Awareness of detoxification, nowadays known as biotransformation, has become an integral part of our daily lives. It is a modern buzz word that is used to promote anything from health food to enhancement of performance in sports. Another lesser known application for detoxification is as a therapy for alleviating symptoms of inborn errors of metabolism. Detoxification is the process where endogenous and xenobiotic metabolites are transformed to less harmful products, in the liver and kidneys, in two phases. Phase 1 detoxification includes oxidation, hydroxylation, dehydrogenation metabolic reduction and hydrolysis. Phase 2 detoxification uses conjugation reactions to increase hydrophillicty of metabolites for excretion in bile and urine. Glycine N-acyltransferse (GLYAT; EC 2.3.1.13) is one of the amino acid conjugation enzymes. There are two variants of human GLYAT. I focused on the full-length mRNA human GLYAT isoform a, with a long term view of using it as a viable therapeutic enzyme for enhanced detoxification of harmful metabolites. I investigated if it is possible to clone and express a biologically active GLYAT. To achieve this goal I used three expression systems: traditional bacterial expression using the pET system; second generation cold shock bacterial expression using the pCOLDTF expression vector to improve solubility of the recombinant protein; and baculovirus expression in insect cells since therein some form of post translation glycosylation of the recombinant protein can occur which might improve solubility and ensure biological activity. The recombinant GLYAT expressed well in all three expression systems but was aggregated and no enzyme activity could be detected. A denature and renature system was also used to collect aggregated recombinant GLYAT and used to try to refold the recombinant protein in appropriate refolding buffers to improve solubility and obtain biological activity. The solubility of the recombinant GLYAT was improved but it remained biologically inactive. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.

Awareness of detoxification, nowadays known as biotransformation, has become an integral part of our daily lives. It is a modern buzz word that is used to promote anything from health food to enhancement of performance in sports. Another lesser known application for detoxification is as a therapy for alleviating symptoms of inborn errors of metabolism. Detoxification is the process where endogenous and xenobiotic metabolites are transformed to less harmful products, in the liver and kidneys, in two phases. Phase 1 detoxification includes oxidation, hydroxylation, dehydrogenation metabolic reduction and hydrolysis. Phase 2 detoxification uses conjugation reactions to increase hydrophillicty of metabolites for excretion in bile and urine. Glycine N-acyltransferse (GLYAT; EC 2.3.1.13) is one of the amino acid conjugation enzymes. There are two variants of human GLYAT. I focused on the full-length mRNA human GLYAT isoform a, with a long term view of using it as a viable therapeutic enzyme for enhanced detoxification of harmful metabolites. I investigated if it is possible to clone and express a biologically active GLYAT. To achieve this goal I used three expression systems: traditional bacterial expression using the pET system; second generation cold shock bacterial expression using the pCOLDTF expression vector to improve solubility of the recombinant protein; and baculovirus expression in insect cells since therein some form of post translation glycosylation of the recombinant protein can occur which might improve solubility and ensure biological activity. The recombinant GLYAT expressed well in all three expression systems but was aggregated and no enzyme activity could be detected. A denature and renature system was also used to collect aggregated recombinant GLYAT and used to try to refold the recombinant protein in appropriate refolding buffers to improve solubility and obtain biological activity. The solubility of the recombinant GLYAT was improved but it remained biologically inactive. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.

