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Hepatitis E virus (HEV) is an important human pathogen, with pigs and likely other animal species serving as natural reservoirs. There are currently four recognized HEV genotypes that infect humans within the genus Hepevirus of the family Hepeviridae. Genotypes 1 and 2 are human viruses that are associated with waterborne and fecal-oral transmission in developing countries, while genotypes 3 and 4 have been identified in humans and other animal species and are zoonotic and endemic in both industrialized and developing countries. In my dissertation research, we identified the first strain of HEV from rabbits in the United States. We subsequently determined the complete genome sequence of the virus. Phylogenetic analyses of the full-length sequence indicated that U.S. rabbit HEV is a distant member of the zoonotic genotype 3, thus raising a potential concern for zoonotic infection. In order to investigate the cross-species potential of rabbit HEV, we then determined its antigenic cross-reactivity with other animal strains of HEV. Additionally, we demonstrated that the novel rabbit HEV could cross species barriers and infect pigs under experimental conditions. Finally, we attempted to determine the risk factors and sources of foodborne HEV infection in the United States. We detected HEV for the first time from non-liver pork commercial products in the United States and demonstrated consumption of undercooked meat a risk factor for HEV infection. HEV sequences of genotype 3 origin were detected from pork products purchased from grocery stores in Southwest Virginia. Approximately 6.3% (21/335) of university students tested seropositive for HEV antibodies and, importantly, those with a history of consuming undercooked meats were 13 times more likely to be seropositive. These results further underscore the importance of cooking pork thoroughly and using proper hygiene when preparing meals. / Ph. D.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an emerging virus of global distribution. At least four distinct genotypes of HEV exist worldwide: genotype 1 and 2 HEV strains are responsible for waterborne epidemics; genotype 3 and 4 HEV strains are responsible for sporadic occurrences of acute hepatitis E. Genotype 3 and 4 HEVs are zoonotic and have a more expanded host range than genotypes 1 and 2 which are restricted to humans. Genotype 3 and 4 HEV isolates obtained from animal tissues are genetically very similar, or identical in some cases, to human HEV recovered from hepatitis E patients. The objectives of this dissertation research were to assess the zoonotic foodborne transmission of HEV and elucidate the viral determinants of HEV host range. To determine the risk of HEV foodborne transmission, 127 packages of commercial pig liver were tested for HEV RNA. Eleven percent of them were positive for HEV RNA and the contaminating virus remained infectious. We also demonstrated that medium-to-rare cooking condition (56°C) does not completely inactivate HEV, although frying and boiling of the contaminated livers inactivated the virus. To reduce the risk of foodborne HEV transmission, commercial pig livers must be thoroughly cooked for consumption. To determine the host range of genotype 4 HEVs, pigs were inoculated with a genotype 4 human HEV. All pigs developed an active HEV infection indicating that genotype 4 human HEVs can cross species barriers and infect pigs. To identify viral determinant(s) of species tropism, ORF2 alone or in combination with its adjacent 5′ junction region (JR) and 3′ non-coding region (NCR), were swapped between genotypes 1 and 4, 3 and 4, and 1 and 3 to produce 5 chimeric viruses. Chimeric viruses containing ORF2 or JR+ORF2+3' NCR from genotype 4 human HEV in the backbone of genotype 3 swine HEV were viable in vitro and infectious in vivo. Chimeric viruses containing the JR+ORF2+3'NCR of genotypes 3 or 4 HEV in the backbone of genotype 1 human HEV were viable in vitro but non-infectious in pigs, suggesting that ORF1 may also be important for host range. / Ph. D.

