Detection and quantitation of HTLV-1 and HTLV-2 mRNA species by real-time RT-PCR.
HTLV-1 and HTLV-2 are highly related delta-retroviruses that infect and transform T-lymphocytes, but have distinct pathogenic properties. HTLV replication and survival requires the expression of multiple gene products from an unspliced and a series of highly related alternatively spliced mRNA species. To date, the comparative levels of all known HTLV-1 and HTLV-2 viral mRNAs in different transformed cell lines and at different stages of virus infection have not been assessed. In this study, we compiled a series of oligonucleotide primer pairs and probes to quantify both HTLV-1 and HTLV-2 mRNA species using real-time RT-PCR. The optimized reaction for detection of each mRNA had amplification efficiency greater than 90% with a linear range spanning 25-2.5 x 10(7) copies. The R(2)'s of all standard curves were greater than 0.97. Quantitation of HTLV mRNAs between different cell lines showed variability (gag/pol>or=tax/rex>env>or=accessory proteins), but the overall levels of each mRNA relative to each other within a cell line were similar. These results provide a method to quantify all specific mRNAs from both HTLV-1 and HTLV-2, which can be used to evaluate further viral gene expression and correlate transcript levels to key stages of the virus life cycle and ultimately, pathogenesis.
Li
Min
M
Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA.
Green
Patrick L
PL
D001402:B-Lymphocytes / Q000821:virology; D002461:Cell Line, Transformed / Q000821:virology; D017931:DNA Primers; D015368:Human T-lymphotropic virus 1 / Q000235:genetics / Q000378:metabolism*; D015367:Human T-lymphotropic virus 2 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013601:T-Lymphocytes / Q000821:virology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins