Harbin: a quantitation PCR analysis tool

Objectives: To enable analysis and comparisons of different relative quantitation experiments, a web-browser application called Harbin was created that uses a quantile-based scoring system for the comparison of samples at different time points and between experiments. Results: Harbin uses the standard curve method for relative quantitation to calculate concentration ratios (CRs). To evaluate if different datasets can be combined the Harbin quantile bootstrap test is proposed. This test is more sensitive in detecting distributional differences between data sets than the Kolmogorov–Smirnov test. The utility of the test is demonstrated in a comparison of three grapevine leafroll associated virus 3 (GLRaV-3) RT-qPCR data sets. Conclusions: The quantile-based scoring system of CRs will enable the monitoring of virus titre or gene expression over different time points and be useful in other genomic applications where the combining of data sets are required

Next-generation sequencing for virus detection : covering all the bases

CITATION: Visser, M., et al. 2016. Next-generation sequencing for virus detection : covering all the base. Virology Journal, 13(1): 85, doi: 10.1186/s12985-016-0539-x.The original publication is available at http://bmcmusculoskeletdisord.biomedcentral.comPublication of this article was funded by the Stellenbosch University Open Access Fund.Background: The use of next-generation sequencing has become an established method for virus detection. Efficient study design for accurate detection relies on the optimal amount of data representing a significant portion of a virus genome. Findings: In this study, genome coverage at different sequencing depths was determined for a number of viruses, viroids, hosts and sequencing library types, using both read-mapping and de novo assembly-based approaches. The results highlighted the strength of ribo-depleted RNA and sRNA in obtaining saturated genome coverage with the least amount of data, while even though the poly(A)-selected RNA yielded virus-derived reads, it was insufficient to cover the complete genome of a non-polyadenylated virus. The ribo-depleted RNA data also outperformed the sRNA data in terms of the percentage of coverage that could be obtained particularly with the de novo assembled contigs. Conclusion: Our results suggest the use of ribo-depleted RNA in a de novo assembly-based approach for the detection of single-stranded RNA viruses. Furthermore, we suggest that sequencing one million reads will provide sufficient genome coverage specifically for closterovirus detection.Publisher's versio

Characterizing Marathon-Induced Metabolic Changes Using 1H-NMR Metabolomics

Although physical activity is a health-promoting, popular global pastime, regular engage-ment in strenuous exercises, such as long-distance endurance running races, has been associated with a variety of detrimental physiological and immunological health effects. The resulting altered physiological state has previously been associated with fluctuations in various key metabolite con-centrations; however, limited literature exists pertaining to the global/holistic metabolic changes that are induced by such. This investigation subsequently aims at elucidating the metabolic changes induced by a marathon by employing an untargeted proton nuclear magnetic resonance (1H-NMR) spectrometry metabolomics approach. A principal component analysis (PCA) plot revealed a natu-ral differentiation between pre- and post-marathon metabolic profiles of the 30-athlete cohort, where 17 metabolite fluctuations were deemed to be statistically significant. These included reduced concentrations of various amino acids (AA) along with elevated concentrations of ketone bodies, glycolysis, tricarboxylic acid (TCA) cycle, and AA catabolism intermediates. Moreover, elevated concentrations of creatinine and creatine in the post-marathon group supports previous findings of marathon-induced muscle damage. Collectively, the results of this investigation characterize the strenuous metabolic load induced by a marathon and the consequential regulation of main energy-producing pathways to accommodate this, and a better description of the cause of the physiological changes seen after the completion of a marathon

The metabolic recovery of marathon runners: an untargeted 1H-NMR metabolomics perspective

