Medical Microbiology

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Open Access
Adaptive immune response in COVID-19 patients and innate immune modulation of SARS-CoV-2
(University of the Free State, 2023) Litabe, Matefo Millicent; Burt, Felicity
In December 2019, a cluster of cases of atypical pneumonia were reported in Wuhan, China. All patients had a history of attending a large seafood market. Surveillance and detection methods established during the 2003 severe acute respiratory syndrome coronavirus (SARS-CoV)-1 outbreak contributed towards the identification of the virus as a novel coronavirus (CoV). The virus was later named SARS-CoV-2 and identified as the causative agent of Coronavirus Disease 2019 (COVID-19). Despite massive attempts to contain the virus in China, the cases rapidly increased and spread globally, and the World Health Organization (WHO) declared COVID-19 a Public Health Emergency of International Concern on 30 January 2020 and characterized the outbreak as a pandemic on 11 March 2020. Assessing immunoglobulin (Ig)-G and neutralizing antibodies is essential to comprehensively evaluate the efficacy and duration of immunity conferred by natural infection and the COVID-19 vaccines. Effectively determining SARS-CoV-2 seroprevalence within a population is important as it helps to improve our understanding of virus circulation dynamics, identify individuals at risk of infection, and the extent of virus exposure in the community. Therefore, this study investigated the duration and kinetics of adaptive immunity, particularly the persistence of IgG and neutralizing antibodies, in patients who recovered from SARS-CoV-2 in the Free State, South Africa. Commercial assays are expensive, and hence two anti-spike (S) inhouse assays, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), were developed and validated for detection of anti-S IgG. A total of 89 serum samples were collected from COVID-19 PCR-confirmed patients between 2-94 days post-symptom onset to validate the assay. A 100 prepandemic samples were used as a negative control panel to determine the cutoff of the assay. A cutoff value of 30% was considered accurate to differentiate between negative and positive samples using a two-graph receiver operating characteristic (TG-ROC). The assays exhibited a sensitivity of 100% for ELISA and 98.8% for IFA when testing samples collected more than one week after the onset of illness. The positive predictive values (ppv) were 92.1% for ELISA and 91.0% for IFA on PCR-confirmed positive samples. The assays were also compared to a commercially available SAPRHA-approved assay, where the ELISA showed a higher ppv of 95.8 %, while the Roche assay was 89.6 %, and the commercial lateral flow was 93.9 %. The two in-house assays also detected IgG antibodies in samples collected from waves in which new variants were circulating, showing that despite mutations of the SARS-CoV-2 S protein, the assay was still sensitive to detect IgG antibodies in circulating variants. This indicates that these assays could be used for surveillance of the South African population. To investigate the duration of anti-S IgG and neutralizing antibodies against SARS-CoV-2, 100 individuals with previously confirmed COVID-19 infection were recruited for the study. The cohort included 64/100 vaccinated and 36/100 unvaccinated individuals. Initial samples were collected between March 2021 and January 2022, with confirmed infection between June 2020 and December 2021. Follow-up samples were collected from 82/100 in 2022 and 62/100 in 2023 of the initial cohort. Samples were tested for anti-S IgG antibodies and neutralizing antibodies against Ancestral strain, Delta, and Omicron variants. A total of 95/100 baseline samples tested positive for anti-S IgG and 79/82 and 53/62 for follow-up samples. A total of 99/100, 78/100, and 72/100 baseline samples tested positive for neutralizing antibodies against the Ancestral strain, Delta, and Omicron variants. A total of 80/82, 63/82, 77/82 in 2022 and 53/62, 50/62, 56/62 in 2023 tested positive for neutralizing antibodies against the Ancestral, Delta, and Omicron variants, respectively. Samples were grouped based on the time (days) the samples were collected post-onset of illness. Results showed that IgG antibodies were significantly higher directly after infection, between 1-180 days, then gradually declined significantly with time. Neutralizing antibody titers against the Ancestral strain and Delta variant were significantly higher early after infection, between 1-180 days, remained relatively stable for an extended period, and then waned gradually before declining significantly. In contrast, although not significant, neutralizing antibodies against the Omicron variant increased with time, but this may be because samples were collected when the Omicron variant was still prevalent. Results also showed that vaccinated individuals had significantly higher antibody titers than unvaccinated, highlighting the importance of continued vaccination. The study shows the longevity of antibodies against SARS-CoV-2, as most individuals still had detectable titers at least 24 months post-acute infection, possibly boosted by vaccination or reinfection. The study also shows that the dynamics of antibodies vary among individuals; most individuals display declining antibody titers with time, and a smaller proportion maintain stable antibodies or a fluctuation of antibodies over time. Traditional medicinal plants have been proposed as promising, cost-effective treatments for SARS-CoV-2, with studies showing the potential to induce protection against different viral infections. The study also investigated the potential of Phela, a traditional medicine prepared from the extracts of four South African plants, and the individual components to modulate the release of cytokines in SARS-CoV-2 Omicron-infected mammalian cells and to investigate the influence of the plant extracts on viral replication. Cells were treated with the plant extracts before or after infection with SARS-CoV-2. Subsequently, cell culture media was collected at 12, 24, 48, and 72 hours post-infection and tested for virus replication and levels of Interleukin (IL)-1β, IL-2Rα, IL-6, IL-10, tumour necrosis factor (TNF)-α, and interferon (IFN)-γ cytokines. There was no statistically significant difference in viral load between infected cells treated with plant extracts compared to infected and untreated cells, showing that the plant extracts may have little to no effect on virus replication. Treatment with plant extracts resulted in significantly lower release of IL-1β, IL-2Rα, and TNF-α, with better response post-treatment than pretreatment, showing that the plant extracts may have the potential to manage cytokine storms and be a potential source of treatment for SARS-CoV-2.
Open Access
Beta-lactam resistance profiles in urinary tract infection among Escherichia coli isolates in Bloemfontein
(University of the Free State, 2005-05) Maqutu, Lennox Makhelane; De Kock, M. J.
English: This study was designed to elucidate the epidemiology, nature and extent of β-lactam resistance in urinary tract infections caused by Escherichia coli isolates in Bloemfontein hospitals. To reach this goal it was necessary to phenotypically characterise and re-identify the E. coli isolates by the Mastascan identification system. Pure cultures were obtained by streaking single colonies onto MacConkey agar containing 50 µg / ml ampicillin. Single colonies were then picked off and inoculated into Mueller-Hinton broth, grown overnight at 37°C and re-streaked onto MacConkey agar containing 50 µg / ml ampicillin. Three colonies were then picked and stored at -20°C in a freeze mixture of 10 % proteose and 10 % glycerol. E. coli isolates were mated to a universal recipient strain (162) in pre-warmed Mueller- Hinton broth. Transconjugants were selected on MacConkey agar supplemented with 50 µg / ml ampicillin and 50 µg / ml nalidixic acid. Lactose-negative colonies resistant to nalidixic acid and ampicillin were inoculated into Mueller-Hinton broth, incubated for six hours and restreaked onto MacConkey agar containing ampicillin. Lactose-negative colonies were picked and considered to be transconjugants. It was found that fifty four (45 %) out of 120 ampicillin-resistant isolates transferred ampicillin-resistance determinants to an E. coli recipient (162) by conjugation. Seventy-five percent of ampicillin-resistant isolates were from female patients, indicating that urinary tract infections are more prevalent in females than in males. The National Committee for Clinical Laboratory Standards* agar dilution method was used to determine minimum inhibitory concentration (MIC) distributions of 12 β-lactam antimicrobial agents (ampicillin, amoxycillin, piperacillin, augmentin, cefoxitin, cefotaxime, cefepime, ceftazidime, cephazolin, ceftriaxone, cefuroxime and imipenem). Strains found to be resistant had MICs that overlapped the range where susceptibility was normally assumed. This was due to inducible β-lactamase producer strains, which go undetected by the Kirby- Bauer disk diffusion method but can be identified by using the Jarlier double-disk method. MIC frequency distributions for the penicillins showed that elevated doses should be administered in order to maximise antibiotic concentration at the site of infection or that a second antibiotic agent or inhibitor should be used in combination therapy. Beta-laetam susceptibility profiles were determined by the Kirby-Bauer disk diffusion method. This method was also used to determine the correlation of MIC values with the inhibition zone diameters in order to predict treatment outcomes from inhibition zone diameters. The Jarlier double-disk technique was used to detect extended-spectrum β-lactamaseproducing organisms and the frequencies at which they occurred. There was no difference between the ratios of ESBL-producers in hospitalised and non-hospitalised patients, although the absolute numbers were different. This was probably due to the 48 hour cut-off point used to define hospitalisation. Samples taken before 48 hours were considered to be nonhospitalised. There were many more female than male patients with urinary tract infections in the Bloemfontein hospitals during the period of the study. The extent of joint resistance to β-lactam antibiotics among E. coli isolates was assessed by comparing two agents at a time. The observed incidence of joint resistance was compared to the rate of double resistance expected if it had been acquired as two independent events. It was found that even amongst two antibiotics that are biologically cross-resistant (ampicillin and augmentin) a close correlation exists between the concentrations of the two agents required to inhibit individual E. coli strains.
