Abstract
Background: The development of an influenza RNA-dependent RNA polymerase (RdRp) inhibitor is required; therefore, a method for evaluating the activity of influenza RdRp needs to be developed. The current method uses an ultracentrifuge to separate viral particles and quantifies RdRp activity with radioisotope-labeled nucleosides, such as 32P-GTP. This method requires special equipment and radioisotope management, so it cannot be implemented in all institutions. We have developed a method to evaluate the activity of RdRp without using ultracentrifugation and radioisotopes. Results: RdRp was extracted from viral particles that were purified from the culture supernatant using magnetic beads. A strand-specific real-time reverse transcription polymerase chain reaction (RT-PCR) method was developed based on the reverse transcription, using tagged primers. Real-time PCR using the tag sequence as the forward primer and a segment-specific reverse primer ensured the specificity for quantifying the mRNA of segments 1, 4, and 5. The temperature, reaction time, and Mg2+ concentration were determined to select the optimum conditions for in vitro RNA synthesis by RdRp, and the amount of synthesized mRNAs of segments 1, 4, and 5 was determined with a detection sensitivity of 10 copies/reaction. In addition, mRNA synthesis was inhibited by ribavirin triphosphate, an RdRp inhibitor, thus indicating the usefulness of this evaluation method for screening RdRp inhibitors.Conclusion: This method makes it possible to analyze the RdRp activity even in a laboratory where ultracentrifugation and radioisotopes cannot be used. This novel method for measuring influenza virus polymerase activity will further promote research to identify compounds that inhibit viral RNA synthesis.
Development of a Method to Evaluate Influenza Virus RNA-dependent RNA Polymerase Activity Through Real-time Reverse Transcription Polymerase Chain Reaction