# Sensitive and Accurate Quantification of Enterovirus-D68 (EV-D68) Viral Loads Using Droplet Digital PCR (ddPCR)

## TL;DR

A new ddPCR assay accurately and sensitively quantifies EV-D68 virus in clinical samples and cell cultures.

## Contribution

A novel EV-D68-specific ddPCR assay with high specificity and sensitivity for viral load quantification.

## Key findings

- The ddPCR assay quantifies EV-D68 RNA copies in a dynamic range of 6.7 × 10−3 to 1.2 × 104 copies/μL.
- The assay is specific to EV-D68 and does not cross-react with other pediatric viruses.
- The method successfully quantified EV-D68 in clinical samples from the 2022 outbreak.

## Abstract

Enterovirus-D68 (EV-D68) is a reemerging virus that has been associated with numerous outbreaks in children in the past 10 years. Most assays examining viral infection kinetics have relied on the use of quantitative RT-PCR (qRT-PCR) assays as an assay of choice. Though valuable, there are inherent limitations that introduce variability, thereby reducing its value when comparing results across the field. Unlike the qRT-PCR assay that uses a standard curve to determine the copy number of viral RNA, the droplet digital PCR assay (ddPCR) directly quantifies the absolute number of copies within a given sample, which in turn makes the assay highly sensitive and accurate. Here, we have developed an EV-D68-specific ddPCR assay that effectively quantifies EV-D68 RNA copies in both cells and supernatants within a dynamic range of 6.7 × 10−3 copies/μL to 1.2 × 104 copies/μL of the sample. The assay was highly specific for a broad range of EV-D68 isolates (Fermon, US/MO/14-18947, US/MO/14-18949, US/KY/14-18953, USA/2018-23088, USA/2020-23336 and EV-D68-infected human nasal turbinate samples from the 2022 outbreak) without cross-reactivity to other viruses such as Enterovirus-A71 (EV-A71), Human Parechovirus (HPeV)-1 and -2, Coxsackievirus (CV)-B1, Human Coronavirus (HCoV)-NL63, SARS-CoV-2, Influenza-A and B, Rhinovirus, and Respiratory Syncytial Virus (RSV)-A2, which are known to cause infection in children. The assay was able to readily quantify EV-D68 in infected cells and supernatants along with nasal turbinate samples collected from children during the 2022 outbreak. Our results suggest that the assay can be readily translated to accurately quantify viral loads in tissues and body fluids such as plasma and lung or nasal aspirates.