Background: Hepatitis E (HEV) is a small, non-enveloped virus that belongs to the viral genus Hepevirus. HEV is a positive sense single-stranded RNA virus and there is insufficient information regarding its replication. This is mainly because the virus has low capacity to grow in normally used cell cultures. Many specific strand assay detection studies have been done in order to understand more about HEV replication. Unfortunately, these assays have the disadvantage of giving false positive results. Aim: The aim of this project was to improve the positive strand assay to increase specificity and eliminate false positivity which is due to high sensitivity of the polymerase chain reaction (PCR). False positivity occurs as remains of transfected material in the cell are amplified. Method: The samples used in this project were swine samples from Sweden and a human sample (plasmid clone of genotype 1) from India. Negative samples, extracted positive samples and transcribed RNA positive sense samples were used. The methods applied were cDNA synthesis, exonuclease I and RNase treatments, DNA purification kits followed by first and nested PCR. Result: The results of this study indicated great improvement of the detection assay especially for the transcribed RNA samples. Best results were obtained at a final concentration of 1.5mM MgCl2 in the mastermix.  Conclusion: Changing the concentration of MgCl2 appeared to have a great effect on PCR specificity. Improving detection assays is very essential as they can be applied in the research field and in public health centers either for diagnosis or tracking disease outbreaks.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Science / ENGLISH ABSTRACT: Recent advances in medical pharmacology have identified the immune-potentiating effects of β-1,3-glucans on mammalian immune systems. Extensive research has identified and described the mechanisms of action and receptor binding specificity of different β-1,3-glucans as well as their structural and functional relationships. Molecular mass, solubility, structural order, degree of branching as well as chemical modification all determine the effectiveness of the β-1,3-glucan immune-modulating activities, which typically include; macrophage activation, antibody adjuvant activities, reduction of LDL-cholesterol, leukocyte mitogenic activities, cytokine and chemokine production as well as antiviral and antitumor activities. Currently β-1,3-glucans have been sold commercially under the name β-glucan, mostly in the form of Betafectin, a genetically modified yeast derived β-1,3-glucan. Recent studies of different β-1,3-glucans have identified the pharmacological activities of paramylon, a Euglena derived β-1,3-glucan. Although paramylon has relatively low immune-stimulating activities, chemical modification of the paramylon granule increased immune-potentiation with specific antimicrobial and anti-HIV activities. Due to these specific immune-potentiating activities, paramylon is novel in terms of both structure as well as functional activity. In terms of biotechnological application, paramylon is greatly favoured as it is synthesized as an insoluble membrane bound granule in the cytosol of Euglena where most plant and fungal β-1,3-glucan synthases are cell membrane bound highly regulated multifunctional complexes, synthesizing β-1,3-glucan as cell wall components. Due to the novel granular nature of paramylon, expression in other systems with genetic modification could potentially further increase immuno-potentiating activities. In this study, different approaches were attempted in order to identify the genes involved in paramylon synthesis including; constructing and screening a Euglena gracilis cDNA library, sequence analysis of the purified proteins as well as transcription analysis of the sequenced transcriptome and genome of E. gracilis. Putative candidates that encode subunits of the paramylon synthase complex have been identified. / AFRIKAANSE OPSOMMING: Onlangse vordering in mediese farmakologie het die immuun-stimulerende effek van β-1,3-glukane op die soogdier immuunsisteem geïdentifiseer. Intensiewe navorsing het die meganisme van die werking en reseptor bindingspesifisiteit van verskillende β-1,3-glukane, asook hulle struktuur en funksionele verhoudings, geïdentifiseer. Die molekulêre massa, oplosbaarheid, strukturele orientasie, mate van vertakking asook chemiese modifikasies bepaal almal die effektiwiteit van die β-1,3-glukaan immuun-modulerende aktiwiteite. Tipiese immuno-moduleringsaktiwiteite sluit makrofaag aktivering, teenliggaampie adjuvant aktiwiteite, verlaging van LDL-cholesterol, leukosiet mitogeniese aktiwiteite, sitokien en chemokien produksie asook anti-virale en antitumor aktiwiteite in. Huidiglik word β-1,3-glukane onder die naam β-glukaan verkoop meestal in die vorm van Betafectin, ‘n geneties gemodifiseerde gis wat van β-1,3-glukaan afkomstig is. Onlangse studies van verskillende β-1,3-glukane het die farmakologiese aktiwiteit van paramylon, ‘n Euglena afkomstige β-1,3-glukaan geïdentifiseer. Alhoewel paramylon relatiewe lae immuun-stimulerende aktiweite toon, verhoog chemiese modifikasies van die paramylon granules immuun-stimulering, spesifiek die anti-mikrobiese en anti-MIV aktiwiteite. Weens hierdie spesifieke immuun-stimulerende aktiweite, word paramylon as nuut beskou veral in terme van beide struktuur asook funksionele aktiwiteit. In terme van biotegnologiese toepassing, verkry paramylon voorkeur aangesien dit as ‘n onoplosbare membraangebonde granule in die sitosol van Euglena gesintetiseer word terwyl meeste plant