Rotavirus infection causes severe gastroenteritis, affecting all children under the age of five regardless of hygiene or water quality. The currently licensed vaccines succeeded in reducing diarrhoea worldwide, but they still have shortcomings, especially the efficacy of the vaccines in developing countries. One of the main reasons for this can be due to the difference in strains, since the strains used to develop the currently licensed vaccines (RotaTeq and Rotarix) were selected from strains circulating in the developed world (G1, G2, G3 and G4), while the main strains present in Africa (G8, G9 and G12) were not included. A second shortcoming of the currently licensed vaccines is the cost of these vaccines. The vaccines are very expensive and most developing countries cannot afford the vaccines as well as the fact that the manufacturing companies cannot produce enough vaccines for all the countries. An attractive alternative to the currently licensed rotavirus vaccines is the non-live vaccine candidate, virus-like particles, which can provide a possible cheaper, safer and efficacious alternative or complement the currently licensed vaccines. Therefore, in this study a South African G9P[6] rotavirus strain, RVA/Humanwt/ ZAF/GR10924/1999/G9P[6], was used to determine whether or not co-expression of the structural proteins VP2 (genome segment 2) and VP6 (genome segment 6) was possible in bacteria and yeast. The South African GR10924 G9P[6] neonatal strain was previously obtained from a stool sample and the nucleotide consensus sequence was determined for both genome segment 2 (VP2) and genome segment 6 (VP6). Bacterial codon optimised coding regions or open reading frames were used in this study. The open reading frames (ORFs) of the genome segments encoding, VP2 and VP6, were cloned into the expression vector pETDuet-1, which allows for the simultaneous expression of two genes in bacteria. The ORF of genome segment 6 was purchased from GeneScript and the ORF of genome segment 2 was obtained from Dr AC Potgieter (Deltamune (Pty) Ltd R&D, South Africa). Compatible restriction enzyme sites were used to sub-clone the ORF of the bacterial codon optimised genome segments into the expression vector. Only the expression of the VP6 protein in bacteria was observed with Coomassie stained SDS-PAGE. The ORFs encoding VP2 (genome segment 2) and VP6 (genome segment 6) of the wild type GR10924 G9P[6] strain were cloned into the wide range yeast expression system vector, pKM173, which allows for the simultaneous expression of more than one gene. Several yeast strains were used in this study namely Kluyveromyces marxianus, Kluyveromyces lactis, Candida deformans, Saccharomyces cerevisiae, Yarrowia lipolytica, Arxula adeninivorans, Hansenula polymorpha and Debaryomyces hansenii. Expression of both proteins was not detected in the several yeast strains, as seen with western blot analysis. DNA extractions were done on two colonies of each yeast strain that were used for western blot analysis to evaluate successful integration into the yeast genomes. Only a few of the colonies contained either both of the genome segments or only one of the two genome segments of interest. To summarise, the simultaneous expression of VP2 and VP6 from rotavirus GR10924 G9P[6] was not successful in bacteria or yeast, but it was possible to soluble express the bacterial codon optimised GR10924 G9P[6] VP6 in bacteria using the pETDuet-1 as expression vector. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

Rotavirus infection causes severe gastroenteritis, affecting all children under the age of five regardless of hygiene or water quality. The currently licensed vaccines succeeded in reducing diarrhoea worldwide, but they still have shortcomings, especially the efficacy of the vaccines in developing countries. One of the main reasons for this can be due to the difference in strains, since the strains used to develop the currently licensed vaccines (RotaTeq and Rotarix) were selected from strains circulating in the developed world (G1, G2, G3 and G4), while the main strains present in Africa (G8, G9 and G12) were not included. A second shortcoming of the currently licensed vaccines is the cost of these vaccines. The vaccines are very expensive and most developing countries cannot afford the vaccines as well as the fact that the manufacturing companies cannot produce enough vaccines for all the countries. An