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Clonagem, expressão da proteína capsidial de Grapevine vírus B (GVB) e produção de anticorpos policlonais e monoclonaisDall'Onder, Leonara Patrícia January 2008 (has links)
GVB é um patógeno agrícola composto por RNA de fita simples de senso positivo, com extremidades 3’ poliadeniladas e tem seu diagnóstico feito por testes biológicos, imunológicos e moleculares. Juntamente com outros vírus, causa a síndrome do “complexo rugoso”, que impede a pega da enxertia, destruindo o floema e matando a planta precocemente. A produção de anticorpos contra uma proteína do capsídeo e o desenvolvimento de teste imunológico são os objetivos deste trabalho. A clonagem da região codificante da proteína do capsídeo do Grapevine Virus B foi obtida por PCR, utilizando primers específicos para região codificante e com sítios de restrição para endonucleases BamHI e NdeI, obtendo-se um amplicon de 594 pb. Este fragmento foi clonado no vetor de expressão pET19b e transformado em Escherichia coli BL21 Codon Plus (DE3) RP (Stratagene). Para a expressão da proteína rGVB1a (24 kDa com cauda de histidina), estabeleceu-se uma indução de 1 mM de IPTG (isopropyl-beta-Dthiogalactopyranoside), mantida sob agitação a 250C por 18 horas. A expressão foi analisada por SDS-PAGE 13% e a presença da rGVB1a foi confirmada por Western blot, usando anticorpo monoclonal anti-histidina. A rGVB1a expressada foi purificada por cromatografia de afinidade a cauda de histidina em resina sepharose-Ni2+. A proteína purificada foi utilizada para imunizar um coelho e sete lotes de três camundongos para obtenção de soro policlonal e monoclonal contra a proteína recombinante. A fusão de células de linfócitos B com mielomas SP2/0 foi realizada, obtendo-se um hibridoma produtor de anticorpo IgG2a que em testes de ELISA e Western blot reconhecem a proteína recombinante. Adicionalmente, testes de Western blot utilizando extrato total de plantas sintomáticas e assintomáticas foram realizados para verificar se estes soros reconheciam a proteína nativa. / GVB is an agricultural pathogen composed by a single-stranded positive sense RNA with poliadenilated 3’ end and its diagnosis is based on biological, immunological and molecular tests. Together with another virus, it causes the rugose wood complex disease which prevents grafting, destroying the phloem and killing the plant early. The objectives of this work are the production of antibodies against the coat protein and the development of an immunological test. Cloning of a coat protein coding gene from Grapevine Virus B was performed by PCR, using specific primers for the coding region with restriction sites for BamHl and Ndel endonucleases, obtaining an amplicon of 594 bp. This fragment was cloned into the pET19b expression vector and transformed with Escherichia coli BL21 Codon Plus (DE3) RP (Stratagene). For expression of rGVB1a protein (24 kDa with histidine tail) a protocol with 1 mM IPTG (isopropyl-beta-D-thiogalactopyranoside), and shaking for 18 hours at 25ºC was established. The expression was analyzed by SDS-PAGE 13% and the presence of rGVB1a was confirmed by Western-blot, using an anti-histidine monoclonal antibody.rGVB1a expressed was purified by histidine tail Ni2+ Sepharose affinity chromatography. Purified protein was used to immunize a rabbit and seven lots of mice to obtain policlonal serum and monoclonal antibody against the recombinant protein. Cell fusions of lymphocyte B and SP2/0 were performed and a hybridoma producer of IgG2a antibody was obtained. This hybridoma recognized the recombinant protein in ELISA and Western blot tests. Additionally, Western blot using the extract of symptomatic and assymptomatic plants were developed to investigate if these serum recognize the native protein.
