Summary:  There is a consensus that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) originated naturally from Bat coronaviruses (CoVs), in particular RaTG13. However, the SARS-CoV-2 host tropism/adaptation pattern has significant discrepancies compared to other CoVs, raising questions concerning the proximal origin of SARS-CoV-2. The flat and non-sunken surface of the sialic acid-binding domain of SARS-CoV-2 spike protein (S protein) conflicts with the general adaptation and survival pattern observed for all other CoVs. Unlike RaTG13, SARS-CoV-2 recombination presumably occurred between the S1/S2 domains of S protein enabling host furin protease utilization. Although millions of recorded cases have been recorded globally, SARS-CoV-2 S protein does not have any apparent further recombination, placing it in conflict with the recombination models of other CoVs.  Similarly, the S protein receptor-binding domain (RBD)  of SARS-CoV-2  has not accumulated high-frequency  non-synonymous substitutions, differentiating SARS-CoV-2 from other CoVs that have positive selection/adaptation mutations in their RBDs.

摘要：有一個共識，即嚴重急性呼吸系統綜合症冠狀病毒2（SARS-CoV-2）自然起源於蝙蝠冠狀病毒（CoV），尤其是RaTG13。然而，與其他CoV相比，SARS-CoV-2宿主的向性/適應性模式存在顯着差異，這引發了有關SARS-CoV-2的近端起源的問題。 SARS-CoV-2穗蛋白（S蛋白）的唾液酸結合結構域的平坦且不凹陷的表面與所有其他CoV所觀察到的一般適應性和存活模式相衝突。與RaTG13不同，SARS-CoV-2重組可能發生在S蛋白的S1 / S2結構域之間，從而可以利用宿主弗林蛋白酶。儘管全球已記錄了數百萬例已記錄病例，但SARS-CoV-2 S蛋白沒有任何明顯的進一步重組，使其與其他CoV的重組模型相衝突。同樣，SARS-CoV-2的S蛋白受體結合域（RBD）並未積累高頻非同義取代，從而使SARS-CoV-2與其他在其RBD中具有正選擇/適應突變的CoV區別開來。

Andersen及其同事記錄了來自BatCoV RaTG13 1,2的SARS-CoV-2的天然來源。 SARS-CoV-2是能夠感染人類的第七種人畜共患冠狀病毒，但也是第一種也是唯一具有大流行潛力的人冠狀病毒（HCoV）。蝙蝠或齧齒動物CoV表現出S蛋白受體結合域（RBD）的某些特定變化 以及S蛋白聚糖結合的N端

 during host tropism/adaptation 4,5. SARS-CoV-2, unlike other CoVs, does not have those signature changes, suggesting that these RBD and NTD subdomains are of very recent origin.  The "Canyon Hypothesis" explains the development of canyons, depression zones or cavities on the surfaces of influenza virus, human rhinovirus, and Meningo viruses 6. In CoVs (except SARS-CoV-2), the S protein NTD domain has several predicted glycan-binding domains, with a common feature being the hidden localization of these glycan binding domains to cavities to limit their access to antibodies and immune cells 5. This pattern of CoVs is thought to be an evolutionary measure to restrict the recognition of these active sites by host immune system 4.     HCoVs can evade detection by host glycan-binding immune receptors. Comparative genomic analysis of six HCoVs with their corresponding native bat or rodent CoVs suggests compatibility with the "Canyon Hypothesis" resulting from various adaptive S protein NTD non-synonymous mutations near or at the glycan binding domain which are predicted to result in these NTD domains being hidden below the protein surface5. The predicted flat, non-sunken pattern of the SARS-CoV-2 S protein NTD glycan binding domains conflicts with this evolutionary host tropism/adaptation strategy 7.  A template-switching mechanism is presumably  responsible for the high rate of RNA recombination in CoVs. In host cells, CoV RNAs show discontinuous RNA synthesis materialized by pauses of the RNA-dependent complex and subsequent jumps to downstream template acceptor sequences. This process results in subgenomic minus strand RNAs which serve as templates for subgenomic mRNAs. Due to the mechanistic similarity to recombination, this process might be at the origin of recombinant CoVs co-opting other CoV or even host related sequences8. Instances include the mouse hepatitis coronavirus S protein NTD sialic acid binding domain, likely arising from recombination of viral RNA with human galectin RNA sequences 8

