l'économie et la santé. Les réservoirs naturels de Coxiella sont principalement les animaux d’élevage qui peuvent contaminer l’environnement en excrétant la bactérie principalement dans les produits de parturition, le mucus vaginal et les fèces. L’Homme s’infecte ensuite par inhalation de pseudo-spores disséminées dans l’environnement. La nature intracellulaire obligatoire de Coxiella a jusqu'ici sévèrement limité son étude et par conséquent, les facteurs de virulence bactériens impliqués dans le développement et la progression de l'infection restent encore largement inconnus. Coxiella se réplique à l'intérieur des cellules hôtes dans une grande vacuole présentant des caractéristiques autolysosomales. Le développement de la vacuole et la survie de Coxiella dans la cellule hôte sont dépendants de la translocation des effecteurs bactériens par un système de sécrétion de type 4 (SST4) Dot/Icm et de la manipulation de nombreuses voies de trafic et de signalisation de la cellule hôte par ces derniers. Notre équipe a généré et criblé la première banque de mutants par transposition de Coxiella, menant ainsi à l'identification d'un nombre important de potentiels déterminants de virulence et de protéines effectrices. Mon projet de thèse est basé sur la caractérisation de deux effecteurs de Coxiella, CvpF et AnkA, provenant de la banque de mutants générée par l’équipe. Les mutants de ces effecteurs présentent des phénotypes de défaut de réplication intracellulaire et de développement de vacuole. Ici, nous démontrons que l’effecteur CvpF est un substrat du système de sécrétion de type 4 Dot/Icm qui localise aux vacuoles contenant Coxiella (CCV). CvpF est également capable d'interagir avec Rab26, conduisant au recrutement du marqueur autophagosomal LC3B aux CCV. Les mutants de cvpF présentent un défaut de réplication in vitro et in vivo, suggérant que le détournement de l'autophagie par cet effecteur est crucial pour la virulence de Coxiella. Comme pour les mutants de cvpF, les mutants ankA présentent le même défaut de réplication in vitro et la protéine AnkA est un substrat du SST4. L'effecteur AnkA contient des motifs de répétition Ankyrin localisés sur son domaine N-terminal. La bactérie induit une hyperfusion des mitochondries de manière dépendante du SST4 et spécifique de l’effecteur AnkA. Nos résultats montrent que AnkA interagit avec Drp1, une protéine motrice impliquée dans la fission mitochondriale et que cette interaction ainsi que l’hyperfusion des mitochondries seraient dépendant du domaine contenant les répétitions Ankyrine. Le mécanisme par lequel AnkA agit sur Drp1 reste à déterminer. Cependant, les effets observés sur la mitochondrie suggèrent que la manipulation de l’organelle par la bactérie promeut le développement de la vacuole et la réplication intracellulaire du pathogène. En conclusion, notre recherche suggère fortement que de nombreux effecteurs de Coxiella manipulent les voies des cellules hôtes pour assurer le développement intracellulaire efficace de ce pathogène. / Intracellular pathogenic bacteria manipulate host cell functions by secreting virulence factors (known as effectors) into the cytoplasm of the infected cell. This process allows the pathogen to proliferate in an otherwise hostile environment. The identification and characterization of the specific effectors of the various pathogens is therefore of crucial importance to counteract bacterial infections. Coxiella burnetii is a Class 3 gram-negative pathogen that causes Q fever, a zoonosis that causes major epidemics with a high impact on the economy and health. The natural reservoirs of Coxiella are mainly farm animals that can contaminate the environment by excreting the bacteria mainly in parturition products, vaginal mucus and feces. Human is then infected by inhalation of pseudo-spores disseminated in the environment. The obligate intracellular nature of Coxiella has so far severely limited its study, and as result, bacterial virulence factors involved in the development and progression of infection remain largely unknown. Coxiella replicates within host cells in a large vacuole with autolysosomal characteristics. The development of vacuole and survival of Coxiella in the host cell depend on the translocation of bacterial effectors by the type 4 Dot / Icm secretion system (SST4B) and the manipulation of many trafficking and signaling pathways of the host cell. Our team has generated and screened the first library of Coxiella transposon mutants, leading to the identification of a significant number of candidate virulence determinants and effector proteins. My thesis project is based on the characterization of two effectors of Coxiella, CvpF and AnkA, from the mutant library generated by the team. Mutants of these effectors exhibit defect in intracellular replication and vacuole development phenotypes. Here, we demonstrate that the effector CvpF is a substrate of the SST4B that localizes to vacuoles containing Coxiella (CCV). CvpF is also able to interact with Rab26, leading to the recruitment of the LC3B autophagosomal marker to CCV. cvpF mutants exhibit in vitro and in vivo replication deficiencies, suggesting that diversion of autophagy by this effector is crucial for Coxiella virulence. As for cvpF mutants, ankA mutants show the same in vitro defect of replication and the protein AnkA is a substrate of the SST4. AnkA contains Ankyrin repetition patterns located on its N-terminal domain. The bacterium induces an AnkA-dependent hyperfusion of mitochondria. Our results show that AnkA interacts with Drp1, a motor protein involved in mitochondrial fission, and that this interaction as well as mitochondrial hyperfusion is dependent on the domain containing Ankyrin-repeat motifs. The mechanism by which AnkA acts on Drp1 remains to be determined. However, the observed effects on mitochondria suggest that the organelle's manipulation by the bacterium promotes the development of the vacuole and the intracellular replication of the pathogen. To conclude, our research strongly suggests that multiple Coxiella effectors manipulate host cell pathways to ensure the efficient intracellular development of this pathogen.

