Rapid Plasmid-Free Generation of Recombinant Positive-Strand RNA Viruses That Use IRES-Mediated Translation Using an Expansion of the Circular Polymerase Extension Reaction (CPER)
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,...
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Bio-protocol LLC
2025-04-01
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| author | Hirotaka Yamamoto Tomokazu Tamura Takausuke Fukuhara |
| author_facet | Hirotaka Yamamoto Tomokazu Tamura Takausuke Fukuhara |
| author_sort | Hirotaka Yamamoto |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-89400e6a911e4161ab72f0425f41af3e |
| institution | DOAJ |
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| language | English |
| publishDate | 2025-04-01 |
| publisher | Bio-protocol LLC |
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| spelling | doaj-art-89400e6a911e4161ab72f0425f41af3e2025-08-20T03:11:21ZengBio-protocol LLCBio-Protocol2331-83252025-04-0115810.21769/BioProtoc.5275Rapid Plasmid-Free Generation of Recombinant Positive-Strand RNA Viruses That Use IRES-Mediated Translation Using an Expansion of the Circular Polymerase Extension Reaction (CPER)Hirotaka Yamamoto0Tomokazu Tamura1Takausuke Fukuhara2Department of Microbiology and Immunology, Faculty of Medicine, Hokkaido University, Sapporo, JapanDepartment of Microbiology and Immunology, Faculty of Medicine, Hokkaido University, Sapporo, JapanInstitute for Vaccine Research and Development (IVReD), Hokkaido University, Sapporo, Japan, One Health Research Center, Hokkaido University, Sapporo, JapanDepartment of Microbiology and Immunology, Faculty of Medicine, Hokkaido University, Sapporo, JapanInstitute for Vaccine Research and Development (IVReD), Hokkaido University, Sapporo, Japan, One Health Research Center, Hokkaido University, Sapporo, Japan, Department of Virology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan, Laboratory of Virus Control, Research Institute for Microbial Diseases, Osaka University, Suita, Japan, AMED-CREST, Japan Agency for Medical Research and Development 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.https://bio-protocol.org/en/bpdetail?id=5275&type=0 |
| spellingShingle | Hirotaka Yamamoto Tomokazu Tamura Takausuke Fukuhara Rapid Plasmid-Free Generation of Recombinant Positive-Strand RNA Viruses That Use IRES-Mediated Translation Using an Expansion of the Circular Polymerase Extension Reaction (CPER) Bio-Protocol |
| title | Rapid Plasmid-Free Generation of Recombinant Positive-Strand RNA Viruses That Use IRES-Mediated Translation Using an Expansion of the Circular Polymerase Extension Reaction (CPER) |
| title_full | Rapid Plasmid-Free Generation of Recombinant Positive-Strand RNA Viruses That Use IRES-Mediated Translation Using an Expansion of the Circular Polymerase Extension Reaction (CPER) |
| title_fullStr | Rapid Plasmid-Free Generation of Recombinant Positive-Strand RNA Viruses That Use IRES-Mediated Translation Using an Expansion of the Circular Polymerase Extension Reaction (CPER) |
| title_full_unstemmed | Rapid Plasmid-Free Generation of Recombinant Positive-Strand RNA Viruses That Use IRES-Mediated Translation Using an Expansion of the Circular Polymerase Extension Reaction (CPER) |
| title_short | Rapid Plasmid-Free Generation of Recombinant Positive-Strand RNA Viruses That Use IRES-Mediated Translation Using an Expansion of the Circular Polymerase Extension Reaction (CPER) |
| title_sort | rapid plasmid free generation of recombinant positive strand rna viruses that use ires mediated translation using an expansion of the circular polymerase extension reaction cper |
| url | https://bio-protocol.org/en/bpdetail?id=5275&type=0 |
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