attractive alternative to the currently licensed rotavirus vaccines is the non-live vaccine candidate, virus-like particles, which can provide a possible cheaper, safer and efficacious alternative or complement the currently licensed vaccines. Therefore, in this study a South African G9P[6] rotavirus strain, RVA/Humanwt/ ZAF/GR10924/1999/G9P[6], was used to determine whether or not co-expression of the structural proteins VP2 (genome segment 2) and VP6 (genome segment 6) was possible in bacteria and yeast. The South African GR10924 G9P[6] neonatal strain was previously obtained from a stool sample and the nucleotide consensus sequence was determined for both genome segment 2 (VP2) and genome segment 6 (VP6). Bacterial codon optimised coding regions or open reading frames were used in this study. The open reading frames (ORFs) of the genome segments encoding, VP2 and VP6, were cloned into the expression vector pETDuet-1, which allows for the simultaneous expression of two genes in bacteria. The ORF of genome segment 6 was purchased from GeneScript and the ORF of genome segment 2 was obtained from Dr AC Potgieter (Deltamune (Pty) Ltd R&D, South Africa). Compatible restriction enzyme sites were used to sub-clone the ORF of the bacterial codon optimised genome segments into the expression vector. Only the expression of the VP6 protein in bacteria was observed with Coomassie stained SDS-PAGE. The ORFs encoding VP2 (genome segment 2) and VP6 (genome segment 6) of the wild type GR10924 G9P[6] strain were cloned into the wide range yeast expression system vector, pKM173, which allows for the simultaneous expression of more than one gene. Several yeast strains were used in this study namely Kluyveromyces marxianus, Kluyveromyces lactis, Candida deformans, Saccharomyces cerevisiae, Yarrowia lipolytica, Arxula adeninivorans, Hansenula polymorpha and Debaryomyces hansenii. Expression of both proteins was not detected in the several yeast strains, as seen with western blot analysis. DNA extractions were done on two colonies of each yeast strain that were used for western blot analysis to evaluate successful integration into the yeast genomes. Only a few of the colonies contained either both of the genome segments or only one of the two genome segments of interest. To summarise, the simultaneous expression of VP2 and VP6 from rotavirus GR10924 G9P[6] was not successful in bacteria or yeast, but it was possible to soluble express the bacterial codon optimised GR10924 G9P[6] VP6 in bacteria using the pETDuet-1 as expression vector. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

'n Onafhanklike, ondersoekende en verantwoordelike media word beskou as een van die belangrikste rolspelers waarvan die behoud van die demokrasie in Suid- Afrika afhanklik is. Die regte en vryhede van die media geniet grondwetlike erkenning en beskerming. As fundamentele regte en onontbeerlike komponente van mediavryheid, waarborg die Grondwet van die Republiek van Suid-Afrika, 1996 aan elkeen die reg op vryheid van uitdrukking (artikel 16) en die reg op toegang tot inligting (artikel 32). Die grondwetlike beskerming van mediavryheid bemagtig die media om (i) openbare aangeleenthede in belang van die publiek aan te spreek, (ii) die staat tot deursigtigheid, openheid en verantwoording op te roep en (iii) as teenwig teen magsmisbruik en wanpraktyke aan regeringskant op te tree. Die reg op mediavryheid is egter nie absoluut afdwingbaar nie en kan gevolglik in die toepassing daarvan beperk word. Die staat beskik oor die gesag om dié reg aan beperkings onderhewig te stel ten einde ander individuele fundamentele regte te beskerm of belangrike openbare belange te dien. Sodanige staatsoptrede is egter slegs moontlik indien dit, weens die oppergesag van die Grondwet (artikel 2), in ooreenstemming met die grondwetlike bepalings geskied. Desnieteenstaande word die media, ondanks die belangrike rol wat hulle ter behoud van die demokrasie in Suid-Afrika vervul en die grondwetlike erkenning en beskerming wat aan mediavryheid verleen word, voortdurend onder groot druk geplaas. Die meeste druk kom van die staat se kant. Die aanname en beoogde implementering van die Protection of State Information Bill, 2010 dien as mees resente voorbeeld hiervan. Staats- en administratiefregtelike beperkings op mediavryheid sal, indien die Wetsontwerp gepromulgeer word, aan die orde van die dag wees. Aangesien die voorgenome beperkings op grond