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突发武汉病毒被修改正丽致命铁证被抓住! 2020-02-07 18:07:34

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Image result for 解放军三军仪仗队第一刀

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关于疫情来源,大体上有几种观点:自然界进化,实验室泄露,敌对

势力投毒,不同群体造成网络空前的纷争。科普者以阴谋论驳斥病毒

人工合成、敌对投毒派等观点,而民族主义者则找寻敌对势力,批判

实验室泄露派是被境外势力利用,这个争论有些乱……

2月2日,武汉病毒所石正丽发表声明:2019年新型冠状病毒是大自然

给人类不文明生活习惯的惩罚,我石正丽用我的生命担保,与实验室

没有关系。奉劝那些相信并传播不良媒体的谣传的人,闭上你们的

臭嘴。同时转发这个打脸消息:印度学者已经决定撤回这篇预印本

文章。

2月5日,石正丽在接受财新记者采访时再次回应争议,希望国家专业

部门来调查,以还团队一个清白。“我自己的话没有说服力,我不能

控制别人的思想和言论。

以下为武小华回应与石正丽的争议,特转发。

关于我质疑石正丽研究员的朋友圈,在我睡了一个小觉后开始漫天飞,

我觉得这并不是一个复杂的问题,而且相当简单。

面对几万人的感染,几万家庭的支离破碎,几百条人命,石研究员

公然撒谎也就算了,还骂这些不幸的人活该,因为是你们自己不文明

习惯的惩罚,请问这些人都是吃蝙蝠吃的吗?

荒唐!而且要质疑你的研究的科学家闭嘴,你已经丧失了最基本一个

科研工作者的最基本要素:实事求是,以及一个科研工作者的社会

底线:人性。

当你说出这样的的话的时候,我真的是被你气的咬牙切齿,那么我

就公开的把你的谎言揭露一下吧,揭露一下你的赤裸裸谎言。

1,从蝙蝠到人,新冠病毒是如何变异的?

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http://www.xilu.com/202002

07/1000010001120545.html


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微信群里微民再次疯传留言 —— 


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美国病毒学家牵头联合中国病毒专家

就曾合作成功提取和鉴定出了一种类SARS

新型冠状嵌合病毒,并且之后又人工制造和

培养出了一种SARS新型冠状重组病毒。


这份研究报告由美国学者和中国学者联合完成,

作者包括美国德克萨斯大学医学分校微生物与

免疫学系 教授Vineet D. Menachery ,中科院

武汉病毒研究所研究员石正丽(Zhengli-Li Shi)

等人,提取发现并又制造培养了一种嵌合型 

SARS样 的新型冠状病毒 ......

......


基于以上这些发现,

上述科学家们最终成功合成了一株具有感染性的

全长SHC014重组病毒,

并同时在体外和体内证实了该病毒的强大复制能力。


成功地人工合成、制造、培养出了

一种新型冠状重组病毒!


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由中国武汉开始爆发的新型冠状病毒肺炎疫情持续引发关注,从 1 月 21 日开始证实新冠病毒肯定有“人传人”之后,一场全民参与的抗疫战役在中国,乃至世界正式打响。目前已知 2019 年 12 月开始,从第一起华南海鲜市场病例被证实,与SARS冠状病毒极为类似的“不明病毒肺炎”新型冠状病毒开始蔓延。

但是钛媒体最近在仔细研究和整理新冠病毒相关研究时也发现,其实早在 5 年前,也就是 2015 年,美国病毒学家牵头联合中国病毒专家就曾合作成功提取和鉴定出了一种类SARS新型冠状嵌合病毒,并且之后又人工制造和培养出了一种SARS新型冠状重组病毒。这次的研究成果也被发表在了2015年的国际顶级科学杂志《Nature》上(下称:Nature论文)。

在今年武汉爆发的新冠病毒肺炎发生之时,回过头来再看这份五年前的研究报告,依然颇觉触目惊心。虽然并不能证明五年前的研究发现和本次发生的武汉肺炎病毒是完全同一的病毒,但是的确都是与Sars极为相近的新冠病毒,在今年1月,有专家认为本次“不明肺炎”疫情,传播速度快、重病率高、难以"防控”的态势,是世界首次发生的时候,实际上,五年前就早有发生,而病毒科学家也早有预知,并且所有预知的内容都与本次发生的疫情状况高度相似。

这份研究报告由美国学者和中国学者联合完成,作者包括美国德克萨斯大学医学分校微生物与免疫学系 教授Vineet D. Menachery ,中科院武汉病毒研究所研究员石正丽(Zhengli-Li Shi)等人,提取发现并又制造培养了一种嵌合型 SARS样 的新型冠状病毒,根据论文所述,该病毒能让小鼠感染上 SARS(非典肺炎),也可以证实这一病毒通过蛋白外壳与体内 RNA 进行结合,感染和传递到人类细胞当中,报告结论就是这种类Sars新冠状病毒,因其高度可人际传播的属性,可能会给人类带来巨大的社会风险。

五年前的预言一语成谶,同时这份研究报告也说明,本次武汉疫情可能不是偶然的,类Sars冠状病毒的变异和传播从未停止,但人类并未重视。正如近日,北卡罗来纳州吉林斯大学全球公共卫生学院的冠状病毒专家兼助理教授蒂莫西·谢汉(Timothy Sheahan)评价武汉新冠肺炎所说,“这不是“一次性”的病毒疫情,很可能会在未来持续发生。”

针对近期沸沸扬扬的关于武汉病毒研究所的猜测和争议,石正丽在其朋友圈发表声明称:“以生命担保,2019新冠病毒与实验室无关,这是大自然对人类不文明生活习惯的惩罚”。

武汉病毒研究所研究员石正丽

武汉病毒研究所研究员石正丽

2015年到底发生了什么?

2015年的Nature论文中阐述了一个背景,即当时在中国有一个叫做马蹄蝠的蝙蝠种群,这是一种在岩洞栖息地里的菊头蝠科蝙蝠群体,它们体内正在流行一种类似SARS的病毒——SHC014-CoV,而当时严重急性呼吸综合征冠状病毒(SARS-CoV)和中东呼吸综合征(MERS)-CoV的出现,突出了跨物种传播事件对人类社会的威胁,故而科学家们希望研究在马蹄蝠中流行的这一病毒的致病潜力。

蝙蝠是许多病毒的自然宿主,包括埃博拉病毒、马尔堡病毒,狂犬病毒、亨德拉病毒、尼帕病毒等。由于蝙蝠特殊的免疫系统,携带病毒却极少出现病症。在漫长的进化历程中,蝙蝠成为了上百种病毒的自然宿主。对于病毒溯源的研究来说,蝙蝠地位很特殊,是重点的关注对象。

中国科研人员已发现,蝙蝠体内一个被称为“干扰素基因刺激蛋白-干扰素”的抗病毒免疫通道受到抑制,这使得蝙蝠刚好能够抵御疾病,却不引发强烈的免疫反应。野生蝙蝠可能会携带很多病毒,但是它们都维持在一个较低的水平上。

于是,Vineet D. Menachery教授等利用SARS-CoV的反向遗传系统,从中提取并鉴定了一种嵌合病毒,该病毒在适应小白鼠的SARS-CoV主干中可表达出蝙蝠冠状病毒SHC014的刺突。结果表明,该新型冠状病毒能利用SARS的人类细胞受体——血管紧张素转换酶II(ACE2)的多个同源基因,在人类呼吸道原代细胞中有效复制,并在体外获得与SARS传染性同等的效果。

此外,体内实验证明,该嵌合病毒被复制在小白鼠肺部后,可导致明显的发病征状。评估显示,现有的基于SARS的免疫治疗和预防模式的效果不佳;单克隆抗体和疫苗方法均未能利用这种新的刺突蛋白来中和免疫CoVs感染。

基于以上这些发现,上述科学家们最终成功合成了一株具有感染性的全长SHC014重组病毒,并同时在体外和体内证实了该病毒的强大复制能力。研究结果表明,这种病毒完全可重现SARS-CoV(SARS冠状病毒)的传播风险。

Nature论文三种范例所

证明的新冠病毒传播原理,

与武汉疫情高度类似


以下都来自钛媒体编辑翻译并整理了上述2015年《Nature》论文中的主要分析内容,传播原理的确与本次武汉新冠肺炎类似,很多结果也类似,例如关于致病性不会比Sars冠状病毒增强、人传人潜在风险极大(传染性大)、老年动物(实验主要以动物进行实验)更易感等。

高致病性冠状病毒SARS冠状病毒的出现,预示着严重呼吸疾病跨物种传播的新时代。全球化为病毒的快速传播带来基础,对全球经济有着巨大影响。

在SARS席卷全球后,甲型流感亚型病毒H5N1、H1N1和H7N9以及MERS冠状病毒(中东呼吸综合征)开始出现,并且可以同样通过动物传染人类,对当地人口带来了死亡的阴影以及经济损失。

尽管公共卫生措施控制了SARS冠状病毒的爆发,但最近的宏基因组研究通过识别近期在中国马蹄蝠中大面积流行的类SARS冠状病毒的病毒序列,发现这些由蝙蝠携带的病毒有可能在未来带来更多的威胁。

但是,病毒序列带来的数据只能为将来识别以及预防类似流行性病毒提供非常有限的洞见。因此,为了分析蝙蝠携带的冠状病毒引起流行性传播的可能性(也即是传染人类的可能性),我们提取了中国马蹄蝠携带的RsSHC014冠状病毒序列中可造成人畜传染的冠状病毒刺穿蛋白,使用适应小白鼠的SARS冠状病毒主链培育出了一种嵌合病毒。

培育出来的混合病毒让我们能够评估该刺穿蛋白在没有经过自然适应性变异的情况下感染人类的能力。通过这个方法,我们对人类气道细胞在活体状态下由SHC014冠状病毒导致的感染特征进行了分析,测试了现有的免疫和治疗方法对SHC014冠状病毒的作用。这个研究方法能对宏基因组学数据进行解读,帮助预防未来可能会出现的病毒,并未将来疫情爆发的可能性做好准备。

SHC014和相关的RsWIV1冠状病毒的序列显示,该类冠状病毒为SARS冠状病毒(图示1a,b)的近亲。但是,SHC014和SARS冠状病毒在结合人类的14残基和SARS冠状病毒受体中存在差异。受体包括5个决定宿主范围的残基:Y442、L472、N479、T487和Y491 。在WIV1中,其中三个残基与Urbani SARS冠状病毒不同,按原本预期,它们不会改变和ACE2的结合。

这一情况由两个假型化实验得到证实。其中一个实验测试了慢病毒属WIV1刺穿蛋白进入细胞释放血管紧张素转换酶II(AC2)的能力。另一实验为复制WIV1冠状病毒(ref. 1)的试管实验。在SHC014中存在14个与ACE2有相互作用的残基,其中有7个残基和SARS冠状病毒的不一样,这包括了所有5个对决定宿主范围起关键作用的残基。

这些变化,以及释放SHC014刺穿蛋白进入细胞的慢病毒假型实验,表明SHC014刺穿蛋白无法与人类的ACE2进行结合。

但是,有研究发现,其他相关的SARS冠状病毒株存在类似变化,这意味着我们需要做进一步的功能测试才能做出更好的判断。因此,我们基于复制功能强、实应小白鼠的SARS冠状病毒主链合成了SHC014刺穿蛋白(该嵌合冠状病毒在后文统称为SHC014-MA15),以研究小白鼠的发病机理和疫苗研究。与现有分子建模和假型实验得出的预测不同,SHC014-MA15保持了活性,并且在Vero细胞中复制致高滴度。

类似于SARS病毒,SHC014-MA15同样需要功能正常的ACE2分子才能进入细胞,并且可以使用与人类、果子狸和蝙蝠直系同源ACE2。为了测试SHC014刺穿蛋白感染人类气道的能力,我们研究了人类上皮气道细胞系Calu-3 2B4对感染的敏感性,发现SHC014-MA15具有强大复制能力,与Urbani SARS病毒相当。

除此之外,人类主要气道上皮(HAR)培养物被感染,两种病毒都展现出了强大的复制能力。总之,这些数据证实了带有SHC014的病毒感染人类气道细胞的能力,并显示出了SHC014冠状病毒具备跨物种传播的潜在威胁。

中美科学家5年前曾提取并制造了类SARS新冠病毒,还预示了巨大社会风险 | 钛媒体科普

图|SARS 样的冠状病毒在人类气道细胞中复制并产生体内的发病机理

为了评估SHC014次突蛋白在体内介导感染中的作用,我们用104个SARS-MA15或SHC014-MA15空斑形成单位 (p.f.u.)感染了10周大小的BALB/c小白鼠(图1e-h)。

