An integrated cell culture-nanopore sequencing (ICC-NanoporeSeq) method for the simultaneous detection of multiple enterovirus types and quantification of infectious Enterovirus B.

ICC-NanoporeSeq recovers the original proportions of 10 enterovirus types from mixed samples and enables type-resolved quantification of infectious Enterovirus B in wastewater extracts.

• Why it matters: Genome-copy monitoring cannot distinguish infectious from non-infectious particles or separate EV types, limiting risk assessment and water-quality surveillance. Type-resolved infectivity is needed for environmental persistence and health implications.
• What they did: The method integrates short-term cell culture amplification with nanopore sequencing of the VP1 gene, adapted from ICC-RTqPCR and optimized from a clinical EV typing workflow. In known-concentration mixtures, it recovered all 10 EV type proportions after bias correction, and in wastewater extracts it detected 45 EV types across five species.
• The result: Type-specific calibration curves from ICC-NanoporeSeq quantify infectious concentrations of six Enterovirus B types, enabling simultaneous, type-resolved infectivity assessment in mixed samples. Compared with ICC-RTqPCR, it removes the need for multiple type-specific PCR primers, supporting expansion to additional EV types.

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