Influenza A variants with reduced susceptibility to baloxavir isolated from Japanese patients are fit and transmit through respiratory droplets

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Abstract

Here we report the isolation of the influenza A/H1N1 2009 pandemic (A/H1N1pdm) and A/H3N2 viruses carrying an I38T mutation in the polymerase acidic protein—a mutation that confers reduced susceptibility to baloxavir marboxil—from patients before and after treatment with baloxavir marboxil in Japan. These variants showed replicative abilities and pathogenicity that is similar to those of wild-type isolates in hamsters; they also transmitted efficiently between ferrets by respiratory droplets.

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Fig. 1: In vitro characterization of four pairs of influenza A/H1N1pdm and A/H3N2 viruses with and without the PA-I38T substitution.
Fig. 2: In vivo characterization of four pairs of influenza A/H1N1pdm and A/H3N2 viruses with and without the PA-I38T substitution.

Data availability

Numerical source data that underlie the graphs shown in Figs. 1 and 2, and Extended Data Figs. 2, 3, 4 and 5 are provided with the paper. The data that support the findings of this study are available from the corresponding authors on reasonable request. Samples were deposited in the Sequence Read Archive of the NCBI, under project accession number PRJNA573567.

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Acknowledgements

We thank S. Watson for scientific editing; Y. Sato and M. Ujie for technical assistance; S. Kurosawa for discussions. This research was supported by Leading Advanced Projects for medical innovation (LEAP) from the Japan Agency for Medical Research and Development (AMED; JP18am001007); Grants-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Science, Sports and Technology (MEXT) of Japan (16H06429, 16K21723 and 16H06434); the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) from AMED (JP19fm0108006); a Research Program on Emerging and Re-emerging Infectious Diseases from AMED (JP19fk0108031, JP19fk0108056, JP19fk0108058 and JP19fk0108066); and the NIAID-funded Center for Research on Influenza Pathogenesis (CRIP, HHSN272201400008C).

Author information

M.Imai, K.I.-H., M.Kiso, J.M., K.Takada and M.Ito performed the in vivo experiments. A.Y. and K.Takada performed the in vitro experiments. N.N., K.Takahashi and H.Hasegawa performed histopathological analysis. Y.S.-T. performed virus isolation. T.J.S.L., J.D., Z.K., D.K. and H.v.B. performed deep-sequencing analysis and interpretation. A.T., H.Hagiwara, N.I., H.K., T.N., N.W., M.Koga, E.A. and D.J. provided clinical samples. M.Imai, M.Y., Y.S.-T., K.I.-H., M.Kiso, N.N., T.J.S.L., H.Hasegawa and Y.K. planned the experiments and/or analysed the data. M.Imai, M.Y., Y.S.-T., K.I.-H., M.Kiso, T.J.S.L. and Y.K. wrote the manuscript.

Correspondence to Masaki Imai or Yoshihiro Kawaoka.

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Competing interests

M.Imai, Y.S.-T., K.I.-H., M.Kiso, J.M., A.Y., K.Takada, M.Ito, N.N., K.Takahashi, T.J.S.L., J.D., Z.K., D.K., H.V.B., A.T., H.Hagiwara, N.I., H.K., T.N., N.W., M.Koga, E.A., D.J. and H.Hasegawa have no competing interests. M.Y. has received a speaker’s honoraria from Daiichi Sankyo. Y.K. has received a speaker’s honoraria from Toyama Chemical and Astellas, grant support from Daiichi Sankyo Pharmaceutical, Toyama Chemical, Shionogi & Co and Kyoritsu Seiyaku, and is a founder of FluGen.

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Extended data

Extended Data Fig. 1 Detection of influenza A/H1pdm and A/H3 virus variants carrying a mutation at amino acid position 38 of PA in patients.

a, b, Respiratory tract specimens were obtained from pediatric (aged 0 to 15 years) and adult (16 years or older) patients before and after baloxavir marboxil therapy and from untreated patients with influenza A/H1pdm (a) and A/H3 (b) virus infections in Japan during the 2018–2019 influenza season. The nucleotide sequences of the PA genes of the viruses in the specimens were determined by means of Sanger sequencing. c, Timeline of the clinical course of the two patients (GR125 and GR142) with influenza A/H3N2 virus infection.

