Alphaviruses Detected in Mosquitoes in the North-Eastern Regions of South Africa, 2014 to 2018

The prevalence and distribution of African alphaviruses such as chikungunya have increased in recent years. Therefore, a better understanding of the local distribution of alphaviruses in vectors across the African continent is important. Here, entomological surveillance was performed from 2014 to 2018 at selected sites in north-eastern parts of South Africa where alphaviruses have been identified during outbreaks in humans and animals in the past. Mosquitoes were collected using a net, CDC-light, and BG-traps. An alphavirus genus-specific nested RT-PCR was used for screening, and positive pools were confirmed by sequencing and phylogenetic analysis. We collected 64,603 mosquitoes from 11 genera, of which 39,035 females were tested. Overall, 1462 mosquito pools were tested, of which 21 were positive for alphaviruses. Sindbis (61.9%, N = 13) and Middelburg (28.6%, N = 6) viruses were the most prevalent. Ndumu virus was detected in two pools (9.5%, N = 2). No chikungunya positive pools were identified. Arboviral activity was concentrated in peri-urban, rural, and conservation areas. A range of Culicidae species, including Culex univittatus, Cx. pipiens s.l., Aedes durbanensis, and the Ae. dentatus group, were identified as potential vectors. These findings confirm the active circulation and distribution of alphaviruses in regions where human or animal infections were identified in South Africa.publishersversionpublishe

West Nile virus, an underdiagnosed cause of acute fever of unknown origin and neurological disease among hospitalized patients in South Africa

DATA AVAILABILITY STATEMENT : Sequencing data presented in this study are openly available on GenBank using the following accession numbers: OL790166, OL790167. Patient data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy reasons.SUPPLEMENTARY MATERIALS : FIGURE S1: Map showing the locations of the 10 public sector hospitals where CSF was sampled from patients experiencing acute neurological disease in 2017 that made up the neurological cohort; FIGURE S2: The three hospitals that make up the ANDEMIA sentinel sites. The urban Gauteng site comprised of Kalafong Provincial Tertiary Hospital and the rural Mpumalanga site, comprised of the Mapulaneng and Matikwane Hospitals, TABLE S1: Details of the flavivirus isolates used in additional neutralization tests, TABLE S2: Details of the PCR, ELISA and VNT results for the WNV positive patients detected in the neurological cohort in 2017 and in the AFDUC cohort, 2019 to 2021, TABLE S3: Demographic data of the AFDUC patients enrolled for West Nile virus molecular testing, 2019 to 2021.West Nile virus (WNV), a mosquito-borne flavivirus, is endemic to South Africa. However, its contribution to acute febrile and neurological disease in hospitalized patients in South Africa is unknown. This study examined two patient cohorts for WNV using molecular testing and IgM serology with confirmation of serological results by viral neutralization tests (VNT) to address this knowledge gap. Univariate analysis was performed using collected demographic and clinical information to identify risk factors. In the first cohort, 219 cerebrospinal fluid (CSF) specimens from patients with acute neurological disease in Gauteng hospitals collected in January to June 2017 were tested for WNV. The study identified WNV in 8/219 (3.65%, 95.00% CI (1.59–7.07)) patients with unsolved neurological infections. The second cohort, from 2019 to 2021, included 441 patients enrolled between January and June with acute febrile or neurological disease from urban and rural sites in Gauteng and Mpumalanga provinces. West Nile virus was diagnosed in 40/441 (9.07%, 95.00% CI (6.73–12.12)) of patients, of which 29/40 (72.50%, 95.00% CI (56.11–85.40)) had neurological signs, including headaches, encephalitis, meningitis, and acute flaccid paralysis (AFP). Notably, most of the cases were identified in children although adolescents and senior adults had a significantly higher risk of testing WNV positive. This suggests a previously underestimated disease burden and that WNV might be underrecognized as a cause of febrile and neurological diseases in hospitalized patients in South Africa, especially in children. This emphasizes the importance of further research and awareness regarding arboviruses of public health concern.The ANDEMIA project funded by a grant from the German Federal Ministry of Education and Research and the G7 Global Health Fund of the Robert Koch Institute.https://www.mdpi.com/journal/virusesam2024Medical VirologySDG-03:Good heatlh and well-bein

