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蝙蝠宿主中新病毒发现及蝙蝠冠状病毒HKU9受体的探索

Novel Virus Discovery in Bat and the Exploration of Receptor of Bat Coronavirus HKU9

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【Author in Chinese】 黄灿平

【Supervisor】 高福舒跃龙

【Author's Information】 中国疾病预防控制中心, 免疫学, 2017, 博士

【Abstract in Chinese】 传染病的暴发(outbreak)和流行(pandemic)严重威胁着人类的健康和生存。在全球化(globalization)和工业化(industralization)背景下,疾病的传播和流行异常迅速。而新病原出现的速度也似乎超过了过去的任何时期。野生动物在人兽共患病(zoonosis)的发生、传播和流行中发挥重要作用。对野生动物所携带病原进行检测、分离与鉴定以及论证其与疾病的相关性,成为当前传染病研究的一个新的热点。随着技术的发展,现代分子生物学方法在病原的发现和鉴定中发挥越来越重要的作用。以宏基因组学(metagenomics)和新一代测序技术(next-generation seqeuncing, NGS)等为代表的分子生物学方法能够从样本中直接分析DNA或RNA遗传物质,在病原鉴定中非常适用。2002年和2012年分别暴发的严重急性呼吸综合征冠状病毒(SARS-CoV)和中东呼吸综合征冠状病毒(MERS-CoV),促使广大研究者密切关注冠状病毒的生物多样性、基因组学和跨物种传播的可能性,尤其是来源于蝙蝠(第二大哺乳动物群体)的冠状病毒。目前已知蝙蝠携带有多种多样的病原体,因此在新发人畜共患病和跨物种传播疾病的监测中,蝙蝠是优先关注的对象。本研究通过泛冠状病毒RT-PCR筛选(pan-coronaviral screening)和新一代测序技术,从棕果蝠(Rousettus leschenaulti)的直肠拭子样本中鉴定出一种新型蝙蝠冠状病毒,将其命名为果蝠冠状病毒GCCDC1 (Rousettus bat coronavirus GCCDC1,Ro-BatCoV GCCDC1)。Ro-BatCoV GCCDC1 在基因组结构和组成上与果蝠冠状病毒HKU9 (Ro-BatCoV HKU9)类似,但是序列和进化分析表明Ro-BatCoV GCCDC1是一种新型蝙蝠冠状病毒。更为引人注意的是,在病毒基因组的3’末端整合有一个独特的基因——p10基因,这个基因在所有已知的冠状病毒中都找不到同源序列,序列和进化分析表明可能来源于一个古老的蝙蝠正呼肠孤病毒。亚基因组mRNA和细胞水平试验表明Ro-BatCoV GCCDC1的p10基因与正呼肠孤病毒的p10基因一样是一个功能基因。在病毒复制周期中,p10基因可能能够促进病毒在细胞与细胞之间转移。这株病毒的发现是单股正链RNA病毒与双股分节段的RNA病毒之间跨科重组的第一次报道。对该冠状病毒的进一步研究能够深入了解病毒间异源重组的机制。蝙蝠冠状病毒HKU9 (Ro-BatCoV HKU9)是一种重要的Beta冠状病毒,系统进化上与MERS-CoV隶属于同一个属。对蝙蝠进行监测的数据表明BatCoV HKU9在蝙蝠群体中广泛流行,因此有必要对这个病毒跨越种间屏障的潜在可能性进行研究。冠状病毒spike蛋白中的受体结合结构域(Receptor binding domain,RBD)识别宿主受体以介导病毒侵入,因此是决定病毒趋向性和传播能力的关键因子。本研究中,通过一系列生物物理和晶体学方法对BatCoV HKU9假定的Spike蛋白RBD进行了研究。表面等离子体共振结果表明HKU9-RBD既不结合SARS-CoV 的受体血管紧张素转化酶 2 (Angiotensin-Converting Enzyme 2,ACE2),也不结合MERS-CoV的受体CD26。我们进一步解析了 HKU9-RBD的分子结构,分析显示该分子结构由一个核心和一个外部亚结构域组成。核心亚结构域的折叠形式与其他Beta冠状病毒RBD的相似,而外部亚结构域在结构上具有独特的特点,仅由一个单一的螺旋组成,这一结构解释了 HKU9-RBD不与ACE2和CD26结合的原因。通过比较目前已知的所有Beta冠状病毒RBD结构,我们进一步提出同源的亚结构域插入结合模式,核心亚结构域和外部亚结构域通过这一模式锚定在一起。通过对HKU9-RBD的结构解析及其与其他Beta冠状病毒的RBD比较,对于阐明Beta冠状病毒受体结合特性及进化规律具有重要参考价值,对于评价HKU9病毒的跨种传播潜力具有重要提示作用。

