Retrospective Genotyping of Enteroviruses Using a Diagnostic Nanopore Sequencing Workflow

Enteroviruses are among the most common viruses pathogenic to humans. They are associated with various forms of disease, ranging from mild respiratory illness to severe neurological diseases. In recent years, an increasing number of isolated cases of children developing meningitis or encephalitis as a result of enterovirus infection have been reported, as well as discrete enterovirus D68 outbreaks in North America in 2014 and 2016. We developed an assay to rapidly genotype enteroviruses by sequencing a region within the VP1 gene using nanopore Flongles. We retrospectively analyzed enterovirus-/rhinovirus-positive clinical samples from the Zurich, Switzerland area mainly collected during two seasons in 2019/2020 and 2021/2022. Respiratory, cerebrospinal fluid, and stool samples were analyzed. Whole-genome sequencing was performed on samples with ambiguous genotyping results and enterovirus D68-positive samples. Out of 255 isolates, a total of 95 different genotypes were found. A difference in the prevalence of enterovirus and rhinovirus infections was observed for both sample type and age group. In particular, children aged 0-4 years showed a higher frequency of enterovirus infections. Comparing the respiratory seasons, a higher prevalence was found, especially for enterovirus A and rhinovirus A after the SARS-CoV-2 pandemic. The enterovirus genotyping workflow provides a rapid diagnostic tool for individual analysis and continuous enterovirus surveillance.