感染SARS-MA15的白鼠在感染后第4天出现体重迅速下降和死亡现象(d.p.i.);而感染了SHC014-MA15的小白鼠体重下降显著(10%),但无死亡(图1e)。

对病毒复制的检测显示,感染了SARS-MA15或SHC014-MA15的小鼠肺部病毒滴度几乎相同(图1f)。感染了SARS-MA15的小白鼠的肺的末梢细支气管和肺实质(图1g)均有较强程度的染色(图1g),而被SHC014-MA15感染的小白鼠的肺抗原染色较弱(图1h);与此相反,在软组织或整体组织评分中未发现抗原染色缺陷,这表示肺部组织对SHC014-MA15的感染情况有异(补充表2)。

感染SARS-MA15的动物体重迅速下降,并死于感染(补充图3a,b)。感染SHC014-MA15可导致动物体重显著下降,但致死率极低。在年轻小白鼠中观测到的组织学和抗原染色模式,在年长些的白鼠中也可观测到(补充表3)。我们使用Ace2−−老鼠进行实验,排除了SHC014-MA15通过另一种感染受体传播的可能性。

该种情况下,小白鼠没有体重下降,或被SHC014-MA15感染后的抗原染色现象(补充图4 a, b和补充表2)。以上这些数据表明,具有SHC014刺突的病毒能够在CoV病毒主干的条件下致使小白鼠体重减轻。

考虑到埃博拉单克隆抗体疗法(例如 ZMApp 10)的临床功效,我们接下来试图确定 SARS-CoV 单克隆抗体对抗 SHC014-MA15 感染的功效。广泛中和针对 SARS 冠状病毒刺突蛋白已经被先前报道,并且是免疫试剂可能人类单克隆抗体。

我们检查了这些抗体对病毒复制的影响(表示为病毒复制的抑制百分比),发现野生型 SARS-CoV Urbani 在相对较低的抗体浓度下被所有四种抗体强烈中和,SHC014-MA15的中和效果有所不同。通过噬菌体展示产生的和逃避突变型的抗体,仅实现抑制 SHC014-MA15 复制(如图 2a)。同样,源自 SARS-CoV 感染患者的记忆 B 细胞的抗体 230.15 和 227.14 也未能阻止SHC014-MA15复制(图 2b,c)。

对于所有三种抗体, SARS 和 SHC014 尖峰氨基酸序列之间的差异对应于 SARS-CoV 逃逸突变体(fm6 N479R; 230.15 L443V; 227.14 K390Q / E)中发现的直接或相邻残基变化,这可能解释了抗体的缺失针对SHC014的中和活性。

最后,单克隆抗体 109.8 能够实现 SHC014-MA15 的 50% 中和,但仅在高浓度(10μg/ ml)时(图2d)。总之,结果表明,针对 SARS-CoV 的广泛中和抗体可能仅对新兴 SARS 样 CoV 菌株(如 SHC014)具有边际功效。

中美科学家5年前曾提取并制造了类SARS新冠病毒,还预示了巨大社会风险 | 钛媒体科普

图|SARS-CoV单克隆抗体对SARS样CoV的疗效折线图

为了评估现有疫苗抵抗 SHC014-MA15 感染的功效,研究人员用双重灭活的完整 SARS-CoV(DIV)疫苗接种了老年小鼠。先前的工作表明,这种疫苗接种方式可以中和并保护年轻小鼠免受同源病毒的攻击;然而,该疫苗未能保护其中还观察到增强的免疫病理的老年动物,这表明由于疫苗接种,动物受到了伤害。在这里,我们发现 DIV 不能在体重减轻或病毒滴度方面提供 SHC014-MA15 的抗攻击保护(图5a,b)。与其他异源组 CoV 的先前报告一致。此外,来自接种 DIV 的老年小鼠的血清也未能中和 SHC014-MA15(如图5c)。

值得注意的是, DIV 疫苗接种导致了强大的免疫病理和嗜酸性粒细胞增多。这些结果证实,DIV 疫苗不能预防 SHC014 感染,并且可能增加老年接种组的疾病。

与用 DIV 疫苗接种相比,将 SHC014-MA15 用作减毒活疫苗显示了针对 SARS-CoV 攻击的潜在交叉保护作用。论文当中的研究人员表示,其用 10 4pfu 的 SHC014-MA15 感染幼鼠,并观察了 28 天。然后,在第 29 天,他们用 SARS-MA15 攻击了小鼠。

尽管在 SHC014-MA15 感染后28 天内产生的抗血清,只有极少的 SARS-CoV 中和反应,但先前用高剂量 SHC014-MA15 感染的小鼠可防御致命剂量的 SARS-MA15 攻击。

在没有二级抗原升压的情况下,28 dpi 表示的抗体滴度的预期峰值,意味着有将随时间而减少针对 SARS-CoV 的保护。在体重减轻和病毒复制方面,在衰老的 BALb/c 小鼠中观察到了类似的结果,表明用致死剂量的SARS-CoV攻击具有保护作用。然而,在某些老年动物中,SHC014-MA1 5感染剂量为 10 4pfu,导致体重减轻和致死率> 10%。

研究人员发现,以较低剂量的 SHC014-MA15(100 pfu)进行疫苗接种不会诱导体重减轻,但也无法保护老年动物免受 SARS-MA15 致命剂量的攻击。总之言之,根据论文的说法,数据表明,SHC014-MA15 攻击可能通过保守的表位赋予针对 SARS-CoV 的交叉保护作用,但所需剂量可诱发发病机理,并不能用作减毒疫苗。

确定 SHC014 尖峰具有介导人类细胞感染并引起小鼠疾病的能力后,我们接下来基于用于SARS-CoV的方法合成了全长SHC014-CoV感染性克隆。在Vero细胞中复制显示SHC014-CoV相对于SARS-CoV没有缺陷;然而,在感染后24小时和48小时,原代HAE培养物中SHC014-CoV显着减弱(P<0.01)。

与SARS-CoV Urbani相比,小鼠的体内感染没有显示出明显的体重减轻,但在全长SHC014-CoV感染的肺部显示出病毒复制减少。总之,这些结果确定了全长SHC014-CoV的生存力,但表明其复制必须与人类呼吸道细胞和小鼠中流行的SARS-CoV的复制相一致,需要进一步的适应。

中美科学家5年前曾提取并制造了类SARS新冠病毒,还预示了巨大社会风险 | 钛媒体科普

图|SHC014-CoV在人呼吸道中复制,但缺乏流行性SARS-CoV的毒力

在 SARS 冠状病毒流行期间,人们很快发现了棕榈科动物和人类冠状病毒之间的联系。基于这一发现,有两种不同观点范例表述。其中,第一种观点认为,流行性 SARS-CoV 起源于蝙蝠病毒,跃迁至小窝并在受体结合域(RBD)内引入了变化,以改善与麝猫 Ace2 的结合,随后在活畜市场上接触人,使人感染了麝香毒株,而该麝香毒株又适合成为流行毒株。

但是,根据发育周期以及数据分析表明,早期人类SARS菌株与蝙蝠菌株的关系似乎比灵猫菌株更为紧密。因此,第二种观点认为蝙蝠直接传播导致SARS-CoV的出现,而棕榈科动物则是继发宿主和持续感染的宿主。对于这两种范例,都认为必须在二级宿主中适应峰值,因为大多数突变预计会在RBD内发生,从而有助于改善感染。两种理论都暗示蝙蝠冠状病毒的库是有限的,宿主范围的突变既是随机的又是罕见的,从而降低了人类未来出现突发事件的可能性。

中美科学家5年前曾提取并制造了类SARS新冠病毒,还预示了巨大社会风险 | 钛媒体科普

图|冠状病毒的出现(传播)范例

尽管Nature论文表示,这一新的研究,并未打破上述说法与范例,但也的确提出了第三种范式,这一论文的作者团队认为,蝙蝠 CoV 病毒细胞为了保持“平衡”的刺突蛋白,能够在不突变的情况下感染人类。通过在SARS-CoV主链中包含SHC014刺突的嵌合病毒在人气道培养物中和在没有RBD适应的小鼠中引起强烈感染的能力可以说明这一假设。

加上以前鉴定的致病冠状病毒主链的观察,上述论文研究结果表明,SARS 样菌株所急需的原材料目前正在动物水库中流通。值得注意的是,尽管全长SHC014-CoV可能需要额外的骨架适应性来介导人类疾病,但有记录的CoV家族中的高频重组事件强调了未来出现的可能性和进一步准备的必要性。

迄今为止,动物种群的基因组学筛选已主要用于鉴定暴发环境中的新型病毒。这里的方法将这些数据集扩展为检查病毒出现和治疗功效的问题。我们认为,具有SHC014尖峰的病毒由于其在原代人类气道培养物中复制的能力而成为潜在的威胁,这是人类疾病的最佳可用模型。另外,在小鼠中观察到的发病机制表明含有SHC014的病毒能够在没有RBD适应的情况下在哺乳动物模型中引起疾病​​。

值得注意的是,相对于SARS-CoV Urbani,与SARS-MA15相比,肺中的向异性和HAE培养物中全长SHC014-CoV的衰减相对于SARS-CoV Urbani而言,提示了ACE2结合以外的因素-包括穗突性,受体生物利用度或拮抗宿主免疫反应中的一部分可能有助于出现。然而,需要对非人类灵长类动物进行进一步测试,以将这些发现转化为人类致病潜能。重要的是,现有治疗方法的失败定义了进一步研究和开发治疗方法的关键需求。有了这些知识,就可以产生监视程序,诊断试剂和有效的治疗方法,从而防止出现组别特异性CoV,例如SHC014,并且可以将其应用于维护相似异质库的其他CoV分支。

在以前出现的模型的基础上,研究人员认为,新型冠状病毒并没有增强致病性。作者表示,预计不会产生嵌合病毒(例如SHC014-MA15)会增加致病性的情况。虽然SHC014-MA15相对于其亲本小鼠适应的SARS-CoV减毒,但类似的研究检查了MA15骨架内野生型Urbani尖峰的CoV的致病性,显示小鼠无体重减轻,病毒复制减少。因此,相对于Urbani峰值–MA15 CoV,SHC014-MA15的发病机理有所改善。

基于这些发现,科学评论小组可能认为类似的研究基于无法冒险进行的循环株构建嵌合病毒,因为不能排除哺乳动物模型中致病性的增加。再加上对小鼠适应株的限制以及使用逃逸突变体开发单克隆抗体的研究,对CoV出现和治疗功效的研究可能会严重受限。这些数据和限制加在一起,代表了新冠病毒研究关注的十字路口。在制定前进的政策时,必须权衡准备和缓解未来爆发的潜力与创造更多危险病原体的风险。

总体而言,研究人员利用实验和分析,对已使用宏基因组学数据来识别由循环蝙蝠SARS状CoV SHC014构成的潜在威胁。由于嵌合的SHC014病毒在人气道培养物中复制的能力,在体内引起发病机理并逃避当前的治疗方法,因此需要针对循环的SARS样病毒的监视和改进的治疗方法。我们的方法还可以利用宏基因组学数据来预测病毒的出现,并将这些知识应用于准备治疗未来出现的病毒感染。

培养并制造出新冠病毒的

实验过程

以下是钛媒体编辑整理了上述论文中科学家们培养并制造出一种新型冠状病毒的实验过程,这一过程被称为“病毒,细胞,体外感染和噬菌斑测定方案”。

科学家将从美国陆军传染病研究所获得的野生型 SARS-CoV(Urbani),适应小鼠的 SARS-CoV(MA15)和嵌合型 SARS 型 CoV 病毒细胞在 Vero E6 培养基细胞上。据悉,这一细胞是根据美国病毒学家杜尔贝科(Dulbecco,Renato) 所研制出改良的 Eagle's 培养基上生长。

另外,获得到的病毒细胞组织,是通过之前研究的信息进行的,其中包括表达 ACE2 直向同源物的 DBT 细胞(Baric实验室,来源未知),这些都在先前被描述为人类和麝猫感染的细胞组织,ACE2 序列是基于从菊头蝠,就是蝙蝠 DBT 细胞当中提取,并从中建立新的病毒培养。

