28 results on '"Burt, Felicity Jane"'
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2. Crimean-Congo Hemorrhagic Fever Virus: An Emerging and Re-emerging Pathogen of Public Health Concern
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Burt, Felicity Jane, Goedhals, Dominique, and Sing, Andreas, editor
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- 2023
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3. Rapid reverse transcriptase recombinase polymerase amplification assay for flaviviruses using non-infectious in vitro transcribed RNA as positive controls
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Bonnet, Elise, van Jaarsveldt, Danelle, and Burt, Felicity Jane
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- 2022
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4. Purification and characterization of soluble recombinant Crimean-Congo hemorrhagic fever virus glycoprotein Gc expressed in mammalian 293F cells.
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Makoah, Nigel Aminake, Litabe, Matefo Millicent, Simo, Fredy Brice Nemg, Maboho, Katlego Keith, and Burt, Felicity Jane
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RAPID diagnostic tests ,GEL permeation chromatography ,HEMORRHAGIC fever ,WESTERN immunoblotting ,AFFINITY chromatography - Abstract
Background: Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonotic disease that presents with severe hemorrhagic manifestations and is associated with significant fatality rates. The causative agent, Crimean-Congo Hemorrhagic Fever Virus (CCHFV), is a high-priority pathogen identified by the World Health Organization with no approved vaccine or specific treatment available. In addition, there is a critical need for enhanced diagnostic tools to improve public health awareness, prevention measures, and disease control strategies. Methods: We designed plasmids to enable the purification of soluble CCHFV glycoprotein Gc expressed in mammalian 293 F cells, followed by purification using affinity and size exclusion chromatography. The purified antigen was analyzed by SDS-PAGE and Western blotting to confirm its reactivity to antibodies from CCHF survivors. Additionally, an in-house indirect ELISA was developed using the purified Gc as a coating antigen. Results: The optimized expression system successfully produced soluble and pure Gc antigen after affinity chromatography. The protein showed specific reactivity with CCHFV-positive serum antibodies in Western blot analysis. The indirect ELISA assay demonstrated high efficacy in distinguishing between CCHFV-positive and -negative serum samples, indicating its potential as a valuable diagnostic tool. Size exclusion chromatography further confirmed the presence of aggregates in our protein preparation. Conclusions: The purified Gc antigen shows promise for developing direct diagnostic assays for CCHFV. The antigen's suitability for subunit vaccine development and its application as bait for monoclonal antibody isolation from survivors could be investigated further. This work lays the foundation for future research into the development of rapid diagnostic tests for field deployment. [ABSTRACT FROM AUTHOR]
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- 2024
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5. Purification and Characterization of Soluble Recombinant Crimean-Congo Hemorrhagic Fever Virus Glycoprotein Gc expressed in mammalian 293F Cells
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Makoah, Nigel Aminake, primary, Litabe, Matefo Millicent, additional, Simo, Fredy Brice Nemg, additional, Maboho, Katlego Keith, additional, and Burt, Felicity Jane, additional
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- 2024
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6. Identification of T cell responses to the nonstructural glycoproteins in survivors of Crimean‐Congo hemorrhagic fever in South Africa
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Maotoana, Makgotso Golda, primary, Burt, Felicity Jane, additional, and Goedhals, Dominique, additional
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- 2023
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7. Chikungunya Virus Diagnosis: A Review of Current Antigen Detection Methods
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Simo, Fredy Brice Nemg, primary, Burt, Felicity Jane, additional, and Makoah, Nigel Aminake, additional
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- 2023
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8. Crimean-Congo Haemorrhagic Fever Virus, an Emerging and Re-Emerging Pathogen
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Burt, Felicity Jane, Goedhals, Dominique, and Sing, Andreas, editor
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- 2015
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9. Perspective Technologies of Vaccination: Do We Still Need Old Vaccines?
