An Integrated Cell Culture - Nanopore Sequencing (ICC-NanoporeSeq) method for the simultaneous detection of multiple enterovirus types and quantification of infectious Enterovirus B
- 1Ecole Polytechnique Federale de Lausanne (EPFL)
Abstract
Enteroviruses (EVs) are ubiquitous contaminants of surface waters, where they can remain infectious for long periods of time. Most methods used for EV monitoring are unable to distinguish between infectious and non-infectious particles or between EV types. Because different types exhibit both distinct environmental persistence and health implications, there is a need for type-resolved infectivity measurements. Here we developed Integrated Cell Culture-Nanopore Sequencing (ICC-NanoporeSeq), a method combining short-term cell culture amplification with Nanopore sequencing of the VP1 gene. The ICC approach was adapted from a previously described ICC-RTqPCR protocol, while the NanoporeSeq workflow was derived from a clinical EV typing protocol and optimized for environmentally circulating EV types. Using samples containing known concentrations of ten EV types, the NanoporeSeq method accurately and reproducibly recovered the original proportions of all EV types after correction of biases. When applied to wastewater extracts, the method detected 45 EV types across five species. Furthermore, type-specific calibration curves generated with ICC-NanoporeSeq enabled quantification of the infectious concentrations of six EV types of the Enterovirus B species, allowing a simultaneous and type-resolved assessment of infectivity in mixed samples. Overall, ICC-NanoporeSeq provides a scalable approach for the parallel analysis of multiple EV types. Compared with the predecessor ICC-RTqPCR method, it eliminates the need for multiple type-specific PCR primers and can therefore be readily expanded to include additional EV types.
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BibTeX
@misc{astorchcardona2026integrated,
title = {{An Integrated Cell Culture - Nanopore Sequencing (ICC-NanoporeSeq) method for the simultaneous detection of multiple enterovirus types and quantification of infectious Enterovirus B}},
author = {A. Astorch-Cardona and S. Rouault and T. Kohn},
howpublished = {bioRxiv},
year = {2026},
month = may,
doi = {10.64898/2026.05.15.725335},
url = {https://www.biorxiv.org/content/10.64898/2026.05.15.725335v2},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - An Integrated Cell Culture - Nanopore Sequencing (ICC-NanoporeSeq) method for the simultaneous detection of multiple enterovirus types and quantification of infectious Enterovirus B
AU - A. Astorch-Cardona
AU - S. Rouault
AU - T. Kohn
PY - 2026
DA - 2026/05/15
PB - bioRxiv
DO - 10.64898/2026.05.15.725335
UR - https://www.biorxiv.org/content/10.64898/2026.05.15.725335v2
AB - Enteroviruses (EVs) are ubiquitous contaminants of surface waters, where they can remain infectious for long periods of time. Most methods used for EV monitoring are unable to distinguish between infectious and non-infectious particles or between EV types. Because different types exhibit both distinct environmental persistence and health implications, there is a need for type-resolved infectivity measurements. Here we developed Integrated Cell Culture-Nanopore Sequencing (ICC-NanoporeSeq), a method combining short-term cell culture amplification with Nanopore sequencing of the VP1 gene. The ICC approach was adapted from a previously described ICC-RTqPCR protocol, while the NanoporeSeq workflow was derived from a clinical EV typing protocol and optimized for environmentally circulating EV types. Using samples containing known concentrations of ten EV types, the NanoporeSeq method accurately and reproducibly recovered the original proportions of all EV types after correction of biases. When applied to wastewater extracts, the method detected 45 EV types across five species. Furthermore, type-specific calibration curves generated with ICC-NanoporeSeq enabled quantification of the infectious concentrations of six EV types of the Enterovirus B species, allowing a simultaneous and type-resolved assessment of infectivity in mixed samples. Overall, ICC-NanoporeSeq provides a scalable approach for the parallel analysis of multiple EV types. Compared with the predecessor ICC-RTqPCR method, it eliminates the need for multiple type-specific PCR primers and can therefore be readily expanded to include additional EV types.
ER -