Chinese Center for Disease Control and Prevention reported that Wuhan novel coronavirus case study in the New England Journal of Medicine

A Novel Coronavirus from Patients with Pneumonia in China, 2019

EDITORIAL

This paper is the Chinese Center for Disease Control and Prevention, Capital Medical University, Hubei Provincial Disease Prevention and Control Center, Chinese Academy of Sciences, Shandong Academy of Medical Sciences in 2020 January 24 jointly published in the New England Journal of Medicine , corresponding author for the Chinese Center for Disease Control and Prevention viral disease control and prevention director virus emergency technical Center sick Tan Wenjie, Chinese Center for disease control and prevention, the main Rengao Fu , China CDC viral disease control and prevention, the party secretary Wu Guizhen.

Summary

December 2019, a group of patients with pneumonia of unknown causes were found and seafood wholesale market in Wuhan, China related. By samples from patients with pneumonia unbiased sequencing, we discovered a previously unknown coronavirus β. Epithelial cells from the human airway to obtain a 2019-nCoV named novel coronavirus, the coronaviruses Sarbecovirus subgenus forms another clades within Orthocoronavirinae subfamily. Different from MERS-CoV and SARS-CoV, 2019-nCoV can infect human coronavirus family, the seventh member. Enhanced ongoing monitoring and further investigation.

Foreword

Emerging pathogens and reproduction is a global public health challenge. Coronavirus is an enveloped RNA virus , it is widely distributed in humans, other mammals and birds, and cause respiratory, intestinal, liver and nervous system diseases . Currently known six kinds of coronaviruses can cause disease in humans. Four viral -229E, OC43, NL63 and HKU1 are common, usually caused by common cold symptoms in individuals with immunocompetent . The other two viruses - coronavirus (SARS-CoV) severe acute respiratory syndrome coronavirus and the Middle East (MERS-CoV) Respiratory Syndrome originated in zoonoses, and sometimes cause fatal diseases. SARS-CoV in 2002 and 2003 in China's Guangdong cause severe acute respiratory syndrome outbreak pathogen. MERS-CoV is caused by the 2012 outbreak of severe respiratory pathogens in the Middle East. Given the height of the epidemic and widespread of coronavirus, frequent recombination and genomic diversity of genes and their ever-increasing interaction of human and animal behavior, due to frequent cross-species infection and accidental spill caused very novel coronavirus It may periodically occur in humans .

In late December 2019, China Wuhan, Hubei several local health agencies have reported a group of patients with pneumonia of unknown causes, epidemiology show local seafood wholesale market related. December 31, 2019, Chinese Center for Disease Control and Prevention sent a rapid response team, coordinated Hubei Province and Wuhan City health department epidemiological and etiological investigation. Here we report the results of this investigation to determine the source of gathered pneumonia, and describes pneumonia outbreak samples from patients with early disease by the Chinese Center for Disease Control and Prevention detected a new type of coronavirus. We also describe the clinical features of patients with pneumonia two of them.

result

1. The patient is described

Three adult patients with severe pneumonia on December 27, 2019 entered a hospital in Wuhan. 1 patient was 49-year-old female patient 2 was 61-year-old male, a 32-year-old male patient 3. 1 and 2 clinical data available to patients. December 23, 2019 the patient reported no underlying chronic diseases 1, the report fever (temperature of 37 ° C to 38 ° C) and cough with chest discomfort. Four days after the onset, cough and chest discomfort in patients 1 increase, but fever reduction; to make a diagnosis of pneumonia based on computed tomography (CT) scan. 1 Career patients seafood wholesale market retailers. 2 patients originally December 20, 2019 report fever and cough; 7 days after the onset of respiratory distress, and deterioration in the next 2 days (see chest X-ray, FIG. 1 ), then began to mechanical ventilation. Patient 2 is a seafood wholesale market regulars. Patients 1 and 3 patients had recovered, and in 2020, January 16 and discharged. Patient 2 2020 January 9 deaths. Biopsy specimens were not obtained.

