非典不可思议？武汉冠状病毒家谱

不明飞行物护理分钟

大流行的COVID-19是由SARS-CoV-2冠状病毒（2019-nCoV）引起的潜在严重的急性呼吸道感染，已在全球正式宣布。关于Habré的许多信息都涉及此主题-始终记住，它既可靠又有用，反之亦然。

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洗手，照顾亲人，尽可能呆在家里并远程工作。

阅读有关以下内容的出版物：冠状病毒 | 远程工作

施正立在武汉病毒研究所的实验室

生物学

RBD CoV2, ACE2.
() 2019-nCov. NTD, N- . RBD, - . RBM, - . SD1, 1. SD2, 2. FP, . HR1, 1. HR2, 2. TM, . IC, .
() RBD 2019-nCov. RBM .
() RBD 2019-nCov, ACE2. ACE2 . RBD 2019-nCoV , RBM — . RBD 2019-nCov . N- ACE2, , .

Overall structure of 2019-nCoV RBD bound with ACE2.
(a) Overall topology of 2019-nCoV spike monomer. NTD, N-terminal domain. RBD, receptor-binding domain. RBM, receptor-binding motif. SD1, subdomain 1. SD2, subdomain 2. FP, fusion peptide. HR1, heptad repeat 1. HR2, heptad repeat 2. TM, transmembrane region. IC, intracellular domain.
(b) Sequence and secondary structures of 2019-nCoV RBD. The RBM is colored red.
() Overall structure of 2019-nCoV RBD bound with ACE2. ACE2 is colored green. 2019-nCoV RBD core is colored cyan and RBM is colored red. Disulfide bonds in the 2019-nCoV RBD are shown as stick and indicated by yellow arrows. The N-terminal helix of ACE2 responsible for binding is labeled.

穿山甲

, , - 2019 . (Manis pentadactyla) 25 (Manis javanica). ; , , . , , , , , , .

Pangolins used in the study were confiscated by Customs and Department of Forestry of Guangdong Province in March-December 2019. They include four Chinese pangolins (Manis pentadactyla) and 25 Malayan pangolins (Manis javanica). These animals were sent to the wildlife rescue center, and were mostly inactive and sobbing, and eventually died in custody despite exhausting rescue efforts. Tissue samples were taken from the lung, lymph nodes, liver, spleen, muscle, kidney, and other tissues from pangolins that had just died for histopathological and virological examinations.

我们收到了2017年8月至2018年1月从18个马来穿山甲（Manis javanica）采集的冷冻组织样本（肺，肠，血液）。这些穿山甲是广西海关在反走私行动中获得的。令人惊讶的是，其RNA的高效测序揭示了43个样本中的六个（两个肺，两个肠，一个肺和肠的混合物，一个血液）中存在冠状病毒。
...
, , , RaTG13 2019-CoV2 (. 1c, d). , 2019-CoV2 - (RBD; 97,4%; . 1c . 2a), 2019-CoV2 RaTG13. RaTG13 2019-CoV2 89,2% RBD. 2019-CoV2 RBD, RaTG13 2019-CoV2 ( 442, SARS-CoV ).

We received frozen tissue (lungs, intestine, blood) samples that were collected from 18 Malayan pangolins (Manis javanica) during August 2017-January 2018. These pangolins were obtained during the anti-smuggling operations by Guangxi Customs. Strikingly, high-throughput sequencing of their RNA revealed the presence of coronaviruses in six (two lung, two intestine, one lung-intestine mix, one blood) of 43 samples. With the sequence read data, and by filling gaps with amplicon sequencing, we were able to obtain six full or nearly full genome sequences — denoted GX/P1E, GX/P2V, GX/P3B, GX/P4L, GX/P5E and GX/P5L — that fall into the 2019-CoV2 lineage (within the genus Betacoronavirus) in a phylogenetic analysis (Figure 1a).
…
More notable, however, was the observation of putative recombination signals between the pangolins coronaviruses, bat coronaviruses RaTG13, and human 2019-CoV2 (Figure 1c, d). In particular, 2019-CoV2 exhibits very high sequence similarity to the Guangdong pangolin coronaviruses in the receptor-binding domain (RBD; 97.4% amino acid similarity; indicated by red arrow in Figure 1c and Figure 2a), even though it is most closely related to bat coronavirus RaTG13 in the remainder of the viral genome. Bat CoV RaTG and the human 2019-CoV2 have only 89.2% amino acid similarity in RBD. Indeed, the Guangdong pangolin coronaviruses and 2019-CoV2 possess identical amino acids at the five critical residues of the RBD, whereas RaTG13 only shares one amino acid with 2019-CoV2 (residue 442, human SARS-CoV numbering).

有趣的是，仅对RBD中同义位点的系统发育分析表明，广东穿山甲的系统发育位置与其余病毒基因组的系统发育位置一致，即它不是2019-CoV2的最近亲缘关系（图2b）。因此，穿山甲冠状病毒和2019-CoV2的RBD中氨基酸的相似性可能是由于选择性介导的趋同进化而不是重组引起的，尽管很难根据可用数据在这些情况之间进行选择。

源文字

Interestingly, a phylogenetic analysis of synonymous sites alone in the RBD revealed that the phylogenetic position of the Guangdong pangolin is consistent with that in the remainder of the viral genome, rather than being the closest relative of 2019-CoV2 (Figure 2b). Hence, it is possible that the amino acid similarity between the RBD of the Guangdong pangolin coronaviruses and 2019-CoV2 is due to selectively-mediated convergent evolution rather than recombination, although it is difficult to choose between these scenarios on current data.

加冕者家谱

我打得很整齐，但是很努力

, , , , , SARS-CoV-2. () COVID-19 . SARS-CoV-2 , .

