Oberste, M. S. | bioRxiv Science

bioRxiv2022

Type-specific EV-D68 real-time RT-PCR assay for the detection of all extant enterovirus D68 strains

Enterovirus D68 (EV-D68) has caused recurring respiratory disease outbreaks in the United States since 2014. The dominant circulating EV-D68 strain has evolved from clade B1 to the more recent B2 and B3 clades. As recurrent outbreaks and continued virus evolution are expected for EV-D68, a robust real-time PCR assay that detects known strains as well as potential emerging strains is critical for national surveillance and clinical diagnostics. We describe a type-specific EV-D68 real-time RT-PCR (rRT-PCR) assay termed CDC2022, which targets sequences encoding conserved amino acid regions of all extant EV-D68 strains. We targeted three motifs conserved among all strains in the last 60 years. The assay achieved 100% (270/270) sensitivity and 100% (344/344) specificity when tested with a collection of 613 respiratory specimens, compared to the gold-standard EV semi-nested VP1 PCR and sequencing assay (snPCR/Seq). CDC2022 gave negative results with 289/289 non-target viruses, including 104 EV A-D isolates, 165 rhinovirus (RV) isolates or clinical specimens, and 14 other common respiratory viruses. The assay can detect as few as 0.28 CCID50 per reaction. An in silico "phylo-primer-mismatch" analysis was performed to visualize primer/probe mismatches and to compare CDC2022 with other EV-D68 rRT-PCR assays, including the previous CDC assay (CDC2015) developed in 2014 for clade B1 strains. It showed that CDC2022 has the fewest primer/probe mismatches among all assays analyzed and is suitable for all clades. We additionally tested 11 EV-D68-positive clinical specimens from 2022 that were confirmed by snPCR/Seq, and all were detected. CDC2022 assay could provide a critical tool for molecular surveillance of EV-D68.

microbiology↗

bioRxiv2019

Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses

Next-generation sequencing is a powerful tool for virological surveillance. While Illumina(R) and Ion Torrent(R) sequencing platforms are used extensively for generating viral RNA genome sequences, there is limited data comparing different platforms. We evaluated the Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5 platforms using a panel of sixteen specimens containing picornaviruses and human caliciviruses (noroviruses and sapoviruses). The specimens were processed, using combinations of three library preparation and five sequencing kits, to assess the quality and completeness of assembled viral genomes, and an estimation of cost per sample to generate the data was calculated. The choice of library preparation kit and sequencing platform was found to impact the breadth of genome coverage and accuracy of consensus viral genomes. The Ion Torrent S5 outperformed the older Ion Torrent PGM platform in data quality and cost, and generated the highest proportion of reads for enterovirus D68 samples. However, indels at homopolymer regions impacted the accuracy of consensus genome sequences. For lower throughput sequencing runs (i.e., Ion Torrent 510 or Illumina MiSeq Nano V2), the cost per sample was lower on the MiSeq platform, whereas with higher throughput runs (Ion Torrent 530 or Illumina MiSeq V2) the cost per sample was comparable. These findings suggest that the Ion Torrent S5 and Illumina MiSeq platforms are both viable options for genomic sequencing of RNA viruses, each with specific advantages and tradeoffs.

microbiology↗