Philosophiae Doctor - PhD / This study has successfully identified transcripts differentially expressed in the salivary gland and midgut and provides candidate genes that are critical to response to parasite invasion. Furthermore, an open-source Glossina resource (G-ESTMAP) was developed that provides interactive features and browsing of functional genomics data for researchers working in the field of Trypanosomiasis on the African continent. / South Africa

Short open reading frames (sORFs) are codon sequences with a start and stop codon within atmost 100 codons. Cells produce many transcripts from them and some sORFs have been found to have function. sORFs have been associated with embryogenesis, myogenesis, immunity and various diseases including cancers. Cell culture screening is a common method to study function in sORFs. By inserting mutations in known sORF locations one can affect their translation by removing start codons, inserting premature stop codons, or removing native stop codons. A new tool set to do this isCRISPR technology, where single guide RNA (gRNA) can be used to make more precise genome edits. Unfortunately, such design is nontrivial and suggests a lot of variants for testing. It results in a back-and-forth testing process involving different available design tools. In this project, a comprehensive way was developed to see and iterate over the many test combinations. This intends to ease the process and decrease the likelihood for errors. The developed solution is a tool that integrates the currently best design tools. It also introduces a method in the form of a new quality summary score that can evaluate the estimated outcomes of the various designed guide variants. The tool was tested, and it was found that the score simplifies and amplifies the earlier usedscore methods. The pipeline is simple to install and use, integrates the currently most actively developed tools, and an installation is as future proof as can be made in a rapidly evolving field.

