Characterisation of visna virus reverse transcriptase: a micro scale reverse transcriptase assay adapted for use with an automated cell harvester.
The reverse transcriptase of the sheep lentivirus visna/maedi virus has been characterised. Optima for magnesium ion concentration (5-10 mM), potassium ion concentration (150 mM) and pH (8.25) for this enzyme are very similar to those previously described for the human immunodeficiency viruses. The assay used for this work makes use of a cell harvester to speed up the processing of multiple samples. It is small scale, requiring 15 microliters of sample, is rapid, and is able to detect virus at titres below 10(3)/ml. Harvesting the assay onto either DEAE paper or using TCA and glass fibre mats make it suitable for use with either tissue culture media or infected cell lysates, but not with body fluids. It has been used to detect cell-associated reverse transcriptase in choroid plexus cells within 36 h of visna infection.
Department of Veterinary Pathology, University of Edinburgh, U.K.
D R
DR
Sargan
N J
NJ
Watt
I
I
McConnell
D000818:Animals; D001331:Automation; D002460:Cell Line; D002845:Chromatography / Q000379:methods; D006041:Goats; D017830:Octoxynol; D011021:Pneumonia, Progressive Interstitial, of Sheep / Q000201:enzymology; D011092:Polyethylene Glycols; D011189:Potassium Chloride; D012194:RNA-Directed DNA Polymerase / Q000032:analysis*; D012680:Sensitivity and Specificity; D012756:Sheep; D013997:Time Factors; D014238:Trichloroacetic Acid / Q000378:metabolism; D014773:Virology / Q000295:instrumentation / Q000379:methods; D014790:Visna-maedi virus / Q000201:enzymology*
D011092:Polyethylene Glycols; D014238:Trichloroacetic Acid; D011189:Potassium Chloride; D017830:Octoxynol; D012194:RNA-Directed DNA Polymerase