Genetic changes of influenza A(H1N1)pdm09 viruses circulating in 2018/2019 season affected subtyping of influenza virus by real-time RT-PCR assay

Aim: Reduced performance of real-time RT-PCR (rRT-PCR) for influenza A(H1N1)pdm09 virus subtyping was observed during the 2018/2019 season. We analysed and compared the primers/probe binding regions in the hemagglutinin (HA) gene to HA sequence of circulating strains. Method: 713 respiratory samples collected from in/outpatients with influenza illness in Lombardy (Northern Italy) during the 2018/2019 season were analysed. Influenza viruses were typed (A/B) and subtyped (H1N1pdm09/H3N2) by rRT-PCR targeting matrix/nucleoprotein and HA gene, respectively, according to international protocols. The expected difference between Ct values obtained from A-typing and H1N1pdm09-subtyping assays [∆(H1-A)Ct] is <5. Full-length HA gene (nt. 1-1778) sequence of A(H1N1)pdm09 circulating strains were obtained and compared to primers/probe sequences used in subtyping rRT-PCR assay. Results: Influenza A viruses were detected in 390/713 (54.7%) specimens, 48.5% (189/390) were A(H1N1)pdm09. 52/189 (27.5%) A(H1N1)pdm09-positive samples showed a ∆(H1-A)Ct≥5 (range: 5.00-14.95). 24/52 A(H1N1)pdm09 with ∆(H1-A)Ct≥5 and 71/137 with ∆(H1-A)Ct<5 were sequenced. Four nucleotide mismatches (C861T+C867T in the forward primer and A897G+A905C in the probe) were detected in 17/24 (70.8%) HA sequences of A(H1N1)pdm09 with ∆(H1-A)Ct≥5, all had a ∆(H1-A)Ct>8. No mismatches were observed among HA sequences of A(H1N1)pdm09 with ∆(H1-A)Ct<5. Conclusions: Four nucleotide mismatches in primers/probe of subtyping rRT-PCR assay were observed in nearly 30% of A(H1N1)pdm09 viruses circulating in Lombardy during the 2018/2019 season. These point mutations reduced the primer/probe binding efficiency affecting the performance of rRT-PCR assay for subtyping. These results emphasised the need of continuously updating the molecular assays for influenza detection considering the constant evolution of influenza viruses.