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.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Grapevine leafroll disease (GLD), caused by the members of the family Closteroviridae, is one of the most economic important viral diseases affecting grapevine. Grapevine leafroll associated virus 3 (GLRaV-3), of the genus Ampelovirus, is the most widespread member of the leafroll associated virus family. To prevent the spread of GLD, management strategies such as rogueing and insect vector control are required to limit crop losses. Alternative control strategies based on genetic modification of the grapevine genome, such as pathogen-derived resistance (PDR), is proven to be effective in conferring resistance to several viruses. Therefore, the focus of this study was to evaluate pathogen-derived resistance strategies for GLRaV-3 using the following two approaches; 1) evaluation of transgenic plants expressing a dysfunctional GLRaV-3 heat shock protein 70 homolog (HSP70h) in order to confer resistance against GLRaV-3, and 2) the construction of artificial microRNAs (amiRNAs) to use as a tool for silencing specific sequences of GLRaV-3 in the grapevine host and the development of an amiRNA-mediated silencing validation system. In the first part of this study, six transgenic plant lines (plant lines #1, #3, #9, #14, #15 and #17) as well as a non-modified plant line, were inoculated with GLRaV-3 by grafting buds of each onto GLRaV-3 infected plant material. After approximately five months, GLRaV-3 virus titres of all grafted plants were quantified relative to two reference genes using RT-qPCR. Results were evaluated by comparing the relative virus titre of each transgenic plant line to that of the non-modified control plant line. Results showed that resistance levels of plant line #3 was significantly enhanced (>99%) and remarkably, plant line #14, showed to be more susceptible to the virus. The second part of the study was the construction and validation of amiRNAs targeting GLRaV-3 sequences. Two 21 nt regions of GLRaV-3 were successfully incorporated into miRNA backbone vvi167b of grapevine. Moreover, target constructs were developed by incorporating corresponding GLRaV-3 target sequences into the 3’ UTR of a green fluorescence protein (GFP) gene. Subsequently, the target constructs were co-infiltrated with the constructed amiRNA in Nicotiana benthamiana and GFP expression levels were quantified to determine the silencing efficiency of the amiRNAs. Results showed that the amiRNAs were successful in silencing the GFP target construct, however, they were not specific in silencing exclusively their corresponding target. These amiRNA constructs are ideal for further viral studies to determine the efficiency of silencing GLRaV-3 in GLD infected grapevines. / AFRIKAANSE OPSOMMING: Wingerd rolblaar siekte (GLD), wat veroorsaak word deur die lede van die familie Closteroviridae, is een van die ekonomies mees belangrike virus siektes van wingerd. Grapevine leafroll-associated virus 3 (GLRaV-3), van die genus Ampelovirus, is die mees wydverspreide lid van die rolblaar geassosieerde virus familie. Om die verspreiding van GLD te voorkom, is bestuur strategieë, soos die verwydering van geïnfekteerde plante en insekvektor beheer, ’n vereiste om oes verliese te beperk. Alternatiewe beheer strategieë gebaseer op genetiese modifikasie van die wingerdgenoom, soos patogeen-afgeleide weerstand (PDR), is bewys om effektief te wees in die verlening van weerstand teen verskeie virusse. Daarom was die fokus van hierdie studie om patogeen-afgeleide weerstand strategieë vir GLRaV-3 te evalueer met behulp van die volgende twee benaderings; 1) die evaluering van transgeniese plante wat 'n disfunksionele GLRaV-3 hitte-skok proteïen 70 homoloog (HSP70h) uitdruk, ten einde weerstand te verleen teen GLRaV-3, en 2) die konstruksie van kunsmatige mikroRNAs (amiRNAs) om te gebruik as 'n instrument vir die ondrukking van spesifieke genoomvolgordes van GLRaV-3 in die wingerd gasheer en die ontwikkeling van ’n stelsel om amiRNA-bemiddelde onderdrukking te bevestig. In die