Die angeborene Immunität ist in der Lage, Pathogene schon beim erstmaligen Eindringen zu erkennen und zu bekämpfen. Haupteffektoren der schnellen, angeborenen Immunantwort sind Makrophagen und polymorphkernige neutrophile Granulozyten. Diese erkennen und phagozytieren Pathogene und koordinieren die weitere Immunantwort durch die Freisetzung von inflammatorischen Mediatoren und Zytokinen. Die Erkennung mikrobieller Bestandteile, wie Lipopolysaccharid (LPS) Gram-negativer Bakterien bzw. Peptidoglykan (PG) und Lipoteichonsäuren (LTA) Gram-positiver Bakterien, führt zur Aktivierung von unterschiedlichen Proteinkinasen, des Transkriptionsfaktors NF-(B und zur Freisetzung von Zytokinen. Mitglieder der Toll-Proteinfamilie, sogenannte Toll-like-Rezeptoren (TLR), wurden kürzlich als Rezeptoren auf Immunzellen identifiziert, die für die Erkennung solcher mikrobieller Bestandteile verantwortlich sind. Während TLR-4 der LPS-Erkennung dient, und TLR-2 und -6 verschiedene Liganden von Gram-positiven Bakterien binden, blieb die Frage der Erkennung von LTA und verwandten Glykolipiden strittig. Sowohl TLR-2 als auch TLR-4 wurden für diese Rolle diskutiert. Zielsetzung dieser Arbeit war, die Rolle von TLRs in der LTA- und Glykolipid-Erkennung zu untersuchten. Glykolipide von zwei eng verwandten Treponemen-Spezies, T. maltophilum (TM) und T. brennaborense (TB), sowie neuartig aufgereinigte Lipoteichonsäuren von Staphylococcus aureus (SA) und Bacillus subtilis (BS) wurden eingesetzt, um die nukleäre Translokation von NF-(B in verschiedenen Zellsystemen zu induzieren. Diese Zellstimulationsexperimente wurden mit verschiedenen TLR-2-negativen Zellinien sowie mit Peritonealexsudatzellen TLR-4-defizienter C3H/HeJ-Mäuse durchgeführt. Weitere Informationen lieferten TLR-2-Überexpressions-Experimente sowie Zellstimulationen unter Verwendung von anti-TLR-4-Antikörpern. Die Aktivierung von NF-(B wurde anhand von Gelshifts nachgewiesen. Mit der Überexpression von dominant-negativen Mutanten verschiedener Moleküle der Signalkaskade, mit Kinase-Hemmstoffen und mit Western Blots wurden die intrazellulären Signaltransduktionswege untersucht. Für Glykolipide von T. maltophilum und beide verwendeten Lipoteichonsäuren ließ sich eine klare TLR-2-Abhängigkeit in der Aktivierung von NF-(B und der Induktion von proinflammatorischen Zytokinen zeigen. Die Glykolipide von T. brennaborense hingegen waren überraschender Weise gleichzeitig auch TLR-4-Liganden. Beide untersuchten Glykolipide sowie beide LTAs aktivierten einen Signalweg unter Einbeziehung des Adaptermoleküls MyD88 und der NF-(B-induzierenden Kinase (NIK). Des weiteren konnte der Einfluß der MAP-Kinasen p42/44 und p38 auf die Treponema-Glykolipid- und LPS-induzierte TNF-(-Ausschüttung dargestellt werden. Zusammenfassend zeigen diese Ergebnisse, daß TLR-2 der Hauptrezeptor von Lipoteichonsäuren ist, und TLR-2 und -4 beide Rezeptoren der Treponema-Glykolipide sein können. Diese Ergebnisse sollten dazu beitragen, die molekularen Grundlagen der Reaktionen des Immunsystems auf Gram-positive Bakterien und Treponemen zu verstehen. / The innate immune response to microbial pathogens is able to protect the host after a first pathogen contact. This immediate immune response is largely mediated by macrophages and neutrophils. They recognize and phagocytose pathogens, and coordinate host responses by secreting inflammatory mediators, such as cytokines. The recognition of lipopolysaccharide (LPS) of Gram-negative bacteria, or peptidoglycan (PG) and lipoteichoic acids (LTAs) of Gram-positive bacteria leads to the induction of protein-kinases, the transcription factor NF-(B, and subsequently the release of proinflammatory cytokines. Recently, members of the Toll-protein-family, the so-called Toll-like receptors (TLRs) have been found to be involved in immune cell activation by microbial products. While TLR-4 has been identified as the transmembrane signal transducer for LPS, and TLR-2 and -6 for different ligands originating from Gram-positive bacteria, the molecular basis of recognition of lipoteichoic acids and related glycolipids has not been completely understood: Both, TLR-4 and -2 have been postulated as receptors. In order to determine the role of TLRs in immune cell activation by Treponema glycolipids and LTAs experiments involving TLR-2-negative cell lines, macrophages from TLR-4-deficient