Theses and Dissertations (Medical Virology)

Permanent URI for this collectionhttp://hdl.handle.net/2263/32536

Browse

Now showing 1 - 20 of 47

Evaluation of molecular and serological assays for improved detection of orthobunyavirus infections in humans and animals in South Africa
(University of Pretoria, 2024) Venter, Marietjie; Mendes, Adriano; mvdwalt89@gmail.com; Van der Walt, Miné
Over the last few decades, there has been a surge in occurrences of emerging and re-emerging infectious diseases, particularly those caused by viruses. To effectively anticipate and respond to future epidemics, rigorous study and attentive surveillance using numerous indicators are required. The vast majority of new human diseases originated in animals with direct zoonotic or vector-borne transmissions. Arthropod-borne viruses are a significant concern due to increased human-animal interactions, new habitats for vectors, and the expansion of geographic ranges through climate change. The recent development of new orthobunyaviruses underscores this issue, with species nearly doubling from 49 in 2011 to 87 in 2019. Orthobunyaviruses, widespread globally, cause mild to severe infections in humans and animals, raising concerns about public health, zoonotic transmissions, and food security. The goal of this study was to evaluate molecular and serological assays for enhanced orthobunyavirus detection in humans, horses, and other animals presenting with febrile and neurological disease symptoms in South Africa (SA). In Chapter 2, an assay for detecting Bunyamwera serogroup viruses was developed and optimised, screening human cases of febrile and neurological disease in Gauteng and Mpumalanga. No positive specimens were detected, emphasising the need for further validation and serosurveys to assess exposure in SA. Chapter 3 describes surveillance of orthobunyaviruses in animals, identifying Shamonda virus (SHAV) in an aborted goat foetus, the first detection in SA. Continuous screening is essential to confirm circulation, assess zoonotic potential, and prevent outbreaks. Chapter 4 characterises the SHAV strain, highlighting challenges in obtaining the full genome sequence and emphasising the need for further research on genetic relationships and zoonotic potential. Chapter 5 investigates Shuni virus (SHUV) epidemiology in horses, revealing significant seroprevalence and emphasising the importance of including serological surveillance. The development and validation of a new SHUV IgG ELISA are discussed, highlighting challenges and the need for broader surveillance to identify potential reservoir hosts. This research underscores the importance of continuous and comprehensive surveillance to manage potential outbreaks and enhance public health responses. In conclusion, the findings of this study contribute significantly to understanding orthobunyavirus epidemiology in SA and highlight the need for ongoing surveillance and assay development.
Establishing a workflow for gastroenteritis virus surveillance, and norovirus whole genome characterisation by next-generation sequencing technologies
(University of Pretoria, 2024-05-27) Mans, Janet; Van Zyl, Walda B; u17043876@tuks.co.za; van Gaalen, Kerri-Anne
Acute gastroenteritis infection is a common illness seen around the world, with most cases being caused by viral pathogens such as rotavirus, norovirus, adenovirus, sapovirus, and astrovirus. Infection causes inflammation of the intestine and stomach, which results in nausea, vomiting, diarrhoea, and dehydration. In most cases, the acute diarrheal disease can resolve itself. However, some infections may lead to significant morbidity and, in extreme cases, death. According to the Centers for Disease Control, viral gastroenteritis infections account for over 200000 deaths per year globally. It is believed that children are potential reservoirs for new emerging viral strains due to their vulnerable immune systems. Thus, it is imperative to do routine gastroenteritis surveillance to allow researchers and vaccine developers to stay up to date with the genetic diversity currently circulating. The norovirus GII.4 genotype has been the predominant strain circulating the globe for the past 20 years, with a new GII.4 variant emerging every few years, allowing it to replace the pre-existing circulating strain. Other genotypes such as GI.5, GI.7, GII.3, GII.6, GII.2, and GII.17 have also been regularly detected in children ( 5 years of age). The lack of complete genome sequence data of non-GII.4 and new emerging GII.4 strains available on the GenBank database illustrates the gap in our knowledge of norovirus diversity and subsequently its epidemiology. In this study, a workflow was established for gastroenteritis virus surveillance in children under the age of five years, and norovirus whole genome characterisation (WGC) by next-generation sequencing technologies. Between January 2022 and July 2023, diarrhoeal stool specimens from hospitalised children < 5 years (115/362) and hospitalised individuals > 5 years (17/362) were screened for five gastroenteritis viruses using the Seegene Allplex™ GI Virus plus assay, which allowed for the detection of adenovirus, sapovirus, rotavirus, astrovirus, and norovirus. From the screening results, adenovirus was the most common virus detected (59%), followed by rotavirus (21%) and norovirus (12%). However, the screening kit used to detect the gastroenteritis viruses screened for all types of adenovirus. Thus, to differentiate gastroenteritis infections from others, a second screening assay targeting adenovirus types F40/F41 needs to be done. In addition, 58 norovirus-positive stool specimens were obtained from a private sector diagnostic laboratory screened for co-infections with the multiplex screening assay. Norovirus GI and GII strains detected were subsequently genotyped based on Sanger sequencing of a ~570 bp region spanning the 3’ end of the RNA-dependent RNA polymerase gene and the 5’ end of the capsid gene. Noroviruses genotypes belonging to genogroup (G)II was more common than genotypes of GI. Genotype GII.4 predominated, being detected in 29.9% of genotyped specimens, followed by GII.17, detected in 16.4%, and GII.1 in 9.1%. In addition to detecting the classic GII.4[P31] Sydney 2012 strain, the recently reported novel GII.4 San Francisco variant was also identified. The GII.4 San Francisco strain was detected more frequently (18.2%) than the GII.4[P31] Sydney 2012 strain (7.6%). Only two GI strains were detected, namely GI.3[P13] and GI.7[P7]. Near complete genome sequencing of selected norovirus strains was achieved by amplifying the genome in 12 overlapping fragments. Both Illumina NextSeq sequencing and Oxford Nanopore singleplex and multiplex sequencing were used. Overall, Illumina sequencing was the most cost-effective method for whole genome sequencing and produced more data than Oxford Nanopore. However, Illumina sequence data contained multiple gaps throughout the sequence. Oxford Nanopore eliminated this limitation which allowed for more certainty of any indels or mutations occurring within the sequence data and better identification. When comparing Oxford Nanopore singleplex and multiplexing sequencing methods, singleplex sequencing is significantly more expensive, however it produces more data than multiplexing. Nevertheless, both Oxford nanopore sequencing methods produce the same quality data. Comprehensively, this study’s results illustrate that norovirus is among the top three causes of viral gastroenteritis infections. The two most common variants detected were GII.4 and GII.17, which had a high percentage identity with other norovirus isolates circulating the globe, including the novel GII.4 San Francisco strain. Viral gastroenteritis surveillance also revealed which viruses were most prominent in children under the age of five years in the Pretoria, Gauteng area of South Africa. The age groups which were most vulnerable to infection, differences between the public and private sector and seasonality. By achieving near-complete norovirus genome sequencings allowed for more accurate identification and will facilitate the management of laboratory diagnostics, identification of structural variants throughout the genome, and contribute knowledge of current stains to norovirus vaccine development, prevention and disease management strategies.
Establishment of serological assays to investigate the incidence of West Nile and Wesselsbron flavivirus infections in Africa
(University of Pretoria, 2023) Venter, Marietjie; Mendes, Adriano; clourens101@gmail.com; Lourens, Carla
The burden of WNV and association with neurological disease was identified through syndromic surveillance in animals and humans using available serological tools. WSLB virus was identified in two samples (an equine and a bird) in South Africa for the first time since 2008. Additionally, ELISA’s were developed testing for IgG and IgM antibodies in equines, humans, cattle, and a species-independent assay for WNV and WSLB using the NS1 antigen, which differs from the commercial ELISA’s available that targets the E protein. The human WNV IgM and IgG ELISA showed very promising results, while the equine WNV ELISA’s had quite high background and this could be due to the different target antigen or the use of a baculovirus expressed protein with commercial antibodies. The cattle WNV IgM ELISA also showed good results and can possibly be implemented in the lab. The WSLB ELISA’s were only developed and optimized with rabbit sera, since a clinical sample lacked neutralizing antibodies for use in the assay. Unfortunately, the baculovirus expression of the NS1 protein was unsuccessful.
