人冠状病毒HKU1在HAE细胞模型中的复制和生物学特性研究

As one of the most important pathogens of humans, human coronavirus HKU1 (HCoV-HKU1) can cause acute respiratory tract illness, especially in Children. But little is known about the replication and characterization of HCoV-HKU1, as all attempts to culture a clinical isolate of HCoV-HKU1 have failed in standard transformed cell cultures. Well differentiated human airway epithelial cell culture system (HAE) is an vitro model of the human airway epithelium. HAE recapitulate the morphological and physiological features of the airway epithelium in vivo and is comprised of ciliated, non-ciliacted mucin-secreting and basal epithelial cells, and it is the only one cell culture system to successfully propagate HCoV-HKU1 in vitro.As there is no HCoV-HKU1 stain isolated in China and little is known about the characterization of HCoV-HKU1 in China, we use HAE system, which has been established in our lab, to culture the clinical isolates of HCoV-HKU1.Now we have gained one strain of HCoV-HKU1. We will continue the isolation of HCoV-HKU1 from clinical material to get more stains of HCoV-HKU1 in China. Meanwhile, we will get the complete genome sequence of HCoV-HKU1 stains and subsequent passage HCoV-HKU1 stains in HAE to know about the basic characterization of HCoV-HKU1, such as the replication kinetics, the cellular localization and so on.Our work will provide basic data about pathogenisis and diagnosis of human coronaviruses.

人冠状病毒是重要的呼吸道感染病原，其中HCoV-HKU1可引起人重症肺炎。因常规细胞培养技术难于获得HCoV-HKU1分离株，国际上对不同HCoV-HKU1临床感染毒株的复制及生物学特性的研究甚少。分化良好的人呼吸道上皮细胞（HAE）是冠状病毒的天然宿主细胞，也是目前世界上唯一能够分离培养HCoV-HKU1的细胞。针对我国尚无HCoV-HKU1毒株和缺乏HCoV-HKU1生物学特性研究的现状，本课题组将前期已经建立的HAE培养体系应用于急性呼吸道感染（特别是重症肺炎）患者中HCoV-HKU1的分离鉴定，获得了一株HCoV-HKU1临床分离株。我们拟将进一步扩大临床样本中HCoV-HKU1的分离工作，研究 HCoV-HKU1在HAE模型中的复制特点；通过序列分析研究其遗传变异规律；并通过细胞生物学研究方法，阐明其亚细胞定位和细胞内转运等生物学特性，为人冠状病毒致病性研究及科学诊治奠定基础。

背景：HCoV-HKU1可引起人重症肺炎，因常规细胞培养难于获得HCoV-HKU1分离株，国际上对HCoV-HKU1的复制及生物学特征研究甚少。.内容：建立了HCoV-HKU1的分离培养的细胞模型HAE，并从重症呼吸道感染患儿呼吸道样本中分离获得了中国大陆第一株HCoV-HKU1 BJ-01毒株（GenBank accession No. KT779555）。项目系统研究了HCoV-HKU1 BJ-01的基因组特点、遗传稳定性、形态学及细胞内发生等生物学特性，并重点研究了HCoV-HKU1 BJ-01在HAE的复制。.结果：HCoV-HKU1 BJ-01属A基因亚型，序列与美国2009年流行株相近，在HAE细胞中具有遗传稳定性；病毒具有典型冠状病毒形态，在纤毛细胞中形成包涵体结构；测定HCoV-HKU1 BJ-01的复制动力曲线表明：103copies/ml的HCoV-HKU1 BJ-01感染HAE 96h，病毒释放达到高峰；在正常HAE模型及IL-13诱导的炎性模型中的病毒复制没有显著的改变；以相同剂量（105copies/ml） HCoV-OC43共感染HCoV-HKU1 BJ-01，同时感染与单独感染复制动力没有显著差别；相差24h感染，两种病毒出现了相互干扰现象，即先感染的病毒抑制了后感染病毒的复制，与单独感染相比均抑制了10倍左右；此外项目扩展研究了宿主对HCoV-HKU1 BJ-01复制的影响因素，结合非编码RNA及小RNA测序与抗体芯片的结果，筛查来自宿主的影响病毒复制的关键分子。.意义：本项目获得一株具有自主知识产权的HCoV-HKU1 BJ-01毒株，对该毒株序列、形态、遗传稳定性等多方面进行了鉴定，特别是分别从病毒自身、其他病毒干扰、宿主三方面深入探讨了HCoV-HKU1 BJ-01复制及影响因素，是国际上为数不多的关于HCoV-HKU1复制的基础研究。