eerste deel van hierdie studie, is ses transgeniese plant lyne (plant lyne # 1, # 3, # 9, # 14, # 15 en # 17) sowel as 'n nie-gemodifiseerde gesonde plant lyn, geïnokuleer met GLRaV- 3 deur enting van ogies van elk op GLRaV-3 besmette plantmateriaal. Na ongeveer vyf maande, is GLRaV-3 virus konsentrasies van alle ingeënte plante gekwantifiseer relatief tot twee verwysing gene deur gebruik te maak van tru-transkripsie kwantitatiewe PCR (RTqPCR). Resultate is geëvalueer deur die relatiewe virus konsentrasie van elke transgeniese plant lyn te vergelyk met dié van die nie-gemodifiseerde kontrole lyn. Resultate het getoon dat weerstand vlakke van plant lyn # 3 beduidend verbeter is (> 99%) en merkwaardig is plant lyn # 14 bewys om meer vatbaar vir die virus te wees. Die tweede deel van die studie was die konstruksie en bevestiging van kunsmatige mikroRNAs (amiRNAs) wat GLRaV-3 genoomvolgordes teiken. Twee 21 nt streke van GLRaV-3 is suksesvol geïnkorporeer in die ruggraat van wingerd mikroRNA vvi167b. Verder is teiken konstrukte ontwikkel deur die inkorporering van ooreenstemmende GLRaV-3 teiken genoomvolgordes in die 3'UTR (3’ ongetransleerde area) van 'n groen fluoressensie proteïen (GFP) geen. Daarna is die teiken konstrukte gesamentlik geïnfiltreer met die gekonstrueerde amiRNA in Nicotiana benthamiana en GFP uitdrukkingsvlakke is gekwantifiseer deur die onderdrukkingsdoeltreffendheid van die amiRNAs te bepaal. Resultate het getoon dat die amiRNAs suksesvol was in die onderdrukking van die GFP teiken konstruk, maar hulle was egter nie-spesifiek in die eksklusiewe onderdrukking van die ooreenstemmende teiken. Hierdie amiRNA konstrukte is ideaal vir verdere virus studies om die doeltreffendheid van GLRaV-3 onderdrukking in GLD besmette wingerdstokke te bepaal.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-11-28T13:39:39Z No. of bitstreams: 1 Monique Bezerra Nascimento.pdf: 1822147 bytes, checksum: 8f2d6b8a88935d10d34decf26f6730b3 (MD5) / Made available in DSpace on 2016-11-28T13:39:39Z (GMT). No. of bitstreams: 1 Monique Bezerra Nascimento.pdf: 1822147 bytes, checksum: 8f2d6b8a88935d10d34decf26f6730b3 (MD5) Previous issue date: 2015-02-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The grapevine (Vitis spp.) virus diseases affect severely the production, the quality of the grape and reduce the life of the vineyards. In Brazil, among them, the complexes of rugose wood caused by Grapevine virus A (GVA), Grapevine virus B (GVB) and Grapevine virus D (GVD) and the leafroll, caused by Grapevine rupestris stem pitting-associated virus (GRSPaV) and Grapevine leafroll-associated virus -1 to -4, -4 strain 5 (GLRaV-1 to -4, -4 strain 5) are very important. The first part of the present work had the objectives of evaluating the effects of these viruses on agronomic traits, such as, fresh weight of the bunch, °Brix, weight of pruned branches, stem diameters of rootstock and canopy, in symptomatic and asymptomatic plants in Niagara Rosada and Merlot vineyards, and to survey commercial nurseries for GRSPaV, GVA, GVB, Grapevine fleck virus (GFkV) and GLRaV-3. All plants were indexed by real-time RT-PCR. Asymptomatic infected grapevines, however, were affected negatively by the viral infection, but to a lesser intensity in comparison with the symptomatic ones. The nurseries presented a high incidence of GRSPaV and GVA, being characterized as sources of inoculum for vineyards formed with these materials. The second part of the work aimed the molecular characterization of isolates of the following viruses: Prunus necrotic ringspot virus (PNRSV), agent of rose mosaic (Rosa sp.), obtained from rose; GRSPaV, GVA and GLRaV-2, from symptomatic and asymptomatic grapevine plants. Despite the economic importance of PNRSV for stone fruit trees in Brazil, only peach isolates have been molecular characterized in Brazil, it was observed high percentages of identity, among the isolates GRSPaV, GVA and GLRaV-2, which made it difficult to explain the variation in symptom expression or their absence, based on the sequence differences. In this study, it was conducted, for