Department of Microbiology and Immunology, Faculty of Medicine, Hokkaido University, Sapporo, Japan.
Institute for Vaccine Research and Development (IVReD), Hokkaido University, Sapporo, Japan.
J Clin Microbiol. 2024 Jul 16;62(7):e0004224. doi: 10.1128/jcm.00042-24. Epub 2024 Jun 14.
Rapid characterization of the causative agent(s) during a disease outbreak can aid in the implementation of effective control measures. However, isolation of the agent(s) from crude clinical samples can be challenging and time-consuming, hindering the establishment of countermeasures. In the present study, we used saliva specimens collected for the diagnosis of SARS-CoV-2-a good example of a practical target-and attempted to characterize the virus within the specimens without virus isolation. Thirty-four saliva samples from coronavirus disease 2019 patients were used to extract RNA and synthesize DNA amplicons by PCR. New primer sets were designed to generate DNA amplicons of the full-length spike (S) gene for subsequent use in a circular polymerase extension reaction (CPER), a simple method for deriving recombinant viral genomes. According to the S sequence, four clinical specimens were classified as BA. 1, BA.2, BA.5, and XBB.1 and were used for the generation of recombinant viruses carrying the entire S gene. Additionally, chimeric viruses carrying the gene encoding GFP were generated to evaluate viral propagation using a plate reader. We successfully used the RNA purified directly from clinical saliva samples to generate chimeric viruses carrying the entire S gene by our updated CPER method. The chimeric viruses exhibited robust replication in cell cultures with similar properties. Using the recombinant GFP viruses, we also successfully characterized the efficacy of the licensed antiviral AZD7442. Our proof-of-concept demonstrates the novel utility of CPER to allow rapid characterization of viruses from clinical specimens.
Characterization of the causative agent(s) for infectious diseases helps in implementing effective control measurements, especially in outbreaks. However, the isolation of the agent(s) from clinical specimens is often challenging and time-consuming. In this study, saliva samples from coronavirus disease 2019 patients were directly subjected to purifying viral RNA, synthesizing DNA amplicons for sequencing, and generating recombinant viruses. Utilizing an updated circular polymerase extension reaction method, we successfully generated chimeric SARS-CoV-2 viruses with sufficient replication capacity and antigenicity. Thus, the recombinant viruses generated in this study were applicable for evaluating the antivirals. Collectively, our developed method facilitates rapid characterization of specimens circulating in hosts, aiding in the establishment of control measurements. Additionally, this approach offers an advanced strategy for controlling other (re-)emerging viral infectious diseases.
在疾病爆发期间快速鉴定病原体有助于实施有效的控制措施。然而，从粗临床样本中分离病原体可能具有挑战性和耗时，阻碍了对策的建立。在本研究中，我们使用了为 SARS-CoV-2 诊断而采集的唾液样本-这是一个实际目标的很好例子-并尝试在不进行病毒分离的情况下对样本中的病毒进行特征鉴定。使用 34 份来自 2019 年冠状病毒病患者的唾液样本，通过 PCR 提取 RNA 并合成 DNA 扩增子。设计了新的引物组，生成全长刺突（S）基因的 DNA 扩增子，用于随后进行环状聚合酶延伸反应（CPER），这是一种衍生重组病毒基因组的简单方法。根据 S 序列，将四个临床样本分为 BA.1、BA.2、BA.5 和 XBB.1，并用于生成携带全长 S 基因的重组病毒。此外，生成携带 GFP 基因编码的嵌合病毒，使用平板读数器评估病毒的复制情况。我们成功地使用直接从临床唾液样本中纯化的 RNA，通过我们更新的 CPER 方法生成携带全长 S 基因的嵌合病毒。嵌合病毒在细胞培养中表现出强大的复制能力，具有相似的特性。使用重组 GFP 病毒，我们还成功地描述了已获许可的抗病毒药物 AZD7442 的功效。我们的概念验证证明了 CPER 在快速鉴定来自临床样本的病毒方面的新颖用途。
鉴定传染病的病原体有助于实施有效的控制措施，特别是在爆发期间。然而，从临床样本中分离病原体通常具有挑战性和耗时。在这项研究中，直接对来自 2019 年冠状病毒病患者的唾液样本进行病毒 RNA 纯化、测序用 DNA 扩增子合成以及重组病毒生成。利用更新的环状聚合酶延伸反应方法，我们成功地生成了具有足够复制能力和抗原性的嵌合 SARS-CoV-2 病毒。因此，本研究中生成的重组病毒可用于评估抗病毒药物。总之，我们开发的方法促进了对宿主循环标本的快速特征鉴定，有助于建立控制措施。此外，该方法为控制其他（再）出现的病毒性传染病提供了一种先进的策略。
