Phylogenetic characterisation of the Palyam serogroup orbiviruses and development of a real-time RT-PCR

The Palyam serogroup of the genus Orbivirus and family Reoviridae are arthropodborne viruses that have been isolated in Africa, Australia and Asia. They are associated with abortion and teratogenesis in cattle and other ruminants. There are currently 13 serotypes recognized by the International Committee on Taxonomy of Viruses (ICTV) including Palyam, Kasba, Vellore, Abadina, D’Aguilar, Nyabira, CSIRO Village, Marrakai, Gweru, Bunyip Creek, Petevo, Marondera and Kindia. Although Palyam viruses had been isolated previously, it was only after an outbreak of congenital abnormalities in cattle in Japan from November 1985 to April 1986 that their pathogenic importance began to be investigated. Of the 13 different serotypes that have been identified, the full genome sequence of only one, Kasba, has been published. Sequences for certain genome segments of the serotypes from Japan, Australia and Zimbabwe are available but not the complete genome data. The few molecular studies that have been done on the Palyam serogroup viruses, focused mainly on Kasba virus and little is known about the other serotypes. In general, not much is published on Palyam viruses, their occurrence or prevalence, and the impact of their epidemiology in South Africa or elsewhere is unknown. The objective of this project was to perform phylogenetic analysis of the different serotypes of the Palyam viruses to enable a better understanding of the genomic features of the Palyam serogroup of orbiviruses, their relation to each other as well as to other orbiviruses. The study is presented in two parts. The aim of the first part was to obtain the full genome sequences of the different Palyam serotypes and Apies River virus, as well as selected field isolates from Africa in order to perform phylogenetic analysis. The aim of the second part was to develop a rapid diagnostic test to detect the Palyam viruses. In the first part, the viruses were propagated and after full-length amplification of cDNA (FLAC) the amplicons were sequenced on an Illumina® Mi-Seq sequencer, using the Nextera XT DNA sample preparation kit and 300-bp paired-end V3 Illumina chemistry. Sequence data generated by Illumina sequencing were analyzed using the CLC Genomics Main workbench, version, 8.0.1. De novo assembly of sequence reads was performed and contig sequences prepared. Sequences were aligned and converted into nexus and phylips files. Data-display networks (neighbour-networks) were constructed with SplitsTree 4 and the phylips files were used to initiate model estimation via jModel test2, by using the online portal Cipres Science gateway. Bayesian analyses was performed in MrBayes version 3. During analysis of the amino acid sequences of the separate genes of the Palyam serogroup serotypes, the gene encoding Viral Protein (VP) 7 (Segment 7) was found to be the most conserved. The amino acid sequences for VP2 and VP5 showed the highest degree of variation, with VP2 being the most variable of the two. Phylogenetic analysis indicated that the Palyam virus group was most closely related to AHSV, and EEV showed the most distant evolutionary relationship to the Palyam viruses. When comparing the different serotypes within the Palyam serogroup viruses, a high degree of sequence identity was found for isolates from the same geographical region. The phylogenetic analysis revealed two clades, which were supported by strong bootstrap values of 100 and a posterior probability value of 1. The African serotypes were all very closely related in one clade, with identical sequences for several gene segments. The second clade contained the Australian and Asian serotypes and one African serotype, Petevo. The high percentage sequence identity (85.6% - 77,5%) that exists between the viruses from Australia and Asia may suggest that there has been some gene flow between the serotypes. It was clear from the sequence data that the geographical origin of Palyam serogroup viruses played an important role in the development of the different serotypes. In the second part of the study, the sequence data obtained in the phylogenetic analysis was used to develop primers and a probe to detect all the Palyam serogroup serotypes in a real-time RT-PCR. The same viruses used in the first part of the study as well as other orbiviruses were propagated and RNA was extracted and tested in a real-time RT-PCR. The real-time RT-PCR was able to detect all the Palyam serogroup serotypes, but further validation is necessary for it to be used as a diagnostic test. The sequence data generated during this study could enable further investigation into molecular evolution of the Palyam serogroup viruses such as reassortment, genetic drift and intragenic recombination. The developed real-time RT-PCR could be a valuable diagnostic tool for both the detection and exclusion of Palyam serogroup viruses during outbreaks involving relevant symptoms.Dissertation (MSc)--University of Pretoria, 2018.Veterinary Tropical DiseasesMScUnrestricte

