Retrospective Surveillance of Wastewater To Examine Seasonal Dynamics of Enterovirus Infections

Enterovirus infections are often not tracked or reported to health officials. This makes it hard to know how many people in a community are infected with these viruses at any given time. Here, we explored enterovirus in municipal wastewater to look at this issue. We show that enteroviruses are present year-round in municipal wastewater at levels of up to 800,000 genomic copies per liter. We estimate that, on average, 2.8% of the people contributing to the wastewater shed enterovirus daily. Sequence analysis of the viral capsid protein 4 gene shows that 8 enterovirus types are key drivers of seasonal trends. Populations of Enterovirus A members peak in the spring, while Enterovirus B types are most prevalent during the summer and fall months and Enterovirus C members influence the winter months. Enterovirus D was observed sporadically and did not influence seasonal trends. ABSTRACT Enteroviruses are RNA viruses that are responsible for both mild gastroenteritis and mild respiratory illnesses as well as debilitating diseases such as meningitis and myocarditis. The disease burden of enteroviruses in the United States is difficult to assess because most infections are not recorded. Since infected individuals shed enterovirus in feces and urine, surveillance of municipal wastewater can reveal the diversity of enteroviruses circulating in human populations. Therefore, monthly municipal wastewater samples were collected for 1 year and enteroviruses were quantified by reverse transcriptase quantitative PCR and identified by next-generation, high-throughput sequencing. Enterovirus concentrations ranged from 3.8 to 5.9 log10 equivalent copies/liter in monthly samples. From the mean monthly concentration, it can be estimated that 2.8% of the contributing population was shedding enterovirus daily. Sequence analysis showed that Enterovirus A and Enterovirus B alternate in predominance, with Enterovirus B comprising over 80% of the reads during the summer and fall months and Enterovirus A accounting for >45% of the reads in spring. Enterovirus C was observed throughout the year, while Enterovirus D was present intermittently. Principal-component analysis further supported the date corresponding to enterovirus seasonal trends as CVA6 (Enterovirus A) was predominant in the spring months; CVB3, CVB5, and E9 (Enterovirus B) were predominant in the summer and fall months; and CVA1, CVA19, and CVA22 (Enterovirus C) and EV97 (Enterovirus B) were predominant in winter. Rhinoviruses were also observed. Wastewater monitoring of human enterovirus provided improved insight into the seasonal patterns of enteroviruses circulating in communities and can contribute to understanding of enterovirus disease burden. IMPORTANCE Enterovirus infections are often not tracked or reported to health officials. This makes it hard to know how many people in a community are infected with these viruses at any given time. Here, we explored enterovirus in municipal wastewater to look at this issue. We show that enteroviruses are present year-round in municipal wastewater at levels of up to 800,000 genomic copies per liter. We estimate that, on average, 2.8% of the people contributing to the wastewater shed enterovirus daily. Sequence analysis of the viral capsid protein 4 gene shows that 8 enterovirus types are key drivers of seasonal trends. Populations of Enterovirus A members peak in the spring, while Enterovirus B types are most prevalent during the summer and fall months and Enterovirus C members influence the winter months. Enterovirus D was observed sporadically and did not influence seasonal trends.