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Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays
Published on: September 26, 2011
A new one-step RT-PCR method for virus quantitation in murine AIDS
Anna Casabianca1, Chiara Orlandi, Alessandra Fraternale
1Institute of Biological Chemistry Giorgio Fornaini, University of Urbino, Via Saffi, 2, 61029 (PU), Urbino, Italy.
Abstract:
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.
Insights
Researchers developed a quantitative RT-PCR assay to detect both defective murine leukemia virus (BM5d) and its helper virus (BM5e) in mice. This method aids in studying murine AIDS (MAIDS) and evaluating antiretroviral drug efficacy.
Area of Science:
- Virology
- Immunology
- Animal Models
Background:
- Murine AIDS (MAIDS) is induced by a defective murine leukemia virus (BM5d) requiring a helper virus (BM5e).
- This animal model shares similarities with human AIDS and is crucial for antiretroviral drug testing.
- Detecting both viruses quantitatively is challenging due to endogenous viral sequences in mice.
Purpose of the Study:
- To develop a reliable method for quantitative detection of BM5d and BM5e viruses.
- To enable accurate analysis of viral load during MAIDS progression and treatment.
Main Methods:
- A specific one-step RT-PCR assay was designed for co-amplification of BM5d or BM5e with ss-actin (internal standard).
- Standard curves using cloned cDNA sequences ensured analysis within the exponential reaction phase.
- The assay demonstrated a dynamic range of at least four-log-units.
Main Results:
- The RT-PCR method accurately quantified BM5d and BM5e viral RNA.
- The ratio of viral RNA to ss-actin was determined in lymph nodes and spleen.
- Quantitative information was obtained on virus-specific cellular transcripts during disease and therapy.
Conclusions:
- A sensitive and specific RT-PCR assay for simultaneous quantification of BM5d and BM5e has been established.
- This assay is valuable for studying MAIDS pathogenesis and assessing antiretroviral therapies.
- The method overcomes challenges posed by endogenous viral sequences in mice.
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