PMMoV and Coliphages in Wastewater: Prominence and Limitations as Viral Indicators
The use of PMMoV and coliphages as indicators in water quality evaluation and potable reuse have been increasingly recognized as a robust, cost-effective, and safe method for assessing water safety and treatment effectiveness. With regards to PMMoV, related studies can be traced back to 2009 when it was identified as correlating with pathogens within the US wastewater system. 24 Initially, it was perceived as a robust indicator of fecal pollution predominantly in coastal environments. Subsequent studies expanded its utility, assessing its prevalence and persistence in river water and sewage samples.25, 26 From 2014 to 2018, PMMoV's use was sparsely observed in a few US coastal cities, primarily serving as a control for enterovirus management during water treatment processes.27, 28 Especially by 2019, the application of monitoring PMMoV as an indicator of wastewater pollution and a gauge for treatment process efficacy had significantly expanded.29 It became a mainstay in public health management, serving as a predominant endogenous wastewater virus biomarker for evaluating water quality and fecal contamination. 18, 29-31
Our observation is consistent with previous studies that have found PMMoV showed a constant high concentration at 6-log to 8-log level during a 13-month study period (Figures 1-4 & Supplemental Figure 1). Our results also are in agreement with previous studies that identified an increase in PMMoV levels in wastewater during wet weather conditions like precipitation events.32 This increase is often attributed to augmented sewage flow and runoff, which can transport the virus from the environment into wastewater treatment plants.32-34 Additionally, several studies have demonstrated that PMMoV has a dietary origin (e.g., pepper and seasoning blends), found in human feces, and it has also been detected together with other pathogens in 72% of raw and 67% of treated wastewater samples.3, 26, 33 Thus, PMMoV has several limitations that highlight the need to identify additional or alternative indicators. Prior studies have claimed that PMMoV may not be a suitable viral indicator because it is a plant virus not specifically associated with fecal contamination.35 While PMMoV can be present in sewage and other environmental samples, it is not exclusive to human or animal feces and can also be found in soil, water, and plants. This makes it difficult to use PMMoV as a reliable indicator of fecal contamination in water or food samples. In contrast, other viruses (e.g., norovirus and adenovirus) are more specific to fecal contamination and are commonly used as water or food safety indicators.26, 36, 37
Bacteriophages have widely been used as indicators of water quality and treatment efficacy (e.g., after removal of viruses during the wastewater treatment processes); moreover, their detection in wastewater, surface waters, and sand columns for longer periods than enteroviruses along with their resistance to purification and disinfection processes, close resemblance to enteroviruses, and easy detectability make them effective surrogates to assess the behavior and fate of viruses in water systems.5, 8, 38, 39 They have several advantages as viral indicators, including their close morphological and biological properties to enteroviruses, their presence in water environments (when enteroviruses are present), their resistance to purification and disinfection processes, and their detectability by simple, rapid, and inexpensive methods;39-46 furthermore, they are specific for fecal contamination, non-pathogenic, and do not multiply in water environments.39-46 Coliphages have been used as models for studying the behavior and survival of enteroviruses in the environment, as well as the resistance of human viruses to water treatment and disinfection processes (e.g., chlorination and UV irradiation).6, 38, 47, 48 They have also been used to evaluate the virological risk associated with the reuse of sludge in agriculture (including wetlands) and as indicators of thermal inactivation of pathogens in biosolids.44, 45, 49
While coliphages have been widely used as indicators of water quality and treatment efficacy, their use has some limitations compared to PMMoV. For instance, they may not always be present in water environments where enteroviruses are present;50 further, the concentration of coliphages may vary and may not always be correlated with the presence or abundance of human viruses.50 This lack of correlation between coliphages and specific viral pathogens can limit their effectiveness as indicators of viral pollution.51 Furthermore, some studies have found that coliphages may not be as specific for fecal contamination as initially thought.52 There is evidence that coliphages can replicate in the water environment, resulting in inaccurate reflection of fecal microbial contamination, which can lead to the detection of coliphages that originated outside the gut, confounding their use as indicators of fecal contamination.52 Bacteriophage-based viral detection and quantification methods may lack sensitivity or exhibit poor specificity, further limiting their suitability for widespread water quality management applications.51 Coliphages have been reported in lower concentrations than other viral targets which can limit their usefulness in certain applications, thus requiring larger sample volumes or concentration techniques for detection, which is more time- and cost-consuming.53, 54
Occurrence of ToBRFV in Wastewater and its Potential as a Viral Indicator
Despite the use of PMMoV and bacteriophages, our study showed that ToBRFV can serve as a better indicator for wastewater treatment and propose its use for future virus outbreaks. When comparing ToBRFV to PMMoV, coliphages, and other plant/fruit viruses, we saw an increased predominance of ToBRFV, which also has a strong correlation with other pathogens, including betacoronaviruses (e.g., SARS-CoV-2) (Figures 1 & 2). ToBRFV was first described in 2018, after which it has been well established, primarily in the agricultural space, regarding its emerging prevalence and effects on local and global tomato production output.9, 55-57 To date, however, no study reports the use, or possibility for use, of ToBRFV for the improvement of public health and infectious disease surveillance or being used to evaluate the water quality and wastewater treatment efficacy.
