Medical Microbiology

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Open Access
Strain characterisation, antibiotic resistance and Meca Gene analysis of Staphylococci
(University of the Free State, 1999-12) Vorster, Alvera; Chalkley, L. J.
S. aureus, is undoubtedly the most pathogenic of the Staphylococcus species, having the ability to produce invasive and toxigenic infections. Historically, the less virulent coagulase-negative staphylococci (ENS) were regarded as clinically insignificant contaminants but they have become increasingly implicated as opportunistic nosocomial pathogens. The increasing frequency of methicillin and multiple-antibiotic resistance in staphylococci over the last four decades has seriously compromised therapeutic options. The study was designed to (a) identify and type staphylococcal species, (b) undertake standardized antimicrobial susceptibility testing, and (c) determine the prevalence of methicillin resistance in staphylococcal isolates. Presumptive staphylococcal strains were isolated from the diagnostic microbiology laboratories of Pelonomi (147 strains) and Universitas (144 strains) hospitals. Subsequently, these strains were identified using conventional biochemical methods. Species-specific PCR identification assays were performed on selected ENS strains. Antimicrobial susceptibilities were determined for 13 clinically available antibiotics on 144 staphylococcal isolates and on selected strains for 5 developmental agents. RAPD and plasmid profiles were generated to assess possible epidemiological strain relatedness. For the detection of methicillin resistance in staphylococci the following methods were used: (a) oxacillin MICs detecting phenotypic methicillin resistance levels (b) a multiplex-PCR detecting the mecA gene, and (c) a slide agglutination test (MASTALEX-MRSA) detecting PBP2' production. The inclusion of bile-aesculin agar plates and a bacitracin susceptibility test into the diagnostic laboratory protocol for the identification of staphylococci would reduce misidentification of non-staphylococcal isolates by 12.8%. Colony morphology in combination with the coagulase test could be instrumental in the improved differentiation of S. aureus from CNS. Although expensive, when a rapid and fairly comprehensive identification of CNS species is required, the STAPH ID 32 API system was found to be satisfactory. Due to the apparent inaccuracy of the PCR identification assay based on API, its use in the clinical microbiology laboratory would be argued against; although if standardized and expanded it could be considered for future incorporation in routine practice. The presence of unique RAPD profiles for each specific Staphylococcus species suggests RAPD profiling could offer a molecular identification technique for the majority of commonly isolated CNS in the clinical microbiology laboratory. Good typeability was observed for Primer I and III in CNS strains, however, for S. aureus strains, poor typeability and discrimination was observed. It has been found by other researchers that longer oligonucleotide primers (>10 bp in length) are more efficient for S. aureus strain typing, but to the contrary in the present study primers ERIC 1 and 2 were totally unsatisfactory. Combined susceptibility data and plasmid profile analysis revealed strain relatedness in S. haemolyticus isolates but RAPD Primers I and III indicated otherwise. All staphylococcal strains isolated were vancomycin-susceptible. Of the staphylococci isolated in the Universitas hospital, 34.3% were oxacillin-resistant. Similarly, 30.1% staphylococci isolated in Pelinomi hospital were oxacillinresistant. Resistance to ciprofloxacin, erythromycin, gentamicin and clindamycin was found in 49% of oxacillin-resistant staphylococci. In comparison to the other quinolones tested, moxifloxacin showed superior activity against oxacillin-resistant CNS. The glycylcyclines, LY333328 and Q/D may well be considered excellent alternatives to vancomycin for the treatment of MRSA. Of the developmental agents investigated, linezolid showed consistent in vitro activity against all staphylococci. The inadequacy of a single diagnostic method for the detection of methicillin resistance in staphylococci was evident when comparing (a) susceptibility data, (b) multiplex-PCR for mecA gene detection, and (c) PBP2' detection. None of these methods was seen to correlate with each other at the 100%-level. The detection of PBP2' was rapid although, in comparison to mecA gene detection and antimicrobial susceptibility tests, inaccurate for the identification of methicillin resistance in staphylococci. DNA sequencing of a fragment of the mecA gene in selected staphylococcal strains revealed minimal sequence variation. This was an indication that variable levels of methicillin resistance in staphylococci can be attributed to different mechanisms of methicillin resistance or variations in the expression of the mecA gene, rather than mutations within the gene itself. The low sequence variation observed in the mecA gene is primarily responsible for initial assumptions of a clonal origin for methicillinresistance in staphylococci. As of yet, pharmaceutical companies have failed to produce an analogous antimicrobial agent to β-Iactam agents that would be able to specifically target PBP2'. The development of such an agent would be instrumental in the reduction of glycopeptide selection pressure. It is imperative that correct identification, strain typing, susceptibility testing and methicillin resistance detection is performed to direct therapy and epidemiologically monitor methicillin-resistant strain types.