Widespread Reassortment Contributes to Antigenic Shift in Bluetongue Viruses from South Africa

Bluetongue (BT), a viral disease of ruminants, is endemic throughout South Africa, where outbreaks of different serotypes occur. The predominant serotypes can differ annually due to herd immunity provided by annual vaccinations using a live attenuated vaccine (LAV). This has led to both wild-type and vaccine strains co-circulating in the field, potentially leading to novel viral strains due to reassortment and recombination. Little is known about the molecular evolution of the virus in the field in South Africa. The purpose of this study was to investigate the genetic diversity of field strains of BTV in South Africa and to provide an initial assessment of the evolutionary processes shaping BTV genetic diversity in the field. Complete genomes of 35 field viruses belonging to 11 serotypes, collected from different regions of the country between 2011 and 2017, were sequenced. The sequences were phylogenetically analysed in relation to all the BTV sequences available from GenBank, including the LAVs and reference strains, resulting in the analyses and reassortment detection of 305 BTVs. Phylogenomic analysis indicated a geographical selection of the genome segments, irrespective of the serotype. Based on the initial assessment of the current genomic clades that circulate in South Africa, the selection for specific clades is prevalent in directing genome segment reassortment, which seems to exclude the vaccine strains and in multiple cases involves Segment-2 resulting in antigenic shift

"Akademisches Schwarmverhalten“ und globale Notlagen : Gleichberechtigte Süd-Nord-Forschungspartnerschaften zur Förderung einer hochwertigen Bildung in unterschiedlichen Kontexten und Kulturen

In this article we apply an Afrocentric Resilience Theory (relationship-resourced resilience) to the domain of education research partnerships. We posit academic flocking as an equitable research partnership approach aimed at developing education knowledge that responds to collective distress and supports collective quality education. We provide support for our supposition regarding academic flocking by overviewing the mutually beneficial development of an online, home-based learning resource with relevance in two transnational contexts and cultures, South Africa and the United States of America. Whereas the context of the argument is a COVID-19 related global need for evidence-based education resources, conceptually we draw on lenses of resilience and emancipatory, democratising methodology to make sense of academic flocking as a fundamental structure for research partnership equity and relevant education innovation. As such, academic flocking holds value as a transformative alternative for South-North researcher partnerships in generating useful, quality educational innovations to address critical needs.In diesem Beitrag wenden wir eine afrozentrische Resilienztheorie (beziehungsgestützte Resilienz) auf einen Ansatz der Bildungsforschungspartnerschaften an. Wir stützen uns auf die Annahme, dass „akademisches Flocking“ (Schwarmverhalten) eine Grundlage für gleichberechtigte Forschungspartnerschaften bildet. Der Ansatz zielt darauf ab, Wissen über Bildung zu generieren, das auf kollektive Notlagen reagiert und kollektive Prozesse der Qualitätsbildung unterstützt. Wir untermauern unsere Annahme über „akademisches Flocking“, indem wir einen Überblick über die für beide Seiten vorteilhafte Entwicklung einer Online-Lernressource geben, die zu Hause eingesetzt werden kann. Dieser Ansatz wurde in zwei transnationalen Kontexten und Kulturen – Südafrika und den Vereinigten Staaten von Amerika – erprobt. Kontext der Argumentation ist der von der COVID-19-Pandemie verstärkt sichtbar gewordene globale Bedarf an der Bereitstellung evidenzbasierter Bildungsressourcen. Konzeptionell stützen wir uns auf resilienztheoretische Sichtweisen und eine emanzipatorische, demokratiefördernde Perspektive, wodurch „akademisches Flocking“ als ein vielversprechender Ansatz für ausgewogene Forschungspartnerschaften zur Begleitung relevanter Bildungsinnovation sichtbar wird. Insbesondere bei Süd-Nord-Forschungspartnerschaften bietet sich hier eine nützliche, qualitativ hochwertige Grundlage für die kollaborative Entwicklung von wissenschaftlich unterstützten Bildungsinnovationen.https://link.springer.com/journal/11618hj2023Educational Psycholog