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Isaguliants, Maria, primary and Burt, Felicity Jane, additional
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- 2022
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10. Validation of laboratory developed serology assays for detection of IgG antibody to severe acute respiratory syndrome coronavirus 2 in the South African population
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Litabe Matefo, van Vuuren Cloete, Bester Philip Armand, Goedhals Dominique, Potgieter Samantha, Frater John, Thompson Craig, Wright Daniel, Lambe Theresa, Gupta Sunetra, Brink Maréza, van Jaarsveldt Danelle, and Burt Felicity Jane
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South Africa ,SARS-CoV-2 ,Virology ,Immunoglobulin G ,Spike Glycoprotein, Coronavirus ,COVID-19 ,Humans ,Enzyme-Linked Immunosorbent Assay ,Antibodies, Viral ,Sensitivity and Specificity - Abstract
Serological assays for detection of IgG, IgM or IgA against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) play an important role in surveillance, antibody persistence, vaccine coverage and infection rate. Serological assays, including both ELISA and rapid lateral flow assays, are available commercially but the cost limits their accessibility for low resource countries. Although serological assays based on mammalian-expressed SARS-CoV-2 spike protein have been previously described these assays need to be validated using samples from local populations within the continent, or country, in which they will be used. Interpretation of results could be influenced by differences in specificity and potential for pre-existing cross-reactive antibodies. In this study, we investigated two laboratory developed serological assays, an enzyme linked immunosorbent assay (ELISA) and an immunofluorescent assay (IFA), developed using recombinant SARS-CoV-2 spike protein, for use in South African populations. The tests were compared with commercially available and South Africa Health Products Regulatory Authority (SAPHRA) approved assays. A panel of 100 residual diagnostic serum samples, collected prior to the pandemic, were tested on three separate occasions to determine a suitable cut-off value for differentiation of positive from negative samples. Specificity of 96 % and 100 % for ELISA and IFA respectively was demonstrated. A total of 82/89 serum samples collected between days 2-94 after onset of illness from patients with a positive molecular result were positive for IgG antibody. The sensitivity of the laboratory developed assays on samples collectedone week after onset of illness was shown to be 100 % and 98.8 % for ELISA and IFA respectively. Positive predictive values were 92.1 % for ELISA and 91.0 % for IFA using characterization of samples as positive based on confirmation of infection using RT-PCR. Serum samples (n = 62) collected from RT-PCR positive patients infected with either ancestral, or emerging variants such as Beta or Delta, tested positive for IgG antibody (62/62) using the laboratory developed assays confirming application of the assays regardless of currently circulating variant during the time of evaluation. High concordance was demonstrated between the laboratory developed assays and the commercial immunoassay among samples collected from South African populations, although the small sample size, especially for the comparison with commercial assays, must be noted. If all quality assurance controls are in place, the use of local laboratory developed assays for high-throughput screening in resource-constrained environments is a realistic alternative option.
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- 2022
11. Immunogenicity of a DNA-Based Sindbis Replicon Expressing Crimean–Congo Hemorrhagic Fever Virus Nucleoprotein
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Tipih, Thomas, primary, Heise, Mark, additional, and Burt, Felicity Jane, additional
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- 2021
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12. Crimean-Congo Haemorrhagic Fever Virus, an Emerging and Re-Emerging Pathogen
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Burt, Felicity Jane, primary and Goedhals, Dominique, additional
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- 2014
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13. Crimean–Congo Hemorrhagic Fever Virus: Advances in Vaccine Development
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Tipih, Thomas, primary and Burt, Felicity Jane, additional
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- 2020
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14. Epitope-mapping of the glycoprotein from Crimean-Congo hemorrhagic fever virus using a microarray approach
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Fritzen, Amanda, primary, Risinger, Christian, additional, Korukluoglu, Gulay, additional, Christova, Iva, additional, Corli Hitzeroth, Arina, additional, Viljoen, Natalie, additional, Burt, Felicity Jane, additional, Mirazimi, Ali, additional, and Blixt, Ola, additional
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- 2018
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15. Construction and Immunogenicity of Modified mRNA-Vaccine Variants Encoding Influenza Virus Antigens.