As novel or variant strains of HEV continue to evolve rapidly both in humans and other animals, it is important to develop a rapid pre-sequencing screening method to select field isolates for further molecular characterization. Two heteroduplex mobility assays (HMA) were developed to genetically differentiate field strains of swine HEV and avian HEV from known reference strains. It was shown that the HMA profiles generally correlate well with nucleotide sequence identities and with phylogenetic clustering between field strains and the reference swine HEV or avian HEV strains. Therefore, by using different HEV isolates as references, the HMA developed in this study can be used as a pre-sequencing screening tool to identify variant HEV isolates for further molecular epidemiological studies. Our previous study showed that avian HEV antibody is prevalent in apparently healthy chickens. A prospective study was conducted on a known seropositive but healthy chicken farm. Avian HEV was identified from the healthy chicken flock. Avian HEV isolates recovered from the healthy chicken share 70-97% nucleotide sequence identities with those isolates which cause hepatitis-splenomegaly (HS) syndrome based on partial helicase and capsid gene regions. Recovery of identical viruses from the experimentally inoculated chickens in the subsequent transmission study further confirmed our field results. The capsid gene of avian HEV isolates from chickens with HS syndrome were also characterized and found to be heterogeneic, with 76-100% nucleotide sequence identities to each other. The study indicates that avian HEV is enzootic in chicken flocks and spread subclinically among chicken populations, and that the virus is heterogeneic. As HEV can not be propagated <i>in vitro</i>, in order to further characterize avian HEV, an infectious viral stock with a known infectious titer must be generated. Bile and feces collected from specific-pathogen-free (SPF) chickens experimentally infected with avian HEV were used to prepare an avian HEV infectious stock. The infectivity titer of this infectious stock was determined, by intravenously inoculating one-week old SPF chickens, to be 5 x 10^4.5 50% chicken infectious doses (CID₅₀) per ml. Seroconversion, viremia as well as fecal virus shedding were observed in the inoculated chickens. Contact control chickens also became infected via direct contact with inoculated ones. Avian HEV infection in chickens was found to be dose-dependent. To determine if avian HEV can infect across species, one-week old SPF turkeys were intravenously inoculated each with 10^4.5(CID₅₀) of avian HEV. The inoculated turkeys seroconverted to avian HEV antibodies at 4-8 weeks postinoculation (WPI). Viremia was detected at 2-6 WPI, and fecal virus shedding at 4-7 WPI in inoculated turkeys. This is the first demonstration of cross-species infection by avian HEV. Little is known regarding the characteristics of the small ORF3 protein largely due to the lack of a cell culture system for HEV. To characterize the small protein, the ORF3 proteins of avian HEV and swine HEV were expressed in <i>Escherchia coli</i>, and purified by BugBuster His-Bind Purification System. Western blot analysis showed that avian HEV ORF3 protein is unique and does not share common antigenic epitopes with those of swine HEV and human HEV. However, swine HEV (genotype 3) and human HEV (genotype 1) ORF3 proteins cross-react with each other antigenically. To determine if the ORF3 protein is a virion protein, infectious stocks of avian HEV and swine HEV were first generated in SPF chickens and pigs, respectively. Virions were subsequently purified by sucrose density gradient centrifugation and virion proteins were characterized by SDS-PAGE and Western blot analysis. Two major forms of ORF2 proteins of avian HEV were identified: a 56 kDa and an 80 kDa proteins. Multiple immunoreactive forms of ORF2 proteins of swine HEV were also observed: 40 kDa, 53 kDa, 56 kDa and 72 kDa. However, the ORF3 protein was not detected from the native virions of avian HEV or swine HEV. These findings provide direct evidence that ORF2 indeed encodes a structural protein of HEV, whereas ORF3 does not. To search for other potential animal reservoirs for HEV, the prevalence of IgG anti-HEV antibody was determined in field mice caught in chicken farms to assess the possibility of mice as a potential reservoir for HEV infection in chickens. Three different recombinant HEV antigens derived from avian HEV, swine HEV, and human HEV were used in the ELISA assays. The anti-HEV seropositive rates in wild field mice (<i>Mus musculus</i>), depending upon the antigen used, are 15/76 (20%), 39/74 (53%), and 43/74 (58%), respectively. HEV RNA was also detected from 29 fecal and/or serum samples of mice. The HEV sequences recovered from field mice shared 72-100% nucleotide sequence identities with each other, 73-99% sequence identities with avian HEV isolates, and 51-60% sequence identities with representative strains of swine and human HEVs. However, attempts to experimentally infect laboratory mice (Mus musculus) with the PCR-positive fecal materials recovered from the wild field mice were unsuccessful. We also attempted to experimentally infect 10 Wistar rats each with avian HEV, swine HEV, and an US-2 strain of human HEV, respectively. However, the inoculated rats did not become infected as evidenced by the lack of viremia, virus shedding in feces or seroconversion. These data suggest that mice caught in chicken farms are infected by a HEV-like virus, but additional work is needed to determine the origin of the mouse virus as well as the potential role of rodents in HEV transmission. In summary, we developed two HMAs which are useful for differentiation and identification of variant strains of swine and avian HEVs. We genetically identified and characterized an avian HEV strain from apparently healthy chickens in seropositive flocks. We showed that avian HEV can cross species barriers and infect turkeys. Our data indicated that avian and swine HEV ORF2 genes encode structural proteins, whereas ORF3 genes do not. Evidence in this study also showed that HEV or HEV-like agent exists in field mice on a chicken farm. / Ph. D.

