Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Grapevine leafroll-associated virus 3 (GLRaV-3) is the type member of the genus Ampelovirus in the family Closteroviridae and is considered to be the main contributing agent of grapevine leafroll disease (GLD) worldwide. A metagenomic sequencing study of a grapevine leafroll-diseased vineyard led to the discovery of a new variant of GLRaV-3 in South Africa. This new variant was most related to a New Zealand isolate, NZ-1. In this study, we sequenced two isolates, GH11 and GH30, of the new variant group of GLRaV-3. These isolates have less than 70% nucleotide (nt) identity to other known GLRaV-3 variants, indicating that they should be considered variants of a different strain of GLRaV-3. We propose that the GLRaV-3-like virus identified in this study be grouped together with NZ-1 and some Napa Valley isolates as Group VI of GLRaV-3. This study also provided further evidence that next-generation sequencing is an invaluable approach to identify novel viruses and variants, in that the draft sequence generated with bioinformatic tools in this study was 98% identical to the GH11 sequence generated using Sanger sequencing. The study further confirmed that the industry standard ELISA is still an effective GLRaV-3 diagnostic method and that it is able to detect all known variant groups of GLRaV-3. However, this assay is not able to differentiate between GLRaV-3 variant groups. In the current study therefore, a real-time RT-PCR was designed that is able to detect GLRaV-3 variant groups I, II, III and VI, using a single primer pair targeting the Hsp70h gene of GLRaV-3. If high-resolution melting (HRM) curve analysis is added to the real-time RT-PCR, it is possible to differentiate between variant groups based on three melting point intervals. The RT-PCR HRM assay provides a more sensitive and rapid tool to detect and differentiate between different GLRaV-3 variant groups. Finally, a multiplex RT-PCR was designed to differentiate between the variant groups present in South Africa. This multiplex RT-PCR offers a validation method for the RT-PCR HRM and provides an end-point PCR alternative for variant identification. In order to investigate the spread and impact of different GLRaV-3 variants in vineyards, sensitive diagnostic techniques are a necessity. The abovementioned tools will contribute to the understanding of the pathogenesis of GLD and aid epidemiological studies to investigate how these different GLRaV-3 variant groups are spreading, the association of specific GLRaV-3 variants to disease symptoms and the mealybug vector transmission efficiency for each GLRaV-3 variant. / AFRIKAANSE OPSOMMING: Grapevine leafroll-associated virus 3 (GLRaV-3) is ’n lid van die genus Ampelovirus in die familie Closteroviridae en word beskou as die hoof bydraende faktor van wingerd-rolbladsiekte wêreldwyd. ’n Metagenomiese studie het bewys dat daar ’n nuwe variant van GLRaV-3 bestaan wat nog nie voorheen in Suid Afrika opgespoor kon word met die huidige opsporingsmetodes nie. Hierdie nuwe variant was naaste verwant aan ’n Nieu-Seelandse isolaat, NZ-1. In hierdie studie is die genoomvolgorde van twee isolate, GH11 en GH30, van hierdie nuwe GLRaV-3 variant groep bepaal. Hierdie twee isolate was minder as 70% identies aan ander GLRaV-3 variante, wat daarop dui dat hulle as variante van ’n nuwe virus-ras beskou behoort te word. Ons beveel aan dat hierdie GLRaV-3-verwante virus geklassifiseer word saam met die NZ-1 isolaat en ander isolate uit Kalifornië, as groep VI van GLRaV-3. Hierdie studie het ook verdere bewyse verskaf dat volgende-generasie volgordebepalingstegnologie ’n waardevolle benadering is om nuwe virusse en variante te identifiseer, deurdat die huidige studie gewys het dat die voorlopige volgorde, wat gegenereer is deur bioinformatika-instrumente, 98% identies was aan die GH11 volgorde wat met Sanger volgordebepaling verkry was. Hierdie studie het ook gevind dat die industrie-standaard ELISA, nog steeds ’n effektiewe GLRaV-3 diagnostiese metode is en wel infeksies, veroorsaak deur al die variant-groepe, sal kan identifiseer. Die ELISA toets is egter nie in staat om te onderskei