Potential pandemic viruses in wastewater

Human viruses do not replicate in the environment; therefore, for a virus to be transferred via the urban water cycle, it must be introduced to water through human bodily fluids and then retain its infectivity until another person comes into contact with the water (Fig. 1). Studies on the presence and fate of viruses in aqueous environments are often limited to enteric viruses, as are regulations, such as those specified by the USEPA Surface Water Treatment Rule and Groundwater Treatment Rule.²¹ Wastewater can contain a number of human viruses outside of the common enteric viruses; untreated wastewater sludge and Class B biosolids, for example, contained genes from coronaviruses in more that 80% of the samples, and rubella virus at lower frequencies.¹⁹
 

Human viruses enter municipal wastewater when they are shed in the feces and urine of infected individuals. A number of human enteric viruses have been quantified in municipal wastewater by qPCR and culture methods, with reported concentrations as high as 10⁹ genome copies per liter (Table 2). For those viruses that have not been studied in wastewater, existing information on their presence in human fecal and urine samples may shed light on their potential importance in the water cycle. Feces and urine samples are rarely collected for illnesses perceived as non-enteric, but genes of the respiratory viruses RSV, human rhinovirus, coronaviruses, and seasonal influenza have been detected in stool samples.^22–25 In many cases, the presence of respiratory virus genes in feces is thought to stem from a patient swallowing virus-laden nasal secretions. Unfortunately, the PCR techniques commonly used to detect viruses in human samples do not relay infectivity information. To be of concern in the urban water cycle, an entire infective virus particle, and not just pieces of viral genome, would need to be present in wastewater. Indeed, infective SARS-CoV and avian influenza virus particles have been detected in fecal or intestinal tract samples of infected individuals.^26–28

Influenza viruses

Influenza viruses are enveloped viruses with segmented, single-stranded RNA genomes. They infect humans and other animals, such as birds and pigs. Influenza viruses have been responsible for four human pandemics in the last century, including the 1918 H1N1 “Spanish Flu” pandemic, the 1957–58 H2N2 “Asian Flu” pandemic, the 1968–69 H3N2 “Hong Kong Flu” pandemic, and the 2009 H1N1 “Swine Flu” pandemic. These pandemics were instigated by the emergence of novel strains to which humans lacked immunity. Novel strains emerge in humans when animal influenza viruses either mix with human viruses or cross directly to humans (e.g., avian influenza viruses).

Human influenza viruses

Seasonal human influenza viruses constantly circulate in human populations around the globe causing seasonal epidemics. The viruses slowly mutate as they circulate via a phenomenon called antigenic drift. The illnesses associated with the seasonal influenza viruses typically involve respiratory tract symptoms and fever. Gastrointestinal symptoms are occasionally experienced, especially in children.²⁹ Transmission of seasonal human influenza occurs through droplet, airborne, and contact transmission modes; however, the relative importance of these three modes of transmission remains unclear.³⁰ As for the potential transmission of human seasonal influenza via wastewater, viral RNA has been detected in human stool samples^24,31–33 and has also been detected in municipal wastewater.^34,35 Levels in feces ranged from 4.9 × 10³ to 8.0 × 10⁷ PCR copies per gram of stool. In one rare case, infective human influenza virus was cultured from human feces,³⁶ but this was suspected to be due to the patient swallowing virus-laden nasopharyngeal secretions, or the spillover of viruses in the blood to other organs.³² Influenza virus receptors are not present on normal human intestinal cells, thus it is unlikely that the viruses infect and replicate in gut cells.³² Based on the available evidence in the literature, high concentrations of infective human seasonal influenza viruses are unlikely in municipal wastewater, even during large outbreaks.

Avian influenza viruses

Birds can be infected by either low pathogenic influenza or highly pathogenic influenza. Whereas low pathogenic avian influenza viruses cause minor illness in birds and are rarely fatal, highly pathogenic avian influenza viruses spread rapidly in birds and are highly virulent. On rare occasions, the highly pathogenic avian influenza viruses have crossed directly over to humans, often when humans were in close proximity to large numbers of domesticated birds. Avian influenza virus infections in humans have had high associated fatality rates (Table 1), but recent avian influenza viruses in humans have not undergone sustained human-to-human transmission. There are serious concerns that an avian influenza will cross over to humans in the future and mutate in a way that allows the virus to spread easily from human to human. This hypothetical event could lead to a deadly avian influenza pandemic in humans like the Spanish influenza pandemic in the early 20th century.

