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Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China
Please cite this article in press as: Wu et al., Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.02.001
Cell Host & Microbe
Commentary Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China Aiping Wu,1,7,9 Yousong Peng,2,9 Baoying Huang,3,9 Xiao Ding,1,7,9 Xianyue Wang,1,7 Peihua Niu,3 Jing Meng,1,7 Zhaozhong Zhu,2 Zheng Zhang,2 Jiangyuan Wang,1,7 Jie Sheng,1,7 Lijun Quan,4 Zanxian Xia,5,8 Wenjie Tan,3,* Genhong Cheng,6,* and Taijiao Jiang1,7,* 1Center for Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China 2College of Biology, Hunan Provincial Key Laboratory of Medical Virology, Hunan University, Changsha 410082, China 3Key Laboratory of Medical Virology, National Health and Family Planning Commission, National Institute for Viral Disease Control and Prevention, China CDC, Beijing 102206, China 4School of Computer Science and Technology, Soochow University, Suzhou, China 5Department of Cell Biology, School of Life Science, Central South University, Changsha 410013, China 6Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, USA 7Suzhou Institute of Systems Medicine, Suzhou, Jiangsu 215123, China 8Hunan Key Laboratory of Animal Models for Human Diseases, Hunan Key Laboratory of Medical Genetics & Center for Medical Genetics, School of Life Science, Central South University, Changsha 410013, China 9These authors contributed equally *Correspondence: [email protected] (W.T.), [email protected] (G.C.), [email protected] (T.J.) https://doi.org/10.1016/j.chom.2020.02.001
An in-depth annotation of the newly discovered coronavirus (2019-nCoV) genome has revealed differences between 2019-nCoV and severe acute respiratory syndrome (SARS) or SARS-like coronaviruses. A systematic comparison identified 380 amino acid substitutions between these coronaviruses, which may have caused functional and pathogenic divergence of 2019-nCoV. A novel coronavirus (CoV) named ‘‘2019 novel coronavirus’’ or ‘‘2019-nCoV’’ by the World Health Organization (WHO) is responsible for the recent pneumonia outbreak that started in early December, 2019 in Wuhan City, Hubei Province, China (Huang et al., 2020; Zhou et al., 2020; Zhu et al., 2020). This outbreak is associated with a large seafood and animal market, and investigations are ongoing to determine the origins of the infection. To date, thousands of human infections have been confirmed in China along with many exported cases across the globe (China CDC, 2020). Coronaviruses mainly cause respiratory and gastrointestinal tract infections and are genetically classified into four major genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus (Li, 2016). The former two genera primarily infect mammals, whereas the latter two predominantly infect birds (Tang et al., 2015). Six kinds of human CoVs have been previously identified. These include HCoV-NL63 and HCoV229E, which belong to the Alphacoronavirus genus; and HCoV-OC43, HCoVHKU1, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV), which belong to the Beta-
coronavirus genus (Tang et al., 2015). Coronaviruses did not attract worldwide attention until the 2003 SARS pandemic, followed by the 2012 MERS and, most recently, the 2019-nCoV outbreaks (China CDC, 2020; Song et al., 2019). SARS-CoV and MERS-CoV are considered highly pathogenic (Cui et al., 2019), and it is very likely that both SARS-CoV and MERS-CoV were transmitted from bats to palm civets (Guan et al., 2003) or dromedary camels (Drosten et al., 2014), and finally to humans (Cui et al., 2019). The genome of coronaviruses, whose size ranges between approximately 26,000 and 32,000 bases, includes a variable number (from 6 to 11) of open reading frames (ORFs) (Song et al., 2019). The first ORF representing approximately 67% of the entire genome encodes 16 non-structural proteins (nsps), while the remaining ORFs encode accessory proteins and structural proteins (Cui et al., 2019). The four major structural proteins are the spike surface glycoprotein (S), small envelope protein (E), matrix protein (M), and nucleocapsid protein (N). The spike surface glycoprotein plays an essential role in binding to receptors on the host cell and determines host tropism (Li, 2016; Zhu et al., 2018). The spike proteins of SARS-CoV and MERS-CoV bind