van hulle potensiële ongrondwetlikheid wyd gekritiseer word, is dit die vernaamste doel van hierdie studie om die grondwetlike grense waarbinne die staat mediavryheid mag beperk, te omlyn en ondersoek in te stel na die trefwydte en grondwetlikheid van die staat se gesag om beperkings op mediavryheid te plaas. Deur die omvang van staatsgesag in die algemeen en in besonder rakende die beperking van mediavryheid, aan die hand van die relevante grondwetlike voorskrifte te ondersoek, is bevind dat alle staatsoptrede streng deur die Grondwet gereguleer word. Alhoewel die reg op mediavryheid vatbaar is vir regulering en beperking, is tot die slotsom gekom dat die Grondwet ingevolge artikels 33 en 36 duidelike grense vir die beperking daarvan deur die staat stel. Alle uitvoerende en administratiewe staatsorgane wat kragtens wetgewing en uit hoofde van die diskresionêre bevoegdhede wat aan hulle verleen word, die reg op mediavryheid beperk, moet aan die vereistes vir regverdige administratiewe optrede voldoen. Bygevolg moet alle administratiewe besluite en handelinge wat die reg op mediavryheid beperk, regmatig, redelik en prosedureel billik wees en met die verskaffing van voldoende skriftelike redes gepaard gaan. Sowel staatsregtelike as administratiefregtelike beperkings op mediavryheid moet voldoen aan die vereistes wat die algemene beperkingsklousule vir die grondwetlike beperking van fundamentele regte stel. Enige beperking van die reg op mediavryheid moet dus kragtens 'n algemeen geldende regsvoorskrif geskied en moet, met in agneming van die artikel 36-sleutelfaktore, as redelik en regverdigbaar binne 'n oop en demokratiese samelewing gebaseer op menswaardigheid, gelykheid en vryheid plaasvind. Enige staats- en administratiefregtelike beperkings op mediavryheid wat nie aan hierdie grondwetlike vereistes voldoen nie, is gevolglik ongrondwetlik. Nadat die bepalings ter beperking van mediavryheid deur die Protection of State Information Bill, 2010 krities ontleed is, is bevind dat dit nie daarin sal slaag om die grondwetlike toets te slaag nie. / LLM, North-West University, Potchefstroom Campus, 2015

'n Onafhanklike, ondersoekende en verantwoordelike media word beskou as een van die belangrikste rolspelers waarvan die behoud van die demokrasie in Suid- Afrika afhanklik is. Die regte en vryhede van die media geniet grondwetlike erkenning en beskerming. As fundamentele regte en onontbeerlike komponente van mediavryheid, waarborg die Grondwet van die Republiek van Suid-Afrika, 1996 aan elkeen die reg op vryheid van uitdrukking (artikel 16) en die reg op toegang tot inligting (artikel 32). Die grondwetlike beskerming van mediavryheid bemagtig die media om (i) openbare aangeleenthede in belang van die publiek aan te spreek, (ii) die staat tot deursigtigheid, openheid en verantwoording op te roep en (iii) as teenwig teen magsmisbruik en wanpraktyke aan regeringskant op te tree. Die reg op mediavryheid is egter nie absoluut afdwingbaar nie en kan gevolglik in die toepassing daarvan beperk word. Die staat beskik oor die gesag om dié reg aan beperkings onderhewig te stel ten einde ander individuele fundamentele regte te beskerm of belangrike openbare belange te dien. Sodanige staatsoptrede is egter slegs moontlik indien dit, weens die oppergesag van die Grondwet (artikel 2), in ooreenstemming met die grondwetlike bepalings geskied. Desnieteenstaande word die media, ondanks die belangrike rol wat hulle ter behoud van die demokrasie in Suid-Afrika vervul en die grondwetlike erkenning en beskerming wat aan mediavryheid verleen word, voortdurend onder groot druk geplaas. Die meeste druk kom van die staat se kant. Die aanname en beoogde implementering van die Protection of State Information Bill, 2010 dien as mees resente voorbeeld hiervan. Staats- en administratiefregtelike beperkings op mediavryheid sal, indien die Wetsontwerp gepromulgeer word, aan die orde van die dag wees. Aangesien die voorgenome beperkings op grond van hulle potensiële ongrondwetlikheid wyd gekritiseer word, is dit die vernaamste doel van hierdie studie om die grondwetlike grense waarbinne die staat mediavryheid mag beperk, te omlyn en ondersoek in te stel na die trefwydte en grondwetlikheid van die staat se gesag om beperkings op mediavryheid te plaas. Deur die omvang van staatsgesag in die algemeen en in besonder rakende die beperking van mediavryheid, aan die hand van die relevante grondwetlike voorskrifte te ondersoek, is