Advances in wastewater analysis revealing the co-circulating viral trends of noroviruses and Omicron subvariants.

Co-presence of enveloped and non-enveloped viruses is common both in community circulation and in wastewater. Community surveillance of infections requires robust methods enabling simultaneous quantification of multiple viruses in wastewater. Using enveloped SARS-CoV-2 Omicron subvariants and non-enveloped norovirus (NoV) as examples, this study reports a robust method that integrates electronegative membrane (EM) concentration, viral inactivation, and RNA preservation (VIP) with efficient capture and enrichment of the viral RNA on magnetic (Mag) beads, and direct detection of RNA on the beads. This method provided improved viral recoveries of 80 ± 4 % for SARS-CoV-2 and 72 ± 5 % for Murine NoV. Duplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays with newly designed degenerate primer-probe sets offered high PCR efficiencies (90-91 %) for NoV (GI and GII) targets and were able to detect as few as 15 copies of the viral RNA per PCR reaction. This technique, combined with duplex detection of NoV and multiplex detection of Omicron, successfully quantified NoV (GI and GII) and Omicron variants in the same sets of 94 influent wastewater samples collected from two large wastewater systems between July 2022 and June 2023. The wastewater viral RNA results showed temporal changes of both NoV and Omicron variants in the same wastewater systems and revealed an inverse relationship of their emergence. This study demonstrated the importance of a robust analytical platform for simultaneous surveillance of enveloped and non-enveloped viruses in wastewater. The ability to sensitively determine multiple viral pathogens in wastewater will advance applications of wastewater surveillance as a complementary public health tool.