http://virological.org/t/the-proximal-origin-of-sars-cov-2/398
SARS-CoV-2的近期起源(BY Google translate)
克里斯蒂安·安德森（Kristian G.Andersen）1,2 *，安德鲁·朗巴特（Andrew Rambaut）3，伊恩·利普金（W.Ian Lipkin）4，爱德华·霍姆斯（Edward C.Holmes）5和罗伯特·F·加里（Robert F.Garry）6,7
1美国斯克里普斯研究所（Scripps Research Institute）免疫学和微生物学系，美国加利福尼亚州92037。
2Scripps Research Translational Institute，拉荷亚，CA 92037，美国。
3英国爱丁堡爱丁堡大学进化生物学研究所。
4美国纽约哥伦比亚大学梅尔曼公共卫生学院感染与免疫中心。
5澳大利亚悉尼大学生命与环境科学学院和医学学院玛丽·巴希尔传染病与生物安全研究所。
6杜兰大学医学院，美国路易斯安那州新奥尔良微生物学和免疫学系。
7 Zalgen Labs，LCC，美国马里兰州日耳曼敦。
*通讯作者：
克里斯蒂安·安德森（Kristian G.Andersen）,免疫学和微生物学系, 斯克里普斯研究所，拉荷亚，CA 92037，美国。
自中国湖北省武汉市首次报道新型肺炎（COVID-19）以来，关于致病性病毒SARS-CoV-2的起源一直存在大量讨论和不确定性。现在，SARS-CoV-2感染在中国非常普遍，每个省都有病例。截至2020年2月14日，已经确诊了64,473起此类病例，其中1,384人死于该病毒。由于报告的轻度和无症状病例有限，这些官方病例数可能被低估了，并且该病毒显然能够有效地进行人与人之间的传播。基于可能传播到医疗体系较弱的国家，世界卫生组织宣布COVID-19疫情为国际关注的突发公共卫生事件（PHEIC）。目前尚无针对该疾病的疫苗或特异性疗法。
SARS-CoV-2是已知感染人类的??冠状病毒科的第七名成员。其中三种病毒，SARS CoV-1，MERS和SARS-CoV-2，可以引起严重的疾病。四，HKU1，NL63，OC43和229E与轻度呼吸道症状有关。本文中，我们回顾了从可用基??因组序列数据的比较分析中可以推断出SARS-CoV-2的起源和早期进化的方法。特别是，我们提供了有关SARS-CoV-2基因组中显着特征的观点，并讨论了可能出现这些特征的场景。重要的是，该分析提供了证据，表明SARS-CoV-2不是实验室构建物也不是有意操纵的病毒。
下文描述的α-和β-冠状病毒（Coronaviridae家族）的基因组比较确定了SARS-CoV-2基因组的两个显着特征：（i）基于结构模型和早期生化实验，SARS-CoV-2似乎针对与人ACE2受体结合; （ii）SARS-CoV-2的高度可变的穗状蛋白（S）通过插入十二个核苷酸在S1和S2边界具有一个多碱基（弗林蛋白酶）切割位点。另外，该事件导致在多碱基切割位点附近获得了三个预测的O-连接的聚糖。
SARS-CoV-2受体结合结构域的突变
SARS-CoV和SARS相关冠状病毒的刺突蛋白中的受体结合域（RBD）是病毒基因组中变化最大的部分。 RBD中的六个残基似乎对于与人ACE2受体结合并确定宿主范围至关重要。使用基于SARS-CoV的Urbani应变的坐标，它们是Y442，L472，N479，D480，T487和Y4911。 SARS-CoV-2中的相应残基为L455，F486，Q493，S494，N501和Y505。与它最密切相关的病毒Ra Rh13相比，SARS-CoV-2中的这六个残基中有五个是突变的，它是从Rhinolophus affinis bat采样而来的，与?96％相同（图1a）。根据建模1和生化实验3,4，SARS-CoV-2似乎具有一种RBD，它可能与人，非人灵长类，雪貂，猪和猫以及其他具有高受体的物种对ACE2的亲和力很高。同源性1。相比之下，SARS-CoV-2在与SARS样病毒相关的其他物种（包括啮齿动物和蜂巢）中与ACE2的结合效率可能较低。
