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Case Study // JUL 21 2014

Co-transfection of multiple plasmid DNAs encoding the IBV genome paves way for better flu vaccines

Improved Influenza B Virus (IBV) strain representation for vaccine seed stocks was enabled by primer design and ligation-free cloning approaches

“Recombinant IBVs were generated by cotransfection of eight reverse-genetics plasmids carrying the cDNA of each gene segment into a 293T/MDCK coculture monolayer, as previously described (20–22). Briefly, 0.6 μg of each plasmid was mixed and incubated with 15 μl of TransIT®-LT1 transfection reagent (Mirus Bio, Madison, WI) at 20°C for 20 min and then added to 80% confluent 293T/MDCK cell cocultures in 6-well plates."

- Zhou, et al.1

Background

Three major types of influenza viruses exist based on their antigenicity, namely Influenza A (IAV), B (IBV) and C (ICV). Of these, IAV and IBV are included in yearly flu vaccines that are available for public use; ICV is not included due to its viral tropism. The IAV genome has been the most well studied out of these three sub types, whereas there is significantly less genetic information about IBV. Despite of being a significant human pathogen that undergoes antigenic drift, the few IBV isolates included in the current flu vaccine design do not address the continuously emerging new IBV strains. Complete sequencing of the IBV genome from different isolates can help understand the pathogenesis of IBV and also enable the construction of better vaccines.

In the present case study, TransIT®-LT1 Transfection Reagent is used to co-transfect eight reverse-genetics plasmids into 293T/MDCK coculture monolayers to produce recombinant Influenza B viruses from different sample types and geographical isolates. This establishes the proof of principle for possible high-throughput generation of recombinant IBV strain repertoire that can be used to predict the lineage of IBVs that will emerge in the upcoming influenza seasons.

Universal Influenza B Virus Genomic Amplification Facilitates Sequencing, Diagnostics, and Reverse Genetics
1 Bin Zhou, Xudong Lin, Wei Wang, Rebecca A. Halpin, Jayati Bera, Timothy B. Stockwell, Ian G. Barr and David E. Wentworth, Published ahead of print 5 February 2014, doi: 10.1128/JCM.03265-13, J. Clin. Microbiol. May 2014 vol. 52 no. 5 1330-1337

What is the role of reverse genetics in the study of viruses?

Reverse genetics is an approach commonly applied by virologists to introduce mutations in viral genomes and study their phenotypic effects. Specifically, co-transfection of multiple plasmid DNAs that transcribe segmented influenza virus RNA genomes has been employed for over the past two decades to produce virus reassortments that have helped understand the life cycle and evolution of influenza. For a comprehensive review of influenza virus reverse genetics methods, please click here.

Can reverse genetics approaches with recombinant IBV strains be used to expedite vaccine seed stock prediction?

Yes. The proof of concept for expediting vaccine design with better IBV representation is essentially accomplished in this article through clever primer design and ligation-free cloning approaches. Computational biology approaches based on short conserved 5' and 3' terminal regions and segment-specific complementarity led to the design of primer sets that could effectively reverse transcribe IBV virus genomes from cell culture isolates as well as clinical specimens that harbor low amounts of the virus. This was verified with >1,000 different IBV genomes spanning past, present, and future IBVs. Furthermore, special terminal sequences were engineered into the amplified IBV isolate genomes to facilitate high-throughput ligation-free cloning to rapidly generate reverse genetics plasmids. These reverse genetics plasmids can then be permuted and co-transfected to create an IBV repertoire that can be utilized as a vaccine seed stock library. This has the potential of tremendously expediting amplification of IBV genomes from actual virus isolates compared to the current time consuming process of growing virus in chicken eggs. It also provides influenza researchers with a tool to better understand the biology of IBV, such as pathogenesis determinants and drug resistance, etc.

View more information on high-titer virus production.

Other Mirus Bio reagents useful in virus production:

  • TransIT-X2® - an animal origin free reagent capable of delivering DNA and/or siRNA with high efficiency in a broad spectrum of cell types; ideal for co-transfection of multiple plasmids or DNA and siRNA.
  • TransIT®-2020 - an animal origin free reagent specifically developed to deliver DNA with high efficiency in difficult-to-transfect cell types; capable of transfecting multiple plasmids.

Free samples of Mirus Bio transfection reagents are available upon request.

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