Patients with hypertension, diabetes, coronary heart disease, cerebrovascular illness, chronic obstructive pulmonary disease, and kidney dysfunction have worse clinical outcomes when infected with SARS-CoV-2, for unknown reasons. The purpose of this review is to summarize the evidence for the existence of elevated plasmin(ogen) in COVID-19 patients with these comorbid conditions. Plasmin, and other proteases, may cleave a newly inserted furin site in the S protein of SARS-CoV-2, extracellularly, which increases its infectivity and virulence.

发现所有具有SARS-CoV-2蛋白同源性大于40％的刺突蛋白都没有弗林蛋白酶切割位点（图1，表1），包括Bat-CoV RaTG13和SARS-CoV（序列同一性为97.4） ％和78.6％）。由于独特的PRRA插入，SARS-CoV-2中的RRAR弗林蛋白酶位点在其家族中是独一无二的。SARS-CoV-2中的此站点几乎不可能从MERS，HCoV-HKU1等演变而来。从数据库中当前可用的序列中，我们很难找到源。也许还有更多的进化中间序列等待发现。

源文字

It was found that all Spike with a SARS-CoV-2 Spike sequence homology greater than 40% did not have a furin cleavage site (Figure 1, Table 1), including Bat-CoV RaTG13 and SARS-CoV (with sequence identity as 97.4% and 78.6%, respectively). The furin cleavage site “RRAR” in SARS-CoV-2 is unique in its family, rendering by its unique insert of “PRRA”. The furin cleavage site of SARS-CoV-2 is unlikely to have evolved from MERS, HCoV-HKU1, and so on. From the currently available sequences in databases, it is difficult for us to find the source. Perhaps there are still many evolutionary intermediate sequences waiting to be discovered.

为了研究主要氨基酸残基序列上的蛋白水解切割是否可以促进细胞融合活性，我们突变了穗状SARS-CoV蛋白，在两个位置之一中创建了弗林蛋白酶识别位点（RRSRR）。

源文字

To investigate whether proteolytic cleavage at the basic amino acid residues, were it to occur, might facilitate cell–cell fusion activity, we mutated the wild-type SARS-CoV glycoprotein to construct a prototypic furin recognition site (RRSRR) at either position.

为了调查SARS-CoV S1-S2和S2'位置作为蛋白水解切割位点的潜在用途，我们首先通过以下突变664-SLLRSTSQSI-SLL RRSRR SI-671（S1-S2）引入了弗林蛋白酶切割识别位点。792-LKPTKRSF-LKRTKRSF-799（S2'）。

源文字

To examine the potential use of the SARS-CoV S1–S2 and S2? positions as sites for proteolytic cleavage, we first introduced furin cleavage recognition sites at these locations by making the following mutations 664-SLLRSTSQSI — SLLRRSRRSI-671 (S1–S2) and 792-LKPTKRSF — LKRTKRSF-799 (S2?).

北京2019

S2' QX , , IBV (WT-IBV). , IBV S2 ( -S2) . IBV .
…
, , FP CoV, , , -, , .

Mutation of the S2' site of QX genotype (QX-type) spike protein (S) in a recombinant virus background results in higher pathogenicity, pronounced neural symptoms and neurotropism when compared with conditions in wild-type IBV (WT-IBV) infected chickens. In this study, we present evidence suggesting that recombinant IBV with a mutant S2' site (furin-S2' site) leads to higher mortality. Infection with mutant IBV induces severe encephalitis and breaks the blood–brain barrier.
…
In summary, our results demonstrate that the furin cleavage site upstream of the FP in S protein is an important site for CoV, modulating entry, cell–virus fusion, adaptation to its host cell, cell tropism and pathogenicity, but not antigenicity.

, HKU4 , HKU4, . , S746R N762A, HKU4.
…
, hPPC hECP HKU4 HKU4 . , HKU4 S1/S2, .

To evaluate the potential genetic changes required for HKU4 to infect human cells, we reengineered HKU4 spike, aiming to build its capacity to mediate viral entry into human cells. To this end, we introduced two single mutations, S746R and N762A, into HKU4 spike. The S746R mutation was expected to restore the hPPC motif in HKU4 spike, whereas the N762A mutation likely disrupted the potential N-linked glycosylation site in the hECP motif in HKU4 spike.
…
Therefore, the reengineered hPPC and hECP motifs enabled HKU4 spike to be activated by human endogenous proteases and thereby allowed HKU4 pseudoviruses to bypass the need for exogenous proteases to enter human cells. These results reveal that HKU4 spike needs only two single mutations at the S1/S2 boundary to gain the full capacity to mediate viral entry into human cells.

简而言之，通过将HEK293T细胞与携带HIV-1基因的质粒，表达Env的Env缺陷型荧光素酶（pNL4-3.luc.RE-）和编码MERS的质粒共转染，获得表达荧光素酶报道基因的假型MERS-CoV-穗状逆转录病毒。 -CoV刺突蛋白。

源文字

Briefly, MERS-CoV-spike-pseudotyped retroviruses expressing a luciferase reporter gene were prepared by cotransfecting HEK293T cells with a plasmid carrying Env-defective, luciferase-expressing HIV-1 genome (pNL4–3.luc.R-E-) and a plasmid encoding MERS-CoV spike protein.

RaTG13来自哪里

, - - (RdRp) (BatCoV RaTG13), Rhinolophus affinis , 2019-CoV2. ( GISAID EPI_ISL_402131). Simplot , 2019-CoV2 RaTG13 (Fig. 1c), 96,2%.

We then found that a short region of RNA-dependent RNA polymerase (RdRp) from a bat coronavirus (BatCoV RaTG13) — which was previously detected in Rhinolophus affinis from Yunnan province — showed high sequence identity to 2019-CoV2. We carried out full-length sequencing on this RNA sample (GISAID accession number EPI_ISL_402131). Simplot analysis showed that 2019-CoV2 was highly similar throughout the genome to RaTG13 (Fig. 1c), with an overall genome sequence identity of 96.2%.