Small-scale movements and foraging areas of Rousettus aegyptiacus in Limpopo Province, South Africa
(University of Pretoria, 2023) Markotter, Wanda; De Vries, Low; Epstein, Jonathan; u16130805@tuks.co.za; Wood, Matthew Roger
Movement is an integral component within the animal kingdom as species respond to internal and external stimuli to ensure their survival. As animals move, they interact with the environment in different ways that help to maintain ecological connectivity. These species interactions are often mutually beneficial such as pollination and seed dispersal. However, there are cases where the interactions between different species have negative implications. The emergence of medically important zoonotic and potentially zoonotic pathogens worldwide has increased over the past 50 years and emphasis has recently been placed on investigating the link between the movement patterns of a host species and potential disease spillover risk. Bats have been recognized as important hosts and especially viral hosts. However, the ecological context of bats as viral hosts is relatively poorly understood and consequently, an important component is often lacking from assessments of viral transmission risk. Bats exploit a wide array of different habitat types but the likelihood for contact between bats and people is increasing as humans encroach on their natural habitats. As such, studying the movements of bats may help understanding their potential role in spillover events by identifying the risk of contact with people or other potentially susceptible host species. This research study aimed to integrate virological and ecological data to provide a better understanding of transmission risk. Specifically, the ecological aspects of bat movement patterns were assessed alongside virological data to improve our understanding of the potential role bats may play in viral transmission events. Bats exhibit wide forms and functions and consequently, their movement patterns may vary according to their specific requirements. There are, however, metrics such as home range size estimations that can be used as a benchmark to assess how the different ecological and biological characteristics of bats are linked to movement patterns. Understanding which traits influence bat movement patterns and how they are linked may enable predictions about a species’ range requirements and provide insight into the potential scope of their species interactions. Similar previous assessments have been performed but with limited sample sizes and a restricted geographical range. Expanding the scope to include bat species around the globe will provide greater species representation with a wider diversity and encompass a broader array of ecological and biological traits. The findings can then be applied as a theoretical framework for species range requirements and movement patterns. This is important for data deficient and threatened species where not much is known about their range requirements. Moreover, it is especially relevant for assessing a species’ interactions with the environment such as seed dispersal or, in the case of zoonotic hosts, potential pathogen spread and transmission. Through a global meta-analysis, specific characteristics of bat species were identified that have a clear relationship with bat home range size. At a local scale, the movement patterns of a known zoonotic viral host, Rousettus aegyptiacus (Egyptian rousette bat), were assessed in the rural village of Fertilis, Limpopo Province, South Africa across a seasonal gradient to determine risk of contact with a human population. The R. aegyptiacus colony inhabits Matlapitsi cave which is within 500m of human dwellings and was specifically selected due to previously being shown to host several zoonotic viruses or viruses with zoonotic potential. The bats were tracked with radiotelemetry for between seven and nine nights every month over a 12-month period to determine their movements which were then compared to human presence. Their movements were further assessed relative to fruit availability that was estimated as the percentage of fruit cover on fruiting trees distributed around the study site. These comparisons were performed throughout the year to assess whether fruit availability influences movement patterns across a seasonal gradient. There were distinct seasonal differences in movement patterns with bats preferentially foraging within residential areas during the dry winter months when subsistence fruit trees provide constant sources of food. These findings are important as the peak in bat activity within residential areas coincides with previously identified periods of high risk of viral shedding. The findings, therefore, suggest that the risk of spillover during this period is high and highlight the importance of incorporating ecological data with virological data for more detailed risk assessments.
Assessment of Bunyavirales activity and circulation in humans, livestock, and peri-domestic rodents in diverse ecologies in Kenya, 2020-2022
(University of Pretoria, 2023) Venter, Marietjie; Sang, Rosemary; Tchouassi, David P.; u19398809@tuks.co.za; Omoga, Dorcus Caroline Achieng
Viruses in the order Bunyavirales are diverse and include zoonotic arthropod- and rodent-borne viruses that cause diseases ranging from mild febrile illnesses to haemorrhagic and/or encephalitis fevers and even death in animals and humans. These viruses contribute significantly to emerging and re-emerging infection threats worldwide as well as neglected tropical diseases as they mainly affect the impoverished and have a long-lasting effect. In tropical and subtropical countries, mainly in Africa, more than 90% of human cases are either undiagnosed or misdiagnosed and treated as other common endemic diseases like malaria. Furthermore, many studies have focused on common arboviruses transmitted by mosquitoes such as the flaviviruses (e.g., yellow fever, Dengue, Chikungunya, Zika viruses), among others, as well as well-known members of the Bunyavirales associated with human outbreaks (Rift Valley Fever virus (RVFV) and Crimean Congo Hemorrhagic Fever virus (CCHFV)). Less known yet potentially harmful arboviruses in the order Bunyavirales are mostly ignored despite being zoonotic or having zoonotic potential with the risk of spreading globally. Despite the associated public and veterinary health, social, and economic importance, the impact of these diseases is largely undetermined due to paucity of active surveillance, poor disease reporting systems, and lack of appropriate diagnosis in affected regions. The prevalence, burden of disease and distribution of most members of the Bunyavirales remains unknown in African countries where they are endemic. Surveillance is thus essential to determine their importance, provide an early warning of their presence, and guide intervention measures to prevent outbreaks. This thesis instituted “One Health” surveillance in selected pastoralist communities of Kenya (Kajiado and Baringo counties) to assess the circulation and viral activity of Bunyavirales among peridomestic rodents, livestock, and humans presenting with febrile illness. Serum samples were analyzed serologically to determine the presence of Bunyavirales neutralizing antibodies as well as culture and molecular methods (RT-PCR and whole genome sequencing and phylogenetic analysis) to isolate and characterise known and novel viruses, respectively using methodologies described in chapter 2. In Chapter 3, we report the findings of the screening experiments including the seroprevalence of RVFV, CCHFV, Ngari virus (NRIV), Ntepes virus (NTPV) and Bunyamwera virus (BUNV) as well as the molecular detection of CCHFV, NTPV; NRIV, BUNV, hantavirus, Shamonda virus (SHAV) and some uncharacterised phleboviruses in livestock and peridomestic rodents. Further characterisation of the most important viruses detected is reported in subsequent chapters (4-7). One of the major findings reported herein includes the isolation and circulation of Ngari virus (NRIV), known to cause outbreaks of haemorrhagic fevers in humans and small ruminants, in apparently healthy cattle, sheep and goats (Chapter 4). Seroprevalence of neutralizing antibodies to the virus ranged 19-52% with growth kinetics on different cells showing efficient replication in cells from sheep and humans and Aedes albopictus but weakly on goat cell lines. Phylogenetic analyses of complete-coding sequences of L, M and S segments of four NRIV isolates showed that the Kenyan viruses clustered with a monophyletic clade that is most closely related to a NRIV sequence from a small ruminant from Mauritania. This is the first detection of NRIV in livestock in Kenya. In Chapter 5, active detection of a Nairovirus, Crimean-Congo haemorrhagic fever (CCHF) virus RNA is demonstrated in sheep and rodent sera as virus exposure is serologically confirmed in these hosts and, cattle, goats, and humans. This virus is the causative agent of CCHF, a fatal viral haemorrhagic fever disease in humans. Among livestock, seroprevalence was lowest in goat (8.1%) and highest in cattle (14%). Phylogenetic analyses of partial sequences of the S segment generated from rodent and sheep revealed a high level of nucleotide sequence identity (96-98%) and a close relationship to other pathogenic strains in the CCHFV Africa 3 lineage. We also report for the first time in Kenya the detection of shrew-borne hantaviruses in Somali shrews (Crocidura somalica) from Baringo county (Chapter 6). The detected hantaviruses closest relative is an African shrew- borne hantavirus, Tanganya viruses (TGNV) with nucleotide identity of 72-80%. Finally, through virus isolation and Next Generation Sequencing (NGS) of samples collected in this study, a novel orbivirus was isolated from cattle which is described in Chapter 7 as an additional finding of this thesis. This followed initial isolation in cell culture from the serum of a clinically sick cow aged 2-3 years, presenting signs of emaciation and lethargy. High throughput sequencing revealed the typical orbivirus genome architecture with ten double-stranded RNA segments and a total size of 18,731 bp. A further detection of Kaptombes virus (KPTV) by RT-PCR in three cattle samples and 5% seroprevalence of neutralizing antibodies to the virus, demonstrate its active circulation. This shows the strength of sequence-independent virus discovery methods to discover new pathogens in surveillance study. Taken together, the findings have provided data on pathogenic bunyavirus prevalence in the two semi-arid regions useful for understanding the virus transmission networks as well as the detection and characterisation of a novel orbivirus. The data herein can be applied in animal health surveillance systems locally to ensure timely and appropriate detection and control as a means of active and continuous animal surveillance and as an early warning sign for zoonotic diseases emergence.
