Sunlight Inactivation of Human Norovirus and Bacteriophage MS2 Using a Genome-Wide PCR-Based Approach and Enzyme Pretreatment

Abstract

Human norovirus (hNoV) is an important etiology of gastrointestinal illness and can be transmitted via ingestion of contaminated water. Currently impractical to culture, hNoV detection is reliant on real-time polymerase chain reaction (RT-PCR)-based methods. This approach cannot distinguish between infective and inactivated viruses because intact regions of the RNA genome can amplify even if the damage is present in other regions of the genome or because intact genetic material is not contained within an infectious virion. Herein, we employ a multiple longamplicon RT-qPCR extrapolation approach to assay genome-wide damage and an enzymatic pretreatment to study the impact of simulated sunlight on the infectivity of hNoV in clear, sensitizerfree water. Using MS2 coliphage as an internal control, the genome-wide damage extrapolation approach, previously successfully applied for UV-254 inactivation, vastly overestimated sunlight inactivation, suggesting key differences in photoinactivation under different spectral conditions. hNoV genomic RNA was more susceptible to simulated sunlight degradation per base compared to MS2 genomic RNA, while enzymatic pretreatment indicated that hNoV experienced more capsid damage than MS2. This work provides practical and mechanistic insight into the endogenous sunlight inactivation of single-stranded RNA bacteriophage MS2, a widely used surrogate, and hNoV GII.4 Sydney, an important health-relevant virus, in clear sensitizer-free water.

Removal and Inactivation of Virus by Ceramic Water Filters Coated with Lanthanum (III)

“…Eq was previously applied to virion inactivation by UV 254 , , and the discrepancy between genome-wide damage extrapolation and MS2 infectivity results by La-coated ceramic materials suggested that the MS2 inactivation process might be different from UV 254 inactivation. For example, the single-hit assumption might be not valid in which the damage of a single amplicon alone may not lead to the complete loss of virion infectivity, and the genome damage might not be random but targeted at specific bases or regions …”

“…Individual amplicon damage was extrapolated to estimate genome-wide damage by combining the RT-qPCR amplification results from six amplicon assays. The proportion of fully intact genomes can be predicted by combining results from amplicons as eq 2: 37,38 Ä…”

“…Individual amplicon damage was extrapolated to estimate genome-wide damage by combining the RT-qPCR amplification results from six amplicon assays. The proportion of fully intact genomes can be predicted by combining results from amplicons as eq : , where N 0 and N are the concentrations of intact genomes before and after La-coated ceramic treatment, respectively; n is the number of amplicons, x 0, i and x i are the concentrations of the intact amplicon i before and after selected La-coated ceramic treatment, respectively.…”

“…For example, the single-hit assumption might be not valid in which the damage of a single amplicon alone may not lead to the complete loss of virion infectivity, and the genome damage might not be random but targeted at specific bases or regions. 38 To distinguish the potential free RNA induced by the Lacoated ceramic treatment from RNA in the intact virus, a set of subsamples were treated with RNase A to remove the free RNA before RNA extraction and quantification. The difference of genome loss between the samples with and without enzyme pretreatment provided direct evidence for the intact virion loss (Figure 4).…”

Investigating the Efficacy of Various Handwashing Methods against Enveloped and Non-Enveloped Viruses

“…RT-qPCR was performed for one genome target for MS2 and one target for Phi6, with targets described in Loeb et al 31 and Ye et al 32 (Table 1), respectively, using a Step-OnePlus TM real-time PCR system. Target size and base pair composition is described in the supplemental materials.…”