The Centers for Disease Control and Prevention (CDC) monitors antiviral susceptibility of viruses submitted to the national surveillance system and those collected in other countries. Nearly all influenza-positive samples undergo next-generation sequencing. We analyzed NA sequences of submitted viruses for substitutions previously associated with reduced susceptibility (5), tested the viruses in an NA inhibition assay, and compared IC₅₀s with a reference IC₅₀ to determine inhibition levels (7).

During May 2023–February 2024, we analyzed 2,039 pH1N1 viruses from the United States (n = 1,274) and 38 other countries (n = 765). Four had the H275Y substitution, indicating low frequency of oseltamivir resistance. Analysis revealed NA substitution I223V in 18 and S247N in 15 viruses; those substitutions confer mildly elevated oseltamivir IC₅₀ (<10-fold). We also detected 17 viruses carrying both substitutions, I223V + S247N (Table 1; Appendix Table 1). As expected, single mutants exhibited normal inhibition by oseltamivir and other NA inhibitors in NA inhibition assay (Table 2). The 6 viruses with I223V + S247N displayed 13- to 16-fold reduced inhibition for oseltamivir and normal inhibition (<4-fold) for other NA inhibitors (Table 2; Appendix Table 2). Both single and dual mutants remained susceptible to the CEN inhibitor baloxavir (Table 2).

Figure 1

Figure 1. Detection of influenza A(H1N1)pdm09 viruses with dual NA-I223V + S247N substitutions through NA inhibitors susceptibility surveillance conducted by the Centers for Disease Control and Prevention and analysis of available sequences...

The dual mutants were collected in the United States and 4 other countries during August–November 2023 (Table 1; Appendix Table 1), which prompted us to explore when dual mutants emerged and how broadly single or dual mutants were circulating worldwide. Analysis of available NA sequences from the GISAID EpiFlu database (https://www.gisaid.org) revealed that pH1N1 viruses with single mutation I223V (n = 110) or S247N (n = 203) were found in many countries (Figure 1, panel A). In addition to the 17 viruses we sequenced, 84 additional dual mutants were identified (total n = 101). Together, they were collected in 15 countries spanning 5 continents (Africa, Asia, Europe, North America, and Oceania) (Figure 1, panel B). The first dual mutant was collected from Canada in May 2023, and the latest were collected from 4 countries (France, the Netherlands, Spain, and the United Kingdom) during January–February 2024. Most dual mutants were detected in the Netherlands (n = 30), France (n = 24), Bangladesh (n = 11), Oman (n = 9), and the United Kingdom (n = 9); fewer were found in Hong Kong (n = 4), Niger (n = 3), Australia (n = 2), Spain (n = 2), and the United States (n = 2). One dual mutant each was detected in Canada, Ethiopia, Maldives, Norway, and Sweden (Figure 1, panel B; Appendix Tables 1, 3).

Figure 2

Figure 2. Tanglegram showing influenza A(H1N1)pdm09 phylogenies for NA gene (left) and HA gene (right) from susceptibility surveillance conducted by the Centers for Disease Control and Prevention and analysis of available sequences...

On the basis of NA phylogenetic analysis, we determined that most single S247N mutants belonged to either subclade C.5 (16%) or the most abundantly sequenced subclade, C.5.3 (80%) (8). All subclade C.5.3 viruses share substitution S200N located at the antibody binding family site VI (Figure 1, panel A). Most single I223V mutants (92%) also belonged to subclade C.5.3 and formed a distinct branch with substitution S366N located at the antibody binding family site III. Within that branch, 2 separate introductions of S247N have occurred, giving rise to dual I223V + S247N mutants that could be divided into distinct groups 1 and 2 (Figure 2). Only 9 dual mutants collected in 6 countries belonged to group 1 and shared an additional substitution R257K. Group 2 encompassed 92 dual mutants from countries with multiple detections (i.e., France, Netherlands, Bangladesh, the United Kingdom, Oman, and Niger) (Figure 1, panel B).

To further characterize the dual mutants, we performed hemagglutinin (HA) phylogenetic analysis. Two major HA clades, 6B.1A.5a.2a (5a.2a) and 6B.1A.5a.2a.1 (5a.2a.1), were seen globally during this period (8,9). Viruses belonging to HA subclades 5a.2a_C.1 (55%–61%) and 5a.2a.1_C.1.1.1 (13%–39%) predominated. Of note, all group 1 dual mutants had HA from 5a.2a_C.1, represented by the previous vaccine prototype virus A/Sydney/5/2021 (Figure 2). Conversely, most group 2 dual mutants had HA from 5a.2a.1_C.1.1.1, represented by the current vaccine prototype virus A/Victoria/4897/2022, and most shared 2 HA changes: R45K in HA1 and L2I in HA2 (Figure 2).

We report the emergence and intercontinental spread of pH1N1 viruses displaying reduced susceptibility to oseltamivir resulting from acquisition of NA-I223V + S247N mutations. Emergence of the dual mutants was also recently noticed by researchers in Hong Kong (10). The dual mutants that we tested retained susceptibility to other approved influenza antiviral drugs, including baloxavir. Analysis of available sequence data revealed that dual mutants have been in global circulation since May 2023; overall detection frequency was low (0.67%, 101/15,003). However, those data may not necessarily represent the actual proportion of what was in circulation because of differences in surveillance and sequencing strategies in each country.

Substitutions at residues 223 or 247 were previously reported and occurred spontaneously in circulating viruses (5,11,12). pH1N1 viruses with S247N circulated in several countries during 2009–2011 (11), and influenza B viruses with I223V (I221V in B numbering) were found in several US states during 2010–2011 (12). Isoleucine at 223 is a highly conserved framework residue in the NA active site. The S247N substitution may alter the hydrogen bonding network of the active site and the conformation of the residue E277 side chain, thereby weakening oseltamivir binding (11). Changes at 223 or 247 are monitored because they can enhance drug resistance by combining with mutations at other residues (11,13,14).

Rapid spread of dual mutants to countries on different continents suggests no substantial loss in their replicative fitness and transmissibility. I223V was shown to alter NA activity (14), and change at 247 may produce a similar effect, which warrants the question whether signature substitution(s) of NA subclade C.5.3 and of the branch to which the dual mutants belong (i.e., S200N, S366N) could serve as prerequisites for emergence of dual mutants. All group 1 dual mutants had an additional substitution R257K, which was previously associated with restoring NA activity of viruses with the H275Y substitution (15). Conversely, most group 2 viruses acquired HA from subclade 5a.2a.1_C.1.1.1 by reassortment, which may have helped to restore the functional HA and NA balance. Acquisition of the antigenically distinct HA could further enhance the spread of group 2 dual mutants. Our study highlights the need to closely monitor evolution of dual mutants because additional changes may further affect susceptibility to antiviral drugs or provide a competitive advantage over circulating wild-type viruses.