Culicoides species abundance and potential overwintering of African horse sickness virus in the Onderstepoort area, Gauteng, South Africa

In South Africa, outbreaks of African horse sickness (AHS) occur in summer; no cases are reported in winter, from July to September. The AHS virus (AHSV) is transmitted almost exclusively by Culicoides midges (Diptera: Ceratopogonidae), of which Culicoides imicola is considered to be the most important vector. The over-wintering mechanism of AHSV is unknown. In this study, more than 500 000 Culicoides midges belonging to at least 26 species were collected in 88 light traps at weekly intervals between July 2010 and September 2011 near horses in the Onderstepoort area of South Africa. The dominant species was C. imicola. Despite relatively low temperatures and frost, at least 17 species, including C. imicola, were collected throughout winter (June–August). Although the mean number of midges per night fell from > 50 000 (March) to < 100 (July and August), no midge-free periods were found. This study,using virus isolation on cell cultures and a reverse transcriptase polymerase chain reaction (RT-PCR) assay, confirmed low infection prevalence in field midges and that the detection of virus correlated to high numbers. Although no virus was detected during this winter period,continuous adult activity indicated that transmission can potentially occur. The absence of AHSV in the midges during winter can be ascribed to the relatively low numbers collected coupled to low infection prevalence, low virus replication rates and low virus titres in the potentially infected midges. Cases of AHS in susceptible animals are likely to start as soon as Culicoides populations reach a critical level

Possible over-wintering of bluetongue virus in Culicoides populations in the Onderstepoort area, Gauteng, South Africa

Several studies have demonstrated the ability of certain viruses to overwinter in arthropod vectors. The over-wintering mechanism of bluetongue virus (BTV) is unknown. One hypothesis is over-wintering within adult Culicoides midges (Diptera; Ceratopogonidae) that survive mild winters where temperatures seldom drop below 10 °C. The reduced activity of midges and the absence of outbreaks during winter may create the impression that the virus has disappeared from an area. Light traps were used in close association with horses to collect Culicoides midges from July 2010 to September 2011 in the Onderstepoort area, in Gauteng Province, South Africa. More than 500 000 Culicoides midges were collected from 88 collections and sorted to species level, revealing 26 different Culicoides species. Culicoides midges were present throughout the 15 month study. Nine Culicoides species potentially capable of transmitting BTV were present during the winter months. Midges were screened for the presence of BTV ribonucleic acid (RNA) with the aid of a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay. In total 91.2% of midge pools tested positive for BTV RNA. PCR results were compared with previous virus isolation results (VI) that demonstrated the presence of viruses in summer and autumn months. The results indicate that BTV-infected Culicoides vectors are present throughout the year in the study area. Viral RNA-positive midges were also found throughout the year with VI positive midge pools only in summer and early autumn. Midges that survive mild winter temperatures could therefore harbour BTV but with a decreased vector capacity. When the population size, biting rate and viral replication decrease, it could stop BTV transmission. Over-wintering of BTV in the Onderstepoort region could therefore result in re-emergence because of increased vector activity rather than reintroduction from outside the region

Complete Genome Sequences of Virus Strains Isolated from Bottle A of the South African Live Attenuated Bluetongue Virus Vaccine

This is a report of the complete genome sequences of plaque-selected isolates of five virus strains included in bottle A of the South African Onderstepoort Biological Products commercial live attenuated bluetongue virus vaccine

Potential link of single nucleotide polymorphisms (SNPs) to virulence of vaccine‐associated field strains of lumpy skin disease virus in South Africa

South Africa is endemic for lumpy skin disease and is therefore reliant on various live attenuated vaccines for the control and prevention of the disease. In recent years, wide‐spread outbreaks of vaccine‐like strains of lumpy skin disease virus (LSDV) were reported internationally, leading to an increase in the generation of full genome sequences from field isolates. In this study, the complete genomes of six LSDVs submitted during active outbreaks in the 1990’s in South Africa were generated. Based on phylogenetic analysis, the six viruses clustered with vaccine strains in LSDV Subgroup 1.1 and are subsequently referred to as vaccine‐associated. The genetic differences between the phenotypically distinct vaccine and vaccine‐associated strains were 67 single nucleotides polymorphisms (SNPs). This study characterised the location and possible importance of each of these SNPs in their role during virulence and host specificity