Circulation, reassortment and transmission of Ngari and Bunyamwera viruses in Northen Kenya
(University of Pretoria, 2015) Venter, Marietjie; Swanepoel, Robert; Sang, Rosemary; odhiamboc@hotmail.com; Otieno, Odhiambo Collins
Kenya has experienced severe arboviral outbreaks of public health concern in the recent past, including yellow fever (YF), Crimean Congo hemorrhagic fever (CCHF), chikungunya, and Rift Valley fever (RVF) among others. Most of these infections are under diagnosed and hence neglected due to non-specific nature of their symptoms. Often they are mistaken for endemic tropical diseases such as malaria and typhoid infections and are only recognized during major outbreaks which result in adverse public health and economic consequences to the affected communities. Ongoing inter-epidemic surveillance in RVF virus hotspots in Kenya has indicated continued intense transmission of Bunyamwera virus (BUNV) in the absence or under low level activity of RVF virus. BUNV belongs to the genus Orthobunyavirus of the family Bunyaviridae. These are segmented RNA viruses whose members have the potential for genetic reassotment and/or drift. Recently, Ngari virus (NRIV), a natural reassortant virus associated with hemorrhagic fever was documented to have emerged from BUNV, which previously was not associated with such symptoms. However, the vectors that are involved in the maintenance and transmission of BUNV and NRIV are diverse and their role in virus maintenance/dynamics is poorly known. It is thus important to investigate the dynamics of BUNV and NRIV in selected transmission foci in an effort to understand their circulation better in order to be able to control and predict outbreaks. In this study, we determined the evolutionary and phenotypic diversity of BUNV and NRIV isolates previously obtained from vectors in parts of Kenya. We have provided genetic sequences of two BUNV and three NRIV isolates which contribute to addressing paucity of genetic sequences associated with this group of viruses. Phylogenetic analysis of these sequences in addition to other sequences in GenBank revealed evidence of geographic/temporal clustering that requires further investigation. Next, we demonstrated that plaque purified phenotypes of selected BUNV and NRIV isolates differ in in vivo growth kinetics and pathogenicity in mice, possibly related to specific mutations within the genome. The phenotypic changes and identification of mutations possibly associated with these changes support further investigation of specific mutations using site directed mutagenesis. In addition, we determined the competence of some of the mosquito species implicated in their transmission, Anopheles gambiae, Aedes aegypti and Culex quinquefaciatus and evaluated the dynamics of their transmission in these vectors. We conclude that Anopheles gambiae is likely a more competent vector for NRIV than Aedes aegypti and is a moderately competent vector for BUNV, which has implications for animal movement in malaria endemic areas where the vector is present. We also report evidence of BUNV transovarial transmission in both Aedes aegypti and Anopheles gambiae with the prevalence of transmission related to the ovarian cycle. Finally, we determined the level of human exposure to these viruses in the transmission foci. Orthobunyavirus-specific antibodies were detected by plaque reduction neutralization test in 89 (25.8%) of 345 persons tested. Multivariable analysis revealed age and residence in North Eastern Kenya as risk factors. In conclusion, we propose that acute febrile disease surveillance needs to be implemented in North Eastern Kenya. This study helps identify the virus strains/populations and the vector species that play a critical role in sustenance and transmission of BUNV and NRIV in different ecosystems in the country. All these are important in understanding virus circulation, potential for emergence and risk to populations in areas of circulation, and will help in making decisions for intervention and management. Generated sequence data in this study contributes to global phylogenetic characterization of Orthobunyaviruses worldwide and their molecular epidemiology. The study also shed light/improve our knowledge on the genetic stability or diversity and evolutionary trends of Orthobunyavirus strains in Kenya.
Occurrence and diversity of tick-borne viruses in the pastoral eco-zone of Ijara District, North Eastern Province of Kenya
(University of Pretoria, 2013-10) Venter, Marietjie; Sang, Rosemary; wesulaolivia@gmail.com; Lwande, Olivia Wesula
Most arbovirus isolations in East Africa have been recorded from mosquitoes but less information is available about tick-borne viruses including Crimean-Congo hemorrhagic fever virus (CCHFV), which is prevalent in Africa with a mortality rate of up to 40%. The study envisioned gaining an in-depth understanding of the circulation, transmission and diversity of tick-borne viruses in Ijara District of North Eastern Kenya, a pastoral ecozone with a defined population size of 19,259 that are served by 8 health facilities where arbovirus activity among mosquitoes, animals and humans is frequently reported. The study aimed at determining the prevalence of CCHF antibodies in humans that attend selected health facilities in Ijara District, identify tick-borne viruses among tick vectors and genetic diversity of the tick-borne viruses circulating among ticks and/or their host animals. A total of 517 human serum samples were screened for the presence of IgG and IgM antibodies to CCHF using CCHF-IgG and IgM ELISA test kits (VectoCrimean-CHF-IgG and IgM ELISA; Vector-Best, Novosibirsk, Russia). A multivariable logistic regression model was used to investigate the exposure to CCHFV in patients enrolled in this study. In this first part of the study, a single patient tested positive for anti-CCHF IgM, while 96 were positive for anti-CCHF IgG, suggesting an overall seroprevalence of CCHFV in Ijara District of 19%. The results indicate the possibility of acute CCHFV infections occurring without being detected in this population. A total of 10,488 ticks were also sampled from livestock and wild animal hosts and processed in 1,520 pools of upto 8 ticks per pool. The sampled ticks were classified to species, processed for virus screening by cell culture using Vero cells, RT-PCR, and sequencing. Bunyamwera (BUN), Dugbe (DUG), Ndumu (NDU), Semliki forest (SF), Thogoto (THO), and West Nile (WN) virus strains were isolated and identified. Phylogenetic analysis based on nucleotide sequences showed that the Kenyan isolates clustered closely to their respective reference strains. Semliki forest virus (SFV) isolate (ATH00510) sequence obtained by 454 sequencing, clustered closely to the Kenyan strains (HQ848388 and JF972635) isolated from mosquitoes sampled in North Eastern Province of Kenya. Ndumu virus (NDUV) (ATH002166) was similar to the Ugandan strain (JN989958). All BUNV strains isolated in the study clustered distinctively on a tree branching comprising viruses of Bunyamwera serogroup. The two Dugbe viruses detected in this study were highly similar to each other and formed a cluster with the United Kingdom (NC004159 and U15018) strains. Thogoto virus was divergent from strains obtained from Germany, Senegal, Turkey, USA and Vietnam. WNV strains isolated in Kenya clustered relatively close to viruses isolated in Russia, Europe and the United States belonging to lineage 1 of WNV. The identification of WNV Lineage 1 in Ijara District brings in to light the ability of this virus to spread across wide geographical regions taking into consideration that this virus lineage is also found in Europe, America, India and the Middle East. These study findings provide additional evidence on the potential role of ticks and animals (both livestock and wildlife) in the circulation of tick-borne viruses as well as viruses previously known to be mosquito-borne. It also provides a basis in understanding the genetic diversity of arboviruses circulating in Ijara District.