Open Access
Characterization of T cell responses to the non-structural proteins of the M segment in survivors of Crimean-Congo haemorrhagic fever
(University of the Free State, 2019) Maotoana, Makgotso Golda; Goedhals, Dominique; Burt, Felicity Jane
Crimean-Congo haemorrhagic fever orthonairovirus (CCHFV) is one of the most widely distributed arboviruses globally. The disease caused by the virus, Crimean- Congo haemorrhagic fever (CCHF), continues to emerge and re-emerge across the globe. There are currently various vaccines under development for CCHF prevention. The non-structural M protein (NSM), GP38, highly variable mucin-like domain and N-terminus of GC regions in CCHFV have proved to be immunogenic in vaccine studies. Furthermore, both arms of the immune system have been found to be fundamental for protection in mice. However, there is limited information about immunity in patients following natural infection. The aim of the study was to characterize T cell immune responses against the NSM, GP38 and the highly variable mucin-like domain of CCHFV in survivors of CCHF. This was achieved by first identifying immunogenic peptides in the regions of interest and determining the amino acid conservation of the identified peptides. An overlapping peptide library spanning the NSM, GP38 and highly variable mucin-like domain was designed using the South African CCHFV isolate SPU 103/87. The secretion of interferon-gamma (IFN-γ) by peripheral blood mononuclear cells isolated from 12 participants was screened using 24 peptide pools in an IFN-γ enzyme linked immunospot (ELISpot) assay. IFN-γ secretion was detected in eight of the twelve participants. Two participants showed no detectable IFN-γ responses to any of the peptide pools, and another two were excluded from the analysis due to a high background in the negative controls indicating non-specific reactivity. Nine peptides were identified with the IFN-γ ELISpot, including five peptides in the GP38 region and four in the NSM region, thus confirming the immunogenic potential of these regions during natural infection. No immunogenic peptides were identified in the highly variable mucin-like domain, which is possibly because of the high genetic diversity in the region. The identified immunogenic peptides were used to stimulate T cells of participants and a flow cytometry assay was performed to characterize the immune responses, with the focus on detecting the presence of the T cell memory subsets, the expression of CD107a, which is a cytotoxic marker, and the secretion of IFN-γ and tumour necrosis factor-alpha (TNF-α), which are antiviral cytokines. Cytotoxic CD8+ T cells were detected in six participants in response to nine peptides. IFN-γ and TNF-α secretion within the CD4 and CD8 populations were comparable; thus, highlighting the ability of the CD8+ T cell population to secrete antiviral cytokines, even though the population is known to be predominantly cytotoxic. The secretion of IFN-γ was more frequent than TNF-α secretion in both the CD4 and CD8 populations. Polyfunctional T cells were detected with the phenotypes IFN- γ+CD107a+ and IFN-γ+ TNF-α+, in both the CD4 and CD8 populations. Therefore, the results indicate the possibility of efficient antiviral responses upon stimulation with viral epitopes in survivors of infection. Heterogeneous functionality of the T cell memory subsets was observed, however the terminally differentiated (TEMRA) subset was the most dominant and abundant, followed by the naïve (TN), effector memory (TEM), with the least abundant being the central memory (TCM) T cell memory subset. The IFN-γ secretion detected with the IFN-γ ELISpot and the flow cytometry assay was used as basis for comparing the sensitivity of the two techniques. The IFN-γ ELISpot proved to be comparable to the flow cytometry assay. The ELISpot is suited for screening purposes, while the flow cytometry allowed further characterization of the T cell responses. Therefore, it is recommended that these complementary assays be used in combination. In conclusion, T cell epitopes were identified in the NSM and GP38 regions of CCHFV. Polyfunctional T cells were found in both the CD4 and CD8 populations, thus suggesting the presence of effective long-term memory T cells responses in survivors of CCHF.
Open Access
Development and application of molecular assays for mosquito-borne alphaviruses in South Africa
(University of the Free State, 2021) Dimaculangan, Micah; Burt, Felicity Jane
Surveillance of mosquito-borne alphaviruses is critical for the prevention of diseases and the control of outbreaks caused by these viruses, especially with the absence of approved vaccines and antiviral treatments available. Hence, the continual development of rapid and reliable tools for the surveillance of alphaviruses is important. This will aid in the understanding of which viruses are currently circulating with the potential to cause outbreaks. Molecular nucleic acid amplification tests (NAATs), particularly conventional and real-time reverse transcription (RT)-polymerase chain reaction (PCR), are typically employed in epidemiological surveys. In this study, a conventional nested RT-PCR assay was developed to detect alphaviruses in South Africa. In addition, an isothermal amplification technique, specifically a RT-helicase dependent amplification (HDA) assay, which only requires a simple heating device, for instance a heating block, and lateral flow dipsticks/ cassettes for end point detection, was developed to detect alphaviruses currently circulating in South Africa, as an alternative to the RT-PCR assay for application in low resource settings or for field application. The conventional nested RT-PCR assay was able to detect ≥620 copies of RNA compared to the RT-HDA assay which had a minimum limit of detection of 4.8 x 105 copies of RNA. Both assays were tested for theoretical cross-reactivity with other alphaviruses, which include Sindbis virus (SINV) and chikungunya virus (CHIKV) isolates from other regions and genotypes, and isolates from alphaviruses such as Ross River virus (RRV), Barmah Forest virus (BFV), Mayaro virus (MAYV), eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV) and western equine encephalitis virus (WEEV) that are endemic to other parts of world. Alignment of the primers with the sequences of these isolates shows that both assays in theory would be able to detect SINV isolates from northern Europe, taking into account the transcontinental transmission of the virus between South Africa and northern Europe by migratory birds. The conventional nested RT-PCR assay may be able to detect most alphaviruses due to minimal mismatches (0 – 1) detected between the primers and the partial nsP4 sequences of the alphavirus isolates, while the RT-HDA assay may not be well suited to detect other alphaviruses due to the many mismatches (>4) detected between the primers and the partial nsP4 sequences of the alphavirus isolates. Nevertheless, this shows that the RT-HDA is theoretically more specific that the conventional nested RT-PCR assay. The RT-HDA however failed to detect any alphaviruses in the 42 mosquito pools tested, which was not unexpected as the assay could only detect up to 4.8 x 105 copies of RNA. In contrast, the conventional nested RT-PCR assay was able to detect alphaviral RNA in five out of the 42 mosquito pools tested, and the nucleotide sequences were determined to identify the alphavirus species. SINV RNA was detected in three mosquito pools and Middelburg virus (MIDV) was detected in two pools. Phylogenetic analysis was subsequently performed to determine the genetic relationship of these isolates from the Free State with previously published/ reported SINV and MIDV isolates in South Africa, Africa, and around the world. The conventional nested RT-PCR assay developed in this study has shown to be a useful surveillance tool for the detection of mosquito-borne alphavirus infections. Given the low sensitivity determined for the RT-HDA assay, improvements, or alternative rapid and fieldable NAATs should be considered in the future for alphavirus surveillance applications in low resource settings.
