Takashita Emi, Yasui Yoshihiro, Ikegaya Asaka, Saka Kyohei, Maeshiro Noriyuki, Morita Hiroko, Nagata Shiho, Fujisaki Seiichiro, Miura Hideka, Kishida Noriko, Nakamura Kazuya, Shirakura Masayuki, Watanabe Shinji, Hasegawa Hideki
Influenza Research Center, National Institute of Infectious Diseases, Japan Institute for Health Security, 4-7-1 Gakuen, Musashimurayama, Tokyo, 208-0011, Japan.
Aichi Prefectural Institute of Public Health, 7-6 Nagare, Tsuji-cho, Kita-ku, Nagoya, Aichi, 462-8576, Japan.
Antiviral Res. 2025 Jul;239:106173. doi: 10.1016/j.antiviral.2025.106173. Epub 2025 May 3.
Baloxavir marboxil, a cap-dependent endonuclease inhibitor, was approved in Japan in 2018 for the treatment and prophylaxis of influenza. Its active form, baloxavir acid, binds to the polymerase acidic (PA) protein endonuclease domain, inhibiting viral RNA cleavage. PA substitutions (e.g., E23K, I38T, E199G) have been associated with reduced susceptibility to baloxavir. During nationwide monitoring in Japan, we identified influenza A(H1N1)pdm09 and A(H3N2) viruses carrying a PA E199K substitution. Database analysis revealed that PA E199K is rare, detected in only 0.01 % of A(H1N1)pdm09 and A(H3N2) viruses. Because its impact on baloxavir susceptibility has not been reported, here, we characterized PA E199K mutant viruses in vitro. Phenotypic analysis showed a 5.0-5.2-fold increase in baloxavir EC values in PA E199K mutants, indicating reduced baloxavir susceptibility similar to PA E199G. However, replication efficiency of PA E199K mutants was significantly lower than wild-type viruses, suggesting impaired viral fitness. Unlike PA E199G, PA E199K introduces charge and steric changes that may further reduce replication capacity. While PA E199G mutants have led to a community cluster, PA E199K has only been detected sporadically, likely due to its greater impairment of viral replication. The PA E199K mutants were susceptible to neuraminidase inhibitors. Given the increasing global use of baloxavir, continuous monitoring of resistance-associated substitutions is essential for public health and clinical management.
巴洛沙韦酯是一种帽依赖性内切核酸酶抑制剂，于2018年在日本获批用于治疗和预防流感。其活性形式巴洛沙韦酸与聚合酶酸性（PA）蛋白的内切核酸酶结构域结合，抑制病毒RNA切割。PA替代（例如E23K、I38T、E199G）与对巴洛沙韦的敏感性降低有关。在日本的全国监测期间，我们鉴定出携带PA E199K替代的甲型流感病毒（H1N1）pdm09和甲型流感病毒（H3N2）。数据库分析显示，PA E199K很罕见，仅在0.01%的甲型流感病毒（H1N1）pdm09和甲型流感病毒（H3N2）中检测到。由于尚未报道其对巴洛沙韦敏感性的影响，在此，我们在体外对PA E199K突变病毒进行了表征。表型分析显示，PA E199K突变体中巴洛沙韦的EC值增加了5.0 - 5.2倍，表明对巴洛沙韦的敏感性降低，类似于PA E199G。然而，PA E199K突变体的复制效率明显低于野生型病毒，表明病毒适应性受损。与PA E199G不同，PA E199K引入了电荷和空间变化，这可能进一步降低复制能力。虽然PA E199G突变体导致了社区聚集，但PA E199K仅偶尔被检测到，可能是由于其对病毒复制的更大损害。PA E199K突变体对神经氨酸酶抑制剂敏感。鉴于巴洛沙韦在全球的使用日益增加，持续监测与耐药相关的替代对于公共卫生和临床管理至关重要。