<p>A successful flaviviral life cycle involves several coordinated events between viral proteins and host factors. The polyprotein processing at the surface of the ER membrane results in the formation of several replication proteins that bring about changes in the ER membrane making it permissive for viral genome amplification. Non-structural proteins 4A (NS4A) and non-structural protein 4B (NS4B) are two of the most important integral membrane proteins of DENV that are essential part of the viral replicase complex. The cleavage at NS4A-2K-NS4B is temporally and spatially regulated. The cleavage at the N-terminal of 2K is carried out by viral NS2B/3 protease while host signalase cleaves on the C-terminal side at the ER lumen to give rise to a mature NS4B protein. This thesis primarily focuses on demonstrating the function of 2K as an independent peptide rather than simply a signal sequence, and the role 2K plays, when present as 2K-NS4B vs NS4B. Moreover, this thesis has attempted to explore the function of transmembrane domains (TMDs) in replication separating them from their membrane anchor function. This thesis will also describe the development of a ZIKV replicon and its use in screening small molecule inhibitors in the last chapter.</p><p>In Chapter 2 of the thesis, we established 2K as an independent, information carrying peptide rather than just a signal peptide. A strategy involving chimeric virus generation and mutational analysis supported the notion that 2K is rather unique and important for viral replication and infectious particle production. Using an interserotypic 2K chimeric virus, it was established that the 2Ks of DENV are serotype specific, however, they are interchangeable with a huge fitness cost in infectious particle production. We further showed that individual amino acid residues towards then end of h-region and C-terminus of the 2K peptide affect viral replication and infectious particle production. Moreover, it was shown that the 2K peptide consists of a highly conserved ‘DNQL’ region at its N-terminal that plays an important role in viral replication.</p><p>Chapter 3 details the mechanistic aspect of the effects observed in interserotypic 2K chimeric viruses. The interserotypic chimeric viruses were comparable to wild type in replication, however, they were deficient in infectious particle production early in the life cycle. The major change to be noted in the chimeric viruses was the absence of signalase cleavage at the 2K-NS4B junction. We demonstrated that in a virus infected system, 2K-NS4B and NS4B populations are always present which led us to look for any specific functions of the cleaved vs uncleaved 2K-NS4B protein. Using a transcomplementation system where NS4B was presented in the absence of 2K, we showed that particle production can be rescued in the interserotypic 2K chimeric viruses. It was further concluded using NS4B truncations that the property of NS4B to rescue particle production was concentrated in the ER luminal loop. Further, alanine scanning mutagenesis of the conserved residues of ER loop resulted in pinpointing T198 and its involvement in the early stages of viral packaging.</p><p>Chapter 4 examined the role of TMDs of NS4A and NS4B and attempted to define their roles separately from their membrane anchoring functions. Several interserotypic TMD chimeric viruses were generated to address the function of these domains. We concluded that TMD1 and TMD3 of NS4A could be replaced with partial success across the DENV serotypes, whereas, TMD2 was serotype specific. The specificity of TMD2 of NS4A is not contributed by a single amino acid and should be a function of the secondary structure formed by TMD2 as it sits on the inner leaflet of the ER membrane. We demonstrated the variable roles different TMDs of NS4B play in viral replication using a similar strategy of reverse genetics of chimeric viruses. TMD1 of NS4B was replaceable with no to minimal effect, whereas, the remaining four showed variable effect upon substitution. More importantly, we demonstrated how the reorientation of TMD5 of NS4B post NS2B/3 cleavage might vary in different serotypes of DENV using revertant virus obtained from the TMD5 interserotypic chimera. Analysis of interserotypic cytosolic and ER luminal loop chimeras of NS4B pointed to functional conservation of the cytosolic loop between DENV-2 and DENV-3, whereas, the remaining cytosolic loops and the ER loops showed variable level of defects upon substitution, suggesting their functions in serotype-dependent manner.</p><p>Chapter 5 describes the construction and characterization of a ZIKV replicon system and use of it to screen several small molecule inhibitors of the flaviviruses MTase. Several small molecule inhibitors of flavivirus N-7-MTase were designed/synthesized in Dr. Arun K Ghosh’s lab which would target the extra pocket unique to the flavivirus SAM-binding site. We analyzed the docking of a set of these compounds into MTase domain of NS5 of ZIKV, DENV and YFV and screened them for their ability to inhibit replication of ZIKV, DENV and YFV. A huge variation in the activity profile of these compounds were observed against different flaviviruses even though these compounds were targeted against the highly conserved MTase domain of flavivirus NS5. GRL-002- and GRL-004-16-MT specifically inhibited ZIKV replication with low micromolar IC₅₀ value, while these compounds showed little to no effect on DENV and YFV.