A newly developed whole genome sequencing protocol enables early tracking of Enterovirus D68 molecular evolution.

Background Human enterovirus D68 (EV-D68) has been associated with an increase in mild-to-severe pediatric respiratory diseases worldwide. The rate of circulation of this virus is largely underestimated in the population and genetic evolutionary data are usually available only for partial sequences (e.g VP1 region). To achieve a timely genomic surveillance, a reliable, high-throughput EV-68 sequencing assay is required. Here we report an improved high-throughput EV-D68 whole-genome sequencing (WGS) assay performed directly on clinical samples that is suitable for short-read sequencing platforms. Between June and December 2022, a total 37 (1.9%) respiratory samples were EV-D68 positive and together with 52 additional samples with a median cycle threshold of 28.3, ranging from 18 to 36.8 were included in the validation analyses. Overall, all the primers had good performance and no mismatches were detected in more than 85% of sequences (932 WGS dataset). Using a cut-off of Cq <32 in at least 85.5% of samples a WG or PG was obtained, confirming an acceptable positive sequencing rate for the designed method. A total of 65 WGS were obtained and have a mean coverage of 98.4% across the genome, with a median depth of 6158x (range 2815x-7560x). Based on the obtained data, this method is cost effective resulting in an easy-to-perform protocol helpful for tracing the evolution of EV-D68 in protein different from VP1. EV-D68 could become a significant pathogen for public health in the next future, and thus this protocol for whole genome sequencing could help clinical and molecular virologists to be ready for molecular epidemiology surveillance.