Yamamoto Soh, Sudo-Yokoyama Yuka, Ogasawara Noriko, Yokota Shin-Ichi
Department of Microbiology, Sapporo Medical University School of Medicine, Sapporo, Japan.
Department of Microbiology, Sapporo Medical University School of Medicine, Sapporo, Japan; Department of Otolaryngology-Head and Neck Surgery, Sapporo Medical University School of Medicine, Sapporo, Japan.
J Virol Methods. 2023 Jun;316:114715. doi: 10.1016/j.jviromet.2023.114715. Epub 2023 Mar 20.
Murine norovirus (MNV) is used widely as a practical alternative to human norovirus (HuNoV). Plaque-forming assays for MNV are important for developing therapeutic agents against HuNoV infections. Although agarose-overlay MNV assays have been reported, recent improvements in cellulose derivatives suggest that they could be optimized further, particularly with respect to improving the overlay material. To determine which overlay material is optimal for the MNV plaque assay, we compared four typical cellulose derivatives [microcrystalline cellulose (MCC), hydroxyethyl cellulose (HEC), hydroxypropyl methylcellulose (HPMC), and carboxymethyl cellulose (CMC)] with conventional agarose. We found that 3.5% (w/v) MCC-containing medium provided clear round-shaped plaques on RAW 264.7 cells 1 day after inoculation; the visibility of plaques was comparable with that of the original agarose-overlay assay. Removing residual MCC powder from the MCC-overlay assay before fixing was important for obtaining distinct plaques that are clearly countable. Finally, after calculating the plaque diameter as a percentage of well diameter, we found that 12- and 24-well plates were better than other plates for accurate plaque counting. The MCC-based MNV plaque assay is cost-effective and rapid, and produces plaques that are easy to count. Accurate virus quantification using this optimized plaque assay will enable reliable estimation of norovirus titers.
鼠诺如病毒（MNV）被广泛用作人类诺如病毒（HuNoV）的一种实用替代品。MNV的噬斑形成试验对于开发抗HuNoV感染的治疗药物很重要。尽管已有关于琼脂糖覆盖MNV试验的报道，但纤维素衍生物的最新进展表明它们可以进一步优化，特别是在改善覆盖材料方面。为了确定哪种覆盖材料最适合MNV噬斑试验，我们将四种典型的纤维素衍生物[微晶纤维素（MCC）、羟乙基纤维素（HEC）、羟丙基甲基纤维素（HPMC）和羧甲基纤维素（CMC）]与传统琼脂糖进行了比较。我们发现，接种后1天，含有3.5%（w/v）MCC的培养基在RAW 264.7细胞上形成了清晰的圆形噬斑；噬斑的可见性与原始琼脂糖覆盖试验相当。在固定前从MCC覆盖试验中去除残留的MCC粉末对于获得清晰可数的明显噬斑很重要。最后，在将噬斑直径计算为孔直径的百分比后，我们发现12孔板和24孔板比其他板更适合准确计数噬斑。基于MCC的MNV噬斑试验具有成本效益且快速，并且产生易于计数的噬斑。使用这种优化的噬斑试验进行准确的病毒定量将能够可靠地估计诺如病毒滴度。
