Global update on the susceptibilities of influenza viruses to neuraminidase inhibitors and the cap-dependent endonuclease inhibitor baloxavir, 2020-2023

Abstract

Antiviral susceptibility of influenza viruses is monitored by the World Health Organization Global Influenza Surveillance and Response System. This study describes a global analysis of the susceptibility of influenza viruses to neuraminidase (NA) inhibitors (NAIs, oseltamivir, zanamivir, peramivir, laninamivir) and the cap-dependent endonuclease inhibitor (CENI, baloxavir) for three periods (May to May for 2020-2021, 2021-2022 and 2022-2023). In particular, global influenza activity declined significantly in 2020-2021 and 2021-2022 when compared to the pre-pandemic period of COVID-19. Combined phenotypic and NA sequence-based analysis revealed that the global frequency of seasonal influenza viruses with reduced or highly reduced inhibition (RI/HRI) by NAIs remained low, 0.09% (2/2224), 0.12% (27/23465) and 0.23% (124/53917) for 2020-2021, 2021-2022 and 2022-2023, respectively. As in previous years, NA-H275Y in A(H1N1)pdm09 viruses was the most frequent substitution causing HRI by oseltamivir and peramivir. Sequence-based analysis of polymerase acidic (PA) protein supplemented with phenotypic testing revealed low global frequencies of seasonal influenza viruses with reduced susceptibility (RS) to baloxavir, 0.07% (1/1376), 0.05% (9/18380) and 0.12% (48/39945) for 2020-2021, 2021-2022 and 2022-2023, respectively; commonly associated substitutions were PA-I38T/M/L. In Japan, the rate was 3.3% (16/488) during 2022-2023, with 11 A(H3N2) viruses having PA-I38T/M substitutions. For zoonotic viruses, 2.7% (3/111) contained substitutions, one each NA-H275Y, NA-S247N and NA-N295S, associated with RI/HRI NAI phenotypes, and none contained PA substitutions associated with RS to baloxavir. In conclusion, the great majority of seasonal and zoonotic influenza viruses remained susceptible to NAIs and CENI baloxavir.

Keywords: Antiviral; Baloxavir; Influenza; Neuraminidase inhibitor; Polymerase inhibitor; Reduced susceptibility.

Fig. 1

Influenza viruses collected and assessed for NAI susceptibility during the 2020–2021, 2021–2022 and 2022–2023 periods by the WHO CCs. For each period viruses were collected from patients at week 21 through week 20 of the following year. (A) The number of viruses collected by year and week of specimen collection and virus type/subtype or lineage for specimens assessed in the three consecutive periods. Typically, week 21 to week 39 of a year covers the Southern Hemisphere influenza season, while week 40 of a year to week 20 of the following year covers the Northern Hemisphere influenza season. (B) The number of viruses assessed for susceptibility to the four NAIs using NA inhibition assays and/or sequence-based analysis by WHO Region for the three consecutive periods. For 2021–2022 and 2022–2023, two reassortant A(H1N2) viruses were counted among the A(H3N2) viruses because of assessment of N2 NA for NAI susceptibility but these are not shown on the graph.

Fig. 2

Comparison of NAI susceptibility surveillance over 11 periods for phenotypic and/or sequence-based analysis data generated by WHO CCs. (A) Number of viruses tested. For the 2012–2018 periods testing was reported based on NA inhibition assays only. For the subsequent five periods, results of assessment by NA inhibition assays and/or sequence-based analysis were included. (B) The proportion of viruses showing RI/HRI by one or more NAIs over the 2012–2023 periods. Data were compiled from the global studies of viruses analyzed by WHO CCs during the 2012–2013 (Meijer et al., 2014), 2013–2014 (Takashita et al., 2015), 2014–2015 (Hurt et al., 2016), 2015–2016 (Gubareva et al., 2017), 2016–2017 (Lackenby et al., 2018), 2017–2018 (Takashita et al., 2020b), 2018–2020 (Govorkova et al., 2022), and 2020–2023 (current study) periods. For 2021–2022 and 2022–2023, reassortant A(H1N2) viruses were counted among the A(H3N2) viruses because of assessment of N2 NA for NAI susceptibility. Based on sequence-based analysis, viruses were considered RI or HRI when the NA contained an amino acid substitution for which the NA marker table posted on the WHO website and/or current testing data showed an IC₅₀ fold change of >10 (for A viruses) or >5 (for B viruses) compared to period median IC₅₀ values by subtype/lineage, WHO-CC and used method. When a particular amino acid substitution listed as RI or HRI at the WHO website was phenotypically confirmed NI in the genetic background of current viruses, all with such an NA amino acid substitution were considered as displaying NI. These were A(H3N2) viruses with NA-N329K/R or NA-S331R substitutions and B/Victoria lineage viruses with NA-K360E substitution.

