应用气-液两相呼吸道类器官从中国临床住院患儿中分离鉴定人副流感病毒1/2/3  

Isolation1andd Identification of Human Parainfluenza Virus 1/2/3 from Hospitalized Children in China Using An Air-liquid Interface Human Airway Epithelial Organoid

作　　者：余玙璠 吴伟波[3] 牛培华 宋敬东[1,2] 霍威邦 胡月超 刘俊彤 魏岚 邓瑶[1,2] 杨扬[3] 朱娜[1,2] 谭文杰[1,2] YU Yufan;WU Weibo;NIU Peihua;SONG Jingdong;HUO Weibang;HU Yuechao;LIU Juntong;WEI Lan;DENG Yao;YANG Yang;ZHU Na;TAN Wenjiel(National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 100052,China;Key Laboratory of Medical Viruses and Viral Diseases,National Institute for Viral Disease Control and Prevention,Chinese Center for Diseases Control and Prevention,Beijing 102206,China;Shenzhen Key Laboratory of Pathogenic Microbes and Immunity,National Center for Clinical Medicine of Infectious Diseases Shenzhen Third People'sHospital,Shenzhen 518112,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,传染病溯源预警和智能决策全国重点实验室,北京100052 [2]中国疾病预防控制中心病毒病预防控制所,国家卫生健康委医学病毒和病毒病重点实验室,北京102206 [3]深圳市病原微生物与免疫重点实验室,国家感染性疾病临床医学研究中心,深圳市第三人民医院,深圳518112

出　　处：《病毒学报》2024年第2期274-282,共9页Chinese Journal of Virology

基　　金：国家自然科学基金(项目号:82072296),题目:新型冠状病毒SARS-CoV-2与人冠状病毒NL63、HKU1在呼吸道上皮类器官模型HAE中的生物学特性比较。

摘　　要：人副流感病毒(Human parainfluenza viruses,hPIVs)是引起人类呼吸道感染的重要病原体之一,目前我国相关临床毒株资源有限,本研究旨在获得近期流行、背景明晰的hPIVs临床分离株。应用原代人呼吸道上皮细胞分化的气-液界面呼吸道上皮类器官(Air-liquid interface human airway epithelial organoid,HAE),从深圳市第三人民医院2016~2017年住院患儿hPIVs阳性咽拭子标本中分离获得hPIV1/2/3,分别命名为hPIV1/C-Tan/SZ01/2016、hPIV2/C-Tan/SZ01/2017、hPIV3/C-Tan/SZ01/2017。通过逆转录荧光定量PCR (Reversetranscriptionquantitative polymerase chain reaction, RT-qPCR)检测病毒核酸扩增、免疫荧光检测病毒蛋白在HAE的表达、负染及超薄切片透射电镜观察病毒形态和细胞定位,并对传代过程中病毒的活性、遗传稳定性和基因组特征进行分析。结果表明基于HAE分离hPIVs可以高效获得hPIVs临床分离株,所获毒株具有典型的病毒形态特征。三种hPIVs在HAE中复制良好且遗传稳定,病毒滴度均可达到约107 TCID50/100μL。对分离获得的hPIV1/2/3临床分离株进行全基因组及HN基因进行序列测定,结果表明:hPIV1全长为15568bp,属于hPIV1 C亚型;hPIV2全长15694bp,属于hPIV2 A3亚型;hPIV3全长15443bp,属于hPIV3 C3亚型,提示3株临床分离株与我国普遍流行的基因型相一致。综上,本研究应用HAE从近期住院患儿临床样本中成功分离获得生物学特性和基因组背景清晰且遗传稳定的hPIV1/2/3毒株。这些临床流行毒株资源的分离鉴定,为hPIVs的感染特点和致病机制的深入研究及抗病毒药物筛选和疫苗研发奠定了基础。Human parainfluenza viruses(hPIVs)are one of the most important pathogens causing human respiratory tract infections.The resources of relevant clinical strains in China are limited.This study aimed to obtain recently prevalent clinically isolated hPIVs with clear background.Primary human respiratory epithelial cells differentiated from human airway epithelial(HAE)organoid with air-liquid interface were applied to isolate hPIV1/2/3 from hPIVs-positive throat swab specimens of hospitalized children in Shenzhen Third People's Hospital from 2016 to 2017,named hPIV1/C-Tan/SZ01/2016,hPIV2/C-Tan/SZ01/2017,and hPIV3/CTan/SZ01/2017,respectively.hPIV1/C-Tan/SZ01/2017 was detected by fluorescence reverse transcription-quantitative polymerase chain reaction(RT-qPCR)to detect viral nucleic acid amplification.Immunofluorescence was employed to measure expression of viral proteins in HAE Transmission electron microscopy(TEM)of negative staining and ultrathin sections was carried out to observe viral morphology and cellular localization.We also analyzed viral activity,genetic stability,and genomic characterization during TEM.The results showed that the isolation of hPIVs based on HAE could efficiently obtain clinical isolates of hPIVs,and the obtained strains had typical viral morphological features.The three hPIVs replicated well and were genetically stable in HAE,and viral titers could reach to 107 TCID50/100μL.The whole genome and HN gene of hPIV1/2/3 clinical isolates were sequenced.hPIV1,with a total length of 15,568 bp,belonged to hPIV1 C isoform.hPIV2,with a total length of 15,694 bp,belonged to the hPIV2 A3 isoform.hPIV3,with a full length of 15443 bp,belonged to hPIV3 C3 isoform.The results suggested that these three clinical isolates were consistent with the genotypes prevalent in China.In conclusion,we applied HAE to successfully isolate hPIV1/2/3 strains with clear biological characteristics and genomic background,as well as genetic stability,from clinical samples of recently hospitalized children.The isolation and

关 键 词：人副流感病毒 病毒分离鉴定 人呼吸道上皮类器官 

分 类 号：R373.1[医药卫生—病原生物学]