Abstract
We used wastewater-based epidemiology and amplicon-based long-read high-throughput sequencing for surveillance of enteroviruses (EVs) in Maricopa County, Arizona, Southwest United States. We collected 48 samples from 13 sites in three municipalities between 18 June and 1 October 2020, and filtered (175 mL each; 0.45 µm pore size) and extracted RNA from the filter-trapped solids. The RNA was converted to cDNA and processed through two workflows (Sanger sequencing (SSW) and long-read Illumina sequencing (LRISW)) each including a nested polymerase chain reaction (nPCR) assay. We subjected the ~350 bp amplicon from SSW to Sanger sequencing and the ~1900–2400 bp amplicon from LRISW to Illumina sequencing. We identified EV contigs from 11 of the 13 sites and 41.67% (20/48) of screened samples. Using the LRISW, we detected nine EV genotypes from three species (Enterovirus A (CVA4, EV-A76, EV-A90), Enterovirus B (E14) and Enterovirus C (CVA1, CVA11, CVA13, CVA19 and CVA24)) with Enterovirus C representing approximately 90% of the variants. However, the SSW only detected the five Enterovirus C types. Similarity and phylogenetic analysis showed that multiple Enterovirus C lineages were circulating, co-infecting and recombining in the population during the season despite the SARS-CoV-2 pandemic and the non-pharmaceutical public health measures taken to curb transmission.
Highlights
IntroductionEnteroviruses (EVs) are non-enveloped, positive-sense, single-stranded RNA viruses that belong to the genus Enterovirus, family Picornaviridae, order Picornavirales
It is important to emphasize that only the 25 samples that yielded amplicons (~350 bp, as viewed through an agarose gel imager) suggestive of EV presence in the Sanger sequencing workflow (SSW) were subjected to the long-read Illumina sequencing workflow (LRISW) (Tables 2 and S1)
We set out to determine whether EVs were circulating in three municipalities in Maricopa County, AZ, USA over the course of 105 days (18 June to 1 October) in 2020, during the historical annual peak of EV transmission
Summary
Enteroviruses (EVs) are non-enveloped, positive-sense, single-stranded RNA viruses that belong to the genus Enterovirus, family Picornaviridae, order Picornavirales. There are over 300 serologically distinct genotypes categorized into 15 species (Enterovirus A to L and Rhinovirus A–C) [1]. The polioviruses (serotypes 1, 2 and 3) are the best studied members of the genus Enterovirus and are members of species Enterovirus C alongside 22 other serotypes, including coxsackievirus (CV)A1, CVA11, CVA13, CVA19 and CVA24. As a result of establishing a correlation between EV serotypes and their VP1 gene 4.0/). Sequence [2], EV typing by serological assays has been replaced by VP1 gene, complete capsid, or whole-genome sequence analysis [3]. In the USA, EVs cause approximately 15 million infections and tens of thousands of hospitalizations [4] with a wide range of clinical manifestations that range from mild (e.g., runny nose and fever) to severe presentations such as intra-uterine infection with fetal death and acute flaccid myelitis (AFM) [5,6]
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