Recently, there have been several examples of human-associated viruses discovered by metagenomic methods. Below we review some notable examples of virus detection in the water environment following their discovery by metagenomics and the potential to use these viruses as water quality monitoring tools.

Bocavirus

Human bocavirus (HBoV) is a member of the parvovirus family first described in 2005 as an agent of respiratory infection using an early metagenomic approach.38 HBoV is a non-enveloped virus with single-stranded 5Kb DNA genome. As HBoV is often detected concurrently with established pathogens, the role of HBoV as a causative agent in disease is unresolved due to the lack either of a simplified cell culture system or animal model.57 HBoV was detected in fecal samples from children who have diarrhea regardless of the presence of respiratory infection, indicating that HBoV may be both an enteric and respiratory pathogen.58

HBoV has been detected in wastewater and environmental waters. Table 1 shows a summary of HBoV detection in wastewater samples and the specific assays used. HBoV was detected using qPCR in 100, 81, 79, and 51% of untreated wastewater samples from Egypt, the United States, Italy, and Norway, respectively.59,60,61,62 HBoV was also detected by viral metagenomics in raw sewage from Nepal and the United States.63 In a 2009 study in Germany, 40% of river water samples contained HBoV at concentrations between 3 × 101 and 2 × 103 genome copies/L.64 Previous environmental detection of HBoV have employed multiple detection methods, including qPCR65 and Luminex66 assays.

Table 1 Previous studies detecting HBoV in wastewater and associated PCR primers Full size table

The study of 28 children hospitalized during a sewage-contaminated drinking water outbreak in Finland highlighted the potential for environmental spread of HBoV.67 In three of the four cases multiple viruses were detected; but in one case, HBoV was the only virus detected, suggesting that HBoV was the primary cause of symptoms.67

The evidence suggests that the metagenomic discovery of HBoV identified an unrecognized but likely minor source of waterborne viral disease; however, the utility of HBoV as a viral water quality-monitoring tool may be limited by method specificity to wastewater less than 100% in the majority of previous studies.

Cosavirus

Human cosavirus (HCoSV), a genus in the Picornaviridae family, was first identified in 2008 in stool samples of Pakistani children with non-polio acute flaccid paralysis through the use of viral metagenomics.68,69 HCoSV has a 7.3 Kb single-stranded RNA genome and is genetically diverse.70 HCoSV was previously detected in stool samples from multiple regions throughout the world.71,72 The presence of HCoSV strongly correlates with diarrhea;70 however, HCoSV has also been detected in the stool of healthy individuals.71 Its higher concentrations in those without gastroenteritis complicates the understanding of its pathogenesis.72 HCoSV was also previously detected in pig feces, albeit at lower concentrations than found in infected humans.73

Table 2 shows a summary of HCoSV detection in wastewater samples and the specific assays used. HCoSV was detected using qPCR in 71 and 25–38% of untreated sewage samples in Japan and the United States, respectively.59,74,75 HCoSV was also detected by viral metagenomics in raw sewage from Nepal.63 HCoSV has also been detected using qPCR in treated wastewater samples in the US, France, and Japan, with 1–2 log 10 removal following treatment.74,75,76 HCoSV was previously detected using qPCR in 29% of river samples in Japan and 12% of tributary samples in France at lower concentrations than in wastewater samples.74,76 Notably, HCoSV was recently detected using qPCR at relatively higher concentrations than observed in other environmental waters in the Bagmati River, Nepal, highlighting the importance of geographic variability in marker evaluation.77

Table 2 Previous studies detecting HCoSV in wastewater and associated PCR primers Full size table

HCoSV may be a causative agent of waterborne viral disease; however, its variable specificity to wastewater (25–71%) across studies and presence in animal sources (pig) may limit its application for viral water quality monitoring. Notably, the recent detection of elevated HCoSV concentrations in environmental waters in Nepal points to a greater potential for application in specific geographic regions.

Cross-assembly phage

Cross-assembly phage, or “CrAssphage”, is a bacteriophage that was discovered in 2014 by metagenomic data mining of human fecal microbiome sequence data.39 It has a dsDNA, 97Kb genome.39 The study found that the CrAssphage genome was present in 73% of available human fecal metagenomes, and that CrAssphage was more abundant in the human gut than all other known human gut phages combined.39 The phage was predicted to be a Bacteroides bacteriophage by co-occurrence profiling,39 and recent successful cultivation of CrAssphage has confirmed this prediction.78 Recent work has identified CrAssphage as the prototypical virus in a new viral family.79

CrAssphage has been successfully detected in both sewage and environmental waters. Table 3 shows a summary of CrAssphage detection in wastewater samples. CrAssphage was detected using metagenomics in 100% of wastewater samples surveyed and was most abundant in samples from North America and Europe and less abundant in samples from Africa and Asia.80 Subsequently, CrAssphage qPCR assays were developed, and CrAssphage was detected in 100% of untreated wastewater samples from throughout the United States and Spain at average concentrations of greater than 109 genome copies/L.49,81,82 CrAssphage was also recently detected with 100% occurrence in untreated wastewater samples from Australia.83 CrAssphage appears to be highly human-associated; however, previous studies have had positive CrAssphage detections in seagull, dog, chicken, cat, and cow fecal samples, albeit at decreased concentrations to those found in human sewage.49,82,84 Recent studies have also successfully deployed CrAssphage markers to detect fecal pollution in environmental waters.84,85 Finally, a recent study exploring CrAssphage diversity found CrAssphage in sewage samples globally.86

Table 3 Previous studies detecting CrAssphage in wastewater and associated PCR primers Full size table

High detection rates and high specificity to human wastewater as well as the high abundance in sewage suggest the significant potential of CrAssphage as a viral water quality indicator and could be a valuable addition to the microbial source tracking toolbox. However, further demonstrations are necessary, including detection in multiple environments, correlation with viral pathogens, and geographic diversity, abundance, and exploration if different specific animal variants of CrAssphage exist.