en fungus β-1,3-glukaan sintases hoogs gereguleerde multifunksionele selmembraan gebonde komplekse is wat β-1,3-glukaan asook ander selwand komponente sintetiseer. Weens die unieke granulêre natuur van paramylon, sal uitdrukking in ander sisteme ‘n moontlike industrie skep waar deur die transgeniese uitdrukking van granulêre paramylon verdere verbetering van die immuun-stimulerings aktiwiteite kan lei. In hierdie studie is verskillende benaderings aangewend om die gene wat by paramylon sintese betrokke is te identifiseer, dit sluit in die konstruksie en sifting van ‘n E. gracilis cDNS biblioteek, aminosuur volgorde analise van gesuiwerde proteiene asook die transkripsionele analise van die volgorde van die transkriptoom en genoom van E. gracilis. Moontelike kandidate wat vir die subeenhede van die paramylon syntase kompleks kodeer is geïdentifiseer.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19016号 / 農博第2094号 / 新制||農||1029(附属図書館) / 学位論文||H27||N4898(農学部図書室) / 31967 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 河田 照雄, 教授 谷 史人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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Regelmäßige Bonituren haben gezeigt, dass virusverdächtige Symptome an Stieleichen, die zu etwa 90 % als chlorotische Ringflecken auftreten, im nord- und mitteldeutschen Raum weit verbreitet sind. In der vorliegenden Arbeit sollte der Erreger dieser Symptome isoliert und näher charakterisiert werden. Aus zwei Blattproben mit chlorotischen Ringflecken konnten stäbchenförmige Viruspartikeln mit einer Länge von ca. 450 nm isoliert und auf krautige Indikatoren übertragen werden. In einer RT-PCR mit Hüllprotein bzw. Transportprotein-sequenzspezifischen Primern wurden diese als Tobacco mosaic virus (TMV)- bzw. Tomato mosaic virus (ToMV)- Isolate identifiziert. Eine Infektion der Stieleichen mit weiteren bekannten Viren von Gehölzen, wie dem Cherry leaf roll virus (CLRV) oder dem Erreger der Ebereschenringfleckigkeit konnte mittels ELISA und RT-PCR ausgeschlossen werden. DsRNAs der Größen 1.5 und 1.6 kb sowie 1.8 und 2.0 kb konnten symptomunabhängig aus Rindengewebe, Knospen und Blättern von Stieleichen isoliert werden. Mit Hilfe der RT-DOP-PCR und der cDNA-Klonierung gelang es, Teile des 1.5/1.6 kb dsRNA-Moleküls zu charakterisieren. Die Sequenz von 479 Aminosäuren (1437 Nukleotiden) wies eine Identität von 56 % zur RNA-abhängigen RNA-Polymerase (RdRp) des Beet cryptic virus 3 (BCV 3) auf. Der spezifische Nachweis dieser Sequenz gelang mittels RT-PCR sowohl in dsRNA-Proben, als auch in angereicherten Nukleokapsiden symptomloser und symptomatischer Stieleichen. In Nested-PCR-Analysen konnte das Fragment jedoch nicht nur in Gesamt-RNA von Stieleichen, sondern auch in Gesamt-RNA und DNA verschiedenster gesunder Pflanzen amplifiziert werden. Phylogenetische Vergleiche mit ausgewählten RdRps viralen und pflanzlichen Ursprungs zeigten die engste Verwandtschaft der Stieleichen-dsRNA-Sequenz zu den Partitiviren, zu denen sich neben BCV 3 auch die endogene dsRNA aus Pyrus und aus Chloroplasten von Bryopsis gruppiert. Diese Erkenntnisse lassen in der charakteristischen Doppelbande von 1.5/1.6 kb das Vorliegen einer endogenen dsRNA vermuten. Hiermit ist in dieser Arbeit das Auftreten verschiedener Viren in Eichen nachgewiesen worden, von denen die meisten höchstwahrscheinlich nicht im direkten ursächlichen Zusammenhang mit der chlorotischen Ringfleckigkeit der Eiche stehen. / Ratings of oak populations revealed that around 90 % of all oak trees affected by viruslike symptoms showed chlorotic ringspots and that these symptoms are widely spread in oaks in north and central Germany. In this study the putative agent of these symptoms should be isolated and specified. Rod-shaped particles with a length of 450 nm were recovered from two different samples of leaves displaying chlorotic ringspots by mechanical inoculation of herbaceous indicator plants. These particles were identified to be Tobacco mosaic virus (TMV)- and Tomato mosaic virus (ToMV)- isolates by RT-PCR analyses of the coat- and movement protein genes. Infections with other well known viruses of forest trees, like Cherry leaf roll virus (CLRV) and the agent causing ringspots in European mountain ash, were excluded by ELISA and RT-PCR. DsRNA fragments of 1.5 and 1.6 kb as well as 1.8 and 2.0 kb were extracted from leaves, inner bark and bulbs of all symptomatic and asymptomatic samples of common oak. The nucleotide sequence of the 1.5 and 1.6 kb dsRNA fragment was partially characterised by reverse transcription degenerated oligonucleotide primed (DOP)-PCR and cDNA cloning. The obtained nucleotide sequence of 1437 nt encoding a putative protein of 479 amino acids revealed an identity of 56 % with the RNA-dependent RNA polymerase (RdRp) of Beet cryptic virus 3 (BCV 3). PCR amplification of the RdRp coding nucleotide sequence was possible using a number of different dsRNA samples as well as concentrated nucleocapside preparations. The same sequence was also amplified successfully by Nested-PCR not only in total RNA extracted from symptomatic and asymptomatic oak samples but also from total RNA and DNA of diverse plants. Phylogenetic analysis revealed further similarities to RdRp´s of endogenous dsRNA of Pyrus and chloroplasts of Bryopsis, both members of the Partitiviridae as well as BCV 3. These results strongly indicate that the 1.5/1.6 kb dsRNA of oak is endogenous dsRNA. In summary, it has been shown that oaks in Germany are commonly infected by a variety of different viruses most of them possibly unrelated to the wide-spread ringspot symptoms of oaks.