Development of a universal primer set for the identification of enterovirus serotypes using next-generation sequencing.

Enteroviruses have been recognized as major etiological agents of viral myocarditis, a significant contributor to sudden cardiac death. However, the causative virus genomes are rarely identified in forensic autopsy specimens, because extensive nucleic acid degradation compromises analysis. The aim of this study was to develop a next-generation sequencing (NGS)-based method for detecting and identifying the serotype of enteroviruses, which are one of the primary viral causes of myocarditis. We developed a primer set using sequences from 21 representative enterovirus serotypes. The sensitivity of the method was assessed using 13 artificial enteroviral DNA constructs. To confirm the clinical performance of our proposed method, a subset of nasal specimens was analyzed independently by a commercial laboratory using the BioFire Respiratory Panel 2.1 microarray-based viral screening assay, and the results were compared with those obtained using our method. Our method was able to detect as few as 10 copies of artificial DNA and successfully identified all 13 evaluated serotypes, even within a mixture sample. Compared with the BioFire Respiratory Panel 2.1 microarray-based assay, our NGS-based method demonstrated concordant results in 12 out of 14 evaluated cases, showing comparable detection sensitivity. In a trial application of the method, we were also able to detect and serotype enteroviruses in frozen myocardial samples from three forensic myocarditis cases. Overall, the findings of this study suggest that the NGS-based method developed here is a promising diagnostic approach for identifying the causative pathogens of viral myocarditis in forensic autopsy cases.