Unlike seasonal human influenza, the highly pathogenic avian influenza strains are transmitted via the fecal-oral route in birds and bind with receptors on avian gut tissues (i.e., gastrointestinal tract infections).⁵² When the highly pathogenic H5N1 virus crossed over from birds to humans in 2004, many patients experienced severe diarrhea,^28,53 viruses were present in stool samples,²⁸ and the viruses infected and replicated in human gut tissues.⁵⁴ Genes of the novel avian influenza H7N9 virus that emerged in humans in 2013 were also frequently detected in stool samples.^8,55 Although not identified as an avian influenza, the H1N1 illness in humans also exhibited unusually high gastrointestinal symptoms,^36,56 infective virus particles in feces,⁵⁷ and efficient replication in intestinal cells.⁵⁸

Taken together, the frequency of gastrointestinal symptoms, the prevalence of virus particles in the patient stool samples, and the ability of the viruses to replicate in gastrointestinal tract cells, highlights the potential importance of the urban water cycle in the control of avian influenza viruses in humans. Thus far, humans infected with avian influenza viruses, such as H5N1 and H7N9, have been limited in their ability to transmit the illness to other humans. Public health officials, however, fear that a new strain will emerge in humans that spreads easily from person to person; should this occur, humans would not have immunity to the illness and effective vaccines would take time to prepare.⁵⁹ It seems likely that under potential outbreak or pandemic scenarios, avian influenza virus particles would enter municipal wastewater, possibly at high concentrations.

Coronaviruses

SARS-CoV emerged in Hong Kong in 2003 and caused severe lower respiratory illness that exhibited high mortality rates. During one cluster of 319 cases at an apartment complex in Hong Kong,^8,60 the outbreak spread via aerosolized fecal particles in the air ducts of the apartment complex. Despite being associated primarily with respiratory illness, SARS-CoV RNA was often detected in the feces of infected individuals, with the detection frequencies in different cohorts ranging from 16% to 97%.^26,61 Diarrhea symptoms were experienced in 23% to 73% of patients, with higher frequency during the first week of the illness.^26,62,63 SARS-CoV RNA levels peaked in fecal samples at 9 to 14 days after the onset of the illness,⁶¹ and in one case, fecal samples tested positive for up to 73 days following the illness onset.²⁶ The presence of SARS-CoV in feces may be due to its ability to replicate in the small and large intestines, as evidenced by infective viruses in lower intestine samples.²⁶
 

In addition to SARS-CoV, there are five other known human coronaviruses. MERS-CoV emerged in Saudi Arabia in 2012, and at the time of this writing, has resulted in 1110 confirmed cases and 456 deaths.⁷ MERS-CoV RNA was detected at low levels in the stool and urine of one infected individual (~10³ gene copies per mL),⁶⁴ but absent in stool samples from two other infected individuals.⁶⁵ Likewise, human coronavirus HKU1 (HKU1-CoV) RNA has also been detected in stool samples,⁶⁶ in some cases from patients exhibiting gastrointestinal symptoms.⁶⁷ In a recent metagenomic study of wastewater, HKU1-CoV was identified in sludge samples collected from different wastewater treatment plants in the U.S., although confirmation with PCR was not conducted.¹⁹ Unfortunately, the infectivity state of the HKU1 viruses in stool and wastewater samples cannot be assessed, as there is no tissue culture system compatible with HKU1. The remaining three human coronaviruses (HCoVNL63, HCoV-OC43, and 

HCoV-229E), have been detected in stool samples^25,67,68 but are not believed to play a significant role in infections of the gastrointestinal tract. Based on the available data, the human coronaviruses that are circulating at this point are unlikely to pose significant threats in the urban water cycle. That being said, it is quite plausible that a highly virulent new coronavirus like SARS-CoV will emerge in the future and pose challenges for the water and wastewater industries.