C3H/HeJ-mice, cells overexpression TLR-2, and inhibitory TLR-4 antibodies were performed. The induction of NF-(B was assessed by electrophoretic mobility shift assays. Glycolipids of two related Treponema species, T. maltophilum (TM) and T. brennaborense (TB), and LTAs from Staphylococcus aureus (SA) and Bacillus subtilis (BS) were investigated for induction of nuclear translocation of NF-(B in different cell systems. Glycolipids from T. maltophilum and both LTAs studied revealed TLR-2-dependency in induction of NF-(B and proinflammatory cytokines. Surprisingly, glycolipids from T. brennaborense were found to be TLR-4-ligands. Furthermore an involvement of the signaling molecules MyD88 and NIK in cell stimulation by LTAs and glycolipids was revealed by dominant-negative overexpression experiments. The induction of TNF-( by Treponema glycolipids furthermore was dependent on activation of MAP kinases p42/44 and p38, as indicated by specific kinase inhibitors. Tyrosinephosporylation of the p42/44 kinase induced by Treponema glycolipids were detected by western blots. In summary, the results presented here indicate that TLR-2 is the main receptor for LTAs. Both TLR-2 and -4 serve as receptors for Treponema glycolipids. These results may potentially contribute to explain immune responses to Gram-positive bacteria and treponemes.

LBP vermittelt im Wirtsorganismus die direkte Immunantwort auf bakterielle Liganden wie das Lipopolysaccharid (LPS) von Gram-negativen oder Lipopeptide von Gram-positiven Bakterien. In dieser Arbeit wurde die Funktionsweise von LBP weiter aufgeklärt. Im ersten Teil der Arbeit wurde eine natürlich vorkommende Mutation des LBP (c998t), die an Position 333 zu einem Austausch der Aminosäure Prolin zu Leucin führt, hinsichtlich ihrer Auswirkungen auf Struktur und Funktionalität des Proteins untersucht. Westernblot-Analysen des rekombinant hergestellten Proteins und humaner Seren von Mutationsträgern weisen auf einen Zerfall des mutierten Proteins hin. Es kommt zu einer Beeinträchtigung der Bindung bakterieller Liganden und einer deutlichen Reduktion der LBP-vermittelten Zytokinausschüttung von Immunzellen. Der hier untersuchte Polymorphismus hat eine Allelfrequenz von 0,072 in einer gesunden europäischen Population. Genotypanalysen von Patientengruppen zeigten, dass es durch die Mutation zu einer deutlich erhöhten Mortalität bei Patienten mit septischen Komplikationen und einer durch Gram-negative Erreger verursachten Pneumonie kommt. Unsere Ergebnisse zur eingeschränkten Funktion des LBP-c998t bieten eine erste Erklärung dafür, wie diese Mutation vermutlich die Fähigkeit, Krankheiten zu bewältigen, beeinträchtigt. Innerhalb dieser Arbeit ging es um die Analyse der Bindung von bakteriellen Liganden an LBP. Dabei wurde eine potentiell gemeinsame Bindungsstelle für Liganden untersucht, die von Gram-positiven und Gram-negativen Bakterien stammen und später von den Toll-like Rezeptoren (TLRs) 2 und -4 erkannt werden. Dazu wurden Bindungsversuche zwischen Lipopeptiden und LPS mit einer zweiten LBP-Variante (LBP-E94/95) durchgeführt. Beim LPS führt dies zu einem Bindungsverlust. Auch für die Lipopeptide war durch die Mutationen die Interaktion mit LBP beeinträchtigt, was die These einer gemeinsamen Bindungsstelle von TLR2- und TLR4-Liganden an das Protein weiter unterstützt. / LBP enhances the innate immune reaction against bacterial ligands like LPS from gram negative or lipopeptides from gram positive bacteria in the host. Here we investigated the function of LBP using two recombinant mutants of the protein. The first part of this work examines a natural occurring mutation of LBP (c998t) leading to an amino acid exchange of proline to leucine at position 333 with regard to the impact on structure and function of the protein. Western blot analyses of the recombinant protein and sera obtained from individuals differing in the LBP genotype indicate the disaggregation of the mutated protein. Thereby binding of bacterial ligands to LBP is diminished and the LBP mediated cytokine secretion of immune cells is reduced. The gene polymorphism leading to the occurrence of the mutation is present with an allelic frequence of 0.072. A recent study has shown that this LBP-SNP led to a higher mortality in patients with septic complications and gram negative pneumonia. The results presented