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Biomedical subjects

L H Nel

Publications and source records attributed to L H Nel.

At least 19 recordsLinked to original sources

Mongoose rabies in southern Africa: a re-evaluation based on molecular epidemiology.

Relative to the developed world, rabies has been poorly studied in the vast African continent. The southern African countries of Zimbabwe and South Africa, however, are known to sustain a great diversity of lyssaviruses, with large biological variations amongst genotype 1 (rabies viruses) at present more apparent here than elsewhere on the continent. One recognized biotype of rabies virus in the subcontinent appears to be specifically adapted to a variety of mongooses, belonging to the Viverrinae subfamily (family Herpestidae) and are commonly referred to as viverrid viruses, although the term mongoose rabies would be more correct, considering the taxonomic status of the host species involved. It was our objective to study the genetic relationships of 77 rabies virus isolates of this mongoose biotype, isolated in South Africa and Zimbabwe, towards elucidation of the molecular epidemiology of this interesting group of African viruses. In our study of a 592 nucleotide sequence encompassing the cytoplasmic domain of the glycoprotein and the G-L intergenic region of the viral genomes, we provide the first comprehensive data on the molecular epidemiology of these viruses and indicate a history of extended evolutionary adaptation in this geographical domain. The molecular epidemiological observations reported here are highly unlikely to be limited to the small geographical areas of South Africa and Zimbabwe and illustrate the need for lyssavirus surveillance in the rest of sub-Saharan Africa and throughout the entire continent.

Adaptation, Biological↗

Segment specific inverted repeat sequences in bluetongue virus mRNA are required for interaction with the virus non structural protein NS2.

Computational secondary structure prediction of all 10 Bluetongue virus (BTV-10) RNA transcripts and mutant inverted repeat transcripts were performed. Transcripts with intact 5' and 3' inverted repeat sequences, all indicated base-pairing between the 5' and 3' ends when optimal folding parameters were applied. Secondary structure analysis of the mutant transcripts lacking the inverted repeat sequences indicated alterations in the secondary structures resulting from altered base-pairing. The importance of the inverted repeat sequences in RNA--protein binding was subsequently investigated. Deletion mutant clones lacking the 5' and/or 3' inverted repeat sequences have been constructed. A baculovirus recombinant expressing the BTV NS2 protein and radioactively synthesized RNA transcripts were subjected to nitrocellulose RNA--protein binding assays. The cumulative results suggested that the inverted repeat deletion mutants display weaker binding compared to BTV-10 segment 8 with intact 5' and 3' inverted repeat sequences. Inverted repeats may influence RNA--protein binding by altering the secondary structure of the RNA and consequently the specific NS2 protein-binding sites may no longer be available.

Base Sequence↗

A comparison of DNA vaccines for the rabies-related virus, Mokola.

Mokola virus, a rabies-related virus, has been reported to date from the African continent only. Like rabies virus, it is highly pathogenic, causes acute encephalitis, and zoonotic events have been documented. Although believed to be rare, there has been an unexplained increase in the number of isolations of the virus in South Africa in recent years. We have cloned and sequenced the glycoprotein (G) and nucleoprotein (N) genes from a South African Mokola virus, and used these in the construction of different DNA vaccines for immunization against Mokola virus. Four vaccines, utilizing different promoters and DNA backbone compositions, were generated and compared for efficacy in protection against Mokola virus. In one of these, both the Mokola virus G and N genes were co-expressed. Two of the single G-expressing DNA vaccines (based on pSG5 and pCI-neo, respectively) protected laboratory mice against lethal challenge, despite major differences in their promoters. However, neither vaccine was fully protective in a single immunization only. Serological assays confirmed titers of virus-neutralizing antibodies after immunization, which increased upon booster vaccine administration. A third construct (based on pBudCE4) was less effective in inducing a protective immune response, despite employing a strong CMV enhancer/promoter also used in the pCI-neo plasmid. Dual expression of Mokola virus G and N genes in pBudCE4 did not enhance its efficacy, under the conditions described. In addition, no significant utility could be demonstrated for a combined prime-boost approach, as no cross-protective immunity was observed against rabies or Mokola viruses from the use of pSG5-mokG or vaccinia-rabies glycoprotein recombinant virus vaccines, respectively, even though both vaccines provided 60-100% protection against homologous virus challenge.

Animals↗

Identification methods for Legionella from environmental samples.