to different host receptors via different receptor-binding domains (RBDs). SARSCoV uses angiotensin-converting enzyme 2 (ACE2) as one of the main receptors (Ge et al., 2013) with CD209L as an alternative receptor (Jeffers et al., 2004), whereas MERS-CoV uses dipeptidyl peptidase 4 (DPP4, also known as CD26) as the primary receptor. Initial analysis suggested that 2019-nCoV has a close evolutionary association with the SARSlike bat coronaviruses (Zhou et al., 2020). Here, based on the first three determined genomes of the novel coronavirus (2019-nCoV), namely Wuhan/ IVDC-HB-01/2019 (GISAID accession ID: EPI_ISL_402119) (HB01), Wuhan/IVDCHB-04/2019 (EPI_ISL_402120) (HB04), and Wuhan/IVDC-HB-05/2019 (EPI_ ISL_402121) (HB05), an in-depth genome annotation of this virus was performed with a comparison to related coronaviruses, including 1,008 human SARSCoV, 338 bat SARS-like CoV, and 3,131 human MERS-CoV, whose genomes were published before January 12, 2020 (release date: September 12, 2019) from Virus Pathogen Database and Analysis Resource (ViPR) (http://www.viprbrc. org/) and NCBI. Comparison of genomes of these three strains showed that they are almost
Cell Host & Microbe 27, March 11, 2020 ª 2020 Published by Elsevier Inc. 1
Please cite this article in press as: Wu et al., Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.02.001
Cell Host & Microbe
Commentary IVDC-HB-01/2019 (~29.8kb)
A
5’UTR
N
7a 8b
3a E M
pp1ab S
pp1a
3’UTR p6
3b
9b orf14
7b
3860 1
nsp1 181
nsp4
nsp3
nsp2 819
nsp5
2764 7096
nsp16
nsp15
6799
6453
3264
nsp13
nsp14
3570
4141
nsp7 nsp8 nsp9 nsp10 4392 3943 4254 4393
nsp12 5325
5926
B
nsp6
C
whole genome
pp1a
MERS-CoV
3a
2019-nCoV
pp1ab
spike
envelope
matrix
SARS-like bat CoV Human SARS-CoV
Alphacoronavirus Betacoronavirus Gammacoronavirus Deltacoronavirus
HB-01/2019 SARS-like bat CoV
7a MERS-CoV
8b
nucleocapsid
Other CoVs
Human SARS-CoV
Figure 1. Genome composition and phylogenetic tree for 2019-nCoV (A) Schematic diagram of the genome organization and the encoded proteins of pp1ab and pp1a for the IVDC-HB-01/2019 (HB01) strain. The largest gene, namely the orf1ab, encodes the pp1ab protein that contains 15 nsps (nsp1-nsp10 and nsp12-nsp16). The pp1a protein encoded by the orf1a gene also contains 10 nsps (nsp1-nsp10). Structural proteins are encoded by the four structural genes, including spike (S), envelope (E), membrane (M), and nucleocapsid (N) genes. The accessory genes are distributed among the structural genes. The protein-encoding genes of the genome of 2019-nCoV were predicted by the online servers of GeneMarkS (http://exon.gatech.edu/GeneMark/genemarks.cgi) and ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/) with manual check. (B) Phylogenetic relationship based on the whole genome for the HB01 strain and other coronaviruses. All viral strains were classified by the genus and the type, which are presented on the left and right schematic phylogenetic trees, respectively. The four genera of the coronaviruses, including Alphacoronavirus (red), Betacoronavirus (blue), Gammacoronavirus (green), and Deltacoronavirus (violet) are blocked in the left phylogenetic tree. The MERS coronavirus (brown), the SARS-like bat coronavirus (violet), human SARS coronavirus (light blue), and the HB01 strain (red) are highlighted by lines of different colors in the right phylogenetic tree. (C) Schematic phylogenetic trees of individual genes for the HB01 strain. The coronavirus species were colored in the same way as (B). The amount of the strains in the phylogenetic clade is denoted by the area of the circles.
identical, with only five nucleotide differences in the genome of ~29.8 kb nucleotides (Figure S1). The 2019-nCoV genome was annotated to possess 14 ORFs encoding 27 proteins (Figure 1A and Tables S1A and S1B). The orf1ab and orf1a genes located at the 50 -terminus of the genome respectively encode the pp1ab and pp1a proteins, respectively. They together comprise 15 nsps including nsp1 to nsp10 and nsp12 to nsp16 (Figure 1A and Table S1B). The 30 -terminus of the genome contains four structural proteins (S, E, M, and N) and eight accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b, and orf14). At the amino acid level, the 2 Cell Host & Microbe 27, March 11, 2020
2019-nCoV is quite similar to that of SARS-CoV, but there are some notable differences. For example, the 8a protein is present in SARS-CoV and absent in 2019-nCoV; the 8b protein is 84 amino acids in SARS-CoV, but longer in 2019nCoV, with 121 amino acids; the 3b protein is 154 amino acids in SARS-CoV, but shorter in 2019-nCoV, with only 22 amino acids (Table S1A). Further studies are needed to characterize how these differences affect the functionality and pathogenesis of 2019-nCoV. As shown in a phylogenetic tree based on whole genomes (Figures 1B and S2) with the Molecular Evolutionary Genetics