here, showing the negative impact on the function of LBP due to the mutation, may therefore be a first explanation on how this mutation affects the ability of people to deal with disease. Within this work binding of ligands to LBP was also explored. It was investigated whether ligands which are later recognized by Toll-like receptors (TLRs) 2 and – 4 share a common binding site on LBP. Assays with immobilized lipopeptides and LPS were performed with a second mutated LBP (LBP-E94/95). LPS binding to LBP is diminished completely. Here we showed that binding of lipopeptide to LBP is affected likewise, furthermore supporting the hypothesis of a common binding site for TLR2- and TLR4- ligands.

Les cellules endothéliales (EC) constituent une première barrière physique à la dissémination de virus pléiotropiques circulant par voie hématogène mais leur contribution à la défense innée anti-virale est peu connue. Des dysfonctions des EC de la barrière hémato-encéphalique (BMEC) et des sinusoïdes hépatiques (LSEC) ont été rapportées dans des neuropathologies et des hépatites aiguës ou chroniques d’origine virale, suggérant que des atteintes à leur intégrité contribuent à la pathogenèse. Les sérotypes de coronavirus de l’hépatite murine (MHV), se différenciant par leur capacité à induire des hépatites et des maladies neurologiques de sévérité variable et/ou leur tropisme pour les EC, représentent des modèles viraux privilégiés pour déterminer les conséquences de l’infection des EC sur la pathogenèse virale. Lors d’infection par voie hématogène, le sérotype MHV3, le plus virulent des MHV, induit une hépatite fulminante, caractérisée par une réponse inflammatoire sévère, et des lésions neurologiques secondaires alors que le sérotype moins virulent, MHV-A59, induit une hépatite modérée sans atteintes secondaires du système nerveux central (SNC). Par ailleurs, le sérotype MHV3, à la différence du MHV-A59, démontre une capacité à stimuler la production de cytokines par la voie TLR2. Les variants atténués du MHV3, les virus 51.6-MHV3 et YAC-MHV3, sont caractérisés par un faible tropisme pour les LSEC et induisent respectivement une hépatite modérée et subclinique. Compte tenu de l’importance des LSEC dans le maintien de la tolérance hépatique et de l’élimination des pathogènes circulants, il a été postulé que la sévérité de l’hépatite et de la réponse inflammatoire lors d’infections par les MHV est associée à la réplication virale et à l’altération des propriétés tolérogéniques et vasculaires des LSEC. Les désordres inflammatoires hépatiques pourraient résulter d’une activation différentielle du TLR2, plutôt que des autres TLR et des hélicases, selon les sérotypes. D’autre part, compte tenu du rôle des BMEC dans la prévention des infections du SNC, il a été postulé que l’invasion cérébrale secondaire par les coronavirus est reliée à l’infection des BMEC et le bris subséquent de la barrière hémato-encéphalique (BHE). À l’aide d’infections in vivo et in vitro par les différents sérotypes MHV, chez des souris ou des cultures de BMEC et de LSEC, nous avons démontré, d’une part, que l’infection in vitro des LSEC par le sétotype MHV3, à la différence des variants 51.6- et YAC-MHV3, altérait la production du facteur vasodilatant NO et renversait leur phénotype tolérogénique en favorisant la production de cytokines et de chimiokines inflammatoires. Ces dysfonctions se traduisaient in vivo par une réponse inflammatoire incontrôlée et une dérégulation du recrutement intrahépatique de leucocytes, favorisant la réplication virale et les dommages hépatiques. Nous avons aussi démontré, à l’aide de souris TLR2 KO et de LSEC dont l’expression du TLR2 a été abrogée par des siRNA, que la sévérité de l’hépatite et de la réponse inflammatoire induite par le sérotype MHV3, dépendait en partie de l’induction et de l’activation préférentielle du TLR2 par le virus dans le foie. D’autre part, la sévérité de la réplication virale au foie et des désordres dans le recrutement leucocytaire intrahépatique induits par le MHV3, et non par le MHV-A59 et le 51.6-MHV3, corrélaient avec une invasion virale subséquente du SNC, au niveau de la BHE. Nous avons démontré que l’invasion cérébrale du MHV3 était associée à une infection productive des BMEC et l’altération subséquente des protéines de jonctions serrées occludine, VE-cadhérine et ZO-1 se traduisant