Les virus zoonotiques sont une menace pour les humains à cause de leur capacité à passer des réservoirs animaux à l'homme. La rage est provoquée par un virus zoonotique (Virus de la Rage) qui a de multiples réservoirs animaux. La rage est contractée lors de la morsure par un animal infecté et est incurable et létale après l'apparition des premiers symptômes. Un défi visant à éradiquer la rage chez les chiens à l'horizon 2030 a été proposé. Il imposera de stopper la transmission du virus aux populations de chiens à partir des autres réservoirs animaux. Pour cela, il est essentiel de comprendre les mécanismes de transmission entre hôtes potentiels du virus. Dans notre thèse, nous faisons l'hypothèse que le passage d'un hôte à un autre est lié à la diversité des populations virales chez un hôte donné, appelée "hétérogénéité virale".Pour étudier cette hétérogénéité virale, des méthodes de séquençage des populations virales ont été développées. La transmission du virus de la rage entre chiens a été analysée et un évènement de transmission entre chien et renard a été étudié. Une plus importante hétérogénéité virale a été observée chez le renard après sa contamination par le chien en comparaison avec d'autres renards infectés par des congénères de la même espèce. Ceci suggère que l'hétérogénéité virale est importante dans le phénomène de transmission inter-espèce. Ces résultats sont importants pour améliorer notre compréhension de l'évolution du virus de la rage chez un nouvel hôte et pourront aider les efforts d'éradication de la maladie. / Zoonotic viruses are a threat to humans, jumping from animal reservoirs into humans. Rabies is caused by rabies virus (RABV), a zoonotic virus, with many animal reservoirs. Rabies is contracted from a bite of infected animal and once symptoms appear, death is inevitable. A challenging target date of 2030 to eliminate rabies in dogs has been set. One challenge will be stopping RABV re-entering the dog population from other animal reservoirs. Understanding how RABV switches hosts is important to prevent it happening. In this thesis, I hypothesise that successful host switching is due to the diverse population of viruses within the host termed ‘viral heterogeneity’. To investigate viral heterogeneity, methods to sequence the virus populations within clinical samples were developed. Transmission of RABV within dogs was analysed and a host shift event from dogs to foxes was investigated. High viral heterogeneity was seen in foxes after the host shift than in other foxes, suggesting it is important for a successful host shift. These data will be important to improve our understanding of how viruses evolve in new hosts, helping governments to eradicate disease.

Members of the family Paramyxoviridae are non-segmented, negative-strand RNA viruses. A large and diverse host species are infected by paramyxoviruses, including avian, porcine, canine, bovine, equine, ovine, reptiles, aquatic species and humans. In the last few decades, many novel paramyxoviruses have emerged causing catastrophic illnesses in different aquatic and terrestrial species of animals and some of them also made the species jump to humans. Two novel paramyxoviruses 81-19252 (Texas81) and 92-7783 (ISU92) were isolated in the 1980s and 1990s from the brain of pigs that experienced respiratory and central nervous system disease from South and North Central United States. To understand their importance as swine pathogens, molecular characterization and pathogenicity studies were undertaken. The complete genome of Texas81 virus was 15456 nucleotides (nt) and ISU92 was 15480 nt in length consisting of six non-overlapping genes coding for the nucloeo- (N), phospho- (P), matrix (M, fusion (F), hemagglutinin-neuraminidase (HN) and large polymerase (L) proteins in the order 3'-N-P/C/V-M-F-HN-L-5'. The features related to virus replication and found to be conserved in most members of Paramyxoviridae were also found in swine viruses. These include: conserved and complementary 3â leader and 5â trailer regions, trinucleotide intergenic sequences, highly conserved gene start and gene stop signal sequences. The length of each gene of these two viruses was similar except for the F gene, in which ISU92 had an additional 24 nt "U" rich 3â untranslated region (UTR). The P gene of these viruses were predicted to express the P protein from the primary transcript and edit a portion of its mRNA to encode V and D proteins and the C protein was expected to be expressed from alternate translation initiation from the