Analysis (MEGA) (version 7.0), the 2019nCoV is in the same Betacoronavirus clade as MERS-CoV, SARS-like bat CoV, and SARS-CoV. The phylogenetic tree falls into two clades. The Betacoronavirus genus constitutes one clade, while the Alphacoronavirus, Gammacoronavirus, and Deltacoronavirus genera constitute the other clade. The 2019-nCoV is parallel to the SARS-like bat CoVs, while the SARS-CoVs are descended from the SARS-like bat CoVs, indicating that 2019-nCoV is closer to the SARS-like bat CoVs than the SARS-CoVs in terms of the whole genome sequence. Tables S1C and S1D also show that the genome
Please cite this article in press as: Wu et al., Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.02.001
Cell Host & Microbe
Commentary nsp1
nsp2 267
272
293
294
316
324
337
350
354
368
374
390
417
442
449
456
531
553
559
594
601
605
608
635
645
649
L
N
N
I
T
L
D
C
D
V
T
A
I
H
E
E
H
G
N
K
S
A
E
E
V
N
L
I
V
L
F
G
V
I
C
S
S
K
K
K
V
T
E
H
N
L
V
P
M Q
D
H
Y
N
D
R
T
G
Q
D
I
S
V
V
L
Q
L
A
L
L
G
1064
655
259
T
S
652
211 A
G
595
166 S
E
539
158 Q
Y
535
157 F
I
386
154 Y
T
283
114 I
L
280
56 V
2019-nCoV
224
38 V
SARS/SARS-like CoVs A
nsp3
nsp2 812
827
893
902
965
975
1027
1042
1057
1081
1104
1124
1178
1218
1271
E
P
T
Q
A
K
M M V
T
D
S
I
I
E
L
M
T
T
Q
S
N
V
Y
N
V
K
F
S
V
I
Q
D
D
S
E
G
Q
L
V
E
N
V
V
D
M V
C
S
E
Q
C
I
H
G
L
A
N
E
L
E
A
880
L
1274
806
G
L
945
798
T
Q
888
764
E
K
849
753
R
R
838
727
K
Q
808
726
H
A
807
724
T
I
776
722
V
R
748
710
K
A
745
701
G
E
728
696
A
A
712
687
F
V
711
685
T
K
666
G
661 2019-nCoV SARS/SARS-like CoVs
nsp3 1399
1420
1512
1522
1537
1553
1619
1623
1662
1677
1679
1682
1692
1706
1718
1734
1736
1742
1744
1758
1779
1795
1813
1817
1822
1825
1837
1856
1863
1889
2105
2137
2142
2156
2165
I
E
V
A
T
K
Q
I
Y
F
Y
N
N
A
A
T
P
E
C
Y
F
D
C
Q
F
K
K
Q
K
T
S
K
S
I
C
L
T
A
I
C
A
V
D
M V
P
R
E
V
H
L
F
S
G
S
V
A
A
D
S
H
L
E
A
K
L
R
Q
E
Q
L
N
T
M V
Y
I
K
V
C
F
2623
2641
2695
1791
1345 V
L
1525
1339 V
C
1444
1298 A
I
1394
1296 L
2019-nCoV
1355
1295 V
SARS/SARS-like CoVs D
nsp3 2233
2238
2254
2280
2303
2314
2316
2336
2431
2442
2453
2501
2517
2553
2584
2596
2618
2647
2660
2673
2709
2710
2718
2738
2754
2759
2309
2228
N
N
F
V
M Y
T
T
S
I
F
W R
R
K
V
I
S
S
A
N
A
N
F
E
Q
Y
S
I
A
F
L
V
A
K
T
L
I
L
S
D
A
V
Y
L
K
R
L
L
P
A
T
D
S
G
V
D
H
F
N
V
S
Y
I
I
S
V
2274
2222
L V
2019-nCoV
2266
2218
2177 L
SARS/SARS-like CoVs F
K
ORF1ab
nsp4
nsp5
2938
2943
2944
2946
2947
2998
3030
3038
3053
3056
3066
3082
3097
3116
3129
3133
3135
3154
3171
3196
3197
3221
3349
3465
3530
3548
3549
L
T
M
T
D
V
I
V
V
A
E
S
V
M
I
V
V
R
S
F
T
V
L
M V
T
L
I
Y
S
D
V
M
V
V
S
A
L
V
M
L
D
G
I
V
L
I
S
S
E
L
I
V
I
L
K
N
A
A
A
F
F
S
I
V
H
T
E
T
L
L
L
A
T
I
nsp8
3627
3628
3668
3670
3678
3704
3715
3737
3751
3757
3769
3791
3847
4188
4276
L
S
F
M
F
D
V
F
K
G
L
I
V
G
F
T
V
F
I
T
S
F
A
L
I
V
A
C
L
L
E
A
Y
R
A
I
V
I
A
L
C
I
Y
V
N
N
Y
P
M
3957
nsp14
4625
4657
5131
5132
5161
5164
5176
5939
6024
6057
6059
6062
6145
6219
6244
6270
6299
6321
6418
6435
T
T
V
Y
K
T
D
F
T
S
S
V
E
D
S
S
A
I
T
I
A
D
S
S
L
V
D
V
A
I
L
C
H
E
Y
N
A
A
I
D
E
T
N
S
V
S
V
S
E
H
A
Q
I
nsp16
6543
6567
6569
6570
6574
6607
6623
6631
6653
6675
6703
6710
6715
6716
6799
6830
6833
6936
6951
6956
7014
7063
7070
7074
D
I
A
P
F
V
A
Y
Q
E
L
F
F
E
S
D
S
T
N
I
Q
R
G
L
S
I
I
E
A
S
S
L
K
S
F
E
Q M S
L
K
A
E
N
V
H
L
K
K
N
Y
E
V
1216
S1 subunit
RBD
S2 subunit
SD 575
726
872
875
879
882
884
1070
1133
T
A
E
R
S
I
T
I
Q
S
A
I
S
A
V
I
S
F
S
D
Q
Q
V
S
V
A
A
S
A
A
S
I
V
37
127
619
209
215
227
228
S
C
S
S
Y
H
V
V
T
Y
G
E
E
N
A
71
Q V
T
V
Q
L
M M
L
L
L
C
P
V
I
L
I
E
P
S
R
Y
A
V
SARS/SARS-like CoVs I
68
2019-nCoV SARS/SARS-like CoVs
2019-nCoV
9b
67
ORF14
T
66
H
I
T
65
T
T
K
I
M
63
A
E
A
P
T
H
52
D
N
T
E
L
L
60
S
D
N
2019-nCoV
F
46
G
S
SARS/SARS-like CoVs T
I T
39
2019-nCoV SARS/SARS-like CoVs
7b
2019-nCoV SARS/SARS-like CoVs
40
I
3
T
L
32
I
T
217
V
R
26
K
T
103
V
25
2019-nCoV SARS/SARS-like CoVs
3b
23
205
L
C
43
200
L
I
40
166
V
Q
5
147
H
L
38
112
I
H
334
78
R
A
111
30
S
N
107
26
D
74
22
I L
72
10 2019-nCoV SARS/SARS-like CoVs
21
572
A
Q
16
570
L
N
14
560
H
K
11
519
N
T
6
430
S
V
5
438
K
E
719
417
N
P
693
354
A
K
689
348
Q
646
271
S N
50
112
S
SARS/SARS-like CoVs L
2019-nCoV
7088
6494 L
6831
6482 T A
6459 F
SARS/SARS-like CoVs Y
2019-nCoV
N
6218
4618
N
V
6156
4590
I
V
5158
4498
K
L
4673
4490
I
G
4617
4458
D
E
4455
D
4454 2019-nCoV SARS/SARS-like CoVs
nsp15
7a
4391
3624
V
4115
3622
I F
4087
3606
L M
4074
3593
3677
3587
3583
3576 I
nsp12
3a
nsp9 nsp10
SARS/SARS-like CoVs V
2019-nCoV
S
A
3145
2873
I L
nsp6
3130
2865
3080
2833
3075
2796
2825
2791
2781
2774
L F
2019-nCoV
2770
2768 N
SARS/SARS-like CoVs T
K
Figure 2. Amino Acid Substitutions of 2019-nCoV against SARS and SARS-like Viruses All 27 proteins encoded by 2019-nCoV have been aligned against SARS-CoVs and SARS-like bat CoVs using the FFT-NS-2 algorithm in MAFFT (version v7.407) (The number of aligned proteins were listed in Table S1E). An amino acid substitution was defined as an absolutely conserved site in the group of SARS and SARS-like CoVs but different from that of 2019-nCoV. In total, 380 amino acid substitutions have been identified between the amino acid sequences of 2019nCoV (HB01) and the corresponding consensus sequences of SARS and SARS-like CoVs.