Laboratories responsible for Legionella diagnostics around the world use a number of different culturing methods of non-equivalent sensitivities and specificities, to detect Legionella species in environmental samples. Specific countries usually standardize and use one approved method. For example, laboratories in Australia use the Australian Standard (AS) method and those in Europe, the International Standard method (ISO). However, no standard culturing methods have been established in South Africa to date. As a result, there is uncertainty about the true prevalence and most common species of Legionella present in the South African environment. In an attempt to provide guidelines for the development of a standard method specific for South Africa, the ISO, AS and a most probable number method were evaluated and compared. In addition, the effect of sample re-incubation with autochthonous amoebae on culture outcome was studied. Samples were collected from four environments, representing industrial water, mine water and biofilm. The samples were concentrated by membrane filtration and divided into three portions and cultured without pretreatment, after acid treatment and after heat treatment, on four culture media namely alphaBCYE, BMPA, MWY and GVPC agar. A selective approach, incorporating heat treatment, but not acid treatment, combined with culture on alphaBCYE and GVPC or MWY, was most appropriate for legionellae detection in the samples evaluated. Legionellae were cultured from 82% of the environmental samples we evaluated. In 54% of the samples tested, legionellae were present in numbers equal to or exceeding 10(2) colony-forming units per milliliter (cfu/ml). Legionella pneumophila serogroups (SGs) 1-14 were the most prevalent species and were present as single, or a combination of two or more SGs in a number of samples tested. Re-incubation of sample concentrates with autochthonous amoebae improved the culturability of legionellae in 50% of cultures on alphaBCYE and 25% on GVPC.

Biofilms↗

Molecular epidemiology of canid rabies in Zimbabwe and South Africa.

The epidemiology of rabies in southern Africa is complex, due to a large number of vector species and the presence of at least two distinct biotypes of the virus. Our objective was to contribute to the understanding of the epidemiology of rabies in the southern African subcontinent by studying the genetic relationship of 89 rabies virus isolates from this region. In this study, we have focused on an analysis of viruses that cycle in canid host species (canid biotype) throughout South Africa and Zimbabwe. By phylogenetic analysis of the cytoplasmic domain of the glycoprotein and the non-coding G-L intergenic region, all the southern African canid viruses were found to be closely related and no apparent general distinction could be made between them. Although there was a minor degree of phylogenetic branching, with certain branches associated with cycles defined by species, location and time, the phylogenetic pattern indicated that canid rabies in southern Africa is derived from a single virus lineage, which has spread opportunistically within whatever canid host population is ecologically capable of sustaining prolonged cycles. This molecular epidemiological study presents the first comprehensive comparison of rabies viruses from South Africa and Zimbabwe and has demonstrated the need for multinational approaches towards the control of this important zoonotic disease in Africa.

Animals↗

PCR-based detection of the transovarial transmission of Uruguayan Babesia bovis and Babesia bigemina vaccine strains.

Bovine babesiosis is responsible for serious economic losses in Uruguay. Haemovaccines play an important role in disease prevention, but concern has been raised about their use. It is feared that the attenuated Babesia bovis and Babesia bigemina vaccine strains may be transmitted by the local tick vector Boophilus microplus, and that reversion to virulence could occur. We therefore investigated the possibility that these strains could be transmitted via the transovarial route in ticks using a Babesia species-specific polymerase chain reaction (PCR) assay. DNA was extracted from the developmental stages of the tick vector that had fed on calves immunized with the haemovaccine. It was possible to detect Babesia DNA not only in adult ticks, but also in their eggs and larvae. In addition, it was shown that calves infested with larvae derived from eggs laid by ticks fed on acutely infected calves, were positive for Babesia using PCR. Caution should therefore be shown with the distribution of the haemovaccine in marginal areas. It is still advisable that suitable tick control measures be used to prevent transovarial transmission and the potential risk of attenuated Babesia reverting to virulence.

Animals↗

Development of a diagnostic one-tube RT-PCR for the detection of Rift Valley fever virus.

Diagnosis of Rift Valley fever (RVF) is based on serology and virus isolation. The disadvantages of the former include poor sensitivity, high cost, risks associated with using infectious virus as antigen, the lengthy duration of ELISA as well as cross-reactivity with other Phleboviruses. We developed, optimised and evaluated a one-tube reverse-transcription-polymerase chain reaction (RT-PCR) for the detection of Rift Valley fever virus (RVFV) in ruminants. The PCR primers for this assay were designed to anneal to a region within the M segment of the virus genome, encoding glycoproteins G1 and G2. A PCR amplicon of 363 bp was obtained. The sensitivity of the assay was determined to be 0.25 TCID50. This test should allow for the early and rapid detection of RVFV in both serum and whole blood. In addition, it could facilitate the quantification of antigen for the manufacture of current vaccines.

Animals↗

Genetic heterogeneity of SAT-1 type foot-and-mouth disease viruses in southern Africa.