作者表示,这一实验本身是伪型实验过程,与使用基于 HIV 的伪病毒实验类似,先前使用武汉病毒研究所提供的 ACE2 直系表达的同源物种的 HeLa 细胞进行检查,HeLa 细胞在最低必需培养基补充有 10% FCS(Gibco公司,CA)以及 MEM(Gibco公司,CA)中生长,得出下图在Vero E6,DBT,将Calu-3和2B4原代人呼吸道上皮细胞的生长曲线。

实验物,也就是仿制的肺,则是在北卡罗来纳大学机构审查委员会批准下采购的,而这种肺并不是活体的肺,是在 HAE 培养的人肺。该 HAE 培养物代表高度分化的人气道上皮,其中包含纤毛和非纤毛上皮细胞以及杯状细胞,培养物应在气液界面上生长数周,才拿到该实验当中。

而后,将细胞用 PBS 溶液进行洗涤,并用病毒接种或在 37℃ 的 PBS 溶液中模拟稀释 40 分钟。将细胞洗涤 3 次,并加入新鲜培养基以表示时间 “0”。在每个所述的时间点收获三个或更多的生物重复样品,所有病毒的培养均在生物安全级别(BSL)为 3 级的实验室中进行。

人员和环境方面,将冗余的生物细胞放进对应的安全柜中,并有电扇进行散热。所有人员都穿着特别的卫生强化防护服,为了实验过程的健康,相关科研人员使用 3M 电动呼吸器(Breathe Easy,3M)进行呼吸,并严格佩戴双手套,使得实验在安全状况下进行。

序列聚类和结构建模

实验中,从 Genbank 或 Pathosystems 资源整合中心(PATRIC)下载具有代表性的 CoV S1 结构域的全长基因组序列和氨基酸序列,并与 ClustalX 进行比对,通过系统分析使用 100 个自举法或使用 PhyML(https://code.google.com/p/phyml/)封装。

事实上,这一过程主要是将数据进行比对,对病毒的未来变异进行管控,形成一定的数据模型。

接着,科研人员使用 PhyML 软件包生成最大可能性数值树。比例尺代表核苷酸取代。仅标记引导程序支持高于 70% 的节点。该树显示 CoV 分为三个不同的系统发育组,分别定义为 α-CoV,β-CoV 和 γ-CoV。对 于β-CoV,经典子群群集标记为 2a,2b,2c 和 2d,对于 α-CoV,标记为 1a 和 1b。使用 Modeller(Max Planck Institute Bioinformatics Toolkit)生成结构模型,以基于晶体结构 2AJF(蛋白质数据库)生成 SARS RBD 的 SHC014 和 Rs3367 与 ACE2 的同源性模型。在 MacPyMol(1.3版)中可视化和操作同源模型。

SARS样嵌合病毒的构建

如前所述,野生型和嵌合病毒均来自 SARS-CoV Urbani 或相应的小鼠适应性(SARS-CoV MA15)感染性克隆(ic)病毒细胞。通过提取含有 SHC014 刺突序列的质粒,并连接到 MA15 感染性克隆的 E 和 F 质粒中。设计该克隆并从 Bio Basic 购买六种连续 cDNA,并使用侧接独特的 II 类限制性核酸内切酶位点(BglI)的公开序列。

之后,研究人员对其扩增,切除,连接和纯化含有野生型,嵌合 SARS-CoV 和 SHC014-CoV 基因组片段的质粒。然后进行体外转录反应以合成全长基因组 RNA,如先前所述将其转染到 Vero E6 细胞中。收获转染细胞的培养基,并用作后续实验的种子库。在用于这些研究之前,通过序列分析证实了嵌合和全长病毒。嵌合突变体和全长 SHC014-CoV 的合成构建,这一新的 SARS样嵌合病毒标准,需要得到北卡罗莱纳大学机构生物安全委员会和关注双重用途研究委员会的批准。

小鼠体内感染

事实上,这份实验需要根据人体,来了解抵抗力以及病毒形成活动,多数据下,形成小鼠体内感染的数据分析。

论文中称,其从 Harlan Laboratories 订购了雌性、10 周龄和 12 个月大的 BALb/c 白变种实验室老鼠。将动物带入 BSL3 实验室,并使其在感染前适应 1 周。对于感染和减毒活疫苗接种,将用氯胺酮和甲苯噻嗪的混合物对小鼠进行麻醉,并在鼻腔感染时用 50μl 磷酸盐缓冲液(PBS)或稀释的病毒经鼻内感染,每个时间点,每只三或四只小鼠,感染组每剂量都按照条件定量。对于个别小鼠,研究人员可以酌情决定是否将小鼠数据排除在外,包括不吸入全部剂量,鼻腔冒泡或通过口部感染引起的感染。

感染后,在任何动物实验中均未使用致盲法,且动物未随机分组。对小鼠进行接种疫苗,通过足垫注射为年轻和老年小鼠接种 20μl 的含明矾或模拟 PBS 的 0.2μg 双灭活 SARS-CoV 疫苗;然后在 22 天后以相同的方案加强小鼠免疫体质。对于所有组,按照实验方案,在实验过程中每天监测动物的疾病临床体征(驼背,毛皮松动和活动减少)。在头 7 天里,每天监测体重是否减轻,此后继续进行体重监测,直到动物恢复至初始初始体重或连续 3 天显示体重增加为止。所有体重下降超过其初始体重 20% 的小鼠都被喂食并每天监测多次,只要它们处于 20% 的临界值以下即可。

按照实验方案,立即处死体重超过其初始体重 30% 的小鼠。根据研究人员的说法,任何被认为垂死,或不太可能恢复的小鼠,将使用异氟烷过量进行安乐死,并通过颈脱位确认死亡,这种处理方式使用 UNC 机构动物护理和使用委员会(IACUC)批准的方案。

组织学分析

在实验结束后,取出左肺,浸没在 10% 福尔马林缓冲液(Fisher)中,不充气 1 周。将组织包埋在石蜡中,并由 UNC Lineberger 综合癌症中心组织病理学核心设施制备 5μm 切片。为了确定抗原染色的程度,使用市售的多克隆 SARS-CoV 抗核衣壳抗体(Imgenex)对切片进行病毒抗原染色,并以盲法方式对气道和实质进行染色,使用带有 Olympus DP71 相机的 Olympus BX41 显微镜捕获图像。

病毒中和测定和统计分析

在这一部分当中,噬斑减少中和效价测定法与先前表征针对 SARS-CoV 的抗体进行。简而言之,就是中和抗体或血清,并将其进行两次连续稀释,与 100pfu 不同感染性克隆 SARS-CoV 菌株在 37°C 温度下孵育 1 小时。然后将病毒和抗体以 5×10 5 孔 Vero E6 细胞添加到另一孔板中,并重复多次(n≥2)。

在 37°C 下孵育 1 小时后,研究人员需要在培养基中铺上 3 ml 0.8% 琼脂糖。在将板在 37℃ 下孵育 2 天,用中性红染色 3 小时并计数噬菌斑。计算噬菌斑减少的百分比为(1-((带有抗体的噬菌斑数量/没有抗体的噬菌斑数量))×100。

实验结束后进行统计分析,所有实验均与两个实验组(两种病毒,或已接种和未接种的队列)进行对比。因此,病毒的显著差异是通过在各个时间点进行检验确定的。最终的数据以正态分布,在每个比较的组别中,放置出相似的方差。(本文首发钛媒体App)

原论文地址:

https://www.nature.com/articles/nm.3985#auth-14

参考来源:

https://microbiology.utmb.edu/faculty/vineet-d-menachery-phd

https://www.the-scientist.com/news-opinion/lab-made-coronavirus-triggers-debate-34502

https://www.nature.com/articles/d41586-020-00262-7


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 实验室里人工合成制造培养

  的冠状病毒引发巨大争议


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Ralph Baric, an infectious-disease researcher at the University of North Carolina at Chapel Hill, last week (November 9) published a study on his team’s efforts to engineer a virus with the surface protein of the SHC014 coronavirus, found in horseshoe bats in China, and the backbone of one that causes human-like severe acute respiratory syndrome (SARS) in mice. The hybrid virus could infect human airway cells and caused disease in mice, according to the team’s results, which were published in Nature Medicine.

The results demonstrate the ability of the SHC014 surface protein to bind and infect human cells, validating concerns that this virus—or other coronaviruses found in bat species—may be capable of making the leap to people without first evolving in an intermediate host, Nature reported. They also reignite a debate about whether that information justifies the risk of such work, known as gain-of-function research. “If the [new] virus escaped, nobody could predict the trajectory,” Simon Wain-Hobson, a virologist at the Pasteur Institute in Paris, told Nature.

In October 2013, the US government put a stop to all federal funding for gain-of-function studies, with particular concern rising about influenza, SARS, and Middle East respiratory syndrome (MERS). “NIH [National Institutes of Health] has funded such studies because they help define the fundamental nature of human-pathogen interactions, enable the assessment of the pandemic potential of emerging infectious agents, and inform public health and preparedness efforts,” NIH Director Francis Collins said in a statement at the time. “These studies, however, also entail biosafety and biosecurity risks, which need to be understood better.”

Baric’s study on the SHC014-chimeric coronavirus began before the moratorium was announced, and the NIH allowed it to proceed during a review process, which eventually led to the conclusion that the work did not fall under the new restrictions, Baric told Nature. But some researchers, like Wain-Hobson, disagree with that decision.

The debate comes down to how informative the results are. “The only impact of this work is the creation, in a lab, of a new, non-natural risk,” Richard Ebright, a molecular biologist and biodefence expert at Rutgers University, told Nature.

But Baric and others argued the study’s importance. “[The results] move this virus from a candidate emerging pathogen to a clear and present danger,” Peter Daszak, president of the EcoHealth Alliance, which samples viruses from animals and people in emerging-diseases hotspots across the globe, told Nature.


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bats are an important source of highly lethal zoonotic viruses, such as Hendra, Nipah, Ebola and Marburg viruses16.

Here we report on a series of fatal swine disease outbreaks in Guangdong province, China, approximately 100 km from the location of the purported index case of SARS. Most strikingly, we found that the causative agent of this swine acute diarrhoea syndrome (SADS) is a novel HKU2-related coronavirus that is 98.48% identical in genome sequence to a bat coronavirus, which we detected in 2016 in bats in a cave in the vicinity of the index pig farm. This new virus (SADS-CoV) originated from the same genus of horseshoe bats (Rhinolophus) as SARS-CoV.

From 28 October 2016 onwards, a fatal swine disease outbreak was observed in a pig farm in Qingyuan, Guangdong province, China, very close to the location of the first known index case of SARS in 2002, who lived in Foshan (Extended Data Fig. 1a). Porcine epidemic diarrhoea virus (PEDV, a coronavirus) had caused prior outbreaks at this farm, and was detected in the intestines of deceased piglets at the start of the outbreak. However, PEDV could no longer be detected in deceased piglets after 12 January 2017, despite accelerating mortality (Fig. 1a), and extensive testing for other common swine viruses yielded no results (Extended Data Table 1). These findings suggested that this was an outbreak of a novel disease. Clinical signs are similar to those caused by other known swine enteric coronaviruses1718 and include severe and acute diarrhoea and acute vomiting, leading to death due to rapid weight loss in newborn piglets that are less than five days of age. Infected piglets died 2–6 days after disease onset, whereas infected sows suffered only mild diarrhoea and most sows recovered within two days. The disease caused no signs of febrile illness in piglets or sows. The mortality rate was as high as 90% in piglets that were five days or younger, whereas in piglets that were older than eight days, the mortality dropped to 5%. Subsequently, SADS-related outbreaks were found in three additional pig farms within 20–150 km of the index farm (Extended Data Fig. 1a) and, by 2 May 2017, the disease had caused the death of 24,693 piglets at these four farms (Fig. 1a). In farm A alone, 64% (4,659 out of 7,268) of all piglets that were born in February died. The outbreak has abated, and measures that were taken to control SADS included separation of sick sows and piglets from the rest of the herd. A qPCR test described below was used as the main diagnostic tool to confirm SADS-CoV infection.

Fig. 1: Detection of SADS-CoV infection in pigs in Guangdong, China.

figure1

a, Records of daily death toll on the four farms from 28 October 2016 to 2 May 2017. b, Detection of SADS-CoV by qPCR. The y axis shows the log(copy number per 106 copies of 18S rRNA). n = 12 sick piglets, 5 sick sows, 16 recovered sows and 10 healthy piglets. c, Tissue distribution of SADS-CoV in diseased pigs. n = 3. Data are mean ± s.d.; dots represent individual values. d, Detection of SADS-CoV antibodies. n = 46 sows from whom serum was first taken in the first three weeks of the outbreak (First bleed), n = 8 sows from whom serum was taken again (Second bleed) at more than one month after the onset of the outbreak, n = 8 sera from healthy pig controls, n = 35 human sera from pig farmers.