of 2019-nCoV has the highest similarity with that of a SARS-like bat CoV (MG772933). In comparison, 2019-nCoV is distant from and less related to the MERS-CoVs. In terms of the encoded proteins of pp1ab, pp1a, envelope, ma-
trix, accessory protein 7a, and nucleocapsid genes, phylogenetic analyses showed that the 2019-nCoV is closest to the SARS-like bat CoVs (Figure 1C and Table S1D). Regarding the spike gene, the 2019-nCoV is closest to the bat
CoVs, while the 3a and 8b accessory genes are both closest to the SARSCoVs. Although phylogenetic analyses for the whole genome and individual genes clearly show that the 2019-nCoV is most closely related to SARS-like bat Cell Host & Microbe 27, March 11, 2020 3
Please cite this article in press as: Wu et al., Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.02.001
Cell Host & Microbe
Commentary viruses (Figures 1B and 1C), we did not find a single strain of a SARS-like bat virus that harbors all proteins with the most similarity to counterparts of the 2019nCoV (Figures 1B and 1C). Given the close relationship between 2019-nCoV and SARS-CoVs or SARSlike bat CoVs (Figures 1B and 1C), an examination of the amino acid substitutions in different proteins could shed light into how 2019-nCoV differs structurally and functionally from SARS-CoVs. In total, there were 380 amino acid substitutions between the amino acid sequences of 2019-nCoV (HB01) and the corresponding consensus sequences of SARS and SARS-like viruses (Figure 2 and Tables S1E and S1F). No amino acid substitutions occurred in nonstructural protein 7 (nsp7), nsp13, envelope, matrix, or accessory proteins p6 and 8b (Table S1F). Respectively, 102 and 61 amino acid substitutions are located in nsp3 and nsp2. In addition, 27 amino acid substitutions were found in the spike protein with a length of 1,273 amino acids, including six substitutions in the RBD at amino acid region 357-528 and six substitutions in the underpinning subdomain (SD) at amino acid region 569-655. Moreover, four substitutions (Q560L, S570A, F572T, and S575A) in the C-terminal of the receptor-binding subunit S1 domain (Figure 2) are situated in two peptides previously reported to be antigens for SARSCoV (Guo et al., 2004). Due to very limited knowledge of this novel virus, we are unable to give reasonable explanations for the significant number of amino acid substitutions between the 2019-nCoV and SARS or SARS-like CoVs. For example, no amino acid substitutions were present in the receptor-bind-
4 Cell Host & Microbe 27, March 11, 2020
ing motifs that directly interact with human receptor ACE2 protein in SARS-CoV (Ge et al., 2013), but six mutations occurred in the other region of the RBD. Whether these differences could affect the host tropism and transmission property of the 2019-nCoV compared to SARS-CoV is worthy of future investigation. SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j.chom.2020.02.001. ACKNOWLEDGMENTS This work was supported by the National Key Plan for Scientific Research and Development of China (2016YFD0500301 and 2016YFC1200200), CAMS Initiative for Innovative Medicine (CAMS-I2M and 2016-I2M-1-005), the National Natural Science Foundation of China (U1603126), the Central Public-Interest Scientific Institution Basal Research Fund (2016ZX310195, 2017PT31026, and 2018PT31016), and NIH R01AI069120 (United States). REFERENCES China CDC (2020). Tracking the Epidemic. http:// weekly.chinacdc.cn/news/TrackingtheEpidemic. htm?from=timeline#Beijing%20Municipality% 20Update. Cui, J., Li, F., and Shi, Z.L. (2019). Origin and evolution of pathogenic coronaviruses. Nat. Rev. Microbiol. 17, 181–192. Drosten, C., Kellam, P., and Memish, Z.A. (2014). Evidence for camel-to-human transmission of MERS coronavirus. N. Engl. J. Med. 371, 1359–1360. Ge, X.Y., Li, J.L., Yang, X.L., Chmura, A.A., Zhu, G., Epstein, J.H., Mazet, J.K., Hu, B., Zhang, W., Peng, C., et al. (2013). Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature 503, 535–538. Guan, Y., Zheng, B.J., He, Y.Q., Liu, X.L., Zhuang, Z.X., Cheung, C.L., Luo, S.W., Li, P.H., Zhang, L.J.,
