Environmental waters are monitored for fecal pollution to protect public health and water resources. Traditionally, general fecal-indicator bacteria are used; however, they cannot distinguish human fecal waste from other animal pollution sources. Recently, a novel bacteriophage, crAssphage, was discovered by metagenomic data mining and reported to be abundant in and closely associated with human fecal waste. To confirm bioinformatic predictions, 384 primer sets were designed along the length of the crAssphage genome. Based on initial screening, two novel crAssphage qPCR assays (CPQ_056 and CPQ_064) were designed and evaluated in reference fecal samples and water matrices. The assays exhibited high specificities (98.6%) when tested against an animal fecal reference library, and crAssphage genetic markers were highly abundant in raw sewage and sewage-impacted water samples. In addition, CPQ_056 and CPQ_064 performance was compared to HF183/BacR287 and HumM2 assays in paired experiments. Findings confirm that viral crAssphage qPCR assays perform at a similar level to well-established bacterial human-associated fecal-source-identification approaches. These new viral-based assays could become important water quality management and research tools.

Microbiomes are vast communities of microbes and viruses that populate all natural ecosystems. Viruses have been considered the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared to other environments. Here we investigate the origin, evolution, and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboratory, we obtained DNA sequences of crAssphage from over one-third of the world's countries, and showed that its phylogeography is locally clustered within countries, cities, and individuals. We also found colinear crAssphage-like genomes in both Old-World and New-World primates, challenging genomic mosaicism and suggesting that the association of crAssphage with primates may be millions of years old. We conclude that crAssphage is a benign globetrotter virus that may have co-evolved with the human lineage and an integral part of the normal human gut virome.

Recent outbreaks involving enveloped viruses, such as Ebola virus, have raised questions regarding the persistence of enveloped viruses in the water environment. Efforts have been made to find enveloped virus surrogates due to challenges investigating viruses that require biosafety-level 3 or 4 handling. In this study, the enveloped bacteriophage Phi6 was evaluated as a surrogate for enveloped waterborne viruses. The persistence of Phi6 was tested in aqueous conditions chosen based on previously published viral persistence studies. Our results demonstrated that the predicted T (time for 90% inactivation) of Phi6 under the 12 evaluated conditions varied from 24 min to 117 days depending on temperature, biological activity, and aqueous media composition. Phi6 persistence was then compared with persistence values from other enveloped viruses reported in the literature. The apparent suitability of Phi6 as an enveloped virus surrogate was dependent on the temperature and composition of the media tested. Of evaluated viruses, 33%, including all conditions considered, had T values greater than the 95% confidence interval for Phi6. Ultimately, these results highlight the variability of enveloped virus persistence in the environment and the value of working with the virus of interest for environmental persistence studies.

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The SARS-CoV-2 lineages B.1.1.7 and 501.V2, which were first detected in the United Kingdom and South Africa, respectively, are spreading rapidly in the human population. Thus, there is an increased need for genomic and epidemiological surveillance in order to detect the strains and estimate their abundances. Here, we report a genomic analysis of SARS-CoV-2 in 48 raw wastewater samples collected from three wastewater treatment plants in Switzerland between July 9 and December 21, 2020. We find evidence for the presence of several mutations that define the B.1.1.7 and 501.V2 lineages in some of the samples, including co-occurrences of up to three B.1.1.7 signature mutations on the same amplicon in four samples from Lausanne and one sample from a Swiss ski resort dated December 9 - 21. These findings suggest that the B.1.1.7 strain could be detected by mid December, two weeks before its first verification in a patient sample from Switzerland. We conclude that sequencing SARS-CoV-2 in community wastewater samples may help detect and monitor the circulation of diverse lineages.