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Updated: Jun 17, 2026
11:31
Generation of Recombinant Influenza Virus from Plasmid DNA
Published on: August 3, 2010
Improved and simplified recombineering approach for influenza virus reverse genetics
Summary
Researchers developed new plasmid vectors for faster cloning of all eight influenza A virus genome segments using homologous recombination. This simplifies reverse genetics, aiding in rapid analysis and vaccine development for novel influenza strains.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Conventional reverse genetics for influenza virus relies on DNA ligation into vectors, a process that can be time-consuming.
- Efficient cloning of all eight influenza virus genome segments is crucial for reverse genetics studies.
Purpose of the Study:
- To describe the construction and application of novel plasmid vectors for cloning influenza A virus genome segments via homologous recombination.
- To establish a more efficient method for influenza virus reverse genetics.
Main Methods:
- Construction of two plasmid vectors, pLLBA and pLLBG, featuring opposing RNA polymerase I and II transcription units and a recombination cassette with conserved influenza virus terminal promoters.
- Cloning of each influenza gene segment into the appropriate linearized vector using homologous recombination in E. coli.
Main Results:
- The novel vectors facilitate direct cloning by recombination, which is simpler and faster than traditional restriction digestion and ligation.
- Successful cloning and rescue of various influenza viruses using the new vector system were achieved.
Conclusions:
- The developed plasmid vector system offers a streamlined approach for influenza virus reverse genetics.
- This method has the potential to accelerate the analysis of novel influenza strains and expedite vaccine development.
Related Concept Videos
00:57Viral Recombination
Cells are sometimes infected by more than one virus at once. When two viruses disassemble to expose their genomes for replication in the same cell, similar regions of their genomes can pair together and exchange sequences in a process called recombination. Alternatively, viruses with segmented genomes can swap segments in a process called reassortment.
02:53Conservative Site-specific Recombination and Phase Variation
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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