Open Access
The development of a method for the detection and estimation of CCHF virus RNA in tick species
(University of the Free State, 2000-05) Du Preez, Patrick Hendrik; Pretorius, G. H. J.; Janse van Rensburg, M. N.
Crimean Congo haemorrhagic fever (CCHF), caused by a RNA virus, is a tick-borne viral zoonosis occurring in Europe, Asia and Africa. The fatality rate is ±30%. Rapid and accurate diagnosis is essential. The aim of this study was to develop a reverse transcription-polymerase chain reaction (RT-PCR) with internal control for the detection of CCHF RNA. Primers were selected for a region in the nucleocapsid - gene of the S segment. The internal control was constructed by ligating this PCR product into a pGEMEX-1 vector. Sequencing of the PCR product (381 bp) revealed two unique restriction sites, BIn I and BstE II which were used to delete a fragment of 59 bp. The shortened PCR-product was re-inserted into E. coli. T3 RNA polymerase produced plasmid derived RNA (322 bp) was used to spike specimens. Standard RT-PCR was then performed. The minimum concentration of target RNA the RTPCR can detect was estimated to be 4 x 10-5 pmol RNA, giving more or less the same sensitivity as the PCR alone. The size difference of 59 bp is enough to distinguish between the full-length and the deletion variant inserts when visualised and therefore provides an internal control. RT-PCR on fifty Hyalomma ticks was negative. The CCHF virus was probably not present or at concentrations below detection level, as RT-PCR of control CCHF virus RNA confirmed the accuracy of the method. RT-PCR allows rapid detection of CCHF virus RNA. The constructed internal control precludes the use of Dugbe virus, an antigenically related nairovirus.
Open Access
Development of detection assays for sindbus virus and investigating in vitro infection of mammalian cells
(University of the Free State, 2013-08) Hanekom, Hermanus Albertus; Burt, F. J.
Sindbis virus (SINV) is a member of the Alphavirus genus and belongs to the family Togaviridae. The virus has a positive sense RNA genome of 11700 bases which encodes for both structural and non structural proteins. Infections are frequently diagnosed based on clinical, epidemiological and laboratory criteria. Laboratory confirmation is essential as SINV infections must be distinguished from various conditions that share similar clinical manifestations. The most frequently used methods for identification are haemagglutination inhibition, enzyme-linked immunosorbent assay, plaque reduction neutralization tests as well as conventional in-vitro neutralization assays. Serological assays for the detection of SINV are not readily available commercially and due to the non-specific symptoms caused by SINV infection the number of infections per annum may be under diagnosed. The purpose of this study was to develop serological assays such as ELISA and a novel neutralization assay that could be used in serological surveys for the detection of IgG antibodies against SINV. Furthermore to develop assays that could be used to determine the level of viral replication in mammalian cells for characterizing infection in mammalian cells as well as investigate the influence of interferon on viral replication and look for evidence of apoptosis caused by SINV infection. An in house ELISA was developed and used to screen 146 sera for IgG antibodies against SINV. The in-vitro neutralization assay is the gold standard for serology and 43 samples in total were tested in both the ELISA and the in-vitro neutralization assay. Analysis and comparison of the results obtained using the in-house ELISA and the neutralization assay indicated that the sensitivity of the ELISA was 68.9% and the specificity of the in house ELISA was 78.57 - 85.71% depending on the use of the percentage positive or optical density values to differentiate positive and negative samples. A forward and reverse primer for the amplification of a conserved 181bp region of the nsp2 gene encoding the nsp2 protein of SINV were designed along with a TaqMan hydrolysis probe to be used in a real time quantitative TaqMan PCR. The infection of mammalian cells, human macrophages and HeLa cells, was determined by measuring viral loads with a real time quantitative TaqMan RT-PCR. Two strains of SINV were used in attempts to infect macrophages, a strain from Egypt and a strain from South Africa. Small increases in viral load suggested possible low levels of viral replication but were considered insufficient to warrant further investigation and insufficient to investigate occurrence of antibody dependent enhancement of disease in macrophages. The mechanism possibly interfering with replication of virus in the human macrophages was investigated. Supernatant fluid samples from macrophage infections were tested for the release of interferon gamma which could inhibit viral replication. There were nine to fifteen fold differences in the concentration of 2 interferon gamma detected in the supernatant fluid at baseline and 24h after infection. HeLa cells were treated with similar concentrations of human interferon gamma at different time intervals. Pretreatment and concurrent treatment with infection showed reduced levels of viral load compared with no treatment or delay in treatment. Hence the suggestion that interferon could have played a role in inhibiting viral replication in the human macrophages. DNA was extracted from HeLa cells infected with SINV and the DNA fragments separated through agarose gel electrophoreses. There were multiple bands visible in the infected samples whereas the negative control did not show multiple bands, only one large band of genomic DNA. The presence of multiple DNA fragments in infected cells and absence of those fragments from uninfected cells were suggestive of virus induced apoptosis.
Open Access
Development of molecular and serological assays for diagnosis and surveillance of Crimean-Congo haemorrhagic fever virus
(University of the Free State, 2015-05) Pieters, Danelle; Burt, F. J.; Jansen van Vuren, P.
Crimean-Congo haemorrhagic fever virus (CCHFV) an arthropod-borne virus associated with haemorrhagic disease in humans. The global distribution of CCHFV correlates with that of ticks from the Hyalomma genus. CCHFV infection is diagnosed by detection of viral nucleic acid using reverse-transcription polymerase-chain-reaction (RT-PCR) or other molecular assays, by virus isolation from infected cell culture or suckling mouse brain or by detection of anti-CCHFV antibodies using enzyme-linked immunosorbent assay (ELISA) or immunofluorescence assay (IFA). High biocontainment facilities are required for virus isolation and preparation of whole virus native antigen for use in serological assays. Currently, treatment is limited to supportive therapy. CCHFV is currently emerging and re-emerging in many regions, which emphasize the requirement for safe, reliable and inexpensive assays to increase diagnostic capacity and monitor emergence of the virus. A nucleic acid sequence-based amplification (NASBA) molecular assay for detection of CCHFV ribonucleic acid (RNA) was developed. The assay can be performed without the requirement for sophisticated laboratory equipment. A commercially available enzyme mixture and buffer were compared with a more cost effective and easier to obtain in-house enzyme mixture and amplification buffer. Specificity of the NASBA assays were determined by testing viral RNA extracted from Vero cell culture infected with genetically diverse southern African CCHFV strains. A total of 41/48 samples tested were positive. Sensitivity of the NASBA assays was determined using dilutions of viral RNA and transcribed RNA to detect minimal copy number that could be amplified. The NASBA assay was able to detect at least 3.7 RNA copies. Diagnostic application of the NASBA assays was investigated by amplifying RNA extracted from clinical samples and the results compared with two commercial real-time RT-PCR assays. A total of 20/22 samples tested positive using the NASBA whereas the commercially available assays were able to amplify 22/22 samples. Subsequently, the inhibitory effect of sera on the amplification of CCHFV RNA using the NASBA assay was investigated using sera spiked with transcribed RNA. Two expression systems were investigated for the expression of recombinant CCHFV nucleocapsid protein (NP) for use in serological assays. The baculovirus expression system was initially investigated. The open reading frame of the S segment of a CCHFV strain was codon optimized for expression in insect cells. A pFastBac HT B transfer vector containing the optimized CCHFV NP gene was prepared and used to transform DH10Bac™ Escherichia coli cells to transpose the optimized CCHFV NP gene to a bacmid. The recombinant bacmid was utilized to transfect Spodoptera frugiperda 9 cells. The cell lysates were analysed, however, no expression of the CCHFV NP could be confirmed. A mammalian expression system was subsequently investigated. A pcDNATM 3.1D/V5-His-TOPO.CCHFV.NP construct was used to transfect baby hamster kidney-21 cells. Expression of CCHFV NP was detected in transiently transfected cells using IFA and serum collected from a convalescent CCHFV patient. To profile the immune response against CCHF viral proteins, 15 sera collected from convalescent patients at various times after onset of illness were tested for antibody against CCHFV NP and glycoproteins (GP) using commercially available slides. The antigen slides were prepared from transfected cells expressing recombinant CCHFV NP and GP. Antibody against CCHFV GP and NP were detected in all samples. End point titers of anti-CCHFV NP and GP were determined for two serum samples. Commercially available slides are expensive and therefore have limited application for testing large numbers. Application of in-house antigen slides prepared from transfected cells expressing CCHFV NP were tested using IFA and 14 sera collected from convalescent CCHFV patients. All sera tested positive, suggesting that preparation of a stable cell line expressing CCHFV NP is warranted for application in detection of antibody against CCHFV.
