Measurement of reverse transcriptase of feline immunodeficiency virus by poly A-linked colorimetric assay.
The method of the poly A-linked colorimetric reverse transcriptase assay (PAC-RTA) was developed and evaluated for the measurement of Mg(2+)-dependent reverse transcriptase (RT) activity of feline immunodeficiency virus (FIV). PAC-RTA was first evaluated for the detection of RT activity in the culture supernatant of FIV Petaluma strain. The detection limit of RT activity by PAC-RTA was about 10-fold better than that by the conventional non-radioisotopic RT assay kit. Then, PAC-RTA was evaluated for the indication of FIV isolation from cats naturally infected with FIV. FIV was isolated from peripheral blood mononuclear cells of 9 FIV-seropositive cats. The time course appearance of RT activity measured by PAC-RTA corresponded with the analysis of FIV antigen expression by indirect immunofluorescence. Finally, PAC-RTA evaluated the drug susceptibility of FIV. MYA-1 cells (feline T-lymphoblastoid cells) were infected with FIV and were cultured in the presence of various concentrations of anti-human immunodeficiency virus agents such as azidothymidine (AZT) or dextran sulfate. An inverse relationship between the RT activities and the concentrations of these agents in the culture supernatant was confirmed by PAC-RTA. PAC-RTA is easy to perform without using radioactive materials, and one plate can handle 96 samples at one time. By monitoring the RT activity, this assay is a useful method for FIV studies such as viral replication and drug susceptibility.
Saito
T
T
Department of Virology, Kanagawa Prefectural Public Health Laboratory, Yokohama, Japan.
Suzuki
K
K
Imai
M
M
Inaba
Y
Y
D000818:Animals; D002415:Cats; D003124:Colorimetry; D004353:Drug Evaluation, Preclinical / Q000379:methods; D016089:Immunodeficiency Virus, Feline / Q000201:enzymology* / Q000302:isolation & purification; D007124:Immunoenzyme Techniques / Q000662:veterinary; D007963:Leukocytes, Mononuclear / Q000821:virology; D011061:Poly A; D012367:RNA, Viral / Q000032:analysis; D012194:RNA-Directed DNA Polymerase / Q000032:analysis*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014779:Virus Replication
D012367:RNA, Viral; D011061:Poly A; D012194:RNA-Directed DNA Polymerase