Takumi-Tanimukai Yuka, Yamamoto Soh, Ogasawara Noriko, Nakabayashi Sayaka, Mizuta Katsumi, Yamamoto Keisuke, Miyata Ryo, Kakuki Takuya, Jitsukawa Sumito, Sato Toyotaka, Tsutsumi Hiroyuki, Kojima Takashi, Takano Kenichi, Yokota Shin-Ichi
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.
Department of Microbiology, Sapporo Medical University School of Medicine, Sapporo, Japan.
J Virol Methods. 2022 Jun;304:114528. doi: 10.1016/j.jviromet.2022.114528. Epub 2022 Mar 28.
Quantifying proliferative virus particles is one of the most important experimental procedures in virology. Compared with classical overlay materials, newly developed cellulose derivatives enable a plaque-forming assay to produce countable clear plaques easily. HEp-2 cells are widely used in plaque assays for human respiratory syncytial virus (RSV). It is crucial to use an overlay material to keep HEp-2 cell proliferation and prevent RSV particles from spreading over the fluid. Among four cellulose derivatives, carboxymethyl cellulose sodium salt (CMC), hydroxypropyl methylcellulose (HPMC), microcrystalline cellulose (MCC), and hydroxyethyl cellulose (HEC), we found that HPMC was the optimal overlay material because HPMC maintained HEp-2 cell proliferation and RSV infectivity. Although MCC was unsuitable for RSV, it assisted the plaque-forming by human metapneumovirus in TMPRSS2-expressing cells. Therefore, depending on the cells and viruses, it is necessary to use different overlay materials at varying concentrations.
对增殖性病毒颗粒进行定量是病毒学中最重要的实验程序之一。与传统的覆盖材料相比，新开发的纤维素衍生物能使蚀斑形成试验轻松产生可数的清晰蚀斑。人喉表皮样癌细胞（HEp-2细胞）广泛用于人呼吸道合胞病毒（RSV）的蚀斑试验。使用覆盖材料以维持HEp-2细胞增殖并防止RSV颗粒在液体中扩散至关重要。在四种纤维素衍生物，即羧甲基纤维素钠盐（CMC）、羟丙基甲基纤维素（HPMC）、微晶纤维素（MCC）和羟乙基纤维素（HEC）中，我们发现HPMC是最佳的覆盖材料，因为HPMC能维持HEp-2细胞增殖和RSV感染性。虽然MCC不适用于RSV，但它有助于在表达跨膜丝氨酸蛋白酶2（TMPRSS2）的细胞中由人偏肺病毒形成蚀斑。因此，根据细胞和病毒的不同，有必要使用不同浓度的不同覆盖材料。