Source data

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A sample collected from the small intestine of a diseased piglet was analysed by metagenomics analysis using next-generation sequencing (NGS) to identify potential aetiological agents. Of the 15,256,565 total reads obtained, 4,225 matched sequences of the bat CoV HKU2, which was first detected in Chinese horseshoe bats in Hong Kong and Guangdong province, China19. By de novo assembly and targeted PCR, we obtained a 27,173-bp CoV genome that shared 95% sequence identity to HKU2-CoV (GenBank accession number NC_009988). Thirty-three full genome sequences of SADS-CoV were subsequently obtained (8 from farm A, 5 from farm B, 11 from farm C and 9 from farm D) that were 99.9% identical to each other (Supplementary Table 1).

Using qPCR targeting the nucleocapsid gene (Supplementary Table 2), we detected SADS-CoV in acutely sick piglets and sows, but not in recovered or healthy pigs on the four farms, nor in nearby farms that showed no evidence of SADS. The virus replicated to higher titres in piglets than in sows (Fig. 1b). SADS-CoV displayed tissue tropism of the small intestine (Fig. 1c), as observed for other swine enteric coronaviruses20. Retrospective PCR analysis revealed that SADS-CoV was present on farm A during the PEDV epidemic, where the first strongly positive SADS-CoV sample was detected on 6 December 2016. From mid-January onwards, SADS-CoV was the dominant viral agent detected in diseased animals (Extended Data Fig. 1b). It is possible that the presence of PEDV early in the SADS-CoV outbreak may have somehow facilitated or enhanced spillover and amplification of SADS. However the fact that the vast majority of piglet mortality occurred after PEDV infection had become undetectable suggests that SADS-CoV itself causes a lethal infection in pigs that was responsible for these large-scale outbreaks, and that PEDV does not directly contribute to its severity in individual pigs. This was supported by the absence of PEDV and other known swine diarrhoea viruses during the peak and later phases of the SADS outbreaks in the four farms (Extended Data Table 1).

We rapidly developed an antibody assay based on the S1 domain of the spike (S) protein using a luciferase immunoprecipitation system21. Because SADS occurs acutely and has a rapid onset in piglets, serological investigation was conducted only in sows. Among 46 recovered sows tested, 12 were seropositive for SADS-CoV within three weeks of infection (Fig. 1d). To investigate possible zoonotic transmission, serum samples from 35 farm workers who had close contact with sick pigs were also analysed using the same luciferase immunoprecipitation system approach and none were positive for SADS-CoV.

Although the overall genome identity of SADS-CoV and HKU2-CoV is 95%, the S gene sequence identity is only 86%, suggesting that the previously reported HKU2-CoV is not the direct progenitor of SADS-CoV, but that they may have originated from a common ancestor. To test this hypothesis, we developed a SADS-CoV-specific qPCR assay based on its RNA-dependent RNA polymerase (RdRp) gene (Supplementary Table 2) and screened 591 bat anal swabs collected between 2013 and 2016 from seven different locations in Guangdong province (Extended Data Fig. 1a). A total of 58 samples (9.8%) tested positive (Extended Data Table 2), all were from Rhinolophus spp. bats that are also the natural reservoir hosts of SARS-related coronaviruses3,4,5,6,7,8,9,10. Four complete genome sequences with the highest RdRp PCR-fragment sequence identity to that of SADS-CoV were determined by NGS. They are very similar in size (27.2 kb) compared to SADS-CoV (Fig. 2a) and we tentatively call them SADS-related coronaviruses (SADSr-CoV). Overall sequence identity between SADSr-CoV and SADS-CoV ranges from 96 to 98%. Most importantly, the S protein of SADS-CoV shared more than 98% sequence identity with sequences of two of the SADSr-CoVs (samples 162149 and 141388), compared to 86% with HKU2-CoV. The major sequence differences among the four SADSr-CoV genomes were found in the predicted coding regions of the S and NS7a and NS7b genes (Fig. 2a). In addition, the coding region of the S protein N-terminal (S1) domain was determined from 19 bat SADSr-CoVs to enable more detailed phylogenetic analysis.

Fig. 2: Genome and phylogenetic analysis of SADS-CoV and SADSr-CoV.

figure2

a, Genome organization and comparison. Colour-coding for different genomic regions as follows. Green, non-structural polyproteins ORF1a and ORF1b; yellow, structural proteins S, E, M and N; blue, accessory proteins NS3a, NS7a and NS7b; Orange, untranslated regions. The level of sequence identity of SADSr-CoV to SADS-CoV is illustrated by different patterns of boxes: Solid colour, highly similar; Dotted fill, moderately similar; Dashed fill, least similar. b, Phylogenetic analysis of 57 S1 sequences (33 from SADS-CoV and 24 from SADSr-CoV). Different colours represent different host species as shown on the left. Scale bar, nucleotide substitutions per site.

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The phylogeny of S1 and the full-length genome revealed a high genetic diversity of alphacoronaviruses among bats and strong coevolutionary relationships with their hosts (Fig. 2b and Extended Data Fig. 2), and showed that SADS-CoVs were more closely related to SADSr-CoVs from Rhinolophus affinis than from Rhinolophus sinicus, in which HKU2-CoV was found. Both phylogenetic and haplotype network analyses demonstrated that the viruses from the four farms probably originated from their reservoir hosts independently (Extended Data Fig. 3), and that a few viruses might have undergone further genetic recombination (Extended Data Fig. 4). However, molecular clock analysis of the 33 SADS-CoV genome sequences failed to establish a positive association between sequence divergence and sampling date. Therefore, we speculate that either the virus was introduced into pigs from bats multiple times, or that the virus was introduced into pigs once, but subsequent genetic recombination disturbed the molecular clock.

For viral isolation, we tried to culture the virus in a variety of cell lines (see Methods for details) using intestinal tissue homogenates as starting material. Cytopathogenic effects were observed in Vero cells only after five passages (Extended Data Fig. 5a, b). The identity of SADS-CoV was verified in Vero cells by immunofluorescence microscopy (Extended Data Fig. 5c, d) and by whole-genome sequencing (GenBank accession number MG557844). Similar results were obtained by other groups2223.

Known coronavirus host cell receptors include angiotensin-converting enzyme 2 (ACE2) for SARS-related CoV, aminopeptidase N (APN) for certain alphacoronaviruses, such as human (H)CoV-229E, and dipeptidyl peptidase 4 (DPP4) for Middle East respiratory syndrome (MERS)-CoV24,25,26. To investigate the receptor usage of SADS-CoV, we tested live or pseudotyped SADS-CoV infection on HeLa cells that expressed each of the three molecules. Whereas the positive control worked for SARS-related CoV and MERS-CoV pseudoviruses, we found no evidence of enhanced infection or entry for SADS-CoV, suggesting that none of these receptors functions as a receptor for virus entry for SADS-CoV (Extended Data Table 3).

To fulfill Koch’s postulates for SADS-CoV, two different types of animal challenge experiments were conducted (see Methods for details). The first challenge experiment was conducted with specific pathogen-free piglets that were infected with a tissue homogenate of SADS-CoV-positive intestines. Two days after infection, 3 out of 7 animals died in the challenge group whereas 4 out of 5 survived in the control group. Incidentally, the one piglet that died in the control group was the only individual that did not receive colostrum due to a shortage in the supply. It is thus highly likely that lack of nursing and inability to access colostrum was responsible for the death (Extended Data Table 4). For the second challenge, healthy piglets were acquired from a farm in Guangdong that had been free of diarrheal disease for a number of weeks before the experiment, and were infected with the cultured isolate of SADS-CoV or tissue-culture medium as control. Of those inoculated with SADS-CoV, 50% (3 out of 6) died between 2 and 4 days after infection, whereas all control animals survived (Extended Data Table 5). All animals in the infected group suffered watery diarrhoea, rapid weight loss and intestinal lesions (determined after euthanasia upon experiment termination, Extended Data Tables 45). Histopathological examination revealed marked villus atrophy in SADS-CoV inoculated farm piglets four days after inoculation but not in control piglets (Fig. 3a, b) and viral N protein-specific staining was observed mainly in small intestine epithelial cells of the inoculated piglets (Fig. 3c, d).

Fig. 3: Immunohistopathology of SADS-CoV infected tissues.

figure3

ad, Sections of jejunum tissue from control (ac) and infected (bd) farm piglets four days after inoculation were stained with haematoxylin and eosin (ab) or rabbit anti-SADSr-CoV N serum (red), DAPI (blue) and mouse antibodies against epithelial cell markers cytokeratin 8, 18 and 19 (green) in (cd). SADS-CoV N protein is evident in epithelial cells and deeper in the tissue of infected piglets, which exhibit villus shortening. Scale bars, 200 μm (ab) and 50 μm (cd). The experiment was conducted three times independently with similar results.

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The current study highlights the value of proactive viral discovery in wildlife, and targeted surveillance in response to an emerging infectious disease event, as well as the disproportionate importance of bats as reservoirs of viruses that threaten veterinary and public health1. It also demonstrates that by using modern technological platforms, such as NGS, luciferase immunoprecipitation system serology and phylogenetic analysis, key experiments that traditionally rely on the isolation of live virus can be performed rapidly before virus isolation.










Methods

Sample collection

Bats were captured and sampled in their natural habitat in Guangdong province (Extended Data Fig. 1) as described previously4. Faecal swab samples were collected in viral transport medium (VTM) composed of Hank’s balanced salt solution at pH 7.4 containing BSA (1%), amphotericin (15 μg ml−1), penicillin G (100 units ml−1) and streptomycin (50 μg ml−1). Stool samples from sick pigs were collected in VTM. When appropriate and feasible, intestinal samples were also taken from deceased animals. Samples were aliquoted and stored at –80 °C until use. Blood samples were collected from recovered sows and workers on the farms who had close contact with sick pigs. Serum was separated by centrifugation at 3,000g for 15 min within 24 h of collection and preserved at 4 °C. Human serum collection was approved by the Medical Ethics Committee of the Wuhan School of Public Health, Wuhan University and Hummingbird IRB. Human, pigs and bats were sampled without gender or age preference unless indicated (for example, piglets or sows). No statistical methods were used to predetermine sample size.

Virus isolation

The following cells were used for virus isolation in this study: Vero (cultured in DMEM and 10% FBS); Rhinolophus sinicus primary or immortalized cells generated in our laboratory (all cultured in DMEM/F12 and 15% FBS): kidney primary cells (RsKi9409), lung primary cells (RsLu4323), lung immortalized cells (RsLuT), brain immortalized cells (RsBrT) and heart immortalized cells (RsHeT); and swine cell lines: two intestinal porcine enterocytes cell lines, IPEC (RPMI1640 and 10% FBS) and SIEC (DMEM and 10% FBS), three kidney cell lines PK15, LLC-PK1 (DMEM and 10% FBS for both) and IBRS (MEM and 10% FBS), and one pig testes cell line, ST (DMEM and 10% FBS). All cell lines were tested free of mycoplasma contamination, species were confirmed and authenticated by microscopic morphologic evaluation. None of the cell lines was on the list of commonly misidentified cell lines (by the ICLAC).

Cultured cell monolayers were maintained in their respective medium. PCR-positive pig faecal samples or the supernatant from homogenized pig intestine (in 200 μl VTM) were spun at 8,000g for 15 min, filtered and diluted 1:2 with DMEM supplemented with 16 μg ml−1 trypsin before addition to the cells. After incubation at 37 °C for 1 h, the inoculum was removed and replaced with fresh culture medium containing antibiotics (below) and 16 μg ml−1 trypsin. The cells were incubated at 37 °C and observed daily for cytopathic effect (CPE). Four blind passages (three-day interval between every passage) were performed for each sample. After each passage, both the culture supernatant and cell pellet were examined for the presence of virus by RT–PCR using the SADS-CoV primers listed in Supplementary Table 2. Penicillin (100 units ml−1) and streptomycin (15 μg ml−1) were included in all tissue culture media.

RNA extraction, S1 gene amplification and qPCR

Whenever commercial kits were used, the manufacturer’s instructions were followed without modification. RNA was extracted from 200 μl of swab samples (bat), faeces or homogenized intestine (pig) with the High Pure Viral RNA Kit (Roche). RNA was eluted in 50 μl of elution buffer and used as the template for RT–PCR. Reverse transcription was performed using the SuperScript III kit (Thermo Fisher Scientific).

