Letteratura scientifica selezionata sul tema "Horses virus diseases molecular genetics"

St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) belong to the Japanese encephalitis antigenic complex (Flavivirusgenus,Flaviviridaefamily). They show antigenic close relationships and share many similarities in their ecology. Both are responsible for serious human diseases. The aim of this study was to investigate the presence of neutralizing antibodies to these viruses in horses from Uruguay. To do this, 425 horse sera were collected in 2007 and analyzed by plaque reduction neutralization tests. As a result, 205 sera (48.2%) were found positive for SLEV, with titers ranging between 10 and 80. Two sera remained inconclusive, since they showed low titers to WNV and SLEV (10 and 20), not allowing us to demonstrate activity of WNV in our territory. This is the first report of circulation of SLEV in horses in Uruguay.

West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) are flaviviruses responsible for severe neuroinvasive infections in humans and horses. The confirmation of flavivirus infections is mostly based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs). These tests suffer from poor specificity, mainly due to antigenic cross-reactivity among flavivirus members. Robust diagnosis therefore needs to be validated through virus neutralisation tests (VNTs) which are time-consuming and require BSL3 facilities. The flavivirus envelope (E) glycoprotein ectodomain is composed of three domains (D) named DI, DII, and DIII, with EDIII containing virus-specific epitopes. In order to improve the serological differentiation of flavivirus infections, the recombinant soluble ectodomain of WNV E (WNV.sE) and EDIIIs (rEDIIIs) of WNV, JEV, and TBEV were synthesised using theDrosophilaS2 expression system. Purified antigens were covalently bonded to fluorescent beads. The microspheres coupled to WNV.sE or rEDIIIs were assayed with about 300 equine immune sera from natural and experimental flavivirus infections and 172 nonimmune equine sera as negative controls. rEDIII-coupled microspheres captured specific antibodies against WNV, TBEV, or JEV in positive horse sera. This innovative multiplex immunoassay is a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases.

The genus Orthobunyavirus (family Peribunyaviridae, order Bunyavirales) comprises over 170 named mosquito- and midge-borne viruses, several of which cause severe disease in animals or humans. Their three-segmented genomes enable reassortment with related viruses, which may result in novel viruses with altered host or tissue tropism and virulence. One such reassortant, Schmallenberg virus (SBV), emerged in north-western Europe in 2011. Shuni virus (SHUV) is an orthobunyavirus related to SBV that is associated with neurological disease in horses in southern Africa and recently caused an outbreak manifesting with neurological disease and birth defects among ruminants in Israel. The zoonotic potential of SHUV was recently underscored by its association with neurological disease in humans. We here report a reverse genetics system for SHUV and provide first evidence that the non-structural (NSs) protein of SHUV functions as an antagonist of host innate immune responses. We furthermore report the rescue of a reassortant containing the L and S segments of SBV and the M segment of SHUV. This novel reverse genetics system can now be used to study SHUV virulence and tropism, and to elucidate the molecular mechanisms that drive reassortment events.

Vesicular stomatitis virus (VSV), which belongs to the Vesiculovirus genus of the family Rhabdoviridae, is a well studied livestock pathogen and prototypic non-segmented, negative-sense RNA virus. Although VSV is responsible for causing economically significant outbreaks of vesicular stomatitis in cattle, horses, and swine, the virus also represents a valuable research tool for molecular biologists and virologists. Indeed, the establishment of a reverse genetics system for the recovery of infectious VSV from cDNA transformed the utility of this virus and paved the way for its use as a vaccine vector. A highly effective VSV-based vaccine against Ebola virus recently received clinical approval, and many other VSV-based vaccines have been developed, particularly for high-consequence viruses. This review seeks to provide a holistic but concise overview of VSV, covering the virus’s ascension from perennial agricultural scourge to promising medical countermeasure, with a particular focus on vaccines.