Guan, Y.J., et al. (2003). Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 302, 276–278. Guo, J.P., Petric, M., Campbell, W., and McGeer, P.L. (2004). SARS corona virus peptides recognized by antibodies in the sera of convalescent cases. Virology 324, 251–256. Huang, C., Wang, Y., Li, X., Ren, L., Zhao, J., Hu, Y., Zhang, L., Fan, G., Xu, J., Gu, X., et al. (2020). Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. https://doi.org/10.1016/s0140-6736(20)30183-5. Jeffers, S.A., Tusell, S.M., Gillim-Ross, L., Hemmila, E.M., Achenbach, J.E., Babcock, G.J., Thomas, W.D., Jr., Thackray, L.B., Young, M.D., Mason, R.J., et al. (2004). CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus. Proc. Natl. Acad. Sci. USA 101, 15748–15753. Li, F. (2016). Structure, Function, and Evolution of Coronavirus Spike Proteins. Annu. Rev. Virol. 3, 237–261. Song, Z., Xu, Y., Bao, L., Zhang, L., Yu, P., Qu, Y., Zhu, H., Zhao, W., Han, Y., and Qin, C. (2019). From SARS to MERS, Thrusting Coronaviruses into the Spotlight. Viruses 11, E59. Tang, Q., Song, Y., Shi, M., Cheng, Y., Zhang, W., and Xia, X.Q. (2015). Inferring the hosts of coronavirus using dual statistical models based on nucleotide composition. Sci. Rep. 5, 17155. Zhou, P., Yang, X.-L., Wang, X.-G., Hu, B., Zhang, L., Zhang, W., Si, H.-R., Zhu, Y., Li, B., Huang, C.L., et al. (2020). Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin. bioRxiv. https://doi.org/10.1101/2020.01.22.914952. Zhu, Z., Zhang, Z., Chen, W., Cai, Z., Ge, X., Zhu, H., Jiang, T., Tan, W., and Peng, Y. (2018). Predicting the receptor-binding domain usage of the coronavirus based on kmer frequency on spike protein. Infect. Genet. evol. 61, 183–184. Zhu, N., Zhang, D., Wang, W., Li, X., Yang, B., Song, J., Zhao, X., Huang, B., Shi, W., Lu, R., et al.; China Novel Coronavirus Investigating and Research Team (2020). A Novel Coronavirus from Patients with Pneumonia in China, 2019. N. Engl. J. Med. https://doi.org/10.1056/NEJMoa2001017.
Cell Host & Microbe
Commentary Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China Aiping Wu,1,7,9 Yousong Peng,2,9 Baoying Huang,3,9 Xiao Ding,1,7,9 Xianyue Wang,1,7 Peihua Niu,3 Jing Meng,1,7 Zhaozhong Zhu,2 Zheng Zhang,2 Jiangyuan Wang,1,7 Jie Sheng,1,7 Lijun Quan,4 Zanxian Xia,5,8 Wenjie Tan,3,* Genhong Cheng,6,* and Taijiao Jiang1,7,* 1Center for Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China 2College of Biology, Hunan Provincial Key Laboratory of Medical Virology, Hunan University, Changsha 410082, China 3Key Laboratory of Medical Virology, National Health and Family Planning Commission, National Institute for Viral Disease Control and Prevention, China CDC, Beijing 102206, China 4School of Computer Science and Technology, Soochow University, Suzhou, China 5Department of Cell Biology, School of Life Science, Central South University, Changsha 410013, China 6Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, USA 7Suzhou Institute of Systems Medicine, Suzhou, Jiangsu 215123, China 8Hunan Key Laboratory of Animal Models for Human Diseases, Hunan Key Laboratory of Medical Genetics & Center for Medical Genetics, School of Life Science, Central South University, Changsha 410013, China 9These authors contributed equally *Correspondence: [email protected] (W.T.), [email protected] (G.C.), [email protected] (T.J.) https://doi.org/10.1016/j.chom.2020.02.001
An in-depth annotation of the newly discovered coronavirus (2019-nCoV) genome has revealed differences between 2019-nCoV and severe acute respiratory syndrome (SARS) or SARS-like coronaviruses. A systematic comparison identified 380 amino acid substitutions between these coronaviruses, which may have caused functional and pathogenic divergence of 2019-nCoV. A novel coronavirus (CoV) named ‘‘2019 novel coronavirus’’ or ‘‘2019-nCoV’’ by the World Health Organization (WHO) is responsible for the recent pneumonia outbreak that started in early December, 2019 in Wuhan City, Hubei Province, China (Huang et al., 2020; Zhou et al., 2020; Zhu et al., 2020). This outbreak is associated with a large seafood and animal market, and investigations are ongoing to determine the origins of the infection. To date, thousands of human infections have been confirmed in China along with many exported cases across the globe (China CDC, 2020). Coronaviruses mainly cause respiratory and gastrointestinal tract infections and are genetically classified into four major genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus (Li, 2016). The former two genera primarily infect mammals, whereas the latter two predominantly infect birds (Tang et al., 2015). Six kinds of human CoVs have been previously identified. These include HCoV-NL63 and HCoV229E, which belong to the Alphacoronavirus genus; and HCoV-OC43, HCoVHKU1, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV), which belong to the Beta-