P gene as in Respiroviruses. Sequence specific features related to virus replication and host specific amino acid signatures in P, F, HN and L proteins indicated that these viruses probably originated from bovine parainfluenzavirus 3. Pairwise comparisons of deduced amino acid sequences of swine viral proteins with members of Paramyxoviridae and phylogenetic analysis based on individual genes as well as predicted amino acid sequences suggested that these viruses were novel members of the genus Respirovirus of the Paramyxovirinae subfamily and genotype A of bovine parainfluenzavirus type 3. The mild clinical signs and undetectable gross and microscopic lesions observed in swine parainfluenzavirus (sPIV3)-infected pigs indicate the inapparent nature of these viruses in pigs. Limited seroprevalence studies in serum samples collected from pig farms in Minnesota and Iowa in 2007-2008 by indirect ELISA revealed that sPIV3 are not circulating in these farms. The mild pathogenicity of sPIV3 can facilitate its development as a vaccine vector. The screening ELISA developed by us could be used to detect seroprevalence of sPIV3 in animal and human populations. / Master of Science

Microsatellites are strings of short DNA motifs (≤6 bp) repeated in tandem across genomes of both prokaryotes and eukaryotes. In 20 years, they became popular genetic markers, successfully employed in the field of genetic mapping and gene hunting, as well as to address various biological questions at the individual, family, population and species level. However, evolutionary and demographic inferences from microsatellite polymorphism are hampered by controversy and ambiguity in the mutational processes of microsatellite sequences. Drawing on new data from genome projects, I review in Chapter 1 the concept of a microsatellite life cycle, which hypothesizes that microsatellites follow a life cycle from birth, through expansion, contraction, death and potentially resurrection. To document and understand this integrative concept of evolution, which could help improve current models of microsatellite evolution, there is an implicit need to study the evolution of microsatellites above the species level. A prerequisite of such comparative studies is therefore to find microsatellite loci that are conserved between different species. The near or full completion of many vertebrate genomes and their alignment against one another offer the ultimate approach to find genomic elements conserved over a large evolutionary scale. In Chapter 2, I present a new comprehensive method to find conserved microsatellites in whole genomes. Using the multiple-alignment of the human genome against those of 11 mammalian and five non-mammalian vertebrates, I examine the genomewide conservation of microsatellites, and challenge the general assumption that microsatellites are too labile to be maintained in distant species. In Chapter 3, I present similar results using the alignment of the newly sequenced platypus genome against those of three mammals, the chicken and the lizard, and incorporate these data into the framework created by the 17-genome analysis. This enlarged dataset was ground for attempting to reconstruct a vertebrate phylogeny from the presence/absence of microsatellites in the different genomes. Maximum parsimony analyses resulted in a tree much similar to that of the current view of the vertebrate phylogeny, while Bayesian analyses showed some discrepancies. This work opens a way for novel theoretical developments regarding the inference of ancestral states of microsatellites. In Chapter 4, I show how knowledge on conserved microsatellite sites can help for the development of a set of comparative primers useful across the Mammalia; implementing a similar protocol, nine conserved dinucleotide repeats were genotyped in 20 unrelated individuals of 18 species (nine sister species) encompassing the mammalian phylogeny, including marsupials and monotremes, and four microsatellites were sequenced in 4 individuals per species. My results emphasize conserved microsatellites as a new resource for genetic mapping and population studies. Finally, in Chapter 5, I recount the unexpected extent of structural change among mammalian orthologous microsatellites, including change of complexity, motif replacement and overall length variability. Altogether, these findings provide a comprehensive framework that may help in many areas of research, including molecular ecology, genome mapping, population genetics, and genome and microsatellite evolution.