2013年5月至2013年7月，蝙蝠在中国云南四个州的五个县的不同地区被捕。

源文字

Bats were captured from various locations in five counties of four prefectures of Yunnan Province, China, from May to July 2013.

武汉1

最重要的是，我们报告了首次从Vero E6细胞中从蝙蝠粪便中分离出的样本中分离出活病毒SL-CoV（蝙蝠SL-CoV-WIV1），该病毒具有典型的冠状病毒形态，具有99.9％的基因组同一性Rs3367并使用ACE2人，灵猫和马蹄形的骨头进入细胞。初步的体外测试表明，WIV1还具有较广泛的物种嗜性。

源文字

Most importantly, we report the first recorded isolation of a live SL-CoV (bat SL-CoV-WIV1) from bat faecal samples in Vero E6 cells, which has typical coronavirus morphology, 99.9% sequence identity to Rs3367 and uses ACE2 from humans, civets and Chinese horseshoe bats for cell entry. Preliminary in vitro testing indicates that WIV1 also has a broad species tropism.

云南其他菌株

1999年：首次嵌合体冠状病毒

使用定向RNA重组，我们构建了一种冠状病毒小鼠肝炎病毒（MHV）的突变体，其中脊柱形的胞外域被猫传染性腹膜炎病毒蛋白的高度趋异的胞外域所取代。所得的嵌合病毒（称为fMHV）获得了感染猫​​细胞的能力，同时丧失了感染培养物中小鼠细胞的能力。

源文字

Using targeted RNA recombination, we constructed a mutant of the coronavirus mouse hepatitis virus (MHV) in which the ectodomain of the spike glycoprotein (S) was replaced with the highly divergent ectodomain of the S protein of feline infectious peritonitis virus. The resulting chimeric virus, designated fMHV, acquired the ability to infect feline cells and simultaneously lost the ability to infect murine cells in tissue culture.

小鼠冠状病毒中仅有少量突变，可通过体外钝化使小鼠冠状病毒从小鼠限制性嗜性转变为扩大的宿主范围。我们研究的一种此类病毒获得了两个假定的硫酸乙酰肝素结合位点，同时在其他地方保留了弗林蛋白酶位点。

源文字

Only a relatively few mutations in its spike protein allow the murine coronavirus to switch from a murine-restricted tropism to an extended host range by being passaged in vitro. One such virus that we studied had acquired two putative heparan sulfate-binding sites while preserving another site in the furin-cleavage motif.

MHV/pi23 — , 23 600 , MHV/BHK, HS- S1 , MHV/BHK, , HS- . MHV/pi23 , , MHV/BHK. HS- , , , S, HR1 (Fig. 1), . S, .

MHV/pi23, a virus obtained after 23 of the 600 passages that resulted in MHV/BHK, also contains a putative HS-binding site in the S1 domain at the same position as in MHV/BHK, albeit as a smaller insertion, while it lacks the putative HS-binding site immediately upstream of the fusion peptide. MHV/pi23 does infect nonmurine cells to some extent but much less efficiently than MHV/BHK. In addition to the multiple HS-binding sites, however, mutations found in other parts of the S protein, such as the HR1 domain and the putative fusion peptide (Fig. 1), might also contribute to the efficient entry into nonmurine cells. We are currently in the process of determining the S protein mutations that are required for the extended host range phenotype.

为了更好地理解MERS-CoV物种的适应性，我们使用了次优DPP4变异体来研究病毒适应性。病毒在表达该DPP4变体的细胞上传代导致病毒刺突蛋白中突变的积累，从而增加了复制。

源文字

To better understand the species adaptability of MERS-CoV, we identified a suboptimal species-derived variant of DPP4 to study viral adaption. Passaging virus on cells expressing this DPP4 variant led to accumulation of mutations in the viral spike which increased replication.

（F）在不同传代中MERS-CoV刺突蛋白中单突变和双突变的出现方案。
（G）MERS-CoV刺突蛋白中突变的位置。

源文字

(F) Schematic of single and double mutation emergence in MERS-CoV spike over different passages.
(G) Location of mutations within MERS-CoV spike.

巴里克如何为所有人点亮道路

II 59 (MHV-A59). , MHV 31,5 . , MHV-A59 ~ 31,5 ... , , , … , , .

源文字

A novel method was developed to assemble a full-length infectious cDNA of the group II coronavirus mouse hepatitis virus strain A59 (MHV-A59). Seven contiguous cDNA clones that spanned the 31.5-kb MHV genome were isolated. The ends of the cDNAs were engineered with unique junctions and assembled with only the adjacent cDNA subclones, resulting in an intact MHV-A59 cDNA construct of ?31.5 kb in length. The interconnecting restriction site junctions that are located at the ends of each cDNA are systematically removed during the assembly of the complete full-length cDNA product, allowing reassembly without the introduction of nucleotide changes… The method has the potential to be used to construct viral, microbial, or eukaryotic genomes approaching several million base pairs in length and used to insert restriction sites at any given nucleotide in a microbial genome.

SARS-2003

, , SARS-CoV Urbani SARS ( SARS-CoV), , . , , … SARS-CoV , .

Using a panel of contiguous cDNAs that span the entire genome, we have assembled a full-length cDNA of the SARS-CoV Urbani strain, and have rescued molecularly cloned SARS viruses (infectious clone SARS-CoV) that contained the expected marker mutations inserted into the component clones. Recombinant viruses replicated as efficiently as WT virus and both were inhibited by treatment with the cysteine proteinase inhibitor… Availability of a SARS-CoV full-length cDNA provides a template for manipulation of the viral genome, allowing for the rapid and rational development and testing of candidate vaccines and therapeutics against this important human pathogen.

SARS-2006

Urbani SARS-CoV (BAC). . , Escherichia coli.
…
SARS-CoV E.coli DH10B 200 , ( ). .