Klassevirus

Human Klassevirus, also known as Salivirus, was first discovered by metagenomic sequencing of a novel picornavirus in pediatric stool samples in the United States.37,87 Klassevirus has a 6–8 Kb single-stranded RNA genome. Serological evidence has identified humans as the host for Klassevirus.88

Table 4 summarizes Klassevirus detection in wastewater samples. Klassevirus was detected using qPCR in 14.6, 57, and 50% of wastewater samples in the United States, South Korea, and Japan, respectively.89,90,91 Notably, using an improved qPCR assay, Klassevirus was detected in 93% of wastewater samples with a maximum concentration of 9.7 × 106 genome copies/L in Japan.92 Klassevirus was also detected by viral metagenomics in untreated sewage from Nepal.63 Klassevirus was also detected using qPCR in 44, 29, and 42–50% of treated wastewater samples from France, Japan, and the United States, respectively.76,89,92 Finally, Klassevirus was detected using qPCR in nine out of 56 river water samples in Japan (16% detection rate) and two of six samples (33%) in Barcelona river water and in one of six samples (16%) in Rio de Janeiro known to be contaminated with sewage.92,93

Table 4 Previous studies detecting klassevirus in wastewater and associated PCR primers Full size table

Klassevirus has been observed globally in wastewater, treated wastewater, and in environmental waters, characteristics necessary for a human fecal pollution indicator. The development of an improved assay by Haramoto et al. that increased detection rates in sewage from ~50 to >90%92 increases the potential to utilize Klassevirus in water quality monitoring applications; however, the lower concentrations in sewage (<107 genome copies/L) than other proposed viral indicators may limit its further development.

Pepper mild mottle virus

PMMoV is a plant pathogen that infects a wide variety of pepper cultivars. PMMoV has an approximately 6.3 Kb single-stranded RNA genome.94 PMMoV was not discovered by viral metagenomics, but is included in this discussion as PMMoV was first proposed as a viral indicator of fecal pollution95 following the metagenomic observation of the abundance of PMMoV in feces.96 The presence of PMMoV in feces and wastewater is due to consumption of infected pepper products. PMMoV is mainly specific to human feces but has previously been detected in chicken, geese, seagull and cow fecal samples.95 The authors posited that this was due to animal consumption of products containing peppers.95 A recent manuscript reviewed PMMoV in viral water quality monitoring.97

Table 5 summarizes of PMMoV detection in wastewater. The original Rosario et al. study found PMMoV using qPCR in 100% of untreated wastewater samples from throughout the United States at concentrations of greater than 109 genome copies /L.95 Subsequent studies using qPCR have found 100% detection rates in untreated sewage in the United States, Costa Rica, and Vietnam.98,99 An exception to this high detection rate in sewage is a previous study in South Korea with a PMMoV detection rate of 57%.90 PMMoV was also found in wastewater in Australia100 and Germany101.

Table 5 Previous studies detecting pepper mild mottle virus in wastewater and associated PCR primers Full size table

Studies have also investigated the removal of PMMoV in water and wastewater treatment. Observed PMMoV removal using qPCR in wastewater treatment plants in the United States and Germany varied from <1 to 3.7 log 10 .101,102 Limited to no removal was observed in Bolivian wastewater treatment ponds103 and surface flow wetlands in the United States.104 For drinking water processes, 1–2 log 10 removal was removed by coagulation and filtration in Thailand,105 and reverse-osmosis removed PMMoV to below the LOD106. PMMoV was proposed as a viral indicator in wastewater reuse.107

PMMoV has also been detected in environmental waters using qPCR, including 97% of river samples in Germany,101 94% of surface water samples and 38% of groundwater samples in Vietnam,99 76% of surface water source samples in Japan,108 and 33% of beach water samples in Australia.100 Conversely, PMMoV was not detected in any Costa Rican surface water samples despite the detection of FIB and other MST markers, and 100% PMMoV detection in untreated wastewater.98 Finally, PMMoV was found in 85% of Karst groundwater samples in Mexico (all samples positive for total coliforms)109 and PMMoV was the most commonly detected virus in groundwater at artificial groundwater recharge site.110 Observed PMMoV are genetically diverse in the environment.108

The abundance of PMMoV in wastewater and high specificity suggests its high potential as a fecal pollution indicator. One potential challenge may be the detection of PMMoV in chicken, seagull, cow, and goose feces, limiting its human-specificity;97 however, the previously demonstrated specificity is above the widely-accepted 80% threshold. In addition, the high detection rates in environmental waters suggest that PMMoV is persistent, potentially limiting its application to detect recent pollution events, although further research is necessary.