Open Access
Die voorkoms van giste tydens hoenderproduksie
(University of the Free State, 2000-12) Laubscher, Willem Diederik Froneman; Viljoen, B. C.
Afrikaans: Die verhoging in die plasing van die aantal hoenders per oppervlakte verhouding in moderne pluimvee boerdery praktyke, skep In klimaat waar mikrobiese groei (insluitend patogeniese mikroorganismes) gestimuleer word. Die stimulering van mikroorganismes word veralopgemerk in respiratories geassosieerde vrektes in groeihokke. Op grond van die verhoging in vrektes van jong kuikens is daar gekyk na die mikrobiese populasies teenwoordig in die tracheas van groeihok kuikens met spesiale karakterisering van die gis populasies teenwoordig. Die data het aangetoon dat die tracheas van jong braaikuikens hoofsaaklik besmet word deur die teenwoordigheid van gis species. Ten spyte van lae populasies in die eerste weke na uitbroeiing, ontwikkel die gispopulasies vinnig tot essensieel 'n klimaks populasie tydens die vierde week in die groeihokke. Die karakterisering van die gispopulasies het daarop gedui dat die belangrikste species blyk as volg te wees: Debaryomyces hansenii, Candida b/ankii, Toru/aspora de/brueckii en Trichosporon beige/ii. Omdat giste ook 'n belangrike bydrae maak tot die bederf van hoenderkarkasse, en deel uitmaak van die normale mikrobiologiese populasie teenwoordig op die eetbare hoenderkarkasse en hoenderstukke, is die voorkoms van hierdie flora verder deur die prosesseringsaanleg gevolg. Alhoewel giste proporsioneel in kleiner getalle voorkom as bakterieë, kan die giste as sekondêre populasie ontwikkel wanneer die bakteriese populasie geinhibeer word soos gevind nadat die karkasse onderdompel is in chloorbevattende immersie-wentelverkoelings baddens tydens prosessering. Cryptococcus en Candida species domineer voor verkoeling van die karkasse tydens ontvering en ontweiding terwyl Debaryomyces species oorheers na verkoeling en tydens bevriessing. Die data dui verder daarop dat die implementasie van chloor as ontsmettingsmiddel tydens verkoeling geen invloed gehad het op die voorkoms van die gisspecies nie. Die bronne van giskontaminasie in die onmiddelike omgewing van die slag pale het dieselfde tipes van giste opgelewer as gevind op die karkasse. Weens die abnormale hoë getalle giste gevind op die karkasse, en die beperkte invloed van die ontsmettingsmiddels tydens verkoeling op die getalle van giste is verskillende bruikbare ontsmettingsmiddels in die immersie-wentelverkoelings bad ondersoek. Goeie inhiberende en dodings resultate is verkry met behulp van meeste van die produkte. Slegs chloor dioksied het egter In genoegsame dodingseffek gehad op Salmonella species en In betekenisvolle inhibering van gisgetalle.
Open Access
The distribution, patient characteristics, therapy and patient outcome in culture positive invasive mold infections in a tertiary hospital in the Free State province, South Africa
(University of the Free State, 2019) Van der Westhuizen, B.; Coovadia, Y.; Potgieter, S.; Abrahams, M. S.
Introduction Fungi, including molds, are increasingly recognized as important pathogens carrying a high morbidity and mortality in critically ill and immune compromised patients and our understanding of these diseases remain incomplete, largely due to the lack of surveillance data. This study aimed to better quantify the distribution, patient characteristics, risk factors, therapy and treatment outcome in culture positive invasive mold infections at Universitas Academic Hospital in the Free State province, South Africa. Methods All culture positive mold isolates cultured from sterile specimens were identified retrospectively from 1 July 2014 to 30 June 2017. Laboratory and clinical data were reviewed for those that met the inclusion criteria. Results A total of 48 isolates were included in this study. There was a similar distribution between males and females and the mean age was 40.5 years. Aspergillus species were the most commonly isolated mold. The most common risk factors identified were HIV infection with a median CD4 of 88.5 cells/μl followed by hematological conditions. The treatment strategies in our study group were heterogeneous with 73.1% (19/26) of patients treated with antifungal therapy alone, 19.2% (5/26) with surgery alone and 7.7% (2/26) with a combined medical and surgical approach. Many patients received no treatment 45.8% (22/48). The overall mortality was 25% (12/48). Conclusions The diagnosis of invasive mold infections remains a challenge. In the current study, molds were found to cause serious infections, especially in at risk patients. Despite treatment with appropriate antifungal agents, the associated mortality rate was still high. This study contributes to the growing knowledge on the distribution, patient characteristics and outcomes of invasive mold infections, particularly in patients in the Free State, and lays the foundation for further research in the field of invasive mold infections.
Open Access
Drug resistance mutations in newly diagnosed HIV-infected infants and in children and adolescents with virological failure on ART
(University of the Free State, 2020-12) Barakzai, Iqra; Goedhals, Dominique
Introduction and aim: South Africa has never experienced an epidemic of the same extent and magnitude as the one with human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS).The roll out of antiretroviral therapy (ART) and other interventions, such as prevention of mother to child transmission (PMTCT), has greatly reduced the incidence of new HIV infections among children. However, early initiation of ART among children has increased their overall exposure to antiretrovirals (ARVs), resulting in an increased prevalence of acquired drug resistance (ADR) on treatment. Presence of drug resistant virus further complicates clinical management of HIV infection, resulting in fewer present and future treatment options. Therefore, drug resistance testing has been recommended in selecting appropriate treatment regimens among children and adolescents diagnosed with virological failure. The aim of the current study was to determine and characterise drug resistance mutations in newly diagnosed HIV-infected children and in children and adolescents with virological failure on ART. Methods: To detect drug resistance mutations, the present study developed and validated a Sanger sequencing assay using dried blood spot (DBS) samples. The Sanger assay was used to perform HIV drug resistance testing on DBS samples testing positive on the early infant diagnosis (EID) platform. Lastly, a retrospective analysis was performed of resistance data from samples of children and adolescents submitted to the routine diagnostic laboratory. Results: A high level of concordance was found among major mutations detected using the Sanger DBS and plasma assays. Since all the discordant mutations were either minor or existed as mixtures, the interpretation of resistance profiles was found to be identical. The DBS assay was then used to detect pre-treatment drug resistance (PDR) among samples from children on the EID platform. Overall, PDR was detected among 73% of the children, with the majority of the children showing resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) (72%). Dual class resistance was most prevalent to NNRTIs and NRTIs (11% [33/308]), followed by NNRTIs + protease inhibitors (PIs) (0.6% [2/308]). Triple class resistance (NNRTI + NRTI + PI) was very rare and was only detected in two children. Protease resistance was very rare among the children and none of the children displayed high-level resistance against any PI. Results from children (0 to 9 years) and adolescents (10 to 19 years) with virological failure while on ART indicated that most of the children were resistant to NRTIs (86% [79/92]), followed by NNRTIs (75% [69/92]) and PIs (28% [26/92]). Among adolescents, prevalence of resistance to NNRTIs (87% [146/167]) was the highest, followed by NRTIs (66% [111/167]) and PIs (30% [51/167]). Dual class resistance to NRTIs + NNRTIs was the most common among both children (44% [41/92]) and adolescents (38%, [63/167]). Triple class resistance (NNRTI+NRTI+PI) was detected in 18% of children (17/92) and 19% of adolescents (32/167). Conclusion: The prevalence of infants, children and adolescents experiencing treatment failure due to the presence of transmitted, acquired and PDR is increasing. While the implementation of virological monitoring and genotypic testing play an essential role in identifying and managing drug resistance among children and adolescents experiencing treatment failure, these services are limited in many developing countries. The current study supports the implementation of HIV drug resistance testing for national surveys and optimal clinical management of HIV-infected children and adolescents.