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
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
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
Molecular epidemiology of Mycobacterium tuberculosis strains from the Free State and Northern Cape provinces, South Africa
(University of the Free State, 2004-05) Mokhethi, Sehloho Zacharia; Van der Spoel van Dijk, Anneke
Background. Tuberculosis is increasing in the Free State and Northern Cape provinces of South Africa, but it is not clear how much of the disease is caused by reactivated latent infection and how much is attributed to interpersonal transmission. The discovery of the transposable DNA insertion sequence, IS6110, provided the desired polymorphism among different strains to track routes of transmission, study the degree of inter-person transmission versus reactivation, to detect laboratory contamination and disease outbreaks. Alternative methods include spoligotyping and the mycobacterial intergenic repetitive units or variable number of tandem repeats (MIRU-VNTR). Sustained studies performed on a small area in the Western Cape Province and some mines in the Gauteng Province of South Africa have found person-to-person transmission of tuberculosis to be high in these populations. In addition, resistance determinants to key antituberculosis drugs have remained unknown among tuberculosis causative organisms circulating in the Free State and Northern Cape. Thus, extensive DNA fingerprinting and gene mutation studies are needed to address these problems. Methods. An area in the Free State suitable for long-term surveillance studies was defined using available information from the governmental database, the 1996 census statistics, and tuberculosis (TB) case loads and transfer data obtained from the National Tuberculosis Database. Each clinic’s catchment information was provided by clinic managers and the population movement data from a 2002 student project. Sputum samples were collected and Mycobacterium tuberculosis isolated from tuberculosis positive patients from the defined area (Gamadi). Isoniazid resistant isolates received from a representative sample from the Free State and a few strains from the Northern Cape Province were also included in the study. IS6110-directed restriction-fragment-length polymorphism (RFLP) analysis was performed on all isolates and drug susceptibility testing (indirect proportion method) done on the Gamadi isolates. Subtyping of identical strains (RFLP clusters) and some of the isolates with less than six IS6110 bands was done using spoligotyping and the MIRU-VNTR typing. DNA sequencing analysis of the katG and rpoB genes was done in resistant isolates and a rapid PCR-based restriction enzyme katG gene mutation detecting method evaluated. Results. An area characterised by extreme poverty (unemployment rate 69.0%), a relatively young population (69.0% below 35 years) of 61534 and with high incidence of tuberculosis (840/100 000) suitable for long -term surveillance studies was identified in the Free State. The area is served by three clinics and a hospital and is situated near the rural town of Thaba Nchu in the Free State province. Eighty eight M. tuberculosis isolates and a mycobacterium-other-than-tuberculosis (MOTT) were isolated from the 286 sputum specimens collected from the Gamadi area. Only two M. tuberculosis isolates tested isoniazid (INH) resistant and no rifampicin (RIF) resistant isolates were found. The MOTT was resistant to INH (0.2, 1 and 5 µg/ml) and to RIF. Standard IS6110-based DNA fingerprinting of 84 of 88 (96.5%) isolates from the defined area was performed. Four of the isolates were cultured from duplicate sputum specimens provided by four patients. Two of these had identical fingerprint patterns to the first isolate of the patient and two had a different profile. The latter pair could be attributed to laboratory error. IS6110 sequences were not detected in six isolates. Fourteen isolates had less than six IS 6110 hybridisation bands and four strains were in clusters. The remaining 57 (88.9%) strains had distinct RFLP profiles with more than six bands. The number of IS6110 copies varied from seven to 21. A total of five strains was distributed in two clusters, one with two and the other with three members. Thirteen family groups, clustered at 65.0% on