Biomedical subjects

L H Nel

Publications and source records attributed to L H Nel.

27 records · Page 2Linked to original sources

PubMed1996

Identification of a short domain within the non-structural protein NS2 of epizootic haemorrhagic disease virus that is important for single strand RNA-binding activity.

The role that a conserved amino acid motif, found in the non-structural protein NS2 of orbiviruses, plays in the interaction of this protein with single stranded (ss) RNA was investigated by mutation analysis of the NS2 of epizootic haemorrhagic disease virus. An NS2 mutant in which this motif (amino acids 75 to 83) was deleted was expressed in Spodoptera frugiperda cells by a recombinant baculovirus and found to be unable to bind to poly(U)-Sepharose. The deletion mutant also differed from wild-type NS2 in that it did not appear to be complexed with ssRNA in cells infected with the baculovirus recombinant. Furthermore, the deletion exerted an adverse effect on the ability of NS2 to form inclusion bodies in the cytoplasm of baculovirus-infected insect cells. To further characterize the role of this motif in RNA-binding, specific residues within the region were substituted by site-directed mutagenesis and the mutants were expressed in Escherichia coli as fusion proteins. Analysis of the different mutant proteins indicated that in each case ssRNA-binding was impaired relative to that of the wild-type NS2 control. The degree of impairment corresponded to the number of amino acid substitutions and the largest effects were associated with non-conserved substitutions. It is suggested that the conserved motif is an important structural determinant in the interaction of NS2 with ssRNA.

Amino Acid Sequence↗

PubMed1995

Molecular epidemiology of rabies virus in South Africa: evidence for two distinct virus groups.

In order to derive phylogenetic relationships between rabies virus isolates from different geographical locations and host species in South Africa, two genome regions of the virus, viz. the cytoplasmic domain of the glycoprotein and the G-L intergenic region (pseudogene), were sequenced. A high level of nucleic acid sequence conservation indicated a close phylogenetic relationship between virus isolates from domestic dogs, jackals and bat-eared foxes, i.e. Canidae. These isolates appeared to be distinct from but closely related to European strains of rabies virus. However, a phylogenetically distinguishable and distant group, which contained isolates from mongooses (i.e. Viverridae) was identifiable. The latter group appears to be distantly related to European and vaccine strains of rabies virus and may have evolved uniquely on the central plateau of South Africa. Our data also indicate that spillover from mongooses (or other viverrids) to canid hosts occurs occasionally.

Animals↗

PubMed1994

Comparison of the expression and phosphorylation of the non-structural protein NS2 of three different orbiviruses: evidence for the involvement of an ubiquitous cellular kinase.

The non-structural protein NS2 of epizootic haemorrhagic disease (EHD), bluetongue (BT) and African horsesickness (AHS) viruses has each been expressed to high levels using a baculovirus vector gene expression system. It was found that the recombinant baculovirus-expressed EHDV NS2 protein was resolved as a doublet following PAGE. Peptide mapping of these protein bands indicated that they were identical. The difference in the sizes of the NS2 protein bands could not be attributed to the phosphorylation of NS2 or other posttranslational modification such as N-glycosylation and remains obscure. The EHDV, BTV and AHSV baculovirus-expressed NS2 proteins were all phosphorylated in vitro without the addition of an exogenous kinase. An unphosphorylated form of EHDV NS2, obtained by expressing the NS2 gene as a fusion protein in Escherichia coli cells, could be phosphorylated in vitro by a protein kinase associated with the cytoplasm of insect cells. The phosphorylated version of this protein was found to be significantly less efficient in binding ssRNA, compared to the unphosphorylated version.

Animals↗

PubMed1993

Molecular epidemiology of rabies virus in South Africa.

Nucleic acid sequence analysis was used to determine the phylogenetic relationships amongst rabies viruses isolated from typical canid hosts such as bat-eared fox, jackal and dog in South Africa (SA). Geographical factors were taken into account in the selection of isolates and three different regions within the genomes of the isolates were compared for their use as phylogenetic indicators. The three genome regions, being the cytoplasmic domain of the G-gene, the G-L intergenic pseudogene and the antigenic domain II of the N-gene were found to differ in terms of the of nucleic acid conservation, but produced similar results when analyzed phylogenetically. The SA canid isolates were found to be closely related and could clearly be distinguished from all other rabies virus groups for which sequence data is available. In addition four SA mongoose rabies isolates were studied which were shown to be distant from the SA canid rabies virus group as well as from any other rabies viruses (or group) for which sequence data is available. Our results also indicate that spillover between the distinct canid and viverrid host reservoirs may occur.

Animals↗

PubMed1991

A comparison of the nucleotide sequences of cognate NS2 genes of three different orbiviruses.

The genes encoding nonstructural protein NS2 of African horsesickness virus (AHSV) and epizootic hemorrhagic disease virus (EHDV) were cloned, sequenced, and compared to the NS2 gene of bluetongue virus (BTV). Nucleotide similarity ranged from 53 to 60%. The length of the proteins varied from 376 amino acids (EHDV) to 365 amino acids (AHSV). The N-terminal half of NS2 is more conserved (+/- 58% similarity) among the three orbiviruses, while the C-terminal half contains a 120 amino acid region of low similarity (18%). The variable region has a high content of alpha-helix conformation and a hydrophilic character. A short region of 9 amino acids contains 5 amino acids that are either similar or identical in single-stranded RNA binding proteins of BTV, EHDV, AHSV, reovirus and rotavirus.