Open Access
Evaluating the role of efflux pumps in bacterial disinfectant resistance
(University of the Free State, 2022) Staats, Gunther Johann; Bragg, R. R.
The global rise of antibiotic resistance could lead to the advent of a post-antibiotic era, where disinfectants and biosecurity will be vital parts to control microbial proliferation. The SARS-CoV-2 pandemic has shown how important proper biosecurity and disinfection protocols are to control disease outbreaks. Disinfectant resistance has the potential to alter every aspect of disease control, as this phenomenon impacts human life from food security to healthcare systems. Antimicrobial resistance at its core originates from the presence and regulation of specific genes within the genome of a microorganism able to combat/resist specific action of an antimicrobial. This study focuses on investigating whether specific resistance determinants are responsible for the insusceptibility of a highly resistant isolate, Serratia sp. HRI. This isolate has high levels of disinfectant resistance; therefore, it provides an opportune chance to study if a specific mechanism is responsible. To achieve this the genomes of the Serratia sp. HRI and its closest related type strain were investigated for efflux pump genes. The efflux pump genes were predicted using an automatic annotation pipeline. The predictions revealed a plethora of resistance efflux genes mostly harbouring multidrug functioning. Additionally, disinfectant-specific efflux pump genes were identified (emrE, sugE, qacA, qacE, and ssmE). Susceptibility testing using three disinfectants revealed how the resistance levels between the two Serratia isolates differed. Further investigation using efflux pump inhibitors (EPIs) showed how specific families of efflux pumps confer resistance in both isolates. Time-kill analyses over an extended period showed how Serratia sp. HRI tolerates long-term disinfection. Using EPI reserpine, the efflux pump activity was determined during long-term disinfection. The results showed that concentration-dependent recruitment of efflux pumps was seen by Serratia sp. HRI. At low disinfectant concentrations, another mechanism was responsible for the survivability of the bacteria. Using liquid chromatography with tandem mass spectrometry it was established that disinfectant levels introduced with Serratia sp. HRI decreased over time. Suggesting that the HRI isolate has some mechanism to alter the structure of the disinfectant, such as metabolism or degradation pathways. This work highlights the role of efflux pumps in disinfectant resistance and the potential of other mechanisms to be involved. Future research will include gene deletion and expression studies to fully determine the efflux pump reliance for disinfectant resistance. The work completed in this thesis added to the knowledge of efflux-mediated disinfectant resistance. This work also highlighted the potential role of metabolism/degradation in resistance to low-level disinfection.
Open Access
The evaluation of a continual disinfection program on a commercial broiler chicken farm
(University of the Free State, 2022) Beauzec, Deon; Bragg, R.; Carlie, S.
The rise of antibiotic resistance as a global health threat has been linked to overuse of antibiotics, in particular, in the agricultural sector. A post-antibiotic world is fast approaching where antibiotics may be completely ineffective. To delay and prepare for this reality, alternative solutions to the use of antibiotics in animal production are required. Biosecurity describes some of the oldest techniques related to animal health, including infectious agent destruction and exclusion. Novel solutions to biosecurity must be investigated, however the possible loss of production related to halting antibiotic use is a major risk to food security. This study focused on assessing a continuous disinfection program using Virukill®, a modified- didecyldimethylammonium chloride-based disinfectant, for post-placement cleaning and disinfection, direct spray, and administration in water supply, at a commercial scale. This work provides a proof of concept for industrial application of a continuous disinfection program as a biosecurity measure in poultry production. Through bacterial and viral evaluation methods it was established that the continuous disinfection program was equal or better than standard aldehyde disinfectants in reducing viral loads. Organisms that survived the cleaning and disinfection process were also isolated and identified with molecular methods. The possibility of disinfectant resistance was also investigated but no evidence of resistance was observed. It was established that Virukill® was more effective than DDAC and BC in inhibiting the growth of organism isolated. Through growth performance studies this work establishes for the first time that the Virukill® continuous disinfection program improves performance of broilers, and a correlation was established between cleaning efficacy and performance. This work provides a viable alternative biosecurity-based alternative to the use of antibiotics in broiler production.
Open Access
Evaluation of constructed recombinant mengoviruses and other HIV vaccine candidates in murine and primate models
(University of the Free State, 2001-03) Van der Ryst, Elna; Smith, M. S.; Borman, A. M.; Botha, P. L.
The development of an effective vaccine against HIV is a formidable challenge. The overall objective of this work was to evaluate different HIV -1 vaccine approaches in primate and murine models. In a first approach recombinant Mengoviruses expressing several HIV -1 and SIV gene products were evaluated for their immunogenicity in mice and macaques. Results indicated that Mengovirus recombinants expressing HIV -1 Nef or SIV CTL epitopes are weak immunogens. This was disappointing in light of the promising results previously obtained using other Mengovirus recombinants and indicated that the nature of the insert might play an important role in the immunogenicity of Mengovirus recombinants. As a second approach, protection of chimpanzees from intravenous and vaginal challenge by immunisation with a recombinant canarypox virus expressing the HIV-ll1lB/LAI gp 120rrM, gag and protease genes was evaluated. In animals challenged by the iv route protection from homologous challenge was seen in one of two animals and this correlated with the neutralising antibody levels. One of five females resisted a total of 3 vaginal challenges, while two further animals resisted 2 challenges. However, only low levels of HIV-l-specific neutralising antibodies were present at time of challenge. This suggests that neutralising antibodies may have little importance for protection from mucosal infection in chimpanzees, in contrast with what was seen for iv challenge. Finally, macaques were immunised with a primary isolate of HIV -1 in order to evaluate the breadth of the immune response induced by HIV-1 in its "native" state. The animals developed moderate to high titers of total anti-HIV -1 antibodies as measured by EIA, which was mainly Gag directed. However, no antibodies capable of neutral ising HIV -1BX08 were demonstrated, and sera From the animals induced strong facilitation of HIV -1 replication in PBMC, raising the concern that whole virus based HIV vaccines might induce facilitating antibodies that can result in Facilitation of transmission and/or evolution of disease.