SARS-CoV-2 S蛋白中第486位残基的苯丙氨酸（F）对应于SARS-CoV Urbani菌株中的L472。值得注意的是，在SARS-CoV细胞培养实验中，L472突变为苯丙氨酸（L472F）5，据预测，苯丙氨酸对于SARS-CoV RBD与人ACE2受体6的结合是最佳的。然而，蝙蝠的几个SARS样冠状病毒中也存在此位置的苯丙氨酸（图1a）。尽管这些分析表明SARS-CoV-2可能能够以高亲和力结合人ACE2受体，但相互作用并不被认为是最佳的。此外，SARS-CoV-2的RBD中的几个关键残基与先前描述的最适合人ACE2受体结合的残基不同6。与这些计算预测相反，最近的结合研究表明，SARS-CoV-2
多元切割位点和O-连接聚糖
SARS-CoV-2的第二个显着特征是在刺蛋白的两个亚基S1和S2（图1b）8,9的连接处的刺蛋白中有一个预测的多元切割位点（RRAR）。除了两个碱性精氨酸和一个在切割位点的丙氨酸外，还插入了一个脯氨酸。因此，完全插入的序列是PRRA（图1b）。脯氨酸插入产生的强烈转角预计会导致在多碱基切割位点侧翼的S673，T678和S686中添加O-连接的聚糖。以前在相关谱系Bβ冠状病毒中未发现多碱基切割位点，这是SARS-CoV-2的独特特征。一些人β冠状病毒，包括HCoV-HKU1（谱系A），具有多碱基切割位点，以及在S1 / S2切割位点附近具有预测的O-连接聚糖。
尽管尚不清楚SARS-CoV-2中多碱基切割位点的功能后果，但SARS-CoV的实验表明，在S1 / S2交界处改造这样的位点可增强细胞-细胞融合，但不会影响病毒的进入10。多元裂解位点允许弗林蛋白酶和其他蛋白酶有效裂解，并且可以在选择快速复制和传播病毒的条件下（例如高密度鸡群）在禽流感病毒血凝素（HA）蛋白的两个亚基的连接处获得）。 HA在细胞-细胞融合和病毒进入中的功能与冠状病毒S蛋白相似。通过插入或重组获得HA中的多元切割位点，可将低致病性禽流感病毒转化为高致病性形式11-13。在细胞培养物中或动物反复传代后，还观察到流感病毒HA获得了多价切割位点14,15。同样，无毒的新城疫病毒分离株在鸡的连续传代过程中通过在融合蛋白亚基的交界处逐渐获得一个多价裂解位点而成为高致病性16。三种预测的O-连接聚糖的潜在功能尚不清楚，但是它们可以产生一个“粘蛋白样结构域”，该结构域可以屏蔽SARS-CoV-2穗蛋白上的潜在表位或关键残基。需要进行生化分析或结构研究，以确定是否利用了预测的O-连接的聚糖位点。
图1.（a）SARS-CoV-2刺突蛋白接触残基的突变。将SARS-CoV-2的突突蛋白（上图）与最密切相关的SARS样CoV和SARS-CoV-1进行比对。与ACE2受体接触的刺突蛋白中的关键残基在SARS-CoV-2和SARS-CoV Urbani菌株中均标有蓝色框。 （b）获得多元裂解位点和O-连接的聚糖。多元裂解位点标记为灰色，三个相邻的预测的O-连接的聚糖标记为蓝色。多元裂解位点和O-连接的聚糖都是SARS-CoV-2特有的，以前在谱系B beta冠状病毒中没有见过。显示的序列来自NCBI GenBank，登录号为MN908947，MN996532，AY278741，KY417146，MK211376。穿山甲冠状病毒序列是从SRR10168377和SRR10168378（NCBI BioProject PRJNA573298）18,19产生的共有序列。
SARS-CoV-2起源的理论
