All the recombinant PR8/ANS1, PR8/BNS1, PR8/CNS1, and PR8/DNS1 viruses were successfully recovered, using plasmid-based reverse genetics techniques, and the identity of the rescued viruses was confirmed by immunofluorescence (Figure 2C) and Western blot (Figure 2D) analysis at 14 h post-infection (p.i.)in MDCK-infected [multiplicity of infection (MOI) = 2] cells, using specific antibodies against the HA epitope tag fused to NS1, and the viral NP protein. This evidence concerns the gene TTC4 and infection.