Identification and molecular characterization of Shamonda virus in an aborted goat fetus in South Africa

Viruses in the Orthobunyavirus genus, Peribunyaviridae family, are associated with encephalitis, birth defects and fatalities in animals, and some are zoonotic. Molecular diagnostic investigations of animals with neurological signs previously identified Shuni virus (SHUV) as the most significant orthobunyavirus in South Africa (SA). To determine if other orthobunyaviruses occur in SA, we screened clinical specimens from animals with neurological signs, abortions, and acute deaths from across SA in 2021 using a small (S) segment Simbu serogroup specific TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR). Positive cases were subjected to Sanger sequencing and phylogenetic analysis to identify specific viruses involved, followed by next-generation sequencing (NGS) and additional PCR assays targeting the medium (M) segment and the large (L) segment. In total, 3/172 (1.7%) animals were PCR positive for Simbu serogroup viruses, including two horses with neurological signs and one aborted goat fetus in 2021. Phylogenetic analyses confirmed that the two horses were infected with SHUV strains with nucleotide pairwise (p-) distances of 98.1% and 97.6% to previously identified strains, while the aborted goat fetus was infected with a virus closely related to Shamonda virus (SHAV) with nucleotide p-distances between 94.7% and 91.8%. Virus isolation was unsuccessful, likely due to low levels of infectious particles. However, phylogenetic analyses of a larger fragment of the S segment obtained through NGS and partial sequences of the M and L segments obtained through RT-PCR and Sanger sequencing confirmed that the virus is likely SHAV with nucleotide p-distances between 96.6% and 97.8%. This is the first detection of SHAV in an aborted animal in SA and suggests that SHAV should be considered in differential diagnosis for abortion in animals in Southern Africa.The Poliomyelitis Research Foundation; the German Federal Ministry of Education and Research and the Long-Term EU-Africa Research and Innovation Partnership on Food and Nutrition Security and Sustainable Agriculture (LEAP-Agri).https://www.mdpi.com/journal/pathogensam2024Medical VirologyParaclinical SciencesSDG-03:Good heatlh and well-bein

Potential mosquito vectors for Shuni virus, South Africa, 2014–2018

Shuni virus is associated with neurologic and febrile illness in animals and humans. To determine potential vectors, we collected mosquitoes in South Africa and detected the virus in species of the genera Mansonia, Culex, Aedes, and Anopheles. These mosquitoes may be associated with Shuni virus outbreaks in Africa and emergence in other regions

Genetic Differences in the Immediate Transcriptome Response to Stress Predict Risk-Related Brain Function and Psychiatric Disorders

Depression risk is exacerbated by genetic factors and stress exposure; however, the biological mechanisms through which these factors interact to confer depression risk are poorly understood. One putative biological mechanism implicates variability in the ability of cortisol, released in response to stress, to trigger a cascade of adaptive genomic and non-genomic processes through glucocorticoid receptor (GR) activation. Here, we demonstrate that common genetic variants in long-range enhancer elements modulate the immediate transcriptional response to GR activation in human blood cells. These functional genetic variants increase risk for depression and co-heritable psychiatric disorders. Moreover, these risk variants are associated with inappropriate amygdala reactivity, a transdiagnostic psychiatric endophenotype and an important stress hormone response trigger. Network modeling and animal experiments suggest that these genetic differences in GR-induced transcriptional activation may mediate the risk for depression and other psychiatric disorders by altering a network of functionally related stress-sensitive genes in blood and brain

Investigation of the molecular epidemiology of West Nile virus at the human-animal interface in South Africa

