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<p>Seasonal human coronaviruses (sHCoVs), made up of alpha (HCoV-229E and HCoV-NL63) and betacoronaviruses (HCoV-OC43 and HCoV-HKU1), are endemic respiratory pathogens that have been circulating in human populations for decades. Despite their long-standing presence, their genomic diversity remains poorly characterized. This is mainly due to the limited availability of complete genome sequences, especially for HCoV-HKU1. This study presents a comprehensive molecular epidemiological analysis of sHCoVs in Slovenia over an 11-year period (2012–2022) based on 2,365 typed clinical samples. To close the gap in genomic data, virus-specific primer schemes for amplicon-based whole-genome sequencing of all four sHCoV types were developed and validated. These schemes achieved high genome recovery rates in samples with RNA concentrations exceeding 30 cp/µL (Ct ≈ 32), with the most consistent performance observed for HCoV-229E and HCoV-OC43. For HCoV-NL63 and HCoV-HKU1, comparable genome recovery was observed only at higher RNA concentrations, exceeding 50 cp/µL and 200 cp/µL, respectively. Comparative sequencing using Illumina and Oxford Nanopore Technologies (ONT) platforms showed highly consistent genome coverage and variant detection across all sHCoV types. Importantly, the protocols facilitated successful genome recovery even from archived specimens, underscoring their suitability for both retrospective and prospective genomic surveillance. This study expands the global repository of sHCoV genomes and provides a valuable methodological framework for high-resolution monitoring of their genetic evolution and epidemiological dynamics.</p>

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Development of type-specific amplicon schemes for whole-genome sequencing of seasonal human coronaviruses from clinical samples

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年7月21日

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  • Anja Ošep,
  • Hannah Goldswain,
  • Alen Suljič,
  • Rok Kogoj,
  • Miša Korva,
  • Miroslav Petrovec,
  • Julian A. Hiscox,
  • Tina Uršič

摘要

Seasonal human coronaviruses (sHCoVs), made up of alpha (HCoV-229E and HCoV-NL63) and betacoronaviruses (HCoV-OC43 and HCoV-HKU1), are endemic respiratory pathogens that have been circulating in human populations for decades. Despite their long-standing presence, their genomic diversity remains poorly characterized. This is mainly due to the limited availability of complete genome sequences, especially for HCoV-HKU1. This study presents a comprehensive molecular epidemiological analysis of sHCoVs in Slovenia over an 11-year period (2012–2022) based on 2,365 typed clinical samples. To close the gap in genomic data, virus-specific primer schemes for amplicon-based whole-genome sequencing of all four sHCoV types were developed and validated. These schemes achieved high genome recovery rates in samples with RNA concentrations exceeding 30 cp/µL (Ct ≈ 32), with the most consistent performance observed for HCoV-229E and HCoV-OC43. For HCoV-NL63 and HCoV-HKU1, comparable genome recovery was observed only at higher RNA concentrations, exceeding 50 cp/µL and 200 cp/µL, respectively. Comparative sequencing using Illumina and Oxford Nanopore Technologies (ONT) platforms showed highly consistent genome coverage and variant detection across all sHCoV types. Importantly, the protocols facilitated successful genome recovery even from archived specimens, underscoring their suitability for both retrospective and prospective genomic surveillance. This study expands the global repository of sHCoV genomes and provides a valuable methodological framework for high-resolution monitoring of their genetic evolution and epidemiological dynamics.