SARS-CoV-2不可能通过实验室处理现有的SARS相关冠状病毒而出现。如上所述，SARS-CoV-2的RBD针对人ACE2受体的结合进行了优化，结合的有效结合溶液不同于已经预测的结合溶液。此外，如果已经进行了遗传操作，则可以预期将使用可用于β冠状病毒的几种反向遗传系统之一。但是，事实并非如此，因为遗传数据表明SARS-CoV-2并非源自任何先前使用的病毒主链17。相反，我们提出了两种可以合理解释SARS-CoV-2起源的方案：（i）人畜共患病转移之前在非人类动物宿主中的自然选择，以及（ii）人畜共患病转移之后在人类中的自然选择。我们还讨论了在传代过程中进行选择是否会引起相同的观察特征。
在动物宿主中进行选择
由于许多早期的COVID-19病例与武汉的华南海鲜和野生动植物市场有关，因此该地点可能存在动物来源。鉴于SARS-CoV-2与蝙蝠类似SARS的CoV（尤其是RaTG13）的相似性，蝙蝠充当SARS-CoV-2的宿主是有道理的。但是，重要的是要注意，先前在人类中爆发的冠状病毒包括直接接触蝙蝠以外的动物，包括带有遗传上与SARS-CoV-1或分别为MERS-CoV。以此类推，与SARS-Cov-2密切相关的病毒可能正在传播
对人类的隐秘适应
SARS-CoV-2的祖先也有可能从非人类动物跃迁到人类，具有上述基因组特征是通过在随后的人与人之间的传播过程中进行适应而获得的。我们推测，一旦（同时或连续）获得了这些适应措施，它将使疫情得以爆发，从而产生足够大且异常的肺炎病例群，从而触发最终发现它的监视系统。
到目前为止，所有测序的SARS-CoV-2基因组都具有很好的适应性RBD和多碱基切割位点，因此是从具有这些特征的共同祖先获得的。穿山甲中存在一种与SARS-CoV-2中非常相似的RBD，这意味着即使我们还没有确切的非人类祖细胞病毒，它也可能已经存在于跳跃到人类的病毒中。 。这使得多价切割位点插入发生在人与人之间的传播过程中。以甲型流感病毒HA基因为例，需要特定的插入或重组事件才能使SARS-CoV-2成为流行病原体。
使用当前可用的基因组序列数据估算SARS-CoV-2的最新共同祖先（tMRCA）的时间，表明病毒在2019年11月下旬至12月上旬出现，20,21与最早的回顾性确诊病例一致。因此，这种情况假定在最初的人畜共患病转移事件与多碱基切割位点的获取之间存在一段无法识别的人类传播时期。如果以前有许多人畜共患病事件在很长一段时间内产生人与人之间传播的短链（所谓的“口吃链”），则可能会有足够的机会。这基本上是阿拉伯半岛MERS-CoV的情况，其中所有人类病例都是病毒从单峰骆驼反复跳出的结果，产生了单个感染或传播的短链最终得以解决。迄今为止，在过去8年中发生了2499例病例之后，还没有出现人类适应症，这使MERS-CoV不能在人群中扎根。
我们如何测试SARS-CoV-2的秘密传播是否能使人类适应？储存血清样品的基因组学研究可能会提供重要信息，但鉴于病毒血症的时间相对较短，因此可能无法在历史样品中检测到低水平的SARS-CoV-2循环。回顾性血清学研究可能会提供参考，并且已经进行了一些此类研究。有人发现动物进口贸易商对冠状病毒的血清阳性率为13％，而另一人指出，中国南方一个村庄的居民中有3％对这些病毒呈血清阳性24。有趣的是，武汉的200名居民没有显示冠状病毒的血清反应活性。然而，至关重要的是，这些研究无法区分阳性血清反应是由于先前感染SARS-CoV-1还是-2。应该进行进一步的回顾性血清学研究，以确定先前人类在不同地理区域接触过β-冠状病毒的程度，尤其是使用可以区分多种β-冠状病毒的测定方法。
通过时的选择
BSL-2多年来在世界各地的多个实验室中进行了有关蝙蝠SARS样冠状病毒在细胞培养和/或动物模型中传代的基础研究25。还记录了在BSL-2密闭环境下工作的实验室人员在实验室获得SARS-CoV-1的实例29,30。因此，我们必须考虑有意或无意释放SARS-CoV-2的可能性。从理论上讲，SARS-CoV-2有可能在适应细胞培养传代过程中获得观察到的RBD突变位点，正如在SARS-CoV5和MERS-CoV31研究中所观察到的那样。然而，如果有功能，则获得多元裂解位点或O-连接的聚糖的说法反对这种情况。仅在细胞培养物或动物中长时间传播低致病性禽流感病毒后，才观察到新的多元切割位点。此外，通过细胞培养或动物传代产生SARS-CoV-2将需要事先分离具有非常高遗传相似性的祖病毒。然后，要在细胞培养物或具有类似于人的ACE-2受体的动物（例如雪貂）中进行大量传代程序，就需要随后的多碱基切割位点的产生。还怀疑在细胞培养传代中是否会发生O-连接聚糖的产生，因为这种突变通常表明免疫系统的参与，这在体外是不存在的。