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Starostina, Ekaterina V., Sharabrin, Sergei V., Antropov, Denis N., Stepanov, Grigory A., Shevelev, Georgiy Yu., Lemza, Anna E., Rudometov, Andrey P., Borgoyakova, Mariya B., Rudometova, Nadezhda B., Marchenko, Vasiliy Yu., Danilchenko, Natalia V., Chikaev, Anton N., Bazhan, Sergei I., Ilyichev, Alexander A., Karpenko, Larisa I., Isaguliants, Maria G., and Burt, Felicity Jane
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INFLUENZA A virus ,INFLUENZA viruses ,GREEN fluorescent protein ,DNA vaccines ,VIRAL antibodies - Abstract
Nucleic acid-based influenza vaccines are a promising platform that have recently and rapidly developed. We previously demonstrated the immunogenicity of DNA vaccines encoding artificial immunogens AgH1, AgH3, and AgM2, which contained conserved fragments of the hemagglutinin stem of two subtypes of influenza A—H1N1 and H3N2—and conserved protein M2. Thus, the aim of this study was to design and characterize modified mRNA obtained using the above plasmid DNA vaccines as a template. To select the most promising protocol for creating highly immunogenic mRNA vaccines, we performed a comparative analysis of mRNA modifications aimed at increasing its translational activity and decreasing toxicity. We used mRNA encoding a green fluorescent protein (GFP) as a model. Eight mRNA-GFP variants with different modifications (M0–M7) were obtained using the classic cap(1), its chemical analog ARCA (anti-reverse cap analog), pseudouridine (Ψ), N6-methyladenosine (m6A), and 5-methylcytosine (m5C) in different ratios. Modifications M2, M6, and M7, which provided the most intensive fluorescence of transfected HEK293FT cells were used for template synthesis when mRNA encoded influenza immunogens AgH1, AgH3, and AgM2. Virus specific antibodies were registered in groups of animals immunized with a mix of mRNAs encoding AgH1, AgH3, and AgM2, which contained either ARCA (with inclusions of 100% Ψ and 20% m6A (M6)) or a classic cap(1) (with 100% substitution of U with Ψ (M7)). M6 modification was the least toxic when compared with other mRNA variants. M6 and M7 RNA modifications can therefore be considered as promising protocols for designing mRNA vaccines. [ABSTRACT FROM AUTHOR]
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- 2021
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16. Mucosal Influenza Vector Vaccine Carrying TB10.4 and HspX Antigens Provides Protection against Mycobacterium tuberculosis in Mice and Guinea Pigs.
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Sergeeva, Mariia, Romanovskaya-Romanko, Ekaterina, Zabolotnyh, Natalia, Pulkina, Anastasia, Vasilyev, Kirill, Shurigina, Anna Polina, Buzitskaya, Janna, Zabrodskaya, Yana, Fadeev, Artem, Vasin, Andrey, Vinogradova, Tatiana I., Stukova, Marina A., Isaguliants, Maria G., and Burt, Felicity Jane
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MYCOBACTERIUM tuberculosis ,GUINEA pigs ,INFLUENZA vaccines ,TUBERCULOSIS ,ANTIGENS - Abstract
New strategies providing protection against tuberculosis (TB) are still pending. The airborne nature of Mycobacterium tuberculosis (M.tb) infection assumes that the mucosal delivery of the TB vaccine could be a more promising strategy than the systemic route of immunization. We developed a mucosal TB vaccine candidate based on recombinant attenuated influenza vector (Flu/THSP) co-expressing truncated NS1 protein NS1(1–124) and a full-length TB10.4 and HspX proteins of M.tb within an NS1 protein open reading frame. The Flu/THSP vector was safe and stimulated a systemic TB-specific CD4+ and CD8+ T-cell immune response after intranasal immunization in mice. Double intranasal immunization with the Flu/THSP vector induced protection against two virulent M.tb strains equal to the effect of BCG subcutaneous injection in mice. In a guinea pig TB model, one intranasal immunization with Flu/THSP improved protection against M.tb when tested as a vaccine candidate for boosting BCG-primed immunity. Importantly, enhanced protection provided by a heterologous BCG-prime → Flu/THSP vector boost immunization scheme was associated with a significantly reduced lung and spleen bacterial burden (mean decrease of 0.77 lg CFU and 0.72 lg CFU, respectively) and improved lung pathology 8.5 weeks post-infection with virulent M.tb strain H37Rv. [ABSTRACT FROM AUTHOR]
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- 2021
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17. Strategies for Immunomonitoring after Vaccination and during Infection.
