Kadoya Syun-Suke, Sano Daisuke
Department of Civil and Environmental Engineering, Tohoku University.
Department of Civil and Environmental Engineering, Tohoku University; Department of Frontier Science for Advanced Environment, Graduate School of Environmental Studies, Tohoku University;
J Vis Exp. 2019 Jan 28(143). doi: 10.3791/58821.
Rotavirus is the main etiological factor for infantile diarrhea. It is a double-stranded (ds) RNA virus and forms a genetically diverse population, known as quasispecies, owing to their high mutation rate. Here, we describe how to measure the specific growth rate and the cell-binding ability of rotavirus as its phenotypes. Rotavirus is treated with trypsin to recognize the cell receptor and then inoculated into MA104 cell culture. The supernatant, including viral progenies, is collected intermittently. The plaque assay is used to confirm the virus titer (plaque-forming unit: pfu) of each collected supernatant. The specific growth rate is estimated by fitting time-course data of pfu/mL to the modified Gompertz model. In the assay of cell-binding, MA104 cells in a 24-well plate are infected with rotavirus and incubated for 90 min at 4 °C for rotavirus adsorption to cell receptors. A low temperature restrains rotavirus from invading the host cell. After washing to remove unbound virions, RNA is extracted from virions attached to cell receptors followed by cDNA synthesis and reverse-transcription quantitative PCR (RT-qPCR). These protocols can be applied for investigating the phenotypic differences among viral strains.
轮状病毒是婴儿腹泻的主要病因。它是一种双链（ds）RNA病毒，由于其高突变率，形成了一个基因多样化的群体，称为准种。在这里，我们描述了如何测量轮状病毒的比生长速率和细胞结合能力作为其表型。轮状病毒用胰蛋白酶处理以识别细胞受体，然后接种到MA104细胞培养物中。间歇性收集包括病毒子代的上清液。空斑试验用于确认每个收集的上清液的病毒滴度（空斑形成单位：pfu）。通过将pfu/mL的时间进程数据拟合到修正的Gompertz模型来估计比生长速率。在细胞结合试验中，用轮状病毒感染24孔板中的MA104细胞，并在4℃下孵育90分钟，以使轮状病毒吸附到细胞受体上。低温抑制轮状病毒侵入宿主细胞。洗涤以去除未结合的病毒粒子后，从附着于细胞受体的病毒粒子中提取RNA，随后进行cDNA合成和逆转录定量PCR（RT-qPCR）。这些方案可用于研究病毒株之间的表型差异。
