Monitoring of antiviral resistance among influenza viruses takes place through a number of surveillance initiatives and ad hoc studies.

The World Health Organisation (WHO) collates data from WHO National Influenza Centres and WHO Collaborating Centres through the Global Influenza Surveillance and Response System (GISRS). Data from European (EU/EEA) countries is collated by The European Surveillance System (TESSy), under the aegis of the European Centre for Disease Prevention and Control (ECDC).

M2 inhibitors

Amantadine and rimantadine target the M2 ion channel of influenza A viruses. Monitoring resistance to M2 inhibitors is based on the detection of amino acid substitutions at residues 26, 27, 30, 31, or 34 (Hayden and Hay 1992), which confer cross-resistance.

Influenza A viruses of both subtypes that are currently circulating in the human population are resistant to amantadine and rimantadine. Resistant A(H3N2) viruses containing S31N in the M2 protein first emerged at the beginning of the 21^st century and subsequently spread worldwide (Bright et al., 2005). The A(H1N1)pdm09 virus derived its ’resistant’ M gene from the Eurasian lineage of swine viruses, which acquired amantadine resistance in the mid-1980s (Smith et al., 2009)

Bright, R. A., Medina, M. J., Xu, X., Perez-Oronoz, G., Wallis, T. R., Davis, X. M., Povinelli, L., Cox, N. J. 2005. Incidence of adamantane resistance among influenza A(H3N2) viruses isolated worldwide from 1994 to 2005: a cause for concern. Lancet. 366:1175-81.

Hayden, F. G., Hay, A. J. 1992. Emergence and transmission of influenza A viruses resistant to amantadine and rimantadine. Curr Top Microbiol Immunol. 176:119-30.

Smith, G. J., Vijaykrishna, D., Bahl, J., Lycett, S. J., Worobey, M., Pybus, O. G., Ma, S. K., Cheung, C. L., Raghwani, J., Bhatt, S., Peiris, J. S., Guan, Y., Rambaut, A. 2009. Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature. 459:1122-5.

Neuraminidase inhibitors

Neuraminidase (NA) inhibitors (NAIs) including zanamivir, oseltamivir, peramivir, and laninamivir were rationally designed based on the NA crystal structure. An enzyme inhibition assay (functional assay) is used to assess susceptibility to NAIs, as cell culture-based assays are not reliable for testing this class of antivirals. Numerous NA amino acid substitutions have been implicated in reduced inhibition by NAIs. Monitoring for the emergence of NAI resistance is conducted using both functional and genotypic assays.

Prior to 2007, surveillance of viruses circulating around the world indicated that the occurrence of resistance or reduced susceptibility to oseltamivir or zanamivir was low. In Japan, which has the highest per capita use of the antivirals, the frequency of detection was less than 1% (Tashiro et al., 2009).

Oseltamivir-resistant seasonal A(H1N1) viruses bearing the H275Y substitution in NA emerged in 2007 and spread globally (Collins et al., 2009). These resistant A(H1N1) viruses were displaced by oseltamivir-sensitive A(H1N1)pdm09 viruses during the 2009 pandemic.

Occurrence of oseltamivir resistance due to H275Y among A(H1N1)pdm09 viruses has been mainly sporadic. There have been a few instances of small clusters of oseltamivir-resistant viruses in immunocompromised patients and healthy adults, indicating the potential of resistant viruses to transmit. Moreover, the local spread of oseltamivir-resistant A(H1N1)pdm09 viruses with H275Y were reported in Australia in 2011 (Hurt et al,. 2012) and in Japan in 2013–2014 (Takashita et al., 2015). However, these viruses retained susceptibility to zanamivir.

Since 2012, surveillance of seasonal influenza viruses detected resistance or reduced susceptibility to NAIs at a frequency of 0.5% – 1.9%, according to the global updates by WHO Collaborating Centres (e.g. Lackenby et al., 2018).