【Abstract】 The outbreak and pandemic of infectious diseases have presented as greatest threat to public health. Under the background of globalization and industralization,pathogens quickly spread all over the world wherever they outbreak. Meanwhile, in recent decades, the rate of occurrence of novel pathogens also seems to exceed any other time in the past.Wildlife plays an important role in the emerging and spreading of zoonoses.Current researches in the public health community give rise more concerns on the detection, isolation and identification of pathogens that wild animals harbor, and the relationship with diseases.The modern molecular biology methods have played a vital role in the clinical detection and identification of pathogens. Metagenomics and next-generation sequencing (NGS) focus on the direct analyses of genetic materials (DNA or RNA)from clinical samples. The advances in technologies play an increasingly important role in the rapid detection and identification of pathogens and further for the prevention and control of diseases.The outbreak of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and the Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 aroused immense attention to biodiversity, genomics and the possibility of cross-species transmission of coronavirus, especially the coronaviruses from bats, the second largest group of mammals. Bats are particularly notable in this respect because they are known to harbor a diverse range of pathogens, and are known to be reservoir hosts of human coronavirus 229E and SARS-CoV, and are closely related to MERS-CoV. As a consequence, bats have been prioritized for surveillance of emerging zoonotic diseases.In the current study, we identified a novel coronavirus, provisionally designated Rousettus bat coronavirus GCCDC1 (Ro-BatCoV GCCDC1), in the rectal swab samples from Rousettus leschenaulti bats by using pan-coronavirus RT-PCR and next-generation sequencing. Although the virus is similar to Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9) in genome characteristics, it is sufficiently distinct to be classified as a new species according to the criteria defined by the International Committee of Taxonomy of Viruses (ICTV). More striking was that Ro-BatCoV GCCDC1 contained a unique gene integrated into the 3’-end of the genome that has no homologs in any known coronavirus, but which sequence and phylogeny analyses indicated most likely originated from the p10 gene of a bat orthoreovirus.Subgenomic mRNA and cellular-level findings demonstrated that the p10 gene is functional and may induce the formation of cell syncytia. This is the first report of heterologous inter-family recombination between a single-stranded, positive-sense RNA virus and a double-stranded segmented RNA virus, and as such provides insights into the fundamental mechanisms of viral evolution.Bat coronavirus (BatCoV) HKU9 is an important betacoronavirus (BetaCoV)that is phylogenetically affiliated to the same genus as MERS-CoV. The bat-surveillance data indicated that BatCoV HKU9 has been widely spreading and circulating in bats. This highlights the necessity of characterizing the virus for its potential of cross-species transmision. The receptor binding domain (RBD) of the coronavirus spike (S) recognizes host receptors to mediate virus entry and is therefore a key factor determining the viral tropism and transmission capacity. In this study, the putative S RBD of BatCoV HKU9 (HKU9-RBD), which is homologous to other BetaCoV RBDs that have been structurally and functionally defined, was characterized via a series of biophysical and crystallographic methods. By surface plasmon resonance, we demonstrated that HKU9-RBD binds to neither the SARS-CoV receptor of ACE2 (Angiotensin-Converting Enzyme 2) nor the MERS-CoV receptor of CD26. We further solved the atomic structure of HKU9-RBD,which is expectedly composed of a core and an external subdomain. The core subdomain fold resembles those of other BetaCoV RBDs; whereas the external subdomain is structurally unique with a single helix, explaining the inertness of HKU9-RBD to react with either ACE2 or CD26. Via comparison of thus-far available RBD structures, we further proposed a homologous inter-subdomain binding mode in BetaCoV RBDs that anchors the core subdomain to the external subdomain. The revealed RBD features would shed light on the BetaCoV evolution route.

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