结论
在全球COVID-19公共卫生紧急情况中，有理由怀疑流行病的起因是什么。对动物病毒如何越过物种边界来如此有效地感染人类的??详细了解将有助于预防未来的人畜共患病事件。例如，如果SARS-CoV-2已预先适应另一种动物，那么即使目前的流行病得到控制，我们也有未来再发生事件的风险。相反，如果我们描述的适应性过程发生在人类中，那么即使我们重复了人畜共患病的转移，除非发生相同系列的突变，它们也不太可能起飞。此外，鉴定出SARS-CoV-2的最亲近的动物亲属将大大有助于病毒功能的研究。确实，RaTG13 bat序列的可用性促进了此处进行的比较基因组分析，有助于揭示RBD中的关键突变以及多碱基切割位点的插入。
此处描述的基因组特征可以部分解释SARS-CoV-2在人类中的传染性和传播性。尽管基因组证据不支持SARS-CoV-2是实验室构建体的观点，但目前尚无法证明或否认本文所述的其起源的其他理论，并且尚不清楚未来的数据是否会帮助解决此问题。确定直接的非人类动物来源并从中获得病毒序列将是揭示病毒起源的最确定的方法。此外，获得有关该病毒的更多遗传和功能数据将是有帮助的，包括受体结合以及多碱基切割位点和预测的O-连接聚糖作用的实验研究。同样，对SARS-CoV-2潜在中间宿主的鉴定以及包括与武汉市场无关的早期病例的测序也将具有很高的信息意义。不管SARS-CoV-2的起源如何，对人类和其他动物进行肺炎的持续监测显然至关重要。
致谢
我们感谢所有为SAISA-CoV-2基因组序列贡献到GISAID数据库（https://www.gisaid.org/ 25）和为Virological.org 16（http://virological.org/ 4）。我们感谢惠康基金会的支持。 ECH由ARC澳大利亚获奖者奖学金（FL170100022）支持。 NIGA授予1U19AI135995-01支持KGA。 AR得到了Wellcome Trust（协作者奖206298 / Z / 17 / Z – ARTIC网络）和欧洲研究理事会（授权协议号725422 – ReservoirDOCS）的支持。
The Proximal Origin of SARS-CoV-2
Kristian G. Andersen1,2*, Andrew Rambaut3, W. Ian Lipkin4, Edward C. Holmes5 & Robert F. Garry6,7
1Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA.
2Scripps Research Translational Institute, La Jolla, CA 92037, USA.
3Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, UK.
4Center for Infection and Immunity, Mailman School of Public Health of Columbia University, New York, New York, USA.
5Marie Bashir Institute for Infectious Diseases and Biosecurity, School of Life and Environmental Sciences and School of Medical Sciences, The University of Sydney, Sydney, Australia.