Open Access
Genetic analysis of human papillomavirus type 11 isolates from patients with recurrent respiratory papillomatosis treated at Universitas Academic Hospital
(University of the Free State, 2021-07) Thuynsma, Corne; Burt, Felicity Jane; Seedat, Riaz
Human papillomavirus type 11 (HPV11) is a causative agent of recurrent respiratory papillomatosis (RRP), a common benign laryngeal neoplasm that presents mainly in children. The genome comprises three regions: the early region (E1, E2, E4, E5a/b, E6 and E7), the late region (L1 and L2), and the upper regulatory region (URR). A sequence-based classification system is primarily used to genotype HPV. The L1 is used for HPV type discrimination, and in combination with the URR, can be used to differentiate between various lineages. However, optimal sub-lineage classification requires whole genome sequencing (WGS). A recent study investigating the genomic diversity of globally circulating HPV11 isolates identified a novel lineage and two novel sub-lineages. It has been proposed that phylogenetic tree topologies using the sequences of concatenated E5a/b- L1-URR genes, a 208bp segment of the E2 gene, and the complete genome generates similar tree topologies. Also, there is currently no published data on the HPV11 intratypic variants circulating in the Free State region. Hence, this study investigated HPV11 intratypic variants circulating in patients with RRP at the Universitas Academic Hospital, and aimed to identify novel (sub)lineages through phylogenetic investigations. The study population included patients diagnosed with RRP caused by HPV11, and sequence data for geographically distinct HPV11 (sub)lineage representatives. The genetic variation of HPV11 isolated from patients with RRP was determined by sequencing the E5a/b, L1, URR and a segment of E2 genes. Four isolates of interest were selected for whole-genome sequencing and phylogenetically analysed to determine the presence of potentially novel isolates. Many nucleic heterogeneities and non-synonymous substitutions were identified in isolates characterised in this study. Phylogenetic analysis of the concatenated L1-URR and E5a/b-L1-URR resolved into lineages A and B; however, sub-lineage classification was unclear. Analysis of the complete genome determined the presence of a lineage B isolate and two isolates of interest. Comparative analysis of genetic variability determined that the concatenated E5a/b-L1-URR could not reliably classify isolates. A segment of the E2 gene could reliably distinguish between all lineages and sub-lineages, suggesting that this gene segment contains stable sub-lineage specific single nucleotide polymorphisms (SNPs) and may serve in sub-lineage identification. In conclusion this study provides the most comprehensive data on the genomic diversity of HPV11 in the Free State to date. Results obtained in the current study support WGS for HPV11 classification below lineage level as a standard, as it generates more information regarding genetic variants.
Open Access
HBV viral load and drug resistance among HIV-HBV co-infected patients: a cross-sectional study in central South Africa
(University of the Free State, 2021-12) Kotze, Jacobus Charles; Goedhals, Dominique
In South Africa, human immunodeficiency virus (HIV) infected individuals co-infected with hepatitis B virus (HBV) do not routinely undergo HBV viral load (VL) testing when on antiretroviral therapy in the public sector treatment programme. We set out to explore whether HIV VL can be used as a proxy for HBV treatment response, since HIV VL testing is routinely performed in HIV/HBV co-infected patients. The clinical utility of HIV VL testing in this context may be impacted by the slower rate of viral decay which has been described for HBV as compared to HIV. In total, 224 patient samples were tested for HBV VL to determine the relatedness between HIV VL and HBV VL results. Samples with detectable HBV VL were sequenced to identify HBV associated drug mutations, hepatitis B surface antigen (HBsAg) mutations and genotype. Chi-square test for independence (χ2) was used to determine the relatedness between the viral loads, which indicated that the two viral loads are related (p-value<0.0001). However, in samples with an undetectable HIV VL, 29.27% (36/123) had a detectable HBV viral load, with 7.32% having an HBV VL >2000 IU/mL which has previously been linked to an increased risk of HBV related complications. Sequencing results showed that 10 samples had lamivudine resistance, however, no tenofovir resistance was detected. Three samples had immune escape mutations, two caused by the HBsAg mutations E164D and I195M and one by the immune-associated escape mutations N131T and D144A. The results from the study show that patients with HIV/HBV co-infection need to be monitored more closely in South Africa regarding HBV treatment response. The extensive use of lamivudine for HIV treatment in South Africa can be a driver of immune escape and further research needs to be done to determine the possible public health impact.
Open Access
Identification of antigen-specific serological cross-reactivity among survivors of Crimean-Congo Haemorrhagic fever
(University of the Free State, 2013) Rangunwala, Azeeza; Burt, F. J.
Abstract not available
Open Access
Identification of antigenic regions and linear B cell epitopes on yellow fever virus
(University of the Free State, 2013-02) Smouse, Shannon Lucrecia; Burt, F. J.
English: Yellow fever virus (YFV) virus is an arthropod-borne virus that causes viral hemorrhagic fever in humans in the tropical parts of both Africa and South America. The virus belongs to the family Flaviviridae, of the genus Flavivirus comprising of approximately 70 viruses. It is transmitted to vertebrates by the bite of an infected female mosquito, primarily the Aedes species. It is a re-emerging pathogen with case-fatality rates that can exceed 50% in humans. YFV can cause an acute febrile illness in humans which can progress to severe disease with hepatic and renal failure. The diagnosis of infection and testing of the immune status of vaccinees require reagents that are prepared in biosafety level (BSL) three and four facilities. Therefore the development of a recombinant antigen that does not require BSL three facilities for preparation and is safe to use, would have an important role in a diagnostic laboratory for detecting antibodies in infected individuals and vaccinees. Despite the availability of a live-attenuated efficacious vaccine, it is not recommended for immunocompromised individuals, thus development of new generation vaccines would have important public health implications. Identification and mapping of antigenic regions and viral epitopes is important for development of subunit vaccines and improved diagnostics. Subunit vaccines focusing on antigens that induce a protective immune response provide a safe approach to the development of vaccines against diseases causing severe and frequently fatal haemorrhagic fevers. The aim of this study was to identify immunodominant viral proteins that induce detectable antibody responses that could be used for developing diagnostic assays and to identify linear B cell epitopes on selected viral proteins. The complete open reading frame of the genes encoding the domain III (EDIII) region of the envelope protein, capsid (C) and NS4a proteins of YFV were amplified, from the 17D strain of YFV, by RT-PCR using primers specifically designed from sequence data retrieved from GenBank. Oligonucleotide primers were modified with BamHI and HindIII restriction enzyme sites that facilitated downstream cloning. Each amplicon was cloned into the pGEM®-T Easy cloning vector using T/A cloning. Each gene was rescued from the recombinant plasmid using BamHI and HindIII restriction enzyme sites and ligated into bacterial expression system, pQE-80L vector. In a previous study, the YFV EDIII gene was cloned into pQE-80L and expressed in JM109 Escherichia coli cells however extremely low yields were obtained. In this study the expression levels were improved using different cell lines and optimizing incubation conditions. An insoluble 13 kDa protein was expressed from the construct and confirmed by Western blot analysis. The protein was expressed with a 6 x Histidine tag that was used to facilitate purification using a Ni2+ column under denaturing conditions. Attempts to express the YFV C and NS4a proteins were not successful and expression was abandoned. In an attempt to improve solubility the YFV EDIII gene was excised from the pGEM®-T Easy vector and subsequently cloned into pCold TF bacterial expression vector. A ~65 kDa soluble protein was expressed from the construct and purified under native conditions. The functional activity of the recombinant antigens in ELISA was compared with whole cell lysate antigen prepared from cell cultures infected with YFV. The biological activity of the recombinant YFV pQE-80L-EDIII antigen was confirmed in immunoassays using serum samples from humans vaccinated with YFV vaccine. Positive sera failed to react in ELISA using pCold TF expressed antigen and this antigen was excluded from further assays. A total of 20/24 serum samples from human vaccinees collected at varying stages after vaccination reacted in an ELISA with the recombinant YFV pQE-80L-EDIII protein and 24/24 reacted in ELISA with whole cell lysate antigen. The EDIII region of the envelope protein was shown to be able to differentiate between West Nile Virus infection and YFV infection in a limited number of convalescent horse sera. The recombinant EDIII protein was used to immunize mice. Serum samples collected from the mice reacted against whole cell lysate antigen in ELISA and was shown to have neutralising antibodies using an in vitro neutralisation assay. Hence the EDIII region of the envelope protein likely induces an important protective immune response. Finally, bioinformatics was used to predict possible epitope regions and using peptide libraries spanning predicted sites, one potential epitopic region was identified in the EDIII protein. Putative epitopic and antigenic regions along the length of the C, NS4a and EDIII proteins of each strain were predicted using the BCPREDS and ABCpred software. In conclusion, the EDIII protein, an immunodominant antigen of YFV, prepared in this study has some potential for differentiation of flavivirus antibodies although it lacks sensitivity for routine diagnosis. A potential epitope, TGHGTVVMQ, from amino acid 21 to 29 on the EDIII protein was identified using bioinformatics and was shown to have reactivity against immune sera. The significance of this epitope needs further investigation. Finally the EDIII region of the YFV protein shows potential as a target region for vaccine development as shown for other flaviviruses but which has not previously been published for YFV.