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Adam, Lucille, Rosenbaum, Pierre, Bonduelle, Olivia, Combadière, Behazine, Isaguliants, Maria G., and Burt, Felicity Jane
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VACCINE effectiveness ,COVID-19 pandemic ,VACCINATION ,COMMUNICABLE diseases ,COVID-19 vaccines - Abstract
Immunomonitoring is the study of an individual's immune responses over the course of vaccination or infection. In the infectious context, exploring the innate and adaptive immune responses will help to investigate their contribution to viral control or toxicity. After vaccination, immunomonitoring of the correlate(s) and surrogate(s) of protection is a major asset for measuring vaccine immune efficacy. Conventional immunomonitoring methods include antibody-based technologies that are easy to use. However, promising sensitive high-throughput technologies allowed the emergence of holistic approaches. This raises the question of data integration methods and tools. These approaches allow us to increase our knowledge on immune mechanisms as well as the identification of key effectors of the immune response. However, the depiction of relevant findings requires a well-rounded consideration beforehand about the hypotheses, conception, organization and objectives of the immunomonitoring. Therefore, well-standardized and comprehensive studies fuel insight to design more efficient, rationale-based vaccines and therapeutics to fight against infectious diseases. Hence, we will illustrate this review with examples of the immunomonitoring approaches used during vaccination and the COVID-19 pandemic. [ABSTRACT FROM AUTHOR]
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- 2021
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18. Development and application of molecular assays for mosquito-borne alphaviruses in South Africa
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Dimaculangan, Micah and Dimaculangan, Micah
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Surveillance of mosquito-borne alphaviruses is critical for the prevention of diseases and the control of outbreaks caused by these viruses, especially with the absence of approved vaccines and antiviral treatments available. Hence, the continual development of rapid and reliable tools for the surveillance of alphaviruses is important. This will aid in the understanding of which viruses are currently circulating with the potential to cause outbreaks. Molecular nucleic acid amplification tests (NAATs), particularly conventional and real-time reverse transcription (RT)-polymerase chain reaction (PCR), are typically employed in epidemiological surveys. In this study, a conventional nested RT-PCR assay was developed to detect alphaviruses in South Africa. In addition, an isothermal amplification technique, specifically a RT-helicase dependent amplification (HDA) assay, which only requires a simple heating device, for instance a heating block, and lateral flow dipsticks/ cassettes for end point detection, was developed to detect alphaviruses currently circulating in South Africa, as an alternative to the RT-PCR assay for application in low resource settings or for field application. The conventional nested RT-PCR assay was able to detect ≥620 copies of RNA compared to the RT-HDA assay which had a minimum limit of detection of 4.8 x 105 copies of RNA. Both assays were tested for theoretical cross-reactivity with other alphaviruses, which include Sindbis virus (SINV) and chikungunya virus (CHIKV) isolates from other regions and genotypes, and isolates from alphaviruses such as Ross River virus (RRV), Barmah Forest virus (BFV), Mayaro virus (MAYV), eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV) and western equine encephalitis virus (WEEV) that are endemic to other parts of world. Alignment of the primers with the sequences of these isolates shows that both assays in theory would be able to detect SINV isolates from northern Europe, tak, National Research Foundation (NRF), Department of Science and Technology (DST), South African Research Chairs Initiative (SARChI), Poliomyelitis Research Foundation
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- 2021
19. Zoonotic diseases in high-risk populations in the Free State province, South Africa
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Van der Westhuizen, Cornelius Gerhardus and Van der Westhuizen, Cornelius Gerhardus
