Researchers developed a new reverse-genetics system to generate influenza A viruses entirely from cloned cDNAs. Human embryonic kidney cells (293T) were transfected with eight plasmids, each encoding viral RNA of A/WSN/33 or A/PR/8/34 viruses, flanked by human RNA polymerase I promoter and mouse RNA polymerase I terminator, along with plasmids encoding viral nucleoprotein and polymerases. This method produced over 10^3 plaque-forming units (pfu) per ml of supernatant at 48 hours post-transfection. Adding plasmids for remaining viral structural proteins increased virus production to 3×10^4–5×10^7 pfu/ml. The system allows efficient production of influenza viruses without helper virus infection, useful for viral mutagenesis studies and vaccine development. The system uses RNA polymerase I to generate viral RNA from cloned cDNAs, enabling the production of infectious influenza viruses. This method is more efficient than previous approaches that required helper virus infection. The system allows for the introduction of mutations into any gene segment and the development of influenza virus-based gene delivery systems. The ability to generate infectious RNA viruses from cloned cDNAs has greatly advanced our understanding of influenza viruses and improved disease control methods. The study demonstrates that the reverse-genetics system can produce influenza A viruses entirely from cloned cDNAs, with high efficiency and the ability to introduce mutations into any gene segment. The system does not require helper virus infection, making it useful for viral mutagenesis studies and vaccine production. The system can also be used to generate reassortant viruses and viruses with mutations in specific genes. The system has potential applications in the development of attenuated live-virus vaccines and gene therapy vectors. The study highlights the importance of reverse-genetics in understanding viral life cycles, protein functions, and pathogenic mechanisms. The system has implications for the study of viral pathogenicity and the development of new vaccines and gene therapy approaches.Researchers developed a new reverse-genetics system to generate influenza A viruses entirely from cloned cDNAs. Human embryonic kidney cells (293T) were transfected with eight plasmids, each encoding viral RNA of A/WSN/33 or A/PR/8/34 viruses, flanked by human RNA polymerase I promoter and mouse RNA polymerase I terminator, along with plasmids encoding viral nucleoprotein and polymerases. This method produced over 10^3 plaque-forming units (pfu) per ml of supernatant at 48 hours post-transfection. Adding plasmids for remaining viral structural proteins increased virus production to 3×10^4–5×10^7 pfu/ml. The system allows efficient production of influenza viruses without helper virus infection, useful for viral mutagenesis studies and vaccine development. The system uses RNA polymerase I to generate viral RNA from cloned cDNAs, enabling the production of infectious influenza viruses. This method is more efficient than previous approaches that required helper virus infection. The system allows for the introduction of mutations into any gene segment and the development of influenza virus-based gene delivery systems. The ability to generate infectious RNA viruses from cloned cDNAs has greatly advanced our understanding of influenza viruses and improved disease control methods. The study demonstrates that the reverse-genetics system can produce influenza A viruses entirely from cloned cDNAs, with high efficiency and the ability to introduce mutations into any gene segment. The system does not require helper virus infection, making it useful for viral mutagenesis studies and vaccine production. The system can also be used to generate reassortant viruses and viruses with mutations in specific genes. The system has potential applications in the development of attenuated live-virus vaccines and gene therapy vectors. The study highlights the importance of reverse-genetics in understanding viral life cycles, protein functions, and pathogenic mechanisms. The system has implications for the study of viral pathogenicity and the development of new vaccines and gene therapy approaches.