The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli.
…
The assembled SARS-CoV infectious cDNA clone was fully stable during its propagation in E. coli DH10B cells for more than 200 generations, considerably facilitating the genetic manipulation of the viral genome (data not shown). The detailed cloning strategy, plasmid maps, and sequences are available upon request.

武汉2007

通过将各种SARS-CoV穗蛋白序列插入SL-CoV蛋白的骨架中，已经构建了许多嵌合穗蛋白。

源文字

A series of S chimeras was constructed by inserting different sequences of the SARS-CoV S into the SL-CoV S backbone.

从这些结果中发现，刺突蛋白BJ01中310至518的区域对于获得刺突蛋白Rp3与人ACE2结合的能力而言是必要和充分的。

源文字

From these results, it was deduced that the region from aa 310 to 518 of BJ01-S was necessary and sufficient to convert Rp3-S into a huACE2-binding molecule.

为了将SARS-CoV中的RBM插入SL-CoV刺突蛋白中，使用刺突蛋白BJ01中424至494个氨基酸的编码区替换Rp3蛋白中的相应区域，从而产生一个嵌合的刺突蛋白基因（CS），命名为CS424 –494。

源文字

For introduction of the RBM of SARS-CoV S into the SL-CoV S, the coding region from aa 424 to 494 of BJ01-S was used to replace the corresponding regions of Rp3-S, resulting in a chimeric S (CS) gene designated CS424–494.

嵌合体2015

SARS-CoV, , SHC014 SARS-CoV. , 2b, SHC014 , SARS (ACE2), in vitro, SARS-CoV. , in vivo . ; , CoV . SHC014 in vitro, in vivo.

源文字

Using the SARS-CoV reverse genetics system, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse-adapted SARS-CoV backbone. The results indicate that group 2b viruses encoding the SHC014 spike in a wild-type backbone can efficiently use multiple orthologs of the SARS receptor human angiotensin converting enzyme II (ACE2), replicate efficiently in primary human airway cells and achieve in vitro titers equivalent to epidemic strains of SARS-CoV. Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis. Evaluation of available SARS-based immune-therapeutic and prophylactic modalities revealed poor efficacy; both monoclonal antibody and vaccine approaches failed to neutralize and protect from infection with CoVs using the novel spike protein. On the basis of these findings, we synthetically re-derived an infectious full-length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo.

（a）分子克隆SHC014-CoV的示意图，该分子克隆以六个连续cDNA片段的形式合成（分别命名为SHC014A，SHC014B，SHC014C，SHC014D，SHC014E和SHC014F），两侧是独特的BglI限制酶位点，可为DNA定向组装1a，1b，尖峰，3，包膜，基质，6-8和核衣壳）。带下划线的核苷酸是限制性内切酶消化后形成的突出序列。

源文字

(a) Schematic of the SHC014-CoV molecular clone, which was synthesized as six contiguous cDNAs (designated SHC014A, SHC014B, SHC014C, SHC014D, SHC014E and SHC014F) flanked by unique BglI sites that allowed for directed assembly of the full-length cDNA expressing open reading frames (for 1a, 1b, spike, 3, envelope, matrix, 6–8 and nucleocapsid). Underlined nucleotides represent the overhang sequences formed after restriction enzyme cleavage.

值得注意的是，在肺中的差动向性相比SARS-MA15和全长SHC014冠状病毒的培养物中的减弱[人上皮呼吸道]相对于SARS-CoV的Urbani的提示，除了结合ACE2，其他因素-包括刺突蛋白的持续合成能力受体的生物利用度或宿主免疫反应的拮抗作用可能会导致毒性。

源文字

Notably, differential tropism in the lung as compared to that with SARS-MA15 and attenuation of full-length SHC014-CoV in [human epithelial airway cell] cultures relative to SARS-CoV Urbani suggest that factors beyond ACE2 binding — including spike processivity, receptor bio-availability or antagonism of the host immune responses — may contribute to emergence.

小鼠SARS-2007

SARS-CoV ( Urbani) BALB/c. (MA15), . , , , . , . , , 15, , . MA15 , ; SARS-CoV (rMA15), MA15. 15 , SARS .

We adapted the SARS-CoV (Urbani strain) by serial passage in the respiratory tract of young BALB/c mice. Fifteen passages resulted in a virus (MA15) that is lethal for mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. Abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. These observations suggest that mice infected with MA15 die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. The MA15 virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant SARS-CoV, these mutations result in a highly virulent and lethal virus (rMA15), duplicating the phenotype of the biologically derived MA15 virus. Intranasal inoculation with MA15 reproduces many aspects of disease seen in severe human cases of SARS.

-2008

, 29,7 — (Bat-SCoV), (SARS), SARS-CoV.
…
, RBDs Bat-SCoV SARS-CoV , RBD Bat-SCoV ( 323–505) RBD SARS-CoV ( 319–518) (27, 28) (GenBank FJ211860), , in vivo (Fig. 1B).

Here, we report the design, synthesis, and recovery of the largest synthetic replicating life form, a 29.7-kb bat severe acute respiratory syndrome (SARS)-like coronavirus (Bat-SCoV), a likely progenitor to the SARS-CoV epidemic.
…
To test whether the RBDs of Bat-SCoV and SARS-CoV were interchangeable, we replaced the Bat-SCoV RBD (amino acid 323–505) with the SARS-CoV RBD (amino acid 319–518) (27, 28) (GenBank accession no. FJ211860), simulating a theoretical recombination event that might occur during mixed infection in vivo (Fig. 1B).

