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diagnostic methods for african horsesickness virus using monoclonal antibodies to structural and non-structural proteins.a panel of 32 hybridoma cell lines secreting monoclonal antibodies (mabs) reactive with african horsesickness virus serotype 4 (ahsv-4) has been developed. four of the mabs recognized the major core antigen vp7, twenty recognized the outer capsid protein vp2 and eight reacted with the non-structural protein ns1. with the vp7-specific mabs a rapid and sensitive double antibody sandwich immunoassay has been developed to detect viral antigen in infected vero cells and in spleen tissue from ahsv-inf ...19921481354
expression of the major core antigen vp7 of african horsesickness virus by a recombinant baculovirus and its use as a group-specific diagnostic reagent.the major core protein, vp7, of african horsesickness virus serotype 4 (ahsv-4), the aetiological agent of a recent outbreak of the disease in southern europe, was expressed in insect cells infected with a recombinant baculovirus containing a cloned copy of the relevant ahsv gene (s7). analyses of its biochemical and antigenic properties confirmed the authenticity of the protein expressed. the high-level expression of vp7 under the control of the strong polyhedrin promoter of autographa californ ...19921378881
evolutionary relationships among the gnat-transmitted orbiviruses that cause african horse sickness, bluetongue, and epizootic hemorrhagic disease as evidenced by their capsid protein sequences.the amino acid sequences of four major capsid proteins of african horse sickness virus (serotype 4, ahsv-4) have been compared with those of bluetongue virus of sheep. epizootic hemorrhagic disease virus of deer, and the phylogenetic relationships established. complete nucleotide sequence analysis of three rna segments (l2, l3, and m6) of ahsv-4 and their encoded products, vp2, vp3, and vp5, together with previously published data for vp7 (roy et al., 1991), have revealed that of the four capsid ...19921329319
the complete sequence of african horsesickness virus serotype 4 (vaccine strain) rna segment 5 and its predicted polypeptide compared with ns1 of bluetongue virus.the complete sequence of rna segment 5 of the african horsesickness virus serotype 4 (ahsv-4) vaccine strain was determined from cdna clones inserted into pbr322. the rna is 1751 bp long (m(r) 1.12 x 10(6)) and contains an open reading frame encoding a protein of 548 amino acids (m(r) 63,122) with a net charge of +0.5 at neutral ph. a comparison of the sequence of ahsv-4 segment 5 with that of segment 6 of bluetongue virus (btv) serotypes 10 and 17 revealed 49.2% and 48.9% nucleotide similarity, ...19921328499
the complete sequence of the group-specific antigen, vp7, of african horsesickness disease virus serotype 4 reveals a close relationship to bluetongue virus.the complete sequence of the s7 rna that codes for the major group-specific coat protein. vp7, of african horsesickness virus serotype 4 (ahsv-4) was determined from cdna analyses and found to be 1179 nucleotides in length. one single open reading frame of 353 codons was observed defining a protein of mr 38,107 with a net charge of -1.5 at neutral ph. comparison of the ahsv-4 vp7 sequence with that of bluetongue virus serotype 10 revealed an overall similarity of 44%, with the amino- and carboxy ...19911646273
identification of a linear neutralization domain in the protein vp2 of african horse sickness virus.overlapping fragments of the outermost capsid protein vp2 of african horse sickness virus serotype 4 (ahsv-4) have been expressed in escherichia coli. horse sera from infected and vaccinated animals, rabbit sera, and mice monoclonal antibodies specific for ahsv were used to screen these fragments for antigenic regions. the screening revealed that the major antigenic domain of the ahsv-4 vp2 is localized in a central region (amino acids 200 to 413) and that both the n-terminal region (aa 1-159) a ...19957542417
neutralizing epitopes of african horsesickness virus serotype 4 are located on vp2.a panel of monoclonal antibodies (mabs) was generated against african horsesickness virus serotype 4 (ahsv/4). three of the mabs (sa6, oh3, and me11) strongly neutralized the homologous virus and a heterologous type 4 isolate. the mabs did not cross-neutralize ahs serotypes 1-3 or 5-9. the mabs immunoprecipitated a viral protein of 108 kda which co-migrated with vp2. pretreatment with sa6 prevented mortality of 71% of day-old mice after intracranial injection of 100 ld50 of ahsv/4, while oh3 and ...19937690505
