【作者】 耿合员；

【导师】 谭文杰；

【作者基本信息】 中国疾病预防控制中心 ， 病原生物学， 2014， 博士

【摘要】 人冠状病毒NL63(Human coronavirus NL63, HCoV-NL63)2004年首次从荷兰被分离鉴定。分子流行病学研究表明,该病毒在多个国家和地区流行,呈全球性分布。该病毒主要感染婴幼儿及免疫功能低下或缺陷的成人,既能感染上呼吸道,引起发热、咳嗽、喉炎等普通感冒症状,又能感染下呼吸道,引起支气管炎、肺炎等急性呼吸道症状。也有研究报道,该病毒感染与川崎病的发生相关。HCoV-NL63是目前已知重要的六种人冠状病毒之一。HCoV-NL63全基因组序列信息截止到2012年5月仅有五株,即3株荷兰株,2株美国株。而该病毒国内株全基因组序列信息、分子结构特征目前还没有相关报道。本研究的首要目的是获得NL63国内株全长基因组序列信息,阐明其分子结构特征,为国内分子流行病学研究提供参考。对NL63国内株遗传变异和系统发生作系统分析,进一步明确HCoV-NL63的基因型以及国内株所在的亚型。另一方面,研究证明HCoV-NL63能够利用与SARS-CoV相同的受体-血管紧张素转换酶2(Angiotensin converting enzyme2, ACE2)入侵宿主细胞。尽管受体相同,但是两者引起的病理反应却有很大差异。本研究拟以NL63病毒作为模式病毒,以HCoV-NL63国内株全基因组序列为基础,构建其全长cDNA的感染性克隆,为深入研究病毒基因组的结构和功能、复制表达调控机理、病毒与宿主的相互作用、致病性等奠定基础。本课题主要研究结果如下：第一,对来自北京儿童医院的32份NL63阳性样本分别进行了总RNA的提取和定量,筛选出病毒拷贝数较高两份样本(编号分别为CBJ037和CBJ123),然后以荷兰株(NL63_Amsterdam)为参考,设计了包含重叠区域、覆盖全长基因组序列的18对引物,通过对病毒RNA提取、RT-PCR、克隆测序并结合3’/5’-RACE技术获得了两株NL63国内株的全基因组序列,阐明了国内株基因组分子结构特征。结合NL63国内株和其他毒株的系统发生分析表明,NL63病毒目前可分为A、B、C、D四个基因型,而国内株处在一个新的基因型,即D型。Bootscan分析表明,国内株与荷兰株和美国株在进化过程中存在着基因重组。第二,在体外通过融合PCR、体外连接的方式获得了NL63国内株全长的cDNA分子。经过体外转录、电转染宿主细胞的方法获得了NL63国内株全长cDNA的感染性克隆。病毒拯救株感染后第7天能够观察到明显的CPE效应,该病毒拯救株命名为ic-NL63。Western blot和IFA实验表明,ic-NL63在宿主细胞内进行了复制和表达。将病毒拯救株连续传代至20代(P20),分别提取P5、P10、P15和P20代的病毒基因组,对引入的分子Marker进行鉴定。结果表明,该分子标记在病毒拯救株传代过程中具有遗传稳定性。第三,为研究ORF3对病毒功能的影响,对其进行了置换,替换为绿色荧光蛋白基因(GFP)。按照同样的方法将带有GFP标签的全长cDNA体外转录本转染宿主细胞,转染后48h即能观察到绿色荧光蛋白的表达。对该重组病毒拯救株连续传代培养,能够观察到CPE效应。Western blot和IFA实验同样表明该重组病毒拯救株在宿主细胞内获得复制和表达,将该重组病毒拯救株命名为ic-NL63-gfp。一步生长曲线实验表明,病毒拯救株ic-NL63和重组病毒拯救株ic-NL63-gfb病毒滴度在感染细胞后第8d达到峰值,分别为105.5TCID50/mL和105TCID50/mL,两者在生长动力学上无显著差异。而荷兰株(NL63_Amsterdam I)病毒滴度在感染后第7d即达到峰值,为106.5TCID50/mL。ic-NL63和ic-NL63-gfp与荷兰株相比在病毒滴度达到峰值的时间上明显滞后且低一个数量级,在生长动力学上存在显著性差异。本研究首次获得了两株HCoV-NL63中国株全基因组序列信息,阐明了其分子结构特征,对其遗传进化作了系统分析,明确了国内株处在一个新的基因型(D型),为NL63国内分子流行病学研究提供参考。构建了人冠状病毒NL63国内株全长cDNA的感染性克隆。通过对ORF3的置换,获得了带GFP标签的重组病毒拯救株,并证明ORF3在细胞培养中为病毒复制所非必需。这为进一步研究NL63病毒基因组的结构和功能、病毒与宿主的相互作用、病毒的致病性以及抗病毒药物筛选等奠定了基础。更多还原