To amplify S1 genes from bat samples, nested PCR was performed with primers designed based on HKU2-CoV (GenBank accession number NC_009988.1)19 (Supplementary Table 2). The 25-μl first-round PCR mixture contained 2.5 μl 10× PCR reaction buffer, 5 pmol of each primer, 50 mM MgCl2, 0.5 mM dNTP, 0.1 μl Platinum Taq Enzyme (Thermo Fisher Scientific) and 1 μl cDNA. The 50-μl second-round PCR mixture was identical to the first-round PCR mixture except for the primers. Amplification of both rounds was performed as follows: 94 °C for 5 min followed by 60 cycles at 94 °C for 30 s, 50 °C for 40 s, 72 °C for 2.5 min, and a final extension at 72 °C for 10 min. PCR products were gel-purified and sequenced.

For qPCR analysis, primers based on SADS-CoV RdRp and N genes were used (Supplementary Table 2). RNA extracted from above was reverse-transcribed using PrimeScript RT Master Mix (Takara). The 10 μl qPCR reaction mix contained 5 μl 2× SYBR premix Ex TaqII (Takara), 0.4 μM of each primer and 1 μl cDNA. Amplification was performed as follows: 95 °C for 30 s followed by 40 cycles at 95 °C for 5 s, 60 °C for 30 s, and a melting curve step.

Luciferase immunoprecipitation system assay

The SADS-CoV S1 gene was codon-optimized for eukaryotic expression, synthesized (GenScript) and cloned in frame with the Renilla luciferase gene (Rluc) and a Flag tag in the pREN2 vector21. pREN2-S1 plasmids were transfected into Cos-1 cells using Lipofectamine 2000 (Thermo Fisher Scientific). At 48 h post-transfection, cells were collected, lysed and a luciferase assay was performed to determine Rluc expression for both the empty vector (pREN2) and the pREN2-S1 construct. For testing of unknown pig or human serum samples, 1 μl of serum was incubated with 10 million units of Rluc alone (vector) or Rluc-S1, respectively, together with 3.5 μl of a 30% protein A/G UltraLink resin suspension (Pierce, Thermo Fisher Scientific). After extensive washing to remove unbounded luciferase-tagged antigens, the captured luciferase amount was determined using the commercial luciferase substrate kit (Promega). The ratio of Rluc-S1:Rluc (vector) was used to determine the specific S1 reactivity of pig and human sera. Commercial Flag antibody (Thermo Fisher Scientific) was used as the positive control, and various pig sera (from uninfected animals in China or Singapore; or pigs infected with PEDV, TGEV or Nipah virus) were used as a negative control.

Protein expression and antibody production

The N gene from SADSr-CoV 3755 (GenBank accession number MF094702), which shares a 98% amino acid sequence identity to the SADS-CoV N protein, was inserted into pET-28a+ (Novagen) for prokaryotic expression. Transformed Escherichia coli were grown at 37 °C for 12–18 h in medium containing 1 mM IPTG. Bacteria were collected by centrifugation and resuspended in 30 ml of 5 mM imidazole and lysed by sonication. The lysate, from which N protein expression was confirmed with an anti-His-tag antibody, was applied to Ni2+ resin (Thermo Fisher Scientific). The purified N protein, at a concentration of 400 μg ml−1, was used to immunize rabbits for antibody production following published methods27. After immunization and two boosts, rabbits were euthanized and sera were collected. Rabbit anti-N protein serum was used 1:10,000 for subsequent western blots.

Amplification, cloning and expression of human and swine genes

Construction of expression clones for human ACE2 in pcDNA3.1 has been described previously528. Human DPP4 was amplified from human cell lines. Human APN (also known as ANPEP) was commercially synthesized. Swine APN (also known as ANPEP), DPP4 and ACE2 were amplified from piglet intestine. Full-length gene fragments were amplified using specific primers (provided upon request). Human ACE2 was cloned into pCDNA3.1 fused with a His tag. Human APN and DPP4, swine APN, DPP4 and ACE2 were cloned into pCAGGS fused with an S tag. Purified plasmids were transfected into HeLa cells. After 24 h, expression human or swine genes in HeLa cells was confirmed by immunofluorescence assay using mouse anti-His tag or mouse anti-S tag monoclonal antibodies (produced in house) followed by Cy3-labelled goat anti-mouse/rabbit IgG (Proteintech Group).

Pseudovirus preparation

The codon-humanized S genes of SADS-CoV or MERS-CoV cloned into pcDNA3.1 were used for pseudovirus construction as described previously528. In brief, 15 μg of each pHIV-Luc plasmid (pNL4.3.Luc.R-E-Luc) and the S-protein-expressing plasmid (or empty vector control) were co-transfected into 4 × 106 HEK293T cells using Lipofectamine 3000 (Thermo Fisher Scientific). After 4 h, the medium was replaced with fresh medium. Supernatants were collected 48 h after transfection and clarified by centrifugation at 3,000g, then passed through a 0.45-μm filter (Millipore). The filtered supernatants were stored at −80 °C in aliquots until use. To evaluate the incorporation of S proteins into the core of HIV virions, pseudoviruses in supernatant (20 ml) were concentrated by ultracentrifugation through a 20% sucrose cushion (5 ml) at 80,000g for 90 min using a SW41 rotor (Beckman). Pelleted pseudoviruses were dissolved in 50 μl phosphate-buffered saline (PBS) and examined by electron microscopy.

Pseudovirus infection

HeLa cells transiently expressing APN, ACE2 or DPP4 were prepared using Lipofectamine 2000 (Thermo Fisher Scientific). Pseudoviruses prepared above were added to HeLa cells overexpressing APN, ACE2 or DPP4 24 h after transfection. The unabsorbed viruses were removed and replaced with fresh medium at 3 h after infection. The infection was monitored by measuring the luciferase activity conferred by the reporter gene carried by the pseudovirus, using the Luciferase Assay System (Promega) as follows: cells were lysed 48 h after infection, and 20 μl of the lysates was taken for determining luciferase activity after the addition of 50 μl of luciferase substrate.

Examination of known CoV receptors for SADS-CoV entry/infection

HeLa cells transiently expressing APN, ACE2 or DPP4 were prepared using Lipofectamine 2000 (Thermo Fisher Scientific) in a 96-well plate, with mock-transfected cells as controls. SADS-CoV grown in Vero cells was used to infect HeLa cells transiently expressing APN, ACE2 or DPP4. The inoculum was removed after 1 h of absorption and washed twice with PBS and supplemented with medium. SARS-related-CoV WIV167 and MERS-CoV HIV-pseudovirus were used as positive control for human/swine ACE2 or human/swine DPP4, respectively. After 24 h of infection, cells were washed with PBS and fixed with 4% formaldehyde in PBS (pH 7.4) for 20 min at room temperature. SARS-related-CoV WIV16 replication was detected using rabbit antibody against the SARS-related-CoV Rp3 N protein (made in house, 1:100) followed by Cy3-conjugated goat anti-rabbit IgG (1:50, Proteintech)7. SADS-CoV replication was monitored using rabbit antibody against the SADSr-CoV 3755 N protein (made in house, 1:50) followed by FITC-conjugated goat anti-rabbit IgG (1:50, Proteintech). Nuclei were stained with DAPI (Beyotime). Staining patterns were examined using confocal microscopy on a FV1200 microscope (Olympus). Infection of MERS-CoV HIV-pseudovirus was monitored by luciferase 48 h after infection.

High-throughput sequencing, pathogen screening and genome assembly

Tissue from the small intestine of deceased pigs was homogenized and filtered through 0.45-μm filters before nucleic acid extraction and ribosomal RNA was depleted using the NEBNext rRNA Depletion Kit (New England Biolabs). Metagenomics analysis of both RNA and DNA viruses was performed. For RNA virus screening, the sequencing library was constructed using Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific). For DNA virus screening, NEBNext Fast DNA Fragmentation & Library Prep Set for Ion Torrent (New England Biolabs) was used for library preparation. Both libraries were sequenced on an Ion S5 sequencer (Thermo Fisher Scientific). An analysis pipeline was applied to the sequencing data, which included the following analysis steps: (1) raw data quality filtering; (2) host genomic sequence filtering; (3) BLASTn search against the virus nucleotide database using BLAST; (4) BLASTx search against the virus protein database using DIAMOND v.0.9.0; (5) contig assembling and BLASTx search against the virus protein database. For whole viral genome sequencing, amplicon primers (provided upon request) were designed using the Thermo Fisher Scientific online tool with the HKU2-CoV and the SADS-CoV farm A genomes as references, and the sequencing libraries were constructed using NEBNext Ultra II DNA Library Prep Kit for Illumina and sequenced on an MiSeq sequencer. PCR and Sanger sequencing was performed to fill gaps in the genome. Genome sequences were assembled using CLC Genomic Workbench v.9.0. 5′-RACE was performed to determine the 5′-end of the genomes using SMARTer RACE 5′/3′ Kit (Takara). Genomes were annotated using Clone Manager Professional Suite 8 (Sci-Ed Software).

Phylogenetic analysis

SADS-CoV genome sequences and other representative coronavirus sequences (obtained from GenBank) were aligned using MAFFT v.7.221. Phylogenetic analyses with full-length genome, S gene and RdRp were performed using MrBayes v.3.2. Markov chain Monte Carlo was run for 20–50 million steps using the GTR+G+I model (general time reversible model of nucleotide substitution with a proportion of invariant sites and γ-distributed rates among sites). The first 10% was removed as burn-in. The association between phylogenies and phenotypes (for example, host species and farms) was assessed by BaTS beta-build2, with the trees obtained in the previous step used as input. For SADS-CoVs, a median-joining network analysis was performed using PopART v.1.7, with ɛ = 0. Phylogenetic analysis of the 33 full-length SADS-CoV genome sequences was performed using RAxML v.8.2.11, with GTRGAMMA as the nucleotide substitution model and 1,000 bootstrap replicates. The maximum likelihood tree was used to test the molecular clock using TempEst v.1.5. Potential genetic recombination events in our datasets were detected using RDP v.4.72.

Animal infection studies

Experiments were carried out strictly in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The use of animals in this study was approved by the South China Agricultural University Committee of Animal Experiments (approval number 201004152).

Two different animal challenge experiments were conducted. Pigs were used without gender preference. In the first experiment, which was conducted before the virus was isolated, we used three-day old specific pathogen-free (SPF) piglets of the same breeding line, cared for at a SPF facility, fed with colostrum (except one). These piglets were bred and reared to be free of PEDV, CSFV, SIV, PCV2 and PPV infections, and were routinely tested for viral infections using PCR. We also conducted NGS to further confirm that these were animals were free of infection of the above viruses before the animal experiment, and to demonstrate that the animals were free of SADS-CoV infection. The intestinal tissue samples from healthy and diseased animals (intestinal samples excised from euthanized piglets, then ground to make slurry for the inoculum and NGS was performed to confirm no other pig pathogens were found in the samples), were used to feed two groups of 5 (control) and 7 (infection) animals, respectively. For the second experiment, isolated SADS-CoV was used to infect healthy piglets from a farm in Guangdong, which had been free of diarrheal disease for a number of weeks. These piglets were from the same breed as those on SADS-affected farms, to eliminate potential host factor differences and to more accurately reproduce the conditions that occurred during the outbreak in the region. Both groups of piglets were cared for at a known pig disease-free facility. Again, qPCR and NGS were used to make sure that there was no other known swine diarrhoea virus present in the virus inoculum or any of the experimental animals. Two groups (6 for each group) of three-day old piglets were inoculated with SADS-CoV culture supernatant or normal cell culture medium as control. NGS and qPCR were used to confirm that there were no other known swine pathogens in the inoculum.

For both experiments, animals were recorded daily for signs of diseases, such as diarrhoea, weight loss and death. Faecal swabs were collected daily from all animals and screened for known swine diarrhoea viruses by qPCR. Weight loss was calculated as the percentage weight loss compared the original weight at day 0 with a threshold of >5%. It is important to point out that piglets when they are three days old tend to suffer from diarrhoea and weight loss when they are taken away from sows and the natural breast-feeding environment even without infection. At experimental endpoints, piglets were humanely euthanized and necropsies performed. Pictures were taken to record gross pathological changes to the intestines. Ileal, jejunal and duodenal tissues were taken from selected animals and stored at –80 °C for further analysis.

