Emergence of the novel PA-D27G mutation conferring reduced baloxavir susceptibility in influenza A viruses circulating in China, 2018–2025

Seasonal influenza A viruses evolve rapidly, yet the emergence and molecular basis of resistance to the polymerase acidic (PA) inhibitor baloxavir marboxil (BXM), which is widely used in China, remain elusive. To address this, 3938 PA gene sequences were collected from influenza patients across mainland China between 2018 and 2025, from the national surveillance network and GISAID. By screening post-market mutations in the N-terminal domain of PA (PAN) that appeared in at least two samples and at a frequency below 50%, twenty-five single-point mutations were identified and additionally six linked mutations potentially associated with drug pressure. The impact of these mutations on BXM sensitivity was subsequently evaluated. Our analysis revealed the emergence of known mutations associated with reduced BXM sensitivity, including L28P, K34R, E198 K, although their prevalence remained low (2/3850, 0.05%). Notably, we identified a novel substitution, D27G, which conferred an approximately 12.4-fold reduction in BXM susceptibility compared with the wild-type virus and exhibited higher replication fitness than the canonical resistance mutation I38T, as demonstrated in human airway organoids. Molecular dynamics simulations further indicated that PA-D27G attenuates the interaction between PA and baloxavir acid, the active form of BXM. Epidemiological analysis showed that D27G mutation remained rare, being detected in four isolates (4/1247, 0.32%) in mainland China, and at a sporadic prevalence (

季节性甲型流感病毒进化迅速，但在中国广泛使用的聚合酶酸性（PA）抑制剂玛巴洛沙韦（baloxavir marboxil, BXM）的耐药性出现及其分子基础仍不明确。为解决这一问题，本研究于2018年至2025年间，通过国家流感监测网络及全球流感数据共享倡议（GISAID）收集了中国内地流感患者的3938条PA基因序列。通过筛选PA的N端结构域（PAN）中至少出现于2份样本、频率低于50%的上市后突变，共鉴定出25种单点突变，同时发现6种可能与药物压力相关的连锁突变。随后评估了这些突变对BXM敏感性的影响。本研究分析发现了已知的与BXM敏感性降低相关的突变，包括L28P、K34R、E198K，尽管其流行率仍较低（2/3850，0.05%）。值得注意的是，本研究鉴定出一种新型替换突变D27G，相较于野生型病毒，该突变可使BXM易感性降低约12.4倍；且在人类气道类器官实验中，其复制适配性优于经典耐药突变I38T。分子动力学模拟进一步表明，PA-D27G突变会减弱PA与BXM的活性形式巴洛沙韦酸之间的相互作用。流行病学分析显示，D27G突变仍较为罕见，在中国内地仅在4株分离株中被检出（4/1247，0.32%），且呈散在流行态势（