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Clonagem, expressão da proteína capsidial de Grapevine vírus B (GVB) e produção de anticorpos policlonais e monoclonaisDall'Onder, Leonara Patrícia January 2008 (has links)
GVB é um patógeno agrícola composto por RNA de fita simples de senso positivo, com extremidades 3’ poliadeniladas e tem seu diagnóstico feito por testes biológicos, imunológicos e moleculares. Juntamente com outros vírus, causa a síndrome do “complexo rugoso”, que impede a pega da enxertia, destruindo o floema e matando a planta precocemente. A produção de anticorpos contra uma proteína do capsídeo e o desenvolvimento de teste imunológico são os objetivos deste trabalho. A clonagem da região codificante da proteína do capsídeo do Grapevine Virus B foi obtida por PCR, utilizando primers específicos para região codificante e com sítios de restrição para endonucleases BamHI e NdeI, obtendo-se um amplicon de 594 pb. Este fragmento foi clonado no vetor de expressão pET19b e transformado em Escherichia coli BL21 Codon Plus (DE3) RP (Stratagene). Para a expressão da proteína rGVB1a (24 kDa com cauda de histidina), estabeleceu-se uma indução de 1 mM de IPTG (isopropyl-beta-Dthiogalactopyranoside), mantida sob agitação a 250C por 18 horas. A expressão foi analisada por SDS-PAGE 13% e a presença da rGVB1a foi confirmada por Western blot, usando anticorpo monoclonal anti-histidina. A rGVB1a expressada foi purificada por cromatografia de afinidade a cauda de histidina em resina sepharose-Ni2+. A proteína purificada foi utilizada para imunizar um coelho e sete lotes de três camundongos para obtenção de soro policlonal e monoclonal contra a proteína recombinante. A fusão de células de linfócitos B com mielomas SP2/0 foi realizada, obtendo-se um hibridoma produtor de anticorpo IgG2a que em testes de ELISA e Western blot reconhecem a proteína recombinante. Adicionalmente, testes de Western blot utilizando extrato total de plantas sintomáticas e assintomáticas foram realizados para verificar se estes soros reconheciam a proteína nativa. / GVB is an agricultural pathogen composed by a single-stranded positive sense RNA with poliadenilated 3’ end and its diagnosis is based on biological, immunological and molecular tests. Together with another virus, it causes the rugose wood complex disease which prevents grafting, destroying the phloem and killing the plant early. The objectives of this work are the production of antibodies against the coat protein and the development of an immunological test. Cloning of a coat protein coding gene from Grapevine Virus B was performed by PCR, using specific primers for the coding region with restriction sites for BamHl and Ndel endonucleases, obtaining an amplicon of 594 bp. This fragment was cloned into the pET19b expression vector and transformed with Escherichia coli BL21 Codon Plus (DE3) RP (Stratagene). For expression of rGVB1a protein (24 kDa with histidine tail) a protocol with 1 mM IPTG (isopropyl-beta-D-thiogalactopyranoside), and shaking for 18 hours at 25ºC was established. The expression was analyzed by SDS-PAGE 13% and the presence of rGVB1a was confirmed by Western-blot, using an anti-histidine monoclonal antibody.rGVB1a expressed was purified by histidine tail Ni2+ Sepharose affinity chromatography. Purified protein was used to immunize a rabbit and seven lots of mice to obtain policlonal serum and monoclonal antibody against the recombinant protein. Cell fusions of lymphocyte B and SP2/0 were performed and a hybridoma producer of IgG2a antibody was obtained. This hybridoma recognized the recombinant protein in ELISA and Western blot tests. Additionally, Western blot using the extract of symptomatic and assymptomatic plants were developed to investigate if these serum recognize the native protein.
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Clonagem, expressão da proteína capsidial de Grapevine vírus B (GVB) e produção de anticorpos policlonais e monoclonaisDall'Onder, Leonara Patrícia January 2008 (has links)
GVB é um patógeno agrícola composto por RNA de fita simples de senso positivo, com extremidades 3’ poliadeniladas e tem seu diagnóstico feito por testes biológicos, imunológicos e moleculares. Juntamente com outros vírus, causa a síndrome do “complexo rugoso”, que impede a pega da enxertia, destruindo o floema e matando a planta precocemente. A produção de anticorpos contra uma proteína do capsídeo e o desenvolvimento de teste imunológico são os objetivos deste trabalho. A clonagem da região codificante da proteína do capsídeo do Grapevine Virus B foi obtida por PCR, utilizando primers específicos para região codificante e com sítios de restrição para endonucleases BamHI e NdeI, obtendo-se um amplicon de 594 pb. Este fragmento foi clonado no vetor de expressão pET19b e transformado em Escherichia coli BL21 Codon Plus (DE3) RP (Stratagene). Para a expressão da proteína rGVB1a (24 kDa com cauda de histidina), estabeleceu-se uma indução de 1 mM de IPTG (isopropyl-beta-Dthiogalactopyranoside), mantida sob agitação a 250C por 18 horas. A expressão foi analisada por SDS-PAGE 13% e a presença da rGVB1a foi confirmada por Western blot, usando anticorpo monoclonal anti-histidina. A rGVB1a expressada foi purificada por cromatografia de afinidade a cauda de histidina em resina sepharose-Ni2+. A proteína purificada foi utilizada para imunizar um coelho e sete lotes de três camundongos para obtenção de soro policlonal e monoclonal contra a proteína recombinante. A fusão de células de linfócitos B com mielomas SP2/0 foi realizada, obtendo-se um hibridoma produtor de anticorpo IgG2a que em testes de ELISA e Western blot reconhecem a proteína recombinante. Adicionalmente, testes de Western blot utilizando extrato total de plantas sintomáticas e assintomáticas foram realizados para verificar se estes soros reconheciam a proteína nativa. / GVB is an agricultural pathogen composed by a single-stranded positive sense RNA with poliadenilated 3’ end and its diagnosis is based on biological, immunological and molecular tests. Together with another virus, it causes the rugose wood complex disease which prevents grafting, destroying the phloem and killing the plant early. The objectives of this work are the production of antibodies against the coat protein and the development of an immunological test. Cloning of a coat protein coding gene from Grapevine Virus B was performed by PCR, using specific primers for the coding region with restriction sites for BamHl and Ndel endonucleases, obtaining an amplicon of 594 bp. This fragment was cloned into the pET19b expression vector and transformed with Escherichia coli BL21 Codon Plus (DE3) RP (Stratagene). For expression of rGVB1a protein (24 kDa with histidine tail) a protocol with 1 mM IPTG (isopropyl-beta-D-thiogalactopyranoside), and shaking for 18 hours at 25ºC was established. The expression was analyzed by SDS-PAGE 13% and the presence of rGVB1a was confirmed by Western-blot, using an anti-histidine monoclonal antibody.rGVB1a expressed was purified by histidine tail Ni2+ Sepharose affinity chromatography. Purified protein was used to immunize a rabbit and seven lots of mice to obtain policlonal serum and monoclonal antibody against the recombinant protein. Cell fusions of lymphocyte B and SP2/0 were performed and a hybridoma producer of IgG2a antibody was obtained. This hybridoma recognized the recombinant protein in ELISA and Western blot tests. Additionally, Western blot using the extract of symptomatic and assymptomatic plants were developed to investigate if these serum recognize the native protein.