the similarity dendogram, each with two to eight strains, but no dominant groups were evident. A cluster of three isolates with five identical IS 6110 bands each was confirmed as one strain by MIRU-VNTR typing while two further isolates (both had three bands of different sizes) were confirmed as different strains by MIRU typing. A total of 37 isoniazid-resistant M. tuberculosis was analysed. DNA fingerprint profiles showed nine isolates with less than six insertions (24.3%). Six of these isolates were from the Free State and three from the Northern Cape Province. Three of these isolates were multidrug resistant. The remaining 28 isolates (75.7%) contained between 9 and 18 copies of the IS6110 insertion sequence. Twenty-six different IS 6110 RFLP types were identified. Only two clusters with two isolates, respectively, were found in each province. Eight clonally related groups (65.0% similarity) with two to four strains were present. Three clusters of two isolates (each with more than six bands) also exhibited identical spoligotype patterns. Spoligotyping of two of three isolates from a fourth cluster (4 RFLP bands each) showed two different banding patterns and all were shown to be different by MIRU-VNTR typing. The fifth cluster (2 bands) was made up of one isolate from each province. Spoligotyping of these strains was identical, but the MIRU was different. One isolate from Bloemfontein had identical IS 6110-RFLP and spoligotyping patterns to a susceptible isolate from Gamadi. Isoniazid resistance in 22/37 isolates was sequence linked to altered nucleotides of codon 315 of the katG gene. Twenty harboured the ACC variant at the codon. One strain carried the AAC mutation at this codon and the other GGC. The remaining 15 carried the wild type (AGC) genotype at this site. Two of the strains harbouring the AGC315ACC mutation belonged to the same IS6110 cluster. Two mutations were found at codon 463 (CGG ® CTG; CGG ® CCG). Thirteen MDR strains were investigated for rpoB gene alterations. Four of these isolates carried no mutations within the 157-bp amplified fragment while the others had various mutations. Analysis of an 808bp fragment of the katG gene from INH-resistant M. tuberculosis isolates after restriction with Msp I agreed with results obtained by sequencing. Thirteen isolates carried a pattern consisting of 228, 153, 146, 109, 79, 65 base pairs with the 153 bp fragment indicating the presence of the wild type AGC at codon 315 of the katG gene. Seventeen isolates demonstrated the 228, 146, 132, 109, 79, 65, 21 profile with the 132 bp fragments indicating the presence of an ACC mutation. Three isolates contained a mixed genotype and were digested into the fragments 228 bp, 153 bp, 146 bp, 132 bp, 109 bp, 79 bp, and 65 bp. Fragments with 146 bp and 65 bp are seen in strains with no mutation (bases CGG) at codon 463, while a 211 bp fragment shows a mutation at this spot. Four strains had the fragments 228, 211, 153, 109, and 79 bp. One strain was digested into six fragments of 228 bp, 211 bp, 132 bp, 109 bp 79 bp and 21 bp containing both a 315 (ACC) and 463 (CTG) codon mutation. Discussion and conclusions. An area consisting of ten villages and characterised by a high incidence of tuberculosis was defined for long-term surveillance studies. Resistance in the area appears to be low and compares favourably to the situation in the Free State. Strains received from this area were highly diverse, but the presence of a cluster of five isolates indicated the need for continuous investigation. Recent transmission of INH resistance in the Free State province is not a significant factor, but since the isolates from the Northern Cape were not representative, no deduction could be made for this province. Resistance to INH is mostly associated with mutation AGC to ACC at codon 315 of the katG gene. The absence of alterations in a proportion of isolates is in agreement with published data implicating the involvement of more genes in causing INH resistance. Resistance to RIF was associated with various point mutations in the 81-bp core region of the rpoB gene. The high proportion of the ACC allele found among INH-resistant strains, cost effectiveness, ease to perform and rapid results, make PCR-RFLP an attractive option for detection of resistance especially in resource-poor countries.
