Pan-Enterovirus Characterization Reveals Cryptic Circulation of Clinically Relevant Subtypes in Arizona Wastewater

Abstract

ABSTRACTBackgroundMost seasonally circulating enteroviruses result in asymptomatic or mildly symptomatic infections. In rare cases, however, infection with some subtypes can result in paralysis or death. Of the 300 subtypes known, only poliovirus is reportable, limiting our understanding of the distribution of other enteroviruses that can cause clinical disease.ObjectiveThe overarching objectives of this study were to: 1) describe the distribution of enteroviruses in Arizona during the late summer and fall of 2022, the time of year when they are thought to be most abundant, and 2) demonstrate the utility of viral pan-assay approaches for semi-agnostic discovery that can be followed up by more targeted assays and phylogenomics.MethodsThis study utilizes pooled nasal samples collected from school-aged children and long-term care facility residents, and wastewater from multiple locations in Arizona during July– October of 2022. We used PCR to amplify and sequence a region common to all enteroviruses, followed by species-level bioinformatic characterization using the QIIME 2 platform. For Enterovirus-D68 (EV-D68), detection was carried out using RT-qPCR, followed by confirmation using near-complete whole EV-D68 genome sequencing using a newly designed tiled amplicon approach.ResultsIn the late summer and early fall of 2022, multiple enterovirus species were identified in Arizona wastewater, with Coxsackievirus A6, EV-D68, and Coxsackievirus A19 composing 86% of the characterized reads sequenced. While EV-D68 was not identified in pooled human nasal samples, and the only reported acute flaccid myelitis case in Arizona did not test positive for the virus, an in-depth analysis of EV-D68 in wastewater revealed that the virus was circulating from August through mid-October. A phylogenetic analysis revealed just one importation into the state led to local circulation.SignificanceThis study further supports the utility of wastewater-based epidemiology to identify potential public health threats. Our further investigations into EV-D68 shows how these data might help inform healthcare diagnoses for children presenting with concerning neurological symptoms.IMPACT STATEMENTMuch of our response to infectious disease threats is reactive, even in the case of seasonally circulating viruses. Wastewater-based epidemiology has been successfully used for mitigation purposes throughout the SARS-CoV-2 pandemic, and has potential to be implemented for other pathogens that can cause severe infections. While vaccines and treatment beyond supportive care are not available for many infections, this method of surveillance can provide situational awareness to public health agencies, medical professionals, and the general population, possibly paving the way for earlier public health response.