Haematoxylin and eosin and immunohistochemistry analysis

Frozen (–80 °C) small intestinal tissues including duodenum, jejunum and ileum taken from the experimentally infected pigs were pre-frozen at –20 °C for 10 min. Tissues were then embedded in optimal cutting temperature (OCT) compound and cut into 8-μm sections using the Cryotome FSE machine (Thermo Fisher Scientific). Mounted microscope slides were fixed with paraformaldehyde and stained with haematoxylin and eosin for histopathological examination.

For immunohistochemistry analysis, a rabbit antibody raised against the SADSr-CoV 3755 N protein was used for specific staining of SADS-CoV antigen. Slides were blocked by incubating with 10% goat serum (Beyotime) at 37 °C for 30 min, followed by overnight incubation at 4 °C with the rabbit anti-3755 N protein serum (1:1,000) and mouse anti-cytokeratin 8+18+19 monoclonal antibody (Abcam), diluted 1:100 in PBST buffer containing 5% goat serum. After washing, slides were then incubated for 50 min at room temperature with Cy3-conjugated goat-anti-rabbit IgG (Proteintech) and FITC-conjugated goat-anti-mouse IgG (Proteintech), diluted 1:100 in PBST buffer containing 5% goat serum. Slides were stained with DAPI (Beyotime) and observed under a fluorescence microscope (Nikon).

Reporting Summary

Further information on experimental design is available in the Nature Research Reporting Summary linked to this paper.

Data availability

Sequence data that support the findings of this study have been deposited in GenBank with accession codes MF094681MF094688MF769416MF769444MF094697MF094701MF769406MF769415 and MG557844. Raw sequencing data that support the findings of this study have been deposited in the Sequence Read Achieve (SRA) with accession codes SRR5991648SRR5991649SRR5991650SRR5991651SRR5991652SRR5991654SRR5991655SRR5991656SRR5991657SRR5991658 and SRR5995595.

Change history

  • 05 April 2018

    The Extended Data Figures and Tables section originally published with this article was missing Tables 1–5. This has now been corrected.


References

  1. 1.

    Olival, K. J. et al. Host and viral traits predict zoonotic spillover from mammals. Nature 546, 646–650 (2017).

  2. 2.

    Guan, Y. et al. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 302, 276–278 (2003).

  3. 3.

    Lau, S. K. et al. Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc. Natl Acad. Sci. USA 102, 14040–14045 (2005).

  4. 4.

    Li, W. et al. Bats are natural reservoirs of SARS-like coronaviruses. Science 310, 676–679 (2005).

  5. 5.

    Ge, X. Y. et al. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature 503, 535–538 (2013).

  6. 6.

    He, B. et al. Identification of diverse alphacoronaviruses and genomic characterization of a novel severe acute respiratory syndrome-like coronavirus from bats in China. J. Virol. 88, 7070–7082 (2014).

  7. 7.

    Yang, X. L. et al. Isolation and characterization of a novel bat coronavirus closely related to the direct progenitor of severe acute respiratory syndrome coronavirus. J. Virol. 90, 3253–3256 (2016).

  8. 8.

    Wu, Z. et al. ORF8-related genetic evidence for Chinese horseshoe bats as the source of human severe acute respiratory syndrome coronavirus. J. Infect. Dis. 213, 579–583 (2016).

  9. 9.

    Wang, L. et al. Discovery and genetic analysis of novel coronaviruses in least horseshoe bats in southwestern China. Emerg. Microbes Infect. 6, e14 (2017).

  10. 10.

    Hu, B. et al. Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus. PLoS Pathog. 13, e1006698 (2017).

  11. 11.

    Drosten, C. et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N. Engl. J. Med. 348, 1967–1976 (2003).

  12. 12.

    Ksiazek, T. G. et al. A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 348, 1953–1966 (2003).

  13. 13.

    Marra, M. A. et al. The genome sequence of the SARS-associated coronavirus. Science 300, 1399–1404 (2003).

  14. 14.

    Peiris, J. S. et al. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361, 1319–1325 (2003).

  15. 15.

    Rota, P. A. et al. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300, 1394–1399 (2003).

  16. 16.

    Wang, L.-F. & Cowled, C. (eds) Bats and Viruses: A New Frontier of Emerging Infectious Diseases 1st edn (John Wiley & Sons, Hoboken, 2015).

  17. 17.

    Dong, N. et al. Porcine deltacoronavirus in mainland China. Emerg. Infect. Dis. 21, 2254–2255 (2015).

  18. 18.

    Sun, D., Wang, X., Wei, S., Chen, J. & Feng, L. Epidemiology and vaccine of porcine epidemic diarrhea virus in China: a mini-review. J. Vet. Med. Sci. 78, 355–363 (2016).

  19. 19.

    Lau, S. K. et al. Complete genome sequence of bat coronavirus HKU2 from Chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome. Virology 367, 428–439 (2007).

  20. 20.

    Chen, J. et al. Molecular epidemiology of porcine epidemic diarrhea virus in China. Arch. Virol. 155, 1471–1476 (2010).

  21. 21.

    Burbelo, P. D. et al. Serological diagnosis of human herpes simplex virus type 1 and 2 infections by luciferase immunoprecipitation system assay. Clin. Vaccine Immunol. 16, 366–371 (2009).

  22. 22.

    Gong, L. et al. A new bat-HKU2-like coronavirus in swine, China, 2017. Emerg. Infect. Dis. 23, 1607–1609 (2017).

  23. 23.

    Pan, Y. et al. Discovery of a novel swine enteric alphacoronavirus (SeACoV) in southern China. Vet. Microbiol. 211, 15–21 (2017).

  24. 24.

    Li, W. et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450–454 (2003).

  25. 25.

    Masters, P. S. & Perlman, S. in Fields Virology Vol. 2 (eds Knipe, D. M. & Howley, P. M.) 825–858 (Lippincott Williams & Wilkins, Philadelphia, 2013).

  26. 26.

    Raj, V. S. et al. Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature 495, 251–254 (2013).

  27. 27.

    Harlow, E. & Lane, D. Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, 1988).

  28. 28.

    Ren, W. et al. Difference in receptor usage between severe acute respiratory syndrome (SARS) coronavirus and SARS-like coronavirus of bat origin. J. Virol. 82, 1899–1907 (2008).

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Acknowledgements

We thank S.-B. Xiao for providing pig cell lines, P. Burbelo for providing the luciferase immunoprecipitation system vector and L. Zhu for enabling the rapid synthesis of the S gene; the WIV animal facilities; J. Min for help with the preparation of the immunohistochemistry samples; and G.-J. Zhu and A. A. Chmura for assistance with bat sampling. This work was jointly supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDPB0301) to Z.-L.S., China Natural Science Foundation (81290341 and 31621061 to Z.-L.S., 81661148058 to P.Z., 31672564 and 31472217 to J.-Y.M., 81572045, 81672001 and 81621005 to Y.-G.T.), National Key Research and Development Program of China (2015AA020108, 2016YFC1202705, AWS16J020 and AWS15J006) to Y.-G.T.; National Science and Technology Spark Program (2012GA780026) and Guangdong Province Agricultural Industry Technology System Project (2016LM1112) to J.-Y.M., State Key Laboratory of Pathogen and Biosecurity (SKLPBS1518) to Y.-G.T., Taishan Scholars program of Shandong province (ts201511056 to W.-F.S.), NRF grants NRF2012NRF-CRP001–056, NRF2016NRF-NSFC002-013 and NMRC grant CDPHRG/0006/2014 to L.-F.W., Funds for Environment Construction & Capacity Building of GDAS’ Research Platform (2016GDASPT-0215) to LBZ, United States Agency for International Development Emerging Pandemic Threats PREDICT project (AID-OAA-A-14-00102), National Institute of Allergy and Infectious Diseases of the National Institutes of Health (Award Number R01AI110964) to P.D. and Z.-L.S.

Reviewer information

Nature thanks C. Drosten, G. Palacios and L. Saif for their contribution to the peer review of this work.

Author information

L.-F.W., Z.-L.S., P.Z., Y.-G.T., and J.-Y.M. conceived the study. P.Z., W.Z., Y.Z., S.M., X.-S.Z., B.L., X.-L.Y., H.G., D.E.A., Y.L., X.L.L. and J.C. performed qPCR, serology and histology experiments and cultured the virus. H.F., Y.-W.Z., J.-M.L., G.-Q.P., X.-P.A., Z.-Q.M., T.-T.H., Y.H., Q.S., Y.-Y.W., S.-Z.X., X.-L.-L.Z., W.-F.S. and J.L. performed genome sequencing and annotations. T.L., Q.-M.X., J.-W.C., L.Z., K.-J.M., Z.-X.W., Y.-S.C., D.L., Y.S., F.C., P.-J.G. and R.H. prepared the samples and carried out animal challenge experiments. Z.-L.S., P.D., L.-B.Z., S.-Y.L. coordinated collection of bat samples. P.Z., L.-F.W., Z.-L.S. and P.D. had a major role in the preparation of the manuscript.

Correspondence to Peter Daszak or Lin-Fa Wang or Zheng-Li Shi or Yi-Gang Tong or Jing-Yun Ma.

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Extended data figures and tables

Extended Data Fig. 1 Map of outbreak locations and sampling sites in Guangdong province, China and the co-circulation of PEDV and SADS-CoV during the initial outbreak on farm A.

a, SADS-affected farms are labelled (farms A–D) with blue swine silhouettes following the temporal sequence of the outbreaks. Bat sampling sites are indicated with black bat silhouettes. The bat SADSr-CoV that is most closely related to SADS-CoV (sample 162140) originated in Conghua. The red flag marks Foshan city, the site of the SARS index case. b, Pooled intestinal samples (n = 5 or more biological independent samples) were collected at dates given on the x axis from deceased piglets and analysed by qPCR. The viral load for each piglet is shown as copy number per milligram of intestine tissue (y axis).

Extended Data Fig. 2 Bayesian phylogenetic tree of the full-length genome and the ORF1a and ORF1b sequences of SADS-CoV and related coronaviruses.

a, Bayesian phylogenetic tree of the full-length genome. b, Bayesian phylogenetic tree of the ORF1a and ORF1b sequences. Trees were constructed using MrBayes with the average standard deviation of split frequencies under 0.01. The host of each sequence is represented as a silhouette. Newly sequenced SADS-CoVs are highlighted in red, bat SADSr-CoVs are shown in blue and previously published sequences are shown in black. Scale bars, nucleotide substitutions per site.

Extended Data Fig. 3 Phylogeny and haplotype network analyses of the 33 SADS-CoV strains from the four farms.

a, Phylogenetic tree constructed using MrBayes. The GTR+GAMMA model was applied and 20 million steps were run, with the first 10% removed as burn in. Viruses from different farms are labelled with different colours. Scale bar, nucleotide substitutions per site. b, Median-joining haplotype network constructed using ProART. In this analysis, ɛ = 0 was used. The size of the circles represents the number of samples. The larger the circle, the more samples it includes.

Extended Data Fig. 4 Recombination analysis for SADS-CoV and related CoVs.