West Nile virus (WNV) is an emerging, neurotropic zoonotic arbovirus that belongs to the Flaviviridae family and the Flavivirus genus. Since its discovery in 1937 in Uganda, Africa, WNV has continued to spread outside of its original geographic range and has now been named the most widespread cause of arboviral neurological disease, occurring on every continent except Antarctica. West Nile virus is maintained in nature through an enzootic bird-mosquito transmission cycle, with Culex (Cx.) univittatus as the principal vector in South Africa. Birds are used as sentinels for WNV in many countries, but in Africa birds seldom die or show signs of disease therefore alternative animals- such as horses- are used as sentinels for WNV. In equines, 90.00 % of symptomatically infected horses will develop neurological disease, and 35 % of infections are fatal. Besides horses, additional vertebrate hosts such as domestic animals, cattle, sheep, goats, and wildlife exist. In humans 80.00 % of WNV infections are asymptomatic, and usually between 1-10.00 % of cases will progress to neurological disease with a fatality rate of 10.00 %, although recent outbreaks in Europe have seen an increase in neurological disease manifestations up to 77.00 % and mortality rates of 40.00 %. This type of data is currently not available for South Africa. This study aimed to investigate the molecular epidemiology of WNV in South Africa using a One-health approach and reports on the active circulation of endemic lineage 2 strains amongst mosquitoes, animals, and humans. The burden of disease and association with neurological disease was identified through syndromic surveillance in animals and humans. Additionally, a next-generation sequencing (NGS) pipeline was developed to sequence WNV full genomes from clinical specimens. Chapter one provides an in-depth review, first starting broad with the topic of flaviviruses, then taking a closer look at WNV highlighting the importance of this virus as a pathogen of medical and veterinary importance. Chapter two describes the optimization of a real-time Pan-flavivirus reverse transcription-polymerase chain reaction (RT-PCR) capable of detecting all members of the Flavivirus genus to identify infections in mosquitoes, animals and humans and the comparison thereof to the historically used assay. In chapter three, flavivirus screening was performed on mosquitoes collected at urban, peri-urban and wildlife sites across the northeastern regions of South Africa. This chapter identified West Nile- and Banzi viruses in 16/1471 (1.08 %, minimum infection rate [MIR] = 0.39) and 2/1471 (0.14 %, MIR = 0.05) pools respectively. Phylogenetic analysis characterised WNV strains as lineage 2. Cytochrome oxidase 1 confirmed morphological identification of potential vector species but also identified new potential vectors for these flaviviruses in South Africa. Minimum infection rates were calculated per site to identify places of increased risk for spillover to humans and animals and identified Kyalami (Gauteng, peri-urban, MIR = 2.53) and the Kruger National Park (wildlife, MIR = 1.22) as ‘hot spots’ for WNV infection in animals and humans. Chapter 4 describes the use of molecular and serological tools in conjunction with syndromic surveillance to identify WNV as the aetiological agent responsible for acute febrile or neurological disease in animals in South Africa. From January 2017 to December 2020, WNV was detected in 11/835 (1.32 %) horses by real-time RT-PCR and in 69/657 (10.50 %) through IgM serodiagnosis that could be confirmed with a microtiter virus neutralization test (micro-VNT). Therefore, a total of 77/835 (9.22 %) acute WNV infections were diagnosed in equines experiencing clinical disease. A further 7/243 (2.88 %) diseased or dead non-equine animals tested positive for WNV by real-time RT-PCR. Phylogenetic analysis confirmed all PCR positive cases to be lineage 2 infections, except for a single lineage 1 case in a lion. Pairwise distance analysis confirmed circulation of endemic lineage 2 strains amongst mosquitoes and animals in South Africa. A geographical correlation between the animals and areas with high mosquito MIR was observed. In chapter 5, a retrospective pilot study identified WNV in 3.65 % of cerebrospinal fluid (CSF) specimens sampled from patients suffering acute undiagnosed neurological disease in Gauteng, South Africa through molecular and serodiagnosis. Phylogenetic analysis of two WNV strains identified in the CSF of two children confirms circulation of endemic neuroinvasive strains between mosquitoes, animals and people in Gauteng, South Africa. Prospective syndromic surveillance focusing on acute febrile diseases of unknown cause (AFDUC), with or without neurological signs, identified WNV as the aetiological agent in 8.26 % of cases through IgM serodiagnosis and virus neutralization assay confirmation. Odds ratio analysis identified clinical signs associated with infection and elucidated socioeconomic and demographic risk factors for WNV infection in South Africa. Patients residing close to the rural Mpumalanga site were more likely to contract WNV infection, which correlates with the high mosquito MIR identified in Chapter three. Finally, chapter 6 describes three next-generation sequencing approaches to obtain full genomes from WNV clinical specimens or cell culture isolates. For the first time in South Africa, full genome sequences originating in mosquitoes and a non-equid animal were obtained. Phylogenetic analysis shows a high nucleotide and amino acid similarity amongst the sequences and to their closest relative, WNV HS101/08, a neuroinvasive strain isolated from the brain of a horse in South Africa. This confirms the active dissemination of neuroinvasive strains between mosquitoes and animals in South Africa, as predicted in the previous chapters. Phylogenetic analysis elucidated that circulating stains were genetically related despite arising from different spatial and temporal parameters. The success of predicting areas of increased WNV spillover to animals and humans using mosquito MIR highlights the benefit of viewing arboviral infections in the context of One-health. Mosquito surveillance is a valuable tool as it allows timely detection of species diversity, distribution, and infection rates which are vital for estimating the risk and transmission of vector-borne disease. Surveillance in humans with AFDUC and/or neurological signs helped to define the burden of disease and indicated an increased risk for WNV associated hospitalisations in summer and late autumn in South Africa suggesting that the virus is still missed likely due to a lack of clinical awareness of the potential risk for more severe disease. Active surveillance at the animal-human interface may aid in predicting outbreaks and may assist in the prevention, diagnosis, and control of these arboviruses at both the local and international levels.Dissertation (MSc (Medical Virology))--University of Pretoria, 2022.Long-term EU- Africa research and innovation Partnership on food and nutrition security and sustainable Agriculture (LEAP-Agri) grant: Research Network (LEARN) on Arboviral Zoonoses which is an EU/NRF facilitated grant (grant number 115574)German Federal Ministry of Education and Research via a program called Research Networks for Health Innovations in Sub-Saharan Africa for the ANDEMIA project (grant number 01KA1606)G7 Global Health Fund of the Robert Koch Institute through Dr Fabian Leendertz (grant number ZMVI1-2517GHP703)Global Disease Detection Centre, US Centers for Disease Control and Prevention, South Africa. This research was supported by the Cooperative Agreement Number, [5 NU2GGH001874-02-00], funded by the Centers for Disease Control and Prevention (CDC)Poliomyelitis research foundation (grant number:19/50)National Research Foundation (grant number 117592)University of Pretoria Postgraduate bursaryMedical VirologyMSc (Medical Virology)Unrestricte

