Human and animal enteric viral markers for tracking the sources of faecal pollution

1.0 Performance Characteristics of the Viral MST Markers

1.1 Overview

The human enteric viruses, adenoviruses, polyomaviruses, and a plant virus, the pepper mild mottle viruses (PMMoVs) have received significant attention as library-independent microbial source tracking (MST) markers of human wastewater due to their high abundance in the faeces and urine of humans, high persistency in environmental waters, and strict host-specificity (Wong et al., 2012). To identify the sources of human wastewater pollution, PCR/qPCR detection of human adenoviruses (HAdVs), human polyomaviruses (HPyVs), and pepper mild mottle viruses (PMMoVs) have been the most commonly used tools in MST studies (Fong et al., 2005; McQuaig et al., 2009; Rosario et al., 2009), whereas, animal viruses such as porcine adenoviruses (PAdVs), bovine adenoviruses (BAdVs), bovine polyomaviruses (BPyVs), chicken/turkey parvoviruses (ChPVs/TuPVs) have also been used at some extent to identify animal wastewater pollution in environmental waters (Hundesa et al., 2006; Carratalá et al., 2012). For the detection of human and animal viral MST markers, PCR and qPCR based assays are used over cell culture assays. Cell culture assays provide information on the infectivity of the viral markers, which may not be considered important from the MST point of view.

The host-sensitivity, host-specificity are important aspects of the characteristics of these viruses as well as the possible correlation between faecal indicator bacteria and viral MST markers, and their relevance to public health risks. Their concentrations of human and animal wastewater-associated viral markers in point sources (raw human wastewater, secondary effluent, treated wastewater and sludge samples), and receiving waters (recreational water, storm water and drinking water reservoirs) are summarized. The application of enteric viruses have been included using relevant MST studies Extending the use of enteric viruses for MST field application through new technologies such as metagenomics, and the importance of efficient virus recovery methods are included.

1.2 Host-sensitivity and -specificity of human viral markers

Host-sensitivity and -specificity are considered as the two most important performance characteristics of MST markers. Non-specific (found in non-target hosts) and non-prevalent (rare) markers tend to yield false positive or negative results in field studies (Stoeckel and Harwood, 2007; Ahmed et al., 2013). It should be pointed out that in PCR-based assays, the precise sequence targeted and the primers/probes used can play a major role in sensitivity and specificity of a given marker/assay as similar sequences may be amplified by poorly designed oligonucleotides. Thus, all assays for a given target may not be equally effective.

The performance characteristics of HPyVs have been evaluated more rigorously than HAdVs. HPyVs are reported to be highly prevalent (100% host-sensitivity) in raw and septic wastewater samples in Australia, New Zealand, Spain and the USA (McQuaig et al., 2006; Ahmed et al., 2010b; Kirs et al., 2011; Rusiñol et al., 2014). McQuaig et al. (2006) developed a PCR assay for the detection of HPyVs in environmental waters. This assay was later upgraded to a TaqMan-based qPCR for the quantification of HPyVs in raw wastewater, human urine and environmental samples (McQuaig et al., 2009). These two PCR/qPCR assays have been most commonly used to determine the host-sensitivity and -specificity of HPyVs in human and non-human wastewater and faecal samples. Merging faecal and wastewater samples from studies (McQuaig et al., 2006; Harwood et al., 2009; McQuaig et al., 2009; Ahmed et al., 2010b; Kirs et al., 2011; Staley et al., 2012a; Hellein et al., 2011), the presence of HPyVs was tested by analysing 841 non-human faecal and wastewater samples. HPyVs were 100% specific to human wastewater and urine samples. Less is known regarding the host-sensitivity and -specificity of other human wastewater associated viruses such as PMMoVs, human enteroviruses (HEVs), human noroviruses group I and II (HNoVs-GI/GII). Some of these viruses although appear to be host-specific, but they have low host-sensitivity. For example, HEVs were detected in 38% of eight wastewater samples (Noble et al., 2003), whereas, HNoVs-GI and HNoVs-GII were detected in 82% of 11 raw wastewater samples (Wolf et al., 2010).