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
Preparation of recombinant antigens for demonstrating antibody responses in patients with Crimean-Congo haemorrhagic fever virus infections
(University of the Free State, 2011-06) Samudzi, Rudo Ruth; Burt, F. J.
Crimean-Congo haemorrhagic fever (CCHF) is a tick-borne viral zoonosis widely distributed in Africa, Asia, Russia and the Balkans. The causative agent, CCHF virus (CCHFV) has the propensity to cause nosocomial infections with a high fatality rate. Cases of CCHF are diagnosed annually in southern Africa. Increasing numbers of cases are seen in regions of Asia and in the past ten years CCHFV has emerged in several countries in the Balkans and re-emergence in south-western regions of the Russian Federation. Diagnosis of CCHFV infections during the acute phase is based on isolation of the virus or amplification of viral RNA. Patients that survive the infection have a demonstrable IgG and IgM antibody response, usually from day 5 to 7 after onset of illness. Current serological diagnostic assays based on ELISA or IF use inactivated virus which requires biosafety level 4 facilities for culturing the virus and therefore limits the number of laboratories that can prepare suitable reagents. Preparation of recombinant antigens would enable laboratories to perform serological diagnosis of CCHFV infections and surveillance studies. The purpose of this study was to prepare a recombinant CCHFV nucleoprotein using a bacterial expression system, to determine if the protein was immunogenic and to determine if the protein was able to detect IgG antibodies in survivors of CCHFV infection. The complete open reading frame of the gene encoding the NP of CCHFV was amplified by RT-PCR using primers specifically designed with restriction sites engineered to the primers to facilitate cloning. The amplicon was cloned into pGEM® T Easy vector using T/A cloning and the gene sequenced to confirm that the correct gene had been amplified and cloned into the vector for downstream cloning and expression applications. Initially we aimed to express the native gene using a bacterial expression system and the NP gene was rescued from the recombinant plasmid and cloned into pQE-80L vector using the BamH1 and Pst1 restriction sites present in the multiple cloning site on the vector. Various attempts were made to express the CCHFV NP protein however no protein was detectable using SDS PAGE methods or Western blot. The nucleotide sequence that we had determined for the open reading frame of our gene encoding the NP was analysed using the Rare Codon Analysis Tool software and we elected to codon optimize the gene for expression in E. coli. The optimized gene was synthesized by GenScript and supplied cloned in the multiple cloning site of pUC57. The optimized gene was excised from pUC57 and cloned into pColdTF bacterial expression vector. A 106 kDa protein was expressed from the construct likely representing the HIS tagged TF chaperone protein fused to the CCHFV NP protein and confirmed by Western blot analysis. A higher yield of the protein was present in the insoluble phase and as optimization of the growth and induction conditions did not significantly alter the insoluble to soluble ratio of the expressed protein, the protein was harvested from the insoluble phase by denaturing, purification and refolding of the protein. The biological activity of the recombinant protein was confirmed using immunoassays and by immunizing mice to determine if the antibodies induced by the recombinant protein could be detected using an antigen prepared from the whole virus. Four of five mice immunized with the recombinant NP had a detectable antibody response using an immunofluorescent assay. Serum samples from acute and convalescent patients collected at varying stages after onset of illness were reacted in a Western blot with the recombinant CCHFV NP protein. The recombinant antigen was able to detect IgG antibody in all the convalescent patient sera except two sera collected on days 14 and 15 during the acute phase. In contrast all the samples were detected using the recombinant antigen in an ELISA. Due to the potential biohazardous nature of samples only samples collected two weeks after onset of illness were tested. The results showed 100% concordance with the results obtained in an ELISA using mouse brain derived antigen. The assay was shown to be reproducible and stability studies showed that four months after preparation the protein was still active. A full validation of the protein using a large panel of serum samples from confirmed CCHF patients is now required. The results suggest that bacterially expressed proteins lacking post translational modifications and folding that occur with mammalian and baculovirus expression can be used in ELISA to detect IgG antibody against CCHFV in human sera which finds application in diagnostics, epidemiologic and surveillance studies.
