Potential indicators of virus transport and removal during soil aquifer treatment of treated wastewater effluent

Metagenomics, or sequencing of the genetic material from a complete microbial community, is a promising tool to discover novel microbes and viruses. Viral metagenomes typically contain many unknown sequences. Here we describe the discovery of a previously unidentified bacteriophage present in the majority of published human fecal metagenomes, which we refer to as crAssphage. Its ~97 kbp genome is six times more abundant in publicly available metagenomes than all other known phages together; comprises up to 90% and 22% of all reads in virus-like particle (VLP)-derived metagenomes and total community metagenomes, respectively; and totals 1.68% of all human fecal metagenomic sequencing reads in the public databases. The majority of crAssphage-encoded proteins match no known sequences in the database, which is why it was not detected before. Using a new co-occurrence profiling approach, we predict a Bacteroides host for this phage, consistent with Bacteroides-related protein homologs and a unique carbohydrate-binding domain encoded in the phage genome,.

Waterborne human enteric viruses, such as noroviruses and adenoviruses, are excreted in the feces of infected individuals and transmitted via the fecal-oral route including contaminated food and water. Since viruses are normally present at low concentrations in aquatic environments, they should be concentrated into smaller volumes prior to downstream molecular biological applications, such as quantitative polymerase chain reaction (qPCR). This review describes recent progress made in the development of concentration and detection methods of human enteric viruses in water, and discusses their applications for providing a better understanding of the prevalence of the viruses in various types of water worldwide. Maximum concentrations of human enteric viruses in water that have been reported in previous studies are summarized to assess viral abundances in aquatic environments. Some descriptions are also available on recent applications of sequencing analyses used to determine the genetic diversity of viral genomes in water samples, including those of novel viruses. Furthermore, the importance and significance of utilizing appropriate process controls during viral analyses are discussed, and three types of process controls are considered: whole process controls, molecular process controls, and (reverse transcription (RT)-)qPCR controls. Although no standards have been established for acceptable values of virus recovery and/or extraction-(RT-)qPCR efficiency, use of at least one of these appropriate control types is highly recommended for more accurate interpretation of observed data.

Accurate indicators of fecal pollution are needed in order to minimize public health risks associated with wastewater contamination in recreational waters. However, the bacterial indicators currently used for monitoring water quality do not correlate with the presence of pathogens. Here we demonstrate that the plant pathogen Pepper mild mottle virus (PMMoV) is widespread and abundant in wastewater from the United States, suggesting the utility of this virus as an indicator of human fecal pollution. Quantitative PCR was used to determine the abundance of PMMoV in raw sewage, treated wastewater, seawater exposed to wastewater, and fecal samples and/or intestinal homogenates from a wide variety of animals. PMMoV was present in all wastewater samples at concentrations greater than 1 million copies per milliliter of raw sewage. Despite the ubiquity of PMMoV in human feces, this virus was not detected in the majority of animal fecal samples tested, with the exception of chicken and seagull samples. PMMoV was detected in four out of six seawater samples collected near point sources of secondary treated wastewater off southeastern Florida, where it co-occurred with several other pathogens and indicators of fecal pollution. Since PMMoV was not found in nonpolluted seawater samples and could be detected in surface seawater for approximately 1 week after its initial introduction, the presence of PMMoV in the marine environment reflects a recent contamination event. Together, these data demonstrate that PMMoV is a promising new indicator of fecal pollution in coastal environments.