The parasite secretome has been shown to play a key role in both pathogenicity and the regulation of host defence, allowing pathogens, such as helminths, to establish a chronic infection within the host. The recently discovered presence of extracellular vesicles within parasite-derived excretory-secretory products introduces a new mechanism of potential cross-species communication. Extracellular vesicles (EVs), such as exosomes, facilitate cellular communication through the transfer of small RNAs, lipids and proteins between cells and organisms across all three kingdoms of life. In addition to their roles in normal physiology, EVs also transport molecules from pathogens to hosts, presenting parasite antigens and transferring infectious agents. Here, I examine secreted vesicles from the murine gastrointestinal nematode Heligmosomoides polygyrus, and their potential role in the host-helminth interactions. Transmission electron microscopy reveals vesicle-like structures of 50- 100 nM in the ultracentrifuged secretory product, and potential evidence of multi-vesicular bodies in the worm intestine. This, coupled with information from the exoproteome, helped support the hypothesis that exosomes originate from the parasite intestinal tract. I have completed a series of studies looking at the fundamental properties of exosome-cell interactions, providing comparative studies between mammalian and H. polygyrus-derived exosomes. I have determined some of the key factors influencing exosome uptake, including time of incubation, cell type and exosome origin. Through microarray analysis of H. polygyrus exosome-treated small intestinal epithelial cells, we see significant gene expression changes, including those involved in the regulation of signalling and the immune response, such as DUSP1 (dual-specificity phosphatase) and IL1RL1 (the receptor for IL-33). The modest reduction of inflammatory cytokine responses by exosomes in small intestinal cell lines was amplified in immune cells, such as macrophages. Exosomes can significantly reduce expression of classical activation markers, as well as inflammatory cytokine production in the macrophage cell line RAW 264.7, and this is further supported by similar responses in bone marrow-derived macrophages. Owing to their suppressive nature, I demonstrate that immunization of mice with an exosome/alum conjugate generates significant protection from a subsequent H. polygyrus larval challenge, as seen through a reduction in egg counts and worm burden. I have investigated the role of the IL33 receptor (IL-33R); a key molecule associated with parasitic resistance that is suppressed by exosomes in type-2 associated immune responses. Uptake of H. polygyrus-derived exosomes by alternatively activated macrophages caused the suppression of type 2 cytokine/protein release and the reduction of key genes associated with this phenotype. In addition, there was also significant repression of both transcript and surface T1/ST2, a subunit of the IL-33R). Using a model of lung inflammation, in vivo studies demonstrate that, in both prophylactic and co-administration experiments, exosomes modulate the innate cellular response. This is represented by changes in the number of innate lymphoid cells (ILCs), bronchoalveolar lavage eosinophils and type-2 cytokine output. In this system, the expression of T1/ST2 on type 2 ILCs was also significantly reduced. I have extended the investigation on exosome-IL-33R responses by using T1/ST2 knockout mice. Despite generating strong antibody responses, vaccination against exosomes could not protect T1/ST2 knockout mice against a subsequent infection. This work suggests that exosomes secreted by nematodes could mediate the transfer and uptake of parasite products into host cells, establishing cross-species communication to suppress the host ‘danger’ or inflammatory response.

Η οικογένεια Tephritidae των Διπτέρων εντόμων περιλαμβάνει είδη με μεγάλη οικονομική σημασία. Τα περισσότερα αποτελούν σημαντικά παράσιτα γεωργικών καλλιεργειών προκαλώντας μεγάλες καταστροφές στην παραγωγή φρούτων και λαχανικών παγκοσμίως. Τα σημαντικότερα παράσιτα ανήκουν στα γένη Anastrepha, Bactrocera, Ceratitis, Dacus και Rhagoletis. Η μελέτη των ειδών αυτών στο επίπεδο της γενετικής και της μοριακής βιολογίας θα μπορούσε να συμβάλλει σημαντικά στην ανάπτυξη μεθόδων βιολογικού ελέγχου των πληθυσμών τους. Με εξαίρεση τη Μεσογειακή μύγα, Ceratitis capitata, που θεωρείται ο καλύτερα μελετημένος οργανισμός της οικογένειας, η πληροφορία για άλλα είδη είναι πολύ περιορισμένη. Στη παρούσα μελέτη εξετάσθηκε η πιθανότητα δια-ειδικής ενίσχυσης μικροδορυφορικών δεικτών που αναπτύχθηκαν στο εργαστήριό μας για τη Μεσογειακή μύγα σε είδη των γενών Anastrephα, Bactrocera και Rhagoletis καθώς και στο είδος C. fasciventris με κύριο στόχο την ανάπτυξη κατάλληλων γενετικών δεικτών που θα μπορούσαν να χρησιμοποιηθούν σε γενετικές