Genetic relationships of 50 SAT-1 type foot-and-mouth disease viruses were determined by phylogenetic analysis of an homologous 417 nucleotide region encoding the C-terminal half of the VP1 gene and part of the 2A segment. Viruses obtained from persistently-infected African buffalo populations were selected in order to assess the regional genetic variation within the host species and compared with ten viruses recovered from recent and historical cases of clinical infection. Phylogenetic reconstructions identified three independently evolving buffalo virus lineages within southern Africa, that correspond with the following discrete geographic localities: (1) South Africa and southern Zimbabwe, (2) Namibia, Botswana and western Zimbabwe, and (3) Zambia, Malawi and northern Zimbabwe. This strict geographic grouping of viruses derived from buffalo was shown to be useful for determining the origin of recent SAT-1 epizootics in livestock. The percentage of conserved amino acid sites across the 50 SAT-1 viruses compared in this study was 50%. Most mutations were clustered within three discrete hypervariable regions, which coincide with the immunogenic G-H loop, H-1 loop and C-terminus region of the protein. Despite the high levels of variation within the primary sequence, secondary structural features appear to be conserved.

Africa South of the Sahara↗

Characterization of major histocompatibility complex DRB diversity in the endemic South African antelope Damaliscus pygargus: a comparison in two subspecies with different demographic histories.

Major histocompatibility complex (MHC) class II locus DRB was investigated by single-strand conformation polymorphism analysis (SSCP) and sequence analysis in the endemic South African antelope, Damaliscus pygargus, of which there are two subspecies. Greater polymorphism was found in the blesbok (D. p. phillipsi) subspecies (n = 44; 22 alleles) than in the bontebok (D. p. pygargus) subspecies (n = 45; 6 alleles). Erosion of allelic diversity in bontebok was most likely the result of two severe bottleneck events caused by hunting pressure and parasitic infection. A majority of the polymorphism observed was found within the peptide binding region (PBR) where dN/dS ratios were higher than for the non-PBR region. This, and the apparent trans-species relationship among alleles in a bovid phylogeny, suggest the evolution of diversity by heterosis or frequency-dependent selection.

Amino Acid Sequence↗

Report of isolations of unusual lyssaviruses (rabies and Mokola virus) identified retrospectively from Zimbabwe.

Rabies isolates that had been stored between 1983 and 1997 were examined with a panel of anti-lyssavirus nucleocapsid monoclonal antibodies. Out of 56 isolates from cats and various wild carnivore species, 1 isolate of Mokola virus and 5 other non-typical rabies viruses were identified. The Mokola virus isolate was diagnosed as rabies in 1993 from a cat. Genetic analysis of this isolate suggests that it falls in a distinct subgroup of the Mokola virus genotype. The 5 non-typical rabies viruses were isolated from honey badgers (Mellivora capensis), African civets (Civettictis civetta) and an unidentified mongoose (Herpestidae). These isolates are representatives of rarely-reported wildlife-associated strains of rabies, probably maintained by the slender mongoose (Galerella sanguinea). These findings indicate that both Mokola virus and the mongoose-associated variant may be more common in Zimbabwe than is apparent from routine surveillance.

Animals↗

The 3A non-structural-protein coding region of the southern African SAT type isolates differs from that of other foot-and-mouth disease viruses.

The 3A non-structural protein of foot-and-mouth disease viruses is a relatively conserved protein comprising 153 amino acids. Recent studies have demonstrated correlation between mutations in the 3A non-structural-protein-coding region, including a 10-amino acid deletion, and attenuation of the viruses in cattle. Although the 3A coding region of several type A, O and C isolates has previously been described, nucleotide sequence data of the 3A coding region of the South African Types (SAT) 1, 2 and 3 viruses are limited. Therefore, the 3A non-structural-coding region of different SAT serotypes was determined, analysed and compared to that of European, South American and Asian isolates. The 3A regions of the SAT isolates investigated differed markedly from that of types A, O, C and Asia-1, but were similar within the group.

Africa South of the Sahara↗

Characterization of the structural-protein-coding region of SAT 2 type foot-and-mouth disease virus.

The South African Territories (SAT) types of foot-and-mouth disease (FMD) virus show marked genomic and antigenic variation in sub-Saharan Africa that is to a large extent geographically determined. This has implications for selection of appropriate vaccine strains as well as the accuracy of laboratory diagnosis. However, adaptation of field isolates as vaccine strains is cumbersome, time consuming and expensive. We propose the construction of recombinant viruses in which specific antigenic determinants can be manipulated. To achieve this goal, the structural-protein-coding region of a SAT 2 vaccine strain, ZIM 7/83/2, was determined and compared with two other known SAT 2 P1 regions. Five hypervariable regions were identified of which four are situated within VP1. The cleavage sites for proteolytic processing differs from serotype A, while the junction between P1/2A is variable within the SAT 2 serotype. These differences could influence the construction of recombinant vaccines.