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The development and characterisation of grapevine virus-based expression vectorsDu Preez, Jacques 03 1900 (has links)
Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Grapevine (Vitis vinifera L.) is a very important agricultural commodity that needs to be
protected. To achieve this several in vivo tools are needed for the study of this crop and the
pathogens that infect it. Recently the grapevine genome has been sequenced and the next
important step will be gene annotation and function using these in vivo tools. In this study the
use of Grapevine virus A (GVA), genus Vitivirus, family Flexiviridae, as transient expression
and VIGS vector for heterologous protein expression and functional genomics in Nicotiana
benthamiana and V. vinifera were evaluated. Full-length genomic sequences of three South
African variants of the virus (GTR1-1, GTG11-1 and GTR1-2) were generated and used in a
molecular sequence comparison study. Results confirmed the separation of GVA variants into
three groups, with group III (mild variants) being the most distantly related. It showed the
high molecular heterogeneity of the virus and that ORF 2 was the most diverse. The GVA
variants GTG11-1, GTR1-2 and GTR1-1 were placed in molecular groups I, II and III
respectively. A collaboration study investigating the molecular divergence of GVA variants
linked to Shiraz disease (SD), described two interesting GVA variants of group II, namely
GTR1-2 and P163-M5 (Goszczynski et al., 2008). The group II variants were found to be
closely linked to the expression of SD. GTR1-2 was isolated from a susceptible grapevine
plant that never showed SD symptoms (Goszczynski 2007). The P163-M5 variant that
resulted in exceedingly severe symptoms in N. benthamiana and is that used as SD positive
control by the grapevine industry, was found to contain a 119 nt insert within the native
ORF2. Comparative analysis performed on the complete nt and aa sequences of group II GVA
variants suggested that the components in the GVA genome that cause pathogenicity in V.
vinifera are more complex (or different) to those that cause pathogenicity in N. benthamiana.
The three South African variants (GTR1-1, GTG11-1 and GTR1-2) were assembled into fulllength
cDNA clones under control of CaMV 35S promoters. After several strategies were
attempted, including a population cloning strategy for GTR1-2, none of the clones generated
were able to replicate in N. benthamiana plants. A single amino acid substitution at position
13 (Tyr/Y Cys/C) in ORF 5 of the GTR1-2 cDNA clone was shown to abolish or reduce
replication of the virus to below a detectable level. Two infectious clones of Israeli variants of
GVA (T7-GVA-GR5 and T7-GVA118, obtained from M. Mawassi) were brought under
control of a CaMV 35S promoter (35S-GVA-GR5 and 35S-GVA118). Both clones were
infectious, able to replicate, move systemically and induce typical GVA symptoms after
agroinfiltration in N. benthamiana. These Israeli clones served as backbone for further experiments in characterisation of transient expression and VIGS vectors. The use of GVA as
gene insertion vector (35S-GVA118) and gene exchange vector (35S-GVA-GR5-
ORF2+sgMP) in N. benthamiana and V. vinifera was compared. The gene insertion vector,
35S-GVA118 was based on the full-length GVA genome. The gene exchange vector, 35SGVA-
GR5- ORF2+sgMP, was constructed in this study by elimination of ORF 2 and
insertion of a sgMP and unique restriction sites to facilitate transgene insertion. In N.
benthamiana both vectors showed similar GUS expression levels and photobleaching
symptoms upon virus-induced NbPDS silencing. In V. vinifera limited GUS expression levels
and VIGS photobleaching symptoms were observed for the gene insertion vector, 35SGVA118.
No GUS expression was observed for the gene exchange vector 35S-GVA-GR5-
ORF2+sgMP in this host. As for silencing, one plant, agroinfiltrated with 35S-GVA-GR5-
ORF2-VvPDS+sgMP, developed photobleaching symptoms in 3 systemic infected leaves
after 4 months. This study showed that GVA can be used as gene insertion and gene exchange
vector for expression and VIGS in N. benthamiana, but in grapevine its use is limited to
expression and silencing of genes in the phloem tissue. It is also the first report that ORF 2 of
GVA is not needed for long distance movement in grapevine.