par une augmentation de la perméabilité de la BHE et l’entrée consécutive du virus dans le cerveau. Dans l’ensemble, les résultats de cette étude mettent en lumière l’importance du maintien de l’intégrité structurale et fonctionnelle des LSEC et des BMEC lors d’infections virales aigües par des MHV afin de limiter les dommages hépatiques associés à l’induction d’une réponse inflammatoire exagérée et de prévenir le passage des virus au cerveau suite à une dissémination par voie hématogène. Ils révèlent en outre un nouveau rôle aggravant pour le TLR2 dans l’évolution de l’hépatite virale aigüe ouvrant la voie à de nouvelles avenues thérapeutiques visant à moduler l’activité inflammatoire du TLR2. / Endothelial cells (EC) act as a physical barrier against invasion by pleiotropic blood borne viruses but their contribution in innate antiviral defense is poorly known. Dysfunctions in blood-brain barrier EC (BMECs) and liver sinusoidal EC (LSECs) have been reported in viral neuropathologies and hepatitis, suggesting that loss of ECs integrity may contribute to the pathogenesis. Mouse hepatitis coronaviruses (MHV), differing in their ability to induce severe to subclinical hepatitis and neurological diseases and / or their tropism for ECs, are relevant viral models to study the consequences of EC infection in viral pathogenesis. Following hematogenous infection, the MHV3 serotype, the most virulent MHV, induces fulminant hepatitis, characterized by severe inflammatory response, followed by neurological damage whereas the less virulent MHV-A59 serotype induces milder hepatitis but does not invade the central nervous system (CNS). In addition, MHV3, in contrast to MHV-A59, shows ability to induce TLR2-dependent cytokine response. The attenuated MHV3 variants, 51.6-MHV3 and YAC-MHV3, are characterized by a weak tropism for LSECs and induce moderated and subclinical hepatitis respectively. Given the importance of LSECs in hepatic tolerance and the elimination of circulating pathogens, it has been postulated that the severity of hepatitis and inflammatory response induced by MHVs correlates with infection and alterations in vascular and tolerogenic properties of LSECs. Hepatic inflammatory disorders may result from differential activation of TLR2, rather than other TLRs and helicases, according to serotypes. Moreover, given the role of BMECs in preventing CNS infections, it has been postulated that secondary cerebral invasion by coronaviruses is related to infection of BMECs and subsequent breakdown of the blood-brain barrier (BBB). Through in vitro and in vivo infections of isolated BMECs, LSECs or mice with the different MHVs, we demonstrated, first, that in vitro productive infection of LSECs by the highly virulent MHV3 serotype, in contrast to 51.6- et YAC-MHV3 variants, altered their production of vasoactive factors and overthrew their intrinsic tolerogenic properties by promoting inflammatory cytokines and chemokines production. These disturbances were reflected in vivo by an uncontrolled inflammatory response and a deregulation of intrahepatic leukocyte recruitment, favoring viral replication and liver damages. We demonstrated, using TLR2 KO mice and LSECs treated with siRNA for TLR2 that the abnormal inflammatory response induced by MHV3 depended in part on preferential induction and activation of TLR2 by the virus on the surface of hepatic cells. Moreover, the severity of the primary viral replication in the liver and disorders in intrahepatic leucocyte recruitment induced by MHV3, but not by MHV-A59 and 51.6-MHV3, correlated with a subsequent brain invasion at the BBB level. Such invasion was related to productive infection of BMECs and subsequent IFN--dependent disruption of tight junction proteins occludin, VE-cadherin and ZO-1, resulting in an increase of BBB permeability and further viral entry into the CNS. Overall, the results of this study highlight the importance of structural and functional integrity of LSECs and BMECs during acute viral infections by MHVs to limit liver damages associated with viral-induced exacerbation of inflammatory response and prevent brain invasion by MHVs following viral spread through the bloodstream. They also reveal a new worsening role for TLR2 in the evolution of acute viral hepatitis paving the way for new therapies targeting TLR2-induced inflammatory activity.

