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D J Roy

Publications and source records attributed to D J Roy.

At least 55 records · Page 3Linked to original sources
PubMed1994

A temporal study of RNAs produced in maedi-visna virus infection of choroid plexus cells.

We have examined the types and cellular distribution of transcripts of maedi-visna virus during infection of choroid plexus fibroblasts. Early in infection (19 hours post infection) only small spliced transcripts are found in the cytoplasm. Little virus specific RNA is detected in the nucleus at this stage. Later in infection structural gene transcripts are detectable in the cytoplasm as well as the nucleus. We have measured the half life of each type of transcript in total cellular RNA late in infection by two different methods (northern blotting or S1 protection assays after alpha-amanitin treatment, or specific hybridisation after pulse chase). Both of these methods suggest half lives for unspliced viral RNAs of 8-12 hours. Although the gel based methods were unsuitable for the accurate determination of half life for the shorter transcripts, they did confirm a long half life for these transcripts: pulse chase measurements suggested that this was similar to that seen for the unspliced transcripts. These observations are consistent with the virus exerting temporal control on protein synthesis by a method analogous to that of the HIV Rev protein.

Animals↗
PubMed1993

Use of recombinant gp135 to study epitope-specific antibody responses to maedi visna virus.

The envelope glycoprotein gp135 of the ovine lentivirus maedi visna virus (MVV) is the main target for neutralising antibody in vivo, however little is known about the specific regions of gp135 which elicit this neutralising response. We have used the polymerase chain reaction (PCR) to generate overlapping fragments of the gp135 gene which have been expressed as fusion proteins in the yeast Ty-VLP system. These fusion proteins have been used to analyse the antibody response to gp135 in MVV infected sheep and we are able to identify at least three distinct regions of gp135 to which antibodies are directed. The approach described in this paper provides a rapid and simple method of generating overlapping fusion proteins with which to carry out epitope mapping studies.

Animals↗
PubMed1993

Expression and characterization of bioactive recombinant ovine TNF-alpha: some species specificity in cytotoxic response to TNF.

We have expressed and partially purified recombinant ovine tumour necrosis factor alpha (rovTNF-alpha) using a yeast Ty, virus like particle, expression system. RovTNF-alpha is at least as active as recombinant human TNF-alpha (rhTNF-alpha) in two different bio-assays performed on ovine material, whilst approximately 1000-fold more rovrTNF-alpha than rhTNF-alpha is required to induce the same level of cytotoxicity in TNF-sensitive murine cell lines L929 and WEHI 164 clone 13. When cytotoxic assays are performed on the porcine TNF sensitive cell line PK(15)-1512, rovTNF-alpha shows about 2 logs greater activity than on murine cells, whilst rhTNF-alpha is about 1 log more active. A monoclonal antibody, raised against rovTNF-alpha, has been used to demonstrate the presence of nanogram amounts of an appropriately sized glycoprotein to be native ovine TNF-alpha in supernants of LPS stimulated ovine alveolar macrophages. These samples show no detectable cytotoxicity to L929 cells, although they show activity attributable to TNF-alpha (through neutralization by a polyclonal antiserum raised to rovTNF-alpha) in an assay on ovine material. The relative lack of activity on murine cells helps to explain previous reports of inability to assay native ovine TNF-alpha using these cells, in spite of their routine use to assay TNF-alpha from several other species. The sequence features in ovine TNF-alpha which might reduce its affinity for the murine TNF type 1 receptor are discussed.

Amino Acid Sequence↗
PubMed1992

Characteristics of the T cell-mediated immune response to maedi-visna virus.

Virus-specific T cell-mediated immunity was investigated in healthy sheep persistently infected with the ruminant lentivirus maedi-visna. Visna-specific lymphocyte proliferation was demonstrated in response to both purified virions and recombinant p25, the major core protein of maedi-visna virus. The responding T cell population in this assay was mainly of the CD4+ phenotype, although in some individuals CD8+ T cells were also shown to respond to visna antigen in this system.

Animals↗
PubMed1992

Expression of maedi-visna virus major core protein, p25: development of a sensitive p25 antigen detection assay.

The gene for the major core protein, p25, of maedi-visna virus (MVV) was cloned using a PCR (polymerase chain reaction) strategy employing primers designed for the insertion of the gene directly into yeast Ty-VLP expression vectors. In this system p25 is expressed as a fusion protein which self-assembles into virus-like particles (VLPs) due to interaction of the Ty A fusion partner. High levels (50-60 mg/l) of p25 fusion protein were produced, and p25 was recovered in soluble and highly pure form following cleavage from the Ty particle by Factor Xa protease digestion. The p25 protein produced in yeast is antigenically authentic, as defined by its reactivity with p25-specific antisera and its ability to elicit antibodies reactive with native viral p25 protein; although the cleaved, soluble form of p25 was found to be considerably more antigenic than the hybrid Ty-p25 VLP. Using this reagent anti-p25 monoclonal and polyclonal antibodies were generated. These sera and the p25 protein have been used to develop a sensitive MVV p25 detection assay. These reagents and assays will facilitate further studies of viral replication and immune response to the virus.

Animals↗
PubMed1992

A simplified method for the detection of maedi-visna virus RNA by in situ hybridization.

A simplified in situ hybridization method for the detection of maedi-visna virus (MVV) RNA in cultured cells using 35S-labelled DNA probes is described. The protocol currently used in this laboratory for the in situ detection of MVV RNA involves paraformaldehyde fixation followed by extensive cellular pretreatment prior to hybridization. It was found that substitution of paraformaldehyde fixation with brief acetone treatment and the removal of subsequent pretreatment steps gave a similar level of hybridization signal to that of our standard protocol. Acetone fixed, non-pretreated samples were used to develop a double labelling procedure in which immunocytochemistry and in situ hybridization were combined to allow the simultaneous detection of visna virus antigens and RNA within the same cell.

Acetone↗