6Tulane University, School of Medicine, Department of Microbiology and Immunology, New Orleans, LA, USA.
7Zalgen Labs, LCC, Germantown, MD, USA.
*Corresponding author:
Kristian G. Andersen
Department of Immunology and Microbiology,
The Scripps Research Institute,
La Jolla, CA 92037,
USA.
Since the first reports of a novel pneumonia (COVID-19) in Wuhan city, Hubei province, China there has been considerable discussion and uncertainty over the origin of the causative virus, SARS-CoV-2. Infections with SARS-CoV-2 are now widespread in China, with cases in every province. As of 14 February 2020, 64,473 such cases have been confirmed, with 1,384 deaths attributed to the virus. These official case numbers are likely an underestimate because of limited reporting of mild and asymptomatic cases, and the virus is clearly capable of efficient human-to-human transmission. Based on the possibility of spread to countries with weaker healthcare systems, the World Health Organization has declared the COVID-19 outbreak a Public Health Emergency of International Concern (PHEIC). There are currently neither vaccines nor specific treatments for this disease.
SARS-CoV-2 is the seventh member of the Coronaviridae known to infect humans. Three of these viruses, SARS CoV-1, MERS, and SARS-CoV-2, can cause severe disease; four, HKU1, NL63, OC43 and 229E, are associated with mild respiratory symptoms. Herein, we review what can be deduced about the origin and early evolution of SARS-CoV-2 from the comparative analysis of available genome sequence data. In particular, we offer a perspective on the notable features in the SARS-CoV-2 genome and discuss scenarios by which these features could have arisen. Importantly, this analysis provides evidence that SARS-CoV-2 is not a laboratory construct nor a purposefully manipulated virus.
The genomic comparison of both alpha- and betacoronaviruses (family Coronaviridae ) described below identifies two notable features of the SARS-CoV-2 genome: (i) based on structural modelling and early biochemical experiments, SARS-CoV-2 appears to be optimized for binding to the human ACE2 receptor; (ii) the highly variable spike (S) protein of SARS-CoV-2 has a polybasic (furin) cleavage site at the S1 and S2 boundary via the insertion of twelve nucleotides. Additionally, this event led to the acquisition of three predicted O-linked glycans around the polybasic cleavage site.
Mutations in the receptor binding domain of SARS-CoV-2
The receptor binding domain (RBD) in the spike protein of SARS-CoV and SARS-related coronaviruses is the most variable part of the virus genome. Six residues in the RBD appear to be critical for binding to the human ACE2 receptor and determining host range1. Using coordinates based on the Urbani strain of SARS-CoV, they are Y442, L472, N479, D480, T487, and Y4911. The corresponding residues in SARS-CoV-2 are L455, F486, Q493, S494, N501, and Y505. Five of these six residues are mutated in SARS-CoV-2 compared to its most closely related virus, RaTG13 sampled from a Rhinolophus affinis bat, to which it is ~96% identical2 (Figure 1a). Based on modeling1 and biochemical experiments3,4, SARS-CoV-2 seems to have an RBD that may bind with high affinity to ACE2 from human, non-human primate, ferret, pig, and cat, as well as other species with high receptor homology1. In contrast, SARS-CoV-2 may bind less efficiently to ACE2 in other species associated with SARS-like viruses, including rodents and civets1.
The phenylalanine (F) at residue 486 in the SARS-CoV-2 S protein corresponds to L472 in the SARS-CoV Urbani strain. Notably, in SARS-CoV cell culture experiments the L472 mutates to phenylalanine (L472F)5, which is predicted to be optimal for binding of the SARS-CoV RBD to the human ACE2 receptor6. However, a phenylalanine in this position is also present in several SARS-like CoVs from bats (Figure 1a). While these analyses suggest that SARS-CoV-2 may be capable of binding the human ACE2 receptor with high affinity, the interaction is not predicted to be optimal1. Additionally, several of the key residues in the RBD of SARS-CoV-2 are different to those previously described as optimal for human ACE2 receptor binding6. In contrast to these computational predictions, recent binding studies indicate that SARS-CoV-2 binds with high affinity to human ACE27. Thus the SARS-CoV-2 spike appears to be the result of selection on human or human-like ACE2 permitting another optimal binding solution to arise. This is strong evidence that SARS-CoV-2 is not the product of genetic engineering.