1. FIG chest

2. The new coronavirus detection and isolation

2019年12月30日，从武汉金银潭医院收集了3份支气管肺泡灌洗液样本。使用RespiFinderSmart-22kit在这些患者的临床标本中未检测到特异性病原体(包括HCoV-229E、HCoV-NL63、HCoV-OC43和HCoV-HKU1)。从患者的支气管肺泡灌洗液中提取RNA作为模板，利用Illumina和纳米孔测序对基因组进行克隆和测序。从单个样本中获得了超过20,000个病毒测序片段，并且大多数重叠群与beta冠状病毒属B分支的基因组相匹配—其中与之前发表的蝙蝠SARS样冠状病毒（bat-SL-CoVZC45，MG772933.1）的全基因组一致性超过85％。使用实时RT-PCR靶向测定泛β-CoV的共有RdRp区也获得了阳性的结果（尽管对于检测到的样品来说其循环数高于34）。利用人气道上皮细胞以及Vero E6和Huh-7细胞系从临床标本中分离获得病毒毒株。分离得到的病毒命名为2019-nCoV。

为了确定是否可以在2019-nCoV感染的人气道上皮细胞中看到病毒颗粒，每天用光学显微镜检查空白感染和2019-nCoV感染的人气道上皮培养物，并于接种后第6天通过透射电子显微镜观察。感染96小时后在人气道上皮细胞的表面层观察到细胞病变作用。利用光学显微镜在焦点中心可以观察到纤毛跳动缺乏（图2）。直到接种后6天，在Vero E6和Huh-7细胞系中都没有观察到特异性的细胞病变作用。

图2. 人气道上皮细胞接种2019-nCoV后的细胞培养图片

电镜下观察2019-nCoV阴性粒子一般呈球形，但有些呈多边形（见图3）。直径在60 - 140 nm之间。病毒颗粒有明显的棘突，大约9 - 12 nm, 导致病毒呈现了日冕状。在人气道上皮超薄切片中可以发现胞外游离的病毒颗粒和胞质膜泡内充满病毒颗粒的包涵体。这些观察到的形态与冠状病毒科相一致。

图3. 2019-nCoV在透射电子显微镜下图片

为了进一步确定病毒特征，对患者临床样本（肺泡灌洗液）和人类呼吸道上皮细胞分离到的病毒用Illumina和Nanopore 平台进行了测序分析。从这3位患者体内都鉴定到了新型冠状病毒。从肺泡灌洗液中获得了2个接近全长的冠状病毒序列（BetaCoV/Wuhan/IVDC- HB-04/2020, BetaCoV/Wuhan/IVDC-HB-05/2020|EPI_ISL_402121），从一位患者体内分离到的病毒得到了全长基因组序列(BetaCoV/Wuhan/IVDC-HB-01/2020|EPI_ISL_402119)。这3个新型冠状病毒全基因组序列已经提交到GASAID(BetaCoV/Wuhan/IVDC-HB-01/2019, accession ID: EPI_ISL_402119; BetaCoV/Wuhan/IVDC-HB-04/2020, accession ID: EPI_ISL_402120; BetaCoV/Wuhan/IVDC-HB-05/2019, accession ID: EPI_ISL_402121)。与之前报道过的蝙蝠体内分离到的SARS样冠状病毒(bat-SL-CoVZC45, MG772933.1)有86.9%的核酸序列一致性。这3个新型冠状病毒基因组聚类在一起，形成了sarbecovirus亚属内的一个独立分枝。这些基因组具有典型的beta冠状病毒属结构：一个5′非翻译区（UTR）、复制酶复合物（orf1ab）、S基因、E基因、M基因、N基因、3′UTR和几个未定义的非结构蛋白开放读码框。