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Zoonotic diseases are infectious diseases transmitted from vertebrate animals to humans and are accountable for more than 60% of all recognized human diseases and 75% of all new or emerging infectious diseases (EID). In South Africa (SA), endemic zoonoses include Mycobacterium bovis (M. bovis), Brucella sp., and Leptospira sp. The prevalence and burden of other pathogens, such as hantaviruses, are unknown. Therefore, identifying high-risk occupations and other risk factors are important for control and preventative measures to decrease the disease burden of these zoonoses. Thus, this study aimed to investigate the incidence rate of M. bovis and Brucella sp. in cattle and farm workers in two different farming communities (communal and commercial), as well as their associated risk factors. This study aimed to document occupational exposure to Brucella sp., Leptospira sp. and hantaviruses across the Free State province, South Africa. Four commercial farms and a rural cattle farming community within the Moqhaka and Ngwathe municipal regions were selected for the purpose of this study. From these farms, sputum and blood specimens were collected from 13 commercial farm workers and 13 communal farm workers. Sputum samples were screened for M. bovis through Mycobacteria Growth Indicator Tube (MGIT) culture. Blood specimens from these 26 farm workers, in addition to 301 archived sera, were screened for Brucella sp., hantaviruses, and Leptospira sp. antibodies using commercially available enzyme-linked immunosorbent assays (ELISA). From the 26 farm workers, no M. bovis was isolated. Out of the 327 sera screened, 35/327 (10.7%) were Brucella sp. IgG positive, 17/327 (5.2%) Leptospira sp. IgM positive, and 38/327 (11.6%) hantavirus IgG positive. A combined total of 321 cattle were screened for M. bovis through tuberculin skin testing (TST); 71 cattle were from communal farms and 250 from commercial farms. Additionally, blood samples collected from 69 and 1793 cattle within the
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- 2021
20. Genetic analysis of human papillomavirus type 11 isolates from patients with recurrent respiratory papillomatosis treated at Universitas Academic Hospital
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Thuynsma, Corne and Thuynsma, Corne
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Human papillomavirus type 11 (HPV11) is a causative agent of recurrent respiratory papillomatosis (RRP), a common benign laryngeal neoplasm that presents mainly in children. The genome comprises three regions: the early region (E1, E2, E4, E5a/b, E6 and E7), the late region (L1 and L2), and the upper regulatory region (URR). A sequence-based classification system is primarily used to genotype HPV. The L1 is used for HPV type discrimination, and in combination with the URR, can be used to differentiate between various lineages. However, optimal sub-lineage classification requires whole genome sequencing (WGS). A recent study investigating the genomic diversity of globally circulating HPV11 isolates identified a novel lineage and two novel sub-lineages. It has been proposed that phylogenetic tree topologies using the sequences of concatenated E5a/b- L1-URR genes, a 208bp segment of the E2 gene, and the complete genome generates similar tree topologies. Also, there is currently no published data on the HPV11 intratypic variants circulating in the Free State region. Hence, this study investigated HPV11 intratypic variants circulating in patients with RRP at the Universitas Academic Hospital, and aimed to identify novel (sub)lineages through phylogenetic investigations. The study population included patients diagnosed with RRP caused by HPV11, and sequence data for geographically distinct HPV11 (sub)lineage representatives. The genetic variation of HPV11 isolated from patients with RRP was determined by sequencing the E5a/b, L1, URR and a segment of E2 genes. Four isolates of interest were selected for whole-genome sequencing and phylogenetically analysed to determine the presence of potentially novel isolates. Many nucleic heterogeneities and non-synonymous substitutions were identified in isolates characterised in this study. Phylogenetic analysis of the concatenated L1-URR and E5a/b-L1-URR resolved into lineages A and B; however, sub-lineage classification was unclear, Poliomyelitis Research Foundation
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- 2021
21. Development of in house assays for detection of Sindbis virus infections
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Kennedy, Nicole and Kennedy, Nicole
