Norovirus infectivity in humans and persistence in water

Abstract

To examine the long-term infectivity of human norovirus in water, 13 study subjects were challenged at different time points with groundwater spiked with the prototype human norovirus, Norwalk virus. Norwalk virus spiked in groundwater remained infectious after storage at room temperature in the dark for 61 days (the last time point tested). The Norwalk virus-seeded groundwater was stored for 1,266 days and analyzed, after RNase treatment, by reverse transcription-quantitative PCR (RT-qPCR) to detect Norwalk virus RNA contained within intact capsids. Norwalk virus RNA within intact capsids was detected in groundwater for 1,266 days, with no significant log(10) reduction throughout 427 days and a significant 1.10-log(10) reduction by day 1266. Purified Norwalk virus RNA (extracted from Norwalk virus virions) persisted for 14 days in groundwater, tap water, and reagent-grade water. This study demonstrates that Norwalk virus in groundwater can remain detectable for over 3 years and can remain infectious for at least 61 days. (ClinicalTrials.gov identifier NCT00313404.).

Fig. 1.

Infectivity and persistence of NV in groundwater. Thirteen genetically susceptible volunteers ingested safety-tested groundwater that was seeded with 8FIIb NV inoculum and stored for the indicated time points. NV-infected (closed circles) and uninfected (open circles) subjects at each time point are indicated. Aliquots were analyzed by RT-qPCR, and NV log₁₀ reductions were calculated (number of genomic equivalent copies/μl at each time point [N_t] divided by the average at day 0 [N₀]). The triangles (samples without RNase treatment) and squares (samples with RNase treatment) represent the means of 2 to 5 replicate samples analyzed in duplicate (i.e., 4 to 10 tubes per sample and time point). The break in the horizontal axis is indicated. Error bars indicate standard deviations. Asterisks correspond to a significant (P < 0.05) NV log₁₀ reduction compared to day 0.

Fig. 2.

Persistence of purified NV RNA in groundwater, tap water, and reagent-grade water. Extracted NV RNA was stored in groundwater, tap water, and reagent-grade water for 14 days. Aliquots were analyzed by RT-qPCR, and NV log₁₀ reductions were calculated (number of genomic equivalent copies/μl at each time point [N_t] divided by the average at day 0 [N₀]). The triangles (groundwater), squares (tap water), and circles (reagent-grade water) represent the means of 2 replicate samples analyzed in duplicate (i.e., 4 tubes per sample and time point). These results are representative of 5 separate experiments. Error bars indicate standard deviations. Asterisks correspond to a significant (P < 0.05) NV log₁₀ reduction compared to day 0 and are associated with groundwater and tap water at days 7 and 14.