the first time, the molecular characterization of the complete genes to the coat protein (CP) and movement protein (MP) of a Brazilian PNRSV isolate obtained from rose. The obtained results demonstrated the high importance of using virus-free propagative materials to achieve the highest agronomic potential of the grapevine cultivars and to reduce losses caused by these pathogens. / As viroses da videira (Vitis spp.) afetam severamente a produção, a qualidade da uva e diminuem a vida útil dos vinhedos. No Brasil, destacam-se, o complexo do lenho rugoso, causados por Grapevine virus A (GVA), Grapevine virus B (GVB) e Grapevine virus D (GVD), e o enrolamento das folhas, causado por Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine leafroll-associated virus -1 ao -4, -4 estirpe 5 (GLRaV-1 ao -4, -4 estirpe 5). A primeira parte do presente trabalho teve como objetivos avaliar os efeitos destes vírus sobre as variáveis agronômicas relacionadas ao vigor da planta, tais como, peso fresco do cacho, °Brix, peso de ramos podados, diâmetros do tronco do porta-enxerto e da copa, em plantas sintomáticas e assintomáticas em vinhedos das cultivares Niágara Rosada e Merlot, além do levantamento de vírus em matrizeiros comerciais para GRSPaV, GVA, GVB, Grapevine fleck virus (GFkV) e GLRaV-3. Todas as plantas foram indexadas por RT-PCR em tempo real. Videiras assintomáticas, porém infectadas, foram afetadas negativamente pela infecção viral, porém em menor intensidade, quando comparadas com as sintomáticas. Os matrizeiros testados apresentaram alta incidência de GRSPaV e GVA, ficando caracterizados como fontes de inóculo destes patógenos para vinhedos formados com esses materiais. Na segunda parte do trabalho, visou-se a caracterização molecular de isolados dos seguintes vírus: Prunus necrotic ringspot virus (PNRSV), agente do mosaico da roseira (Rosa sp.), obtido da roseira; GRSPaV, GVA e GLRaV-2, de videiras sintomáticas e assintomáticas. Apesar da importância econômica do PNRSV para as fruteiras de caroço no Brasil, apenas isolados brasileiros, obtidos de pessegueiro, tinham sido caracterizados molecularmente. Dentro de cada espécie, os isolados de GRSPaV, GVA e GLRaV-2 demonstraram altas porcentagens de identidade, de modo que, as diferenças entre eles, dificilmente, explicariam as variações na expressão e ausência de sintomas. Neste estudo, foi realizada pela primeira vez a caracterização molecular dos genes completos da capa proteica (CP) e da proteína de movimento (MP) de um isolado brasileiro do PNRSV, obtido em roseira no Brasil. Os resultados obtidos demonstraram a utilização de material propagativo livre de vírus como opção para atingir o melhor potencial agronômico das cultivares de videira e reduzir perdas ocasionadas por estes patógenos.

The main aims of diploma thesis were: 1) The sequence analysis of the Czech isolate of Potato virus M (PVM) VIRUBRA 4/009 and phylogenetic analysis of PVM coat proteins sequences 2) The bacterial expression of recombinant triple gene block protein 1 (TGB1) of PVM derived from the Czech isolate VIRUBRA 4/007 3) The construction of expression cassette of Potato leafroll virus (PLRV) coat protein and its transformation into A. tumefaciens for transgenic PLRV resistant plant formation In theoretical part of the thesis the taxonomic classification, morphology, genomic structure and virus transmission are discussed. Furthermore, the main rules concerning the bacterial expression of recombinant proteins and construction of transgenic plants using A. tumefaciens are described. Methodical part is devoted to description of generally used molecular biological and immunochemical methods. The following results were obtained in the thesis: The complete nucleotide sequences of open reading frames coding for three movement proteins (Triple gene block -TGB), coat protein and NA-binding protein of PVM isolate VIRUBRA 4/009; phylogenetic analysis was performed; the TGB1 protein was expressed in bacterial cells and will be used for polyclonal antibodies raising. Finally, the expression cassette containing the PLRV...

