Influenza viruses have a rapid replication cycle, using enzymes without proofreading capacity and generating multiple virus quasispecies during replication. The identification and quantification of these quasispecies populations require time-consuming and expensive cloning and sequencing approaches. In the present study, we developed mutation-specific real-time PCR (RT-PCR) tests for the fine quantification of mutations in a background of wild-type sequences. As a proof-of-concept model, we developed mutation-specific RT-PCR tests to quantify antibody escape mutations during passage under monoclonal antibody (mAb) selection pressure in quasispecies populations of HPAI A/crested eagle/Belgium/01/2004 (H5N1). Mutation-specific RT-PCRs were developed for two mutations (one in HA1 and one in HA2) and validated using plasmids representing either the wildtype sequence or the mutation. The approach achieves a precise and accurate estimation of mutation frequencies on mixed populations in the range of 1% to 99% and does not require standard curves or calibrators. For the HA1 mutations, a directional increase of % G over the passages towards fixation of the G mutation could be observed. On the contrary, as expected from the inaccessibility of the HA2 region to antibodies, the HA2 mutation increased in frequency by factors unrelated to mAb-driven selection. This approach allows in-depth analysis of quasispecies dynamics using large sample sizes. It may also be applied to the dynamics of hot spots of mutations in several genes, such as HA or PB2, and to the early detection of critical changes in the field situation.