To investigate the possible role of the P163-M5 119 nt insertion and the GVA ORF 2 (of
unknown function), in expression of symptoms in plants, ORF 2 of a 35S-GVA-GR5 cDNA
clone was removed and subsequently substituted by the corresponding ORFs of four South
African GVA variants. Upon agro-infiltration into N. benthamiana leaves, all chimaeric GVA
constructs were able to move systemically through the plant. At this stage no correlation
could be found between severity of symptoms, the presence of the P163-M5 insert and the
specific GVA ORF 2 present in the chimaeras, indicating that other factors in the viral
genome or the host plant probably play a crucial role.
This study contributed to the pool of available in vivo tools for study and improvement of the
valuable grapevine crop. It also opened several exciting research avenues to pursue in the near
future. / AFRIKAANSE OPSOMMING: Wingerd (Vitis vinifera L.) is ‘n baie belangrike landboukundige gewas wat beskerm moet
word. Om die rede word verskeie in vivo gereedskap vir die bestudering van die
wingerdplant, en die patogene wat dit infekteer benodig. Die wingerd genoom se volgorde is
bepaal en dus is die volgende logiese stap om die gene te annoteer en funksie daaraan toe te
skryf. In hierdie studie is die gebruik van Grapevine virus A (GVA), genus Vitivirus, familie
Flexiviridae, as tydelike uitdrukking- en virus-geinduseerde geenuitdowingsvektor vir
heteroloë proteïen uitdrukking en funksionele genoomstudies in Nicotiana benthamiana en V.
Vinifera getoets. Vollengte genoomvolgordes van drie Suid-Afrikaanse variante van die virus
(GTR1-1, GTG11-1 en GTR1-2) is gegenereer en in ‘n molekulêre volgorde vergelyking
studie gebruik. Resultate het die verdeling van GVA variante in drie groepe, waar groep III
die verste verwant is, bevestig. Dit het ook gewys dat die virus ‘n baie hoë molekulêre
heterogeniteit het en dat oopleesraam 2 (ORF 2) die mees divers is. ‘n Samewerking studie
waar die molekulêre diversiteit van GVA variante, gekoppel aan Shiraz siekte (SD),
ondersoek is, is twee interessante variante van groep II beskryf, naamlik GTR1-2 en P163-M5
(Goszczynski et al., 2008). Groep II variante is vooraf gevind om nou verwant te wees aan die
ontwikkeling van SD in wingerd. Die GTR1-2 variant is uit ’n vatbare wingerd plant, wat
nooit SD-simptome vertoon het nie, geïsoleer (Goszczynski et al., 2007). In die ORF 2 van
die P163-M5 variant, wat simptome van die ergste graad in N. benthamiana geïnduseer het, en
ook deur die industrie as betroubare SD-positiewe kontrole gebruik word, is ’n 119 nt
invoeging gevind. Die vergelykende analise wat uitgevoer is, het daarop gedui dat die
determinante van patogenisiteit in die GVA genoom moontlik meer kompleks kan wees in V.
vinifera as in N. benthamiana. Die drie Suid-Afrikaanse variante (GTR1-1, GTG11-1 en
GTR1-2) is in afsonderlike vollengte cDNA klone, onder beheer van CaMV 35S promotors,
aanmekaargesit. Nadat verskeie kloneringstrategieë, insluitend ’n populasie kloneringstrategie
vir die GTR1-2 kloon, gebruik is, het geen een van die cDNA klone die vermoë besit om in
N. benthamiana te repliseer nie. ’n Enkele aminosuur substitusie in posisie 13
(Tyr/Y Cys/C) in ORF 5 van die GTR1-2 kloon, het die replisering van die virus tot laer as
’n opspoorbare vlak verlaag. Twee infektiewe klone van Israeliese GVA variante (T7-GVAGR5
en T7-GVA118, verkry van M. Mawassi) is onder beheer van ‘n CaMV 35S promotor
geplaas (35S-GVA-GR5 and 35S-GVA118). Beide klone het na agro-infiltrasie in N.
benthamiana plante gerepliseer, sistemies beweeg en tipiese GVA simptome geinduseer.
Hierdie twee klone het as raamwerk gedien vir verdere eksperimente in karakterisering van tydelike uitdrukkings- en VIGS vektore. Die gebruik van GVA as geen-insvoegingsvektor
(35S-GVA118) en geen-vervangingsvektor (35S-GVA-GR5- ORF2+sgMP) is in N.
