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<p>Human noroviruses (hNoVs) are the leading cause of foodborne gastroenteritis worldwide, yet progress in infectivity-based studies has been hindered by the lack of robust virus purification methods that preserve infectivity. Here, we evaluated porcine gastric mucin (PGM)-coated magnetic beads as a sample preparation tool for concentrating and purifying infectious hNoVs from complex clinical and food matrices for use in two emerging infectivity models, human intestinal enteroids (HIEs) and zebrafish embryos/larvae. Using 18 hNoV strains across multiple genotypes, we observed variable recovery efficiencies (0.03–40%) from stool suspensions, with PGM beads often enhancing viral binding to HIEs but sometimes reducing replication (ten hNoV strains replicated without beads but only seven demonstrated replication with addition of beads), likely due to partial blocking of cell entry. In oyster homogenates spiked with hNoV GII.4[P16], bead aggregation and toxicity issues were mitigated by sample dilution, use of zebrafish larvae instead of embryos, and incorporation of an additional purification step, enabling detection at 10<sup>5</sup> to 10<sup>6</sup> genome copies per reaction. No significant cytotoxicity was detected in HIEs or zebrafish larvae under optimized conditions. Our findings indicate that PGM bead-based purification can be a valuable optional step for infectivity assays when working with samples containing strong inhibitors. This protocol provides a practical bridge between environmental/food hNoV detection and infectivity assessment in advanced model systems.</p>

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Using Porcine Gastric Mucin Coated Magnetic Beads to Concentrate and Purify Infectious Human Noroviruses for Infectivity Assays with Human Intestinal Enteroids and Zebrafish

  • 期刊论文

  • 发表日期：

    2026年1月31日

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  • Malcolm Turk Hsem Tan,
  • Samantha Q. Wales,
  • Dan Li

摘要

Human noroviruses (hNoVs) are the leading cause of foodborne gastroenteritis worldwide, yet progress in infectivity-based studies has been hindered by the lack of robust virus purification methods that preserve infectivity. Here, we evaluated porcine gastric mucin (PGM)-coated magnetic beads as a sample preparation tool for concentrating and purifying infectious hNoVs from complex clinical and food matrices for use in two emerging infectivity models, human intestinal enteroids (HIEs) and zebrafish embryos/larvae. Using 18 hNoV strains across multiple genotypes, we observed variable recovery efficiencies (0.03–40%) from stool suspensions, with PGM beads often enhancing viral binding to HIEs but sometimes reducing replication (ten hNoV strains replicated without beads but only seven demonstrated replication with addition of beads), likely due to partial blocking of cell entry. In oyster homogenates spiked with hNoV GII.4[P16], bead aggregation and toxicity issues were mitigated by sample dilution, use of zebrafish larvae instead of embryos, and incorporation of an additional purification step, enabling detection at 105 to 106 genome copies per reaction. No significant cytotoxicity was detected in HIEs or zebrafish larvae under optimized conditions. Our findings indicate that PGM bead-based purification can be a valuable optional step for infectivity assays when working with samples containing strong inhibitors. This protocol provides a practical bridge between environmental/food hNoV detection and infectivity assessment in advanced model systems.