Collins, P. J., Haire, L. F., Lin, Y. P., Liu, J., Russell, R. J., Walker, P. A., Martin, S. R., Daniels, R. S., Gregory, V., Skehel, J. J., Gamblin, S. J., Hay, A. J. 2009. Structural basis for oseltamivir resistance of influenza viruses. Vaccine 27:6317-23.

Hurt, A.C., Hardie, K., Wilson, N.J., Deng, Y.M., Osbourn, M., Leang, S.K., Lee, R.T., Iannello, P., Gehrig, N., Shaw, R., Wark, P., Caldwell, N., Givney, R.C., Xue, L., Maurer-Stroh, S., Dwyer, D.E., Wang, B., Smith, D.W., Levy, A., Booy, R., Dixit, R., Merritt, T., Kelso ,A., Dalton, C., Durrheim, D., Barr, I.G. 2012. Characteristics of a widespread community cluster of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza in Australia. J Infect Dis. 206:148-57.

Lackenby, A., Besselaar, T. G., Daniels, R. S., Fry, A., Gregory, V., Gubareva, L. V., Huang, W., Hurt, A. C., Leang, S. K., Lee, R. T. C., Lo, J., Lollis, L., Maurer-Stroh, S., Odagiri, T., Pereyaslov, D., Takashita, E., Wang, D., Zhang, W., Meijer, A. 2018. Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors and status of novel antivirals, 2016-2017. Antiviral Res. 157:38-46.

Takashita, E., Kiso, M., Fujisaki, S., Yokoyama, M., Nakamura, K., Shirakura, M., Sato, H., Odagiri, T., Kawaoka, Y., Tashiro, M., 2015. Characterization of a large cluster of influenza A(H1N1)pdm09 viruses cross-resistant to oseltamivir and peramivir during the 2013-2014 influenza season in Japan. Antimicrob. Agents Chemother. 59, 2607e2617.

Tashiro, M., McKimm-Breschkin, J. L., Saito, T., Klimov, A., Macken, C., Zambon, M., Hayden, F. G. 2009. Surveillance for neuraminidase-inhibitor-resistant influenza viruses in Japan, 1996-2007. Antivir Ther 14:751-61

Polymerase inhibitors

The PB1 inhibitor favipiravir is approved in Japan, and the PA inhibitor baloxavir is approved in several countries. Susceptibility to these polymerase inhibitors is assessed using cell culture-based assays and sequence analysis (Gubareva et al., 2019; Koszalka et al., 2019; Takashita et al., 2016, 2018).

Treatment-emergent resistance to baloxavir is associated with amino acid substitutions at residue 38 in the PA protein, which is highly conserved in influenza A and B viruses (Omoto et al., 2018; Gubareva et al., 2019).

In 2018–2019, baloxavir was the most widely used influenza antiviral in Japan. During this period, PA I38X substitutions were detected in A(H1N1)pdm09 and A(H3N2) viruses at 1.5% and 9.5%, respectively. These viruses were collected mainly from baloxavir-treated children <12 years of age. However, a few were from untreated children, indicating human-to-human transmission (Takashita et al., 2019; Imai et al., 2020). In the USA, where baloxavir is only approved for patients aged 12 years and older, no baloxavir-resistant viruses were detected during the same period (Xu et al. 2019).

The replicative fitness of contemporary influenza A viruses with a PA I38X substitution was observed to be relatively unaltered in vitro and in animal models (Checkmahomed et al., 2020, Chesnokov et al., 2019, Imai et al., 2020).

Checkmahomed, L., M’hamdi. Z., Carbonneau, J., Venable, M. C., Baz, M., Abed, Y., Boivin, G. 2020. Impact of the baloxavir-resistant polymerase acid I38T substitution on the fitness of contemporary influenza A(H1N1)pdm09 and A (H3N2) strains. J Infect Dis. 221:63-70.