Introduction: Extreme endurance events may result in numerous adverse metabolic, immunologic, and physiological perturbations that may diminish athletic performance and adversely affect the overall health status of an athlete, especially in the absence of sufficient recovery. A comprehensive understanding of the post-marathon recovering metabolome, may aid in the identification of new biomarkers associated with marathon-induced stress, recovery, and adaptation, which can facilitate the development of improved training and recovery programs and personalized monitoring of athletic health/recovery/performance. Nevertheless, an untargeted, multi-disciplinary elucidation of the complex underlying biochemical mechanisms involved in recovery after such an endurance event is yet to be demonstrated.Methods: This investigation employed an untargeted proton nuclear magnetic resonance metabolomics approach to characterize the post-marathon recovering metabolome by systematically comparing the pre-, immediately post, 24, and 48 h post-marathon serum metabolite profiles of 15 athletes.Results and Discussion: A total of 26 metabolites were identified to fluctuate significantly among post-marathon and recovery time points and were mainly attributed to the recovery of adenosine triphosphate, redox balance and glycogen stores, amino acid oxidation, changes to gut microbiota, and energy drink consumption during the post-marathon recovery phase. Additionally, metabolites associated with delayed-onset muscle soreness were observed; however, the mechanisms underlying this commonly reported phenomenon remain to be elucidated. Although complete metabolic recovery of the energy-producing pathways and fuel substrate stores was attained within the 48 h recovery period, several metabolites remained perturbed throughout the 48 h recovery period and/or fluctuated again following their initial recovery to pre-marathon-related levels

Sequencing and detection of a new strain of grapevine leafroll-associated virus 3 in South Africa

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

Small RNA profiling of grapevine leafroll-associated virus 3 infected grapevine plants