benthamiana en V. vinifera vergelyk. Die geen-invoegingsvektor 35S-GVA118, was op die
vollengte GVA genoom gebasseer. Die geen-vervangingsvektor 35S-GVA-GR5-
ORF2+sgMP, was in hierdie studie gekonstrueer. Dit is gemaak eerstens deur eliminasie van
ORF 2 in die 35S-GVA-GR5 kloon, en tweedens deur die invoeging van ’n subgenomiese
promotor van die beweginsproteïen (sgMP) en unieke beperkings-ensiemsetels om klonering
van transgene te fasiliteer. Beide vektore het in N. benthamiana vergelykbare GUS
uitdrukkingsvlakke en fotobleikende simptome getoon na virus-geinduseerde NbPDS
uitdowing. In V. Vinifera is beperkte GUS uitdrukkingsvlakke en VIGS fotobleikende
simptome opgemerk met die geen-invoegingsvektor, 35S-GVA118. Geen GUS uitdrukking is
in hierdie gasheerplant met die geen-vervangingsvektor opgemerk nie. Slegs een wingerdplant
het fotobleikende simptome, na 4 maande in 3 sistemies geïnfekteerde blare gewys, na agroinfiltrasie
van die 35S-GVA-GR5- ORF2-VvPDS+sgMP konstruk. Hierdie studie het
bevestig dat GVA as geen-invoeging en geen-vervangingsvektor, vir heteroloë proteïenuitdrukking
en VIGS, in N. benthamiana gebruik kan word, maar dit blyk of die gebruik
daarvan in wingerd meer tot die floeëm weefsel beperk is. Hierdie studie wys vir die eerste
keer dat ORF 2 nie nodig is vir langafstand beweging van die virus in wingerd nie.
Om die moontlike rol van die P163-M5 119 nt invoeging en die GVA ORF 2 (met onbekende
funksie), in die uitdrukking van simptome in plante te ondersoek, is ORF 2 van die 35SGVA-
GR5 cDNA kloon verwyder en daaropvolgens vervang met die ooreenstemmende
ORFs van vier Suid-Afrikaanse GVA variante. Na agro-infiltrasie in N. benthamiana blare,
het al die chimeras die vermoë gehad om te repliseer, sistemies te beweeg en simptome te
induseer. Geen korrelasie kon gevind word tussen die graad van simptome, die
teenwoordigheid van die P163-M5 insersie en die spesifieke GVA ORF 2 teenwoordig in die
chimeras nie, wat dus daarop dui dat ander faktore in die virusgenoom of die gasheerplant `n
moontlike belangrike rol kan speel.
Hierdie studie het bygedrae tot die beskikbare poel van in vivo gereedskap vir die bestudering
en verbetering van die kosbare wingerdgewas. Dit het ook talle interessante
navorsingsgeleenthede oopgemaak om in die nabye toekoms te betree.
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Molecular characterization of grapevine virus E in South AfricaDe Koker, Wenhelene Crystal 12 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Grapevine virus E (GVE) is a newly identified virus that has been detected in an established vineyard in South Africa. This virus is a member of the genus Vitivirus, family Flexiviridae. Members of this genus are known to infecte grapevine and are associated with various disease complexes, such as the Rugose wood complex (RWC) and Shiraz disease (SD). However, the role and impact of GVE in South African vineyards are still unknown. It is important to study these viruses to determine how they infect and the possible impact they may have on vine health.
The accurate and early detection of grapevine viruses is the first important step in disease management. In this study, reverse transcription-polymerase chain reaction (RT-PCR), double antibody sandwich enzyme linked immunesorbent assay (DAS-ELISA) and quantitative (q)RT-PCR were used for the detection of GVE in the vineyard (Vitis vinifera cv Merlot) where GVE was first identified in South Africa. Reverse transcription-PCR was used for detection and determining the incidence of GVE. The incidence was as low as 3% in the vineyard surveyed. All the GVE positive plants were co-infected with GLRaV-3 and no disease association could therefore be made. Evaluation of the Bioreba Grapevine virus A (GVA) DAS-ELISA kit showed that it did not detect GVE. No cross-reactivity occurred with epitopes of GVE, confirming this kit to be a valid and specific assay for GVA infection. The relative virus titer of GVE was calculated over the growing season of 2010/2011, using qRT-PCR. No fluctuation in virus titer was observed during that growing season.
Transmission experiments were performed in an attempt to transfer GVE from grapevine to an alternative host. Three different transmission buffers as well as nine different herbaceous plant species, that have shown to be susceptible to several plant viruses in previous studies, were evaluated. In these experiments, GVE could not be transmitted to any of the herbaceous species. To further characterize GVE, chimeric clones were constructed with GVA. The ORF2 and ORF5 of GVE were cloned into previously constructed GVA ORF2 and ORF5 deletion mutants. Construction of the chimeric clones, 35S-GVA-GR5-ΔORF2-GVE-ORF2 and 35S-GVA-118-ΔORF5-GVE-ORF5 were successful and they were evaluated for their infectivity in N. benthamiana. The 35S-GVA-GR5-ΔORF2-GVE-ORF2 chimera was able to infect and replicate in these plants and disease symptoms such as yellowing of veins and leaf curling were observed. Virus, derived from this vector, was detected by TPIA, RT-PCR and DAS-ELISA. The 35S-GVA-118-ΔORF5-GVE-ORF5 chimeric vector was not able to infect N. benthamiana as no disease symptoms were observed in any of the infiltrated plants and virus was not detected with serological analysis and RT-PCR.