West Nile Virus, an Underdiagnosed Cause of Acute Fever of Unknown Origin and Neurological Disease among Hospitalized Patients in South Africa

West Nile virus (WNV), a mosquito-borne flavivirus, is endemic to South Africa. However, its contribution to acute febrile and neurological disease in hospitalized patients in South Africa is unknown. This study examined two patient cohorts for WNV using molecular testing and IgM serology with confirmation of serological results by viral neutralization tests (VNT) to address this knowledge gap. Univariate analysis was performed using collected demographic and clinical information to identify risk factors. In the first cohort, 219 cerebrospinal fluid (CSF) specimens from patients with acute neurological disease in Gauteng hospitals collected in January to June 2017 were tested for WNV. The study identified WNV in 8/219 (3.65%, 95.00% CI (1.59–7.07)) patients with unsolved neurological infections. The second cohort, from 2019 to 2021, included 441 patients enrolled between January and June with acute febrile or neurological disease from urban and rural sites in Gauteng and Mpumalanga provinces. West Nile virus was diagnosed in 40/441 (9.07%, 95.00% CI (6.73–12.12)) of patients, of which 29/40 (72.50%, 95.00% CI (56.11–85.40)) had neurological signs, including headaches, encephalitis, meningitis, and acute flaccid paralysis (AFP). Notably, most of the cases were identified in children although adolescents and senior adults had a significantly higher risk of testing WNV positive. This suggests a previously underestimated disease burden and that WNV might be underrecognized as a cause of febrile and neurological diseases in hospitalized patients in South Africa, especially in children. This emphasizes the importance of further research and awareness regarding arboviruses of public health concern

