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Sequencing and detection of a new strain of grapevine leafroll-associated virus 3 in South Africa
Thesis (MSc)--Stellenbosch University, 2012.
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Stellenbosch : Stellenbosch University
2012
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| _version_ | 1867614109619453952 |
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| access_status_str | Open Access |
| author | Bester, Rachelle |
| author2 | Burger, J. T. |
| author_browse | Bester, Rachelle Burger, J. T. |
| author_facet | Burger, J. T. Bester, Rachelle |
| author_sort | Bester, Rachelle |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2012. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/71743 |
| institution | Stellenbosch University (South Africa) |
| last_indexed | 2026-06-10T12:46:49.061Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2012 |
| publishDateRange | 2012 |
| publishDateSort | 2012 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/71743 Sequencing and detection of a new strain of grapevine leafroll-associated virus 3 in South Africa Bester, Rachelle Burger, J. T. Maree, H. J. Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Grapevine leafroll-associated virus 3 Grapes -- Diseases and pests Molecular variants Theses -- Genetics Dissertations -- Genetics 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. Masters 2012-11-19T11:44:18Z 2012-12-12T08:09:48Z 2012-11-19T11:44:18Z 2012-12-12T08:09:48Z 2012-12 Thesis http://hdl.handle.net/10019.1/71743 Stellenbosch University xv, 74 p. : col. ill. application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Grapevine leafroll-associated virus 3 Grapes -- Diseases and pests Molecular variants Theses -- Genetics Dissertations -- Genetics Bester, Rachelle Sequencing and detection of a new strain of grapevine leafroll-associated virus 3 in South Africa |
| title | Sequencing and detection of a new strain of grapevine leafroll-associated virus 3 in South Africa |
| title_full | Sequencing and detection of a new strain of grapevine leafroll-associated virus 3 in South Africa |
| title_fullStr | Sequencing and detection of a new strain of grapevine leafroll-associated virus 3 in South Africa |
| title_full_unstemmed | Sequencing and detection of a new strain of grapevine leafroll-associated virus 3 in South Africa |
| title_short | Sequencing and detection of a new strain of grapevine leafroll-associated virus 3 in South Africa |
| title_sort | sequencing and detection of a new strain of grapevine leafroll associated virus 3 in south africa |
| topic | Grapevine leafroll-associated virus 3 Grapes -- Diseases and pests Molecular variants Theses -- Genetics Dissertations -- Genetics |
| url | http://hdl.handle.net/10019.1/71743 |
| work_keys_str_mv | AT besterrachelle sequencinganddetectionofanewstrainofgrapevineleafrollassociatedvirus3insouthafrica |