bevind dat alle staatsoptrede streng deur die Grondwet gereguleer word. Alhoewel die reg op mediavryheid vatbaar is vir regulering en beperking, is tot die slotsom gekom dat die Grondwet ingevolge artikels 33 en 36 duidelike grense vir die beperking daarvan deur die staat stel. Alle uitvoerende en administratiewe staatsorgane wat kragtens wetgewing en uit hoofde van die diskresionêre bevoegdhede wat aan hulle verleen word, die reg op mediavryheid beperk, moet aan die vereistes vir regverdige administratiewe optrede voldoen. Bygevolg moet alle administratiewe besluite en handelinge wat die reg op mediavryheid beperk, regmatig, redelik en prosedureel billik wees en met die verskaffing van voldoende skriftelike redes gepaard gaan. Sowel staatsregtelike as administratiefregtelike beperkings op mediavryheid moet voldoen aan die vereistes wat die algemene beperkingsklousule vir die grondwetlike beperking van fundamentele regte stel. Enige beperking van die reg op mediavryheid moet dus kragtens 'n algemeen geldende regsvoorskrif geskied en moet, met in agneming van die artikel 36-sleutelfaktore, as redelik en regverdigbaar binne 'n oop en demokratiese samelewing gebaseer op menswaardigheid, gelykheid en vryheid plaasvind. Enige staats- en administratiefregtelike beperkings op mediavryheid wat nie aan hierdie grondwetlike vereistes voldoen nie, is gevolglik ongrondwetlik. Nadat die bepalings ter beperking van mediavryheid deur die Protection of State Information Bill, 2010 krities ontleed is, is bevind dat dit nie daarin sal slaag om die grondwetlike toets te slaag nie. / LLM, North-West University, Potchefstroom Campus, 2015

Emotions and its effect on the individual’s general functioning are a key concept of humanity. The modern child is confronted with all kinds of emotional developmental tasks with direct influence on his ultimate figuration to adulthood. Emotion is an internal experience in contrast to the fact that various reactions are displayed externally as a result thereof. Children’s behavior demonstrates that which are experienced internally. It might also be a way to conceal especially those emotions. Emotional awareness manifests through demonstration or concealing of inner feelings. The child’s external reactions to inner feelings must be based on knowledge of what is being experienced. Behavior is mostly a reaction to ignorance of the inner experience which might manifest in anxiety attacks, anger and emotional episodes. Empowerment of the child to recognize and experience emotions enables him to gain insight of emotions as a natural part of human nature. This causes him to develop the ability to express emotion in a socially acceptable manner. The process of emotional awareness alerts the child to the fact those specific emotions results in specific bodily experiences. Emotional awareness is an indication of the child’s knowledge of emotions and its impact in emotional, physical and psychological reactions. It provides an explanation for anxieties and fears which enables the child to own these feelings and take control of it. The middle childhood phase is the period that follows the achievement of a mass of developmental skills like the mastery of language, control over bodily functions and cognitive abilities. These abilities are refined in this phase. Emotional awareness and especially concepts of self and the purpose of individuals in the systems that surrounds him evolves. Considerable part of the child’s day during this phase, is spent at school. Emotional wellbeing can thus efficiently be recognized and addressed by the educational system. The educator’s knowledge of emotional awareness will ensure meaningful emotional development of learners. Results obtained from questionaires completed by educators in primary education indicates a need for understanding of problem behavior in children. The need for education on emotional awareness and techniques for development of emotional awareness in learners is also expressed. The focus of this study is thus on the concept of educators regarding the emotional awareness of learners; the role of emotional awareness in the child’s development and the educators knowledge regarding development of emotional skills and promotion of emotional awareness. / Dissertation (MSD (Play Therapy))--University of Pretoria, 2006. / Social Work and Criminology / MSD / unrestricted

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