A new one-step RT-PCR method for virus quantitation in murine AIDS.
The causative agent of murine AIDS (MAIDS) in C57BL/6 mice, is a defective murine leukemia virus (BM5d) that requires the replication-competent helper virus (BM5e). Since this animal model of immunodeficiency, which shows many similarities to human AIDS, is also used to test the efficacy and toxicity of antiretroviral drugs, a method that allows the quantitative detection of both viruses would be very useful also if hampered potentially by endogenous viral sequences usually present in mice. While BM5d alone could induce the disease, the effect of BM5e on the immune system of diseased mice is unclear. A specific and reliable one-step RT-PCR method was developed for the co-amplification, with the same efficiency, of BM5d or BM5e with ss-actin used as an internal standard. The standard curves produced with cloned cDNA sequences (ss-actin and BM5d or BM5e) assure that all samples are analyzed during the exponential phase of the reaction. Using this new assay which provided a dynamic range of at least four-log-unit, the ratio of initial absolute amounts of the virus and ss-actin RNA was determined, obtaining quantitative information on virus-specific cellular-transcript in the lymph nodes and spleen during the natural history of the disease and during therapeutic regimens.
Casabianca
Anna
A
Institute of Biological Chemistry Giorgio Fornaini, University of Urbino, Via Saffi, 2, 61029 (PU), Urbino, Italy.
Orlandi
Chiara
C
Fraternale
Alessandra
A
Magnani
Mauro
M
D000199:Actins / Q000378:metabolism; D000818:Animals; D018076:DNA, Complementary; D003673:Defective Viruses / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004195:Disease Models, Animal; D006378:Helper Viruses / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D009052:Leukemia Virus, Murine / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D016183:Murine Acquired Immunodeficiency Syndrome / Q000821:virology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D014779:Virus Replication
D000199:Actins; D018076:DNA, Complementary; D012333:RNA, Messenger