1.3 Host-sensitivity and -specificity of animal viral markers

Table 2 summarizes the host-sensitivity and -specificity values for animal faecal-associated viral markers in the literature. PAdVs, BAdVs and BPyVs have been used as targets to identify porcine and bovine faecal pollution in field studies. The prevalence of these viruses can be high in slaughterhouse wastewater (a mixture of faeces and urine from thousand animals) compared to a faecal sample from an individual animal. In reality, the chance of slaughterhouse or farm wastewater reaching environmental waters is higher than waste from an individual animal. To date, only three studies have investigated the host-specificity of PAdVs (De Motes et al., 2004; Hundesa et al., 2009; Wolf et al., 2010). Merging faecal samples from these studies, the presence of PAdVs was determined by analysing 87 non-porcine faecal samples. PAdVs were 100% specific to faeces tested. BAdVs also appear to be highly host-specific (100%) when merging 329 non-bovine faecal samples from studies by De Motes et al., 2004; Ahmed et al., 2010a; Wolf et al., 2010; Wong and Xagoraraki, 2010; Ahmed et al., 2013). Little has been published regarding the host-sensitivity and -specificity of ovine polyomaviruses (OPyVs), bovine noroviruses (BNoVs), bovine enteroviruses (BEVs) and porcine teschoviruses (PTVs) (Ley et al., 2002; Wolf et al., 2010; Rusiñol et al., 2013). Nonetheless, these viral markers have shown potential to indicate bovine and porcine faecal pollution in environmental waters. Their application along with commonly used BAdVs, PAdVs and BPyVs in a “toolbox” format may provide additional information on the presence of animal wastewater in environmental water samples.

Human and animal enteric viruses are highly host-specific, with the exception of two studies that reported the occasional presence of host-specific viruses in non-target hosts. For example, BEVs (cattle) have been detected in goose, deer, sheep, goat and horse faecal samples (Ley et al., 2002; Jimenez-Clavero et al., 2005). Similarly, PMMoVs have been detected in chicken and seagull faecal samples (Rosario et al., 2009). The non-specific markers are unreliable for field studies due to the possibility of yielding false positive detection, which may result in the wasted capital investment for mitigation activities. In such scenario, obtaining additional information on the concentrations of non-specific markers in faecal samples may be important. Considering the high concentration (10⁷/10⁸ gene copies per L) of a marker in its host, it is unlikely that if it is detected at a low concentration (10¹/10² gene copies per L) in the non-target host(s) would be a limitation to MST results interpretation (Weidhaas et al., 2010).

Notably, most of the host-sensitivity and -specificity assays have been undertaken in Australia, New Zealand, Spain and the USA. A little is known regarding the host-sensitivity and-specificity of viral markers in other continents such as Asia, Africa and South America. Further host-sensitivity and -specificity testing on the currently used markers should be undertaken prior to their application in new geographical locations. In addition, new assays need to be developed specifically for MST studies, since many currently used qPCR assays used for source tracking purposes have been originally developed to monitor enteric viruses in clinical studies and wastewater treatment efficacy monitoring (Heim et al., 2003; He and Jiang, 2005; Wong et al., 2012). Priority should also be given to develop new assays to detect faecal pollution from a wide range of wild, pet and domesticated animals.

1.4 Correlation among FIB, pathogens and viral MST markers and the relevance to public health risks

An ideal MST marker should correlate with FIB that is used as regulatory standards. Several studies have reported negative correlations between FIB concentrations and viral markers in environmental waters (Fong et al., 2005; Korajkic et al., 2010; Staley et al., 2012a; Ahmed et al., 2013; Sidhu et al., 2013). Several factors such as types of water, dilution effect, turbidity, differences in analytical methods (culture-based vs. qPCR), sources of faecal inputs, differential decay and numbers of samples may account for the lack of correlations observed. Harwood et al., (2009) compared Enterococcus spp. concentrations in undiluted human wastewater and the dilution corresponding to the limit of detection of HPyVs by PCR, with the rationale that the observations should be correlated if the FIB and marker co-vary in the undiluted human wastewater (Harwood et al., 2009). The study found no significant correlation between HPyVs and Enterococcus spp. concentrations in human wastewater and the dilution necessary to achieve the limit of detection. McQuaig et al., (2009) determined the correlations among FIB (faecal coliforms, E. coli and Enterococcus spp.) and HPyVs for human, disinfected and septic wastewater samples. HPyVs were poorly or negatively correlated with all three FIB tested. Poor correlations between FIB and viral MST markers may not necessarily hinder their application as MST tools if the objective of the study is to determine the sources of faecal pollution for mitigation purpose.