Assessment of selected environmental water samples in Kenya for the presence of clinically important enteric viruses
(University of Pretoria, 2013) Taylor, Maureen B.; nick.kiulia@gmail.com; Kiulia, Nicholas Mukaria
Worldwide it is estimated that 1.1 billion people drink unsafe water and 1.7 million deaths are caused by drinking of unsafe water and poor sanitation. Enteric viruses are transmitted by the faecal-oral route and their spread has in part been attributed to the consumption of contaminated food and drinking water. Viruses are a major cause of waterborne disease but the health impact of waterborne viral infections is underestimated. Waterborne illness associated with viruses are more common than those caused by bacteria making it important to estimate the prevalence and diversity of enteric viruses in environmental water sources in order to assess the potential public health risks posed by the contaminated water sources. In Kenya, despite these risks data regarding the occurrence of the viruses in environmental water sources is limited, and in addition, data on the prevalence and molecular epidemiology of enteroviruses (EVs) and noroviruses (NoVs) in the clinical and environmental settings is lacking. Therefore, in this investigation the microbiological quality, the prevalence and molecular epidemiology of selected clinically relevant enteric viruses namely, EVs, NoVs and rotaviruses (RVs) in Kenyan urban and rural high risk water sources, namely the Mboone, Mutoine and Nairobi rivers and water from the Dadaab refugee camp, were investigated. Microbial indicator organisms, namely Escherichia. coli and total coliforms were detected at levels >200 cfu/mℓ in 100% (40/40) of the samples thereby exceeding the World Health Organization (WHO) recommended guidelines for drinking water (<1 most probable number/100 mℓ), suggesting that there is gross faecal contamination in these Kenyan water sources. The RVs were detected in 85% (34/40) of the samples and it was the most predominant virus detected being identified in 83% of the Mboone river samples, 100% of the Mutoine river samples, 83% of the Nairobi river samples and 50% of the household water from the Dadaab refugee camp. The G types detected were G1 and G9 and mixed G1+G9 being detected in the Nairobi river. The P types detected were, P[4], P[6] and P[8], with mixed P types P[4]+P[6], P[4]+P[8], P[6]+P[8] and P[4]+P[6]+P[8] being detected in the three surface water sources. Noroviruses were detected in 63% (25/40) of the selected water samples, with 60% (24/40) of the samples positive for NoV GII and 20% (8/40) for NoV GI. A number of genotypes were identified, namely, NoV GI.1, NoV GI.3 and NoV GI.9 and NoV GII.4, GII.6, GII.12, GII.16 and GII.17 with NoV GII.17 predominating. Enteroviruses were detected in 58% (23/40) of the samples by direct real-time reverse transcriptasepolymerase chain analysis of the recovered virus concentrates and were identified in the three rivers (Mboone, Mutoine and Nairobi rivers) accounting for 50%, 83% and 58% of water samples, respectively, but not in the borehole or household water from Dadaab. No polioviruses (PVs) were identified using the recommended WHO methods designed for identification of PVs and differentiation between vaccine-derived PVs and wild-type PVs in clinical specimens. In conclusion, this is the first comprehensive report on the molecular epidemiology of NoVs in Kenyan water sources. From this study it is evident that further nationwide studies are necessary to fully establish the prevalence, distribution and clinical relevance of enteric viruses in Kenyan water sources.
The development and application of multiplex real-time reverse transcriptase polymerase chain reaction assays for the detection of enteric viruses on berry fruits and in water samples
(University of Pretoria, 2013) Taylor, Maureen Beatrice; gabyderidder@gmail.com; De Ridder, Gabriel Adriaan
The transmission of enteric viruses by food, food products and water remains a wellrecognised, largely underestimated widespread public health problem. Outbreaks of gastroenteritis and hepatitis A due to the consumption of contaminated berry fruits have become a growing phenomenon worldwide. Contamination of fresh produce and other minimally processed foods can be attributed to pre- and post-harvest irrigation and washing water and food handlers. The prevention of such outbreaks relies on the optimisation of adequate methods for the recovery and detection of enteric viruses from food matrices and irrigation water. The aim of this study was to develop and apply optimised multiplex real-time reverse transcriptase-polymerase chain reaction (rt RT-PCR) assays for the detection of selected enteric viruses on berry fruits and in paired associated irrigation waters. In this study quality control measures were implemented by the development and optimisation of an internal amplification control (IAC) for norovirus (NoV) GII to monitor for the success of the amplification process. Mengovirus was used as a process control to validate the recovery and nucleic acid extraction of selected enteric viruses from strawberries and in associated irrigation waters. Three multiplex rt RT-PCR assays for the detection of NoV GI, NoV GII, sapovirus , hepatitis A virus, human astrovirus, human rotavirus and mengovirus were optimised with the IAC. Blackberries and strawberries were used to assess the efficiency of three nucleic acid extraction kits with the most efficient used in further investigations. Three elution buffers based on protein concentration, pH, Tris and elution period were assessed for the recovery of the viruses from the berry fruits. The pH more so than the protein concentration proved to be more effective in the recovery of the selected enteric viruses from the strawberries with no analytical significant differences noted for the two 3% glycine-beef extract (GBE) buffers assessed, irrespective of the parameters considered. During the period September 2010 to August 2011, strawberries and associated irrigation water were collected from which NoV GII, NoV GI and HAV could be recovered using a 3% tris-GBE pH 9.5 elution buffer and a glass-wool absorption elution method, respectively, and detected using optimised singleplex rt RT-PCR assays. The irrigation water samples together with eight surface and three groundwater samples collected from the Limpopo area was retested using the optimised multiplex rt RT-PCR assays. The multiplex rt RT-PCR assays proved to be more efficient in the detection of NoVs than the commercial environmental rt RT-PCR assays with lower detection efficiencies noted for HAV. Commercially obtained strawberries were dipped in polluted surface water, the viruses recovered from both and detected using the optimised multiplex rt RT-PCR assays resulted in the detection of similar viruses on both the strawberries and polluted irrigation water. Norovirus GII.7 and swine NoV GII.18 were identified on the strawberries and in the associated irrigation water, respectively. This is the first report of swine NoVs in South Africa, and begs the question as to the possibility of zoonotic NoV infection. This link between the viruses detected on the surface of the strawberries and in the irrigation water could not be confirmed by typing data. From this study, a functional AC was developed and used in the development and optimisation of three multiplex rt RT-PCR assays which made the gathering of new data of the role of irrigation water as a source of contamination of irrigated berry fruits in SA possible.
Efficiency of glass wool adsorption-elution technique for the recovery of enteric viruses from water
(University of Pretoria, 2013) Taylor, Maureen B.; vruhanya@yahoo.com; Ruhanya, Vurayai
One of the major obstacles to human health relates to unsafe water and poor sanitation. Faecal contamination of source and drinking water introduces enteric pathogens which result in disease outbreaks. Therefore monitoring the occurrence of human pathogens in source water and drinking water is necessary in order to limit the prevalence of environmentally transmitted infectious diseases. Knowledge of pathogen loads in source waters provides the basis for establishing treatment requirements and health standards stipulated by water regulatory authorities and assists in determining the efficacy of water treatment plants. Water quality monitoring and public health assurance is performed routinely by enumerating faecal indicator bacteria. Studies have demonstrated that there is no relationship between current bacterial indicator detection and the presence of enteric pathogenic viruses in treated and source water. There is therefore a need to monitor the levels of pathogenic enteric viruses in surface waters, irrigation water, sewage effluent as well as treated drinking water for public health safety and quality assessment. However due to the low concentration of viruses in water matrices and presence of inhibitors, efficient concentration methods from large quantities of water are essential. The analysis of water for enteric viruses is a two stage process: the first step is to apply efficient viral recovery and concentration procedures from large volumes (10–1000 ℓ) of water followed by viral detection. Glass wool adsorption-elution is a cost-effective and practical viral recovery method for use in resource-limiting settings. The main objective of this study was to determine the efficiency of the glass wool adsorptionelution method for the recovery of viruses of different genera from large water samples (10 ℓ) of different quality by a step-by-step evaluation of its performance using seeding experiments. Standard curves were prepared using quantitative reverse transcription-polymerase chain reactions (RT-PCR)(for RNA viruses) and PCR (for DNA viruses). The efficiency of recovery (EOR) of glass wool between tap water and turbid surface water was compared for six enteric viruses by examining the recovery and loss of viruses at each stage of the process. The generalised linear statistical model was applied to compare the EOR of each virus in each water type and results clearly indicated that the EOR varied for each virus type and was higher for tap water than for turbid surface water for each virus. There was extensive loss of virus in the flow through and this was also higher for the turbid water than the tap water. In this study it was also demonstrated that mengovirus behaved similarly to the pathogenic enteric viruses and was therefore a suitable process control to monitor viral recovery and nucleic acid extraction when recovering and detecting enteric viruses from environmental matrices using glass wool adsorption method. It was also demonstrated that EOR of glass wool for turbid surface water was underestimated as the poor sample quality affected the quantitative molecular detection assays. Adenovirus was shown to be a suitable indicator for virus contamination of water. Modification of the glass wool column preparation did not result in significant difference in EOR but an increase in the amount of glass wool used resulted in reduction in EOR. There were no significant differences between the two polyethylene glycol/sodium chloride (PEG6000/NaCl and PEG8000/NaCl) precipitation methods applied to the secondary concentration of the viruses, but it should be noted that the former has the disadvantage of overnight incubation. The EOR of glass wool was shown to be influenced by pH of the sample. The optimal sample pH for the recovery of hepatitis A virus in turbid surface water was pH 6.0. The study provides valuable new data on the EOR of enteric viruses using the glass wool adsorption-elution technique where virus quantities could be traced from seeding to detection by molecular-based methods.
