The technology disclosed is a Luria-Delbruck fluctuation test for influenza virus mutation rates that scores reversion to fluorescence in a set of 12 mutant green fluorescent proteins (GFP).
Globally, seasonal influenza is a major cause of severe respiratory infections, leading to excessive hospitalizations and 1 million deaths/year. The global influenza market (vaccines, antivirals, and diagnostic products) grew from ~$3.8 billion in 2012 to ~$4 billion in 2013. Sales for influenza diagnostics are expected to increase to $380 million by 2018, up from $312 million in 2013; this reflects an increase of 4%. The influenza diagnostic market is important for the identification of new vaccines to make for seasonal influenza. Many of the existing methods to measuring influenza virus mutation rates are laborious and/or prone to bias. This technology addresses the problems with current technologies by making the technology more quantitative, higher throughput, and extended all 12-mutation classes.
A high-throughput assay for the determination of influenza virus mutation rates using 12 mutant green fluorescent proteins (GFPs)
The technology consists of a plasmid that contains GFP instead of hemagglutinin on influenza A virus segment 4 (ΔHA-GFP), to generate a set of 12 recombinant influenza A viruses that each express a mutant GFP protein. Each of the mutant GFP proteins has a single nucleotide mutation that, with reversion to fluorescence, will highlight a specific mutational class introduced by the viral RdRp during replication. A Luria-Delbrück fluctuation test is used to convert the reversion frequencies to viral mutation rates. Inoculated parallel cultures of MDCK-HA cells and transferred replicated virus to a MDCK cells in a 96-well imaging plate for each of the 12 mutant ΔHA-GFP. The technology can be used to identify compounds that increase viral mutation rates and viruses with altered mutation rates for the development of anti-viral compounds and attenuated vaccines, respectively.
- Identification of compounds that increase viral mutation rates
- Identification of viruses with altered mutation rates
- Can be used with other viral systems beyond influenza
- Precise measurement of mutation rates
- High throughput
- Rapid analysis
- Inclusive to all 12 Influenza mutation classes