From public health point of view, the relationship between viral markers and pathogens is more critical than that of the markers to FIB. One of the important aspects of using viral markers for MST field studies is that their presence indicates potential health risks because some of the viral targets such as HAdVs, HEVs and HNoVs are capable of causing illnesses in humans. Further studies would be required to obtain information on the correlations of viral markers to pathogens to translate the health outcomes of human wastewater pollution in environmental waters.

2.0 Overview of Methods for Recovery of Viral MST Markers from Environmental Matrices

Enteric viruses are relatively difficult to concentrate from environmental waters due to typically low concentrations and their small size (Maier et al., 2008). Although rapid enumeration of viruses by quantitative PCR (qPCR) has the potential to greatly improve water quality analysis and risk assessment, the upstream steps of capturing and recovering viruses from environmental water sources along with removing PCR inhibitors from extracted nucleic acids remain formidable barriers to routine use. A wide range of methods has been developed and used to recover viruses from various types of environmental waters (Casas and Sunen, 2002; Katayama et al., 2002; Lambertini et al., 2008; Nordgren et al., 2009; Rodriguez-Diaz et al., 2009; Bennett et al., 2010; Leskinen et al., 2010). Virus adsorption and elution (VIRADEL) methods such as negatively charged HA membranes (Katayama et al., 2002; Haramoto et al., 2005), positively or negatively charged 1MDS cartridge filters (Lukasik et al., 2000), filterite filters (Wetz et al., 2004), electropositive nanoCeram filters (Lee et al., 2011) and glass wool (Lambertini et al., 2008) have been most commonly used to recover viruses from environmental waters. The mechanisms of these methods have been described in a review paper (Wong et al., 2012).

Besides the VIRADEL methods, virus recovery techniques such as hollow-fiber ultrafiltration (HFUF) has been used widely to recover viruses from environmental waters. Research studies by several groups (Morales-Morales et al., 2003; Hill et al., 2005; Hill et al., 2007; Polaczyk et al., 2008;) reported that HFUF can be effective for higher recovery (50-90%) of viruses, bacteria and parasites from various water matrices. In addition, HFUF is rapid, and it does not require the use of extensive chemicals. The method also simultaneously retains bacteria, protozoa and viruses in a single step, which is an added advantage when analysis of multiple MST markers is required (Kfir et al., 1995; Morales-Morales et al., 2003; Wong et al., 2012).

It has been suggested that capturing viruses on membranes followed by direct nucleic acid extraction may result in higher recoveries compared to protocols that require viral elution from membranes (Wong et al., 2012). Ahmed et al., (2015) compared the efficiency of virus recovery for three rapid methods of concentrating HAdVs and HPyVs from river water samples on HA membranes. Samples were spiked with raw wastewater, and viral adsorption to membranes was promoted by acidification or addition of MgCl₂. Viral nucleic acid was extracted directly from membranes, or viruses were eluted with NaOH and concentrated by centrifugal ultrafiltration. Recovery efficiencies of HAdVs and HPyVs were approximately ten-fold greater for a method that involved direct DNA extraction compared to the frequently-used strategy of viral absorption with added cations (Mg²⁺) and elution with acid, with mean recovery efficiency ranging from 31-78%.

