Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 170-205).

Thesis (Ph. D.)--University of Hawaii at Manoa, 2002. / Includes bibliographical references (leaves 178-190).

has been significant progress in the development of new technologies and methodologies to characterize gene expression. The fluorescent-based real-time reverse transcription (RT) polymerase chain reaction (PCR) is an important tool used for clinical and molecular research, biotechnology and as a diagnostic test. Insulin-like growth factors (IGF-1 and IGF-2) and insulin are ubiquitously expressed and play important roles in the regulation of cell growth, differentiation and the maintenance of cell differentiation in mammals. The IGF system (IGF-1, IGF-2, IGF -1 receptor, IGF-2 receptor and six IGF-binding proteins) and insulin are consequently essential to most aspects of male and female reproduction. IGF-1 is produced in multiple tissues but predominately in the liver, from where it enters the circulation. Insulin is secreted by β-cells of the pancreas’ islets of Langerhans. Both IGF-1 and insulin polypeptides bind to specific cell surface receptors. These receptors are members of the superfamily known as tyrosine protein kinases, and are composed of two α and two β subunits linked by disulfide bonds to form an αβ–αβ heterotetramer. The α subunits include ligand binding sites, whereas the β subunits contain tyrosine kinase activity. The aim of this project was to develop real-time RT-PCR assays for quantification of equine insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor (INS-R) mRNA. The assays were developed using stallion testicular tissue samples, obtained by excisional biopsy, from three horse breeds (Friesan, Thoroughbred and Warmblood). The assays developed were efficient, sensitive and had a broad linear range of detection (seven logs for IGF-1R and six logs for INS-R). The assays worked well in our hands and were both sensitive and specific for the detection of equine IGF-1R and INS-R mRNA in a variety of equine tissues. / Dissertation (MMedVet)--University of Pretoria, 2011. / Production Animal Studies / Unrestricted

4 Abstract The antral follicle provides a specialized microenvironment or niche, which is necessary for production of high quality oocyte. The developmental competence of bovine oocyte is influenced by the follicle size. Oocytes originated from medium or larger follicles (≥6 mm) have greater developmental competence (ability to develop to the blastocyst stage). The changes in cytoplasmic factors, for example mRNAs, could explain differences in oocyte developmental potential. Using Bovine Oligonucleotide microarrays the differences in gene expression profiles of oocytes at germinal vesicle and MII stages from medium (MF, 6-10 mm) or small (SF, 2-5 mm) follicles were characterized. The aim was to find differencies between oocytes diverse developmental competence. The expression fold change between the two experimental groups was in 61 genes. Subsets of 15 differentially expressed genes were validated by quantitative RT-PCR. Before maturation, significant differences were confirmed at the level of ATP5C1, MAP3K13, MTRF1L, TAF1A and UBL5. Subpopulations of oocytes were classified according to atresia of cumulus cells and follicle size. We determined the level of 12 individual transcripts after maturation. ATP5F1 remained stable in all experimental groups of oocytes. The level of BRD7 transcript remained stable...

