Retrospective Genotyping of Enteroviruses Using a Diagnostic Nanopore Sequencing Workflow

Area of Science:

• Virology
• Infectious Diseases
• Molecular Diagnostics

Background:

• Enteroviruses are common human pathogens causing a spectrum of illnesses, from mild respiratory infections to severe neurological diseases like meningitis and encephalitis.
• Recent years have seen increased reports of enterovirus-associated pediatric meningitis/encephalitis and notable enterovirus D68 outbreaks, highlighting the need for effective surveillance.

Purpose of the Study:

• To develop and validate a rapid enterovirus and rhinovirus genotyping assay utilizing nanopore sequencing of the VP1 gene.
• To analyze the prevalence and diversity of enteroviruses and rhinoviruses in clinical samples from Zurich, Switzerland, during the 2019/2020 and 2021/2022 respiratory seasons.

Main Methods:

• Development of a nanopore sequencing-based assay targeting a specific region of the VP1 gene for rapid enterovirus and rhinovirus genotyping.
• Retrospective analysis of 255 enterovirus/rhinovirus-positive clinical samples (respiratory, CSF, stool) collected from 2019 to 2022.
• Whole-genome sequencing was employed for samples with ambiguous genotyping results and for enterovirus D68-positive cases.

Main Results:

• The assay successfully identified 95 distinct enterovirus and rhinovirus genotypes across the analyzed clinical samples.
• Enterovirus and rhinovirus infection prevalence varied significantly by sample type and age group, with a notable higher frequency in children aged 0-4 years.
• A higher prevalence of Enterovirus A and Rhinovirus A was observed in the 2021/2022 season compared to 2019/2020, particularly after the SARS-CoV-2 pandemic.

Conclusions:

• The developed nanopore sequencing workflow offers a rapid and effective tool for the molecular typing of enteroviruses and rhinoviruses.
• This assay facilitates both individual patient diagnostics and continuous epidemiological surveillance of these common viral pathogens.
• Findings underscore the dynamic nature of enterovirus and rhinovirus circulation, with specific genotypes showing increased prevalence in certain age groups and post-pandemic periods.