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Sindbis virus is a mosquito-borne virus associated with chronic arthritis and is endemic in South Africa. It is the prototype virus for the genus Alphavirus in the family Togaviridae. Sporadic outbreaks occur naturally, often related to heavy rainfall and an increase in mosquito populations. The virus causes a mild disease and hence the exact prevalence in South Africa is not known. In addition, the association of Sindbis virus disease and arthritis is not well documented in South Africa. The aim of this study was to investigate Sindbis virus prevalence and develop serological assays for detection of Sindbis virus infection. An in-house ELISA was developed and optimized. The ELISA was used to screen a total of 165 stored serum samples collected from patients attending a local arthritis clinic in Universitas Hospital, 266 stored serum samples from patients with acute febrile illness, suspected of tickbite fever and with no diagnosis, as well as 136 serum samples from high risk populations (horse and stable workers in Bainsvlei). Production of a recombinant antigen of the Sindbis virus E2 protein for use in immunofluourescence assays (IFA) was attempted. An in-house IFA, prepared with Sindbis virus infected cells, was developed. The positive samples were tested using a commercial immunofluorescence assay (IFA), a neutralisation assay and the in-house IFA. The results indicated that 31/165 samples from patients attending arthritis clinic, 13/136 samples from high risk populations, and 25/266 samples from acute febrile illness patients with no diagnosis tested positive for immunoglobulin G (IgG) Sindbis virus antibodies using the in-house ELISA. Commercial IFA results were as follows: 46/69 samples tested positive, 15/69 samples tested negative and 8/69 samples were indeterminate. A total of 65/69 samples tested positive using the neutralisation assay. Sensitivity for the ELISA and commercial IFA was determined and found to be 100% for the ELISA and 70.7% for the commer, National Research Foundation (NRF), Poliomyelitis Research Foundation (PRF)
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- 2019
22. Innate immune signalling induced by Crimean-Congo haemorrhagic fever virus proteins in vitro
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Viljoen, Natalie and Viljoen, Natalie
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Crimean-Congo haemorrhagic fever orthonairovirus (CCHFV) is a tick-borne viral zoonosis associated with haemorrhagic fever in humans. The World Health Organisation identified CCHFV as a priority pathogen for research. The disease is widespread globally with regions of endemicity in Africa, Asia and eastern Europe; however, the emergence and re-emergence of disease in endemic and non-endemic regions is a cause for concern. Additionally, the lack of rapid point-of-care assays, a vaccine or therapeutic interventions approved for use in humans complicates the control and management of disease, which requires an improved understanding of the virus-host interactions. Viruses have co-evolved with their host/s. Dysregulation of the immune response is a common strategy utilised to evade immune detection and clearance. The innate immune response is a robust non-specific response to infection with the aim of limiting virus replication and spread while activating the adaptive immune response that mediates virus clearance and protection. In this study, innate immune modulation by selected non-structural CCHFV proteins, including the NSM protein and ovarian-tumour like (OTU) protease, and Hazara orthonairovirus (HAZV), a possible model for CCHFV, was investigated. The CCHFV NSM protein, encoded on the M-segment, was expressed in vitro to evaluate the ability of the protein to modulate innate immune signalling. In South Africa, isolates containing an M-segment that are genetically related to other South African isolates (non-reassortant) and isolates containing an M-segment that are genetically similar to Asian isolates (reassortant) have been identified. The CCHFV NSM proteins from both reassortant and non-reassortant CCHFV isolates were evaluated. Despite 92,88% amino acid sequence similarity between the reassortant and non-reassortant NSM protein, the non-reassortant NSM protein downregulated key innate immune markers, including DDX58 (RIG-1), IFNB1, IFNAR1 and STAT1, whereas t, Poliomyelitis Research Foundation (PRF), National Research Foundation (NRF), University of the Free State
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- 2019
23. Identification of arboviruses circulating in mosquito populations in the Bloemfontein area, South Africa
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Terblance, Gert Ignatius du Preez and Terblance, Gert Ignatius du Preez
