Evaluation of Viability of Human Norovirus Using Propidium Monoazide (PMA) and Ethidium Monoazide (EMA)

Introduction: As in vitro culture systems have not been developed for the study of noroviruses (NoVs), it is not possible to determine the viability of norovirus by reverse transcription-polymerase chain reaction (RT-PCR) or real-time quantitative RT-PCR (qRT-PCR).

Purpose: The aims of this study were to examine the viability of human norovirus using PMA-combined qRT-PCR or EMA-combined qRT-PCR.

Methods: Human norovirus (hNoV) GII.4 stool was confirmed by qRT-PCR and sequence analysis. This virus stock was aliquoted into 500 µl with 10⁶ copy number/μl concentration. Each virus was heat-treated at room temperature, 70°C, 75°C, 80°C, 85°C, and 90°C in water bath for 90 s. Each sample mixed with 250 µM PMA or 25 µM EMA was stored in a dark room for 10 min. Then, the PMA- or EMA-treated samples were exposed to 40 W LED light at 460 nm wavelength for 15 min. The negative control was not treated with PMA or EMA as well as LED light. hNoV RNA was measured using qRT-PCR, PMA-combined qRT-PCR, and EMA-combined qRT-PCR.

Results: Although the relative quantification of hNoV treated at 70°C, 75°C, 80°C, 85°C, and 90°C showed 0.87, 1.22, 1.64, 1.85, and 2.02 log reduction, qRT-PCR could detect all heat-treated hNoV without the treatment of EMA or PMA. In PMA-combined qRT-PCR, the hNoV treated at 70°C, 75°C, and 80°C reduced 0.86, 1.44, and 2.51 log, respectively. In EMA-combined qRT-PCR, virus aliquot treated at 70°C, 75°C, and 80°C decreased by 1.00, 1.38, and 2.33 log, respectively. Interestingly, hNoV RNA treated at 85°C and 90°C was not detected by both PMA- combined qRT-PCR and EMA-combined qRT-PCR.

Significance: PMA- or EMA-combined qRT-PCR could be useful molecular technique for determining the infectious and non-infectious human norovirus.