Extended Data Fig. 2 Body weight changes and histopathological findings in infected Syrian hamsters.

a, Syrian hamsters were intranasally inoculated with 106 PFU of WT-KK001-I38 (H1N1pdm), MUT-KK001-I38T (H1N1pdm), WT-KK003-I38 (H1N1pdm), or MUT-KK003-I38T (H1N1pdm), or MEM containing 0.3% BSA (mock). Body weights of virus-infected (n=3) and mock-infected animals (n=4) were monitored daily for 6 days. Data are presented as the mean percentages of the starting weight ± SD. P-values were calculated by using pairwise comparisons after a linear mixed model analysis (*P < 0.05; **P < 0.01). Asterisks next to data points depict statistically significant differences between mutant and wild-type viruses. See Methods for more details regarding the statistical analysis. b, Representative pathological images of lungs infected with WT-KK001-I38 (H1N1pdm, n=2), MUT-KK001-I38T (H1N1pdm, n=2), WT-KK003-I38 (H1N1pdm, n=2), MUT-KK003-I38T (H1N1pdm, n=2), WT- KK015-I38 (H3N2, n=2), MUT-KK015-I38T (H3N2, n=2), WT-GR117-I38 (H3N2, n=2), or MUT-GR117-I38T (H3N2, n=2) on Day 6 post-infection. Left panels, hematoxylin and eosin (HE) staining. Scale bars, 200 μm. Right panels, immunohistochemistry (IHC) for influenza viral antigen detection. Scale bars, 100 μm. Source data

Extended Data Fig. 3 Virus titers in respiratory organs of infected mice.

Three mice per group were intranasally inoculated with 106 PFU of WT-KK001-I38 (H1N1pdm), MUT-KK001-I38T (H1N1pdm), WT-KK003-I38 (H1N1pdm), or MUT-KK003- I38T (H1N1pdm). Three mice per group were euthanized on Days 3 and 6 post-infection for virus titration. Virus titers in the nasal turbinates and lungs were determined by use of a plaque assay on hCK cells. Vertical bars show the mean. Points indicate data from individual mice. Source data

Extended Data Fig. 4 Virulence in mice.

Five mice per group were intranasally inoculated with 106 PFU of WT-KK001-I38 (H1N1pdm), MUT-KK001-I38T (H1N1pdm), WT-KK003-I38 (H1N1pdm), or MUT-KK003- I38T (H1N1pdm), or MEM containing 0.3% BSA (mock). Body weights (a) and survival (b) were monitored daily for 11 days. The values for body weights are presented as the mean percentages of the starting weight ± SD. P-values were calculated by using pairwise comparisons after a linear mixed model analysis (*P < 0.05; **P < 0.01). Asterisks next to data points depict statistically significant differences between mutant and wild-type viruses. See Methods for more details regarding the statistical analysis. Source data

Extended Data Fig. 5 Body weight changes of inoculated and exposed ferrets during the transmission study.

Ferrets were intranasally inoculated with 106 PFU of WT- KK001-I38 (H1N1pdm, n=3), MUT-KK001-I38T (H1N1pdm, n=3), WT-GR117-I38 (H3N2, n=5), or MUT-GR117-I38T (H3N2, n=5). One day later, naïve ferrets (exposed ferrets, n=3, n= 3, n=5, and n=5 for WT-KK001-I38, MUT-KK001- I38T, WT-GR117-I38, and MUT-GR117-I38T, respectively) were each placed in a cage adjacent to an infected ferret. a, Body weights of inoculated ferrets were measured on Days 0, 1, 3, 5, 7, 9, and 11 post-infection. b, Body weights of exposed ferrets were measured on Days 0, 3, 5, 7, 9, 11, and 13 post- exposure. Data are presented as the mean percentages of the starting weight ± SD. Source data

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Imai, M., Yamashita, M., Sakai-Tagawa, Y. et al. Influenza A variants with reduced susceptibility to baloxavir isolated from Japanese patients are fit and transmit through respiratory droplets. Nat Microbiol (2019) doi:10.1038/s41564-019-0609-0

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