The potential genetic recombination events were detected using RDP. For each virus strain, different colours represent different sources of the genomes. Source data

Extended Data Fig. 5 Isolation and antigenic characterization of SADS-CoV.

ab, Vero cells are shown 20 h after infection with mock (a) or SADS-CoV (b). cd, Mock or SADS-CoV-infected samples stained with rabbit serum raised against the recombinant SADSr-CoV N protein (red) and DAPI (blue). The experiment was conducted independently three times with similar results. Scale bars, 100 μm. Source data

Extended Data Table 1 List of all known swine viruses tested by PCR at the beginning of the of SADS outbreak investigation on the four farms

Full size table

Extended Data Table 2 List of SADSr-CoVs detected in bats in Guangdong, China

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Extended Data Table 3 Test of SADS-CoV entry and infection in Hela cells expressing known coronavirus receptors

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Extended Data Table 4 Experimental infection of SPF piglets using intestine tissue homogenate

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Extended Data Table 5 Experimental animal infection of farm piglets using cultured SADS-CoV

Full size table

Supplementary information

Supplementary Information

This file contains two supplementary tables. Supplementary table 1 contains a list of nucleotide variants among the 33 SADS-CoV genomes obtained from the four farms. Supplementary table 2 contains a list of PCR primers used in this study

Reporting Summary

Source data

Source Data Figure 1

Source Data Extended Data 4

Source Data Extended Data 5

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Zhou, P., Fan, H., Lan, T. et al. Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat origin. Nature 556, 255–258 (2018). https://doi.org/10.1038/s41586-018-0010-9

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Abstract

The discovery of SARS-like coronavirus in bats suggests that bats could be the natural reservoir of SARS-CoV. However, previous studies indicated the angiotensin-converting enzyme 2 (ACE2) protein, a known SARS-CoV receptor, from a horseshoe bat was unable to act as a functional receptor for SARS-CoV. Here, we extended our previous study to ACE2 molecules from seven additional bat species and tested their interactions with human SARS-CoV spike protein using both HIV-based pseudotype and live SARS-CoV infection assays. The results show that ACE2s of Myotis daubentoni and Rhinolophus sinicus support viral entry mediated by the SARS-CoV S protein, albeit with different efficiency in comparison to that of the human ACE2. Further, the alteration of several key residues either decreased or enhanced bat ACE2 receptor efficiency, as predicted from a structural modeling study of the different bat ACE2 molecules. These data suggest that M. daubentoni and R. sinicus are likely to be susceptible to SARS-CoV and may be candidates as the natural host of the SARS-CoV progenitor viruses. Furthermore, our current study also demonstrates that the genetic diversity of ACE2 among bats is greater than that observed among known SARS-CoV susceptible mammals, highlighting the possibility that there are many more uncharacterized bat species that can act as a reservoir of SARS-CoV or its progenitor viruses. This calls for continuation and expansion of field surveillance studies among different bat populations to eventually identify the true natural reservoir of SARS-CoV.

Introduction

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the aetiological agent responsible for the SARS outbreaks during 2002–2003, which had a huge global impact on public health, travel and the world economy [411]. The host range of SARS-CoV is largely determined by the specific and high-affinity interactions between a defined receptor-binding domain (RBD) on the SARS-CoV spike protein and its host receptor, angiontensin-converting enzyme 2 (ACE2) [679]. It has been hypothesized that SARS-CoV was harbored in its natural reservoir, bats, and was transmitted directly or indirectly from bats to palm civets and then to humans [10]. However, although the genetically related SARS-like coronavirus (SL-CoV) has been identified in horseshoe bats of the genus Rhinolophus [581218], its spike protein was not able to use the human ACE2 (hACE2) protein as a receptor [13]. Close examination of the crystal structure of human SARS-CoV RBD complexed with hACE2 suggests that truncations in the receptor-binding motif (RBM) region of SL-CoV spike protein abolish its hACE2-binding ability [710], and hence the SL-CoV found recently in horseshoe bats is unlikely to be the direct ancestor of human SARS-CoV. Also, it has been shown that the human SARS-CoV spike protein and its closely related civet SARS-CoV spike protein were not able to use a horseshoe bat (R. pearsoni) ACE2 as a receptor [13], highlighting a critical missing link in the bat-to-civet/human transmission chain of SARS-CoV.

There are at least three plausible scenarios to explain the origin of SARS-CoV. First, some unknown intermediate hosts were responsible for the adaptation and transmission of SARS-CoV from bats to civets or humans. This is the most popular theory of SARS-CoV transmission at the present time [10]. Second, there is an SL-CoV with a very close relationship to the outbreak SARS-CoV strains in a non-bat animal host that is capable of direct transmission from reservoir host to human or civet. Third, ACE2 from yet to be identified bat species may function as an efficient receptor, and these bats could be the direct reservoir of human or civet SARS-CoV. Unraveling which scenario is most likely to have occurred during the 2002–2003 SARS epidemic is critical for our understanding of the dynamics of the outbreak and will play a key role in helping us to prevent future outbreaks. To this end, we have extended our studies to include ACE2 molecules from different bat species and examined their interaction with the human SARS-CoV spike protein. Our results show that there is great genetic diversity among bat ACE2 molecules, especially at the key residues known to be important for interacting with the viral spike protein, and that ACE2s of Myotis daubentoni and Rhinolophus sinicus from Hubei province can support viral entry.

Materials and methods

Cell lines and antibodies

HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, USA). Rabbit polyclonal antibodies against ACE2 of R. pearsoni (RpACE2) were generated using R. pearsoni ACE2 protein expressed in Escherichia coli at the Wuhan Institute of Virology following standard procedures.

Bat sample collection and identification

Bats were sampled from their natural habitats in Hubei, Guangxi, Guizhou, Henan and Yunnan provinces in China as described previously [8]. Bat identification was initially determined in the field by morphology and later confirmed in the laboratory by sequencing the mitochondrial cytochrome b gene from samples of blood cells or rectal tissue as described previously [1].

Bat ACE2 amplification and cloning

Total RNA was extracted from bat rectal tissue using TRIzol Reagent (Invitrogen, USA) and treating with RNase-free DNase I at 37°C for 30 min. First-strand cDNA was synthesized from total RNA by reverse transcription with random hexamers. Full-length bat ACE2 fragments were amplified using the forward primer bAF2 (5′-CTTGGTACCATGTCAGGCTCTTYCTGG-3′) and the reverse primer bAR2 (5′-CCGCTCGAGCTAAAAB[G/T/C]GAV[G/A/C]GTCTGAACATCATC-3′). The PCR mixture (25 μL) contained 0.5 μL cDNA, 1.5 mM MgCl2 and 0.2 μM of each primer, and the fragments were amplified using the following parameters: 95°C for 5 min, 35 cycles of 94°C for 30 s, 55°C for 45 s and 68°C for 3 min, with a final elongation step at 68°C for 10 min. All bat ACE2s were cloned into pCDNA3.1 with KpnI and XhoI, and this was verified by sequencing.

Chimeric ACE2 construction

For samples in which full-length ACE2 amplification was unsuccessful, the N-terminal region (1–1,170 bp) was amplified using the forward primer bAF2 and the reverse primer RMR (5′-TTAGCTCCATTTCTTAGCAGGTAGG-3′). Chimeric ACE2 was constructed by combining the N-terminal region of bat ACE2 with the C-terminal portion of human ACE2 at the unique BamHI site (1,070–1,075 bp). The chimera was subsequently cloned into pCDNA3.1 with KpnI and XhoI and sequenced as above.

Construction of bat ACE2 mutants

ACE2 from M. daubentoni was chosen to generate a series of ACE2 mutants using a QuikChange II Site-Directed Mutagenesis Kit (Stratagene, USA). The altered amino acid codon for each mutant is indicated as follows: I27T, N31K, K35E, and H41Y. Mutants were confirmed by sequencing.

Sequence analysis

All bat ACE2s were submitted to GenBank (EF569964, GQ999931–GQ999938). Sequence alignment was performed using ClustalX version 1.83 [15] and corrected manually. A phylogenetic tree based on amino acid (aa) sequences was constructed using the neighbor-joining (NJ) method in MEGA version 4.1. [14].

Analysis of ACE2 expression by western blotting

Lysates of HeLa cells expressing human ACE2 or bat ACE2 were separated on a 4–10% SDS-PAGE gel, followed by transfer to a polyvinylidene difluoride (PVDF) membrane using a semi-dry protein transfer apparatus (Bio-Rad, USA). The membrane was probed with a rabbit polyclonal antibody against the bat ACE2 protein (1:200) at room temperature for 1 h, followed by incubation with alkaline-phosphatase-conjugated goat anti-rabbit IgG (1:1,000) (Chemicon, Australia). The probed proteins were visualized using NBT and BCIP color development (Promega, USA).

Pseudotype virus infection assays

An HIV-1-luciferase pseudotype virus carrying the SARS-CoV BJ01 S protein, HIV/BJ01-S, was prepared as described previously [13]. HeLa cells were seeded onto 96-well plates for 18 h and then transfected with 0.2 μg recombinant plasmid containing bat or human ACE2 using 0.5 μL Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s protocol. At 24 h post-transfection, 30 μL medium containing HIV/BJ01-S was added to each well. At 2–3 h postinfection, unadsorbed viruses were removed, and fresh medium was added. The infection was monitored by measuring luciferase activity, expressed from the reporter gene carried by the pseudovirus, using a luciferase assay kit (Promega, USA). Cells were lysed at 48 h postinfection by adding 20 μL lysis buffer provided with the kit, and 10 μL of the resulting lysates were tested for luciferase activity by the addition of 20 μL luciferase substrate in a Turner Designs TD-20/20 luminometer. Each infection experiment was conducted in triplicate, and all experiments were repeated three times.

Live virus infection assays

Live SARS-CoV infection was carried out under BSL4 conditions at the Australian Animal Health Laboratory (AAHL) as described previously [1617]. Briefly, 48 h after transfection, the time at which expression of the ACE2 receptor on the HeLa cell surface is optimal, 2 × 106 TCID50 of virus was added to the cells for infection. The cells were fixed 24 h later by treatment with 100% methanol for 10 min and washed five times with PBST. The primary antibody, chicken anti-SARS-CoV S (produced against the recombinant S protein expressed in E. coli at AAHL), at a 1:500 dilution in 1% BSA/PBS, was added and incubated with the cells for 1 h at room temperature. An FITC anti-chicken conjugate (Chemicon, Australia) at 1:1,000 in 1% BSA/PBS was added after washing the cells five times and incubated with the cells for 1 h. Infection was monitored by immunofluorescent microscopic analysis.

Results and discussion

Cloning and expression of ACE2 genes from different bat species

ACE2 genes from seven bat species were amplified and cloned (Fig. 1, sFig. 1). Full-length genes were obtained from Rhinolophus ferrumequinum from Hubei province (Rf-HB), R. macrotis from Hubei province (Rm-HB), R. pearsoni from Guangxi (Rp-GX), R. pusillus from Hubei province (Rpu-HB), R. sinicus from Guangxi province (Rs-GX) and R. sinicus from Hubei province (Rs-HB). For the following bat species, amplification of the full-length coding region was not successful, and instead,the N-terminal region was cloned in frame with the C-terminal region of the human ACE2 gene to form a chimeric full-length ACE molecule: R. pearsoni from Guizhou province (Rp-GZ), Myotis daubentonii bat from Yunnan province (Md-YN) and Hipposideros pratti bat from Henan province (Hp-HN). The full-length sequences of bat ACE2 are identical in size to that of hACE2 (805 aa in total). Sequence comparison showed that bat ACE2s are closely related to ACE2s of other mammals and have an aa sequence identity of 80–82% to human and civet ACE2. The aa identity of ACE2 from different bat families ranges from 78 to 84%, and within the genus Rhinolophus, the sequence identity increases to 89–98%. The major sequence variation among bat ACE2s is located in the N-terminal region, which has been identified in structural studies as the SARS-CoV-binding region [67]. A phylogenetic tree was constructed based on the sequences of bat ACE2 (sFig. 2) using the MEGA package [14].

Fig. 1figure1

Sequence alignment of SARS-CoV binding regions of ACE2s from 9 bats, civet and human. The GenBank accession numbers of bat, civet and human ACE2 are as follows: human (NM021804), civet (AY881174), Rf-HB (GQ999931), Rm-HB (GQ999932), Rs-GX (GQ999933), Rp-GX (EF569964), Hp-HN (GQ999934), Rp-GZ (GQ999935), Rs-HB (GQ999936), Md-YN(GQ999937) and Rpu-HB (GQ999938). The alignment was generated using ClustalX v1.83. In black are single, fully conserved residues. In gray are strongly conserved residues. In light gray are weakly conserved residues. Asterisks indicate residues that interact directly with the receptor-binding domain of the SARS-CoV S protein

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All ACE2 genes were cloned into a eukaryotic expression vector and used to transfect HeLa cells. Western blot analysis showed that all ACE2s were expressed efficiently and at very similar levels and were recognized by a rabbit anti-bat ACE2 antibody with an apparent molecular weight of approximately 100–130 kDa (Fig. 2c).