In order to aid the booming wine industry in the state of Virginia, U.S.A., we developed a series of studies to provide a deeper understanding of the viruses and vectors for management of virus diseases and development of better tools for grapevine virus diagnostics. A statewide survey for 14 different grapevine viruses between 2009 and 2014 was conducted: 721 samples were collected from 116 vineyards in the period. Among the 12 viruses identified, Grapevine leafroll associated virus-3 (GLRaV-3), Grapevine rupestris stem-pitting associated virus (GRSPaV), and Grapevine red blotch-associated virus (GRBaV) were most commonly present. A new real-time PCR method for the detection of the V2 gene of GRBaV was developed. The resulting method takes less time for more accurate diagnostics than conventional PCR. Evaluation of insecticide effectiveness on GLRaV-3 vectors (mealybugs) and the spread of GLRaV-3 were examined: Four trials conducted from 2012 to 2014 revealed that despite successful control of mealybugs, GLRaV-3 is spread at a very rapid rate. A new sampling technique for efficient nucleic acid storage and testing was developed: the nitrocellulose membrane-based method allows simpler extraction of nucleic acid and provides a storage medium that can hold viable RNA/DNA at room temperature for up to 18 months. An investigation of multiple virus-infected vines and the impact of these co-infections on grapevine fruit chemistry was conducted. GLRaV-3, GRBaV, GRSPaV, and co-infections of the 3 all negatively impacted Brix, pH, titratable acidity, and anthocyanin levels. / Ph. D.

Grapevine leafroll-associated virus-2 (GLRaV-2), GLRaV-3, and grapevine fleck virus (GFkV) are widespread in grapes around the world. These viruses can cause significant crop loss and affect wine quality by reducing sugar accumulation and compromising skin color. Mealybugs are vectors of grapevine leafroll-associated viruses (GLRaVs). A statewide survey of commercial and wild grapevines in Virginia was conducted during 2009 through 2011. Also, vector management options were tested in two field studies. GLRaV-2, GLRaV-3, and GFkV were detected in 8%, 25%, and 1%, respectively, of over 1,200 vine samples (41 wine grape varieties) from 77 locations, and 64% of vineyards were positive for at least one of the tested viruses. All 100 wild grapevines tested were free of these three viruses, indicating that they are not alternative hosts. The majority of infected vines from commercial vineyards were planted prior to the 1990\'s; however, some new plantings were also found to be positive, indicating movement of the viruses among vineyards and also potential infection prior to planting. The high frequency of virus-infected vines emphasizes the importance of clean plant materials, as well as management of vector insects. The insecticide trials resulted in promising vector control with dinotefuran and spirotetramat; however, acetamiprid and pryrethroid resulted in an increase in mealybug population. This study is the first to examine multiple grape viruses in VA. It will aid in developing better strategies aimed at controlling mealybugs to restrict the movement of viral diseases. / Master of Science in Life Sciences

Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Science / ENGLISH ABSTRACT: Grapevine leafroll-associated virus 3 (GLRaV-3) is the type strain for the genus Ampelovirus, family Closteroviridae. There has been only one report that claimed the complete nucleotide sequence of GLRaV-3 (isolate NY-1, AF037268). Here we report the complete sequence of the South African GLRaV-3, isolate GP18 (EU259806) and show a significantly extended 5’ end. We used RLM-RACE to determine the 5’ end of GP18 and found the 5’ UTR to be 737 nt compared to 158 nt in the NY-1 sequence. This extended UTR was found in all other South African isolates of GLRaV-3 that were tested. In two collaborative studies the existence of the extended 5’ UTR was confirmed and further investigated. In the first study (Coetzee et al., 2010), metagenomic data generated by next generation sequencing (Illumina Genome Analyzer II) was analysed for GLRaV-3 specific sequences. Sequences similar to the GP18 isolate confirmed the sequence of the extended 5’ UTR. In the second study (Jooste et al., 2010), three genetic variants were identified and their respective 5’ UTRs studied. Great