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by a virus of the family Bunyaviridae, genus Phlebovirus. It is responsible for extensive outbreaks of disease in livestock in Africa with significant mortality and economic impact. Virus neutralization is considered the gold standard for confirming Rift Valley fever virus (RVFV) infection but the procedure is time consuming and expensive. Real-time reverse transcription-polymerase chain reaction (rRT-PCR), histopathology, and immunohistochemistry (IHC) are the diagnostic methods most often used in South Africa to confirm or exclude a diagnosis of RVF in necropsied animals. Validated estimates of diagnostic accuracy of these tests, in naturally infected livestock, however, have not been published. The objective of this study was to estimate the diagnostic sensitivity and specificity of rRT-PCR, histopathology, and IHC using Bayesian latent class methods in the absence of a gold standard. A secondary objective was to estimate stratum-specific values based on species, age, degree of specimen autolysis, and the presence/absence of tissue pigments. The Sensitivity (Se) and Specificity (Sp) of qRT-PCR were 97.4% (95% credibility interval (CI): 95.2% - 98.8%) and 71.7% (95% CI: 65% - 77.9%) respectively. The extraordinary analytical sensitivity of PCR makes this test very susceptible to false positive reactions, and thus reduced specificity. This is more likely during large-scale epidemics due to crosscontamination of specimens at necropsy facilities or testing laboratories. The Se and Sp of histopathology were 94.6% (95% CI: 91% - 97.2%) and 92.3% (95% CI: 87.6% - 95.8%) respectively. Single cases of RVF could be confused with acute poisoning with plants, bacterial septicaemias, and viral diseases such as infectious bovine rhinotracheitis and Wesselsbron disease. Most of these conditions, however, can be excluded using histological examination of the liver, special stains, bacterial culture, and toxicological or serological investigations. The Se and Sp of IHC were 97.6% (95% CI: 93.9% - 99.8%) and 99.4% (95% CI: 96.9% - 100%) respectively. Immunohistochemistry is highly specific because characteristic positive immunolabelling of the cytoplasm of hepatocytes can be correlated with the presence of hepatocellular injury typical for RVFV infection. False negative results are sometimes obtained with IHC because of reader error or loss of the antigenic epitopes due to advanced autolysis. Scant positive immunolabelling might be missed or viral proteins might be absent from sections of liver with advanced hepatocellular damage. The stratified analysis suggested differences in test accuracy in foetuses and severely autolysed specimens. The Sp of histopathology in foetuses (83.0%) was 9.3% lower than the value obtained for the sample population (92.3%). Lesions in some foetuses are more subtle and the typical eosinophilic intranuclear inclusions are often difficult to detect. In severely autolysed specimens, the Se of IHC decreased by 16.1% and the Sp of rRT-PCR by 17.4%. There is no plausible biological explanation for this decrease in the Sp of rRTPCR since the RNA of RVFV is resistant to degradation in autolysed tissues. Conversely, the antibody used to detect RVFV using IHC detects epitopes raised against nucleoproteins of the virus and it is possible that viral proteins become too widely dispersed and/or degraded in autolysed tissues to detect by light microscopy. It is possible that the marked decrease in Se of histopathology and IHC in severely autolysed specimens caused an apparent decrease in Sp of rRT-PCR, due to the latent class method. In conclusion, the high estimated Sp (99.4%) of IHC and the low Sp of rRT-PCR (71.3%) suggests that the definitive diagnosis or exclusion of RVF should not rely on a single PCR test and that IHC would be an effective confirmatory test for rRT-PCR positive field cases necropsied during an epidemic. Immunohistochemistry results from severely autolysed specimens, however, should be interpreted with caution and aborted foetuses in areas endemic for RVF should be screened using a variety of tests. The diagnostic Se and Sp of histopathology was much higher than expected confirming the value of routine post mortem examinations and histopathology of liver specimens. The most feasible RVF testing option in areas that do not have suitably equipped PCR laboratories, and where disease is often not detected in livestock until after human cases have been diagnosed, would be routine histopathology screening with IHC confirmation. Key Words: Rift Valley fever; Rift Valley fever virus; Bayesian; latent-class model; real-time reverse transcription-polymerase chain reaction; immunohistochemistry; histopathology; diagnosis; sensitivity; specificity. / Dissertation (MSc)--University of Pretoria, 2014. / gm2014 / Paraclinical Sciences / unrestricted