Fig. 2figure2

Testing of the ability of bat ACE2 proteins to mediate pseudovirus HIV/BJ01-S and live SARS- CoV infection. a HeLa cells transfected with plasmids encoding bat and human ACE2s were infected with pseudovirus HIV/BJ01-S. Infectivity was determined by measuring the activity of reporter luciferase as described in “Materials and methods”. HeLa cells transfected with pcDNA3.1 and human ACE2 were used as the negative and positive controls, respectively. All tests were performed in triplicate, and the experiments were repeated three times. The error bar represents the calculated standard deviation. I27T, N31K, K35E, and H41Y are mutants of MdACE2 that were made using a QuikChange II Site-Directed Mutagenesis Kit. b SARS-CoV live virus infection using the ACE2s from bats as described in “Materials and methods”. HeLa cells transfected with pcDNA3.1 and human ACE2 were used as the negative and positive controls, respectively. c Expression of bat or human ACE2. Lysates from HeLa cells transfected with plasmid expressing human or bat ACE2 were analyzed by western blot. Rabbit anti-bat ACE2 polyclonal antibody (upper panel) or β-actin monoclonal antibody (lower panel) was used as the primary antibody. Lane 1 vector pcDNA3.1 control; lanes 2–10 bat ACE2 from samples Rf-HB, Rm-HB, Rpu-HB, Hp-HN, Rp-HB, Rp-GZ, Rs-GX, Rs-HB and Md-YN; lanes 11–14 Md-YN ACE2 mutant I27T, N31K, K35E and H41Y; lane 15 human ACE2. The abbreviations of bat species are given in the main text

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Functionality of bat ACE2 as an SARS-CoV entry receptor

To examine the susceptibility of different bat ACE2 molecules to SARs-CoV entry, the HIV/BJ01-S pseudovirus system was used to infect HeLa cells transiently expressing bat ACE2 or human ACE2 genes. Among the bat ACE2s, only MdACE2 (MdACE2) and Rs-HB ACE2 demonstrated significant pseudovirus infection, as deduced from the significantly higher level of luciferase activity in comparison to background activity in the negative control (Fig. 2a). Although such assays are not to be viewed as an absolute quantification of receptor activity, it is nevertheless worth noting that MdACE2-mediated infection seemed to be more efficient than with Rs-HB ACE2. In the same context, it is clear that the bat ACE2s were less efficient overall than the human ACE2 in this particular assay system. The biological significance of this observation remains to be determined. The functionality of MdACE2 and Rs-HB ACE2 as SARS-CoV entry receptors was further confirmed by infection with live virus. As shown in Fig. 2b, both bat ACE2 proteins could clearly support SARs-CoV infection. No attempt was made to quantify infection efficiency in this study due to difficulties encountered in conducting experiments under BSL4 conditions.

Structural modeling of bat ACE2 molecules

Homologous structural modeling of human SARS-CoV RBD complexed with MdACE2 supports MdACE2 as a receptor for human SARS-CoV S protein. The crystal structure of human SARS-CoV RBD complexed with hACE2 shows that two salt bridges at the SARS-CoV-hACE2 interface, between hACE2 Lys31 and Glu35 and between hACE2 Lys353 and hACE2 Glu38, are both buried in a hydrophobic environment and contribute critically to the SARS-CoV-hACE2 interactions (Fig. 3a, c) [7]. Disturbance of the formation of either of these salt bridges weakens SARS-CoV-hACE2 binding. The Lys31-Glu35 salt bridge at the SARS-CoV-hACE2 interface becomes an Asn31-Lys35 hydrogen bond at the SARS-CoV-Md-YNACE2 interface (Fig. 3b), which possibly weakens virus-receptor binding but still is largely compatible with the virus-receptor interface. Thr27 on hACE2 supports the Lys31-Gu35 salt bridge through hydrophobic interactions with Tyr475 (Fig. 3a); Ile27 on MdACE2 supports the Asn31-Lys35 hydrogen bond more efficiently than Thr27 through tighter hydrophobic interactions with Tyr475 (Fig. 3b). Moreover, Tyr41 on hACE2 supports the Lys353-Glu38 salt bridge (Fig. 3c); His41 on MdACE2 supports the same salt bridge less efficiently than Tyr41 (Fig. 3d). Overall, MdACE2 is an efficient receptor for SARS-CoV, despite the fact that its receptor activity is lower than that of hACE2.

Fig. 3figure3

Homologous structural modeling of SARS-CoV and Md-YN ACE2 (MdACE2) interactions. a Critical salt bridge between hACE2 Lys31 and Glu35 and the hydrophobic residues surrounding it, based on the experimentally determined crystal structure of SARS-CoV RBD complexed with hACE2 (PDB 2AJF). b Homologous structural modeling of the hydrogen bond between MdACE2 Asn31 and Lys35. The modeling was done in the program O [3]. c Critical salt bridge between hACE2 Lys353 and Glu38 and the hydrophobic residues surrounding it, based on the structure of SARS-CoV RBD complexed with hACE2. d Homologous structural modeling of the salt bridge between MdaACE2 Lys353 and SARS-CoV Glu38 and the hydrophobic residues surrounding it. Structural illustrations were prepared using the program Povscript [2]

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Compared with MdACE2, Rs-HB ACE2 contains Glu31 and Glu35, which are not compatible with each other due to their same negative charges, which disfavor virus-receptor binding. However, Rs-HB ACE2 also contains Thr27 and Tyr41, both of which support SARS-CoV entry by contributing favorably to the hydrophobic interactions at the virus-receptor interface. Thus, Rs-HB is a low-efficiency receptor for SARS-CoV. All of the other bat ACE2 molecules contain combinations of the aforementioned key residues that are completely incompatible with virus–receptor interactions. More specifically, they either contain same-charged residues at the 31 and 35 positions, which repel each other, or contain His41 and Lys27, which disfavor SARS-CoV binding (Fig. 1). In particular, Lys27 on some of these bat ACE2 molecules is incompatible with certain hydrophobic residues, such as Leu443 and Phe460, on SARS-CoV RBD (Fig. 3a, b). Therefore, these bat ACE2 molecules are not receptors for SARS-CoV.

Site-directed mutagenesis analysis

To confirm the above homologous structural analysis, we carried out site-directed mutagenesis on MdACE2. Our results show that mutations E31K, K35E, and I27T all dramatically decrease the receptor activity of MdACE2, whereas mutation H41Y greatly increases its receptor activity (Fig. 2a). Therefore, our mutagenesis data further confirmed that key residues in ACE2 determine the receptor activity of MdACE2.

Our finding that M. daubentoni and R. sinicus could support SARS-CoV infection has important implications in relation to the origin of SARS-CoV. Since all lines of investigation have indicated that ACE2-binding affinity is among the important determinants for SARS-CoV host range, our data would suggest that M. Daubentonii and R. sinicus have the potential to serve as the direct reservoirs for human SARS-CoV or its highly related civet SARS-CoV. To further investigate the potential of M. Daubentonii and R. sinicus as reservoirs for SARS-CoV, more efforts will have to be directed toward widening the surveillance of bats in these families and in different geographical locations.

Another important finding of our current study is the great genetic diversity of bat ACE2 proteins, which is in contrast to the genetically homogenous hACE2 [10]. Sequence variations of bat ACE2, especially in positions that are critical to SARS-CoV binding, such as residues 27, 31, 35, and 41, suggest that, in addition to the Md-YN and Rs-HB ACE2s, there may be many other bats with an ACE2 protein that makes them susceptible to SARS-CoV entry. This again highlights the need for more field surveillance and molecular characterization of different bat ACE2 proteins until the true reservoir host of SARS-CoV is identified and its spillover mechanisms and transmission pathways are fully characterized.

References

  1. 1.

    Cui J, Han N, Streicker D, Li G, Tang X, Shi Z, Hu Z, Zhao G, Fontanet A, Guan Y, Wang L, Jones G, Field HE, Daszak P, Zhang S (2007) Evolutionary relationships between bat coronaviruses and their hosts. Emerg Infect Dis 13:1526–1532

  2. 2.

    Fenn TD, Ringe D, Petsko GA (2003) POVScript+: a program for model and data visualization using persistence of vision ray-tracing. J Appl Crystallogr 36:944–947

  3. 3.

    Jones TA, Zou JY, Cowan SW, Kjeldgaard M (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A 47:110–119

  4. 4.

    Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, Tong S, Urbani C, Comer JA, Lim W, Rollin PE, Dowell SF, Ling AE, Humphrey CD, Shieh WJ, Guarner J, Paddock CD, Rota P, Fields B, DeRisi J, Yang JY, Cox N, Hughes JM, LeDuc JW, Bellini WJ, Anderson LJ (2003) A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 348:1953–1966

  5. 5.

    Lau SK, Woo PC, Li KS, Huang Y, Tsoi HW, Wong BH, Wong SS, Leung SY, Chan KH, Yuen KY (2005) Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc Natl Acad Sci USA 102:14040–14045

  6. 6.

    Li F, Li W, Farzan M, Harrison SC (2005) Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 309:1864–1868

  7. 7.

    Li F (2008) Structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections. J Virol 82:6984–6991

  8. 8.

    Li W, Shi Z, Yu M, Ren W, Smith C, Epstein JH, Wang H, Crameri G, Hu Z, Zhang H, Zhang J, McEachern J, Field H, Daszak P, Eaton BT, Zhang S, Wang LF (2005) Bats are natural reservoirs of SARS-like coronaviruses. Science 310:676–679

  9. 9.

    Li W, Zhang C, Sui J, Kuhn JH, Moore MJ, Luo S, Wong SK, Huang IC, Xu K, Vasilieva N, Murakami A, He Y, Marasco WA, Guan Y, Choe H, Farzan M (2005) Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2. EMBO J 24:1634–1643

  10. 10.

    Li W, Wong SK, Li F, Kuhn JH, Huang IC, Choe H, Farzan M (2006) Animal origins of the severe acute respiratory syndrome coronavirus: insight from ACE2-S-protein interactions. J Virol 80:4211–4219

  11. 11.

    Peiris JS, Lai ST, Poon LL, Guan Y, Yam LY (2003) Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361:1319–1325

  12. 12.

    Ren W, Li W, Yu M, Hao P, Zhang Y, Zhou P, Zhang S, Zhao G, Zhong Y, Wang S, Wang LF, Shi Z (2006) Full-length genome sequences of two SARS-like coronaviruses in horseshoe bats and genetic variation analysis. J Gen Virol 87:3355–3359

  13. 13.

    Ren W, Qu X, Li W, Han Z, Yu M, Zhou P, Zhang SY, Wang LF, Deng H, Shi Z (2008) Difference in receptor usage between severe acute respiratory syndrome (SARS) coronavirus and SARS-like coronavirus of bat origin. J Virol 82:1899–1907

  14. 14.

    Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

  15. 15.

    Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882

  16. 16.

    Tu C, Crameri G, Kong X, Chen J, Sun Y, Yu M, Xiang H, Xia X, Liu S, Ren T, Yu Y, Eaton BT, Xuan H, Wang LF (2004) Antibodies to SARS coronavirus in civets. Emerg Infect Dis 10:2244–2248

  17. 17.

    Yu M, Stevens V, Berry JD, Crameri G, McEachern J, Tu C, Shi Z, Liang G, Weingartl H, Cardosa J, Eaton BT, Wang LF (2008) Determination and application of immunodominant regions of SARS coronavirus spike and nucleocapsid proteins recognized by sera from different animal species. J Immunol Methods 331:1–12

  18. 18.

    Yuan J, Hon CC, Li Y, Wang D, Xu G, Zhang H, Zhou P, Poon LL, Lam TT, Leung FC, Shi Z (2010) Intraspecies diversity of SARS-like coronaviruses in Rhinolophus sinicus and its implications for the origin of SARS coronaviruses in humans. J Gen Virol 91:1058–1062

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Acknowledgments

This work was jointly funded by the State Key Program for Basic Research Grants (2005CB523004, 2010CB530100) from the Chinese Ministry of Science, Technology and the Knowledge Innovation Program Key Project administered by the Chinese Academy of Sciences (KSCX1-YW-R-07) to Z.S. and the CSIRO CEO Science Leader Award to L.-F.W. We thank Gary Crameri and Jennifer Barr for help with live virus infection studies.

Author information

    • Yuxuan Hou

    • , Cheng Peng

    • , Yan Li

    • , Zhenggang Han

    •  & Zhengli Shi

    • Meng Yu

    •  & Lin-Fa Wang

    • Fang Li

Correspondence to Lin-Fa Wang or Zhengli Shi.

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Hou, Y., Peng, C., Yu, M. et al. Angiotensin-converting enzyme 2 (ACE2) proteins of different bat species confer variable susceptibility to SARS-CoV entry. Arch Virol 155, 1563–1569 (2010). https://doi.org/10.1007/s00705-010-0729-6

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Keywords

  • Salt Bridge

  • Severe Acute Respiratory Syndrome

  • ACE2 Gene

  • Pseudotype Virus

  • Severe Acute Respiratory Syndrome Coronavirus





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作者:贪官太多 留言时间:2020-02-14 14:16:20

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作者:Pascal 留言时间:2020-02-07 21:38:58

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作者:Pascal 留言时间:2020-02-07 18:11:41

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