This study was aimed at further characterizing the recently identified virus GVE. Here, insight is given into the prevalence of this virus in the vineyard where it was first identified and attempts to biologically characterize GVE were made. / AFRIKAANSE OPSOMMING: Grapevine virus E (GVE) is „n nuut geïndetifiseerde virus wat onlangs in „n gevestigde wingerd in Suid Afrika opgespoor is. Hierdie virus vorm deel van die genus Vitivirus, familie Betaflexiviridae. Spesies in hierdie genus is bekend vir wingerdinfeksies en word met „n verskeidenheid wingerd siektes geassosieer, soos bv. Rugose wood complex (RWC) en Shiraz siekte (SD). Die rol en impak van GVE is nog onbekend. Dit is dus belangrik om die virus te bestudeer om te bepaal hoe dit infekteer en of dit enige impak het op wingerd gesondheid.
Akkurate en vroeë opsporing van virusse is die eerste belangrike stap vir virussiekte beheer. In hierdie studie word tru-transkripsie (TT) – polimerase ketting reaksie (PKR), dubbel teenliggaam (DAS) -ensiem gekoppelde immuno-absorberende analise (ELISA) en qTT-PKR gebruik vir die opsporing van GVE in die wingerd (Vitis vinifera cv Merlot) waar dit vroeër in Suid Afrika geïdentifiseer was. Vir opsporing en bepaling van verspreiding is TT-PKR gebruik. Daar is bepaal dat 3% van die wingerd met GVE geïnfekteer is. Al die GVE-positiewe stokke het ook positief getoets vir GLRaV-3 en geen assosiasie met siekte simptome kon gemaak word nie. Evaluering van die Bioreba GVA DAS-ELISA met GVE positiewe stokke het nie GVE opgespoor nie. Geen kruisreaktiwiteit het plaasgevind met epitope van GVE nie en dus is die DAS-ELISA ʼn betroubare toets vir GVA infeksie. Die relatiewe virus titer van GVE was ook bepaal oor die groeiseisoen van 2010/2011 deur qTT-PKR te gebruik. Geen fluktuasie in virus titer gedurende die groeiseisoen is waargeneem nie.
Transmissie eksperimente is gedoen om GVE vanaf wingerd na ʼn alternatiewe gasheer oor te dra. Drie verskillende transmissie buffers en tien verskillende sagteplant spesies, wat voorheen vatbaarheid vir plantvirusse getoon het, is gebruik. In die transmissie eksperimente kon GVE nie na enige van die sagteplante oorgedra word nie.
Om GVE verder te karakteriseer is hibried-virusse met GVA gemaak. Die leesraam (ORF) 2 en ORF5 van GVE gekloneer in GVA ORF2 en -ORF5 delesie konstrukte, 35S-GVA-GR5-ΔORF2 en 35S-GVA-118-ΔORF5, onderskeidelik (Blignaut, 2009; Du Preez, 2010). Klonering van die hibried konstrukte, 35S-GVA-GR5-ΔORF2-GVE-ORF2 en 35S-GVA-118-ΔORF5-GVE-ORF5, was suksesvol en is in N. benthamiana geëvalueer. Virus afkomstig van die 35S-GVA-GR5-ΔORF2-GVE-ORF2 hibried konstruk, kon plante suksesvol infekteer en kon repliseer binne hierdie plante. Siektesimptome soos vergeling van die are en rolblaar is ook waargeneem in plante geïnfekteer met hierdie hibried konstruk. Plante is getoets met weefsel afdruk immuno analise (TPIA), TT-PKR en DAS-ELISA en is positief gevind vir virus afkomstig van hierdie konstruk. Die 35S-GVA-118-ΔORF5-GVE-ORF5 hibried kon nie N. benthamiana infekteer nie en geen siektesimptome is waargeneem in enige van die plante geïnfiltreer met hierdie konstruk. Serologiese analise en TT-PKR het ook nie virus in die N. benthamiana plante opgespoor nie.
Die doel van hierdie studie was om GVE te karakteriseer. In hierdie studie word insig gegee oor die verspreiding van hierdie virus in Suid Afrika en pogings is gemaak om GVE biologies te karakteriseer.
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Výskyt virových patogenů na klonech révy vinné (Vitis vinifera L.) českého a zahraničního původuZávodský, Pavel January 2015 (has links)
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.
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A metagenomic approach using next-generation sequencing for viral profiling of a vineyard and genetic characterization of grapevine virus ECoetzee, Beatrix 12 1900 (has links)
Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Science / ENGLISH ABSTRACT: Next-generation sequencing technologies are increasingly used in metagenomic studies, largely
due to the high sequence data throughput capacity and unbiased approach in determining the
genetic composition of an unknown environmental sample. This study investigated the
applicability of the Illumina next-generation sequencing platform for metagenomic sequencing
of grapevine viruses to provide the first complete viral profile, or virome, of a diseased
vineyard.