在宿主向性/適應過程4,5。與其他CoV不同，SARS-CoV-2沒有這些簽名更改，表明這些RBD和NTD子域是最近才起源的。 “峽谷假說”解釋了流感病毒，人類鼻病毒和腦膜炎病毒表面上峽谷，凹陷區域或空腔的形成。6在冠狀病毒（SARS-CoV-2除外）中，S蛋白NTD結構域具有幾種預測的聚糖-結合結構域，其共同特徵是這些聚糖結合結構域在腔中的隱蔽定位，以限制它們對抗體和免疫細胞的訪問。5 CoV的這種模式被認為是一種進化手段，可通過以下方式限制對這些活性位點的識別宿主免疫系統4. HCoV可以逃避宿主聚糖結合免疫受體的檢測。對六種HCoV及其相應的天然蝙蝠或齧齒動物CoV進行的比較基因組分析表明，與聚糖結合域附近或處的各種適應性S蛋白NTD非同義突變導致的“峽谷假說”具有相容性，預計這些突變會導致這些NTD域成為隱藏在蛋白質表面以下5。 SARS-CoV-2 S蛋白NTD聚糖結合結構域的預測的平坦，非凹陷模式與這種進化宿主的向性/適應策略7相衝突。模板轉換機制可能是CoV中RNA重組率很高的原因。在宿主細胞中，CoV RNA表現出不連續的RNA合成，這是由於RNA依賴複合物的暫停以及隨後跳至下游模板受體序列而實現的。該過程產生亞基因組負鏈RNA，其充當亞基因組mRNA的模板。由於與重組的機制相似性，該過程可能是重組CoV選擇其他CoV甚至宿主相關序列的起點。實例包括小鼠肝炎冠狀病毒S蛋白NTD唾液酸結合域，可能是病毒RNA與人半乳糖凝集素RNA序列重組產生的8

The furin recognition motif present at the SARS-CoV2 S1/S2 junction has no analogy in other "linage B" beta-coronaviruses, including neither  pangolin-CoV  nor  RaTG13 1.  This indicates that the S protein S1/S2 junction is not a hot spot for RNA recombination termination that depends on a pattern swapping templates (copy-choice) 8.  Additionally, clinical isolates of SARS-CoV-2 S protein have not indicated any further recombination in this S1/S2 area, suggesting that the addition of a motif for S1/S2 site furin cleavage constituted a unique recombination occurrence. Finally, the CoV-unique insertion of 4 amino-acids creating a novel RRAR furin cleavage site introduces two arginine codons CGG-CGG, whose usage is extremely rare in CoVs, further supporting the hypothesis of a unique recombination occurrence. 

存在於SARS-CoV2 S1 / S2連接處的弗林蛋白酶識別基序與其他“甲型B”β冠狀病毒沒有相似之處，包括穿山甲CoV和RaTG13 1都不存在。這表明S蛋白S1 / S2連接處不熱。 RNA重組終止的位點，取決於模式交換模板（複製選擇）8。此外，SARS-CoV-2 S蛋白的臨床分離株未表明在該S1 / S2區域有任何進一步的重組，表明添加了一個 S1 / S2位點弗林蛋白酶裂解的基序構成獨特的重組發生。 最後，通過CoV獨特地插入4個氨基酸，創建了一個新的RRAR弗林蛋白酶切割位點，引入了兩個精氨酸密碼子CGG-CGG，它們在CoV中極少使用，進一步支持了獨特重組發生的假設。

HCoVs have high-frequency “hot spots” for non-synonymous amino acid replacements that  can  possibly create  positive selection for  host tropism/adaptation, resistance to neutralizing  antibodies, or immune evasion 2.  Interestingly, clinical SARS-CoV-2 isolates to date have only a single high frequency non-synonymous mutation, D614G, in their S protein 9. Based on currently known mutation rates and patterns in clinical isolates of SARS-CoV-2, the S protein does not appear to be a mutational “hot spot” for SARS-CoV-2, unlike other human CoVs.   SARS-CoV-2 is the seventh HCoV, but the first HCoV with pandemic potential. SARS-CoV disappeared without a pandemic, and MERS-CoV is mostly endemic to the Arabian Peninsula with some additional limited traveler infections resulting in outbreaks in South Korea 3,4. These unique features of SARS-CoV-2 raise several questions concerning the proximal origin of the virus that require further discussion. 

HCoV具有非同義氨基酸替代的高頻“熱點”，可能為宿主的嗜性/適應性，對中和抗體的抵抗力或免疫逃避2產生積極的選擇。有趣的是，迄今為止，臨床SARS-CoV-2分離株具有 S蛋白9中只有一個高頻非同義詞突變D614G。根據目前已知的SARS-CoV-2臨床分離株的突變率和模式，S蛋白似乎不是突變的“熱點”。 與SARS-CoV-2不同，它不同於其他人類CoV。 SARS-CoV-2是第七個HCoV，但第一個具有大流行潛力的HCoV。 SARS-CoV在沒有大流行的情況下就消失了，而MERS-CoV在阿拉伯半島是很流行的，另外還有一些有限的旅行者感染，導致了韓國的爆發3,4。 SARS-CoV-2的這些獨特特徵引起了有關病毒近端起源的幾個問題，需要進一步討論。

     論文鏈接：

https://onlinelibrary.wiley.com/doi/epdf/10.1002/jmv.26478