Yersinia enterocolitica es un patógeno Gram-negativo que provoca diversos síndromes gastrointestinales y expresa una panoplia de factores de virulencia, la mayoría regulados por la temperatura. El lipopolisacatido (LPS) es uno de los principales factores de virulencia de las bacterias Gram-negativas patógenas. Además, es una de las moléculas reconocidas por el sistema inmune innato y diana de los péptidos antimicrobianos. Por consiguiente, no es de extrañar que las bacterias modifiquen la estructura de su LPS con el fin de resistir a la defensa del sistema inmune. En esta Tesis Doctoral se han identificado los loci responsables de las modificaciones del lípido A de Y. enterocolitica O:8 (YeO8) y se ha demostrado que están reguladas por la temperatura. Se ha definido un circuito regulatorio complejo en el que intervienen los sistemas PhoP/PhoQ y PmrA/PmrB y en el que RovA y H-NS son piezas centrales. Además se demuestra que el lípido A tiene un papel en la virulencia de YeO8. Por último , se han identificado por primera vez en YeO8 la enzima PmrC, encargada de la adición de fosfoetanolamina al lípido A y la enzima LpxR encargada de la deacilación del lípido A.

Reverse genetics is an innovative molecular biology tool that enables the manipulation of viral genomes at the cDNA level in order to generate particular mutants or artificial viruses. The reverse genetics system for the influenza virus is arguably one of the best illustrations of the potential power of this technology. This reverse genetics system is the basis for the ability to regularly adapt influenza vaccines strains. Today, reverse genetic systems have been developed for many animal RNA viruses. Selection-free reverse genetics systems have been developed for the members of the Reoviridae family including, African horsesickness virus, bluetongue virus and orthoreovirus. This ground-breaking technology has led to the generation of valuable evidence regarding the replication and pathogenesis of these viruses. Unfortunately, extrapolating either the plasmid-based or transcript-based reverse genetics systems to rotavirus has not yet been successful. The development of a selection-free rotavirus reverse genetics system will enable the systematic investigation of poorly understood aspects of the rotavirus replication cycle and aid the development of more effective vaccines, amongst other research avenues. This study investigated the importance of co-expressed rotavirus proteins in the development of a selection-free rotavirus reverse genetics system. The consensus sequences of the rotavirus strains Wa (RVA/Human-tc/USA/WaCS/1974/G1P[8]) and SA11 (RVA/Simian-tc/ZAF/SA11/1958/G3P[2]) where used to design rotavirus expression plasmids. The consensus nucleotide sequence of a human rotavirus Wa strain was determined by sequence-independent cDNA synthesis and amplification combined with next-generation 454® pyrosequencing. A total of 4 novel nucleotide changes, which also resulted in amino acid changes, were detected in genome segment 7 (NSP3), genome segment 9 (VP7) and genome segment 10 (NSP4). In silico analysis indicated that none of the detected nucleotide changes, and consequent amino acid variations, had any significant effect on viral structure. Evolutionary analysis indicated that the sequenced rotavirus WaCS was closely related to the ParWa and VirWa variants, which were derived from the original 1974 Wa isolate. Despite serial passaging in animals, as well as cell cultures, the Wa genome seems to be stable. Considering that the current reference sequence for the Wa strain is a composite sequence of various Wa variants, the rotavirus WaCS may be a more appropriate reference sequence. The rotavirus Wa and SA11 strains were selected for plasmid-based expression of rotavirus proteins, under control of a T7 promoter sequence, due to the fact that they propagate well in MA104 cells and the availability of their consensus sequences. The T7 RNA polymerase was provided by a recombinant fowlpox virus. After extensive transfection optimisation on a variety of mammalian cell lines, MA104 cells proved to be the best suited for the expression rotavirus proteins from plasmids. The expression of rotavirus Wa and SA11 VP1, VP6, NSP2 and NSP5 could be confirmed with immunostaining in MA104 and HEK 293H cells. Another approach involved the codon-optimised expression of the rotavirus replication complex scaffold in MA104 cells under the control of a CMV promoter sequence. This system was independent from the recombinant fowlpox virus. All three plasmid expression sets were designed to be used in combination with the transcript-based reverse genetics system in order to improve the odds of developing a successful rotavirus reverse genetics system. Rotavirus transcripts were generated