Polybasic cleavage site and O-linked glycans
The second notable feature of SARS-CoV-2 is a predicted polybasic cleavage site (RRAR) in the spike protein at the junction of S1 and S2, the two subunits of the spike protein (Figure 1b)8,9. In addition to two basic arginines and an alanine at the cleavage site, a leading proline is also inserted; thus, the fully inserted sequence is PRRA (Figure 1b). The strong turn created by the proline insertion is predicted to result in the addition of O-linked glycans to S673, T678, and S686 that flank the polybasic cleavage site. A polybasic cleavage site has not previously been observed in related lineage B betacoronaviruses and is a unique feature of SARS-CoV-2. Some human betacoronaviruses, including HCoV-HKU1 (lineage A), have polybasic cleavage sites, as well as predicted O-linked glycans near the S1/S2 cleavage site.
While the functional consequence of the polybasic cleavage site in SARS-CoV-2 is unknown, experiments with SARS-CoV have shown that engineering such a site at the S1/S2 junction enhances cell–cell fusion but does not affect virus entry10. Polybasic cleavage sites allow effective cleavage by furin and other proteases, and can be acquired at the junction of the two subunits of the haemagglutinin (HA) protein of avian influenza viruses in conditions that select for rapid virus replication and transmission (e.g. highly dense chicken populations). HA serves a similar function in cell-cell fusion and viral entry as the coronavirus S protein. Acquisition of a polybasic cleavage site in HA, by either insertion or recombination, converts low pathogenicity avian influenza viruses into highly pathogenic forms11-13. The acquisition of polybasic cleavage sites by the influenza virus HA has also been observed after repeated forced passage in cell culture or through animals14,15. Similarly, an avirulent isolate of Newcastle Disease virus became highly pathogenic during serial passage in chickens by incremental acquisition of a polybasic cleavage site at the junction of its fusion protein subunits16. The potential function of the three predicted O-linked glycans is less clear, but they could create a “mucin-like domain” that would shield potential epitopes or key residues on the SARS-CoV-2 spike protein. Biochemical analyses or structural studies are required to determine whether or not the predicted O-linked glycan sites are utilized.
Figure 1. (a) Mutations in contact residues of the SARS-CoV-2 spike protein. The spike protein of SARS-CoV-2 (top) was aligned against the most closely related SARS-like CoVs and SARS-CoV-1. Key residues in the spike protein that make contact to the ACE2 receptor are marked with blue boxes in both SARS-CoV-2 and the SARS-CoV Urbani strain. ( b) Acquisition of polybasic cleavage site and O-linked glycans. The polybasic cleavage site is marked in grey with the three adjacent predicted O-linked glycans in blue. Both the polybasic cleavage site and O-linked glycans are unique to SARS-CoV-2 and not previously seen in lineage B betacoronaviruses. Sequences shown are from NCBI GenBank, accession numbers MN908947, MN996532, AY278741, KY417146, MK211376. The pangolin coronavirus sequences are a consensus generated from SRR10168377 and SRR10168378 (NCBI BioProject PRJNA573298)18,19.
Theories of SARS-CoV-2 origins
It is improbable that SARS-CoV-2 emerged through laboratory manipulation of an existing SARS-related coronavirus. As noted above, the RBD of SARS-CoV-2 is optimized for human ACE2 receptor binding with an efficient binding solution different to that which would have been predicted. Further, if genetic manipulation had been performed, one would expect that one of the several reverse genetic systems available for betacoronaviruses would have been used. However, this is not the case as the genetic data shows that SARS-CoV-2 is not derived from any previously used virus backbone17. Instead, we propose two scenarios that can plausibly explain the origin of SARS-CoV-2: (i) natural selection in a non-human animal host prior to zoonotic transfer, and (ii) natural selection in humans following zoonotic transfer. We also discuss whether selection during passage in culture could have given rise to the same observed features.
