Quantitative micro P30 and reverse transcriptase assays for Moloney murine leukemia virus

Area of Science:

• Virology
• Immunology
• Molecular Biology

Background:

• Moloney murine leukemia virus (MoMLV) is a retrovirus frequently used in research.
• Accurate quantification of viral infectivity is crucial for understanding retroviral replication and infection dynamics.

Purpose of the Study:

• To adapt and validate two assays for quantifying Moloney murine leukemia virus (MoMLV) infectivity in a high-throughput 96-well plate format.
• To enable efficient assessment of retroviral infection efficiency under diverse experimental conditions.

Main Methods:

• Adaptation of an anti-P30 immunohistochemical assay to a 96-well plate format for detecting viral proteins.
• Adaptation of a reverse transcriptase assay to a 96-well plate format for quantifying viral RNA-dependent DNA polymerase activity.
• Correlation of assay results with the number of infectious viral particles.

Main Results:

• Both the adapted immunohistochemical and reverse transcriptase assays demonstrated results directly proportional to the number of infectious viral particles.
• The micro P30 assay provided a direct estimate of infectious centers.
• The reverse transcriptase assay quantified progeny from a single replication cycle.
• The semi-automated nature of these assays facilitates the analysis of large sample numbers.

Conclusions:

• The developed 96-well plate assays provide a reliable and efficient method for estimating the infective titers of Moloney murine leukemia virus (MoMLV) preparations.
• These assays are suitable for examining the efficiency of retroviral/MoMLV infection under varied times or conditions, supporting large-scale investigations.