(B) , SARS-CoV Bat-SCoV. . Bat-SRBD Bat-SCoV, , RBD Bat-SCoV ( Bat-SCoV 323–505) RBD SARS-CoV ( 319–518) ( GenBank FJ211860 ). Bat-SRBD-MA RBD MA15 SARS-CoV Y436H. Bat-SRBM 13 SARS-CoV, ACE2, RBD 323I-429T Bat-SCoV 426R-518D SARS-CoV. Bat-Hinge Bat-SRBM, Bat-SCoV 392L-397E SARS-CoV 388V-393D. Bat-F 1–24057 SARS-CoV ( 855 ) 3'- Bat-SCoV. 1- (P1). ND , .

源文字

(B) Schematic representation showing organization of the SARS-CoV and Bat-SCoV Spike proteins. The engineered Spike proteins are pictured below with the virus name to the left. Bat-SRBD includes all of the Bat-SCoV Spike sequence except that the Bat-SCoV RBD (Bat-SCoV amino acid 323–505) is replaced with the SARS-CoV RBD (amino acid 319–518) (GenBank accession no. FJ211860). Bat-SRBD-MA includes the MA15 Spike RBD change at SARS-CoV aa Y436H. Bat-SRBM includes the minimal 13 SARS-CoV residues critical for ACE2 contact, resulting in a chimeric RBD of Bat-SCoV amino acid 323I-429T and SARS-CoV amino acid 426R-518D. Bat-Hinge is Bat-SRBM sequence, with Bat-SCoV amino acid 392L-397E replaced with SARS-CoV amino acid 388V-393D. Bat-F includes nt 1–24057 of SARS-CoV (to Spike amino acid 855), with the remaining 3? sequence from Bat-SCoV. To the right of the schematic representations, observation of transcript activity and approximate stock titers at passage 1 (P1) are indicated. ND indicates no infectious virus detected by plaque assay.

巴里克2016

SARS-CoV (7), WIV1-CoV, , , , (. S1A). WIV1-CoV, SARS WIV1 (WIV1-MA15, . S1B). … Vero SARS-CoV Urbani, WIV1-MA15 WIV1-CoV.

Using the SARS-CoV infectious clone as a template (7), we designed and synthesized a full-length infectious clone of WIV1-CoV consisting of six plasmids that could be enzymatically cut, ligated together, and electroporated into cells to rescue replication competent progeny virions (Fig. S1A). In addition to the full-length clone, we also produced WIV1-CoV chimeric virus that replaced the SARS spike with the WIV1 spike within the mouse-adapted backbone (WIV1-MA15, Fig. S1B). … To confirm growth kinetics and replication, Vero cells were infected with SARS-CoV Urbani, WIV1-MA15, and WIV1-CoV.

巴里克1990

在整个研究过程中，使用了小鼠肝炎病毒A59株（MHV-A59）。该病毒在连续的小鼠星形细胞瘤细胞系（DBT）中繁殖并克隆了3次。
将
各种温度敏感突变体组合在一起，并接种到感染复数等于10的细胞中。

源文字

The A59 strain of mouse hepatitis virus (MHV-A59) was used throughout the course of this study. Virus was propagated and cloned three times in the continuous murine astrocytoma cell line (DBT).
…
Various combinations of ts mutants were mixed and inoculated onto cells at a multiplicity of infection of 10 each.

巴里克2019

在一起，这些结果表明蛋白酶切割也是用HKU5-CoV感染Vero细胞的主要障碍。进一步看，我们比较了在MERS，PDF2180和HKU5穗状蛋白中S1 / S2，S2边界和内体半胱氨酸蛋白酶位点的预期切割（图6D）（26）。在S1 / S2位点，MERS，乌干达和HKU5保留了RXXR裂解位点，尽管各种内部氨基酸可能会影响其有效性。对于S2序列，MERS和HKU5也保留了RXXR基序。但是，乌干达菌株的尖峰中不存在第一个精氨酸（SNAR），这可能会影响其裂解。

源文字

Together, these results demonstrate that protease cleavage is also the primary barrier to infection of Vero cells with HKU5-CoV. Examining further, we compared the predicted cleavage at S1/S2 border, S2’, and the endosomal cysteine protease site across MERS, PDF2180, and HKU5 spikes (Fig. 6D) (26). For the S1/S2 site, MERS, Uganda, and HKU5 maintain the RXXR cleavage motif, although the different interior amino acids may alter efficiency. For the S2’ sequence, MERS and HKU5 also retain the RXXR motif; however, the Uganda spike lacks the first arginine (SNAR), potentially impacting cleavage.

功能获得：“拒绝无法继续”

, (GOF). (Fig. 4a, b) , SHC014-MA15, , . SHC014-MA15 SARS-CoV, , CoV Urbani MA15, , . , Urbani-MA15 CoV, SHC014-MA15 (. 1). , , , . , . GOF; . , , , .

源文字

In addition to offering preparation against future emerging viruses, this approach must be considered in the context of the US government–mandated pause on gain-of-function (GOF) studies. On the basis of previous models of emergence (Fig. 4a,b), the creation of chimeric viruses such as SHC014-MA15 was not expected to increase pathogenicity. Although SHC014-MA15 is attenuated relative to its parental mouse-adapted SARS-CoV, similar studies examining the pathogenicity of CoVs with the wild-type Urbani spike within the MA15 backbone showed no weight loss in mice and reduced viral replication. Thus, relative to the Urbani spike–MA15 CoV, SHC014-MA15 shows a gain in pathogenesis (Fig. 1). On the basis of these findings, scientific review panels may deem similar studies building chimeric viruses based on circulating strains too risky to pursue, as increased pathogenicity in mammalian models cannot be excluded. Coupled with restrictions on mouse-adapted strains and the development of monoclonal antibodies using escape mutants, research into CoV emergence and therapeutic efficacy may be severely limited moving forward. Together, these data and restrictions represent a crossroads of GOF research concerns; the potential to prepare for and mitigate future outbreaks must be weighed against the risk of creating more dangerous pathogens. In developing policies moving forward, it is important to consider the value of the data generated by these studies and whether these types of chimeric virus studies warrant further investigation versus the inherent risks involved.