Molecular characterisation of hepatitis A virus strains from clinical and environmental sources in South Africa
(University of Pretoria, 2013) Taylor, Maureen B.; Wolfaardt, M.; saidarachida19@yahoo; Said, Rachida
Hepatitis A virus (HAV) is the most common cause of acute viral hepatitis worldwide. Hepatitis A virus is classified in the genus Hepatovirus of the family Picornaviridae. The virion has a 7.5 kb positive-sense single-stranded RNA genome and only one serotype of the virus has been identified so far. There are six genotypes of HAV distinguished by nucleotide sequence analysis of the VP1 region. Genotypes I, II and III, each with their subdivisions A and B, infect humans. Genotypes IV, V and VI infect non-human primates. These genotypes have a distinct geographical distribution. In 1997, it was established that genotype IA and IB prevail in South Africa (SA) with IB being predominant. However, no recent data have been published on the types of HAV circulating in SA even though the virus was detected in surface water sources and on fresh produce at the point of retail. There is an effective vaccine for the prevention of hepatitis A, but recent publications have reported the possible emergence of antigenic escape mutants of HAV. The aim of the study was therefore to establish which genotypes and possibly variants thereof of HAV are circulating the community compared to the genotypes present in water and food sources. To identify the different strains of HAV circulating in SA, detection and characterisation of HAV strains was performed on clinical and environmental sources. A total of 117 clinical specimens and 80 water samples (irrigation, surface and wastewater) and 20 fresh produce (tomatoes, lettuce, cabbage) samples were analysed. Samples were collected in SA and neighbouring countries. Hepatitis A virus was detected in 78% of the clinical specimens and in only 46% of the water samples. None of the fresh produce analysed in the study tested positive for HAV even though some of the produce were irrigated with HAV contaminated water. Molecular characterisation of the detected strains revealed the presence of genotype IB in SA. The SA strains have a unique nucleic acid sequence, namely a R298K mutation within the VP1 region and R71S substitution within the VP1/P2B junction, which distinguishes them from strains detected in the rest of the world and even in neighbouring countries. The characterised strains were also analysed for the presence of previously isolated antigenic escape mutants and/or novel ones. One possible vaccine escape mutant, with a I179T substitution next to the neutralisation residue Y181 in the VP1 region, was identified. A variety of strains with mutation(s) at or around the neutralisation sites were also identified as well as circulation of quasispecies among clinical HAV strains. The study revealed the circulation of a potential new HAV genotype among the SA simian population. The data provided by the study stresses the need to implement a proper surveillance system of the circulation of HAV strains in SA.
Early infant HIV diagnosis and characterization of HIV drug resistance in Gauteng, South Africa
(University of Pretoria, 2021-01) Lukhwareni, Azwidowi; Rossouw, Theresa; u15187030@tuks.co.za; Smit, Odette
Despite the high prevalence of the human immunodeficiency virus (HIV) in South African women of reproductive age, the South African (SA) Prevention of Mother-to-Child Transmission (PMTCT) programme has significantly reduced the incidence of new HIV infections in infants from >20% in 2004 to <2% and overall MTCT approximately >5%. The PMTCT programme, however, faces challenges in terms of early infant diagnosis (EID) because of HIV polymerase chain reaction (PCR) indeterminate results as well as HIV drug resistance (HIVDR) secondary to antiretroviral therapy (ART) exposure of mothers and infants. The National Health and Laboratory Services (NHLS) uses the Roche COBAS®AmpliPrep (CAP)/COBAS®TaqMan® (CTM) HIV-1 Qualitative Test (Roche Molecular Systems, Pleasanton, CA) (CAP/CTM) platform as part of EID. Recently, the Roche cobas® 6800/8800 System has been introduced to test HIV viral load and HIV DNA PCR for EID. The platform is already processing samples for HIV viral load; however, verification for HIV DNA PCR for EID with dried blood samples (DBS) is needed, especially for CAP/CTM HIV PCR indeterminate results (Cycle threshold [Ct]>33 with any relative fluorescent intensity [RFI] value or Ct≤33 and RFI <5 on the CAP/CTM, Ct>38 on the Roche cobas® 6800/8800 System). In addition, HIVDR in newly diagnosed infants significantly limits treatment options. Therefore, the current study verified the Roche cobas® 6800/8800 System against the CAP/CTM system for the detection of HIV in EID and determined the HIVDR prevalence and profiles in infants <6 months, and how this affects current SA PMTCT and EID guidelines. The study comprised 642 DBS samples (235 HIV PCR positive, 193 HIV PCR negative and 214 HIV PCR indeterminate) previously tested on the CAP/CTM assay. Overall, 99.6% (234/235) CAP/CTM HIV PCR positive samples remained positive, while 99.5% (192/193) HIV PCR negative samples remained negative with the Roche cobas® 6800/8800 System. The HIV PCR indeterminate results as detected by the CAP/CTM decreased from 100% (214/214) to 8.4% (18/214) with the Roche cobas® 6800/8800 System. The Roche cobas® 6800/8800 System had a specificity of 99.5% and a sensitivity of 99.6%, but this decreased to 96.3% and 90.8% when HIV PCR indeterminate results were included. The kappa value increased from 0.5, which signifies moderate agreement, to 0.9, which is excellent agreement, when RFI from the CAP/CTM was excluded for result determination. The overall agreement between the two assays, taking only cycle threshold values into account, was 93.8%. As for HIVDR, mutations were detected in 42.9% (24/56) of infants <6 months. The most common non-nucleoside reverse transcriptase inhibitor (NNRTI) mutation causing high-level resistance was K103N (21.4% [12/56]), followed by Y181C and the NRTI mutation, M184V, both in 8.9% (5/56) of infants. Also, major protease inhibitor (PI) mutations, M46L and V82A were detected in one case each (1.8%). In conclusion, the performance of the Roche cobas®6800/8800 System was comparable to the CAP/CTM; however, it detected fewer HIV PCR indeterminate results, thus potentially offering conclusive results in a larger proportion of infants. The detection of high levels of the NNRTI mutation, K103N, emphasises the need for constant surveillance since nevirapine is included as part of the SA PMTCT guidelines and the World Health Organization recommends that NNRTIs should be phased-out of as part of PMTCT once the resistance prevalence exceeds 10%.