Thesis (PhD)--Stellenbosch University, 2016.ENGLISH ABSTRACT: One of the most important viral diseases of grapevine worldwide is grapevine leafroll disease (GLD). A number of viruses from the family Closteroviridae have been associated with this disease, though Grapevine leafroll-associated virus 3 is considered the leading causative agent due to its consistent association with GLD. To better understand the disease and develop effective control strategies, it is necessary to characterise the molecular interactions between the virus and the plant. Small RNA (sRNA) molecules have been shown to play an important role in gene regulation of normal development and defence responses to biotic and abiotic stresses in plants. Therefore, the aim of this study was to characterise the sRNA species in healthy and infected grapevine to contribute to the growing database of sRNAs present in Vitis vinifera. Microarray analysis and next-generation sequencing was used to identify sRNA species in Chardonnay, Chenin blanc, Cabernet Sauvignon and own-rooted Cabernet Sauvignon plants. Differential expression of sRNAs was evaluated to identify sRNAs associated with GLRaV-3 infection. The modulation of the differentially expressed microRNAs (miRNAs) was validated with stemloop RT-qPCR assays. Transcriptome NGS was also performed to validate the differential expression of the predicted miRNA targets, and to identify metabolic pathways modulated in response to GLRaV-3 independently from sRNA regulation. The transcriptome NGS transcripts that were differentially expressed in all cultivar groups, and transcripts that anti-correlated with miRNA expression, were validated with RT-qPCR assays. These highthroughput approaches identified several differentially expressed sRNAs and (target) genes in infected plants. The anti-correlation of miRNA expression and putative target expression were shown for two miRNAs. Cultivar specificity was identified in the sRNA and gene expression analyses, and both approaches identified Chenin blanc-specific responses. This comparison of symptomatic and asymptomatic GLRaV-3-infected plants provides the first insight into the disease symptom inhibition observed in certain cultivars. The differentially expressed genes identified in all cultivar groups, using the NGS transcriptome data, provides a collection of genes displaying a potentially universal molecular response against GLRaV-3. These genes showed strong associations with cell wall biosynthesis and signalling during pathogen recognition. This study has contributed significantly to the knowledge of sRNAs produced in grapevine and significantly extended the existing sRNA reference database for grapevine. The knowledge generated in this study can be utilised as potential targets for grapevine functional studies, and be translated into potential management strategies to control the disease. A better understanding of both the host defence and viral counter-defence strategies can lead to the prevention of virus replication or the impaired ability of the virus to induce pathogenesis in plants.AFRIKAANS OPSOMMING: Een van die belangrikste virussiektes van wingerd wêreldwyd is wingerd-rolblaarsiekte (GLD). 'n Aantal virusse van die familie Closteroviridae hou verband met hierdie siekte, maar Grapevine leafroll-associated virus 3 (GLRaV-3) word beskou as die hoof-bydraende faktor van GLD as gevolg van die korrelasie met GLD. Om die siekte beter te verstaan en effektiewe beheer toe te pas, is dit nodig om die molekulêre interaksie tussen die virus en die plant te ondersoek. Klein RNA (sRNA) molekules het getoon dat hulle 'n belangrike rol speel in die geenregulering van normale plant ontwikkeling, asook tydens die plant se verdediging teen biotiese en abiotiese stresfaktore. Die doel van hierdie studie was dus om die sRNA spesies in gesonde en geïnfekteerde wingerdstokke te karakteriseer en sodoende by te dra tot die snelgroeiende databasis van Vitis vinifera sRNAs. ‘n Mikro-DNA-volgorde-raamwerk analise, asook nuwe-generasie volgordebepaling (NGS) is gebruik om sRNAs spesies te identifiseer in die kultivars Chardonnay, Chenin blanc, Cabernet Sauvignon en eie-gewortelde Cabernet Sauvignon plante. Daarna is die differensiële uitdrukking van sRNAs geëvalueer om sodoende sRNAs te identifiseer wat verbandhou met GLRaV-3 infeksie. Die regulering van die differensiële uitgedrukte mikroRNA’s (miRNAs) is bevestig met stam-lus tru-transkripsie kwantitatiewe polimerase ketting reaksie (stemloop-RT-qPCR) toetse. Transkriptoom-NGS is ook uitgevoer om die differensiële uitdrukking van die miRNAs se voorspelde teikens te valideer en om gemoduleerde metaboliese paaie in reaksie op GLRaV-3, onafhanklik van sRNA regulasie, te identifiseer. Die transkriptoom NGS gene wat differensieël uitgedruk was in al die kultivar groepe, asook die gene wat ’n anti-korrelasie getoon het met miRNA uitdrukking, is bevestig met RT-qPCR toetse. Hierdie hoë-deurset benaderings het verskeie sRNAs en gene geïdentifiseer wat differensieël uitgedruk was in geïnfekteerde plante. Die anti-korrelasie van miRNA uitdrukking en voorspelde teiken uitdrukking is geïdentifiseer vir twee miRNAs. Kultivar-spesifisiteit was geïdentifiseer in beide die sRNA en geenuitdrukking analises en beide benaderings het ook Chenin blanc-spesifieke reaksies geïdentifiseer. Hierdie vergelyking van simptomatiese en asimptomatiese GLRaV-3-geïnfekteerde plante bied die eerste insig in die simtoom-inhibisie wat waargeneem word in sekere kultivars. Die differensieël-uitgedrukte gene wat geïdentifiseer was in alle kultivar groepe, met behulp van die NGS transkriptoom data, bied 'n versameling van gene wat ‘n potensiële universele molekulêre reaksie toon teen GLRaV-3. Hierdie gene het sterk assosiasies met selwand-biosintese en patogeen herkenningseine. Hierdie studie het aansienlik bygedra tot die kennis van wingerd-geassosieerde sRNAs en dra beduidend by tot die uitgebreide sRNA databasis. Die kennis wat gegenereer is in hierdie studie kan gebruik word om teikens te identifiseer vir wingerd funksionele studies en om verwerk te word na potensiële strategieë om die siekte te beheer. 'n Beter begrip van beide die gasheer verdedigings- en die virale teen-verdedigingstrategieë, kan lei tot die voorkoming van virus replikasie of ‘n verlaging in die vermoë van die virus om die siekte in plante te veroorsaak

Citrus Tristeza Virus Genotype Detection Using High-Throughput Sequencing

CITATION: Bester, R., Cook, G. & Maree, H.H. 2021. Citrus Tristeza Virus Genotype Detection Using High-Throughput Sequencing. Viruses, 13(2):1-17. https://doi.org/10.3390/v13020168The original publication is available at https://www.mdpi.com/journal/viruses/aboutThe application of high-throughput sequencing (HTS) has successfully been used for virus discovery to resolve disease etiology in many agricultural crops. The greatest advantage of HTS is that it can provide a complete viral status of a plant, including information on mixed infections of viral species or virus variants. This provides insight into the virus population structure, ecology, or evolution and can be used to differentiate among virus variants that may contribute differently toward disease etiology. In this study, the use of HTS for citrus tristeza virus (CTV) genotype detection was evaluated. A bioinformatic pipeline for CTV genotype detection was constructed and evaluated using simulated and real data sets to determine the parameters to discriminate between false positive read mappings and true genotype-specific genome coverage. A 50% genome coverage cut-off was identified for non-target read mappings. HTS with the associated bioinformatic pipeline was validated and proposed as a CTV genotyping assay.Publishers versio

