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

Abstract

Background: Most 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.

Objective: The 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.

Methods: This 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.

Results: In 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 on this relatively limited dataset revealed just a few importations into the state, with a single clade indicating local circulation.

Significance: This 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.

Keywords: EV-D68; Wastewater-based epidemiology; enteroviruses; pan-enterovirus; phylogenetics.

Figure 1.. Acute Flaccid Myelitis cases in the United States over time.

Initiation of surveillance by the Centers for Disease Control and Prevention was initiated in August 2014 and is shown here through August 2023. This figure was prepared using data from the CDC [15].

Figure 2.. Taxonomic composition bar plot illustrating the species-level composition of enteroviruses in wastewater samples.

Enterovirus subspecies that had at least 1% of the total reads classified as that subspecies are shown in this plot. A more detailed summary of the read classification per sample can be found in Table S2. Samples were sorted by Location Type and Collection Date. Columns showing only black bars indicate samples where no sequencing reads were produced, and white indicates uncharacterized reads

Figure 3.. Trends of EV-D68 in City of Flagstaff and City of Tempe wastewater.

EV-D68 viral loads in City of Flagstaff WWTPs and Tempe Biointel Sampling Basins between July and October 2022. Viral load in both cities was assessed using two different RT-qPCR assays: CDC2022 (A) and D68-Detect (B) with City of Flagstaff in teal and City of Tempe in purple. Assay comparisons for each city can be seen in plots C (Flagstaff) and D (Tempe) with CDC2022 in gray and D68-Detect in orange. A trend line was fitted to each set of data points on each plot coupled with a 99% confidence interval around each trend line. Data points in red boxes represent true zeros in viral load detection that were removed from the trend lines and confidence intervals then reintroduced into the plots.

Figure 4.. Trends of enteroviruses/rhinoviruses in clinical samples.

Percent positivity at Flagstaff Medical Center between June and October 2022 compared to EV-D68 viral load in two City of Flagstaff wastewater treatment plants. Viral load was assessed with the D68-Detect assay. Dotted vertical lines indicate the 1st of each month.

Figure 5.. Genome coverage using the newly developed tiled amplicon design.

The number of sequencing reads, displayed on the y-axis (log-scale), across all positions of each genome are shown. A) NR-49135 (accession MH708882), B) NR-52357 (accession MN246009), and C) NR-55939.

Figure 6.. Subset of global EV-D68 maximum likelihood phylogeny.

69 of 935 publicly available EV-D68 genomes from NCBI and 31 new AZ genomes generated as part of this study. Bootstrap values indicating cluster support are on branches. Full phylogeny can be visualized in Supplementary Figure 1.