tussen GLRaV-3 variant-groepe nie. In hierdie studie is ’n variant-identifiseerbare in-tyd tru-transkripsie polimerase ketting reaksie (PKR) ontwerp wat GLRaV-3 variant-groepe I, II, III en VI kan identifiseer deur middel van ’n enkele inleier-stel wat die GLRaV-3 Hsp70h-geen teiken. As hoë-resolusie smeltingskurwe-analise bygevoeg word by die in-tyd tru-transkripsie PKR, is dit moontlik om te onderskei tussen variant-groepe op grond van drie smeltingspunt intervalle. Die tru-transkripsie hoë-resolusie smeltingskurwe-toets verskaf meer sensitiewe en geoutomatiseerde metodes om GLRaV-3 variant-groepe te identifiseer en te onderskei. ’n Veelvuldige tru-transkripsie PKR is ook ontwerp om tussen variante wat tans in Suid-Afrika aangetref word, te onderskei en te dien as ’n valideringsmetode vir die in-tyd tru-transkripsie hoë-resolusie smeltingskurwe-toets. Sensitiewe en akkurate toetse, soos bogenoemde, is noodsaaklik vir die bestudering van die verspreiding en impak van die verskillende GLRaV-3 variante in wingerd. Hierdie metodes kan gebruik word om kennis ten opsigte van rolblad patogenese te verbreed en om by te dra tot epidemiologiese studies wat ondersoek hoe hierdie variant-groepe versprei, of daar ’n assosiasie bestaan tussen ’n spesifieke variant en siekte-simptome en of daar ’n verskil is in die witluisvektor oordragseffektiwitiet vir elke GLRaV-3 variant.

Plus de 40 ans après la découverte de la technologie des hybridomes, une soixantaine d’anticorps monoclonaux thérapeutiques IgG ou assimilés sont aujourd’hui commercialisés. Leur succès découle de leur humanisation, en particulier celle de la portion Fc qui dérive de différents variants humains naturels, isotypes et allotypes. Depuis quelques années, apparaissent sur le marché de nombreux anticorps comportant des portions Fc artificiellement modifiées dans le but de moduler diverses propriétés pharmacologiques (propriétés cytolytiques, demi-vie, stabilité, etc.), dont certaines ont été particulièrement étudiées suite aux travaux de notre équipe. Les variants Fc sont protégés par des technologies brevetées dont on connaît mal l’étendue, qui ne font pas nécessairement l’objet de publications scientifiques, et dont la raison d’être reste méconnue des chercheurs et plus encore des professionnels de santé. Nous avons donc entrepris de réaliser une cartographie et une analyse fine des brevets traitant des modifications dans la portion Fc des IgG. Cette analyse a été menée de front avec une étude bibliographique détaillée, car les données scientifiques décrites dans les brevets sont toujours à considérer avec prudence, les demandes de brevets n’étant pas revues par des pairs. Nous avons eu l’occasion d’ailleurs d’épingler certaines dérives, comme celle de considérer qu’il pourrait y avoir plus de 4 sous-classes d’IgG dans l’espèce humaine (jusqu’à 19 dans certaines revendications…). / More than 40 years after the discovery of the hybridoma technology, around sixty therapeutic monoclonal antibodies based on IgG or assimilated are marketed today. Their success comes from their humanization, especially of the Fc portion derived from various natural human variants, isotypes and allotypes. For some years, many antibodies artificially modified in their Fc portions have emerged, in order to alter various pharmacological properties (cytolitic properties, half-life, stability, etc.), some of them having been particularly studied following the works of our team. Fc variants are covered by patented technologies of which little is known about the extent, which are not necessarily the subject of scientific publications, and whose purpose remains unknown for researchers and even more for health professionals. We therefore undertook to realize a landscape and a detailed analysis of patents dealing with modifications in the Fc portion of IgG. This analysis has been conducted in front with a detailed literature survey, since the scientific data described in patents must be treated with caution, as patent application are not peer reviewed. We actually point certain abuses, such as to consider that there might be more than four human IgG subclasses (up to 19 in some claims…).