coronavirus genus (Tang et al., 2015). Coronaviruses did not attract worldwide attention until the 2003 SARS pandemic, followed by the 2012 MERS and, most recently, the 2019-nCoV outbreaks (China CDC, 2020; Song et al., 2019). SARS-CoV and MERS-CoV are considered highly pathogenic (Cui et al., 2019), and it is very likely that both SARS-CoV and MERS-CoV were transmitted from bats to palm civets (Guan et al., 2003) or dromedary camels (Drosten et al., 2014), and finally to humans (Cui et al., 2019). The genome of coronaviruses, whose size ranges between approximately 26,000 and 32,000 bases, includes a variable number (from 6 to 11) of open reading frames (ORFs) (Song et al., 2019). The first ORF representing approximately 67% of the entire genome encodes 16 non-structural proteins (nsps), while the remaining ORFs encode accessory proteins and structural proteins (Cui et al., 2019). The four major structural proteins are the spike surface glycoprotein (S), small envelope protein (E), matrix protein (M), and nucleocapsid protein (N). The spike surface glycoprotein plays an essential role in binding to receptors on the host cell and determines host tropism (Li, 2016; Zhu et al., 2018). The spike proteins of SARS-CoV and MERS-CoV bind
to different host receptors via different receptor-binding domains (RBDs). SARSCoV uses angiotensin-converting enzyme 2 (ACE2) as one of the main receptors (Ge et al., 2013) with CD209L as an alternative receptor (Jeffers et al., 2004), whereas MERS-CoV uses dipeptidyl peptidase 4 (DPP4, also known as CD26) as the primary receptor. Initial analysis suggested that 2019-nCoV has a close evolutionary association with the SARSlike bat coronaviruses (Zhou et al., 2020). Here, based on the first three determined genomes of the novel coronavirus (2019-nCoV), namely Wuhan/ IVDC-HB-01/2019 (GISAID accession ID: EPI_ISL_402119) (HB01), Wuhan/IVDCHB-04/2019 (EPI_ISL_402120) (HB04), and Wuhan/IVDC-HB-05/2019 (EPI_ ISL_402121) (HB05), an in-depth genome annotation of this virus was performed with a comparison to related coronaviruses, including 1,008 human SARSCoV, 338 bat SARS-like CoV, and 3,131 human MERS-CoV, whose genomes were published before January 12, 2020 (release date: September 12, 2019) from Virus Pathogen Database and Analysis Resource (ViPR) (http://www.viprbrc. org/) and NCBI. Comparison of genomes of these three strains showed that they are almost
Cell Host & Microbe 27, March 11, 2020 ª 2020 Published by Elsevier Inc. 1
Please cite this article in press as: Wu et al., Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.02.001
Cell Host & Microbe
Commentary IVDC-HB-01/2019 (~29.8kb)
A
5’UTR
N
7a 8b
3a E M
pp1ab S
pp1a
3’UTR p6
3b
9b orf14
7b
3860 1
nsp1 181
nsp4
nsp3
nsp2 819
nsp5
2764 7096
nsp16
nsp15
6799
6453
3264
nsp13
nsp14
3570
4141
nsp7 nsp8 nsp9 nsp10 4392 3943 4254 4393
nsp12 5325
5926
B
nsp6
C
whole genome
pp1a
MERS-CoV
3a
2019-nCoV
pp1ab
spike
envelope
matrix
SARS-like bat CoV Human SARS-CoV
Alphacoronavirus Betacoronavirus Gammacoronavirus Deltacoronavirus
HB-01/2019 SARS-like bat CoV
7a MERS-CoV
8b
nucleocapsid
Other CoVs
Human SARS-CoV
Figure 1. Genome composition and phylogenetic tree for 2019-nCoV (A) Schematic diagram of the genome organization and the encoded proteins of pp1ab and pp1a for the IVDC-HB-01/2019 (HB01) strain. The largest gene, namely the orf1ab, encodes the pp1ab protein that contains 15 nsps (nsp1-nsp10 and nsp12-nsp16). The pp1a protein encoded by the orf1a gene also contains 10 nsps (nsp1-nsp10). Structural proteins are encoded by the four structural genes, including spike (S), envelope (E), membrane (M), and nucleocapsid (N) genes. The accessory genes are distributed among the structural genes. The protein-encoding genes of the genome of 2019-nCoV were predicted by the online servers of GeneMarkS (http://exon.gatech.edu/GeneMark/genemarks.cgi) and ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/) with manual check. (B) Phylogenetic relationship based on the whole genome for the HB01 strain and other coronaviruses. All viral strains were classified by the genus and the type, which are presented on the left and right schematic phylogenetic trees, respectively. The four genera of the coronaviruses, including Alphacoronavirus (red), Betacoronavirus (blue), Gammacoronavirus (green), and Deltacoronavirus (violet) are blocked in the left phylogenetic tree. The MERS coronavirus (brown), the SARS-like bat coronavirus (violet), human SARS coronavirus (light blue), and the HB01 strain (red) are highlighted by lines of different colors in the right phylogenetic tree. (C) Schematic phylogenetic trees of individual genes for the HB01 strain. The coronavirus species were colored in the same way as (B). The amount of the strains in the phylogenetic clade is denoted by the area of the circles.
