In a previous study a 15-mer phosphorothioate oligonucleotide (S-ON) derived from the packaging signal in the 5' end of segment 1 (PB2) of influenza A virus (designated 5-15b) proved markedly inhibitory to virus replication. Here we investigated whether analogous inhibitory S-ONs targeting the 5' end of segments 2 (PB1) and 3 (PA) could be identified and whether viral resistance to S-ONs can be developed. Similar to our earlier result, 20-mer S-ONs reproducing the 5' ends of segments 2 or 3 (complementary to the 3'-coding regions of PB1 and PA, respectively) exerted a powerful antiviral activity against a variety of influenza A virus subtypes in MOCK cells. Serial passage of the A/Taiwan/1/86 H1N1 strain in the presence of S-ON 5-15b or its antisense as5-15b analogue showed that mutant viruses with reduced susceptibility to the S-ON could indeed be generated, although the resistant viruses displayed reduced replicative fitness. Sequencing the resistant viruses identified mutations in the PB1, PB2, PA and M1 genes. Introduction of these changes into the A/PR/8/34 H1N1 strain by reverse genetics, suggested that alterations to RNA function in the packaging regions of segments 2 and 3 were important in developing resistance to S-ON inhibition. However, many of the other sequence changes induced by S-ON treatment were markedly deleterious to virus fitness. We conclude that packaging signals in the influenza A virus polymerase segments provide feasible targets for nucleic acid-based antivirals that may be difficult for the virus to evade through resistance mutations. (C) 2011 Elsevier B.V. All rights reserved.