Introduction: Les cadres de lectures alternatifs (CLA) sont utilisés par de multiples virus afin de générer plusieurs protéines à partir d'une seule séquence nucléotidique. Les épitopes dits « cryptiques », c’est-à-dire les épitopes dérivés de protéines codées dans des CLAs, ont étés dernièrement l’objet de différentes études portant sur la réponse immunitaire antivirale et les lymphocytes T cytotoxiques. Méthodologie: Afin de vérifier le potentiel immunogène d'épitopes encodés dans des CLAs programmés, trois cassettes ont été construites pour mener à l'expression de trois épitopes bien caractérisés (épitope GAG77–85 du virus de l'immunodéficience humaine de type 1; épitope NS31406-1415 du virus de l'hépatite C; épitope core18-27 du virus de l'hépatite B) à partir de trois cadres de lectures superposés. La première cassette permet une initiation alternative de la traduction, la deuxième comprend deux signaux bipartites en tandem permettant un frameshift ribosomique et la troisième est une cassette contrôle. Ces éléments ont été introduits dans des vecteurs adénoviraux. Les virions générés ont servi à immuniser des souris C57BL/6 transgéniques pour HLA-A*0201 et HLA-DR1. La réponse immunitaire induite une semaine post-immunisation a été mesurée par essai ELISpot IFN . Résultats: Dans le contexte de cassettes vaccinales, les peptides dérivés d'une initiation alternative de traduction et de changement de cadre de lecture ribosomique ribosomal peuvent être exprimés et détectés par le système immunitaire dans un modèle animal. Conclusion: Ces expériences suggèrent la possibilité de développer de nouvelles stratégies vaccinales dans le but de prévenir ou de guérir certaines maladies associées aux infections virales chroniques telles que celles causées par le virus de l’immunodéficience humaine et le virus de l’hépatite C. / Introduction: Alternative reading frames (ARFs) are used by multiple viruses in order to generate different proteins from a single nucleotide sequence. Cryptic epitopes, which comprise antigens derived from proteins encoded in ARFs, have recently been the focus of studies pertaining to antiviral immunity and cytotoxic T lymphocytes. Methodology: In order to verify the immunological potential of epitopes encoded in programmed ARFs, three cassettes were constructed to permit the expression of three welldescribed epitopes (GAG77–85 epitope of human immunodeficiency virus type 1; NS31406-1415 epitope of hepatitis C virus; core18-27 epitope of hepatitis B virus) from three overlapping reading frames. The first cassette permits alternative translation initiation, the second cassette includes signals inducing ribosomal frameshifting and the third cassette serves as a control. These elements were introduced into adenoviral vectors. Recombinant adenoviruses were used to immunize C57BL/6 transgenic mice expressing HLA-A*0201 and HLA-DR1. The immune response induced was measured one week following immunization using IFN ELISpot assays. Results: In the context of vaccine cassettes, peptides derived from alternative translation initiation and ribosomal frameshifting can be expressed and detected by the immune system in an animal model. Conclusion: These findings suggest the possibility of designing vaccination strategies in the hope of preventing or curing certain diseases associated with chronic viral infections, such as those caused by human immunodeficiency virus and hepatitis C virus.

The CCAAT binding-factor (CBF) is a transcriptional activator conserved in eukaryotes. The CBF in Saccharomyces cerevisiae is a multimeric heteromer termed the Hap2/3/4/5 complex. Hap4, which contains the activation domain of the complex, is also the regulatory subunit and is known to be transcriptionally controlled by carbon sources. However, little is known about Hap4 regulation. In this report, I identify mechanisms by which Hap4 is regulated, including: (1) transcriptional regulation via two short upstream open reading frames (uORFs) in the 5' leader sequence of HAP4 mRNA; (2) proteasome-dependent degradation of Hap4; and (3) identification of two negative regulators of HAP4 expression, CYC8 and SIN4. I also report differential patterns of Hap4 cellular localization which depends on (1) carbon sources, (2) abundance of Hap4 protein, and (3) presence or absence of mitochondrial DNA (mtDNA).