WRFs, in both rural and urban sites, can use ToBRFV to provide increased confidence in monitoring data as a pooled sample in a study site or region rather than individual samples.13 Importantly, the consistently higher abundance of ToBRFV compared to other virus biomarkers implicates a higher degree of water purification that can be verified with ToBRFV. For instance, using PMMoV or MS2 for wastewater treatment quantification will yield more conservative water treatment efficacy, as any virus with a higher abundance (e.g., ToBRFV and yet-to-be-identified/classified viruses) will remain in the wastewater; thus, using ToBRFV will increase the threshold of detection to ensure all viruses with lower abundances will get removed. The advantage of using a highly abundant viral indicator that is consistently and robustly persistent in the wastewater is that they provide a more accurate assessment of the effectiveness of the wastewater treatment process in removing viral contaminants. ToBRFV can be used as a more sensitive and specific viral indicator, identified in human stool and wastewater, and is more prevalent and abundant than all currently used markers.11 By using ToBRFV as a persistent viral indicator, WRFs globally can better monitor and improve their treatment processes to ensure that they are more effectively removing viral contaminants and producing safer, higher-quality effluent; moreover, this will further help protect public health and the environment by reducing the risk of exposure to harmful viruses and other pathogens.
Challenges
This study has several limitations that should be considered when interpreting the results. While our study assessed the presence of ToBRFV, PMMoV, and bacteriophages in various wastewater systems in Northern NV, USA, it is important to acknowledge that the distribution and persistence of these viral indicators might differ in other geographical regions. Expanding the study to include more diverse locations could provide a broader understanding of the applicability of ToBRFV as a viral indicator in different environments and regions. The abundance and availability of ToBRFV in regions across the US and worldwide have yet to be determined. After determining these concentrations, ToBRFV can be used confidently as a new normalization biomarker, albeit notwithstanding the utility of PMMoV and coliphages.
Furthermore, studies should investigate the performance of ToBRFV in comparison to a broader range of potential viral indicators, including human enteroviruses, to establish a more comprehensive understanding of its effectiveness. Although we utilized two diagnostic sample processing methods (RVP panel and SC-2-enrichment method) to identify and quantify ToBRFV and PMMoV in the wastewater samples, it is possible that the use of alternative methods (e.g., digital droplet PCR) could influence the detection and quantification of these viral indicators. Further research should explore the performance of different detection methods to establish the most reliable and accurate approach for identifying and quantifying ToBRFV in wastewater samples.
Summary
We report the detection of ToBRFV in wastewater samples collected in the Reno-Sparks metropolitan area from November 2021 to November 2022. Our study demonstrated the potential of ToBRFV as a novel viral indicator for assessing water quality and treatment efficacy in water reclamation systems. At our largest collection area site, which represents 80% of the influent from this region, the RVP sample processing method showed ample ToBRFV abundance compared to the currently used indicators (PMMoV and bacteriophages) as well as other plant/fruit viruses. These results are consistent with those from the other 2 sampled WRF sites and 3 sub-sewershed sites, suggesting that ToBRFV could be a more reliable and robust indicator for water quality and treatment efficacy in various geographic settings, which is critical for safeguarding public health in both urban and rural settings. Moreover, using ToBRFV will increase the threshold of detection for all other pathogenic viruses in the wastewater, yielding more accurate evaluation of water purity. Thus, we postulate the future use of ToBRFV as a better biomarker than PMMoV and coliphages for water quality and health risk evaluation, especially in regions with a high abundance of ToBRFV, which may allow for better evaluation and optimization of the water treatment processes, ultimately improving the safety and quality of water supplies for the public. Further research should focus on validating the effectiveness of ToBRFV as a viral indicator in different geographical regions and water systems and comparing its performance to a broader range of potential viral indicators, including human enteroviruses.