Acute lower respiratory tract infection (ALRI) is a major cause of paediatric morbidity and mortality, annually accounting for approximately 2 million childhood deaths worldwide of which up to 90% resides in the developing world. In 12-39% of ALRI cases no aetiological agent is identified, despite comprehensive investigations, thus suggesting that additional unknown agents may be involved. Since 2001 a number of new viruses have been identified that may account for some of these cases including human metapneumovirus (hMPV), human bocavirus (hBoV), and two new coronaviruses (hCoV) NL63 and HKU1. The contribution of the recently identified respiratory viruses to annual seasonal lower respiratory tract disease in Sub-Saharan Africa where human immunodeficiency virus infections may exacerbate respiratory infections is not fully understood. In addition, the role and disease association of many of these viruses as primary or co-infecting pathogens, as well as the underlying factors that may determine the pathogenesis of these viruses, is not yet well defined. Quantitative multiplex real-time RT-PCR assays were developed and validated for the detection of 13 well recognized and newly identified viral causes of ALRI, including respiratory syncytial virus (RSV), influenza viruses A and B, parainfluenza virus (PIV) types 1, 2, and 3, adenovirus, hMPV, hBoV and hCoV-NL63, -HKU1, -229E, and -OC43. The newly designed assays were subsequently used to facilitate the investigation of the contribution of respiratory viruses in patients requiring hospitalisation or attending outpatient visits in public sector hospitals serving the Pretoria area, South Africa. During 2006, the prevalence of the aforementioned respiratory viruses was determined by investigating the well recognized viruses previously diagnosed by routine immunofluorescence assays (IFA) in 737 respiratory specimens as well as viruses retrospectively detected by multiplex real-time RT-PCR in a sample group of 319 specimens. The epidemiology and disease association of these respiratory viruses in children who were predominantly less than 5 years of age with acute respiratory tract infections was investigated. Specimens were received from 2 public sector hospitals in Pretoria, South Africa. In addition, the disease association of each virus as a single or co-infection in human immunodeficiency virus (HIV) infected/exposed and HIV-uninfected children as well as the role of viral load was investigated. The multiplex assays could detect 2.5-25 recombinant plasmid DNA/RNA (in vitro transcribed) copies/μl, with a co-efficient of variation of less than 3.1%. Validation on 91 known positive respiratory specimens indicated similar specificity to IFA or single-round PCRs used in the initial identification of the viruses. Application of the multiplex assays to IFA negative specimens improved the detection of respiratory viruses by up to 44%. In children less than 5 years of age RSV was identified in 35.1%, followed by PIV 3 (8.3%); adenovirus (5.6%); influenza A (4.2%); hMPV (4.2%); hBoV (3.8%); hCoV-NL63 (1.6%); influenza B (1.0%); and PIV1, PIV2, hCoV-OC43, hCoV-229E, hCoV-HKU1 in less than 1% of cases. Co-infections were more common for the new viruses ranging from 58% of hMPV cases to 84% for hCoV-NL63 relative to 27% of RSV cases. Viruses were most frequently identified in children <1-year. RSV activity peaked in autumn and winter, PIV 3 in spring, while influenza A and B were mostly detected in winter. The observed seasonal distribution of hBoV and hMPV was less defined compared to traditional viruses, with both viruses showing variability over the two years. Comparable hospitalisation rates were observed for RSV, hMPV, PIV 3 and adenovirus, where approximately 60% of infected children were hospitalised. In addition, a high frequency of hospitalisations was observed in patients for both hMPV and hBoV in HIV-infected/exposed children. Co-infections occurred at higher frequencies with the new viruses, were more frequently associated with severe disease and were frequent in HIV-infected/exposed patients. Viral load was associated with severe RSV disease (p=0.014) however no significant association was observed for the new viruses as single infections. However, where hMPV occurred as a co-infection, higher viral loads of either hMPV or co-infecting agents occurred in severe cases. This association was also observed for hBoV. Most cases of hCoV-NL63 and hCoV-OC43 were co-infections in hospitalised patients. The newly developed multiplex assays demonstrates an improved sensitivity and scope of detecting respiratory viruses relative to routine antigen detection assays while the quantitative utility may facilitate investigation of the role of co-infections and viral load in respiratory virus pathogenesis. RSV remains the most significant viral cause of paediatric ALRI in South Africa. Viruses not currently included in routine diagnostic assays collectively contributed to 11% of ALRI cases in children <2-years in South African hospitals. / Dissertation (MSc)--University of Pretoria, 2010. / Medical Virology / unrestricted

