Universal influenza B virus genomic amplification facilitates sequencing, diagnostics, and reverse genetics

Area of Science:

• Virology
• Genomics
• Molecular Biology

Background:

• Human influenza B virus (IBV) poses a significant public health threat.
• The genetic diversity of IBV hinders universal genome amplification for sequencing and vaccine development.
• Rapid generation of sequence data and viral gene cloning are crucial for IBV research, diagnostics, and therapeutics.

Purpose of the Study:

• To develop and optimize a universally applicable method for amplifying the complete segmented negative-sense RNA genome of all IBV strains.
• To facilitate next-generation sequencing, advanced genomic diagnostics, and rapid vaccine development for IBV.

Main Methods:

• Developed and optimized a single-tube/well technique for IBV genomic amplification (IBV-GA).
• Engineered special terminal sequences into IBV-GA2 products for ligation-free cloning.
• Applied the technology to amplify and sequence over 1,000 diverse IBV genomes from various sample types.

Main Results:

• Successfully amplified the complete genomes of >1,000 diverse IBV strains using the IBV-GA technique.
• Demonstrated that the method is sensitive, robust, and sequence-independent, amplifying past, present, and future IBVs.
• Enabled ligation-free cloning of IBV-GA2 products for rapid generation of reverse-genetics plasmids.

Conclusions:

• The optimized IBV-GA technology provides a universal, robust, and sensitive solution for amplifying the diverse IBV genome.
• This advancement significantly facilitates next-generation sequencing, genomic diagnostics, and the development of recombinant viruses and vaccines for IBV.
• The sequence-independent nature of IBV-GA ensures its utility against current and emerging IBV strains.