<b> </b>On the other hand, compounds GRL-007-, GRL-0012- and GRL-0015-16-MT demonstrated a dual inhibitory effect against DENV and YFV albeit the CC₅₀ values of the GRL-012 and GRL-015 were concerning. Compounds GRL-007-16-MT showed broad spectrum activity against ZIKV, DENV and YFV even though it was slightly cytotoxic to Vero cells. Moreover, GRL-002-16 was inhibitory to YFV while ineffective against DENV, whereas, GRL-016-16 had the opposite effect. Our results reveal the differential efficacies of the small molecule inhibitors targeting N-7-MTase. The experimental data suggests these compounds have different cytotoxicities in different cell lines and the compounds act in a virus-specific way. Nonetheless, we were able to shortlist some potent compounds for future modifications.</p>

<div> <div> <div> <p>Flaviviruses is a genus within the family Flaviviridae. The genus consists of more than 70 viruses, including important threatening human pathogens such as dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV). These viruses are causative agents for a range of mild to lethal diseases and there are currently no US- licensed therapeutic treatments for infection. The virus genome is a positive-sense, single-stranded RNA, encoding ten viral proteins. Of the ten flavivirus proteins, Non- Structural protein 1 (NS1) remains the most elusive in terms of its functions. To date NS1 has been linked to disease pathology and progression and plays roles in virus replication and assembly. However, little is understood how NS1 orchestrates these functions and how NS1 from different viruses function distinctively from one another. Moreover, flavivirus NS1 has a peculiar ability to associate with lipid membranes. During the life cycle of NS1, the protein travels through the classical secretory path- way, similar to infectious virus particles, and is secreted into the extracellular space as mostly hexameric oligomers containing a lipid core. How the protein binds to lipids and whether such lipid binding is important for NS1 functions and overall flavivirus pathology remain unknown. Using structure-based mutagenesis, we found a group of mutants on WNV NS1, which particularly altered the viral specific infectivity but maintained wild-type level of virus replication. Purified mutated virus particles revealed that the specific infectivity alteration was not because of the particle but interaction of the virus particles and NS1 mutated proteins. Here we demonstrated that specific residues on NS1 were responsible for distinctly roles in NS1 functions and the virus specific infectivity was regulated by NS1 protein. In other structure-base study, we focused on the membrane association ability of NS1. All structure-predicted regions on NS1 were examined for its contribution for the membrane/lipid binding function. This interaction was required for NS1 biology activities including intracel- lular trafficking, oligomerization, and endocytosis. The lipidomes from deletion of each membrane association region revealed differences in lipid classes binding to each region and the composition flexiblity of the lipid cargo of NS1 hexamer. </p> </div> </div> </div>

Malaria is one of the most serious tropical infectious diseases affecting mankind. The prevention of the disease is hampered by the increasing resistance of the parasite to existing chemotherapy and -prophylaxis drugs. The need for novel therapeutic targets and drugs is therefore enormous and the understanding of the biochemistry of the parasite is imperative. The aim of this study was the identification and molecular characterisation of the eDNA of one such metabolic target protein, ornithine decarboxylase (ODC), in the human malaria parasite P. falciparum. The P. falciparum ODC eDNA was isolated by means of a modified RT-PCR technique, RACE. No sequence data were available and the primers used were based on consensus areas identified in the protein sequences from other related organisms. The isolation and identification of the eDNA with degenerate primers was successful in 3' -RACE, but necessitated the optimisation of the eDNA synthesis protocol and the use of total RNA as starting material. The sequence obtained facilitated the application of 5' -RACE with ODC-specific primers based on the 3' -RACE sequence data. The full-length ODC eDNA sequence was obtained by overlap-alignment of various segments. A novel suppression PCR technology was applied during the 5' -RACE in order to create an uncloned eDNA library of amplified cDNAs representing only the mRNA population. The P. falciparum ODC eDNA contains an open reading frame of ---2847 bp and translates to a large 939 amino acid protein. The protein contained large internal insertions and was extended by '""273 N-terminal residues compared to ODCs from other organisms. Several possible signature motifs were identified for phosphorylation, glycosylation and transamidation. The P. falciparum ODC protein seems to contain more hydrophilic and a-helix forming residues. These characteristics should be further investigated after expression of the recombinant protein. The isolation of the P. falciparum ODC eDNA facilitates the validation of this protein as an antimalarial target. / Dissertation (MSc)--University of Pretoria, 1998. / gm2014 / Biochemistry / unrestricted