Equid herpesvirus 5 (EHV-5) is one of two γ-herpesviruses that commonly infect horses worldwide. The objective of the study was to estimate the genetic variability within EHV-5 viruses circulating among horses in Poland. Partial glycoprotein B (gB) sequences from 92 Polish horses from 13 studs throughout Poland were compared to each other and to three EHV-5 sequences from other countries. Despite the overall high level of conservation, considerable variability was observed around the putative furin cleavage site. Based on phylogenetic analysis, the viruses clustered within two major lineages (A and B), with further sub-clustering within group A. The clustering of EHV-5 sequences was independent of age or geographical origin of the sampled horses. Recombination was identified as one of the factors contributing to the genomic heterogeneity. Viruses from unweaned foals were more similar to viruses from other foals at the same stud than to viruses form their dams, suggesting the horizontal transfer and/or evolution of EHV-5 within individual hosts. Our data indicate that the gB sequence is not suitable for tracking the source of EHV-5 infection. Further research is needed to elucidate the importance of the sequence variability around the EHV-5 gB furin cleavage site on the biology of the virus.

In 2006, vesicular stomatitis New Jersey virus (VSNJV) caused outbreaks in Wyoming (WY) horses and cattle after overwintering in 2004 and 2005. Within two weeks of the outbreak onset, 12,203 biting flies and 194 grasshoppers were collected near three equine-positive premises in Natrona County, WY. Insects were identified to the species level and tested by RT-qPCR for VSNJV polymerase (L) and phosphoprotein (P) gene RNA. Collected dipterans known to be competent for VSV transmission included Simulium black flies and Culicoides biting midges. VSNJV L and P RNA was detected in two pools of female Simulium bivittatum and subjected to partial genome sequencing. Phylogenetic analysis based on the hypervariable region of the P gene from black flies showed 100% identity to the isolate obtained from the index horse case on the same premises. This is the first report of VSNJV in S. bivittatum in WY and the first field evidence of possible VSV maintenance in black fly populations during an outbreak.

Sylvatic rabies was present in Slovenia between 1973 and 2013, with the red fox as the main reservoir of the rabies virus. The first oral rabies vaccination (ORV) control program in foxes started in 1988, using the manual distribution of baits. Significant improvement of fox vaccination was achieved with the aerial distribution of baits, starting in 1995 and successfully finished with the final, fifty-ninth vaccination campaign in 2019. Between 1979 and 2019, a total of 86,471 samples were tested, and 10,975 (12.69%) rabies-positive animals were identified. Within the ORV, two different vaccines were used, containing modified live virus strain Street Alabama Dufferin (SAD) B19 and SAD Bern, while the last ORV campaigns were completed in 2019, with a vaccine containing a genetically modified strain of SPBN GASGAS. Molecular epidemiological studies of 95 rabies-positive samples, originating from red foxes, badgers, cattle, dogs, martens, cats, and horses, revealed a low genetic diversity of circulating strains and high similarity to strains from neighboring countries. During the elimination program, few vaccine-induced rabies cases were detected: three in red foxes and one case in a marten, with no epidemiological relevance. Slovenia has been officially declared a country free of rabies since 2016.