Recovery of viruses from environmental water requires filtration on the scale of 1-100 L of the sample. Processing larger volume of samples can be difficult to accomplish in the field, and many methods require expensive, expendable filters that cannot be reused. When large volumes are processed, it is always necessary to use a secondary concentration method. Reconcentration methods such as organic flocculation (Katzenelson et al., 1976), and polyethylene glycol (PEG) precipitation (Lewis and Metcalf, 1988) have some disadvantages, e.g. these methods do not produce consistent recovery efficiency for different viruses and the sample processing time can be lengthy (Lewis and Metcalf, 1988). Alternatively, specifically designed ultrafilters, which retain viruses based on molecular weight cut-off can be used as a secondary concentration step. In a previous study, Centriprep Filter Concentrators provided high and stable recovery yields (74%) of seeded polioviruses (Haramoto et al., 2004). Another study reported the 35% recovery of HAdV 41 through Centricon filters (Wu et al., 2011). Ultracentrifugation is another alternative method for virus concentration from environmental water samples. This method requires minimal sample manipulation and samples can be processed under natural pH and an elution step is not needed. By ultracentrifugation it is possible to concentrate all viruses in a sample, by using a sufficient g-force. A drawback of this method is that fine organic matter present in certain environmental matrices is also concentrated during the ultrafiltration procedure, and can cause PCR inhibition issues during downstream analysis. Alternatively, a number of recent studies have taken the optional approach of concentrating 1-2 L volumes of surface water by membrane filtration to test for enteric viruses (Katayama et al., 2002; Fong et al., 2005; Haramoto et al., 2005; De Paula et al., 2007; Haramoto et al., 2008; Miagostovich et al., 2008; Victoria et al., 2009; Ahmed et al., 2010a; Ahmed et al., 2010b). Application of these methods may be suitable for MST field studies, which warrant delivering the results rapidly for remediation. If the concentration of a chosen viral marker is high in its host, then using a small volume of water sample may not be problematic to its detection in the environment.

One method that accomplishes highly efficient concentration of all viruses has not yet been found. Variations in several factors such as adsorption of viruses to membranes, membrane type, elution buffer, seeding materials (strains vs. wastewater), seeded concentrations, sample type, sample volume and sensitivity of qPCR assays can influence recovery efficiency (Bofill-Mas et al., 2006; Albinana-Gimenez et al., 2009; Li et al., 2010). A good recovery method should fulfil several criteria: it should be simple, rapid, provide high recovery, consistently recover a wide range of viruses, provide a small volume of concentrate, be cost effective and not alter viral community structure (Bosch, 1998; Angly et al., 2006). More studies are needed for the development of new recovery methods and comparing with the existing methods for the effective recovery of viruses from environmental waters.

3.0 Environmental Occurrence and Persistence

3.1 Concentrations of viral MST markers in human wastewater, faeces, and urine

The concentration of a viral marker in its host is an important factor because it is likely that a marker whose concentration is high will be consistently and more easily detected in polluted water samples. Markers whose concentrations are highly variable or low can be difficult to detect in the environment due to factors such as dilution, turbidity and different decay rates. Tables 3 and 4 summarize the concentrations of human and animal markers in target host groups and point sources in the published studies. Several studies have provided quantitative data on HAdVs and HPyVs in raw, treated and septic tank wastewater. The mean concentrations of HAdVs A-F in raw wastewater samples tested in Australia, New Zealand and Spain ranged from 1.0×10⁶ to 8.7×10⁶ gene copies per L. However, the mean concentration of HAdV species C in raw wastewater samples was four orders of magnitude lower than the HAdVs group (species A-F) (Wolf et al., 2010). Such data clearly indicate that application of assays that are targeting one or two species of HAdVs may not be sensitive enough to detect human wastewater pollution in environmental samples. In general, the concentration of HAdVs in secondary wastewater was one to two orders of magnitude lower than raw wastewater, which is expected in effective wastewater treatment processes.

The concentration of HPyVs in human wastewater was similar to HAdVs, although one order of magnitude higher concentration has been reported in wastewater samples from Florida, USA (McQuaig et al., 2009). HPyVs in human urine samples were approximately two orders of magnitude higher than raw wastewater. This is because HPyVs are shed mainly through human urine. Among the human wastewater-associated viral markers, the concentrations of PMMoVs have been reported to be two to three orders of magnitude higher than HAdVs and HPyVs (Rosario et al., 2009). Such high concentrations may results in the overestimation of the magnitude of wastewater pollution in environmental waters; however, they may also be useful for tracing more dilute pollution. More studies would be required to investigate the usefulness of PMMoVs for MST field studies and associated health risks.