Molecular characterisation of human adenoviruses from environmental samples in Tshwane, Gauteng
(University of Pretoria, 2020) Van Zyl, Walda B.; Mans, Janet; u14105447@tuks.co.za; Davids, Michaela
Human adenoviruses (HAdV) are non-enveloped viruses with an icosahedral capsid and a linear double-stranded DNA genome. These viruses belong to the family Adenoviridae and genus Mastadenovirus. An important property of the HAdV is that it is non-enveloped making it highly resistant to detergents and harsh environmental conditions. This virus is grouped in seven species (A-G) with more than 88 genotypes. These seven species are associated with several diseases, such as, respiratory infections, keratoconjunctivitis, urinary infections, hepatitis and gastrointestinal infections. The HAdV is one of the etiological causes of acute gastroenteritis, mainly caused by HAdV-F40 and HAdV-F41. The virus can be transmitted via the faecal-oral route, inhalation of respiratory droplets and direct contact with contaminated environments. The virus is known to be ubiquitous in environments where human contamination is likely to occur such as wastewater treatment plants. These human contaminations could occur through contaminated secretion and excretions within aqueous environments. There is currently a limited amount of information on the HAdV in water Molecular characterisation of human adenoviruses from environmental environments, particularly in Tshwane, Gauteng. Therefore, the aim of the study was to investigate the presence and genotypes of human adenovirus in environmental samples namely raw sewage and treated effluent, using molecular methods. For genotypic characterisation, Sanger sequencing was used on amplicons from 12 HAdV positive samples and next generation sequencing were used on all the amplicons from HAdV positive samples. A total of 150 environmental samples (75 raw sewage and 75 effluent) were collected from two wastewater treatment plants in Tshwane over the study period of 18 months. These environmental samples comprised of 1 L raw sewage and 10 L treated effluent samples. The primary viral recovery for the 1 L raw sewage and 10 L treated effluent samples were performed using skimmed milk flocculation procedure and glass wool adsorption elution technique, respectively. For secondary viral recovery, both environmental samples were subjected to polyethylene glycol/sodium chloride precipitation. Manual extraction was used to extract the nucleic acids from the virus concentrate with mengovirus (MV) used as an extraction control. For the quantification of HAdV, standard curves prepared from known dilutions of HAdV and MV were used. Human adenovirus was detected in 140/150 (93%) of the environmental samples comprising of 69/75 (92%) being raw sewage and 71/75 (95%) being effluent samples. The HAdV concentrations detected in wastewater treatment plant 1 (WWTP 1) ranged from 1.38x105 gc/L to 4.50 x 109 gc/L for raw sewage and 5.08x103 gc/l to 4.30x108 gc/L for effluent. The HAdV concentrations detected in WWTP 2 ranged from 6.84x104 gc/L to 1.69x1012 gc/L for raw sewage and 5.27x103 gc/L to 1.16x108 gc/L for effluent. The HAdV hexon amplification success rate from the nucleic acids was 43/140 (31%). Eighteen HAdV genotypes were successfully characterised using Sanger sequencing. The HAdV-D was the most predominant species in both WWTPs, follow by HAdV-B and HAdV-F. The HAdV-A and HAdV-E species were the least identified. Next generation sequencing identified four times as many genotypes as Sanger sequencing (77 different genotypes). The HAdV-D (types 8, 9, 13, 17, 19, 20, 23, 24, 28, 29, 32, 33, 36, 42, 44, 47, 49, 51, 56, 60, 62, 64, 67 and 81) and HAdV-B (types 2, 3, 7, 11 and 66) were the most predominant species followed by HAdV-F (types 40 and 41), HAdV-A (types 12 and 76), HAdV-E ( type 4) and HAdV-C (type 1). Testing wastewater treatment plants is advantageous as it allows for the detection and identification of HAdV types circulating in the surrounding communities. Due to the large number of species identified using NGS, it is the superior typing method and should be used for future studies. These include strains causing symptomatic and asymptomatic infections. Human adenovirus was detected at comparable frequencies in raw sewage and treated effluent wastewater, with slightly higher detection in effluent samples. However, the viability of these viruses is unknown and should be investigated in further studies. The detection of viruses in wastewater treatment plants are a public health concern as the treated effluent is discharged into rivers, which may be used by communities for domestic and recreational purposes.
Identification and phylogenetic analysis of Aedes species (Diptera: Culicidae) and arboviruses associated with them across tropical and temperate regions of South Africa (2015-2018)
(University of Pretoria, 2020) Venter, Marietjie; Almeida, Antonio Paulo Gouveia; milehna.guarido@up.ac.za; Guarido, Milehna M.
Emerging and re-emerging diseases have increased worldwide in incidence in the past decades. Of these emerging diseases 60.3% are caused by zoonotic pathogens of which 22.8% are arboviruses or arthropod borne viruses. Arboviruses are transmitted by hematophagous insects, especially moquitoes. Multiple factors such as human population growth, climate change and adaptations of certain Aedes mosquito vector species to urban environments and anthropophilic have been attributed to causing this rise in arboviral infections. In Southern Africa, zoonotic arboviruses belonging to the families Flaviviridae (genus Flavivirus), Togaviridae (genus Alphavirus), and those in the order Bunyavirales, family Phenuiviridae: (genus Phlebovirus) and Peribunyaviridae (genus Orthobunyaviruses), have proven, in the past, to be of both medical and veterinary importance. Recent detection of neurological cases in South Africa, most likely, due to flaviviruses, alphaviruses and orthobunyaviruses in the Simbu serogroup, has rekindled interest in these zoonotic diseases. This interest is also warranted because of lack of recent information on arboviral prevalence in mosquito species, distributions, abundance, and ecology, especially of Aedes species, the likely primary vectors of these arboviruses in Southern Africa. To update this lack of information, this study t reports on zoonotic arboviruses circulating in selected areas in the north-eastern provinces of South Africa in mosquitoes with a focus on Aedes. Many Aedes species are morphologically quite difficult to identify especially when they are old, and scales rubbed off in the process of trapping. To aid in the identification of Aedes in this study we provide molecular barcodes for Aedes species occurring in in South Africa and define their phylogenetic relationship with other mainly Afrotropical Aedes mosquitoes based on the cytochrome oxidase I gene sequences. The first Chapter provides a comprehensive review of the literature and describes the importance of arboviruses worldwide and in South Africa, highlighting the role of Aedes mosquitoes as vectors. In Chapter 2, what is known about the broad patterns of Aedes mosquito species diversity, abundance, and distribution in different habitats across selected sites in five different provinces in South Africa is described. The sites selected were chosen because of evidence of neurological cases in humans and animals in recent years likely due to arboviral infections. In total, 61,737 adult mosquitoes were collected from January 2014 to May 2018, using three kinds of carbon dioxide baited trap types About 16% (11,440) were Aedes species, of which, 14 species were recognised or suspected vectors of mosquito-borne diseases because of positive infections, including Aedes mcintoshi which was the most abundant Aedes species captured. The effect of the climatic conditions on the mosquito population dynamics were also investigated. Aedes species were present in the sites following the peak of the rainfall and were mostly captured in temperatures between 18°C and 27°C. Chapter 3 focuses on determining the blood meal source present in engorged Aedes mosquitoes sampled to give an assessment of blood feeding tendencies that would serve useful to determine their vector status. Aedes species were identified feeding on a broad range of livestock, and wildlife, only two specimens were identified as feeding on avian species.Chapter 4 focuses on interpretations of cytochrome oxidase subunit 1 (COI) gene sequences to identify Aedes species in South Africa and to analyse the relationship among the species. A total of 52 COI sequences were aligned representing 21 Aedes species. In several cases these were the first African aedine species uploaded in NCBI GenBank. Neomelaniconion species clustered together, except for Ae. aurovenatus. Finally, the data also suggested that Ae. cumminsii present in South Africa belongs to the subspecies ssp. mediopunctatus. In Chapter 5 results of arboviral infections in Culicidae mosquitoes captured from the selected sites, particularly Aedes species is provided. Arboviral infection or prevalence screening was performed using multiple genus specific polymerase chain reactions (PCR). Alphavirus and Orthobunyavirus were detected in different Culicidae genera, including Aedes, Culex, Anopheles and Mansonia. There were no isolations of pathogenic flaviviruses in mosquitoes. The only alphaviruses detected in mosquitoes were Middelburg, Sindbis and Ndumu viruses during the period of the study. Shuni virus was the only member of Orthobunyavirus genus, detected. Even though, the main aim was to identify pathogenic viruses, several insect-specific viruses belonging to Alphavirus and Flavivirus genera were also detected and these are described in Chapter 6. The numerous arboviruses detected in Culicine mosquitoes, including Aedes species, demonstrate that some species are likely maintaining natural cycling of these arboviruses. Noteworthy, is that mosquito species positive for arboviruses are often the most abundant in the selected sampling locations and that these species blood feed mostly on the larger vertebrates present in the area. Outbreaks possibly occur when the prevalence of certainmosquito species are high due to favourable climatic conditions. Highest arbovirus detections occurred in peri-urban, rural, and conservation areas, indicating that livestock and wildlife likely play an important role in the amplification of these arboviruses. This study highlights the importance of a continues mosquito-based surveillance for arboviruses in South Africa, and the role that Aedes species might be playing in the circulation of these arboviruses. Surveillance for the species that tested positive for pathogenic arboviruses during the arbovirus season may act as an early warning system and can also help to avoid spill over in animals and humans in the area surveyed.
