detection of infectious noroviruses from wastewater and seawater using pemaxtm treatment combined with rt-qpcr

Rapid detection of infectious noroviruses from environmental samples is essential to minimize the risk of norovirus outbreaks associated with environmental transmission. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) methods are rapid and sensitive, but cannot differentiate between infectious and non-infectious noroviruses. In this study, a PEMAXTM treatment followed by RT-qPCR (PEMAXTM-RT-qPCR) method was developed for murine norovirus and norovirus GI/GII, and evaluated for the selective detection of infectious viruses following heat inactivation. The norovirus PEMAXTM-RT-qPCR method was then evaluated for the selective detection of infectious viruses from environmental samples. Following heat-treatment (90 °C for 3 min), the murine norovirus PEMAXTM-RT-qPCR showed at least a 2.04 log10 reduction in detectable virus, compared to a 0.43 log10 reduction for RT-qPCR alone. Under the same conditions, the norovirus PEMAXTM-RT-qPCR showed a 0.34 to 0.98 log10 (GI.3) and 0.63 to 2.06 log10 (GII.4) reduction in detectable viruses, compared to 0.05 to 0.18 log10 (GI.3) and 0.06 to 0.25 log10 (GII.4) for RT-qPCR alone. Evaluation of the norovirus PEMAXTM-RT-qPCR on norovirus-contaminated influent and effluent wastewater, and seawater indicated a high proportion of non-infectious norovirus GI and GII (i.e., 56 to 100% in seawater, 32 to 76% in effluent, and 11 to 79% in influent) was present in samples. While potentially overestimating the amount of infectious noroviruses, this approach has potential to provide better information on viral infectivity than RT-qPCR alone.