3.2 Concentrations of viral MST markers in animal wastewater, faeces, and urine

Compared to human wastewater viral markers, information on the concentration of animal markers is scant. PAdVs and BAdVs in composite slaughterhouse wastewater and dairy manure samples ranged from 1.0×10⁵ to 1.4×10⁷ gene copies per L (Hundesa et al., 2006; Wong and Xagoraraki, 2011). However, in the faeces of individual animals, the concentrations were two to three orders of magnitude lower than in wastewater (De Motes et al., 2004; Wong and Xagoraraki, 2010). Hundesa et al., (2006) developed a BPyV assay for the determination of bovine faecal pollution in the environment. The concentration of BPyVs in slaughterhouse wastewater estimated to be 1.0×10⁴ to 1.0×10⁵ per L. Similar concentrations of BPyVs in bovine urine and slaughterhouse wastewater have been reported in subsequent studies by the same group. In contrast, Wong and Xagoraraki (2011) reported much higher concentrations (1.2×10⁸ to 2.8×10⁹ per L) of BPyVs in dairy manure samples. It is recommended that a marker with high concentrations in its host should be used to determine the sources of faecal pollution in environmental waters (Ahmed et al., 2016). More studies are warranted to determine the concentrations of the viral markers in new geographical locations in order to identify their potential for global utility.

3.3 Concentrations of viral MST markers in the water environment

Tables 5 and 6 summarize the prevalence and concentrations of human and animal viral markers in various environmental matrices. HAdVs are reported to be highly prevalent in environmental water samples collected from Australia, Brazil, Greece, Hungary, Spain, Sweden, New Zealand and the USA. Only a handful of MST studies has provided the concentrations of HAdVs in environmental waters (Table 5). Among these studies, Rusiñol et al., (2014) investigated the concentrations of HAdVs in 792 water samples from diverse watersheds in Greece, Spain, Sweden, Hungary and Brazil. The concentrations of HAdVs were as high as 5.1×10⁷ per L which is similar to that found in wastewater. The prevalence and concentrations of HPyVs are reported in the published articles are similar to HAdVs (Chase et al., 2012; Rusiñol et al., 2014). The high prevalence of these two viruses in the environments could be attributed to the fact that both are double-stranded DNA viruses, which can persist lengthy periods in the environment (Love et al., 2010). Therefore, their presence may not indicate the recent pollution or any adverse health risks. The information on the recent pollution can be obtained by testing human RNA viruses such as PMMoVs or HEVs, which do not persist longer in the environment (Lipp et al., 2007).

Compared to human viral markers, less information is available on the prevalence and concentrations of animal wastewater markers (Table 6). PAdVs, BAdVs and BPyVs have been most commonly used in MST field studies in the South Pacific, Europe and North America. Rusiñol et al., (2014) reported the wide prevalence of PAdVs and BPyVs in a river in Hungary with concentrations as high as 4.2×10⁸ and 3.9×10⁷ per L of water, respectively. Low to moderate prevalence of PAdVs and BPyVs have also been detected in river waters in Spain, Greece, Sweden and Brazil. Other animal viral markers such as BEVs, PTVs, OPyVs have also been detected in various types of surface water samples with low to high prevalence rates.

The presence/absence results of any given marker in a sample should be interpreted with care. Lack of detection does not necessarily indicate the sample is free from pollutants and safe for human exposure. As shown in Tables 4 and 5, viral concentrations vary widely in environmental samples, and Tables 3 and 4 demonstrate the variability in host faecal and wastewater. Therefore, detection of these markers in environmental waters can be difficult due to factors such as dilution, sorption to particulate matters, environmental persistence, loss due to recovery and DNA extraction, and the fact that only a small volume of DNA sample is used for PCR/qPCR analysis (Horswell et al., 2010; Wong et al., 2012). It is therefore, recommended that a “toolbox” approach should be used for the accurate identification of polluting source(s) (Noble et al., 2006; Ahmed et al., 2012; Mauffret et al., 2012). None of the field studies reported the presence of viral markers in soils and sediments. It is well known that these environmental matrices may harbour enteric viruses and other faecal microorganisms (Wheeler et al., 2003; Staggemeier et al., 2015). Viruses associated with particular matter in suspension or solid matrices tend to remain viable for a longer time than if they were dispersed in water (Schenewski and Julich, 2001). These results have implications for the accuracy of MST tools as regulatory standards for the protection of water quality.