Identification and Molecular Characterization of Shamonda Virus in an Aborted Goat Fetus in South Africa

Viruses in the Orthobunyavirus genus, Peribunyaviridae family, are associated with encephalitis, birth defects and fatalities in animals, and some are zoonotic. Molecular diagnostic investigations of animals with neurological signs previously identified Shuni virus (SHUV) as the most significant orthobunyavirus in South Africa (SA). To determine if other orthobunyaviruses occur in SA, we screened clinical specimens from animals with neurological signs, abortions, and acute deaths from across SA in 2021 using a small (S) segment Simbu serogroup specific TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR). Positive cases were subjected to Sanger sequencing and phylogenetic analysis to identify specific viruses involved, followed by next-generation sequencing (NGS) and additional PCR assays targeting the medium (M) segment and the large (L) segment. In total, 3/172 (1.7%) animals were PCR positive for Simbu serogroup viruses, including two horses with neurological signs and one aborted goat fetus in 2021. Phylogenetic analyses confirmed that the two horses were infected with SHUV strains with nucleotide pairwise (p-) distances of 98.1% and 97.6% to previously identified strains, while the aborted goat fetus was infected with a virus closely related to Shamonda virus (SHAV) with nucleotide p-distances between 94.7% and 91.8%. Virus isolation was unsuccessful, likely due to low levels of infectious particles. However, phylogenetic analyses of a larger fragment of the S segment obtained through NGS and partial sequences of the M and L segments obtained through RT-PCR and Sanger sequencing confirmed that the virus is likely SHAV with nucleotide p-distances between 96.6% and 97.8%. This is the first detection of SHAV in an aborted animal in SA and suggests that SHAV should be considered in differential diagnosis for abortion in animals in Southern Africa

Survey of West Nile and Banzi Viruses in Mosquitoes, South Africa, 2011–2018

We collected >40,000 mosquitoes from 5 provinces in South Africa during 2011–2018 and screened for zoonotic flaviviruses. We detected West Nile virus in mosquitoes from conservation and periurban sites and potential new mosquito vectors; Banzi virus was rare. Our results suggest flavivirus transmission risks are increasing in South Africa

Alphaviruses detected in mosquitoes in the North-Eastern regions of South Africa, 2014 to 2018

The prevalence and distribution of African alphaviruses such as chikungunya have increased in recent years. Therefore, a better understanding of the local distribution of alphaviruses in vectors across the African continent is important. Here, entomological surveillance was performed from 2014 to 2018 at selected sites in north-eastern parts of South Africa where alphaviruses have been identified during outbreaks in humans and animals in the past. Mosquitoes were collected using a net, CDC-light, and BG-traps. An alphavirus genus-specific nested RT-PCR was used for screening, and positive pools were confirmed by sequencing and phylogenetic analysis. We collected 64,603 mosquitoes from 11 genera, of which 39,035 females were tested. Overall, 1462 mosquito pools were tested, of which 21 were positive for alphaviruses. Sindbis (61.9%, N = 13) and Middelburg (28.6%, N = 6) viruses were the most prevalent. Ndumu virus was detected in two pools (9.5%, N = 2). No chikungunya positive pools were identified. Arboviral activity was concentrated in peri-urban, rural, and conservation areas. A range of Culicidae species, including Culex univittatus, Cx. pipiens s.l., Aedes durbanensis, and the Ae. dentatus group, were identified as potential vectors. These findings confirm the active circulation and distribution of alphaviruses in regions where human or animal infections were identified in South Africa