the complete sequences of african horsesickness virus serotype 4 (vaccine strain) rna segment 2 and 6 which encode outer capsid protein.the complete sequences of rna segment 2 and segment 6 of african horsesickness virus serotype 4 (ahsv-4) vaccine strain were determined from cdna clones inserted into pbr 322. the rnas of segment 2 and 6 are 3229, 1566 bp long respectively and both contain an open reading frame encoding proteins vp2 and vp5 of 1060, 505 amino acid residues. the estimated molecular weight of vp2 was 124,178 dalton and that of vp5 was 56,793 dalton. their noncoding end sequences were 5'gtttaa . . . and . . . acata ...19948075221
diagnosis of the african horse sickness virus serotype 4 by a one-tube, one manipulation rt-pcr reaction from infected organs.a single tube reverse transcription-polymerase chain reaction (rt-pcr) method for detection of african horse sickness virus (ahsv) in splenic tissues from infected horses is described. double stranded rna was extracted from infected organs of horses and used to produce complementary dna (cdna) with the two primers selected for the pcr. the 1179 bp amplified product (the segment 7 which encodes for vp 7), detected by electrophoresis on agarose gel and ethidium bromide staining, was hydrolysed wit ...19948188813
the complete nucleotide sequence of african horsesickness virus serotype 4 (vaccine strain) segment 4, which encodes the minor core protein vp4.the complete sequence of rna segment 4 of african horsesickness virus serotype 4 (ahsv-4) vaccine strain was determined from the full-length cdna clone inserted into pbr322. the rna is 1978 bp long (m(r) 1.27 x 10(6)) and contains an open reading frame encoding a protein of 642 amino acids (m(r) 75826) with a net charge of +10 at neutral ph. the 5' and 3' termini of ahsv-4 segment 4,5'gtttat... and ...ccttac3', were different from orbivirus characteristic terminal sequences, being 5'gttaaa... an ...19938346671
rapid detection of african horsesickness virus by the reverse transcriptase polymerase chain reaction (rt-pcr) using the amplimer for segment 3 (vp3 gene).the complete sequence of the major core protein (vp3) gene of african horsesickness virus serotype 4 (ahsv-4; vaccine strain) was determined by analysis of a complete cdna clone representing segment 3. the rna was 2,789 bp long and a comparison of its sequence with that of bluetongue virus serotype 10 (btv-10) revealed 58% nucleotide similarity. based on these data, the reverse transcriptase-polymerase chain reaction (rt-pcr) technique was applied to the specific detection of ahsv using a pair o ...19948002793
characterization of african horsesickness virus serotype 4-induced polypeptides in vero cells and their reactivity in western immunoblotting.the structural and non-structural proteins induced by african horsesickness virus serotype 4 (ahsv-4) in infected vero cells were analysed by sds-page. twenty-two virus-induced polypeptides were detected in infected cells by comparison with the polypeptides of mock-infected cells, of which four major (vp2, vp3, vp5 and vp7) and three minor (vp1, vp4 and vp6) structural proteins and four non-structural proteins (p58, p48, p21 and p20) were shown to be virus-coded, as deduced from electrophoretic ...19938423451
the use of african horse sickness virus ns3 protein, expressed in bacteria, as a marker to differentiate infected from vaccinated horses.segment 10 of the double-stranded rna (dsrna) genome from african horse sickness virus serotype 4 (ahsv-4) was cloned and sequenced. the sequence of the coding region showed a total length of 667 bp. nucleotide comparisons showed a 95% sequence similarity between serotypes 4 and 9, and 76% between serotypes 4 and 3. cdna clones containing the coding region were cloned in the vector pet3xb and expressed in escherichia coli. the ns3 gene product was synthesised at very high level as an insoluble f ...19958578859
crystal structure of the top domain of african horse sickness virus vp7: comparisons with bluetongue virus vp7.the baculovirus-expressed core protein vp7 of african horse sickness virus serotype 4 (ahsv-4) has been purified to homogeneity and crystallized in the presence of 2.8 m urea. the x-ray structure has been solved to a 2.3-angstroms (1 angstrom = 0.1 nm) resolution with an rfactor of 19.8%. the structure of ahsv vp7 reveals that during crystallization, the two-domain protein is cleaved and only the top domain remains. a similar problem was encountered previously with bluetongue virus (btv) vp7 (wh ...19968648715
