Developing a qPCR assay for Influenza A/FM/1/47-MA: Cloning of the Mouse-Adapted Influenza M1 Gene

Influenza, or flu, is a contagious respiratory illness caused by influenza viruses.  Human strains of influenza have been adapted to replicate in mouse lung in order to study their pathogenesis.  Interestingly, studies have shown influenza virulence to be determined by several genes.  The virulent mouse-adapted A/FM/1/47 influenza lineage, derived from a human strain associated with an outbreak of flu at Fort Monmouth, New Jersey in 1947, is an ideal candidate for use in mouse models.  Using this A/FM/1/47-MA strain, we have developed a qPCR assay for mouse-passaged influenza by targeting the virus M1 gene.  We first isolated the virus 594 bp M1 gene by reverse-transcriptase RT-PCR and created a standard curve with known copies of this gene in the pCR-2.1 vector.   Virus recovery from the lungs of infected mice will be calculated based on a direct comparison to this standard curve, after correction for the amount of RNA recovered (copies of GAPDH).  We have developed this assay in order to examine virus replication and inflammation in mice primed intranasally with Lactobacillus species (Gabryszewski et al., J Immunol. 2011).   As described in this manuscript, mice primed with lactobacilli respond to an otherwise lethal infection with pneumonia virus of mice (PVM) with augmented survival, reduced granulocyte recruitment, and decreased expression of pro-inflammatory cytokines.  This assay will permit us to extend our studies to a second important respiratory virus pathogen, that of A/FM/1/47-MA influenza.