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
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
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
Preparation and immunogenicity of a candidate replicon based yellow fever vaccine
(University of the Free State, 2014-12) Viljoen, Natalie; Burt, Felicity Jane
English: Yellow fever virus (YFV), a mosquito-borne virus that belongs to the family Flaviviridae and genus Flavivirus, is a significant cause of morbidity and mortality in yellow fever endemic areas, especially in West Africa. In humans, YFV causes yellow fever, a disease characterised by renal failure, jaundice, and/or haemorrhage. The burden of disease is highest in Africa constituting approximately 90% of reported cases worldwide. Despite the availability of highly efficacious live attenuated vaccines against YFV, the estimated prevalence for yellow fever in Africa was 130 000 severe cases and 78 000 deaths for 2013. The available live attenuated vaccines have been contraindicated for use in immunocompromised patients and individuals with hypersensitivity to eggs and/or chicken. Vaccine-associated neurotropic adverse events that result in the development of meningoencephalitis in infants and vaccine-associated viscerotropic adverse events that result in disease resembling wild-type yellow fever have been reported. Vaccine-associated viscerotropic adverse events are associated with fatality rates exceeding 40%. Therefore, there is a need for a safer alternative to complement the use of the available live attenuated vaccines. The aim of this study was to construct a DNA-launched candidate vaccine against YFV and to determine the immunogenicity of the DNA-launched pSinED-lll replicon, which would provide information regarding the applicability of DNA-launched replicons as an approach to vaccine development. The pSinGFP replicon encoding the green fluorescent protein (GFP) was kindly provided by Prof. Mark Heise. Expression of the GFP was confirmed in mammalian cell culture post-transfection with the pSinGFP replicon, thus confirming the functioning of the replicon elements and subsequent expression of the encoded protein. The gene encoding the GFP was excised and replaced with a synthesised codon-optimised gene encoding the YFV ED-lll protein using directional cloning. The nucleotide sequence was confirmed by bidirectional sequencing at the site of insertion and subsequently protein expression was confirmed in selected mammalian cell lines. Protein expression was confirmed by detection of the C-terminal histidine tag by immunofluorescence using anti-His6 mouse monoclonal antibody. Thereafter, the expressed protein was characterised by immunofluorescence using mouse immune sera previously shown to contain anti-YFV ED-lll antibody. Reactivity of the expressed protein with anti-YFV ED-lll antibody substantiated the use of the pSinED-lll replicon in a mouse immunisation study. Mice were immunised intramuscularly according to pre-approved immunisation regimes consisting of DNA only, as well as mixed modal immunisation regimes. Serum samples were collected and spleens were harvested two weeks after the administration of the final immunisations. Serum samples were screened for anti-YFV antibodies by an enzyme-linked immunosorbent assay (ELISA) and immunofluorescence to determine the induction of a humoral immune response. Five of the twenty mice in groups immunised with the DNA-launched pSinED-lll replicon developed low level antibody responses illustrating the potential of the pSinED-lll replicon to induce a humoral immune response. Splenocytes were stimulated in cell culture with concanavalin A, YFV ED-lll protein, or no stimulant to facilitate the detection of cytokine release using ELISA’s. A predominantly T-helper 1 cell-mediated immune response characterised by high level release of interferon-γ post-stimulation with the YFV ED-lll protein in vitro was elicited. The release of interleukin-10 post-stimulation may be associated with the prevention of immunologically mediated damage to the host by preventing an overzealous inflammatory immune response. Antibody-mediated anti-vector immunity should not negatively impact the immunogenicity of the DNA-launched pSinED-lll replicon as antibody directed against the encoded Sindbis virus non-structural proteins was not detected in mouse sera. In conclusion, the pSinED-lll replicon was shown to have the potential to induce both a cell-mediated and a humoral immune response post-immunisation. However, optimisation of the humoral immune response will be required.