Selection in an animal host. As many of the early cases of COVID-19 were linked to the Huanan seafood and wildlife market in Wuhan, it is possible that an animal source was present at this location. Given the similarity of SARS-CoV-2 to bat SARS-like CoVs, particularly RaTG13, it is plausible that bats serve as reservoir hosts for SARS-CoV-2. It is important, however, to note that previous outbreaks of betacoronaviruses in humans involved direct exposure to animals other than bats, including civets (SARS) and camels (MERS), that carry viruses that are genetically very similar to SARS-CoV-1 or MERS-CoV, respectively. By analogy, viruses closely related to SARS-Cov-2 may be circulating in one or more animal species. Initial analyses indicate that Malayan pangolins ( Manis javanica ) illegally imported into Guangdong province contain a CoV that is similar to SARS-CoV-218,19. Although the bat virus RaTG13 remains the closest relative to SARS-CoV-2 across the whole genome, the Malayan pangolin CoV is identical to SARS-CoV-2 at all six key RBD residues (Figure 1). However, no pangolin CoV has yet been identified that is sufficiently similar to SARS-CoV-2 across its entire genome to support direct human infection. In addition, the pangolin CoV does not carry a polybasic cleavage site insertion. For a precursor virus to acquire the polybasic cleavage site and mutations in the spike protein suitable for human ACE2 receptor binding, an animal host would likely have to have a high population density – to allow natural selection to proceed efficiently – and an ACE2 gene that is similar to the human orthologue. Further characterization of CoVs in pangolins and other animals that may harbour SARS-CoV-like viruses should be a public health priority.
Cryptic adaptation to humans. It is also possible that a progenitor to SARS-CoV-2 jumped from a non-human animal to humans, with the genomic features described above acquired through adaptation during subsequent human-to-human transmission. We surmise that once these adaptations were acquired (either together or in series) it would enable the outbreak to take-off, producing a sufficiently large and unusual cluster of pneumonia cases to trigger the surveillance system that ultimately detected it.
All SARS-CoV-2 genomes sequenced so far have the well adapted RBD and the polybasic cleavage site, and are thus derived from a common ancestor that had these features. The presence of an RBD in pangolins that is very similar to the one in SARS-CoV-2 means that this was likely already present in the virus that jumped to humans, even if we don’t yet have the exact non-human progenitor virus. This leaves the polybasic cleavage site insertion to occur during human-to-human transmission. Following the example of the influenza A virus HA gene, a specific insertion or recombination event is required to enable the emergence of SARS-CoV-2 as an epidemic pathogen.
Estimates of the timing of the most recent common ancestor (tMRCA) of SARS-CoV-2 using currently available genome sequence data point to virus emergence in late November to early December 201920,21, compatible with the earliest retrospectively confirmed cases22. Hence, this scenario presumes a period of unrecognised transmission in humans between the initial zoonotic transfer event and the acquisition of the polybasic cleavage site. Sufficient opportunity could occur if there had been many prior zoonotic events producing short chains of human-to-human transmission (so-called ‘stuttering chains’) over an extended period. This is essentially the situation for MERS-CoV in the Arabian Peninsula where all the human cases are the result of repeated jumps of the virus from dromedary camels, producing single infections or short chains of transmission that eventually resolve. To date, after 2,499 cases over 8 years, no human adaptation has emerged that has allowed MERS-CoV to take hold in the human population.
