Porcine retrovirus: optimal conditions for its biochemical detection.
The assay of reverse transcriptase (RT) activity was used to detect the presence of retrovirus in porcine cells. A set of optimal assay conditions was determined to design a sensitive, quantitative and reproducible RT assay for porcine systems. The template-primer poly(rA).oligo(dT) was an absolute requirement. The presence of Mn++ was indispensable, with an optimal concentration of 0.25 mM. Monocations (K+, Na+) at 50 mM greatly enhanced, but their high doses inhibited the reaction. The pH of the medium influenced very much the reaction, especially with non-purified virus samples, with which the RT activity was inhibited at pHs above 8.2. Non-ionic detergents at 1% enhanced several-fold the RT activity. It was also shown that porcine retrovirus could be spontaneously reactivated in porcine cell lines by in vitro long-term propagation and transmitted to pigs by inoculation with virus-producing cells.
Institut National de la Recherche Agronomique, Centre de Recherche de Tours, Nouzilly, France.
L
L
Phan-Thanh
B
B
Kaeffer
E
E
Bottreau
D000818:Animals; D002460:Cell Line; D003902:Detergents / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000494:pharmacology; D008854:Microscopy, Electron; D011188:Potassium / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D012190:Retroviridae / Q000201:enzymology / Q000302:isolation & purification* / Q000648:ultrastructure; D012192:Retroviridae Infections / Q000382:microbiology / Q000635:transmission; D012964:Sodium / Q000494:pharmacology; D013552:Swine; D013698:Templates, Genetic; D014158:Transcription, Genetic
D003902:Detergents; D012333:RNA, Messenger; D008345:Manganese; D012964:Sodium; D012194:RNA-Directed DNA Polymerase; D008274:Magnesium; D011188:Potassium