Surveillance for rabies-related lyssaviruses in South African insectivorous bat species
(University of Pretoria, 2021) Markotter, Wanda; Coertse, Jessica; colyngrobler12@gmail.com; Grobler, Colyn Stefan
Lyssaviruses are bullet shaped negative-sense RNA viruses that are all able to cause the fatal encephalitic disease known as rabies. The genus currently consists of 17 formally recognised viral species with one tentative species awaiting classification. The prototype virus for the Lyssavirus genus is the well-known rabies virus (RABV), while all other species in the genus are classified as rabies-related viruses. In South Africa specifically, RABV, Lagos bat virus (LBV), Duvenhage virus (DUVV), and Mokola virus (MOKV) are known to circulate, with RABV and DUVV associated with human fatalities. Active surveillance on rabies-related lyssaviruses in bats, specifically African insectivorous bat species, is either very sporadic or non-existing, providing an inaccurate overall representation of prevalence, diversity, and geographic distribution. Therefore, we conducted viral nucleic acid surveillance for lyssaviruses in different insectivorous bat species in South Africa. These samples were collected during routine field surveillance and included bats that were found dead, appeared to be displaying abnormal behavior or taken as vouchers specimens as part of bat taxonomic studies. A quantitative real-time reverse transcription polymerase chain reaction assay, capable of detecting the diversity of lyssaviruses were used to test extracted RNA. Three brain samples tested positive and were further characterized by conventional RT-PCR, DNA sequencing and phylogenetic analyses targeting the nucleoprotein gene. One of the positive brains was detected from a Common slit-faced bat (Nycteris thebaica) and the other two positive brains were detected from the Natal long-fingered bat (Miniopterus natalensis). Phylogenetic analysis of the nucleoprotein indicated one detection to be a Duvenhage lyssavirus with the other two detections showing a close relationship with the West Caucasian bat virus species, previously only detected in Eastern Europe. However, a more than 20 % nucleotide divergence indicated it to be a potentially new lyssavirus species, Matlo bat lyssavirus. The virus was successfully isolated using the mouse inoculation test followed by full genome next generation amplicon sequencing. The results of the full genome characterisation further supported the initial findings with concatenated coding regions nucleotide divergence ranging between 16% and 23.7% as well as consistent phylogenetic tree topology groupings identical to initial phylogenetic analyses using multiple evolutionary models. The identification of a putative new lyssavirus highlights the importance of routine lyssavirus surveillance to understand the diversity. Further investigation is required to determine the possible reservoir species since the Natal long-fingered bats are known to co-roost with different bat species in caves. The potential of spillover to humans and other animals is unknown but people often enter these bat roosts for traditional and recreational purposes and bats do come into contact with several animal species including humans during foraging.
Genetic assessment of infectious hepatitis A virus strains detected in selected water sources in Gauteng, South Africa
(University of Pretoria, 2019-12) Taylor, Maureen B.; achidasaid@gmail.com; Rachida, Said
Hepatitis A is a vaccine preventable liver inflammation caused by the hepatitis A virus (HAV). Hepatitis A virus is the most common cause of acute viral hepatitis worldwide that is transmitted via the faecal-oral route with waterborne transmission recognised as a major public health concern. Hepatitis A virus is classified in the genus Hepatovirus of the family Picornaviridae. The stability of HAV regarding pH, temperature and different treatment systems contributes to the virus’ persistence in the environment. The virion of HAV has a 7.5 kb positive-sense single-stranded RNA genome. Nucleotide sequence analysis of the VP1 region has identified six geographically distinct genotypes infecting humans (genotypes I, II and III) and non-human primates (genotypes IV, V and VI). In South Africa (SA), unique HAV IB strains have been detected in surface and wastewater samples, as well as on fresh produce at the point of retail. However, as the viruses were detected by molecular-based assays it is unknown whether the detected strains were still infectious. Although hepatitis A is a notifiable disease in SA there is gross underreporting, no routine surveillance system and a paucity of epidemiological data on HAV strains in circulation. Recently, the pretreatment of water and food samples with intercalating dyes prior to nucleic acid extraction was successfully applied for the quantification of potentially infectious HAV using molecular assays. Given that analysing sewage, wastewater and surface waters would provide a more accurate estimation of the HAV strains circulating in the country, the present study aimed to detect and characterise infectious HAV strains from selected South African water sources. From April 2015 to March 2016, 118 samples consisting of sewage, treated wastewater discharge and a downstream dam water were collected monthly from five wastewater treatment plants (WWTP 1, 2, 3, 4, 5). High titres of HAV were detected in the sewage (1.34 x e5 and 3.70 x e10 genome copies [gc]/litre [L]) and treated discharge (4.74 x e3 to 3.39 x e7 gc/L) samples. None of the dam water samples tested positive for HAV. Genetic characterisation of the detected strains by Sanger sequencing revealed the circulation of HAV IB strains that carried the R298K amino acid change over the VP1 region or the R63K and R71S change over the VP1/P2B junction or the C70S and M104I change over the VP1/P2B junction. The quasispecies dynamic of HAV has been detected in sewage samples. Hepatitis A virus strains carrying amino acid mutation at the immunodominant and neutralisation epitopes were characterised in both the sewage and treated discharge samples. The virus concentrates of HAV-positive sewage and treated discharge samples were treated with a combination of PMA-water (50 μM) and Tween®20 (0.5%) and the quantification of HAV from the samples was repeated. Potentially infectious HAV was quantified from the wastewater samples, with titres ranging up to e6 and e4 gc/L of sewage and treated discharge samples, respectively. Characterisation of these potentially infectious strains by Sanger sequencing confirmed the circulation of HAV strains carrying the R298K amino acid change over the VP1 region or the R63K and R71S change over the VP1/P2B junction or the C70S and M104I change over the VP1/P2B junction. The complete coding sequence, obtained from sewage and treated discharge samples by NGS, confirmed the circulation of HAV strains carrying the R63K and R71S changes but not the C70S and M104I changes over the VP1/P2B junction. The present study provides a methodology for the quantification and genetic characterisation of potentially infectious HAV from wastewaters.
Epidemiology and characterisation of enteric DNA viruses associated with gastroenteritis in children in selected regions of South Africa
(University of Pretoria, 2019) Page, N.A. (Nicola); Taylor, Maureen B.; rembu.netshikweta@gmail.com; Netshikweta, Rembuluwani
Acute gastroenteritis (AGE) is a global public health problem causing considerable morbidity and mortality among infants and children, especially in low-income settings. Viruses including group A rotaviruses (RVA), noroviruses (NoV), adenoviruses (AdV), sapoviruses (SaV) and astroviruses (AstV) are widely acknowledged to be the most common cause of AGE in children. The importance of newly recognised viruses such as human bocavirus (HBoV) as an aetiological agent of AGE is becoming increasingly evident. The aim of this study was to investigate the molecular epidemiology of HAdV and HBoV in children aged ≤5 years hospitalised for AGE in South Africa (SA) from April 2009 to April 2015. Clinical and demographic data, along with stool specimens were collected from hospitalised children who presented with AGE. Real-time polymerase chain reaction (PCR) was used to screen for the presence of enteric DNA viruses. Genotyping was achieved by nucleotide sequence analysis or multiplex PCR. Whole genome sequencing was performed on selected strains to characterise their genetic variation and evolution. Between April 2009 and December 2014, the prevalence of HAdV in hospitalised children with AGE in SA was 18.1% (656/3623); 62.3% of the HAdV_positive children were 7–24 months of age. Human AdV was detected year round. Co-infections were found in 76.3% (222/291) cases of the HAdV_positive specimens with full enteric screening and AstV was detected most frequently as a co-infecting pathogen. Prolonged hospital stay was observed in human immunodeficiency virus (HIV)-infected children with HAdV. Human AdV-F was the most common species identified (254/603, 42.1%), with almost equally distribution of -40 and -41. Recombination breakpoints of the five HAdV41 strains varied in the number and location, indicating different evolution origins. Between April 2009 and April 2015, the prevalence of HBoV in hospitalised children with AGE in SA was 5.6% (212/3765); the majority of which were from children ≤2-year of age (92%, 195/212). Viral co-infections were found in 67% (142/212) of HBoV cases, while in fully screened specimens (virus, bacteria and parasites), 83.1% (74/89) had evidence of co-infections. In all co-infections, only HAdV was significantly associated with HBoV (adjusted Odds Ratio (aOR))=1.68; (95% CI 1.10-2.52; p=0.015) in multivariate analysis. Human BoV infections were reported throughout the year. All four HBoV genotypes were detected with HBoV1 being the most prevalent (79.6% (152/191). The variation in total number of specimens screened for HAdV and HBoV is because HAdV screening was done until December 2014; while HBoV screening was done until April 2015. The current study highlights the genetic diversity of HAdV-40 and -41 strains circulating in SA and suggests possible evolution from inter-strain recombination. Furthermore, the present study highlights the wide spectrum of HBoV genotypes in children with AGE in SA. This study presents the most comprehensive recent data on HAdV diversity in SA, and new baseline data on a HBoV-associated gastroenteritis in a country where no previous report is available.
