Persistence of Human Norovirus (GII) in Surface Water: Decay Rate Constants and Inactivation Mechanisms

Abstract

Human norovirus (HuNoV) is an important cause of acute gastroenteritis and can be transmitted by water exposures, but its persistence in water is not well understood. Loss of HuNoV infectivity in surface water was compared with persistence of intact HuNoV capsids and genome segments. Surface water from a freshwater creek was filter-sterilized, inoculated with HuNoV (GII.4) purified from stool, and incubated at 15 or 20 °C. We measured HuNoV infectivity via the human intestinal enteroid system and HuNoV persistence via reverse transcription-quantitative polymerase chain reaction assays without (genome segment persistence) or with (intact viral capsid persistence) enzymatic pretreatment to digest naked RNA. For infectious HuNoV, results ranged from no significant decay to a decay rate constant ("k") of 2.2 day^-1. In one creek water sample, genome damage was likely a dominant inactivation mechanism. In other samples from the same creek, loss of HuNoV infectivity could not be attributed to genome damage or capsid cleavage. The range in k and the difference in the inactivation mechanism observed in water from the same site could not be explained, but variable constituents in the environmental matrix could have contributed. Thus, a single k may be insufficient for modeling virus inactivation in surface waters.

Keywords: capsid integrity; creek water; genome integrity; virus.

Figure 1

Persistence of HuNoV in filter-sterilized surface water incubated at 15 and 20 °C under dark conditions over time assessed via HIE assay (A) and via RT-qPCR assays targeting short and long HuNoV genome segments in different regions of the genome (color) with (B) and without (C) RNase pretreatment. Decay rate constants (k values) are provided in each plot (units are day^–1); k values that were not significantly different from 0 are denoted as “k < 0.01.” Error bars depict the standard deviation of technical replicates, except for experiments 20-1 and 20-2 for which they depict the standard deviation of experimental replicates. RNase pretreatment was not completed for experiments 15-1 and 20-1.

Figure 2

Variation in k values by experiment. The dashed vertical dashed line denotes 0. Error bars depict the standard error of the first-order decay rate constant (k). The red, open points are not significantly different from 0. No error bars are shown for experiment 15-1 HIE assay because only two data points were used to calculate k and an error could not be calculated.