recombinant baculovirus-synthesized african horsesickness virus (ahsv) outer-capsid protein vp2 provides protection against virulent ahsv challenge.african horsesickness virus serotype 4 (ahsv-4) outer-capsid proteins vp2 or vp2 and vp5, prepared from single or dual recombinant baculovirus expression vectors grown in sf9 insect cells, were administered in different amounts to horses and the neutralizing antibody responses were measured. control and vaccinated horses were challenged with virulent ahsv-4 6 months later and monitored post challenge. the results indicated that two inoculations of extracts containing vp2 and vp5, or vp2 alone, i ...19968811002
effects of domain-switching and site-directed mutagenesis on the properties and functions of the vp7 proteins of two orbiviruses.based on the crystal structure of the vp7 major core protein of bluetongue virus serotype 10 (btv-10) and that of the top domain of the vp7 protein of african horsesickness virus serotype 4 (ahsv-4), chimeras and site-directed mutants of the proteins were constructed and the products analyzed with respect to their properties and functions. chimeras with the central upper domain of btv-10 vp7 replaced by that of ahsv-4 vp7 (construct bab) formed trimers, as did the converse construct (aba). furth ...19979356334
serological and virological responses in mules and donkeys following inoculation with african horse sickness virus serotype 4.two groups, comprising 4 donkeys and 4 mules (group 1) and 4 donkeys and 3 mules (group 2), were used to determine the duration of viraemia and to monitor the development of antibodies following inoculation with african horse sickness virus (ahsv). one group of animals was given a single dose of attenuated ahsv serotype 4 (ahsv 4) vaccine. the second group was inoculated with a virulent field strain of ahsv 4. both groups were subsequently challenged with the virulent field strain of ahsv 4, 51 ...19989785493
full protection against african horsesickness (ahs) in horses induced by baculovirus-derived ahs virus serotype 4 vp2, vp5 and vp7.african horsesickness virus serotype 4 (ahsv-4) outer capsid protein vp2, or vp2 and vp5 plus inner capsid protein vp7, derived from single or dual recombinant baculovirus expression vectors were used in different combinations to immunize horses. when the proteins were purified by affinity chromatography, the combination of all three proteins induced low levels of neutralizing antibodies and conferred protection against virulent virus challenge. however, purified vp2 or vp2 and vp5 in the absenc ...19968683209
future international management of african horse sickness vaccines.three types of african horse sickness (ahs) vaccine, namely adult mouse brain, modified live vaccine and inactivated viral vaccine (ivv) are reviewed. the results of efficacy trials carried out with each vaccine type highlight the advantages of the ivv. vaccination with african horse sickness virus serotype 4 ivv, given as 2 separate doses, provided full protection against subsequent, homologous challenge. the absence of any detectable viraemia after challenge would also prevent infection of ins ...19989785514
antigenic profile of african horse sickness virus serotype 4 vp5 and identification of a neutralizing epitope shared with bluetongue virus and epizootic hemorrhagic disease virus.african horse sickness virus (ahsv) causes a fatal disease in horses. the virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: vp2 and vp5. vp2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. the biological function of vp5, the other component of the capsid, is unknown. in this report, ahsv vp5, expressed in insect cells alone or together with vp2, was able to induce ahsv-specific neutralizing antibodies. ...199910329555
development of competitive elisa for serodiagnosis on african horsesickness virus using baculovirus expressed vp7 and monoclonal antibody.vp7, the sero-group common antigen, of african horsesickness virus (ahsv-4) was expressed in insect cells by recombinant baculovirus. to develop a specific diagnostic method, monoclonal antibody (mab) against vp7 was prepared and investigated as diagnostic reagent with the baculovirus expressed vp7. however, the mab against vp7 of ahsv cross-reacted with chuzan virus by the indirect immunofluorescence assay (ifa), confirming the presence of conserved domain of vp7 among orbiviruses. this study d ...200314500122