Assessing the risk of transmission of yellow fever and Dengue viruses by Aedes (Stegomyia) mosquito populations in Northern Kenya
(University of Pretoria, 2019) Venter, Marietjie; Sang, Rosemary; Tchouassi, David P.; edith.chepkorir@gmail.com; Chepkorir, Edith
East Africa has been experiencing an increase in the occurrence of emerging infectious diseases such as yellow fever (YF) and dengue (DEN). Increasing frequency of YF activity in East Africa constitutes a re-emergence that was not detected for over 40 years. Additionally, DEN outbreaks have also increased in frequency and continue to be detected in Kenya and in neighboring countries like Tanzania, Somalia, Djibouti, Eritrea and South Sudan. The renewed vigor of YF and dengue fever (DF) re-emergence in East Africa presents a new challenge to public health in spite of the availability of a safe and effective vaccine for YF. However, there is need to understand the potential for YF and DEN transmission along the border areas of Kenya, because Kenya is classified among countries with medium to high risk for YF transmission. This classification was mainly based on historical data, proximity to countries reporting recent YF outbreaks, the presence of non-human primates known reservoirs for these viruses, unrestricted human movement and presence of potential vector mosquito species. Both YF and DEN share a similar niche in the ecosystem and are associated with Aedes mosquito species of the subgenus Stegomyia. While the factors leading to the re-emergence of these diseases are poorly understood, a better epidemiologic understanding relating to disease ecology including presence of potential vectors, their host blood feeding preferences, the vector competence in transmission of these viruses and evidence of virus circulation in human population, will guide assessment of disease risk in the target areas and help to prevent or mitigate severe outbreaks in this region.
Surveillance of the rabies-related lyssavirus, Mokola, in small non-volant mammals in South Africa and Mozambique
(University of Pretoria, 2020) Markotter, Wanda; Coertse, Jessica; u13057368@tuks.co.za; McMahon, William Charles
Mokola virus (MOKV), a rabies-related lyssavirus, represents one of 17 recognized species within the Lyssavirus genus, all of which are capable of causing the fatal encephalitic rabies disease. MOKV is exclusively endemic to Africa with only 30 sporadic cases reported since its discovery more than 50 years ago. The reservoir host for MOKV remains unknown, however, several hypotheses have been formulated. Small non-volant mammals (i.e. shrews, sengis and rodents) have been suggested as possible reservoir hosts with previous MOKV isolations from shrews (Crocidura spp.) and a single rodent (Lophuromys sikapusi) providing support of the first lyssavirus species that has an association with small non-volant mammals. To investigate further, nucleic acid- and serological surveillance were conducted in small non-volant mammals from Southern Africa (specifically South Africa and Mozambique). Nucleic acid surveillance with a pan-lyssavirus qRT-PCR assay of 355 brain samples did not identify any new MOKV infections. Serological surveillance using a micro-neutralization test of 287 serum samples identified 37 samples that were positive for the presence of MOKV virus neutralizing antibodies. These positive serum samples indicate previous MOKV exposure and were all collected from Bushveld gerbils (Gerbilliscus leucogaster) in both South Africa (n=36) and Mozambique (n=1). From all of the Bushveld gerbils that were tested, an overall MOKV seropositivity of 87.80% is observed for the gerbils that were caught at Meletse in Limpopo. Since MOKV have been shown to cross-react in serological assay with closely-related lyssaviruses, the seropositivity observed could have been due to exposure of another phylogroup II lyssavirus. Serological evidence of MOKV in this rodent species was previously observed in a study conducted in Zimbabwe in 1988, which raises their profile as a potential MOKV host candidate. Experimental pathogenicity studies support this notion due to significant amounts of MOKV found in their salivary glands that could be sufficient for transmission. To gain further insight of the phylogeny and genetic diversity of MOKV, complete genome sequences of three historic MOKV isolates from South Africa (MOKV 700/70, 229/97, and 12/458) were generated. Future studies are needed to expand surveillance, detection and characterization of lyssaviruses.
The molecular epidemiology and diversity of gastroenteritis viruses in HIV-infected, -exposed and -unexposed children under the age of five years in Pretoria, South Africa
(University of Pretoria, 2020) Mans, Janet; Brauer, Marieke; u12225925@tuks.co.za; Rossouw, Esmari
Viruses are common causes of both endemic and epidemic gastroenteritis, infecting millions of people per year, with norovirus, rotavirus and adenovirus-F as the main causative agents, and sapovirus and astrovirus as contributing viruses. These viruses are highly infectious and most severe in the very young, old, or individuals who are immunocompromised. The viral infection usually causes self-limited gastroenteritis, although chronic infection has been observed in highly immunocompromised patients. African and South-East Asian regions are disproportionally affected by diarrhoeal disease. These regions (especially South Africa) are also more severely affected by human immunodeficiency virus (HIV) infections. It has been suggested that immunocompromised individuals may form part of a reservoir for novel virus variants and recombinants. It should be taken into account that not every person is equally susceptible to infection after pathogen exposure and that not all infected persons develop clinical symptoms (Ramani and Giri, 2019). One host genetic factor that can influence susceptibility to enteric infection is the expression of histo-blood group antigens (HBGAs). Histo-blood group antigens are a major group of complex carbohydrates and are determinants of both human and animal ABO blood groups and the Lewis blood group systems, which are distributed in abundance on the mucosal epithelia of the gastrointestinal tract. Histo-blood group antigens have been proven to influence susceptibility to rotavirus and norovirus infections. Saliva, blood and stool specimens (n=205) have previously been collected from children (≤ 5 years of age) hospitalised with gastroenteritis at Kalafong Provincial Tertiary Hospital from June 2016 to December 2017. Follow up stool specimens were then collected six weeks after enrolment when possible. A descriptive questionnaire was completed by each child’s guardian, giving information on age, residential area, HIV status etc. of the participating child. The stool specimens were screened for six gastroenteritis causing viruses (norovirus GI and –GII, rotavirus, sapovirus, astrovirus and adenovirus) by multiplex PCR. Forty-seven percent (96/205) of specimens tested positive for at least one gastroenteritis causing virus. Rotavirus predominated (46/205), followed by norovirus (32/205), adenovirus (15/205), sapovirus (9/205) and astrovirus (3/205). A total of 27/32 norovirus (GI.3, GII.2, GII.3, GII.4, GII.7, GII.12 and GII.21), 44/46 rotavirus (G1P[8], G2P[4], G2P[6], G3P[4], G3P[8], G8P[4], G8P[6], G9P[6] and G9P[8]) and 8/9 sapovirus (GI.1, GI.2, GII.1, GII.4 and GII.8) strains have been genotyped, of which norovirus GII.4 and rotavirus G3P[4] predominated. A total of 46/205 children submitted a follow up stool specimen to be tested. Of the 46 children, 9 tested positive for norovirus infection with initial stool specimen testing. Follow up screening resulted in 13/46 (28%) specimens testing positive for either norovirus GI or GII, with all patients presenting as asymptomatic. After genotyping it was observed that only one of the follow up specimens were identical to the original sequence genotyped, indicating prolonged shedding. FUT2 genotyping of 205/205 children showed a 71%:29% ratio between secretors and non-secretors. Eighty percent (77/96) of the virus-infected children were secretors whereas only 20% (19/96) were non-secretors. Rotavirus (p<0.01) and norovirus GII.4 (p<0.05) specifically were found to be more prevalent in secretors. In this study, no statistical significance was observed in terms of severity of and susceptibility to gastroenteritis viruses between HIV-infected, HIV-exposed uninfected or HIV-uninfected individuals. Histo-blood group phenotyping has resulted in various combinations, with Le(b) being the most prevalent antigen found. Next generation sequencing was unsuccessful. In future, fresh specimens should be considered for testing, with more funding and time for optimisation of this process and to give adequate results. In summary, gastroenteritis is still a leading cause of childhood morbidity and mortality, with all advancements in understanding the disease helping to decrease the impact of it. This study again reinforced the importance of these viruses, as they are circulating in such high abundance. It also reinforced the concept that susceptibility to noro- and rotavirus infection is affected by the secretor status of a person. This could in future help with better understanding the viral infection mechanisms and in turn help with vaccine development and treatment

Browse

Recent Submissions