Introduction: Les cadres de lectures alternatifs (CLA) sont utilisés par de multiples virus afin de générer plusieurs protéines à partir d'une seule séquence nucléotidique. Les épitopes dits « cryptiques », c’est-à-dire les épitopes dérivés de protéines codées dans des CLAs, ont étés dernièrement l’objet de différentes études portant sur la réponse immunitaire antivirale et les lymphocytes T cytotoxiques. Méthodologie: Afin de vérifier le potentiel immunogène d'épitopes encodés dans des CLAs programmés, trois cassettes ont été construites pour mener à l'expression de trois épitopes bien caractérisés (épitope GAG77–85 du virus de l'immunodéficience humaine de type 1; épitope NS31406-1415 du virus de l'hépatite C; épitope core18-27 du virus de l'hépatite B) à partir de trois cadres de lectures superposés. La première cassette permet une initiation alternative de la traduction, la deuxième comprend deux signaux bipartites en tandem permettant un frameshift ribosomique et la troisième est une cassette contrôle. Ces éléments ont été introduits dans des vecteurs adénoviraux. Les virions générés ont servi à immuniser des souris C57BL/6 transgéniques pour HLA-A*0201 et HLA-DR1. La réponse immunitaire induite une semaine post-immunisation a été mesurée par essai ELISpot IFN . Résultats: Dans le contexte de cassettes vaccinales, les peptides dérivés d'une initiation alternative de traduction et de changement de cadre de lecture ribosomique ribosomal peuvent être exprimés et détectés par le système immunitaire dans un modèle animal. Conclusion: Ces expériences suggèrent la possibilité de développer de nouvelles stratégies vaccinales dans le but de prévenir ou de guérir certaines maladies associées aux infections virales chroniques telles que celles causées par le virus de l’immunodéficience humaine et le virus de l’hépatite C. / Introduction: Alternative reading frames (ARFs) are used by multiple viruses in order to generate different proteins from a single nucleotide sequence. Cryptic epitopes, which comprise antigens derived from proteins encoded in ARFs, have recently been the focus of studies pertaining to antiviral immunity and cytotoxic T lymphocytes. Methodology: In order to verify the immunological potential of epitopes encoded in programmed ARFs, three cassettes were constructed to permit the expression of three welldescribed epitopes (GAG77–85 epitope of human immunodeficiency virus type 1; NS31406-1415 epitope of hepatitis C virus; core18-27 epitope of hepatitis B virus) from three overlapping reading frames. The first cassette permits alternative translation initiation, the second cassette includes signals inducing ribosomal frameshifting and the third cassette serves as a control. These elements were introduced into adenoviral vectors. Recombinant adenoviruses were used to immunize C57BL/6 transgenic mice expressing HLA-A*0201 and HLA-DR1. The immune response induced was measured one week following immunization using IFN ELISpot assays. Results: In the context of vaccine cassettes, peptides derived from alternative translation initiation and ribosomal frameshifting can be expressed and detected by the immune system in an animal model. Conclusion: These findings suggest the possibility of designing vaccination strategies in the hope of preventing or curing certain diseases associated with chronic viral infections, such as those caused by human immunodeficiency virus and hepatitis C virus.

Kaposi's sarcoma-associated herpesvirus (KSHV) expresses numerous intronless mRNAs that are unable to access splicing-dependent cellular mRNA nuclear export pathways. To circumvent this problem, KSHV encodes the open reading frame 57 (ORF57) protein, which orchestrates the formation of an export-competent virus ribonucleoprotein particle comprising the nuclear export complex hTREX, but not the exon-junction complex (EJC). Interestingly, EJCs stimulate mRNA translation, which raises the intriguing question of how intronless KSHV transcripts are efficiently translated. Herein, we show that ORF57 associates with components of the 48S pre-initiation complex and co-sediments with the 40S ribosomal subunits. Strikingly, we observed a direct interaction between ORF57 and PYM, a cellular protein that enhances translation by recruiting the 48S pre-initiation complex to newly exported mRNAs, through an interaction with the EJC. Moreover, detailed biochemical analysis suggests that ORF57 recruits PYM to intronless KSHV mRNA and PYM then facilitates the association of ORF57 and the cellular translation machinery. We, therefore, propose a model whereby ORF57 interacts directly with PYM to enhance translation of intronless KSHV transcripts.

Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs and variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved. This optimized method was used in an adapted ribosome-profiling approach to sequence mRNA footprints protected either by 80S monosomes or polysomes in S. cerevisiae. Contrary to popular belief, we show that 80S monosomes are translationally active as demonstrated by strong three-nucleotide phasing of monosome footprints across open reading frames. Most mRNAs exhibit some degree of monosome occupancy, with monosomes predominating on upstream ORFs, canonical ORFs shorter than ~590 nucleotides and any ORF for which the total time required to complete elongation is substantially shorter than the time required for initiation. Additionally, endogenous NMD targets tend to be monosome-enriched. Thus, rather than being inactive, 80S monosomes are significant contributors to overall cellular translation.