【Abstract】 Human coronavirus NL63(HCoV-NL63) was first identified in Netherlands in2004. Epidemiological studies have shown that this virus is globally distributed. HCoV-NL63mainly infects infants and immunocompromised adults, causing both upper and lower respiratory tract diseases. Clinical symptoms usually manifest as the common cold such as fever, cough and croup. However, it might also cause bronchiolitis, pneumonia even resulting in acute respiratory distress symptoms in some cases. HCoV-NL63is known as one of the six important human coronaviruses.Update to December of2012, there are only five complete genome sequences available in GenBank:three from the Netherlands and two from America. No information is available on the complete genome sequences and phylogenetic analysis of HCoV-NL63derived from China. In this study, our first aim is to sequence and characterize the complete genome sequence of HCoV-NL63derived from China. This may pay a way for molecular epidemiological studies of HCoV-NL63in China.On the other hand, studies have demonstrated that HCoV-NL63share the same receptor (angiotensin converting enzyme2, ACE2) with SARS-CoV for cell entry. However, the clinical manifestations of both viruses are different. We plan to construct the full-length infectious cDNA clone based on the complete genome sequence of HCoV-NL63strains in this study, which might lay a foundation for further study of virus gene functions, replication and virus-host interactions of HCoV-NL63derived from Chinese patients. Furthermore, this reverse genetics platform might be tool or model to characterize the pathogen biology of SARS-CoV infection.The main results of this study are summarized as follows:Firstly, RNAs from32HCoV-NL63positive specimens collected from Beijing Children’s hospital were extracted and quantified respectively, and two positive specimens with high copies were selected for clone and sequencing of full-length genome. Total18pairs of primers were designed according to NL63_Amsterdam reference strains, which covered the complete genome of NL63. By RT-PCR together with3’end and5’end of rapid amplification of cDNA ends (3’/5’RACE), two complete full-length genome sequences were obtained directly from clinical NL63specimens. The genomic structure and phylogenetic analysis of these domestic NL63strains was also characterized. Systematic phylogenetic analysis demonstrated that all known sequences of HCoV-NL63can be genotyping as A, B, C and D, while two domestic HCoV-NL63strains cloned in this study were genotyped as novel group, genotype D. Bootscan analysis indicated that recombination with other strains of HCoV-NL63might be occured during virus circulation and evolution of Chinese HCoV-NL63strains.Secondly, the complete full-length cDNA of NL63domestic strains was assembled by strategy of overlapping PCR and in vitro ligation. The recombined virus, which named as ic-NL63, was successfully rescued after in vitro transcription and electronic transfection of viral mRNAs based on the full-length infectious cDNA clone. The CPE of rescued virus was significantly evident at8d.p.i. Western blot and IFA assays also demostrated that N protein was expressed in infected cells. The introduced molecular marker was identified and genetic stability of rescued ic-NL63during rescued virus propogation was also confirmed.Thirdly, the open reading frame3(ORF3) of HCoV-NL63was replaced with heterologous green fluorescent protein (GFP) for ic-NL63by molecular cloning, which was named as ic-NL63-gfp. And we demonstrated that the ORF3was not essential for the replication of NL63virus in vitro. The growth kinetics of both ic-NL63and ic-NL63-gfp were similar and reached peak at titirs of105/TCID50/mL8d.p.i., which was lower and delayed compared to reference HCoV-NL63strains (NL63_Amsterdam I).In Summary, two full-length genome were cloned and sequenced directly from clinical specimens of Chinese patients. The genomic structure and phylogenetic analysis of these HCoV-NL63strains was also characterized. Systematic phylogenetic analysis demonstrated that NL63virus can be genotyping as A, B, C and D. While domestic strains were formed as a novel group as genotype D. The full-length infectious cDNA clone was successfully assembled and ic-NL63virus was rescued by in vitro transcription and electronic transfection. Furthermore, a novel reporter virus, ic-NL63-gfp in which ORF3was substituted with GFP was also successfully assembled and developed. Our data demonstrated that the ORF3was not essential for the replication of NL63virus in vitro, which might provide a novel good platform for further study of gene functions, replication and pathogenesis of HCoVs.更多还原