using transcriptively active rotavirus SA11 double layered particles (DLPs). MA104 and HEK293H cells proved to be the best suited for the expression of rotavirus transcripts although expression of rotavirus VP6 could be demonstrated in all cell cultures examined (MA104, HEK 293H, BSR and COS-7) using immunostaining. In addition, the expression of transcript derived rotavirus VP1, NSP2 and NSP5 could be confirmed with immunofluorescence in MA104 and HEK 293H cells. This is the first report of rotavirus transcripts being translated in cultured cells. A peculiar cell death pattern was observed within 24 hours in response to transfection of rotavirus transcripts. This observed cell death, however does not seem to be related to normal viral cytopathic effect as no viable rotavirus could be recovered. In an effort to combine the transcript- and plasmid systems, a dual transfection strategy was followed where plasmids encoding rotavirus proteins were transfected first followed, 12 hours later, by the transfection of rotavirus SA11 transcripts. The codon- optimised plasmid system was designed as it was postulated that expression of the DLP-complex (VP1, VP2, VP3 and VP6), the rotavirus replication complex would form and assist with replication and/or packaging. Transfecting codon- optimized plasmids first noticeably delayed the mass cell death observed when transfecting rotavirus transcripts on their own. None of the examined coexpression systems were able to produce a viable rotavirus. Finally, the innate immune responses elicited by rotavirus transcripts and plasmid-derived rotavirus Wa and SA11 proteins were investigated. Quantitative RT-PCR (qRT-PCR) experiments indicated that rotavirus transcripts induced high levels of the expression of the cytokines IFN- α1, IFN-1β, IFN-λ1 and CXCL10. The expression of certain viral proteins from plasmids (VP3, VP7 and NSP5/6) was more likely to stimulate specific interferon responses, while other viral proteins (VP1, VP2, VP4 and NSP1) seem to be able to actively suppress the expression of certain cytokines. In the light of these suppression results, specific rotavirus proteins were expressed from transfected plasmids to investigate their potential in supressing the interferon responses provoked by rotavirus transcripts. qRT-PCR results indicated that cells transfected with the plasmids encoding NSP1, NSP2 or a combination of NSP2 and NSP5 significantly reduced the expression of specific cytokines induced by rotavirus transcripts. These findings point to other possible viral innate suppression mechanisms in addition to the degradation of interferon regulatory factors by NSP1. The suppression of the strong innate immune response elicited by rotavirus transcripts might well prove to be vital in the quest to better understand the replication cycle of this virus and eventually lead to the development of a selection-free reverse genetics system for rotavirus. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014

Reverse genetics is an innovative molecular biology tool that enables the manipulation of viral genomes at the cDNA level in order to generate particular mutants or artificial viruses. The reverse genetics system for the influenza virus is arguably one of the best illustrations of the potential power of this technology. This reverse genetics system is the basis for the ability to regularly adapt influenza vaccines strains. Today, reverse genetic systems have been developed for many animal RNA viruses. Selection-free reverse genetics systems have been developed for the members of the Reoviridae family including, African horsesickness virus, bluetongue virus and orthoreovirus. This ground-breaking technology has led to the generation of valuable evidence regarding the replication and pathogenesis of these viruses. Unfortunately, extrapolating either the plasmid-based or transcript-based reverse genetics systems to rotavirus has not yet been successful. The development of a selection-free rotavirus reverse genetics system will enable the systematic investigation of poorly understood aspects of the rotavirus replication cycle and aid the development of more effective vaccines, amongst other research avenues. This study investigated the importance of co-expressed rotavirus proteins in the development of a selection-free rotavirus reverse genetics system. The consensus sequences of the rotavirus strains Wa (RVA/Human-tc/USA/WaCS/1974/G1P[8]) and SA11 (RVA/Simian-tc/ZAF/SA11/1958/G3P[2]) where used to design rotavirus expression plasmids. The consensus nucleotide sequence of a human rotavirus Wa strain was determined by sequence-independent cDNA synthesis and amplification combined with next-generation 454® pyrosequencing. A total of 4 novel nucleotide changes, which also resulted in amino