Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth in vitro depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here, we developed a simplified quantitative real-time PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 growth from a small amount of cell culture supernatants. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. Using this assay, we screened the activities of a number of compounds that were predicted to alter SARS-CoV-2 entry and replication as well as HIV-1-specific drugs in a proof-of-concept study. We found that E64D (inhibitor of endosomal proteases cathepsin B and L) and apilimod (endosomal trafficking inhibitor) potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) similarly decreased SARS-CoV-2 RNA levels in supernatants, suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (a nonnucleoside reverse transcriptase inhibitor [NNRTI]), amprenavir (a protease inhibitor), and allosteric integrase inhibitor 2 (ALLINI-2) modestly inhibited SARS-CoV-2 replication, albeit the 50% inhibitory concentration (IC₅₀) values were much higher than that required for HIV-1. Taking the data together, this simplified assay will expedite basic SARS-CoV-2 research, be amenable to mid-throughput screening assays (i.e., drug, CRISPR, small interfering RNA [siRNA], etc.), and be applicable to a broad number of RNA and DNA viruses.

中文翻译：

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用于监测细胞培养中 SARS-CoV-2 生长的简化定量实时 PCR 检测。

严重急性呼吸系统综合症冠状病毒 2 (SARS-CoV-2) 在短短几个月内感染了数百万人，导致严重的呼吸道疾病和死亡。体外监测 SARS-CoV-2 生长的分析依赖于耗时且昂贵的 RNA 提取步骤，阻碍了基础研究和药物开发工作的进展。在这里，我们开发了一种简化的实时定量 PCR 检测，它绕过了病毒 RNA 提取步骤，可以从少量细胞培养上清液中监测 SARS-CoV-2 的生长。此外，我们表明这种方法很容易适用于许多其他 RNA 和 DNA 病毒。使用这种分析方法，我们在概念验证研究中筛选了许多预计会改变 SARS-CoV-2 进入和复制的化合物以及 HIV-1 特异性药物的活性。我们发现 E64D（内体蛋白酶组织蛋白酶 B 和 L 的抑制剂）和阿匹莫德（内体运输抑制剂）有效地减少了细胞培养上清液中 SARS-CoV-2 RNA 的数量，并且细胞毒性最小。出奇，我们发现巨胞饮作用抑制剂乙基异丙基阿米洛利 (EIPA) 同样降低了上清液中的 SARS-CoV-2 RNA 水平，这表明进入可能另外由替代途径介导。HIV-1 特异性抑制剂奈韦拉平（一种非核苷逆转录酶抑制剂 [NNRTI]）、安普那韦（一种蛋白酶抑制剂）和变构整合酶抑制剂 2 (ALLINI-2) 适度抑制了 SARS-CoV-2 复制，尽管抑制浓度为 50% （我知道了₅₀ ) 值远高于 HIV-1 所需的值。将数据综合起来，这种简化的检测将加速基本的 SARS-CoV-2 研究，适用于中等通量筛选检测（即药物、CRISPR、小干扰 RNA [siRNA] 等），并适用于广泛的RNA和DNA病毒的数量。