ή και πληθυσμιακές μελέτες των ειδών αυτών. Από τους 102 μικροδορυφορικούς δείκτες που αναλύθηκαν μέσω της PCR, οι 31 (31%) έδωσαν δια-ειδική ενίσχυση σε τουλάχιστον ένα από τα εξεταζόμενα είδη έκτος του γένους Ceratitis. Το αντίστοιχο ποσοστό για την C. fasciventris ήταν 67,75%. Το μέγεθος των προϊόντων αυτών ήταν παρόμοιο ή και ταυτόσημο με το αναμενόμενο στη Μεσογειακή μύγα. Τα αποτελέσματα από την αλληλούχιση των προϊόντων PCR και τη σύγκριση τους με τις αντίστοιχες περιοχές της Μεσογειακής Μύγας δείχνουν ότι η δομή των μικροδορυφορικών μοτίβων αλλά και των μοναδικών περιοχών που τους περιβάλλουν φαίνεται να διατηρείται στη πλειοψηφία των περιπτώσεων για τη C. fasciventris. Σε όλες τις περιπτώσεις που εξετάσθηκαν βρέθηκαν επαναλαμβανόμενα μοτίβα. Η συγκριτική ανάλυση των αλληλουχιών αυτών μπορεί να προσφέρει πολύτιμους γενετικούς δείκτες για τα είδη αυτά αλλά και πληροφορίες για τις φυλογενετικές τους σχέσεις. / Tephritidae family of Diptera includes species of great economical importance. Most of them are significant parasites of agriculture causing severe damages by attacking fruits and vegetables world wide.The most important pests belong to the genera Anastrepha, Bactrocera, Ceratitis, Dacus and Rhagoletis. Studies of these species on genetic and molecular level could contribute significantly to the development of methods for their biological control. Except for the Mediterranean fruit fly, Ceratitis capitata, which is the best understood fruit fly pest at the genetic/molecular level, limited information exists for other species. In the present work we examined the possibility of cross-species amplification of microsatellite markers that were developed in our laboratory for C. capitata. Cross-species analyses were performed on species belonging to the genera Anastrepha, Bactrocera and Rhagoletis as well as C. fasciventris, aiming at the development of suitable markers that could be used in genetic and/or population studies of these species. A total of 102 microsatellites markers were examined through PCR. Thirty one of them (31%) showed cross-species amplification in at least one of the analyzed species. The percentage for C. fasciventris was 67.75%. The majority of the products were similar or identical in size to those expected in C. capitata. Sequencing analyses of the PCR products are when compared to the Medfly markers demonstrate that the structures of the repeat motifs and their flanking sequences are maintained in most studied cases that involve C. fasciventris. In every case studied repeat motifs were found. This analysis can provide not only useful genetic markers for the analyzed species but also information for their phylogenetic relationships.

Microsatellites are strings of short DNA motifs (≤6 bp) repeated in tandem across genomes of both prokaryotes and eukaryotes. In 20 years, they became popular genetic markers, successfully employed in the field of genetic mapping and gene hunting, as well as to address various biological questions at the individual, family, population and species level. However, evolutionary and demographic inferences from microsatellite polymorphism are hampered by controversy and ambiguity in the mutational processes of microsatellite sequences. Drawing on new data from genome projects, I review in Chapter 1 the concept of a microsatellite life cycle, which hypothesizes that microsatellites follow a life cycle from birth, through expansion, contraction, death and potentially resurrection. To document and understand this integrative concept of evolution, which could help improve current models of microsatellite evolution, there is an implicit need to study the evolution of microsatellites above the species level. A prerequisite of such comparative studies is therefore to find microsatellite loci that are conserved between different species. The near or full completion of many vertebrate genomes and their alignment against one another offer the ultimate approach to find genomic elements conserved over a large evolutionary scale. In Chapter 2, I present a new comprehensive method to find conserved microsatellites in whole genomes. Using the multiple-alignment of the human genome against those of 11 mammalian and five non-mammalian vertebrates, I examine the genomewide conservation of microsatellites, and challenge the general assumption that microsatellites are too labile to be maintained in distant species. In Chapter 3, I present similar results using the alignment of the newly sequenced platypus genome against those of three mammals, the chicken and the lizard, and incorporate these data into the framework created by the 17-genome analysis. This enlarged dataset was ground for attempting to reconstruct a vertebrate phylogeny from the presence/absence of microsatellites in the different genomes. Maximum parsimony analyses resulted in a tree much similar to that of the current