Leaf material was harvested from 44 randomly selected vines in a leafroll-diseased vineyard in
South Africa. Sample material was pooled and double-stranded RNA extracted. The dsRNA was
sequenced as a paired-end sequencing run using the Illumina sequencing-by-synthesis
technique, and more than 19 million sequence reads, equivalent to approximately 837
megabases of metagenomic sequence data, were obtained. Of these data, approximately 400
megabases could be assembled into 449 scaffolds, using the de novo assembler Velvet. These
scaffolds were subjected to BLAST searches against the NCBI databases and top hit scores were
used for virus identification. Based on the BLAST results, suitable sequences were selected from
the NCBI database and used as reference sequence in MAQ mapping assemblies.
The bioinformatic analyses allowed for the determination of the virus species present, the most
prominent variants, and the relative abundance of each. Four known grapevine viral pathogens
were identified. Grapevine leafroll-associated virus 3, representing 59% of the analyzed short
read sequence data, was identified as the most prominent virus species. Three variants of this
virus were detected: GP18 was the most abundant, followed by a minor Cl766/NY1 variant and
a potential novel grapevine leafroll-associated ampelovirus. A single Grapevine rupestris stem
pitting ]associated virus variant, similar to SG1, and a Grapevine virus A variant, a member of
molecular group III, were identified. This study is also the first to report the presence of
Grapevine virus E (GVE) in South African vineyards. Grapevine virus E was further genetically characterized and the genome sequence of GVE
isolate SA94 determined. The GVE SA94 genome sequence, 7568 nucleotides in length, is the
first complete genome sequence for the virus species. The genome organization of GVE SA94 is
typical of vitiviruses, but in contrast to other RNA viruses, the AlkB domain is located within the
helicase domain in open reading frame 1 (ORF 1). Grapevine virus E SA94 shares nearly 100%
nucleotide identity with the Japanese TvP15 isolate and GVE 3404, a de novo scaffold generated
from the metagenomic sequence data.
Bioinformatic analysis of metagenomic sequence data further revealed the presence of three
fungus-infecting viral families, Chrysoviridae, Totiviridae and the unclassified dsRNA virus,
Fusarium graminearum dsRNA mycovirus 4. A virus from the family Chrysoviridae, similar to
Penicillium chrysogenum virus, was the second most abundant virus detected.
We demonstrated the successful application of a short read sequencing technology, such as the
Illumina platform, for viral profiling of an infected vineyard. To our knowledge this is the first
application of the Illumina technology for this purpose. / AFRIKAANSE OPSOMMING: Volgende-generasie tegnologie om basis volgordes van nukleiensure te bepaal, word al meer
gebruik in metagenomiese studies. Dit is veral weens die hoe data-omset kapasiteit en
onbevooroordeelde aanslag in die bepaling van die genetiese samestelling van onbekende
omgewingsmonsters. Hierdie studie het die aanwending van die Illumina volgende-generasie
volgorde-bepalingsplatform in 'n metagenomiese studie van wingerdvirusse, ondersoek. Dit het
ten doel gehad om die eerste volledige virus profiel, of viroom, van 'n geinfekteerde wingerd
saam te stel.
Blaarmateriaal is verkry vanaf 44 lukraak-gekose wingerdstokke in 'n rolblad-geinfekteerde
wingerd in Suid-Afrika. Monster materiaal is saamgevoeg en dubbelstring-RNS geekstraheer.
Die dubbelstring-RNS is onderwerp aan gepaarde-ent volgorde-bepaling deur gebruik te maak
van die Illumina volgorde-bepaling-deur-sintese tegniek. Meer as 19 miljoen volgorde reekse,
ekwivalent aan ongeveer 837 megabasisse volgorde data, is verkry. Van hierdie data kon
ongeveer 400 megabasisse saamgevoeg word in 449 konstrukte ("scaffolds"), deur gebruik te
maak van die de novo samesteller Velvet. Hierdie konstrukte is onderwerp aan BLAST soektogte
teen die NCBI databasisse en die hoogste trefslag-telling is gebruik vir virus identifikasie. Op
grond van die "BLAST" resultate is geskikte volgordes geselekteer vanaf die NCBI databasis en
gebruik as verwysingvolgordes in MAQ kartering-analises.