Open Access
Immune responses to Crimean-Congo haemorrhagic fever virus and molecular characterization of viral isolates
(University of the Free State, 2014-02) Goedhals, Dominique; Burt, F. J.; Paweska, J.
Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne virus belonging to the family Bunyaviridae, genus Nairovirus. The distribution of the virus correlates with that of the principal vector, ticks belonging to the genus Hyalomma. This includes areas in Africa, Asia, Eastern Europe and the Middle East, with recent emergence in Turkey, Greece and India. CCHFV is associated with haemorrhagic fever in humans, with a case fatality rate of up to 30%. Current patient management relies on supportive therapy and administration of ribavirin, but the efficacy of this antiviral drug is controversial. Although an inactivated vaccine has been used in Eastern Europe and the former Soviet Union, it has not been accepted for widespread use. An understanding of immune correlates is therefore needed to guide further development of therapeutic and preventative interventions. This study aimed to investigate immune responses in survivors of CCHF in South Africa, focusing on the presence of detectable memory T lymphocyte responses and the identification of epitopic regions within the nucleoprotein and glycoproteins. In order to ensure applicability of identified epitopes to geographically distinct isolates, viral sequence diversity was also investigated by means of next generation sequencing and phylogenetic studies. A synthetic overlapping peptide library was used to screen for interferon gamma production by peripheral blood mononuclear cells from survivors of CCHFV infection in ELISPOT assays. Ten potential epitopic regions were identified, the majority of which were located on the nucleoprotein with only two regions identified on the glycoprotein GC in a single patient. Long-lived memory CD8+ T cell responses were detected in survivors of CCHF up to 13 years after infection. These findings indicate the presence of effective long term cellular immune responses which could be modulated through vaccination and gives an indication of epitopic regions that should be considered in candidate vaccines and testing vaccine immunogenicity. The presence of detectable memory responses in the absence of reexposure or chronic infection will allow future studies to fully characterize T cell responses in survivors. With an expanding area of CCHFV endemicity, safe, sensitive and specific serological assays are required for diagnostic and serosurveillance purposes. As the biosafety level 4 facilities required to culture the virus are lacking in many endemic areas, alternative means of producing reagents for diagnostic assays are needed which will not pose a safety risk to laboratory workers. The use of synthetic peptides in serological assays is one such alternative approach. In addition, identification of immunodominant epitopic regions may have application in vaccine development if they induce protective immunity. The peptide library was used to screen for antibodies recognizing human defined linear B cell epitopic regions in survivors of CCHFV infection by means of an enzyme-linked immunosorbent assay (ELISA). Two potential epitopic regions were identified on the GC glycoprotein with reactivity in 13 – 14 of 15 patients tested. Further investigation will be required to determine whether these epitopic regions also correlate with immune protection and to identify non-contiguous B cell epitopes which are likely to play an important role in antibody induction during natural infection with CCHFV. With new foci of CCHFV infections emerging in recent years, it is important to ascertain whether genomic variation will influence applicability of vaccine candidates and diagnostic assays in distinct geographic areas. Next generation sequencing techniques were used to obtain complete genome sequences for ten southern African CCHFV isolates. This is the first application of next generation sequencing technology to CCHFV isolates and proved to be a rapid and cost effective alternative to standard Sanger sequencing which can be effectively applied to the approximately 20kb CCHFV genome. The phylogenetic results confirmed that although there is extensive variability among geographically distinct CCHFV isolates at a genomic level, conserved areas are present which could be targeted for vaccine development and diagnostic purposes. The genetic variability seen results from point mutations and segment reassortment, which was shown to occur commonly in southern African CCHFV isolates. Despite the extensive variation in primary sequence, at a protein level, the motifs involved in protein function are well conserved. Prediction software analysis confirmed the presence of conserved OTU-like cysteine protease and RNA dependent RNA polymerase (RdRp) domains in the L segment of diverse southern African CCHV isolates. The RdRp is essential for viral replication while the OTU-like protease likely plays a role in immune evasion and therefore affects viral pathogenicity. Analysis of the M segment showed conservation of the basic protein coding strategy, with two structural and three non-structural glycoproteins. However, amino acid variation was notable across all predicted proteins but particularly in the variable mucin-like domain which is thought to play a role in viral pathogenicity. This study identifies targets for further investigation of viral pathogenicity which may include in vivo studies in animal models and mutagenicity assays.
Open Access
Immunogenicity and serological applications of flavivirus ED III proteins and multiplex RT-PCR for detecting novel Southern African viruses
(University of the Free State, 2015-01) Mathengtheng, Lehlohonolo; Burt, Felicity; University of the Free State, Grow Our Own Timber Fellowship
English: West Nile virus (WNV) is endemic to southern Africa but the true burden of disease associated with WNV infection remains unknown in this region. The presence of the mosquito-borne Wesselsbron virus (WESSV) has also been established in southern Africa. Although not considered a serious human pathogen, WESSV has been associated with encephalitis in humans. No routine testing is performed for WESSV diagnosis in South African patients and hence, similar to WNV infections, the virus remains unreported and overlooked. The presence of tick-borne flaviviruses in southern Africa on the other hand, has not been established despite the presence of suitable vectors. A challenge associated with serological identification of flaviruses is the high level of cross-reactivity between members of flaviviruses and the impracticality of using neutralization assays. Serological assays using reagents that can be handled in a biosafety level 2, or lower facility, were developed and evaluated for the detection and differentiation of tick- and mosquito-borne flaviviruses in the Free State province of South Africa. A total of 2393 serum samples from a variety of species including humans, cattle and sheep were tested using Kunjin virus (KUNV) cell lysate antigen for the detection of anti-flavivirus antibodies in an indirect IgG enzyme-linked immonosorbent assay (ELISA). To further differentiate positive reactors on KUNV assay for antibodies against tick- or mosquito-borne flaviviruses, recombinant envelope domain III (r-EDIII) proteins of Langat virus (LGTV), WNV and WESSV were expressed in a bacterial expression system and used in ELISA. A total of 722 samples were positive on the KUNV assay of which 71, 457 and 431 were positive on the r-LGTVEDIII, r-WNVEDIII and r-WESSVEDIII assays, respectively. A total of 70 samples were reactive on the KUNV assay but not on any of the other assays, suggesting that there are other flaviviruses circulating in the Free State province for which specific r-EDIII assays were not available. Collectively, the results suggest a strong presence of flaviviruses co-circulating in the Free State province with an abundance of mosquito-borne flaviviruses. There is evidence suggesting the presence of tick-borne flaviviruses but it has yet to be confirmed. The EDIII protein is a useful tool that can be utilized in the detection and differentiation of flaviviruses in resource-limited laboratories. Vertebrate hosts play a role in the maintenance and circulation of flaviviruses and, although not involved in the direct transmission of tick- and mosquito-borne flaviviruses, form a link for virus transmission between vectors. In addition to rodent involvement in maintenance of flaviviruses, rodents have also been implicated in the transmission of other medically significant viruses such as arenaviruses, lyssaviruses and hantaviruses. Arboviruses and viral heamorrhagic fevers are among the most pathogenic and devastating disease agents in many parts of the world. It is therefore important for surveillance of such pathogens to be conducted as they may result in considerable public health implications. Molecular assays were developed for the detection of a selected number of arboviruses and viral heamorrhagic fevers, specifically Crimean-Congo haemorrhgaic fever virus (CCHFV), mosquito-borne