identical, with only five nucleotide differences in the genome of ~29.8 kb nucleotides (Figure S1). The 2019-nCoV genome was annotated to possess 14 ORFs encoding 27 proteins (Figure 1A and Tables S1A and S1B). The orf1ab and orf1a genes located at the 50 -terminus of the genome respectively encode the pp1ab and pp1a proteins, respectively. They together comprise 15 nsps including nsp1 to nsp10 and nsp12 to nsp16 (Figure 1A and Table S1B). The 30 -terminus of the genome contains four structural proteins (S, E, M, and N) and eight accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b, and orf14). At the amino acid level, the 2 Cell Host & Microbe 27, March 11, 2020
2019-nCoV is quite similar to that of SARS-CoV, but there are some notable differences. For example, the 8a protein is present in SARS-CoV and absent in 2019-nCoV; the 8b protein is 84 amino acids in SARS-CoV, but longer in 2019nCoV, with 121 amino acids; the 3b protein is 154 amino acids in SARS-CoV, but shorter in 2019-nCoV, with only 22 amino acids (Table S1A). Further studies are needed to characterize how these differences affect the functionality and pathogenesis of 2019-nCoV. As shown in a phylogenetic tree based on whole genomes (Figures 1B and S2) with the Molecular Evolutionary Genetics
Analysis (MEGA) (version 7.0), the 2019nCoV is in the same Betacoronavirus clade as MERS-CoV, SARS-like bat CoV, and SARS-CoV. The phylogenetic tree falls into two clades. The Betacoronavirus genus constitutes one clade, while the Alphacoronavirus, Gammacoronavirus, and Deltacoronavirus genera constitute the other clade. The 2019-nCoV is parallel to the SARS-like bat CoVs, while the SARS-CoVs are descended from the SARS-like bat CoVs, indicating that 2019-nCoV is closer to the SARS-like bat CoVs than the SARS-CoVs in terms of the whole genome sequence. Tables S1C and S1D also show that the genome
Please cite this article in press as: Wu et al., Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.02.001
Cell Host & Microbe
Commentary nsp1
nsp2 267
272
293
294
316
324
337
350
354
368
374
390
417
442
449
456
531
553
559
594
601
605
608
635
645
649
L
N
N
I
T
L
D
C
D
V
T
A
I
H
E
E
H
G
N
K
S
A
E
E
V
N
L
I
V
L
F
G
V
I
C
S
S
K
K
K
V
T
E
H
N
L
V
P
M Q
D
H
Y
N
D
R
T
G
Q
D
I
S
V
V
L
Q
L
A
L
L
G
1064
655
259
T
S
652
211 A
G
595
166 S
E
539
158 Q
Y
535
157 F
I
386
154 Y
T
283
114 I
L
280
56 V
2019-nCoV
224
38 V
SARS/SARS-like CoVs A
nsp3
nsp2 812
827
893
902
965
975
1027
1042
1057
1081
1104
1124
1178
1218
1271
E
P
T
Q
A
K
M M V
T
D
S
I
I
E
L
M
T
T
Q
S
N
V
Y
N
V
K
F
S
V
I
Q
D
D
S
E
G
Q
L
V
E
N
V
V
D
M V
C
S
E
Q
C
I
H
G
L
A
N
E
L
E
A
880
L
1274
806
G
L
945
798
T
Q
888
764
E
K
849
753
R
R
838
727
K
Q
808
726
H
A
807
724
T
I
776
722
V
R
748
710
K
A
745
701
G
E
728
696
A
A
712
687
F
V
711
685
T
K
666
G
661 2019-nCoV SARS/SARS-like CoVs
nsp3 1399
1420
1512
1522
1537
1553
1619
1623
1662
1677
1679
1682
1692
1706
1718
1734
1736
1742
1744
1758
1779
1795
1813
1817
1822
1825
1837
1856
1863
1889
2105
2137
2142
2156
2165
I
E
V
A
T
K
Q
I
Y
F
Y
N
N
A
A
T
P
E
C
Y
F
D
C
Q
F
K
K
Q
K
T
S
K
S
I
C
L
T
A
I
C
A
V
D
M V
P
R
E
V
H
L
F
S
G
S
V
A
A
D
S
H
L
E
A
K
L
R
Q
E
Q
L
N
T
M V
Y
I
K
V
C
F
2623
2641
2695
1791
1345 V
L
1525
1339 V
C
1444
1298 A
I
1394
1296 L
2019-nCoV
1355
1295 V
SARS/SARS-like CoVs D
nsp3 2233
2238
2254
2280
2303
2314
2316
2336
2431
2442
2453
2501
2517
2553
2584
2596
2618
2647
2660
2673
2709
2710
2718
2738
2754
2759
2309
2228