African Horse Sickness Virus↗

PubMed1991

A comparison of different genomic probes in the detection of virus-specified RNA in Orbivirus-infected cells.

Different 32P-labelled genomic probes of bluetongue virus (BTV), epizootic haemorrhagic disease virus (EHDV) and equine encephalosis virus (EEV) were compared with respect to the detection of virus-specified RNA in infected cells. The probe derived from the genome segment that encodes nonstructural protein NS1 was found to be the most sensitive, detecting virus-specified RNA in glutaraldehyde-fixed cells as early as 2-3 h p.i. This comparison was based on the observation that the NS1 gene probe required a smaller number of infected cells to produce a positive hybridization signal than the other nucleic acid probes. The only exception was the EHDV NS2 gene probe which appeared to be as sensitive as the NS1 gene probe. The advantage of using the NS1 gene probe was particularly evident in the analysis of cells infected at very low multiplicities of infection. At a multiplicity of infection of 1 x 10(-5) plaque forming units/cell, virus-specified RNA could be detected 48 h after infection. The greater sensitivity of the NS1 gene-specific probe is ascribed to the fact that its target, the NS1 mRNA, is transcribed more frequently than the other target viral mRNAs. The major application of the cell-hybridization method is the rapid detection of small quantities of infectious virus particles.

Animals↗

PubMed1991

A characterization of the nonstructural protein from which the virus-specified tubules in epizootic haemorrhagic disease virus-infected cells are composed.

The complete nucleotide sequence of segment 6 of epizootic haemorrhagic disease virus serotype 2 (Alberta) which encodes nonstructural protein NS1 was determined from a cDNA clone containing a full-length copy of the gene. The gene was found to be 1806 bp in length, constituted by one open reading frame of 1656 bp which is flanked by 5' and 3' noncoding regions of 32 and 118 bp, respectively. The conserved 5' and 3' terminal hexanucleotide sequences were identical to those of BTV-10. The 5' noncoding nucleotide sequences of cognate genome segments 4, 6 and 8 were found to be highly conserved in EHDV-2 and BTV-10. The 3' noncoding regions are less conserved but share common characteristics. The predicted EHDV-2 NS1 gene product, a 552 amino acid polypeptide, is predominantly hydrophobic and has a net charge of +5 at neutral pH. Comparison to its BTV-10 counterpart revealed a high degree of homology. Regions of high amino acid similarity were shown to correlate with hydrophobic domains on the proteins whilst regions of lower amino acid similarity corresponded with the hydrophilic domains. Thirteen conserved cysteine residues of which the majority occurred in hydrophobic regions with more than 80% amino acid similarity were identified.

Amino Acid Sequence↗

PubMed1991

Synthesis of the virus-specified tubules of epizootic haemorrhagic disease virus using a baculovirus expression system.

The formation of virus-specific tubules is one of the most characteristic features in the orbivirus infection cycle, yet little is known about their role in virus replication. The tubuli are composed of a major nonstructural protein, NS1. We have investigated the expression of the NS1-encoding gene of epizootic haemorrhagic disease virus serotype 2 (Alberta-strain) by producing a recombinant Autographa californica nuclear polyhedrosis virus (AcNPV). Prior to cloning in the baculovirus transfer vector, pAcYM1 and cotransfection with AcNPV DNA, the NS1 gene was tailored by means of a polymerase chain reaction method to remove G/C tails. The baculovirus recombinant, AcNPV-EHDV2 NS1, expressed large amounts of a 55 K protein which could be purified by sucrose gradient sedimentation as a tubular complex. It appeared that the tubules could break up into 50 nm diameter circular units, which in turn were composed of approximately 16 subunits. The circular units appeared to be hollow and stacked on top of one another (100 units/micron tubule length), giving the tubules a segmented, ladderlike appearance. A large excess of EHDV2-specific tubuli could also be demonstrated in AcNPV-EHDV2 NS1-infected Spodoptera frugiperda cells by electron microscopic examination of thin sections. With pulse-labelling experiments it was shown that, regardless of the level of NS1 expression, the majority of NS1 synthesized in a 30 min period could only be recovered in a particulate form.

Animals↗

PubMed1990

A comparison of different cloned genome segments of epizootic haemorrhagic disease virus as serogroup-specific probes.

The eight largest double-stranded (ds) RNA genome segments of epizootic haemorrhagic disease virus (EHDV) serotype 2 (Alberta strain) have been cloned. Of these, segments 4, 5, 6, 7, and 8 are represented by clones that correspond in size to those predicted for full-length clones. The different clones were used as nucleic acid probes to study the nucleic acid homology of cognate genes of four different EHDV serotypes. The results indicated that the 4 isolates may be subdivided in two geographic groups which include Ibaraki virus as the sole member of one group and EHDV1 (New Jersey), EHDV2 (Alberta) and EHDV6 (XBM 67--isolated in South Africa) as a second. Genome segments 1, 3, 4, 6, and 8 were found to be highly conserved with more than 90% homology amongst cognate genes of three of the members of the EHDV serogroup. Of these, segments 1 and 3 showed the largest degree of homology with cognate genes of members of the BTV serogroup. Segment 6 and 8 probes of EHDV2 (Alberta) on the other hand did not hybridize with BTV dsRNA under conditions of moderate to low stringency and are recommended for use as EHDV group-specific probes.

Autoradiography↗