人鼻粘膜类器官冠状病毒感染模型可用于抗病毒药物的筛选和评价

Development of a new platform for testing antiviral drugs using coronavirus-infected human nasal mucosa organoids

目的利用人鼻粘膜类器官建立冠状病毒(CoV)感染模型,并用于药物抗病毒作用体外评价,探讨人类鼻粘膜类器官病毒感染模型用于病毒研究和抗病毒药物开发的可行性。方法使用人源鼻粘膜类器官,测试其对SARS-CoV-2和HCoV-OC43假病毒的易感性。在P3实验室中,评估鼻粘膜类器官对SARS-CoV-2原始株及4种变异株的感染情况,优化感染条件。通过荧光定量PCR检测不同时间点培养基中病毒含量,并利用免疫荧光法定位新冠病毒衣壳蛋白,以确定病毒感染细胞的类型。在优化后的鼻粘膜病毒感染模型中,分析已知具有抑制新冠病毒作用的化合物,如卡莫司他和佛手柑素,单因素方差分析推断其在类器官中的抑制效果并预测其机制。结果通过培养体系和感染条件的优化,使用SARS-CoV-2不同亚型感染鼻粘膜类器官,结果显示,2~24 h受感染鼻粘膜类器官上清液中的病毒载量明显增加,鼻粘膜类器官可被SARS-CoV-2,HCoV-OC43假病毒感染(P<0.001)。真病毒感染实验证实,SARS-CoV-2原始株及4种变异株,均能稳定感染鼻粘膜类器官,证实病毒感染模型的建立。卡莫司他和佛手柑素,在鼻粘膜类器官中均剂量依赖性抗病毒作用(P<0.0001)。结论人鼻粘膜类器官能够作为新冠病毒感染模型,用于抗病毒药物筛选和评价,特别是经鼻腔给药方式的提供了理想的体外研究模型。

Objective To establish a coronavirus(CoV)infection model using human nasal mucosa organoids for testing antiviral drugs and evaluate the feasibility of using human nasal mucosa organoids with viral infection as platforms for viral research and antiviral drug development.Methods Human nasal mucosa organoids were tested for susceptibility to SARS-CoV-2 and HCoV-OC43 pseudoviruses.In a P3 laboratory,nasal mucosa organoids were infected with the original strain of SARS-CoV-2 and 4 variant strains,and the infection conditions were optimized.The viral loads in the culture supernatants were measured at different time points using RT-qPCR,and immunofluorescence assay was employed to localize SARS-CoV-2 nucleocapsid protein to determine the type of the infected cells.In the optimized nasal mucosa viral infection model,the antiviral effects of camostat and bergamot extract(which were known to inhibit SARS-CoV-2)were tested and the underlying molecular mechanisms were explored.Results In the optimized nasal mucosa organoid models infected with SARS-CoV-2 and HCoV-OC43 pseudoviruses,the viral load in the culture supernatants increased significantly during the period of 2 to 24 h following the infection,which confirmed infection of the organoids by both of the pseudoviruses.The nasal mucosa organoids could be stably infected by the original SARS-CoV-2 strain and its 4 variant strains,validating successful establishment of the viral infection model,in which both camostat and bergamot extract exhibited dose-dependent antiviral effects.Conclusions Human nasal mucosa organoids with SARS-CoV-2 infection can serve as platforms for screening and testing antiviral drugs,particularly those intended for nasal administration.