Aim: To develop an immunomagnetic assay for the isolation of circulating tumor cells (CTCs) followed by the analysis of a multimarker panel, which will enable the characterization of these malignant cells with high accuracy. Patients and Methods: Peripheral blood (PB) was collected from 32 metastatic breast cancer patients and 42 negative controls. The antibodies BM7 and VU1D9 were used for immunomagnetic tumor cell enrichment. A real-time reverse transcription-polymerase chain reaction (RT-PCR) approach for the markers KRT19, SCGB2A2, MUC1, EPCAM, BIRC5 and ERBB2 was used for CTC detection and characterization. Results: The positivity rates for each marker were as follows: 46.9% for KRT19, 25.0% for SCGB2A2, 28.1% for MUC1, 28.1% for EPCAM, 21.9% for BIRC5, and 15.6% for ERBB2. After the creation of individualized cutoffs, the sensitivity and specificity of the combined marker gene panel increased to 56.3% and 100%, respectively. Interestingly, 27.0% of the HER2-negative tumor patients showed ERBB2 mRNA-positive CTCs. Conclusions: The described technique can be used to measure CTCs with great accuracy. The use of a multimarker panel for the characterization of CTCs may provide real-time information and be of great value in therapy monitoring. / Ziel: Entwicklung eines immunomagnetischen Verfahrens zur Isolierung zirkulierender Tumorzellen (CTCs) in Kombination mit einer molekularen Multimarkeranalyse für die hochspezifische Identifizierung maligner Zellen. Patientinnen und Methoden: Peripheres Blut (PB) von 32 Patientinnen mit metastasiertem Mammakarzinom und von 42 gesunden Kontrollen wurde für die immunomagnetische Tumorzellanreicherung mit den Antikörpern BM7 und VU1D9 genutzt. Eine Real-Time Reverse Transkription Polymerase-Kettenreaktion (RT-PCR)-Methodik mit den Markern KRT19, SCGB2A2, MUC1, EPCAM, BIRC5 und ERBB2 wurde für den CTC-Nachweis und die Tumorzellcharakterisierung entwickelt. Ergebnisse: Für die einzelnen Marker wurden die folgenden Positivitätsraten ermittelt: 46,9% für KRT19, 25,0% für SCGB2A2, 28,1% für MUC1, 28,1% für EPCAM, 21,9% für BIRC5 und 15,6% für ERBB2. Nach der Bestimmung individualisierter Cut-off-Werte ergab sich für den kombinierten Multimarkernachweis eine Sensitivität und Spezifität von 56,3% bzw. 100%. Bemerkenswert war der Befund, dass 27,0% der HER2-tumornegativen Patientinnen ERBB2-mRNA-positive CTCs aufwiesen. Schlussfolgerung: Die hier beschriebene Methodik bestimmt CTCs mit hoher Spezifität. Die molekulare Multimarkeranalyse liefert wertvolle Real-Time-Informationen für personalisierte Behandlungsmodalitäten. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

A via sexual é a principal forma de transmissão inter-humana da infecção pelo vírus da imunodeficiência humana (HIV). Com o incremento do número de mulheres infectadas por esse agente retroviral, o estudo de particularidades da biologia do vírus no trato genital feminino adquiriu maior importância. Com o objetivo de avaliar a excreção genital do HIV ao longo do ciclo menstrual, coletaram-se, nas diversas fases de dois ciclos, lavados cervicovaginais de 17 mulheres soropositivas para essa infecção e acompanhadas em serviço ambulatorial especializado de São Paulo. O RNA viral livre foi quantificado por RT-PCR e o DNA proviral por PCR em tempo real, empregando sistema TaqMan. Avaliou-se ainda a carga viral plasmática de HIV, o número de células CD4+ periféricas e presença de co-infecção genital. Detectou-se excreção de RNA-HIV e de DNA proviral, respectivamente, em 18,8% e 31,3% das pacientes. Todas as pacientes que apresentaram excreção de RNA viral também exibiram a de DNA proviral, incluindo paciente com viremia de HIV indetectável. Não houve variação significativa da excreção genital do vírus durante o ciclo menstrual. Em 6 dessas pacientes, que apresentaram co-infecção genital previamente à admissão no estudo, avaliou-se também a excreção genital de HIV quando da co-infecção. Em 2 casos, a excreção genital do DNA-HIV foi superior na vigência de co-infecção causada por Streptococcus sp e Ureaplasma. Não se observou excreção de RNA viral livre nas pacientes co-infectadas. Os resultados obtidos podem contribuir para o entendimento do potencial de transmissibilidade sexual da infecção por HIV e reiteram a necessidade de adesão às práticas de sexo protegido para evitar sua transmissão inter-humana / The sexual route is the main means of transmission of the human immunodeficiency virus (HIV). With the increasing numbers of HIV-infected women, the investigation of particular biological features of HIV infection in the genital tract has become more important. To evaluate HIV genital shedding during the menstrual cycle, we collected cervicovaginal lavages (CVL) from 17 women, assisted at an HIV outpatient clinic in São Paulo, in different hormonal phases during 2 cycles. HIV-RNA and proviral DNA shedding were quantified using RT-PCR and a TaqMan real-time PCR assay, respectively. In addition, patients were screened for genital coinfections and had their HIV plasma viral loads and CD4+ cell counts assessed. Cell-free HIV-RNA and proviral DNA shedding were found in 18.8% and 31.3% of women. All patients who shed HIV-RNA were also shown to present detectable proviral DNA in their CVL, including one woman with undetectable HIV plasma viral load. No significant difference in viral shedding was seen among menstrual cycle phases. Six patients from the cohort, who exhibited genital coinfections previous to admission to the study, had their HIV genital shedding compared at time of coinfection and after its resolution. In two of them proviral DNA shedding was higher at the time of coinfection, caused by Streptococcus sp and Ureaplasma. No cell-free HIV-RNA shedding was detected in coinfected patients. Our results may contribute to the understanding of HIV sexual infectivity from women and emphasize the need for adherence to protected sexual practices in order to avoid viral transmission.