— , , BSL-4, . ; , . « ».
…
. BSL-4 ; , , , .

, , BSL-4 — , , — . « , , », — .

BSL-4 . , , , , . , , .

« », — . , : « , , ».

特雷凡说，最重要的是，中国对BSL-4实验室的投资可以成为向世界证明该国具有竞争力的一种方式。他说：“这是生物学中的重要地位标志，无论是否需要它们。”

源文字

Future plans include studying the pathogen that causes SARS, which also doesn’t require a BSL-4 lab, before moving on to Ebola and the West African Lassa virus, which do. Some one million Chinese people work in Africa; the country needs to be ready for any eventuality, says Yuan. “Viruses don’t know borders.”
…
The plan to expand into a network heightens such concerns. One BSL-4 lab in Harbin is already awaiting accreditation; the next two are expected to be in Beijing and Kunming, the latter focused on using monkey models to study disease.
Lina says that China’s size justifies this scale, and that the opportunity to combine BSL-4 research with an abundance of research monkeys — Chinese researchers face less red tape than those in the West when it comes to research on primates — could be powerful. “If you want to test vaccines or antivirals, you need a non-human primate model,” says Lina.
But Ebright is not convinced of the need for more than one BSL-4 lab in mainland China. He suspects that the expansion there is a reaction to the networks in the United States and Europe, which he says are also unwarranted. He adds that governments will assume that such excess capacity is for the potential development of bioweapons.
“These facilities are inherently dual use,” he says. The prospect of ramping up opportunities to inject monkeys with pathogens also worries, rather than excites, him: “They can run, they can scratch, they can bite.”
Trevan says China’s investment in a BSL-4 lab may, above all, be a way to prove to the world that the nation is competitive. “It is a big status symbol in biology,” he says, “whether it’s a need or not.”

然而，为了将这些发现转化为人类的致病潜能，需要对非人类灵长类动物进行进一步测试。重要的是要注意，缺乏可用的治疗剂决定了进一步研究和开发治疗方法的关键需求。有了这些知识，就可以开发出监视程序，诊断试剂和有效的治疗方法，以防止出现2b组特异性冠状病毒，例如SHC014，并可以应用于支持相似异质库的其他冠状病毒分支。

源文字

However, further testing in nonhuman primates is required to translate these finding into pathogenic potential in humans. Importantly, the failure of available therapeutics defines a critical need for further study and for the development of treatments. With this knowledge, surveillance programs, diagnostic reagents and effective treatments can be produced that are protective against the emergence of group 2b–specific CoVs, such as SHC014, and these can be applied to other CoV branches that maintain similarly heterogeneous pools.

关于启蒙精神准备多少奇迹般的流行...

只有一名科学家对该病毒起作用，雷耶斯说实验室怀疑该科学家在11月不小心将试管扔了进去。
...
加尔维斯顿生物实验室的安全措施最严格，因为它正在研究BSL-4生物安全材料，即没有疫苗或药物的危险传染病。BSL-4材料包括瓜纳里托，埃博拉和天花。

源文字

Only one scientist worked with the virus, and Reyes said the lab suspects that scientist accidentally threw the vial away in November.
…
Galveston biolab requires the most stringent safety measures because it studies biosafetly level BSL-4 materials, or dangerous infectious diseases that have no vaccines or cures. BSL-4 materials include Guanarito, Ebola and smallpox.

H1N1 1918 , ( 1947 ), 1957 «» H2N2, . H1N1, -, , 20 . 1969 H3N2 H2N2 .

1977 . H1N1 , 1978 . , 1977 . H1N1 - , , . , , , H1N1 1977 H1N1, 1949–1950 , , .
…
2009–2010 . , H1N1 1977 : « — A H1N1, 1977 ».
…
, 1977 . H1N1, , « » (Kung 1978). , , , 1970- ( ) . .

, , 1977–78 . - , , -, H1N1 1978 . H1N1, , . 1976–77 20 H1N1 1957, . 1977 , .

Human influenza H1N1 viruses appeared with the 1918 pandemic, and persisted, slowing accumulating small changes in its genome (with a major change in 1947), until the H2N2 “Asian” flu appeared in 1957, causing a worldwide pandemic. H1N1 influenza virus then apparently became extinct, and was not isolated for 20 years. In 1969 the “Hong Kong” H3N2 virus replaced the H2N2 virus, and is still circulating.
In September 1977 an H1N1 influenza virus was isolated from human infections in the Far East region of the Soviet Union, and in early 1978 the Chinese reported they had isolated H1N1 virus in May of 1977 in northeast China adjacent to the Soviet outbreak. Using the early genetic tools available at the time, the 1977 H1N1 virus was found to be closely related to H1N1 human influenza viruses circulating in 1949–1950, but not to those circulating earlier or later.
…
Only since 2009–2010 did major papers begin to state directly the 1977 emergence of H1N1 influenza was a laboratory related release: “The most famous case of a released laboratory strain is the re-emergent H1N1 influenza A virus which was first observed in China in May of 1977 and in Russia shortly thereafter.”
…
The speculation that the 1977 release may have been related to H1N1 vaccine research is supported by the observation that in the initial outbreaks in China, nine of the ten viral isolates expressed “temperature sensitivity” (Kung 1978). Temperature sensitivity normally an uncommon trait, but one that was in the 1970s (and still is) a fundamental trait for making live attenuated influenza vaccines. Temperature sensitivity generally occurs only after a series of substantial laboratory manipulations and selections.
Interestingly, further investigation indicated the circulating strains in 1977–78 were often comprised of mixed temperature-sensitive and normal components, and that temperature sensitivity apparently disappeared from the post-1978 H1N1 lineage rapidly. Escape of a mid-protocol population of H1N1 virus undergoing laboratory selection for temperature sensitive mutants would provide such a mixed population. In 1976–77 laboratory personnel in their late teens or early 20s would not have been exposed to pre-1957 H1N1 influenza viruses, and been susceptible to laboratory infections. The low severity of the 1977 pandemic might be in part due to the temperature sensitivity of the virus, a trait that limits virus replication in pulmonary tissues.

