Preparing for the Next Influenza Season: Monitoring the Emergence and Spread of Antiviral Resistance

Abstract

Purpose: The relaxation of pandemic restrictions in 2022 has led to a reemergence of respiratory virus circulation worldwide and anticipation of substantial influenza waves for the 2022/2023 Northern Hemisphere winter. Therefore, the antiviral susceptibility profiles of human influenza viruses circulating in Germany were characterized.

Methods: Between October 2019 (week 40/2019) and March 2022 (week 12/2022), nasal swabs from untreated patients with acute respiratory symptoms were collected in the national German influenza surveillance system. A total of 598 influenza viruses were isolated and analyzed for susceptibility to oseltamivir, zanamivir and peramivir, using a neuraminidase (NA) inhibition assay. In addition, next-generation sequencing was applied to assess molecular markers of resistance to NA, cap-dependent endonuclease (PA) and M2 ion channel inhibitors (NAI, PAI, M2I) in 367 primary clinical samples. Furthermore, a genotyping assay based on RT-PCR and pyrosequencing to rapidly assess the molecular resistance marker PA-I38X in PA genes was designed and established.

Results: While NAI resistance in the strict sense, defined by a ≥ 10-fold (influenza A) or ≥5-fold (influenza B) increase of NAI IC₅₀, was not detected, a subtype A(H1N1)pdm09 isolate displayed 2.3- to 7.5-fold IC₅₀ increase for all three NAI. This isolate carried the NA-S247N substitution, which is known to enhance NAI resistance induced by NA-H275Y. All sequenced influenza A viruses carried the M2-S31N substitution, which confers resistance to M2I. Of note, one A(H3N2) virus displayed the PA-I38M substitution, which is associated with reduced susceptibility to the PAI baloxavir marboxil. Pyrosequencing analysis confirmed these findings in the original clinical specimen and in cultured virus isolate, suggesting sufficient replicative fitness of this virus mutant.

Conclusion: Over the last three influenza seasons, the vast majority of influenza viruses in this national-level sentinel were susceptible to NAIs and PAIs. These findings support the use of antivirals in the upcoming influenza season.

Keywords: antiviral resistance; baloxavir marboxil; cap-dependent endonuclease; genotypic assay; influenza viruses; molecular resistance marker; neuraminidase; phenotypic assay; surveillance.

Figure 1

NAI susceptibility of influenza type A viruses. The A(H1N1)pdm09-S247N isolate displayed an up to 7-fold increase of inhibitory concentration (IC50) for all three neuraminidase inhibitors.

Figure 2

Neuraminidase amino acid sequence alignment of A(H1N1)pdm09 isolate (A/NRW/142/2019) carrying the NA-S247N substitution and of vaccine strain A/Brisbane/02/2018 as wild-type reference.

Figure 3

Genotypic analysis of M2 ion channel of influenza A viruses based on next generation sequencing data. Alignment of M2 ion channel amino acid sequences of A(H1N1)pdm09 and A(H3N2) influenza viruses. All analyzed viruses as well as the reference viruses A/Brisbane/02/2018 and A/Kansas/14/2017 carry an asparagine (N) at position 31 (marked with an arrow), which is associated with resistance to adamantanes.

Figure 4

Genotypic analysis of influenza virus PA based on next generation sequencing data. Amino acid sequence alignment of cap-dependent endonuclease (PA) of A(H3N2) isolate (A/BLN/9/2020) carrying the PA-I38M substitution and vaccine strain A/Kansas/14/2017 as wild-type reference.

Figure 5

Pyrosequencing analysis of original specimen. An RT-PCR assay with subsequent pyrosequencing (PSQ) to detect the substitution of isoleucine to methionine at amino acid position 38 (I38M) of the influenza A(H3N2) cap-dependent endonuclease was designed and applied to (A) influenza virus (original specimen) carrying substitution I38M in cap-dependent endonuclease (PA) (B) cell-cultured A(H3N2) PA-I38M, and (C) reference virus A(H3N2) A/Hong Kong/1/2022. The primary sample and the cell cultured virus showed the nucleotide sequence ATG coding for methionine, the sensitive wild-type virus showed nucleotide codon ATA coding for isoleucine.