Viruses | Free Full-Text | Use of Capsid Integrity-qPCR for Detecting Viral Capsid Integrity in Wastewater

The use of wastewater-based surveillance (WBS) to monitor community-level health via the detection of harmful disease-causing pathogens, excreted in faeces and urine, has greatly increased in recent years [1]. For example, many studies now exist showing the utility of WBS to detect viruses, such as norovirus [2], polio and non-polio enterovirus [3,4,5], adenovirus [6,7,8], hepatitis A [9,10], hepatitis E [11,12], and respiratory viruses such as coronaviruses, influenza viruses, and respiratory syncytial virus [13,14,15]. During the COVID-19 pandemic, WBS was widely implemented for population-level surveillance of SARS-CoV-2 by research groups and laboratories globally [16,17,18,19,20,21,22], further showcasing the usefulness of WBS for disease monitoring and public health policy implementation.

Viruses in wastewater are typically detected by first concentrating and precipitating the viruses, extracting their nucleic acids, and then performing molecular techniques on the samples such as RT-qPCR (real-time quantitative polymerase chain reaction), dd-PCR (digital droplet PCR), or next generation sequencing. However, these methods cannot distinguish between intact potentially infectious viruses and degraded non-infectious viral particles; rather, they detect genetic markers from viruses regardless of viability. Thus, while the data gathered by these techniques provide an overview of infection levels in the community, it does not indicate the viral stability or the potential for infectivity if an individual comes into contact with sewage-contaminated water. This aspect of WBS is becoming more important, given the increased discharge of untreated sewage into rivers and seas and the associated risks to human and environmental health (i.e., One Health) [23,24].

Cell culture is the gold standard for virus viability testing. However, it requires specialised equipment like incubators, inverted microscopes, centrifuges, plate readers, hemacytometers, cryogenic storage, CO₂, media, and other consumables [25]. In addition, it can take 4–7 days to generate infectivity data, causing delays in the implementation of public health interventions. It also requires staff with expertise to maintain the cell lines. Furthermore, many human respiratory viruses can only be cultured in biosecurity level (BLS) 3 laboratories, which are usually not available in environmental monitoring facilities. These requirements can be limiting factors, especially in lower- or middle-income countries (LMICs), where, in addition, frequent power outages may be common. Furthermore, isolating viruses from wastewater for cell culture assays is extremely challenging. Viruses are often present at low viral loads; so, large volumes (>10 l) of wastewater must be concentrated prior to culturing. Wastewater also contains debris and microbes that can contaminate and destroy cell cultures used for virus viability assays. Therefore, methods like filtration are needed to remove these contaminants without damaging viable viruses [26]. Antibiotics also may need to be added to the culture media to reduce microbial activity, albeit at levels that do not compromise the cell line [27]. Therefore, simpler methods like capsid integrity-PCR/qPCR (ci-PCR or ci-qPCR) (also referred to as viability qPCR/RT-qPCR) are needed to assess the potential infectivity of viruses in wastewater samples.

Capsid integrity-PCR/qPCR has become an important technique in virology for selectively detecting potentially infectious viral particles. Photo-reactive monoazide dyes such as ethidium monoazide (EMA), propidium monoazide (PMA), and PMAxx^® penetrate damaged viral capsids with exposed genomes, which are incapable of cell attachment and entry but are excluded from viruses with intact capsids that maybe capable of host cell infection. After addition of the dye to a wastewater viral concentrate, the sample is exposed to light at which point the dye modifies any exposed nucleic acids, blocking their subsequent amplification by PCR [28]. In contrast, the genomes of viruses with intact capsids that are potentially infectious can be successfully amplified. This allows selective quantification of the encapsulated viral genomes versus the non-infectious nucleic acids present in the sample. Capsid integrity-PCR using monoazide-based dyes has been applied to differentiate infectious and non-infectious viruses including influenza-A virus [29], norovirus [30], and SARS-CoV-2 [31]. Furthermore, the quantification of encapsulated viral genomes has provided insights into viral persistence in the environment [32,33] and the disinfection efficiency [34,35,36]. It is worth noting that the capsids of viruses inactivated using UV are still intact, and as a result, ci-qPCR is not suitable for assessing UV disinfection [36]. However, the sensitivity and specificity of this approach continues to be improved through optimising dye types, the concentration of dye used, incubation times, and light exposure length [37].

Many studies have utilised ci-qPCR to assess the capsid integrity of viruses in contaminated drinking water and food, as reviewed [38]; however, the use of ci-qPCR in wastewater monitoring has received much less attention. Of the wastewater studies that do exist, ci-qPCR has been used to assess the capsid integrity of adenovirus [39], enterovirus [39,40], norovirus GI and GII [30,32,40,41], rotavirus [32,39], hepatitis A virus [10,42], and SARS-CoV-2 [31,32]. Human enteric virus genomes (adenovirus, enterovirus, and norovirus) in wet and dry wastewater-derived struvite have also been shown to remain encapsulated using intercalating dyes [43]. We hypothesize that wastewater-based ci-qPCR can provide improved estimates of public health exposure risks compared to conventional PCR, especially regarding the potential infection risks posed by sewage discharges into the environment. This study therefore aims to optimise and assess the usefulness of ci-RT-qPCR using PMAxx dyes alongside conventional wastewater monitoring via RT-qPCR, for important viruses of public health concern including adenovirus (AdV), enterovirus (EV), hepatitis A (HAV), influenza A virus (IAV), influenza B virus (IBV), norovirus GI (NoV GI), norovirus GII (NoV GII), and SARS-CoV-2. The optimisation of PMAxx dye for a range of viral targets is also presented.