A via sexual é a principal forma de transmissão inter-humana da infecção pelo vírus da imunodeficiência humana (HIV). Com o incremento do número de mulheres infectadas por esse agente retroviral, o estudo de particularidades da biologia do vírus no trato genital feminino adquiriu maior importância. Com o objetivo de avaliar a excreção genital do HIV ao longo do ciclo menstrual, coletaram-se, nas diversas fases de dois ciclos, lavados cervicovaginais de 17 mulheres soropositivas para essa infecção e acompanhadas em serviço ambulatorial especializado de São Paulo. O RNA viral livre foi quantificado por RT-PCR e o DNA proviral por PCR em tempo real, empregando sistema TaqMan. Avaliou-se ainda a carga viral plasmática de HIV, o número de células CD4+ periféricas e presença de co-infecção genital. Detectou-se excreção de RNA-HIV e de DNA proviral, respectivamente, em 18,8% e 31,3% das pacientes. Todas as pacientes que apresentaram excreção de RNA viral também exibiram a de DNA proviral, incluindo paciente com viremia de HIV indetectável. Não houve variação significativa da excreção genital do vírus durante o ciclo menstrual. Em 6 dessas pacientes, que apresentaram co-infecção genital previamente à admissão no estudo, avaliou-se também a excreção genital de HIV quando da co-infecção. Em 2 casos, a excreção genital do DNA-HIV foi superior na vigência de co-infecção causada por Streptococcus sp e Ureaplasma. Não se observou excreção de RNA viral livre nas pacientes co-infectadas. Os resultados obtidos podem contribuir para o entendimento do potencial de transmissibilidade sexual da infecção por HIV e reiteram a necessidade de adesão às práticas de sexo protegido para evitar sua transmissão inter-humana / The sexual route is the main means of transmission of the human immunodeficiency virus (HIV). With the increasing numbers of HIV-infected women, the investigation of particular biological features of HIV infection in the genital tract has become more important. To evaluate HIV genital shedding during the menstrual cycle, we collected cervicovaginal lavages (CVL) from 17 women, assisted at an HIV outpatient clinic in São Paulo, in different hormonal phases during 2 cycles. HIV-RNA and proviral DNA shedding were quantified using RT-PCR and a TaqMan real-time PCR assay, respectively. In addition, patients were screened for genital coinfections and had their HIV plasma viral loads and CD4+ cell counts assessed. Cell-free HIV-RNA and proviral DNA shedding were found in 18.8% and 31.3% of women. All patients who shed HIV-RNA were also shown to present detectable proviral DNA in their CVL, including one woman with undetectable HIV plasma viral load. No significant difference in viral shedding was seen among menstrual cycle phases. Six patients from the cohort, who exhibited genital coinfections previous to admission to the study, had their HIV genital shedding compared at time of coinfection and after its resolution. In two of them proviral DNA shedding was higher at the time of coinfection, caused by Streptococcus sp and Ureaplasma. No cell-free HIV-RNA shedding was detected in coinfected patients. Our results may contribute to the understanding of HIV sexual infectivity from women and emphasize the need for adherence to protected sexual practices in order to avoid viral transmission.

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.