view of the vertebrate phylogeny, while Bayesian analyses showed some discrepancies. This work opens a way for novel theoretical developments regarding the inference of ancestral states of microsatellites. In Chapter 4, I show how knowledge on conserved microsatellite sites can help for the development of a set of comparative primers useful across the Mammalia; implementing a similar protocol, nine conserved dinucleotide repeats were genotyped in 20 unrelated individuals of 18 species (nine sister species) encompassing the mammalian phylogeny, including marsupials and monotremes, and four microsatellites were sequenced in 4 individuals per species. My results emphasize conserved microsatellites as a new resource for genetic mapping and population studies. Finally, in Chapter 5, I recount the unexpected extent of structural change among mammalian orthologous microsatellites, including change of complexity, motif replacement and overall length variability. Altogether, these findings provide a comprehensive framework that may help in many areas of research, including molecular ecology, genome mapping, population genetics, and genome and microsatellite evolution.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: This thesis explores the use of networks as a means to visualise, interpret and mine MS-based proteomics data. A network-based approach was applied to a quantitative, cross-species LCMS/ MS dataset derived from two yeast species, namely Saccharomyces cere- visiae strain VIN13 and Saccharomyces paradoxus strain RO88. In order to identify and quantify proteins from the mass spectra, a workflow consisting of both custom-built and existing programs was assembled. Networks which place the identifed proteins in several biological contexts were then constructed. The contexts included sequence similarity to other proteins, ontological descriptions, proteins-protein interactions, metabolic pathways and cellular location. The contextual, network-based representations of the proteins proved effective for identifying trends and patterns in the data that may otherwise have been obscured. Moreover, by bringing the experimentally derived data together with multiple, extant biological resources, the networks represented the data in a manner that better represents the interconnected biological system from which the samples were derived. Both existing and new hypotheses based on proteins relating to the yeast cell wall and proteins of putative oenological potential were investigated. These proteins were investigated in light of their differential expression between the two yeast species. Examples of proteins that were investigated included cell wall proteins such as GGP1 and SCW4. Proteins with putative oenological potential included haze protection factor proteins such as HPF2. Furthermore, differences in capacity for maloethanolic fermentation between the two strains were also investigated in light of the protein data. The network-based representations also allowed new hypotheses to be formed around proteins that were identified in the dataset, but were of unknown function. / AFRIKAANSE OPSOMMING: Hierdie studie verken die gebruik van netwerke om proteonomiese data te visualiseer, te interpreteer en te ontgin. 'n Netwerkgebaseerde benadering is gevolg ter ontleding van 'n kwantitatiewe LC-MS/MS datastel wat afkomstig was van twee gis-spesies nl, Saccharomyces cerevisiae ras VIN1 en Saccharomyces paradoxus ras RO88. Die massaspektra is met bestaande en selfgeskrewe rekenaarprogramme verwerk om 'n werkvloei saam te stel ter identifisering en kwantifisering van die betrokke proteïene. Hierdie proteïene is dan aan bestaande biologiese databasisse gekoppel om die proteïene in biologiese konteks te plaas. Die gekontekstualiseerde is dan gebruik om biologiese netwerke van die data te bou. Die kontekste beskou onder meer lokalisering van selaktiwiteite, ontologiese beskrywings, ooreenkomste in aminosuur-volgordes en interaksies met bekende proteïene asook assosiasie en verbintenisse met metaboliese paaie. Hierdie kontekstuele, netwerk-gebaseerde voorstelling van die betrokke prote- ïene het effektief duidelike data-tendense en patrone opgelewer wat andersins nie opmerkbaar sou wees nie. Daarby het die kombinering van eksperimentele data en bestaande biologiese bronne 'n beter perspektief aan die data-analise verleen. Beide bestaande en nuwe hipoteses tov gis-selwandproteïene en prote ïene met moontlike wynkundige potensiaal is ondersoek in die lig van hul differensiële uitdrukking in die twee gis-spesies. Voorbeelde wat ondersoek is sluit in selwandproteïene soos GGP1 en SCW4 asook waasbeskermingsfaktorproteïen HPF2. Verskille tov kapasiteit mbt malo-etanoliese gisting is ook gevind. Die netwerk-gebaseerde voorstellings het ook aanleiding gegee tot die formulering van nuwe hipoteses mbt datastel-proteïene waarvan die funksies tans onbekend is.