diversity was observed between the 5’ UTRs of the different genetic variants, however within a variant the 5’ UTR was found to be highly conserved. Grapevine leafroll-associated virus 3 is a positive sense, single stranded RNA virus that has been shown, like other closteroviruses, to produce subgenomic (sg) RNAs during replication. These sgRNAs are deployed for the expression of the ORFs on the 3’ half of the genome. In this study a dsRNA blot confirmed the presence of three, 3’ coterminal sgRNAs species [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] in GLRaV-3-infected plant material when using a probe directed at the coat protein gene. The specific 5’ terminal nucleotides for these sgRNAs as well as four additional sgRNAs [sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] were determined by RLM-RACE for GLRaV-3 isolate GP18. The construction of a GLRaV-3 mini-replicon, analogous to RNA1 of Lettuce infectious yellows virus, for the evaluation of putative sg-promoters is also described. / AFRIKAANSE OPSOMMING: Grapevine leafroll-associated virus 3 (GLRaV-3) is ‘n lid van die Closteroviridae familie en die hooflid vir die genus Ampelovirus. Tot dusver was daar net een studie wat die volledige nukleïensuurvolgorde van GLRaV-3 gerapporteer het (isolaat NY-1, AF037268). In hierdie studie rapporteer ons die volledige volgorde van ‘n Suid-Afrikaanse GLRaV-3, isolaat nl. GP18 (EU259806) wat noemenswaardig langer is aan die 5’ kant. RLM-RACE is gebruik om die 5’ eindpunt van GP18 te bepaal en daar is gevind dat die 5’ ongetransleerde streek (UTR) 737 nt lank is in vergelyking met die 158 nt van die NY-1 volgorde. Die verlengde 5’ UTR is gevind in alle Suid-Afrikaanse monsters wat getoets is. Die verlengde 5’ UTR is bevestig en verder bestudeer tydens twee samewerkingsprojekte. In die eerste studie (Coetzee et al., 2010), is metagenomiese data gegenereer deur volgende-generasie volgordebepaling (Illumina Genome Analyzer II) en geanaliseer vir GLRaV-3 spesifieke volgordes. Volgordes soortgelyk aan die GP18 isolaat het die verlengde 5’ UTR volgorde bevestig. In die tweede studie (Jooste et al., 2010), is drie genetiese variante van GLRaV-3 geidentifiseer en hulle onderskeie 5’ UTR volgordes bepaal en bestudeer. Daar is groot diversiteit tussen die 5’ UTRs van die verskillende genetiese variante gevind, maar tussen isolate van dieselfde variant is die volgordes gekonserveerd. Grapevine leafroll-associated virus 3 is ‘n positiewe-sin, enkelstring RNA virus wat al voorheen bewys is om, soos ander closterovirusse, subgenomiese (sg) RNAs te produseer tydens replisering. Hierdie sgRNAs word ingespan vir die uitdrukking van die ORFs op die 3’ helfte van die virusgenoom. In hierdie studie is ‘n dsRNA klad gebruik om die voorkoms van 3’ ko-terminale sgRNAs [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] te bevestig in GLRaV-3 geinfekteerde plantmateriaal deur gebruik te maak van ‘n peiler teen die kapsiedproteïengeen. Die spesifieke 5’ terminale nukleotiedes vir hierdie sgRNAs sowel as vier additionele sgRNAs [sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] is bepaal deur gebruik te maak van RLM-RACE op die GLRaV-3 isolaat GP18. Die konstruksie van ‘n GLRaV-3 mini-repliserings konstruk, analoog aan die RNA1 van Lettuce infectious yellows virus, vir die evaluasie van moontlike sg-promotors word ook beskryf.

The thesis deals with the occurrence of viral pathogens on grape - Chardonnay clones. Monitored and evaluated clones were 8, 95, 96 (foreign) on rootstocks 1103 Paulsen, SO4, Kober 5 BB and 110 Richter and VP-155/6-VP 161/6, PO-158/7 and PO-160 / 1 (Czech) on the rootstock Kober 5 BB. All plants have a controlled origin. The experiment was conducted in 2013 on the test sites in the cadastral Perná. At the beginning of vegetation were recorded values on 1 herbaceous plant -- sprouting and not-sprouting buds. During vegetation were the plants observed. From the monitored plants were harvested grapes and following parameters were checked: number and weight of the grapes, weight of berries and the stem. Furthermore, before leaf the leaves were sampled for subsequent ELISA test for viral diseases Grapevine fanleaf virus, Arabis mosaic virus, Grapevine fleck virus, Grapevine leafroll-associated virus 1 and 3, Grapevine virus A. All values were evaluated by statistical program Statistica 10.