Chesnokov, A., Patel, M. C., Mishin, V. P., De La Cruz, J. A., Lollis, L., Nguyen, H. T., Dugan, V., Wentworth, D. E., Gubareva, L. V. 2019. Replicative fitness of seasonal influenza A viruses with decreased susceptibility to baloxavir. J Infect Dis. pii: jiz472.

Gubareva, L.V., Mishin, V.P., Patel, M.C., Chesnokov, A., Nguyen, H.T., De La Cruz, J., Spencer, S., Campbell, A.P., Sinner, M., Reid, H., Garten, R., Katz, J.M., Fry, A.M., Barnes, J., Wentworth, D.E. 2019. Assessing baloxavir susceptibility of influenza viruses circulating in the United States during the 2016/17 and 2017/18 seasons. Euro Surveill. 24(3):pii=1800666.

Imai, M., Yamashita, M., Sakai-Tagawa, Y., Iwatsuki-Horimoto, K., Kiso, M., Murakami, J., Yasuhara, A., Takada, K., Ito, M., Nakajima, N., Takahashi, K., Lopes, T. J. S., Dutta, J., Khan, Z., Kriti, D., van Bakel, H., Tokita, A., Hagiwara, H., Izumida, N., Kuroki, H., Nishino, T., Wada, N., Koga, M., Adachi, E., Jubishi, D., Hasegawa, H., Kawaoka, Y. 2020. Influenza A variants with reduced susceptibility to baloxavir isolated from Japanese patients are fit and transmit throught respiratory droplets. Nat Microbiol. 5:27-33.

Koszalka, P., Tilmanis, D., Roe, M., Vijaykrishna, D., Hurt, A.C. 2019. Baloxavir marboxil susceptibility of influenza viruses from the Asia-Pacific, 2012-2018. Antiviral Res. 164:91-96.

Omoto, S., Speranzini, V., Hashimoto, T., Noshi, T., Yamaguchi, H., Kawai, M., Kawaguchi, K., Uehara, T., Shishido, T., Naito, A., Cusack, S. 2018. Characterization of influenza virus variants induced by treatment with the endonuclease inhibitor baloxavir marboxil. Sci Rep. 8:9633.

Takashita, E., Ejima, M., Ogawa, R., Fujisaki, S., Neumann, G., Furuta, Y., Kawaoka, Y., Tashiro, M., Odagiri, T. 2016. Antiviral susceptibility of influenza viruses isolated from patients pre- and post-administration of favipiravir. Antiviral Res. 132:170-7.

Takashita, E., Ichikawa, M., Morita, H., Ogawa, R., Fujisaki, S., Shirakura, M., Miura, H., Nakamura, K., Kishida, N., Kuwahara, T., Sugawara, H., Sato, A., Akimoto, M., Mitamura, K., Abe, T., Yamazaki, M., Watanabe, S., Hasegawa, H., Odagiri, T. 2019. Human-to-Human Transmission of Influenza A(H3N2) Virus with Reduced Susceptibility to Baloxavir, Japan, February 2019. Emerg Infect Dis. 25:2108-2111.

Takashita, E., Morita, H., Ogawa, R., Nakamura, K., Fujisaki, S., Shirakura, M., Kuwahara, T., Kishida, N., Watanabe, S., Odagiri, T. 2018. Susceptibility of Influenza Viruses to the Novel Cap-Dependent Endonuclease Inhibitor Baloxavir Marboxil. Front Microbiol. 9:3026.

Xu, X., Blanton, B., Abd Elal, A. I., Alabi, N., Barnes, J., Biggerstaff, M., Brammer, L., Budd, A. P., Burns, E., Cummings, C. N., Garg, S., Kondor, R., Gubareva, L., Kniss, K., Nyanseor, S., O’Halloran, A., Rolfes, M., Sessions, W., Dugan, V. G., Fry, A. M., Wentworth, D. E., Stevens, J., Jernigan D. 2019. Update: Influenza Activity in the United States During the 2018–19 Season and Composition of the 2019–20 Influenza Vaccine. MMWR. 68:544-51.

UPDATED 8 February 2020