Fig. 3

Column-scatter plots of log-transformed 50% inhibitory concentration (IC₅₀) fold-change values for NAIs. Overall, 1447/2015, 5287/9899 and 10558/19030 viruses assessed for NAI susceptibility were tested phenotypically for one or more NAIs for 2020–2021, 2021–2022 and 2022–2023 periods, respectively. Data are presented by virus subtype or lineage [(A) A(H1N1)pdm09; (B) A(H3N2); and (C) B/Victoria-lineage] with NAI (labelled on the x-axis: oseltamivir, zanamivir, peramivir, and laninamivir) and log-transformed IC₅₀ fold-changes compared to the period median on the y-axis. The boxes indicate the 25th–75th percentiles, and the whiskers stretch to the lowest and highest values within 1.5 times the interquartile region (IQR) value from both the 25th and 75th percentile values, respectively (Tukey's definition). The y-axes have been split into three compartments according to the thresholds recommended by the WHO-AVWG for NI (<10-fold for type A viruses; <5-fold for type B viruses), RI (10- to 100-fold for type A viruses; 5- to 50-fold for type B viruses), and HRI (>100-fold for type A viruses; >50-fold for type B viruses). NA amino acid substitutions are shown for viruses displaying RI or HRI phenotypes that have been sequenced. For several viruses with RI phenotype that have been sequenced, no amino acid substitution marker associated with this phenotype was identified. For another number of viruses with RI phenotype no sequence was available for evaluation. Viruses showing NI but carrying amino acid substitutions previously associated with RI or HRI by one or more other NAI are not indicated in the figure. These were A(H3N2) viruses with NA-N329K/R or NA-S331R substitutions and B/Victoria lineage viruses with NA-K360E substitution. Amino acid position numbering is specific to A subtype and B type. Most viruses were tested for susceptibility to oseltamivir and zanamivir; only a subset was tested for susceptibility to peramivir and laninamivir.

Fig. 4

Influenza viruses collected and assessed for baloxavir susceptibility during the 2020–2021, 2021–2022 and 2022–2023 periods by the WHO CCs. For each period viruses were collected from patients at week 21 through week 20 of the following year. (A) The number of viruses collected by year and week of specimen collection and virus type/subtype or lineage for specimens assessed in the three consecutive periods. Typically, week 21 to week 39 of a year covers the Southern Hemisphere influenza season, while week 40 of a year to week 20 of the following year covers the Northern Hemisphere influenza season. (B) The number of viruses assessed for susceptibility to baloxavir using phenotypic assays and/or sequence-based analysis, by WHO Region for the three consecutive periods. One A(H1N2) virus was included in the baloxavir susceptibility assessment but are not shown in the graph.

Fig. 5

Comparison of baloxavir susceptibility surveillance over six periods for phenotypic and/or sequence-based analysis data generated by WHO CCs. (A) Number of viruses tested. For the 2017–2018 period, baloxavir susceptibility assessment was reported based on sequence-based analysis only. For the 2018–2023 periods, results of phenotypic testing by cell culture-based assays and/or sequence-based analysis were included. (B) The proportion of viruses showing RS to baloxavir over the 2017–2023 periods. Data were compiled from the global studies of viruses during the 2017–2018 (Takashita et al., 2020b), 2018–2020 (Govorkova et al., 2022) and 2020–2023 (current study) periods. For 2017–2018, data from all PA sequences available from public sequence databases; for 2018–2020, data from all PA sequences available from public databases and phenotypic testing data from WHO CCs; for 2020–2023, data from all PA sequences and phenotypic testing data generated by WHO CCs. Viruses characterized by sequence-based analysis were considered RS when the PA contained amino acid substitutions for which the WHO website and/or current testing data showed an >3-fold EC₅₀ fold-change compared to baloxavir period median EC₅₀ values by subtype/lineage, WHO-CC and method. When a particular PA amino acid substitution listed as RS in the WHO website was phenotypically confirmed NS in the genetic background of current viruses, all with such an amino acid substitution were considered NS (e.g., PA-L28P in A(H3N2) viruses).

Fig. 6

Column-scatter plots of log-transformed 50% effective concentration (EC₅₀) fold-change values for baloxavir. The phenotypic susceptibility of influenza viruses to baloxavir was tested by cell culture–based assays, focus-reduction assay (FRA), high-content imaging neutralization test (HINT), or influenza replication inhibition neuraminidase-based assay (IRINA). Overall, 126/1244, 766/6516, and 1054/11172 viruses assessed for baloxavir susceptibility were tested phenotypically for the 2020–2021, 2021–2022 and 2022–2023 periods, respectively. Data are presented by virus subtype or lineage [labelled on the x-axis: A(H1N1)pdm09; A(H3N2); and B/Victoria-lineage] and log-transformed EC₅₀ fold changes compared to the period median on the y-axis. The boxes and whiskers are as defined in Fig. 3. An arbitrary cut-off of >3-fold increase in the EC₅₀ of a test virus compared to period median EC₅₀ was used for reporting viruses with RS to baloxavir. PA amino acid substitutions are shown for viruses displaying a RS phenotype that have been sequenced. Of seven A(H3N2) and eleven B/Victoria-lineage viruses showing an RS phenotype, the PA sequences did not show known markers associated with RS to baloxavir. Amino acid position numbering is specific to type A and B viruses.