尽管2019-nCoV与在蝙蝠中检测到的某些β-冠状病毒相似（图4），但它与SARS-CoV和MERS-CoV不同。来自武汉的三种2019-nCoV冠状病毒与两种蝙蝠衍生的SARS样毒株ZC45和ZXC21共同构成了sarbecovirus亚种B系。来自人类的SARS-CoV病毒株和从中国西南地区收集的蝙蝠的SARS样冠状病毒形成了sarbecovirus亚属内的另一个进化枝。由于在2019-nCoV和beta冠状病毒的其他成员之间保守复制酶结构域（ORF 1ab）中的序列一致性小于90％，因此作为武汉病毒性肺炎的可能病原体2019-nCoV是一种属于冠状病毒科sarbecovirus亚属的新型beta冠状病毒。

图4.2019-nCoV和正冠状病毒亚科中其他乙型冠状病毒基因系统发育树分析图

讨论

我们报告了一种新的冠状病毒（2019-nCoV），该病毒已于2019年12月和2020年1月在中国武汉的住院患者中被鉴定出。该病毒的存在证据包括通过全基因组测序在三名患者的支气管肺泡灌洗液中进行鉴定，直接PCR和培养。该冠状病毒可能引起的疾病被称为“新型冠状病毒感染的肺炎”（NCIP）。完整的基因组已提交给GASAID。系统发育分析表明，2019-nCoV属于beta冠状病毒属，其中包括在人，蝙蝠和其他野生动物中发现的冠状病毒（SARS-CoV、蝙蝠SARS状CoV等）。我们报道了病毒的分离以及对其特定细胞病变效应和形态的初步描述。

多年来分子生物学技术已成功用于鉴定传染原。无偏倚的高通量测序是发现病原体的有力工具。下一代测序和生物信息学正在改变我们应对传染病暴发的方式，加深了我们对疾病发生和传播的理解，加速了对病原体的识别并促进了数据共享。我们在本报告中描述了利用分子技术和无偏倚DNA测序来发现导致在中国武汉的三名患者中引起严重肺炎的一种新型的β冠状病毒。

尽管建立人呼吸道上皮细胞培养物是劳动密集型的，但它们似乎是分析人呼吸道病原体的有价值的研究工具。我们的研究表明，通过呼吸道分泌物最初在人气道上皮细胞培养物上增殖，然后用透射电子显微镜和对培养上清进行全基因组测序，成功地用于观察和检测新的人冠状病毒，而这可能是传统方法无法做到的。

我们还需要进一步建立可准确、快速鉴别呼吸道未知病原体的方法。基于在此研究中得到的3条完整基因组，我们设计了几个针对2019-nCoV基因组的ORF1ab、N和E基因区域的特异且敏感的分析方法，可以检测出临床样本中的病毒RNA。引物序列和标准操作程序已与世界卫生组织共享，用于在全球和中国监测和检测2019-nCoV感染。最新的数据显示，中国已有830人检测出2019-nCoV。

虽然我们的研究没有完全遵循科赫法则，但我们的分析提供了2019-nCoV在武汉暴发的证据。我们还需提供有关2019-nCoV引起武汉疫情的其他证据，包括在患者的肺组织中用免疫组化方法检测到2019-nCoV抗原，从同一患者两个时间点的血清标本中检测出可证实血清转化的抗病毒IgM和IgG抗体，以及来自动物（猴子）实验的致病性证据。至关重要的是进行流行病学调查，以确定感染的传播方式、繁殖间隔期和感染的临床症状谱，从而为制定预防、控制和制止2019-nCoV传播的战略提供指导。

Reference

Zhu Na, Zhang Dingyu, Wang Wenling, et al. A Novel Coronavirus from Patients with Pneumonia in China, 2019.NEJM,January 24, 2020 DOI: 10.1056/NEJMoa2001017

撰文：李杰常熟理工学院生物与食品工程学院

责编：刘永鑫中科院遗传发育所

Written on the back

To encourage readers to communicate quickly solve difficult research, we established a "metagenome" professional discussion groups, there are currently already at home and abroad 5000+ frontline researchers added. Participate in discussions, get professional answers, please share this article to a circle of friends, and scan code plus editor friend to take you into the group, be sure Remarks "Name - Unit - research - Title / Year." Technical problems to seek help, first read "How elegant question" learning problem-solving ideas, still within the end of the discussion group to solve the problem is not private chat, help peers.