Les virus de l’enroulement de la vigne (Grapevine leafroll-associated virus, GLRaV) sont répandus mondialement et transmis à la vigne uniquement par cochenilles (Coccoidea). En France, l’enroulement viral affecte particulièrement les vignobles des régions septentrionales.L’approche biologique de la vection a montré la capacité de Phenacoccus aceris à transmettre à la vigne les GLRaV-1, -3, -4, -5, -6, -9 et ceux du bois strié Grapevine virus A et B. Cette étude est la première démonstration de la transmission du GLRaV-6 et confirme l’absence de spécificité des cochenilles dans la transmission des Ampelovirus. Les larves néonates de P. aceris et de Neopulvinaria innumerabilis représentent un stade de développement efficace pour la transmission de ces virus. En conséquence, leurs capacités vectrices, associées à leur fort potentiel de dissémination anémophile, impliquent un risque important de dispersion naturelle de ces virus dans un vignoble infesté. Les relevés sur quatre parcelles distinctes montrent que Parthenolecanium corni, Pulvinaria vitis, Heliococcus bohemicus et P. aceris sont communes, chaque vignoble différant par la diversité spécifique, le taux de ceps infestés et l’abondance des cochenilles. L'étude épidémiologique prouve le rôle des cochenilles dans la dispersion de l’enroulement viral dans les vignobles septentrionaux. A Bonzon, la responsabilité de P. aceris dans la diffusion rapide du GLRaV-1 est mise en évidence. Cette découverte représente la première preuve en Europe d’une dispersion naturelle du GLRaV-1. A Marsannayla-Côte, l’incidence du GLRaV-1 reste faible, la colonie de P. aceris ne semblant avoir qu’un rôle très limité dans la diffusion de la maladie. L'épidémiologie moléculaire à Bonzon révèle une diversité génétique importante du GLRaV-1 à l’échelle parcellaire et fournit pour la première fois des données sur le polymorphisme génétique d'une population de GLRaV-1 ayant été dispersée par des cochenilles. / Grapevine leafroll viruses (Grapevine leafroll-associated virus, GLRaV) are present worldwide and transmitted to grapevine only by scale insect vectors (Coccoidea). In France, leafroll disease is present in all vine-growing areas, particularly in north-eastern regions. The biological approach of transmission allowed us to show the capacity of the mealybug Phenacoccus aceris to transmit the viruses GLRaV-1, -3, -4, -5, -6, -9 and the rugose wood viruses Grapevine virus A and B. This study represents the first evidence of the transmission of GLRaV-6 and confirms the absence of mealybug specificity in the transmission of Ampelovirus. First instar nymphs of P. aceris and of Neopulvinaria innumerabilis represent a very efficient development stage in the transmission of leafroll and rugose wood viruses. As a consequence, their vector capacities associated with the high potential of dispersal of these nymphs imply an important risk of natural spread of viruses in an infested vineyard. The entomological monitoring on 4 plots shows that Parthenolecanium corni, Pulvinaria vitis, Heliococcus bohemicus and P. aceris are common in vineyards, each site differing by the specific diversity, the level of infested stocks and the abundance of scale insects on stocks. The epidemiological study proves the role of scales insects in the dispersal of leafroll disease in the vineyards of north-eastern France. In Bonzon, the major role of P. aceris in the rapid spread of the GLRaV-1 is demonstrated. This finding represents the first report in Europe of a natural spread of GLRaV-1. In Marsannay-la-Côte, the incidence of the GLRaV-1 remains low and the colony of P. aceris, not associated to grapevine, seems to have only a very limited role in the disease spread. The molecular epidemiology study in Bonzon reveals an important genetic diversity of GLRaV-1 within a signle plotand supplies for the first time information on the genetic polymorphism of a GLRaV-1population being spread by scale insects.