Pepper Mild Mottle Virus as an Indicator of Fecal Pollution

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.Existing wastewater treatment practices are not always effective at removing the large number of pathogens (bacteria, protists, and viruses) present in human feces (17, 42, 47-49, 51). Therefore, wastewater discharges into the environment can have a negative impact on human health. Recreational waters throughout the United States are monitored for the presence of fecal pollution as a means of limiting public exposure to pathogens in areas impacted by wastewater discharges (44). The presence of pathogenic viruses in aquatic environments is an important parameter to consider in the evaluation of water quality. However, the bacterial indicators currently used to detect fecal contamination, such as fecal coliforms and enterococci, often do not correlate with the presence of feces-associated viruses and other pathogens (5, 10, 26, 33, 37, 51). In response, several researchers have proposed the use of viral indicators as a more effective method for monitoring wastewater contamination and the associated risks to public health (11, 14, 31).To date, the majority of the proposed viral indicators of fecal pollution are enteric viruses transmitted via the fecal-oral route (4). Enteric viruses present in raw sewage (including members of the families Adenoviridae, Caliciviridae, Picornaviridae, and Reoviridae and of the genus Anellovirus) have been used in several previous studies to identify fecal pollution in the environment (7, 8, 11, 12, 13, 18, 19, 27, 28, 32-36, 38, 50, 51). Of the enteric viruses that have been used as indicators, only the adenoviruses were ubiquitously found in raw sewage samples collected throughout the United States (41). Picobirnaviruses and Torque teno virus are abundant in raw sewage from some regions and thus have also been proposed as indicator viruses (15, 41). However, one potential problem with the use of human viruses as indicators is that their abundance in wastewater depends on the degree of infection and shedding in the human population at any given time.In addition to viruses infecting humans, other viruses shed in feces may be useful for indicating wastewater pollution. The plant pathogen Pepper mild mottle virus (PMMoV) was the most abundant virus found in a metagenomic survey of RNA viruses from human feces (52). PMMoV is a positive-sense, single-stranded RNA virus that belongs to the Tobamovirus genus and infects hot, bell, and ornamental peppers (Capsicum spp.) (9). The nonenveloped, rod-shaped PMMoV virions are extremely stable (9) and have been demonstrated to retain their infectivity for plants after passage through the human gut (52). PMMoV originates from processed pepper products (e.g., hot sauce and curry) and is excreted in human feces at concentrations of 1 million to 1 billion viruses per g (dry weight) (52). Since the presence of PMMoV in human feces is dietary in origin, this plant pathogen may be more abundant in the healthy human population than viruses that cause human disease.This study analyzed the presence of PMMoV in raw sewage and treated wastewater samples collected from wastewater treatment facilities throughout the coastal United States. To determine if PMMoV is a human-specific indicator useful for tracking the source of fecal pollution, fecal samples from numerous animals were tested for this virus. Finally, the presence of PMMoV in marine environments exposed to wastewater was determined and compared to that of other microbial indicators. The results of this work demonstrate that PMMoV is a promising indicator of fecal pollution.