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.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009.
Grapevine Vitivirus A (GVA), genus Vitivirus, family Flexiviridae is a well characterised single-stranded RNA virus that has been implicated in the grapevine diseases, Kober stem grooving and Shiraz disease. The virus infects both its host, Vitis vinifera and the experimental model plant, Nicotiana spp.. Biological studies performed on the virus in its herbaceous host, Nicotiana benthami- ana, revealed that many divergent variants of the virus exists in South Africa and can induce di erent symptoms in the model plant. Further molecular analysis divided the variants into three molecular groups based on molecular heterogeneity and nucleotide identity. The establishment of an infectious full-length cDNA clone of GVA contributed towards the elucidation of gene functions for 4 of the 5 open reading frames (ORF's), and indicated ORF5 as the pathogenicity determinant within the genome. Further studies also showed that ORF5 encodes for a nucleic acid binding protein that exhibits suppression activity of a plants' natural virus silencing mechanism. Many proteins that have previously been identi ed as the pathogenicity determinant within a viral genome have been found to encode for suppression activity. Although suppression activity has been elucidated within the ORF5 of the Italian cDNA clone of GVA, IS 151, no such study has yet been performed on the divergent South African variants of GVA. Three variants, GTR1-1, GTR1- 2 and GTG11-1, which represent each of the molecular groups (Group III, II and I), were selected for this study. The aim of this study was to visually elucidate suppression activity of RNA transgene silencing by the ORF5's of GTR1-1, GTR1-2 and GTG11-1 in a transient expression assays in transgenic N. benthamiana (line 16c). Pathogenicity studies for these variants were also performed. The ORF5 of the infectious full-length clone, GVA118, which can also serve as an expression vector, was deleted and provided with restriction enzyme sites into which the respective ORF5s and the marker genes, GFP and GUS could be cloned directionally. Infectivity, symptom development and systemic movement were compared between the di erent full length clones after co-in ltration in N. benthamiana. Preliminary results obtained in this study failed to visually indicate any suppression activity encoded by the ORF5 of GTR1-1, GTR1-2 and GTG11-1. The deletion of ORF5 within GVA118 was successful and rendered the infectious full length clone asymptomatic. Directional cloning of the ORF5 of GTR1-1 into the unique restriction enzymes provided previously, resulted in much milder symptoms than those observe for GTR1-2 and GTG11-1. No GFP and GUS accumulation could be detected. This study has established an infectious full-length cDNA clone, pBINSN-e35SGVA118 ORF5-1-1-pA, that can possibly induce much milder symptoms in the herbaceous host, N. benthamiana. This construct can be further characterised as a possible expression vector of foreign proteins in herbaceous hosts and grapevine.

Corrigendum inserted at the back. Includes bibliographical references (leaves 133-158). Ch. 1. General introduction -- ch. 2. General materials and methods -- ch. 3. Biological characterisation of Australian PSbMV isolates -- ch. 4. Developing nucleic acid based diagnostics for PSbMV -- ch. 5. Detection of PSbMV isolates by RT-PCR and RFLP analysis -- ch. 6. Developing an internal control for PSbMV RT-PCR -- ch. 7. Molecular analysis of the PSbMV VPG -- ch. 8. PSbMV sequence and phylogenetic analysis -- ch. 9. General discussion Sixteen pea seed borne mosaic virus (PSbMV) isolates were collected between 1995 and 1998. These isolates were biologically distinct yet serologically indistinguishable. The conclusion is that PSbMV is widespread and occurs at a low incidence in Australia. Reports sequence information on new isolates of PSbMV which has allowed genomic regions to be identified which distinguish PSbMV pathotypes and isolates; and, to the development of PSbMV nucleic acid hybridisation and RT-PCR assays.