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
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
Preparation of recombinant antigen for serological detection of African hantaviruses
(University of the Free State, 2017-07) Damane, Deborah Rethabile; Burt, Felicity Jane
Unlike other members of the Bunyaviridae family, hantaviruses are transmitted to humans through direct exposure or inhalation of virus contaminated urine or droppings from their reservoir hosts. Hantaviruses were first discovered in 1976 with the identification of Hantaan virus (HNTV) from the reservoir Apodemus agarius in Asia and later in North America. In 2006, Sangassou virus (SANGV) was the first to be isolated in Africa in the African house mouse, Hylomyscus sinus and subsequently followed by the identification of ten more African hantaviruses in both rodent and insectivore hosts through reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence assay (IFA). Hantaviruses are a public health concern with annual cases of disease reported to be approximately 200,000 per year, with most cases reported in Asia. In Africa, disease associated with hantaviruses is not well defined. Culturing the virus and preparing reagents using native virus requires the use of biosafety level (BSL) 3 or 4 laboratories limiting the number of facilities with capability to prepare serological assays. Hence, the use of recombinant antigens that are safe to use in a BSL 1 laboratory that have application as serological tools for surveillance are required. The aim of the study was to develop serological assays to test for antibodies against hantaviruses in human serum samples collected in the Free State, South Africa using a recombinant nucleocapsid protein (NP) of SANGV as a representative of African hantaviruses. Transiently transfected cells were used to prepare antigen slides for IFA and expressed protein was used in an in-house enzyme linked immunosorbent assay (ELISA). In-house assays and commercially available ELISA kits were used to screen human serum samples. There are limited seroprevalence studies performed in Africa to detect IgG antibodies against hantaviruses in humans and no commercial serological assays are available using an African antigen. Hence, it was considered that the preparation of a recombinant African hantavirus antigen based on SANGV could have application in serological surveillance studies. The S gene segment of the SA14 strain of SANGV was modified and codon optimized for enhanced expression and detection. The construct was sequenced and aligned to the native S gene. It was used to transfect baby hamster kidney cells (BHK-21). Expression of a 50kDA protein was confirmed by SDS-PAGE and Western blot assay. Antigen slides were prepared from transfected cells fixed on 12 well chamber slides. Positive controls from the commercially available ELISA kits were used in the IFA. Four of the 176 serum samples tested gave a positive test. For the in-house ELISA, protein was harvested from T75 culture flasks. The antigen was tested using positive and negative controls from the commercial ELISA kits. A suitable differentiation between positive and negative samples was not detectable despite attempts to optimize the in house ELISA. It is likely that the protein yield was insufficient for the ELISA and further attempts, beyond the scope of this project, to increase the protein yield will be investigated using a stable cell line. Commercially available ELISA kits comprising of HNTV, Dobrava (DOBV) and Puumala (PUUV) recombinant NP antigens for Europe and Asia group and Andes virus (ANDV) and SNV for the American group were used to screen acute human sera in the laboratory. Positive reactors were identified using both kits. The significance of the results is difficult to interpret as there was lack of concordance. However, it does suggest that hantaviruses are to be found in this area and that the use of a homologous antigen for serological surveillance is essential. Results confirmed some serological cross-reactivity between heterologous Asian, American and African hantaviruses and a potential application for an African hantavirus as a tool for surveillance.