First report of citrus virus A in citrus in South Africa

High-throughput sequencing (HTS) of citrus indicator hosts, originally inoculated from field samples and showing transient chlorotic flecking or oak leaf patterns, revealed the presence of the first South African variant of citrus virus A (CiVA). This virus was first identified in citrus in Italy and was classified as a member of the second species (Coguvirus eburi) of the genus Coguvirus within the order Bunyavirales. The South African sequence variants of CiVA share 95.26-95.55% (RNA1) and 94.82-97.85% (RNA2) nucleotide sequence identity with the CiVA sequences from the Italian isolate. The discovery of CiVA in South African citrus orchards indicates a wide distribution of CiVA and further investigations are required to determine the association of CiVA with citrus disease symptoms. &nbsp

Utilisation of a mitochondrial intergenic region for species differentiation of fruit flies (Diptera: Tephritidae) in South Africa

Abstract Background Fruit flies (Diptera: Tephritidae) comprise species of agricultural and economic importance. Five such fruit fly species are known to affect commercial fruit production and export in South Africa: Ceratitis capitata, Ceratitis cosyra, Ceratitis rosa, Ceratitis quilicii, and Bactrocera dorsalis. Management practices for these pests include monitoring, application of pest control products, post-harvest disinfestation measures and inspection of consignments both prior to shipment and at ports of entry. In activities relating to monitoring and inspection, accurate identification of these pests to species level is required. While morphological keys for adult stages of these fruit fly species have been well developed, morphological keys for earlier life stages remain problematic. In instances where closely related species cannot be reliably distinguished morphologically, there is a need for molecular tools to assist in identifying these five fruit fly species during surveillance practices, where sequencing-based approaches would be beneficial. Results Two complete mitochondrial genomes were assembled for each fruit fly species investigated using high throughput sequencing data generated in this study. A single primer set was designed to amplify a region between tRNAile and tRNAmet. The amplicon consists of a partial segment of tRNAile, intergenic region I (tRNAile - tRNAgln), the complete sequence of tRNAgln, intergenic region II (tRNAgln - tRNAmet), and a partial segment of tRNAmet. PCR amplicons were generated for 20 specimens of each species, five of which were colony adult males, five colony larvae, and 10 wild, trap-collected specimens. Upon analysis of the amplicon, intergenic region I was identified as the most informative region, allowing for unambiguous identification of the five fruit fly species. The similarity in intergenic region II was too high between C. rosa and C. quilicii for accurate differentiation of these species. Conclusion The identity of all five fruit flies investigated in this study can be determined through sequence analysis of the mitochondrial intergenic regions. Within the target amplicon, intergenic region I (tRNAile - tRNAgln) shows interspecific variation sufficient for species differentiation based on multiple sequence alignment. The variation in the length of intergenic region I is proposed as a potential tool for accurately identifying these five fruit flies in South Africa

Identification of Interactions between Proteins Encoded by Grapevine Leafroll-Associated Virus 3

The roles of proteins encoded by members of the genus Ampelovirus, family Closteroviridae are largely inferred by sequence homology or analogy to similarly located ORFs in related viruses. This study employed yeast two-hybrid and bimolecular fluorescence complementation assays to investigate interactions between proteins of grapevine leafroll-associated virus 3 (GLRaV-3). The p5 movement protein, HSP70 homolog, coat protein, and p20B of GLRaV-3 were all found to self-interact, however, the mechanism by which p5 interacts remains unknown due to the absence of a cysteine residue crucial for the dimerisation of the closterovirus homolog of this protein. Although HSP70h forms part of the virion head of closteroviruses, in GLRaV-3, it interacts with the coat protein that makes up the body of the virion. Silencing suppressor p20B has been shown to interact with HSP70h, as well as the major coat protein and the minor coat protein. The results of this study suggest that the virion assembly of a member of the genus Ampelovirus occurs in a similar but not identical manner to those of other genera in the family Closteroviridae. Identification of interactions of p20B with virus structural proteins provides an avenue for future research to explore the mechanisms behind the suppression of host silencing and suggests possible involvement in other aspects of the viral replication cycle