Amino Acid Sequence↗

Removal of waterborne human enteric viruses and coliphages with oxidized coal.

Human enteric viruses and coliphages have been detected in water that has undergone what is generally considered adequate treatment, including chlorination. Because small numbers of virus particles are needed for the initiation of a productive virus infection, the presence of any number of virus particles in water resources will always be of concern. In this investigation the ability of oxidized coal to remove viruses from water was investigated. The oxicoal product was found to be able to remove not only coliphages, but also various pathogenic human viruses from seeded water sources. Removal was dependent upon the type of virus, the period of exposure, and the concentration of oxidized coal.

Coal↗

A nucleotide-specific polymerase chain reaction assay to differentiate rabies virus biotypes in South Africa.

Antigenic and nucleotide sequence analyses have shown that two distinct biotypes of rabies virus are circulating in South Africa. One of these typically infects members of the family Canidae, while the other comprises a heterogeneous group of apparently indigenous viruses, infecting members of the Viverridae family. In recent times, it has become evident that a considerable amount of cross-infection may occur and the manifestation of viverrid rabies in non-viverrid animals in particular appears to have become more commonplace. Consequently, the need to rapidly distinguish between rabies virus biotypes has become increasingly important in efforts to monitor the epidemiology of rabies in the southern African region. In this study, a nested polymerase chain reaction (PCR) assay was developed to distinguish between these two groups of rabies viruses. Consensus oligonucleotides were used to amplify the cytoplasmic domain of the rabies virus glycoprotein and the adjacent intergenic region. The resultant amplicon was subsequently used as template in second round heminested PCR in the presence of type-specific primers, thereby successfully generating amplicons of characteristic size for each biotype.

Animals↗

Stable protein-RNA interaction involves the terminal domains of bluetongue virus mRNA, but not the terminally conserved sequences.

The interaction of bluetongue virus (BTV) proteins with viral RNA was investigated in vitro by means of a biochemical approach. By subjecting cytoplasmic extracts from virus-infected baby hamster kidney cells and in vitro synthesized radiolabeled RNA to ultraviolet cross-linking assays, we demonstrated that, of all the BTV proteins, NS2 becomes most intimately associated with the labeled viral RNA. Competition binding studies indicated that NS2 has the greatest affinity for the 3' region of the viral transcripts. By analyzing the binding efficiency of NS2 to mutant RNA transcripts which lacked the fully conserved 5'- and/or 3'-terminal hexanucleotides, we have established that these sequences are not necessary for optimal binding. The specificity of the NS2-RNA interaction was investigated by competition experiments with unlabeled BTV-specific homologous and heterologous competitor RNAs as well as with viral double-stranded RNA (dsRNA). Although apparent differences in the ability of NS2 to bind to the different RNA transcripts were observed, it did not bind to the dsRNA.

Animals↗

Comparative sequence analysis and expression of the M6 gene, encoding the outer capsid protein VP5, of African horsesickness virus serotype nine.

The entire nucleotide and deduced amino acid sequence of the M6 gene of African horsesickness virus (AHSV) serotype nine has been determined from four overlapping cDNA clones. The gene was found to be 1566 bp long, encoding a protein of 505 amino acids with a molecular weight of 56 737 Da and a nett charge of - 1 at neutral pH Comparative sequence analysis of the deduced amino acid sequence with the VP5 protein of AHSV-4, showed that only 81% of amino acids were conserved in type and position, although alternating regions of lower and higher conservation was identified by alignment of the primary sequences of different orbiviral VP5 proteins. Antigenically authentic AHSV-9 VP5 was also expressed in a baculovirus expression system and the expressed protein was shown to react specifically with anti-AHSV-9 as well as AHSV-3 serum in Western blot analysis.

African Horse Sickness Virus↗

Site-specific mutations in the NS2 protein of epizootic haemorrhagic disease virus markedly affect the formation of cytoplasmic inclusion bodies.

The importance of a conserved amino acid motif in the nonstructural protein NS2 of different orbiviruses was investigated with regard to virus inclusion body (VIB) formation. A number of epizootic haemorrhagic disease virus NS2 deletion and substitution mutants were prepared and expressed as baculovirus recombinants. Deletion of the motif or substitution of at least three residues within the region, had a detrimental effect on VIB formation in insect cells. Furthermore, these NS2 mutants were not complexed with single-stranded RNA in infected cells and appeared to be cytotoxic. Cumulatively, the results suggest that this motif is an essential structural determinant for NS2 function.

Amino Acid Sequence↗