How could we test whether cryptic spread of SARS-CoV-2 enabled human adaptation? Metagenomic studies of banked serum samples could provide important information, but given the relatively short period of viremia it may be impossible to detect low level SARS-CoV-2 circulation in historical samples. Retrospective serological studies potentially could be informative and a few such studies have already been conducted. One found that animal importation traders had a 13% seropositivity to coronaviruses23, while another noted that 3% residents of a village in Southern China were seropositive to these viruses24. Interestingly, 200 residents of Wuhan did not show coronavirus seroreactivity. Critically, however, these studies could not have distinguished whether positive serological responses were due to a prior infection with SARS-CoV-1 or -2. Further retrospective serological studies should be conducted to determine the extent of prior human exposure to betacoronaviruses in different geographic areas, particularly using assays that can distinguish among multiple betacoronaviruses.
Selection during passage. Basic research involving passage of bat SARS-like coronaviruses in cell culture and/or animal models have been ongoing in BSL-2 for many years in multiple laboratories across the world25-28. There are also documented instances of the laboratory acquisition of SARS-CoV-1 by laboratory personnel working under BSL-2 containment29,30. We must therefore consider the possibility of a deliberate or inadvertent release of SARS-CoV-2. In theory, it is possible that SARS-CoV-2 acquired the observed RBD mutations site during adaptation to passage in cell culture, as has been observed in studies with SARS-CoV5 as well as MERS-CoV31. However, the acquisition of the polybasic cleavage site or O-linked glycans - if functional - argues against this scenario. New polybasic cleavage sites have only been observed after prolonged passaging of low pathogenicity avian influenza virus in cell culture or animals. Furthermore, the generation of SARS-CoV-2 by cell culture or animal passage would have required prior isolation of a progenitor virus with a very high genetic similarity. Subsequent generation of a polybasic cleavage site would have then required an intense program of passage in cell culture or animals with ACE-2 receptor similar to humans (e.g. ferrets). It is also questionable whether generation of the O-linked glycans would have occurred on cell culture passage, as such mutations typically suggest the involvement of an immune system, that is not present in vitro .
Conclusions
In the midst of the global COVID-19 public health emergency it is reasonable to wonder why the origins of the epidemic matter. A detailed understanding of how an animal virus jumped species boundaries to infect humans so productively will help in the prevention of future zoonotic events. For example, if SARS-CoV-2 pre-adapted in another animal species then we are at risk of future re-emergence events even if the current epidemic is controlled. In contrast, if the adaptive process we describe occurred in humans, then even if we have repeated zoonotic transfers they are unlikely to take-off unless the same series of mutations occurs. In addition, identifying the closest animal relatives of SARS-CoV-2 will greatly assist studies of virus function. Indeed, the availability of the RaTG13 bat sequence facilitated the comparative genomic analysis performed here, helping to reveal the key mutations in the RBD as well as the polybasic cleavage site insertion.
The genomic features described here may in part explain the infectiousness and transmissibility of SARS-CoV-2 in humans. Although genomic evidence does not support the idea that SARS-CoV-2 is a laboratory construct, it is currently impossible to prove or disprove the other theories of its origin described here, and it is unclear whether future data will help resolve this issue. Identifying the immediate non-human animal source and obtaining virus sequences from it would be the most definitive way of revealing virus origins. In addition, it would be helpful to obtain more genetic and functional data about the virus, including experimental studies of receptor binding and the role of the polybasic cleavage site and predicted O-linked glycans. The identification of a potential intermediate host of SARS-CoV-2, as well as the sequencing of very early cases including those not connected to the Wuhan market, would similarly be highly informative. Irrespective of how SARS-CoV-2 originated, the ongoing surveillance of pneumonia in humans and other animals is clearly of utmost importance.
Acknowledgements
We thank all those who have contributed SARS-CoV-2 genome sequences to the GISAID database (https://www.gisaid.org/ 25) and contributed analyses and ideas to Virological.org 16 (http://virological.org/ 4). We thank the Wellcome Trust for supporting this work. ECH is supported by an ARC Australian Laureate Fellowship (FL170100022). KGA is supported by NIH grant 1U19AI135995-01. AR is supported by the Wellcome Trust (Collaborators Award 206298/Z/17/Z – ARTIC network) and the European Research Council (grant agreement no. 725422 – ReservoirDOCS).
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