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 (Genetics))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: South Africa is regarded as one of the top wine producing countries in the world. One of the threats to the sustainability of the wine industry is viral diseases of which Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine virus A (GVA) are considered to be the most important and wide spread. Scion material is regularly tested for viruses; however scion material is often grafted onto rootstocks that have questionable phytosanitary status. Virus detection in rootstocks is challenging due to low and varying titres, but is imperative as a viral control mechanism. An additional viral control mechanism is the use of transgenic grapevine material which offers resistance to grapevine infection. The objective of this project was to establish a detection system using real time PCR (qPCR) techniques, to accurately and routinely detect GLRaV-3 and GVA in rootstock propagation material. qPCR would furthermore be used to perform molecular characterisation of transgenic plants containing a GLRaV-3 antiviral ΔHSP-Mut construct. A severely infected vineyard (Nietvoorbij farm) in the Stellenbosch area was screened throughout the grapevine growing season to investigate virus prevalence throughout the season and to determine the optimal time for sensitive virus detection. A large scale screening of nursery propagation material for GLRaV-3 infection was also conducted. The qRT-PCR results were compared to DAS-ELISA results to compare the efficacy and sensitivity of the two techniques. For the severely infected vineyard, the ability to detect GLRaV-3 increased as the season progressed towards winter. qRT-PCR was more sensitive and accurate in detecting GLRaV-3 than DASELISA, as the latter technique delivered numerous false positive results later in the season. The best time to screen for GLRaV-3 in the Western Cape region was from the end of July to September. For the nursery screenings, our qRT-PCR results were compared to the results of the DAS-ELISA performed by the specific nurseries. No GLRaV-3 infection was detected in the specific samples received from the two different nurseries. The results for all the samples correlated between the two techniques. This confirms that the propagation material of these nurseries has a healthy phytosanitary status with regards to GLRaV-3. However, the detection of GVA in the severely infected vineyard yielded inconsistent results. Detection ability fluctuated throughout the season and no specific trend in seasonal variation and virus titre fluctuation could be established. The highest percentage of GVA infected samples were detected during September, April and the end of July. Previously published universal primers were used for the detection of GVA, but further investigation indicated that they might not be suitable for sensitive detection of specific GVA variants present in South Africa. Vitis vinifera was transformed with a GLRaV-3 antiviral construct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) and qRT-PCR were utilised as alternative methods for molecular characterisation of transgenic plants. The qPCR and Southern blot results correlated for 76.5% of the samples. This illustrated the ability of qPCR to accurately estimate transgene copy numbers. Various samples were identified during qRT-PCR amplification that exhibited high mRNA expression levels of the transgene. These samples are ideal for further viral resistance studies. This study illustrated that the versatility of real time PCR renders it a valuable tool for accurate virus detection as well as copy number determination.