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Globally there are more than 3 500 different species of mosquito. Many of these are known to be the primary insect vectors of many medically important diseases. Adequate surveillance programs should be put in place to develop effective control strategies and to prevent outbreaks of disease. For a surveillance programme to be effective, mosquito vectors need to be identified accurately. This is done through combining morphological, molecular and environmental data to get more accurate identification results. Currently the diversity of mosquito populations circulating in the Bloemfontein area is not well defined. Mosquitoes were captured from three different sites in the Bloemfontein area. A total of 318 mosquitoes were collected in four different genera. A total of ten different species were identified using morphological identification. Six specimens could only be identified to genus level, because of extensive damage to their external anatomy. Representative specimens were selected from selected species. These included Anopheles squamosus, Culex theileri, Aedes aegypti, Mansonia uniformis and two Aedes subgenus Ochlerotatus species. The Ochlerotatus species include Ochlerotatus harrisoni and Ochlerotatus juppi. DNA was extracted from these mosquitoes and sequenced bidirectionally making use of the barcoding primers, HCO2198 and LCO1490. Anopheles squamosus and Aedes aegypti were identified successfully using the barcoding primers. The primers were less useful for obtaining adequate sequence data for genetic identification of Ochlerotatus spp., Culex theileri and Mansonia uniformis and it is proposed that additional sequence data be obtained subsequent to cloning of fragments. The field caught mosquitoes were sorted and pooled, according to species, capture site and capture date. An RT-qPCR assay was developed to detect Sindbis virus (SINV) using a primer and probe set specifically targeting a region of the nsp2 gene. Another RT-qPCR assay was developed to detect We, National Research Foundation (NRF), Poliomyelitis Research Foundation, University of the Free State
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- 2019
24. The development and validation of a reverse transcription recombinase polymerase amplification assay for detection of flaviviruses
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Bonnet, Elisabeth Hendrika and Bonnet, Elisabeth Hendrika
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Flaviviruses have been of clinical importance since ancient times. Five flaviviruses are known to occur or have been identified historically in South Africa (SA) namely, West Nile virus (WNV), Usutu virus (USUV), Wesselsbron virus (WSLV), Spondweni virus (SPOV) and Banzi virus (BANV). Medically significant flaviviruses, WNV and WSLV, are known to occur annually in SA. Development of isothermal assays, such as recombinase polymerase amplification (RPA), plays an important role in performing surveillance studies and increasing diagnostic capacity for emerging viral pathogens in limited resource settings. In their native form, WNV and WSLV can only be handled in a biosafety laboratory level 3 and this restricts laboratories that lack such resources, hence transcribed RNA controls were successfully prepared for WNV, USUV and WSLV to develop and validate a RT-RPA for the detection of flaviviruses. A lateral-flow RT-RPA was developed by identifying theoretical cross reactivity between the probe and primer candidates by sequence alignments of the conserved NS5 protein of WNV, USUV and WSLV. It was determined that a few mismatches were present between WNV and USUV in the probe binding region and in the reverse primer, as well as between WNV and WSLV, hence different probe and reverse primer regions were identified for WNV/USUV and WSLV. A limitation of the study was the selection of a reference strain of WNV belonging to lineage 1 as a lineage 2 isolate would have been a more suitable representative of SA lineages. Nonetheless, RNA from SA isolate 93/01 was amplified using the RT-RPA. The sensitivity of the assay was determined by diluting RNA control ten-fold, and was found that the WNV RT-RPA could detect WNV and USUV transcribed RNA diluted 109 fold, whereas the WSLV RT-RPA detected WSLV transcribed RNA diluted 1010 fold. Testing RNA from other arboviruses suggested that despite the binding tolerability of the assay there was good specificity as no other arboviruses were, National Research Foundation (NRF), Poliomyelitis Research Foundation (PRF)
- Published
- 2019
25. Epitope-mapping of the glycoprotein from Crimean-Congo hemorrhagic fever virus using a microarray approach
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- 2018