Open Access
PhyloPi: an affordable, purpose built phylogenetic pipeline for the HIV drug resistance testing facility
(2018) Bester, Phillip A.; De Vries, Andrie; Riekert, S. J. P. K.; Steegen, Kim; Van Zyl, Gert; Goedhals, Dominique
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
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
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.
Open Access
Whole-genome analyses of rotavirus strains circulating pre- and post- Rotateq™ vaccine introduction in Rwanda
(University of the Free State, 2020) Rasebotsa, Sebotsana Paula; Nyaga, Martin; Sabiu, Saheed
𝑬𝒏𝒈𝒍𝒊𝒔𝒉 Children living in developing countries are constantly faced with the burden of diarrheal infections that account for over 1.6 million death cases globally. Rotavirus group A (RVA) has been identified as one of the viruses implicated in most viral-induced diarrhoeal infections in children less than five years worldwide. In Rwanda, over 3500 RVA related mortality cases were reported yearly prior to the implementation of the RotaTeq® vaccine in 2012 to overcome this burden, which led to a significant decrease in rotavirus infections. Africa has a huge diversity of rotavirus strains compared to other developed continents especially Europe and North America, thus requiring a deeper understanding of this phenomenon. This study aimed at characterizing all the 11-segments of RVA strains circulating in Rwanda pre- and post-vaccine introduction as part of the World Health Organization (WHO) supported African rotavirus pilot surveillance program. The study was based on 158 rotavirus positive samples that were collected from children presenting symptoms associated with rotavirus infection between 2011 and 2016. The rotavirus double-stranded ribonucleic acid (dsRNA) was extracted from the viral particles and converted into complementary deoxyribonucleic acid (cDNA) prior to library preparation for whole-genome sequencing with an Illumina MiSeq platform. Several bioinformatics tools were utilized to construct phylogenetic trees and the proteins structures. From the sequenced samples, 36 samples were identified as G1P[8] strains, and five samples were reassortant strains. Ten G1P[8] strains were identified pre-vaccine introduction while 26 were identified post-vaccine introduction. Thirty-five of the G1P[8] strains expressed pure Wa-like genome constellations, while one of the strains that was identified in 2012 exhibited a genome constellation typical of a RotaTeq® vaccine strain. On the other hand, the five reassortant strains were identified post-vaccine introduction between 2013-2015. Whole-genome analysis revealed that the G4P[4], G9P[4] and one G12P[8] reassortant strains exhibited both the Wa-like and the DS-1-like genome constellations while two G12P[8] strains had all the three genogroup constellations. Furthermore, the phylogenetic analysis of most of the G1P[8] strains revealed that they segregated according to their vaccination status; strains identified pre-vaccine introduction clustered together while post-vaccine strains also formed a separate cluster. The five reassortant strains were closely related to human RVA strains in all the gene segments and RotaTeq® vaccine strains in the VP1, VP2, NSP2, NSP4, and NSP5 gene segments. Analysis of the neutralization epitopes and cytotoxic T-lymphocytes (CTL) of the G1P[8] strains revealed multiple amino acid substitutions, with some changes influencing the change in polarity thus deemed to be radical in nature. A similar trend was also observed in the reassortant strains, with 27 amino acid substitutions in the VP7 epitope region and only three substitutions in the VP4 epitope region. Changes observed in these epitope regions have the potential of generating vaccine-escape mutants that may undermine the effectiveness of the rotavirus vaccine with time. Whole-genome sequencing has proven to provide information that could have been missed when looking only at the outer capsid proteins. It is thus important to continue conducting rotavirus whole-genome studies to unpack the hidden information behind the huge diversity of rotavirus strains in African countries such as Rwanda. ___________________________________________________________________

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