Yamamoto Hirotaka, Tamura Tomokazu, Fukuhara Takausuke
Department of Microbiology and Immunology, Faculty of Medicine, Hokkaido University, Sapporo, Japan.
Institute for Vaccine Research and Development (IVReD), Hokkaido University, Sapporo, Japan.
Bio Protoc. 2025 Apr 20;15(8):e5275. doi: 10.21769/BioProtoc.5275.
Reverse genetics systems in virology are technologies used to generate recombinant viruses, enabling the manipulation of viral genes. Recombinant viruses facilitate the investigation of pathogenesis and the development of antivirals. In studies of positive-sense single-stranded RNA (ssRNA) viruses, a reverse genetics approach typically uses infectious viral cDNA clones derived from bacterial artificial chromosomes and plasmids or from the in vitro ligation of viral cDNA fragments. However, these methods are time-consuming, involve complex procedures, and do not always successfully generate recombinant viruses. Possible reasons for unsuccessful outcomes include i) viral sequences exhibiting toxicity in bacterial systems, ii) the duplication of viral genes observed in some strains, complicating the acquisition of correct cDNA clones, and iii) certain cell lines being highly susceptible to infection but difficult to transfect with nucleotides. For these reasons, a simple and rapid reverse genetics system is needed to accelerate research on ssRNA viruses. The circular polymerase extension reaction (CPER) method offers a solution by eliminating the need for molecular cloning in bacteria, enabling the generation of recombinant viruses over a shorter timeframe. This method has been widely adopted for the study of ssRNA viruses, including SARS-CoV-2 and flaviviruses. Recently, we expanded the CPER method for ssRNA viruses using internal ribosome entry site (IRES)-mediated translation. This protocol details the experimental procedures, using bovine viral diarrhea virus as an example-one of the most challenging viruses for generating viral cDNA clones because of the factors listed above. Key features • Rapid generation of recombinant positive-strand RNA viruses. • The CPER method eliminates the need for molecular cloning in bacteria, enabling the rapid generation of recombinant viruses. • The CPER method for ssRNA viruses enables efficient translation of viruses using IRES by incorporating the gene cassette of RNA Pol-I promoters and terminators.
病毒学中的反向遗传学系统是用于产生重组病毒的技术，可实现对病毒基因的操控。重组病毒有助于发病机制的研究以及抗病毒药物的开发。在正链单链RNA（ssRNA）病毒的研究中，反向遗传学方法通常使用源自细菌人工染色体和质粒或病毒cDNA片段体外连接的感染性病毒cDNA克隆。然而，这些方法耗时、程序复杂，且并非总能成功产生重组病毒。结果不成功的可能原因包括：i）病毒序列在细菌系统中表现出毒性；ii）在某些毒株中观察到病毒基因重复，使得获取正确的cDNA克隆变得复杂；iii）某些细胞系极易感染但难以用核苷酸转染。由于这些原因，需要一种简单快速的反向遗传学系统来加速对ssRNA病毒的研究。环化聚合酶延伸反应（CPER）方法提供了一种解决方案，它无需在细菌中进行分子克隆，能够在更短的时间内产生重组病毒。该方法已被广泛应用于包括严重急性呼吸综合征冠状病毒2（SARS-CoV-2）和黄病毒在内的ssRNA病毒研究。最近，我们利用内部核糖体进入位点（IRES）介导的翻译扩展了用于ssRNA病毒的CPER方法。本方案详细介绍了实验步骤，以牛病毒性腹泻病毒为例——由于上述因素，它是产生病毒cDNA克隆最具挑战性的病毒之一。
关键特性
• 快速产生重组正链RNA病毒。
• CPER方法无需在细菌中进行分子克隆，能够快速产生重组病毒。
• 用于ssRNA病毒的CPER方法通过整合RNA聚合酶I启动子和终止子的基因盒，利用IRES实现病毒的高效翻译。