and tick-borne flaviviruses, as well as hantaviruses. To date, the presence of hantaviruses have not been confirmed in southern Africa despite their emergence in the western and eastern parts of Africa in recent years. In our study, serum samples of patients presenting with a tick-bite and febrile illness without diagnosis were screened for hantavirus IgG antibodies using commercial assays that represent the American and Eurasian hantavirus species. The overall seropositivity rate obtained was 10% and 6% for assays representing the Eurasia and America hantavirus species, respectively. The emergence of hantaviruses in Africa and their seroprevalence in the Cape region of South Africa as well as in our study warranted the development of a molecular assay to further investigate the presence of these viruses in southern Africa. In order to achieve this, a real-time RT-PCR was designed and optimized. The assay was designed by identifying in-house primers targeting the partial region of the S-segment of hantaviruses and hydrolysis probes targeting the inner region of the amplicon. The probes were based on nucleotide sequences targeting the Murinae-associated hantaviruses for the HNLS probe, Sigmodontinae- and Arvicolinae-associated hantaviruses for the ASPRB probe, as well as the SANGV probe for the African hantavirus Sangassou virus. The flavivirus RT-PCR targeted the NS5 region with a probe shown to successfully detect RNA samples that represent eight different flavivirus species. The hantavirus primers and probes were evaluated using RNA transcribed from synthetic genes representing the different hantaviral genotypes and subsequently reverse transcribed cDNA. The limit of detection was determined to range from ~160 to ~17 copies of DNA for the various hantaviral probes and flavivirus probe. In addition, a conventional multiplex PCR assay aimed at detecting CCHFV and flavivirus RNA in samples collected from undiagnosed patients presenting with a tick-bite and febrile illness was developed by using nested primers targeting the partial region of the genome of the S-segment of CCHFV and hemi-nested primers targeting the partial region of the NS5 gene of flaviviruses. When clinical samples from patients with known tick-bites, mild disease and no diagnosis were screened, a patient was restrospectively diagnosesd as having a CCHFV infection. This result highlights the need for awareness to arboviruses and viral hemorrhagic fevers in mild cases that may easy be overlooked but constitute a significant public health risk. Similarly, there needs to be an increase in awareness for travelers to South Africa at risk of returning to their country with an exotic viral haemorrhagic fever, highlighting the need for increased awareness and increased diagnostic capacity for arboviruses. Finally the current lack of registered human vaccines warrants continued investigation of the immunogenicity of selected viral proteins. The recombinant antigens developed for serological purposes were further employed in this study to determine the immunogenicity of the envelope domain III proteins of WNV and LGTV in a mouse model. Small molecule antigens or weakly immunogenic antigens frequently require an adjuvant to stimulate a stronger immune response. In addition, adjuvants can shift an immune response towards a Th1 or Th2 response as required based on immune correlates of protection. Groups of mice were immunized with purified r-WNVEDIII or r-LGTVEDIII protein alone, r-WNVEDIII or r-LGTVEDIII protein in combination with one of three adjuvants, including saponin, Titermax® gold and Alhydrogel® or one of the three adjuvants without a flavivirus protein. In the absence of any adjuvant the results from WNV protein alone were inconclusive whereas a strong IgG1 response was induced by LGTV EDIII. Briefly, protein alone or mixed with alum elicited a predominantly Th2 response whereas protein in combination with saponin or Titermax® gold induced a mixed Th1 and Th2 response. Mice immunized with r-WNVEDIII reacted against KUNV native antigen indicating that the protein was expressed in conformation exposing epitopes that are required to induce a detectable antibody response. The formulation of the WNV and LGTV proteins with different adjuvants produced similar results with a shift in response depending on the adjuvant. Despite an absence of being able to assess cell mediated responses using antigen stimulated splenocytes and profiling cytokine production as initially planned, the results do confirm that r-WNVEDIII and r-LGTVEDIII proteins are immunogenic in the absence of complete E protein, with ability to induce detactable antibody when formulated with adjuvant and that different adjuvants are able to have an immunomodulatory influence on the type of response induced.
Open Access
Immunogenicity of Sindbis based replicons for Crimean-Congo hemorrhagic fever virus
(University of the Free State, 2019-02) Tipih, Thomas; Burt, Felicity
Introduction and Aim: Crimean-Congo hemorrhagic fever virus (CCHFV) infrequently causes hemorrhagic fever in humans with a case fatality rate of 30%. Currently, there is neither an internationally approved antiviral drug nor vaccine against the virus. In a move aimed at averting future epidemics, the World Health Organization has added the virus to the list of priority infectious organisms. The aim of the study was to investigate mechanisms of immunogenicity of Sindbis replicons encoding CCHFV glycoproteins and nucleoproteins for future development of an efficacious vaccine. Methodology: Genes encoding the complete open reading frames of the CCHFV nucleoprotein and glycoprotein precursor proteins of South African strains were amplified by the reverse transcription polymerase chain reaction technique and cloned into a Sindbis virus replicon vector. Sanger sequencing and next-generation sequencing were carried out to confirm gene sequences. Nucleoprotein and glycoprotein expression were demonstrated by transfecting baby hamster kidney cells and human embryonic kidney cells. Vaccine construct self-replication rates were assessed by transfecting BHK-21 cells and assaying for CCHFV RNA using gene-specific primers. Apoptosis induction in transfected BHK-21 cells was determined by measuring the enrichment of nucleosomes in the cytoplasm using an ELISA. Groups of three NIH mice were immunized with 100 μg of vaccine constructs three times intramuscularly three weeks apart with plasmid constructs pSinCCHF-31S, pSinCCHF-52S and pSinCCHF-52M. To augment cytokine responses the adjuvant poly (I:C) was co-inoculated with pSinCCHF-52S and pSinCCHF-52M separately. In addition, the constructs pSinCCHF-52M and pSinCCHF-52S were co-immunised with and without poly(I:C) to induce a response against both proteins simultaneously. Two weeks after receiving the third dose mice were sacrificed and blood was collected for determination of humoral immune responses while harvested splenocytes were stimulated with a CCHFV antigen for cytokine responses. Results: Two vaccine constructs (pSinCCHF-31S and pSinCCHF-52S) expressing CCHFV nucleoprotein and a construct (pSinCCHF-52M) expressing CCHFV glycoprotein were prepared. Recombinant protein expression was demonstrated by immunofluorescence assays targeting the histidine tag fused to the CCHFV proteins. Further confirmation of protein expression was performed by immunofluorescence assays using serum from CCHF survivors. All prepared vaccine constructs transcribed CCHFV RNA, as demonstrated by detection of protein using immunofluorescent antibody assays, and induced apoptosis in transfected cells. Immunized mice responded with the production of high titers of CCHFV IgG NP specific antibodies and higher levels of IgG2a in comparison to IgG1 responses were observed in responders suggesting a predominant Th1 antibody response. CCHFV IgG GP specific antibodies were not induced in vaccinated mice. Vaccine construct pSinCCHF-52S resulted in higher secretion of IL-2, (p = 0.0495) IFN-γ (p = 0.0369) and TNF-α (p = 0.0495) relative to immunisation with pSinGFP. An enhanced secretion of IFN-γ and IL-2 (p = 0.0463) was observed from splenocytes from mice co-immunised with pSinCCHF-52S and pSinCCHF-52M while vaccinating with pSinCCHF-52M increased IL-2 secretion (p = 0.0463). Co-administration of pSinCCHF-52M and pSinCCHF-52S constructs augmented IFN-γ (p = 0.0463) secretion. Co-inoculation of vaccine constructs with adjuvant poly (I:C) did not enhance cytokine secretion. Conclusion: The study demonstrated the expression of CCHFV nucleoproteins and glycoproteins by a Sindbis virus vector in mammalian cells. Vaccination of mice with construct pSinCCHF-52S induced type 1 immunity. Immunoglobulin G subtyping demonstrated IgG2a/IgG1 >1 as well as significantly higher IL-2, IFN- γ and TNF- α. Immunisation with pSinCCHF-31S and pSinCCHF-52M did not elicit specific antibody production and cytokines responses were weak. Further studies in CCHFV susceptible animals are necessary to determine whether the immune responses generated by vaccinating with pSinCCHF-52S are protective. However, this study shows the utility of Sindbis replicons in vaccine development against CCHFV.