4.0 Future Applications and Directions

The currently used MST viral tools rely on detecting or quantifying a single viral marker in a water sample. This is a significant limitation for better management of water quality. Detecting a single marker does not rule out the presence of potential faecal pollution from other sources. Viral community analysis may ultimately allow for a more comprehensive assessment of source contributions by identifying multiple sources of faecal pollution in a single sample. This could be advantageous for scenarios where the pollution sources are not known or mixed sources of pollution impacting waterways. Microarray technology where hundreds of probes targeting multiple markers and pathogens can be tested simultaneously by hybridization makes it a powerful tool for MST studies. Li et al. (2015) developed a custom microarray targeting pathogens, MST markers and antibiotic resistance genes. The array was able to detect HAdVs, bacteriophage, bocavirus, influenza C, HNoVs, HPyVs, PMMoVs and torqueteno viruses (TTVs) in human wastewater and animal faeces. The authors also noted that the host-sensitivity values of human markers were somewhat low, ranging from 21 to 33%. However, the host-specificity values were much higher ranging from 83 to 90%. Therefore, further studies are warranted in order to improve the detection sensitivity of this technology.

4.1 Final Conclusions

Application of enteric viruses for MST field studies is promising due to their high host-specificity. Among the human wastewater-associated viral markers, HAdVs and HPyVs have been shown to be useful tools for tracking human wastewater in environmental waters. High persistence of HAdVs in the environment, and the fact that some serotypes can be pathogenic, make it a very useful viral marker. However, based on the performance characteristics, we recommend the use of both HAdVs and HPyVs in a tandem fashion for the accurate and sensitive detection of human wastewater pollution in environmental waters.

Little research has been undertaken on the host-specificity and -sensitivity of most animal viruses, particularly OPyVs, BNoVs, BEVs and PTVs. Some animal markers were also found to cross-react with faecal samples from non-target hosts. Further evaluation of performance characteristics should be undertaken to determine the broader applicability of these markers. Non-specific markers may still be useful for source tracking if information is available on the contributing sources and if possible testing should be accompanied with additional markers in a toolbox format.

Limited numbers of PCR/qPCR-based assays have been developed, and field tested. Several currently used qPCR assays have been originally developed to monitor enteric viruses in clinical samples and wastewater monitoring. Focus should be given on the development of new assays specifically for MST field studies. Priority should also be given to develop new assays to detect faecal pollution from wild animals, pets, birds, and domesticated animals.

Quantitative data on the occurrence of human and animal viruses in their hosts is limited. Such data is important to determine the suitability of a marker for detecting faecal pollution in environmental waters. Baseline concentrations which are appropriate to detect faecal pollution and also indicate health risks to some extent need to be established.

The high prevalence and concentrations of human wastewater associated markers in environmental waters in Australia, New Zealand, Spain, USA and several other European countries indicate chronic human wastewater pollution in environmental waters. The application of these tools is encouraged in continents such as Asia and Africa where wastewater pollution due to improper sanitation and gastrointestinal diseases is a major concern.

Regulatory and public health concerns mandate that more studies should be undertaken to gain an understanding of how viral markers correlate with FIB and pathogens. The absence of correlations does not necessarily impede the utility of these markers to identify faecal pollution and potential for mitigation of faecal pollution.

The most significant challenge associated with the field application of viral markers is effective, quantitative recovery of these viruses from environmental samples. Recent developments in virus recovery methods indicate that small volume (1 to 2 L) of an environmental water sample can be analysed for their potential presence making the analysis more rapid and cost-effective. Also recent developments in qPCR technology such as digital droplet PCR will enable sensitive detection of these viruses from environmental waters. Incorporation of quantitative microbial risk assessment (QMRA) with MST science will improve our understanding of the relative public health risks associated with human vs. animal faecal pollution.