AFRIKAANSE OPSOMMING: Suid Afrika word geag as een van die top wyn produserende lande ter wereld. Die volhoubaarheid van die wynbedryf word onder andere bedreig deur virus-infeksies. Grapevine leafroll associated virus 3 (GLRaV-3) en Grapevine virus A (GVA) is van die mees belangrike virusse wat siektes veroorsaak in Suid-Afrikaanse wingerde. Wingerd bo-stok materiaal word gereeld getoets vir hierdie virusse, maar hierdie materiaal word meestal geënt op onderstokmateriaal waarvan die virus status onbekend is. Virus opsporing in onderstokke word egter gekompliseer deur baie lae en variërende virus konsentrasies, maar opsporing in voortplantingsmateriaal is ‘n noodsaaklike beheermeganisme vir virus-infeksie. Die doel van die projek was om ‘n opsporingsisteem te ontwikkel via kwantitatiewe PCR (qPCR) tegnieke vir akkurate en gereelde toetsing van GLRaV-3 en GVA in onderstokmateriaal. qPCR sal ook verder gebruik word vir molekulêre karakterisering van transgeniese plante wat ‘n GLRaV-3 antivirale ΔHSP-Mut konstruk bevat. ‘n Hoogs geïnfekteerde wingerd was regdeur die seisoen getoets om seisoenale fluktuasies in viruskonsentrasie te ondersoek en om die optimale tydstip vir sensitiewe virus opsporing te bepaal. ‘n Grootskaalse toetsing van kwekery voortplantingsmateriaal vir GLRaV-3 infeksie was ook uitgevoer. Die qRT-PCR resultate is met die DAS-ELISA resultate vergelyk om die effektiwiteit en sensitiwiteit van die twee tegnieke te vergelyk. Vir die hoogs geïnfekteerde wingerd het die GLRaV-3 opsporing toegeneem met die verloop van die seisoen tot en met winter. qRT-PCR was meer sensitief en akkuraat as DAS-ELISA in die opsporing van GLRaV-3, weens verskeie vals positiewe resultate wat later in die seisoen deur die laasgenoemde tegniek verkry is. Die beste tyd om vir GLRaV-3 te toets is vanaf einde Julie tot September. Tydens die kwekery toetsings was qRT-PCR resultate met die DAS-ELISA resultate van die spesifieke kwekerye vergelyk. Geen GLRaV-3 infeksie was waargeneem in die spesifieke monsters wat vanaf die kwekerye ontvang is nie. Die resultate van die twee tegnieke het ooreengestem vir al die monsters wat v getoets is. Dit het bevestig dat die voortplantingsmateriaal van hierdie kwekerye gesonde fitosanitêre status met betrekking tot GLRaV-3 gehad het. Die opsporing van GVA in die geïnfekteerde wingerd het egter wisselvallige resultate gelewer. Opsporing van die virus het ook regdeur die seisoen gefluktueer en geen spesifieke neiging in seisoenale opsporingsvermoë kon gemaak word nie. Die hoogste persentasie GVA geïnfekteerde monsters was waargeneem tydens September, April en die einde van Julie. Voorheen gepubliseerde universele inleiers was gebruik vir die opsporing van GVA, maar verdere ondersoeke het getoon dat hierdie inleiers nie noodwendig geskik is vir sensitiewe opsporing van GVA variante wat teenwoordig is in Suid-Afrika nie. Vitis vinifera was getransformeer met ‘n GLRaV-3 antivirale konstruct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) en qRT-PCR was ingespan as alternatiewe metodes vir molekulêre karaterisering van transgeniese plante. Die qPCR en Southern-klad resultate het ooreengestem vir 76.5% van die monsters. Dit illustreer die vermoë van qPCR om akkurate kopie-getalle van transgene te bepaal. Verskeie plante is geïdentifiseer tydens qRT-PCR amplifisering wat hoë vlakke van transgeen mRNA uitdrukking getoon het. Hierdie monsters is ideaal vir verdere virus weerstandbiedendheids studies. Hierdie studie het die veelsydigheid van real time PCR bewys en getoon dat dit ‘n kosbare tegniek is vir akkurate virus opsporing sowel as kopie-getal bepaling.