26. Epitope-mapping of the glycoprotein from Crimean-Congo hemorrhagic fever virus using a microarray approach
- Published
- 2018
27. Preparation of recombinant antigen for serological detection of African hantaviruses
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Damane, Deborah Rethabile and Damane, Deborah Rethabile
- Abstract
Unlike other members of the Bunyaviridae family, hantaviruses are transmitted to humans through direct exposure or inhalation of virus contaminated urine or droppings from their reservoir hosts. Hantaviruses were first discovered in 1976 with the identification of Hantaan virus (HNTV) from the reservoir Apodemus agarius in Asia and later in North America. In 2006, Sangassou virus (SANGV) was the first to be isolated in Africa in the African house mouse, Hylomyscus sinus and subsequently followed by the identification of ten more African hantaviruses in both rodent and insectivore hosts through reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence assay (IFA). Hantaviruses are a public health concern with annual cases of disease reported to be approximately 200,000 per year, with most cases reported in Asia. In Africa, disease associated with hantaviruses is not well defined. Culturing the virus and preparing reagents using native virus requires the use of biosafety level (BSL) 3 or 4 laboratories limiting the number of facilities with capability to prepare serological assays. Hence, the use of recombinant antigens that are safe to use in a BSL 1 laboratory that have application as serological tools for surveillance are required. The aim of the study was to develop serological assays to test for antibodies against hantaviruses in human serum samples collected in the Free State, South Africa using a recombinant nucleocapsid protein (NP) of SANGV as a representative of African hantaviruses. Transiently transfected cells were used to prepare antigen slides for IFA and expressed protein was used in an in-house enzyme linked immunosorbent assay (ELISA). In-house assays and commercially available ELISA kits were used to screen human serum samples. There are limited seroprevalence studies performed in Africa to detect IgG antibodies against hantaviruses in humans and no commercial serological assays are available using an African antigen. Hence, it, National Research Foundation (NRF), Polio Research Foundation (PRF), University of the Free State (UFS)
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- 2017
28. Molecular assays for detecting human papillomavirus in head and neck squamous cell carcinoma
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Sekee, Tumelo Robert and Sekee, Tumelo Robert
- Abstract
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide and is traditionally associated with alcohol and tobacco. However over the past few decades there has been a decrease in smoking and drinking but still an increase in incidence of HNSCC with reports across South America, Europe and Asia. The increase in incidence is now attributed to human papillomavirus (HPV), an etiological agent of cervical cancer. HPV belongs to the Papillomaviridae family and over 150 HPV types have been identified. HPVs can be grouped into three groups based on the association with cancer; high risk HPV (HR-HPV) types which are associated with cancer, low risk HPV (LR-HPV) types which are not associated with cancer and possible cancer causing HPV types. Little is known about the association between HPV and HNSCC in South Africa (SA) with few studies conducted in Northern Transvaal and to our knowledge none in the Free State. Additionally, there is no standardized method that can be used for the detection of HPV in HNSCC. Therefore the aims of this study were to investigate molecular assays that can be used for detection of HPV types circulating in the Free State province, SA and to develop a method that can be used to determine transcriptionally active HPV. Three molecular assays were compared by screening for HPV DNA in a total of 74 tissue biopsies from patients with confirmed head and neck tumours. A nested polymerase chain reaction (PCR) that targets part of the L1 region, an E6 multiplex hemi-nested type specific PCR using type specific primers for HPV types -6,-11, -16, -18, -31, -33, -45, -58 and -84 that target the E6 region and the Roche linear array (LA) that target part of the L1 region. To investigate the performance of the Roche LA assay, the PCR targeting the L1 region was repeated on selected samples using modified primers PGMY11/09 and GP5+/6+ (nested PCR). A total of 4/74 (5.4%) samples tested positive for HPV DNA by nested PCR and sequ
- Published
- 2016
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