Evaluation of Three Viral Capsid Integrity qPCR Methods for Wastewater-Based Viral Surveillance

Abstract

Capsid Integrity qPCR (CI-qPCR) assays offer a promising alternative to cell culture-based infectivity assays for assessing pathogenic human virus viability in wastewater. This study compared three CI-qPCR methods: two novel (Crosslinker, TruTiter) and one established (PMAxx dye). These methods were evaluated on heat-inactivated and non-heat-inactivated 'live' viruses spiked into phosphate-buffered saline (PBS) and wastewater, as well as on viruses naturally present in wastewater samples. The viral panel included Human adenovirus 5 (HAdV), enterovirus A71 (EV), hepatitis-A virus (HAV), influenza-A H3N2 (IAV), respiratory syncytial virus A2 (RSV), norovirus GI, norovirus GII, and SARS-CoV-2. All three methods successfully differentiated between degraded, heat-inactivated, and live viruses in PBS. While all three methods were comparable for HAdV and norovirus GI, PMAxx detected significantly lower gene copies for EV and IAV. In spiked wastewater, PMAxx yielded significantly lower gene copies for all heat-inactivated viruses (HAdV, EV, HAV, IAV, and RSV) compared to the Crosslinker and TruTiter methods. For viruses naturally present in wastewater (un-spiked), no significant difference was observed between PMAxx and TruTiter methods. Intact, potentially infectious viruses were detected using both PMAxx and TruTiter on untreated and treated wastewater samples. A comparative analysis of qPCR data and TEM images revealed that viral flocculation of IAV may interfere with capsid integrity assays using intercalating dyes. In summary, our findings not only advance the development of more effective methods for assessing viral viability in wastewater, but also highlight the potential of CI-qPCR techniques to enhance early warning systems for emerging pathogens, thereby strengthening public health preparedness and response strategies.

Keywords: Capsid integrity; Environmental surveillance; Pathogen viability; Public health risk; Viral persistence; Wastewater-based epidemiology.

Fig. 1

Comparison of viral integrity assays for detecting live (non-heat-inactivated) and heat-inactivated viruses in PBS. Gene copies (gc l⁻¹) of HAdV, EV, IAV, and norovirus GI before and after treatment with Crosslinker, TruTiter, or PMAxx assays relative to the untreated control (qPCR)

Fig. 2

Quantification (gc l⁻¹) of heat-inactivated and live (non-heat-inactivated) viruses (HAdV, EV, HAV, IAV and RSV) in spiked wastewater either before (control, qPCR) or after the application of three different capsid integrity pretreatment methods (Crosslinker, TruTiter and PMAxx)

Fig. 3

Viral gene copies (gc l⁻¹) in wastewater influent and effluent measured after pretreatment with either the TruTiter or PMAxx viral integrity methods. The pretreatment methods were used to assess the integrity of HAdV, EV, norovirus GI, norovirus GII, and SARS-CoV-2 in comparison to samples receiving no pretreatment (qPCR, control). Outliers in the data are indicated with an ×

Fig. 4

Impact of filtration and heat inactivation on the detection of HAdV and IAV spiked into wastewater. Gene copy concentrations (gc l⁻¹) of live and heat-inactivated (HI) HAdV (green) and IAV (blue) in filtered (diagonal line) and non-filtered wastewater, compared to non-spiked controls. All samples were pretreated with PMAxx dyes before nucleic acid extraction. Error bars represent the standard error of the mean

Fig. 5

Transmission electron microscopy images of live (non-heat-inactivated) and heat-inactivated HAdV and IAV spiked into wastewater. Panel A—Wastewater control image. Panel B—Live HAdV in non-filtered wastewater. Panel C—Live IAV (blue arrow) floccing and binding to particulate/hydrophobic matter (orange arrow) in filtered wastewater. Panel D—Live IAV (blue arrow) floccing in filtered wastewater. Panel E—Live IAV (Blue arrow) bound to particulate (orange arrow) in filtered wastewater. Panel F—Degraded HAdV (green arrow), bound to particulate (orange arrow) in filtered wastewater. Panel G and H—Heat-inactivated IAV (Blue arrow) floccing. Panel I—heat-inactivated IAV (green arrow)