Molecular methods used to assess the capsid integrity of viruses present in wastewater are needed to provide more in-depth risk analysis of human exposure to wastewater [38], especially where sewage is discharged into the environment, as it may contain intact potentially infectious viruses [56]. It may also be relevant for undertaking quantitative microbial risk assessments (QMRA) to estimate the potential viral exposure of operators at wastewater treatment plants or those working within the sewage network [57]. As evidenced, EV, NoV GI, and NoV GII capsids remain intact, and as a result are potentially infectious.

In this study, we further advance the use of intercalating dyes for viral capsid integrity-RT-qPCR assays used in WBS applications. PMAxx dye at 50 μM and 100 μM concentrations was selected based on a review by Leifels et al. 2021 [38], where over half of the research papers reported dye concentrations of 50 μM and 100 μM to be sufficient for detecting damaged viral capsids. Due to the increased cost associated with using 200 μM of PMAxx dye, and the fact that 100 μM was deemed sufficient, we decided to assess lower concentrations of the dye. The heat-inactivated viruses exposed to PMAxx dye had a lower amplification resulting in lower gc/ml values than that of the live and no-dye-treated heat-inactivated viruses, a result consistent with other studies that assessed the effectiveness of PMAxx on heat treated Norovirus GI and GII and HAV [42,58]. In this study, we used heat inactivation to damage the viral capsid and compared the results to corresponding samples that did not undergo heat inactivation. Exposing viruses to high temperatures (>50 °C) causes them to become non-infectious and is a well-established method, as evidenced in cell culture assays [59]. However, using lower temperatures (60] and similarly for NoV on vegetables [58]. However, the concentration of PMAxx dye treatment required varies depending on the sample type and what compounds maybe present in the sample [30]. Therefore, initial optimisation is required before the use of PMAxx in capsid integrity assays, across the sample type and target viruses.

Once the concentration of PMAxx dye, effective on several viruses, was determined, we tested our assay on wastewater samples for more targets (AdV, EV, HAV, IAV, IBV, NoV GI, NoV GII, and SARS-CoV-2). Wastewater samples were negative for AdV, HAV, IAV, and IBV. Therefore, we compared the RT-qPCR and ci-RT-qPCR data for EV, NoV GI, NoV GII, and SARS-CoV-2, which were detected in the wastewater samples. SARS-CoV-2 was detected in all samples by RT-qPCR only, showing that SARS-CoV-2 did not remain viable in the wastewater samples tested; as a result, the log reductions were not calculated, as no intact SARS-CoV-2 viruses were detected. The integrity of SARS-CoV-2 in wastewater samples has previously been investigated using PMAxx dyes and cell culture [31]. SARS-CoV-2 that had been spiked into wastewater had strongly (>3 log) or moderately (>1 log) reduced infectivity, and encapsulated SARS-CoV-2 RNA was always detected at a lower titre than total RNA in wastewater [31]. The reduced integrity of SARS-CoV-2 in wastewater is likely due to the enveloped capsid, which is less stable than that of non-enveloped viruses (e.g., NoVs, EV) [61], a result which mirrors the known modes of SARS-CoV-2 transmission. EV, NoV GI, and NoV GII were detected by RT-qPCR and ci-RT-qPCR. EV showed the lowest log reductions between RT-qPCR and ci-RT-qPCR, suggesting the viral particles remain intact and potentially infectious in wastewater. The EV RT-qPCR assay used in this study is a non-specific assay that detects a highly conserved region of the 62 nonpolio enteroviruses and three poliovirus types characterised at the time the assay was developed [62]. Therefore, the exact enterovirus strains detected are unknown. Norovirus GI and GII were detected in all samples, with NoV GII at higher gene copies. The log reductions for NoV GI and GII were 30], while PMA dyes have been used to detect NoV GI and GII in contaminated drinking water [63], freshwater [29], and sewage samples [64]; again, these viruses remain intact and pose a human health risk.

Reductions in viral gene copies for inactivated viruses were observed in our assays, and our results show that PMAxx dyes are suitable for use in WBS; however, there are limitations to the approach, and where possible, steps should be taken to avoid these. Intercalating dyes such as PMAxx rely on a photolysis step; therefore, the sample matrix needs to be considered, as shown when detecting viruses in shellfish [30], as when a sample has a high volume of suspended solids, light penetration maybe insufficient to induce the reaction. The floccing of viruses in the sample may also need to be considered, as damaged viruses in the middle of viral aggregations maybe protected from the dye, and samples may need to be agitated to limit this. In addition, the dyes cannot differentiate between non-viable viruses that have an intact capsid and live infectious viruses, as seen with UV treatment [36,39]. Furthermore, the target virus, concentration of dye, length of dye incubation period, and length of light exposure all need to be considered [38]. Therefore, while ci-RT-qPCR gives a better estimation of the number of viable virus particles than RT-qPCR alone, an overestimation of viable viruses within a sample may still occur.