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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

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

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

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)

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

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

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)

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)

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

English: Yellow fever virus (YFV), a mosquito-borne virus that belongs to the family Flaviviridae and genus Flavivirus, is a significant cause of morbidity and mortality in yellow fever endemic areas, especially in West Africa. In humans, YFV causes yellow fever, a disease characterised by renal failure, jaundice, and/or haemorrhage. The burden of disease is highest in Africa constituting approximately 90% of reported cases worldwide. Despite the availability of highly efficacious live attenuated vaccines against YFV, the estimated prevalence for yellow fever in Africa was 130 000 severe cases and 78 000 deaths for 2013. The available live attenuated vaccines have been contraindicated for use in immunocompromised patients and individuals with hypersensitivity to eggs and/or chicken. Vaccine-associated neurotropic adverse events that result in the development of meningoencephalitis in infants and vaccine-associated viscerotropic adverse events that result in disease resembling wild-type yellow fever have been reported. Vaccine-associated viscerotropic adverse events are associated with fatality rates exceeding 40%. Therefore, there is a need for a safer alternative to complement the use of the available live attenuated vaccines. The aim of this study was to construct a DNA-launched candidate vaccine against YFV and to determine the immunogenicity of the DNA-launched pSinED-lll replicon, which would provide information regarding the applicability of DNA-launched replicons as an approach to vaccine development. The pSinGFP replicon encoding the green fluorescent protein (GFP) was kindly provided by Prof. Mark Heise. Expression of the GFP was confirmed in mammalian cell culture post-transfection with the pSinGFP replicon, thus confirming the functioning of the replicon elements and subsequent expression of the encoded protein. The gene encoding the GFP was excised and replaced with a synthesised codon-optimised gene encoding the YFV ED-lll protein using directional clon, Afrikaans: Geelkoorsvirus (GKV), ‘n muskiet-oordraagbare virus wat behoort tot die familie Flaviviridae en genus Flavivirus, is ‘n belangrike oorsaak van morbiditeit en mortaliteit in geelkoors endemiese gebiede, veral in Wes-Afrika. In die mens veroorsaak GKV, geelkoors, ‘n siekte wat gekenmerk word deur nierversaking, geelsug en/of bloeding. Die las van die siekte is die hoogste in Afrika waar ongeveerd 90% van aangemeldte gevalle wêreldwyd voorkom. Ten spyte van die beskikbaarheid van hoogs effektiewe lewend verswakte entstowwe teen GKV is die beraamde voorkoms van geelkoors in Afrika vir 2013 steeds 130 000 ernstige gevalle en 78 000 sterftes. Die beskikbare lewend verswakte entstowwe is teenaangedui vir gebruik in immuunonderdrukte pasiënte en individue met eier en/of hoenderallergieë. Entstof-verwante neurotropiese newe-effekte wat lei tot die ontwikkeling van meningoenkefalitis/breinvliesontsteking in babas en entstof-verwante viserotropiese newe-effekte wat lei tot siekte wat natuurlike infeksie met GKV naboots is aangemeld. Entstof-verwante viserotropiese newe-effekte word geassosieer met ‘n sterftekoers wat 40% oorskry. Daar is dus ‘n behoefte vir ‘n veiliger alternatief wat die gebruik van die beskikbare lewend verswakte entstowwe kan aanvul. Die doel van hierdie studie was die voorbereiding van ‘n DNS-geloodsde replikon teen GKV en om die immunogenisiteit van die DNS-geloodsde pSinED-lll replikon te bepaal wat insae sal verskaf tot die toepaslikheid van DNS-geloodsde replikons vir entstof ontwikkeling. Die pSinGFP replikon enkodeer die groen fluoresserende proteïen (GFP) en is goedkunstiglik deur Prof. Mark Heise verskaf. Die uitdrukking van die GFP is bevestig in geselekteerde soogdierselle na transfeksie met die pSinGFP replikon en gevolglik was die werking van die replikon elemente en die uitdrukking van die kodeerde proteïen bevestig. Die geen wat die GFP kodeer was verwyder uit die pSinGFP replikon en vervang met ‘n vervaardigde kodon-geoptimiseerde, National Health Laboratory Service Research Trust, National Research Foundation (NRF), Poliomyelitis Research Foundation, University of the Free State, School of Medicine