Enhanced replication of a contemporary avian influenza A H9N2 virus in human respiratory organoids

H9N2 is currently the second most common avian influenza A virus subtype infecting humans. Monitoring viral phenotypic and genotypic adaptation to humans is crucial for risk assessment. Here, we compared the replication of an H9N2 human isolate collected in 2024 (A/HK/2346/2024) to a human isolate collected in 1999 (A/HK/1073/1999). In Madin Darby canine kidney (MDCK) cells, A/HK/2346/2024 and A/HK/1073/1999 replicated to 8 and 5 log10 plaque-forming units (PFU) per ml, respectively. In both human nasal and lung organoids, A/HK/2346/2024 replicated to 6 log10 PFU/ml, but A/HK/1073/1999 failed to replicate in either organoid. The infection rates of both ciliated and non-ciliated cells and the ratios of infected 2,6/2,3 cells were higher for A/HK/2346/2024 than A/HK/1073/1999. Apart from the mammalian adaptive substitutions that were present in the nasopharyngeal specimen collected on day 1 post-symptom onset (pso) (HA-D183N/D190 T/Q192R/Q226L; NA-del62-64; PB2-A588V/K702R; PB1-I368V; PA-K356R/S409N; M1-R95K), the mammalian-adaptive substitution PB2-D253N emerged de novo on day 7 pso. Analysis of all human (n = 96) and avian influenza (n = 14,762) H9N2 deposited at GISAID showed the dominance of several human-adaptive substitutions in H9N2 strains collected from humans in recent years. In summary, we demonstrated that a recent H9N2 virus is more adapted to humans, and is able to replicate to high titres in both upper and lower human respiratory tract which may confer higher person-to-person transmissibility and virulence. Our study underscores the importance of human organoid-based phenotypic monitoring and inter/intrahost genotypic monitoring for assessing the zoonotic risk of avian influenza viruses.

H9N2是目前第二大常见的可感染人类的甲型禽流感病毒亚型。监测病毒对人类宿主的表型与基因型适应性，对于风险评估至关重要。本研究对比了2024年分离的人类H9N2毒株（A/HK/2346/2024）与1999年分离的人类H9N2毒株（A/HK/1073/1999）的复制能力。在马-达二氏犬肾（Madin Darby canine kidney, MDCK）细胞中，A/HK/2346/2024与A/HK/1073/1999的病毒滴度分别达到每毫升8和5 log10空斑形成单位（plaque-forming units, PFU）。在人类鼻腔与肺类器官中，A/HK/2346/2024的复制滴度可达6 log10 PFU/ml，而A/HK/1073/1999在两类类器官中均无法实现复制。相较于A/HK/1073/1999，A/HK/2346/2024对纤毛细胞与非纤毛细胞的感染率，以及受感染细胞中2,6/2,3唾液酸受体的比例均更高。除症状出现后第1天（post-symptom onset, pso）鼻咽标本中携带的哺乳动物适应性突变位点（HA-D183N/D190T/Q192R/Q226L；NA-del62-64；PB2-A588V/K702R；PB1-I368V；PA-K356R/S409N；M1-R95K）外，哺乳动物适应性突变PB2-D253N在症状出现后第7天新发出现。对存放在全球共享禽流感数据倡议组织（Global Initiative on Sharing All Influenza Data, GISAID）的所有人类H9N2毒株（n=96）与禽流感H9N2毒株（n=14762）的分析显示，近年来从人类体内分离的H9N2毒株中，多个人类适应性突变位点占据主导地位。综上，本研究证实近期分离的H9N2病毒对人类的适应性更强，可在人类上下呼吸道中达到较高的病毒滴度，这或许使其具备更强的人际传播能力与致病力。本研究强调了基于人类类器官的表型监测，以及宿主内与宿主间的基因型监测，在评估禽流感病毒人畜共患风险中的重要性。