cloning and expression of the m5 rna segment encoding outer capsid vp5 of epizootic hemorrhagic disease virus japan serotype 2, ibaraki virus.the complete nucleotide sequence of a cdna clone representing the m5 rna segment of epizootic hemorrhagic disease virus japan serotype 2 (ehdv-2), ibaraki virus, was determined. the m5 segment is 1641 base pairs long with the single open reading frame which predicts a polypeptide of 527 amino acids. the comparison of the amino acid sequence of the vp5 with those of ehdv-1, bluetongue virus serotype 10, and african horse sickness virus serotype 4 revealed that the protein shared 67%, 57% and 42% ...200010770603
definition of neutralizing sites on african horse sickness virus serotype 4 vp2 at the level of peptides.the antigenic structure of african horse sickness virus (ahsv) serotype 4 capsid protein vp2 has been determined at the peptide level by pepscan analysis in combination with a large collection of polyclonal antisera and monoclonal antibodies. vp2, the determinant for the virus serotype and an important target in virus neutralization, was found to contain 15 antigenic sites. a major antigenic region containing 12 of the 15 sites was identified in the region between residues 223 and 400. a second ...200111562535
african horse sickness virus serotype 4 antigens, vp1-1, vp2-2, vp4, vp7 and ns3, induce cytotoxic t cell responses in vitro.it was shown in a previous study that proliferating cd8+ t cells could be detected in immune horse peripheral blood mononuclear cells (pbmc) when stimulated with african horse sickness virus serotype 4 (ahsv4). in this study the cytotoxicity of cd8+ t cells were tested by using the fluorescent antigen-transfected target cells-cytotoxic t lymphocytes (fatt-ctl) assay, for both the virus and its individual proteins expressed in escherichia coli. this ctl assay measures the killing of viral protein ...201627063332
b-cell epitopes of african horse sickness virus serotype 4 recognised by immune horse sera.identifying antigenic proteins and mapping their epitopes is important for the development of diagnostic reagents and recombinant vaccines. b-cell epitopes of african horse sickness virus (ahsv) have previously been mapped on vp2, vp5, vp7 and ns1, using mouse, rabbit and chicken monoclonal antibodies. a comprehensive study of the humoral immune response of five vaccinated horses to ahsv-4 antigenic peptides was undertaken. a fragmented-genome phage display library expressing a repertoire of ahs ...201728281773
immune gene expression profiling of pbmc isolated from horses vaccinated with attenuated african horsesickness virus serotype 4.development of african horsesickness (ahs) subunit vaccines will have to include a rational approach that uses knowledge of how the virus interacts with the host immune system. the global in vivo immune response induced by attenuated ahsv serotype 4 in horses was characterised using transcriptome sequencing. pbmc were collected with 24h intervals for four days after inoculation and four days after a second boost, 21 days later. transcriptome data were normalised to the day 0 naïve transcriptome ...201626382058
virus-specific cd8⁺ t-cells detected in pbmc from horses vaccinated against african horse sickness virus.african horsesickness (ahs) is an infectious but noncontagious viral disease affecting all species of equidae. the recall immune response of ahsv naïve horses immunised with an attenuated african horsesickness virus serotype 4 (ahsv4) was characterised using immune assays including elispot, real-time pcr (qpcr) and flow cytometry. the recall immune response detected in pbmc isolated from three inoculated horses showed an upregulation of circulating b lymphocytes that correlated with elevated il- ...201222365333
an african horse sickness virus serotype 4 recombinant canarypox virus vaccine elicits specific cell-mediated immune responses in horses.a recombinant canarypox virus vectored vaccine co-expressing synthetic genes encoding outer capsid proteins, vp2 and vp5, of african horse sickness virus (ahsv) serotype 4 (alvac(®)-ahsv4) has been demonstrated to fully protect horses against homologous challenge with virulent field virus. guthrie et al. (2009) detected weak and variable titres of neutralizing antibody (ranging from <10 to 40) 8 weeks after vaccination leading us to hypothesize that there could be a participation of cell mediate ...201222763149
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