The Protective Effect of Virus Capsids on RNA and DNA Virus Genomes in Wastewater

Abstract

Virus concentrations measured in municipal wastewater help inform both the water treatment necessary to protect human health and wastewater-based epidemiology. Wastewater measurements are typically PCR-based, and interpreting gene copy concentrations requires an understanding of the form and stability of the nucleic acids. Here, we study the persistence of model virus genomes in wastewater, the protective effects provided by the virus capsids, and the relative decay rates of the genome and infectious viruses. In benchtop batch experiments in wastewater influent at 25 °C, extraviral (+)ssRNA and dsDNA amplicons degraded by 90% within 15-19 min and 1.6-1.9 h, respectively. When encapsidated, the T₉₀ for MS2 (+)ssRNA increased by 424× and the T₉₀ for T4 dsDNA increased by 52×. The (+)ssRNA decay rates were similar for a range of amplicon sizes. For our model phages MS2 and T4, the nucleic acid signal in untreated wastewater disappeared shortly after the viruses lost infectivity. Combined, these results suggest that most viral genome copies measured in wastewater are encapsidated, that measured concentrations are independent of assay amplicon sizes, and that the virus genome decay rates of nonenveloped (i.e., naked) viruses are similar to inactivation rates. These findings are valuable for the interpretation of wastewater virus measurements.

Keywords: BCoV; MS2; T3; T4; amplicon; bacteriophage; decay; infectivity.

Figure 1

Extraviral (+)ssRNA and dsDNA decay in untreated wastewater at 25 °C. C represents the concentration of the viral nucleic acids in gene copies μL^–1 at time t in hours, and C₀ represents the initial concentration of the viral nucleic acids at time = 0 based on the amount of stock that was spiked into the wastewater. Initial concentrations in wastewater were ∼10⁹ gc mL^–1 for MS2 (+)ssRNA and ∼10⁸ gc mL^–1 for BCoV (+)ssRNA, T3 dsDNA, and T4 dsDNA. Open symbols represent measurements that were below the limit of quantification and are plotted as the limit of quantification for each virus assay. Error bars represent the standard error for three experimental replicates conducted in wastewater samples collected on different days.

Figure 2

MS2 (+)ssRNA (left) and T4 dsDNA (right) encapsidated genome decay in untreated wastewater replicates A–F at 25 °C. C refers to the concentration of the viral nucleic acids in gene copies μL^–1 at time t in hours. C₀ represents the initial concentration of the viral nucleic acids at time = 0 based on the number of gene copies measured immediately after the stocks were added to the sample. Initial concentrations in wastewater were ∼10⁹ gc mL^–1 for MS2 and ∼10⁸ gc mL^–1 for T4. Open symbols represent measurements that were below the limit of quantification (LOQ) and are plotted as the limit of quantification for each virus assay. The data points for T4 dsDNA at time points >200 h were below LOQ and are not shown to keep x-axis scales consistent.

Figure 3

Effect of three different amplicon sizes on encapsidated MS2 genome persistence in untreated wastewater at 25 °C. C refers to the concentration of the viral nucleic acids in gene copies μL^–1 at time t in hours. C₀ represents the initial concentration of the viral nucleic acids at time = 0 based on the amount of gene copies measured immediately after the stocks were added to the sample. Experiments were run in duplicate with two different wastewater samples collected on different days.

Figure 4

Infectivity loss and genome decay for MS2 (panels A–C) and T4 (panels D–F) in untreated wastewater at 25 °C. C refers to the concentration of the viral nucleic acids in gene copies μL^–1 at time t in hours. C₀ represents the initial concentration of the viral nucleic acids at time = 0 based on the amount of gene copies measured immediately after the stocks were added to the sample. N refers to the concentration of infectious viruses in PFU mL^–1 at time t in hours. N₀ represents the initial concentration of the infectious virus at time = 0 based on the number of plaques measured immediately after the stocks were added to the sample. Open symbols represent measurements that were below the limit of quantification and are plotted for each virus assay. We present the triplicate experimental replicates for each virus in three different wastewater samples in different panels for clarity.