acid changes, were detected in genome segment 7 (NSP3), genome segment 9 (VP7) and genome segment 10 (NSP4). In silico analysis indicated that none of the detected nucleotide changes, and consequent amino acid variations, had any significant effect on viral structure. Evolutionary analysis indicated that the sequenced rotavirus WaCS was closely related to the ParWa and VirWa variants, which were derived from the original 1974 Wa isolate. Despite serial passaging in animals, as well as cell cultures, the Wa genome seems to be stable. Considering that the current reference sequence for the Wa strain is a composite sequence of various Wa variants, the rotavirus WaCS may be a more appropriate reference sequence. The rotavirus Wa and SA11 strains were selected for plasmid-based expression of rotavirus proteins, under control of a T7 promoter sequence, due to the fact that they propagate well in MA104 cells and the availability of their consensus sequences. The T7 RNA polymerase was provided by a recombinant fowlpox virus. After extensive transfection optimisation on a variety of mammalian cell lines, MA104 cells proved to be the best suited for the expression rotavirus proteins from plasmids. The expression of rotavirus Wa and SA11 VP1, VP6, NSP2 and NSP5 could be confirmed with immunostaining in MA104 and HEK 293H cells. Another approach involved the codon-optimised expression of the rotavirus replication complex scaffold in MA104 cells under the control of a CMV promoter sequence. This system was independent from the recombinant fowlpox virus. All three plasmid expression sets were designed to be used in combination with the transcript-based reverse genetics system in order to improve the odds of developing a successful rotavirus reverse genetics system. Rotavirus transcripts were generated using transcriptively active rotavirus SA11 double layered particles (DLPs). MA104 and HEK293H cells proved to be the best suited for the expression of rotavirus transcripts although expression of rotavirus VP6 could be demonstrated in all cell cultures examined (MA104, HEK 293H, BSR and COS-7) using immunostaining. In addition, the expression of transcript derived rotavirus VP1, NSP2 and NSP5 could be confirmed with immunofluorescence in MA104 and HEK 293H cells. This is the first report of rotavirus transcripts being translated in cultured cells. A peculiar cell death pattern was observed within 24 hours in response to transfection of rotavirus transcripts. This observed cell death, however does not seem to be related to normal viral cytopathic effect as no viable rotavirus could be recovered. In an effort to combine the transcript- and plasmid systems, a dual transfection strategy was followed where plasmids encoding rotavirus proteins were transfected first followed, 12 hours later, by the transfection of rotavirus SA11 transcripts. The codon- optimised plasmid system was designed as it was postulated that expression of the DLP-complex (VP1, VP2, VP3 and VP6), the rotavirus replication complex would form and assist with replication and/or packaging. Transfecting codon- optimized plasmids first noticeably delayed the mass cell death observed when transfecting rotavirus transcripts on their own. None of the examined coexpression systems were able to produce a viable rotavirus. Finally, the innate immune responses elicited by rotavirus transcripts and plasmid-derived rotavirus Wa and SA11 proteins were investigated. Quantitative RT-PCR (qRT-PCR) experiments indicated that rotavirus transcripts induced high levels of the expression of the cytokines IFN- α1, IFN-1β, IFN-λ1 and CXCL10. The expression of certain viral proteins from plasmids (VP3, VP7 and NSP5/6) was more likely to stimulate specific interferon responses, while other viral proteins (VP1, VP2, VP4 and NSP1) seem to be able to actively suppress the expression of certain cytokines. In the light of these suppression results, specific rotavirus proteins were expressed from transfected plasmids to investigate their potential in supressing the interferon responses provoked by rotavirus transcripts. qRT-PCR results indicated that cells transfected with the plasmids encoding NSP1, NSP2 or a combination of NSP2 and NSP5 significantly reduced the expression of specific cytokines induced by rotavirus transcripts. These findings point to other possible viral innate suppression mechanisms in addition to the degradation of interferon regulatory factors by NSP1. The suppression of the strong innate immune response elicited by rotavirus transcripts might well prove to be vital in the quest to better understand the replication cycle of this virus and eventually lead to the development of a selection-free reverse genetics system for rotavirus. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014