Met die bioinfomatika analises kon die virus spesies teenwoordig, asook die mees prominente
variante en relatiewe voorkoms van elk, bepaal word. Vier bekende virus wingerdpatogene is
geidentifiseer. Grapevine leafroll-associated virus 3, verteenwoordig deur 59% van die
geanaliseerde kort-reeks volgorde data, is identifiseer as die mees prominente virus spesie. Drie
variante van die virus is in die wingerdmonster opgespoor: GP18 kom die mees algemeen voor,
gevolg deur 'n CL-766/NY1 variant en 'n potensiele nuwe wingerd rolblad-geassosieerde
ampelovirus. 'n Enkele Grapevine rupestris stem pitting-associated virus variant, soortgelyk aan
SG1, en 'n Grapevine virus A variant, 'n lid van molekulere groep III, is geidentifiseer. Hierdie
studie is ook die eerste om die teenwoordigheid van Grapevine virus E (GVE) in Suid-Afrikaanse
wingerde te rapporteer. Grapevine virus E is verder geneties gekarakteriseer en die genoomvolgorde van GVE isolaat
SA94 is bepaal. Die GVE SA94 genoomvolgorde, 7568 nukleotiede lank, is die eerste volledige
genoomvolgorde vir hierdie virus spesie. Die genoomorganisasie is tipies van vitivirusse, maar
in kontras met ander RNA virusse is die AlkB domein binne-in die helikase domein van
oopleesraam 1 (ORF 1) geleë. Grapevine virus E SA94 deel byna 100% nukleotied identiteit met
die Japannese TvP15 isolaat en GVE 3404, 'n de novo konstruk gegenereer vanaf die
metagenomiese volgorde data.
Bioinformatika analises van die metagenomiese volgorde data het verder die teenwoordigheid
van drie swam-infekterende virus families, die Chrysoviridae, Totiviridae en ongeklassifiseerde
dubbelstring-RNS virus, Fusarium graminearum dsRNA mycovirus 4, aangetoon. 'n Virus van die
Chrysoviridae familie, soortgelyk aan Penicillium chrysogenum virus, het die tweede meeste
voorgekom in die wingerd monster.
Hierdie studie demonstreer die suksesvolle toepassing van 'n kort reeks volgorde-bepalingstegnologie
soos die Illumina platform, vir die opstel van 'n virusprofiel van 'n
geinfekteerde wingerd. Sover ons kennis strek is hierdie die eerste aanwending van die Illumina
tegnologie vir hierdie doel.
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Biologie de la vection de l'ampélovirus GLRaV-1 et du vitivirus GVA par la cochenille Phenacoccus aceris / Transmission biology of the ampelovirus GLRaV-1 and the GVA by the mealybug Phenacoccus acerisAlliaume, Antoine 19 February 2016 (has links)
L’enroulement de la vigne cause des pertes de rendement et de la qualité des vins au niveau mondial. Il est causé par quatre espèces de Grapevine leafroll-associated virus(GLRaVs) ; GLRaV-1, -2, -3, -4-like. Si le GLRaV-2 (genre Closterovirus) ne possède pas de vecteur connu, les trois autres espèces (genre Ampelovirus) sont transmises par cochenilles(Coccoidea) qui contribuent à leur dispersion dans et entre les vignobles. Les vignobles de la France septentrionale (Alsace, Bourgogne, Champagne) sont les plus impactés par l’enroulement viral. Ce travail a porté sur le rôle vecteur de Phenacoccus aceris, espèce connue pour son efficacité de transmission et de dispersion des ampélovirus, ainsi que de vitivirus souvent associés. Les interactions cellulaires et moléculaires entre virus et cochenille restent peu connues. Une approche pluridisciplinaire combinant entomologie,virologie, biologie cellulaire et moléculaire a été développée pour étudier la biologie de la vection du GLRaV-1 et du Grapevine virus A (GVA) par P. aceris. Des expériences de transmission ont montré que ces virus sont transmis selon le mode semi-persistant non circulant. L’étude préliminaire du comportement alimentaire de P. aceris sur vigne par électropénétrographie a révélé une activité similaire à celle d'autres espèces de cochenilles déjà décrites et suggère un effet de l’infection sur le comportement alimentaire. L’anatomie des pièces buccales de P. aceris, organes directement impliqués dans la transmission et la rétention de virus non-circulants a été décrite et une méthode basée sur l’acquisition de virus purifié sur membrane a été développée pour rechercher les sites de rétention virale dans le vecteur. / Grapevine leafroll disease affects grape yield and wine quality worldwide. It is caused by four species of Grapevine leafroll-associated virus (GLRaVs) (GLRaV-1, -2, -3, -4-like).While GLRaV-2 (genus Closterovirus) has no known vector, the other three (genus Ampelovirus) are transmitted by mealybugs (Coccoidea) and thus prone to be dispersed within and between vineyards. In north-eastern France (Alsace, Bourgogne et Champagne),vineyards are more impacted by Grapevine leafroll disease. This thesis focusses on the vector role of the species Phenacoccus aceris, known for its efficiency in transmission and dissemination of ampeloviruses, as well as often associated vitiviruses. Molecular and cellular interactions between viruses and mealybugs remain poorly known. A multidisciplinary approach, combining entomology, virology, molecular and cellular biology, was developed to analyse the vector biology of GLRaV-1 and Grapevine virus A (GVA) by P. aceris.Transmission experiments showed that GLRaV-1 and GVA transmission follows the semipersistent non-circulative mode. A preliminary study of P. aceris feeding behavior on grape using electropenetrography revealed an activity similar to that of other mealybug species already described and suggested a potential effect of infection on Ph. aceris feeding behavior. The anatomy of mouth parts, directely implied in transmission and retention of non circulative viruses was described and a method for membrane acquisition of purified virus was developed to search for virus retention sites within the vector.
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