电报消息人士说，他们应该对WIV实验室的严重安全问题，特别是与蝙蝠冠状病毒的合作提出警告。使馆官员呼吁美国对这个实验室给予更多关注，并提供更多支持。
...
« WIV , , », — 19 2018 , , , WIV. ( .)
WIV的中国研究人员得到了得克萨斯大学医学系加尔维斯顿国家实验室和美国其他组织的帮助，但中国人寻求其他帮助。电报认为美国应向武汉实验室提供进一步的支持，主要是因为对蝙蝠冠状病毒的研究很重要，但也很危险。

源文字

Sources familiar with the cables said they were meant to sound an alarm about the grave safety concerns at the WIV lab, especially regarding its work with bat coronaviruses. The embassy officials were calling for more U.S. attention to this lab and more support for it, to help it fix its problems.
…
“During interactions with scientists at the WIV laboratory, they noted the new lab has a serious shortage of appropriately trained technicians and investigators needed to safely operate this high-containment laboratory,” states the Jan. 19, 2018, cable, which was drafted by two officials from the embassy’s environment, science and health sections who met with the WIV scientists. (The State Department declined to comment on this and other details of the story.)
The Chinese researchers at WIV were receiving assistance from the Galveston National Laboratory at the University of Texas Medical Branch and other U.S. organizations, but the Chinese requested additional help. The cables argued that the United States should give the Wuhan lab further support, mainly because its research on bat coronaviruses was important but also dangerous.

人造痕迹

SARS-CoV Bat-SCoV.
(A) SARS-CoV Bat-SCoV ( GenBank FJ211859) . () nsp ORF1ab ( , - ; , nsp5 [3C- ]). , , A F. , Bat-SCoV, SARS-CoV, , Bat-SCoV 2 , Bat-E1 Bat-E2, SARS-E.

Schematic representation of SARS-CoV and Bat-SCoV variants.
(A) Schematic representation of SARS-CoV and Bat-SCoV (GenBank accession no. FJ211859) genomes and reverse genetics system. (Top) Arrowheads indicate nsp processing sites within the ORF1ab polyprotein (open arrowheads, papain-like proteinase mediated; filled arrowheads, nsp5 [3C-like proteinase] mediated). Immediately below are the fragments used in the reverse genetics system, labeled A through F. The fragments synthesized to generate Bat-SCoV exactly recapitulate the fragment junctions of SARS-CoV with the exception that the Bat-SCoV has 2 fragments, Bat-E1 and Bat-E2, which correspond to the SARS-E fragment.

, - , SARS-CoV (24, 33, 53) . , Bat-F A-E SARS-CoV F Bat-SCoV, Bgl-NotI. Bat-SCoV Bat-SRBD, Bat-SRBM Bat-Hinge , 7 Bat-SCoV, BglI Bat-A, Bat-B, Bat-C Bat -D, BglI AflII Bat-E1 Bat-E2 BglI NotI Bat-F. , . m7Message mMachine (Ambion), Vero (24, 53).

Viruses containing PCR-generated insertions within the viral coding sequence were produced by using the SARS-CoV assembly strategy (24, 33, 53) with the following modifications. Briefly, for Bat-F virus, full-length cDNA was constructed by ligating restriction products from SARS-CoV fragments A–E and Bat-SCoV fragment F, which required a BglI-NotI digestion. For Bat-SCoV and Bat-SRBD, Bat-SRBM, and Bat-Hinge, plasmids containing the 7 cDNA fragments of the Bat-SCoV genome were digested by using BglI for Bat-A, Bat-B, Bat-C, and Bat-D, BglI and AflII for Bat-E1 and Bat-E2, and BglI and NotI for Bat-F. Digested, gel-purified fragments were simultaneously ligated together. Transcription was driven by using a T7 mMessage mMachine kit (Ambion), and RNA was electroporated into Vero cells (24, 53).

在组装完整的全长cDNA产物的过程中，系统地去除了位于每个cDNA末端的限制性酶切位点的连接位点，从而可以重新组装而无需修饰核苷酸。

源文字

The interconnecting restriction site junctions that are located at the ends of each cDNA are systematically removed during the assembly of the complete full-length cDNA product, allowing reassembly without the introduction of nucleotide changes.

为了快速组装共有克隆，我们使用了IIS类限制性核酸内切酶，将其切入不对称区域并留下不对称末端。这些酶产生特定的独特突出端，提供两个cDNA的无缝连接，随后失去限制性位点。

源文字

To rapidly assemble consensus clones, we used class IIS restriction endonucleases that cut at asymmetric sites and leave asymmetric ends. These enzymes generate strand-specific unique overhangs that allow the seamless ligation of two cDNAs with the concomitant loss of the restriction site.

2.2。重组病毒的
产生如前所述[20，28]，通过牛痘重组获得了含有带有弗林蛋白酶位点S2'的刺突蛋白的重组病毒rYN-S2 / RRKR。简而言之，使用Seamless Assembly试剂盒（Invitrogen，Carlsbad，CA，USA）产生了S′弗林蛋白酶位点质粒，并将其转染到被含有YN-βS-GPT基因的牛痘病毒感染的CV-1细胞中。使用时间优势选择系统，通过同源重组将弗林蛋白酶-S2位点引入YN cDNA [25]。

源文字

2.2. Generation of Recombinant Virus
Recombinant rYN-S2/RRKR virus containing an S protein with the furin-S2? site was generated by vaccinia recombination, as described previously [20,28]. Briefly, plasmid with the furin-S2? site was generated using the Seamless Assembly kit (Invitrogen, Carlsbad, CA, USA) and transfected into CV-1 cells infected by vaccinia virus containing the genome of YN-?S-GPT. Furin-S2’ site was introduced into the YN cDNA by homologous recombination using the transient dominant selection system [25].