Widespread reassortment contributes to antigenic shift in Bluetongue viruses from South Africa

DATA AVAILABIITY: Information on the country of origin, year of isolation, and GenBank accession number for each of the 98 genomes used in this study is provided in Supplementary Table S1.Bluetongue (BT), a viral disease of ruminants, is endemic throughout South Africa, where outbreaks of different serotypes occur. The predominant serotypes can differ annually due to herd immunity provided by annual vaccinations using a live attenuated vaccine (LAV). This has led to both wild-type and vaccine strains co-circulating in the field, potentially leading to novel viral strains due to reassortment and recombination. Little is known about the molecular evolution of the virus in the field in South Africa. The purpose of this study was to investigate the genetic diversity of field strains of BTV in South Africa and to provide an initial assessment of the evolutionary processes shaping BTV genetic diversity in the field. Complete genomes of 35 field viruses belonging to 11 serotypes, collected from different regions of the country between 2011 and 2017, were sequenced. The sequences were phylogenetically analysed in relation to all the BTV sequences available from GenBank, including the LAVs and reference strains, resulting in the analyses and reassortment detection of 305 BTVs. Phylogenomic analysis indicated a geographical selection of the genome segments, irrespective of the serotype. Based on the initial assessment of the current genomic clades that circulate in South Africa, the selection for specific clades is prevalent in directing genome segment reassortment, which seems to exclude the vaccine strains and in multiple cases involves Segment-2 resulting in antigenic shift.Technology Innovation Agency of South Africa: E Venter Seed Fund.https://www.mdpi.com/journal/virusesVeterinary Tropical Disease

Promoting critical-analytic thinking through teacher discourse moves and pedagogical principles : the case of a rural South African secondary school

This article reports a case study in a rural South African school on promoting critical-analytic thinking through teacher discourse moves and pedagogical principles. The study investigated the use of teacher discourse moves and pedagogical principles as a component of the Quality Talk model. The Qualitative research methodology and a case study design that entailed the use of interviews, classroom observations and document analysis were used. Data was gathered from an English teacher and 52 Grade 8 students. The data was coded using Quality Talk model indicators and analysed thematically. The findings revealed evidence that teacher training and support in the use of a range of teacher discourse moves and pedagogical principles could enhance students’ development of critical-analytic thinking. It is therefore recommended that teacher training in the use of pedagogical approaches that enhance the development of critical-analytic thinking should be incorporated in professional development programmes.https://www.ajol.info//index.php/jlthj2021Educational Psycholog

Seminal transmission of lumpy skin disease virus in heifers

It is known that lumpy skin disease virus (LSDV) can be shed in bull semen following infection and that artificial insemination (AI) poses a biosecurity risk. It is however not known whether the use of LSDV infected semen in AI poses a biosecurity risk. The aims of the current study were to investigate whether LSDV, transmitted through semen, can infect cows and embryos.. Two controlled trials were performed simultaneously. Eleven (11) young beef heifers, naïve to LSDV, were synchronized using an OvSynch protocol and inseminated with fresh semen spiked with a field strain of LSDV on day 0. Six (6) of the heifers were superovulated on Day 1 using PMSG, and embryos were flushed from these heifers on Day 6. Blood and serum samples were collected from Day 4 until Day 27 to determine the presence of LSDV by PCR and virus isolation, and the presence of antibodies against LSDV by SNT. The first clinical signs of LSD were noticed on Day 10, followed by severe generalized LSD in 3 heifers, and mild LSD in 2 more heifers. Two heifers were humanely euthanized due to severe unresponsive stranguria. LSDV was detected by PCR, virus isolation or electron microscopy in blood, embryos and organs of experimentally infected animals, and 8 heifers had seroconverted by Day 27. Two control animals were not affected. This is the first report of experimental seminal transmission of LSDV in cattle.NRF. Project number FA 200704250000.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1865-16822015-10-31hb201