Thesis (MSc (Genetics))--University of Stellenbosch, 2010.
Includes bibliography.
ENGLISH ABSTRACT: The first aim of this study was to reliably and rapidly detect Grapevine rupestris stem pittingassociated virus (GRSPaV) in grapevine. This was achieved by screening 94 grapevines using crude plant extracts in both quantitative and conventional reverse transcription polymerase chain reaction (RT-PCR). The second aim was to establish a technique capable of differentiating GRSPaV sequence variants. The application of this technique is for the largescale screening of diseased vines to associate sequence variants of GRSPaV with disease symptoms. Nested quantitative polymerase chain reaction and high resolution melting assays (qPCR-HRM) were developed for three regions of the GRSPaV genome (coat protein, RNAdependant RNA-polymerase and triple gene block movement protein). The qPCR-HRM technique using the high saturation dye, EvaGreen™, and the Rotor-Gene™ 6000 analyzer was validated with a panel of sixteen sequence-characterized viral isolates. Diluted RT-PCR products and cloned cDNA gave the most consistent amplification plots and dissociation profiles. RT-PCR products generated from total RNA extracts were used as template for qPCR-HRM assays and for direct sequencing of sixteen samples in the three aforementioned regions. The average amplification efficiency for qPCR was 1.52±0.04. Auto-calling of userdefine genotypes was performed at a confidence interval of 70%. Phylogenetic analysis of the three regions of the GRSPaV genome was performed with published GenBank sequences to confirm the HRM data. The dominant sequence variants found in the South African sample set radiated with Group II, reference full-length variant GRSPaV-SG1. GRSPaV-infected samples can in future be subjected to qPCR-HRM assays developed during this study. This can be performed to establish similarity to known genotypes and therefore phylogenetic groups. Mixed infection of sequence variants and quasi-species were a common occurrence. The assay will be useful in establishing correlation of specific genotypes to different phenotypical expression of viral disease. This could provide insight into the etiology of diseases associated with GRSPaV.
AFRIKAANSE OPSOMMING: Die eerste doel van hierdie studie was om die virus wat met Rupestris-stamverpitting (Grapevine rupestris stem pitting-associated virus of “GRSPaV”) in wingerd verbind is, vinnig en betroubaar op te spoor. Dit is bereik deur 94 wingerdstokke vir die teenwoordigheid van die virus te toets met beide kwantitatiewe en konvensionele trutranskripsie polimerase kettingreaksies (RT - PCR) vanaf ongesuiwerde plant-ekstraksies. Die tweede doel was die daarstelling van ’n tegniek om onderskeid te tref tussen variante van GRSPaV met verskillende nukleotiedvolgordes. Hierdie tegniek kan op groot skaal gebruik word om ge-affekteerde wingerdstokke te toets om sodoende siektesimptome met spesifieke variante van GRSPaV te verbind. Ge-neste kwantitatiewe polimerase-kettingreaksies (qPCR) en hoë-resolusie smelt-analises (HRM) is ontwikkel vir drie streke van die GRSPaV-genoom (mantelproteïen, RNS-afhanklike RNS-polimerase en trippelgeenblok bewegingsproteïen). Die tegniek van qPCR-HRM met die hoë-versadingingskleurstof EvaGreen™ en die Rotor- Gene™ 6000 ontleder se geldigheid is bevestig deur vergelyking met ’n paneel van sestien virus-isolate waarvan die volgorde reeds bepaal is. Verdunde RT-PCR-produkte en gekloneerde DNS het die mees konsekwente amplifikasie-uitstipping en dissosiasieprofiele opgelewer. RT-PCR-produkte wat vanuit totale RNS-ekstrakte verkry is, is as templaat vir qPCR-HRM-analises gebruik. Dieselfde produkte is ook gebruik, om die volgorde van sestien monsters in drie streke direk te bepaal. Die gemiddelde amplifikasiedoeltreffendheid van die qPCR was 1.52±0.04. Gebruiker-gedefinieerde genotipes is deur middel van outooproeping teen ’n vertroue-interval van 70% uitgevoer. Filogenetiese analises vir drie streke van die GRSPaV-genoom is uitgevoer met gepubliseerde GenBank-volgordes om die HRMdata te bevestig. Die dominante volgorde-variante in die stel Suid-Afrikaanse monsters het ooreengestem met Groep II, vollengte-verwysingsvariant GRSPaV-SG1. Monsters wat met GRSPaV besmet is kan in die toekoms onderwerp word aan die qPCR-HRM-analises wat in hierdie studie ontwikkel is. Dit kan uitgevoer word om ooreenkomste met bekende genotipes te bepaal, en dus ook met filogenetiese groepe. Die besmetting van plante met meer as een volgorde-variant het algemeen voorgekom. Die kwasi-spesies populasie-struktuur van die virus het ook gedurig na vore gekom. Die toets sal nuttig wees in die bepaling van korrelasies tussen spesifieke genotipes en verskillende fenotipiese voorkomste van virussiektes. Dit kan insig verleen in die etiologie van siektes wat met GRSPaV verbind word.

African horse sickness (AHS) is a viral disease with high mortality rates, vectored by the Culicoides midge and affecting members of the Equidae family. AHS is endemic to South Africa, and, as a result, affects export and international competitiveness in equine trade, and impacts significantly on the South African racehorse and performance horse industries. AHS also has devastating consequences for rural and subsistence equine ownership. The protocol developed in this dissertation has the potential to serotype and confirm the AHS virus within a few hours at significantly less cost than current methods. It will ease the financial and time constraints of studying an outbreak in real time and has the potential to solve many of the unknown factors surrounding AHS, particularly and most importantly, the role that each serotype plays in outbreaks and the form of the disease contracted by horses.
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.