GeneArt 1 4 , , 30- . E.coli .
• — 4 ( 13 ); ,
• — , , , 90% .
• —
• — - -,
• — , , .

The GeneArt Seamless Cloning and Assembly Kit enables the simultaneous and directional cloning of 1 to 4 PCR fragments, consisting of any sequence, into any linearized vector, in a single 30-minute room temperature reaction. The kit contains everything required for the assembly of DNA fragments, and their transformation into E. coli for selection and growth of recombinant vectors.
• Speed and Ease?—?Clone up to 4 DNA fragments, with sequence of your choice, simultaneously in a single vector (up to 13 Kb); no restriction digestion, ligation or recombination sites required
• Precision and Efficiency — Designed to let you clone what you want, where you want, in the orientation you want, and achieve up to 90% correct clones with no extra sequences left behind
• Vector Flexibility — Use our linear vector or a vector of your choice
• Free Tools — Design DNA oligos and more with our free web-based interface that walks you step-by-step through your project
• Diverse Applications — Streamline many synthetic biology and molecular biology techniques through the rapid combination, addition, deletion, or exchange of DNA segments

密码子首选项

但是对自然界中人造性的反驳又如何呢？

, SARS-CoV-2 ACE2 , , , RBD SARS-CoV [ SARS — ..] . , SARS-CoV-2 ACE2, , ACE2, . , SARS-CoV-2 .

While the analyses above suggest that SARS-CoV-2 may bind human ACE2 with high affinity, computational analyses predict that the interaction is not ideal and that the RBD sequence is different from those shown in SARS-CoV to be optimal for receptor binding. Thus, the high-affinity binding of the SARS-CoV-2 spike protein to human ACE2 is most likely the result of natural selection on a human or human-like ACE2 that permits another optimal binding solution to arise. This is strong evidence that SARS-CoV-2 is not the product of purposeful manipulation.

, , , , -. , , SARS-CoV-2 - .

Furthermore, if genetic manipulation had been performed, one of the several reverse-genetic systems available for betacoronaviruses would probably have been used. However, the genetic data irrefutably show that SARS-CoV-2 is not derived from any previously used virus backbone.

, O- . in vitro in vivo. , SARS-CoV-2 - , . ACE2, , .

The acquisition of both the polybasic cleavage site and predicted O-linked glycans also argues against culture-based scenarios. New polybasic cleavage sites have been observed only after prolonged passage of low-pathogenicity avian influenza virus in vitro or in vivo. Furthermore, a hypothetical generation of SARS-CoV-2 by cell culture or animal passage would have required prior isolation of a progenitor virus with very high genetic similarity, which has not been described. Subsequent generation of a polybasic cleavage site would have then required repeated passage in cell culture or animals with ACE2 receptors similar to those of humans, but such work has also not previously been described.

史正立2020

, SARS-CoV-2 , SARS-CoV, , SARS-CoV-2 .
…
, β- S1/S2, . , SARS-CoV , L . S1/S2 SARS-CoV .

In this study, we have shown that SARS-CoV-2 exhibits much higher capacity of membrane fusion than SARS-CoV, suggesting that the fusion machinery of SARS-CoV-2 is an important target for development of coronavirus fusion inhibitors.
…
Generally, β-B coronaviruses lack the S1/S2 furin-recognition site, and their S proteins are uncleaved in the native state. For example, SARS-CoV enters into the cell mainly via the endosomal membrane fusion pathway where its S protein is cleaved by endosomal cathepsin L and activated. Inducing the S1/S2 furin-recognition site could significantly increase the capacity of SARS-CoV S protein to mediate cellular membrane surface infection.

这就是终点，美丽的朋友

哪些动物是SARS-CoV-2的人畜共患病携带者？
尚未发现此类动物。有证据表明穿山甲可能是中间宿主，但穿山甲病毒与SARS-CoV-2的同源性仅为88–98％。为了进行比较，首例SARS的果子狸和浣熊冠状病毒株与2003年SARS-CoV株具有99.8％的同一性。换句话说，我们谈论的是2003年麝香，浣熊和人类之间的几种突变。穿山甲[CoV2毒株]的核苷酸变化超过3,000个，因此穿山甲决不能成为贮藏物种。绝对没有机会。

源文字

What is the reservoir species of SARS-CoV-2?
They have not identified the actual reservoir species. Reports show that pangolins are potentially the intermediate host, but pangolin viruses are 88–98% identical to SARS-CoV-2. In comparison, civet and racoon dog strains of SARS coronaviruses were 99.8% identical to SARS-CoV from 2003. In other words, we are talking about a handful of mutations between civet strains, racoon dog strains and human strains in 2003. Pangolin [strains of CoV2] have over 3000 nucleotide changes, no way they are the reservoir species. Absolutely no chance.

UPD：RaTG13和Cov2基因组差异的约4％

. 10-4 , -. , 6 . in vitro , , , .

We obtained an estimate of the spontaneous mutation rate of ca. 10^-4 substitutions per site or lower, a value within the typically accepted range for RNA viruses. A roughly threefold increase in mutation rate and a significant shift in mutation spectrum were observed in samples from patients undergoing 6 months of interferon plus ribavirin treatment. This result is consistent with the known in vitro mutagenic effect of ribavirin and suggests that the antiviral effect of ribavirin plus interferon treatment is at least partly exerted through lethal mutagenesis.

UPD2：RaTG13-与RaBtCoV / 4991相同的菌株吗？

jiang江自治区是中国云南南部普Pu市管辖的自治区。维基百科