Recombinant influenza viruses with the single amino acid substitution (N3A) within the NP 366 peptide, ASNENMETM, were generated using the eight-plasmid reverse genetics system [50] . The substitution was first incorporated by site directed mutagenesis using PCR primers encoding N3A 366 peptide, ASAENMETM, and the opposite primer encoding NP. Recombinant PCR products encoding N3A 366 were digested with BsmB1 and ligated into the alkaline phosphatase treated pHW2000 vector. Recombinant viruses (HK-NPN3A and PR8-NPN3A) were rescued following transfection of MDCK-293T cell coculture with the eight plasmids encoding influenza segments. Viruses were then amplified in the allantoic cavity of 10-day old embryonated chicken eggs, and the viral titre determined by plaque assay of allantoic fluid infecting monolayers of MDCK cells. There was any evidence of altered fitness in vivo for the HKNPN3A virus, as the kinetics of virus growth and clearance following i.n. challenge of naïve B6 mice were found to be equivalent for the wt HK and mutant viruses (Fig S3A) . Similarly, the levels of CTL activity found using target cells infected with HKNPN3A or the HK wt viruses were comparable, and the same was seen for peptide pulsed cells, suggesting that antigen presentation of NPN3A peptide remains constant (Fig S3 BC) .