N
N
F
V
M Y
T
T
S
I
F
W R
R
K
V
I
S
S
A
N
A
N
F
E
Q
Y
S
I
A
F
L
V
A
K
T
L
I
L
S
D
A
V
Y
L
K
R
L
L
P
A
T
D
S
G
V
D
H
F
N
V
S
Y
I
I
S
V
2274
2222
L V
2019-nCoV
2266
2218
2177 L
SARS/SARS-like CoVs F
K
ORF1ab
nsp4
nsp5
2938
2943
2944
2946
2947
2998
3030
3038
3053
3056
3066
3082
3097
3116
3129
3133
3135
3154
3171
3196
3197
3221
3349
3465
3530
3548
3549
L
T
M
T
D
V
I
V
V
A
E
S
V
M
I
V
V
R
S
F
T
V
L
M V
T
L
I
Y
S
D
V
M
V
V
S
A
L
V
M
L
D
G
I
V
L
I
S
S
E
L
I
V
I
L
K
N
A
A
A
F
F
S
I
V
H
T
E
T
L
L
L
A
T
I
nsp8
3627
3628
3668
3670
3678
3704
3715
3737
3751
3757
3769
3791
3847
4188
4276
L
S
F
M
F
D
V
F
K
G
L
I
V
G
F
T
V
F
I
T
S
F
A
L
I
V
A
C
L
L
E
A
Y
R
A
I
V
I
A
L
C
I
Y
V
N
N
Y
P
M
3957
nsp14
4625
4657
5131
5132
5161
5164
5176
5939
6024
6057
6059
6062
6145
6219
6244
6270
6299
6321
6418
6435
T
T
V
Y
K
T
D
F
T
S
S
V
E
D
S
S
A
I
T
I
A
D
S
S
L
V
D
V
A
I
L
C
H
E
Y
N
A
A
I
D
E
T
N
S
V
S
V
S
E
H
A
Q
I
nsp16
6543
6567
6569
6570
6574
6607
6623
6631
6653
6675
6703
6710
6715
6716
6799
6830
6833
6936
6951
6956
7014
7063
7070
7074
D
I
A
P
F
V
A
Y
Q
E
L
F
F
E
S
D
S
T
N
I
Q
R
G
L
S
I
I
E
A
S
S
L
K
S
F
E
Q M S
L
K
A
E
N
V
H
L
K
K
N
Y
E
V
1216
S1 subunit
RBD
S2 subunit
SD 575
726
872
875
879
882
884
1070
1133
T
A
E
R
S
I
T
I
Q
S
A
I
S
A
V
I
S
F
S
D
Q
Q
V
S
V
A
A
S
A
A
S
I
V
37
127
619
209
215
227
228
S
C
S
S
Y
H
V
V
T
Y
G
E
E
N
A
71
Q V
T
V
Q
L
M M
L
L
L
C
P
V
I
L
I
E
P
S
R
Y
A
V
SARS/SARS-like CoVs I
68
2019-nCoV SARS/SARS-like CoVs
2019-nCoV
9b
67
ORF14
T
66
H
I
T
65
T
T
K
I
M
63
A
E
A
P
T
H
52
D
N
T
E
L
L
60
S
D
N
2019-nCoV
F
46
G
S
SARS/SARS-like CoVs T
I T
39
2019-nCoV SARS/SARS-like CoVs
7b
2019-nCoV SARS/SARS-like CoVs
40
I
3
T
L
32
I
T
217
V
R
26
K
T
103
V
25
2019-nCoV SARS/SARS-like CoVs
3b
23
205
L
C
43
200
L
I
40
166
V
Q
5
147
H
L
38
112
I
H
334
78
R
A
111
30
S
N
107
26
D
74
22
I L
72
10 2019-nCoV SARS/SARS-like CoVs
21
572
A
Q
16
570
L
N
14
560
H
K
11
519
N
T
6
430
S
V
5
438
K
E
719
417
N
P
693
354
A
K
689
348
Q
646
271
S N
50
112
S
SARS/SARS-like CoVs L
2019-nCoV
7088
6494 L
6831
6482 T A
6459 F
SARS/SARS-like CoVs Y
2019-nCoV
N
6218
4618
N
V
6156
4590
I
V
5158
4498
K
L
4673
4490
I
G
4617
4458
D
E
4455
D
4454 2019-nCoV SARS/SARS-like CoVs
nsp15
7a
4391
3624
V
4115
3622
I F
4087
3606
L M
4074
3593
3677
3587
3583
3576 I
nsp12
3a
nsp9 nsp10
SARS/SARS-like CoVs V
2019-nCoV
S
A
3145
2873
I L
nsp6
3130
2865
3080
2833
3075
2796
2825
2791
2781
2774
L F
2019-nCoV
2770
2768 N
SARS/SARS-like CoVs T
K
Figure 2. Amino Acid Substitutions of 2019-nCoV against SARS and SARS-like Viruses All 27 proteins encoded by 2019-nCoV have been aligned against SARS-CoVs and SARS-like bat CoVs using the FFT-NS-2 algorithm in MAFFT (version v7.407) (The number of aligned proteins were listed in Table S1E). An amino acid substitution was defined as an absolutely conserved site in the group of SARS and SARS-like CoVs but different from that of 2019-nCoV. In total, 380 amino acid substitutions have been identified between the amino acid sequences of 2019nCoV (HB01) and the corresponding consensus sequences of SARS and SARS-like CoVs.
of 2019-nCoV has the highest similarity with that of a SARS-like bat CoV (MG772933). In comparison, 2019-nCoV is distant from and less related to the MERS-CoVs. In terms of the encoded proteins of pp1ab, pp1a, envelope, ma-
trix, accessory protein 7a, and nucleocapsid genes, phylogenetic analyses showed that the 2019-nCoV is closest to the SARS-like bat CoVs (Figure 1C and Table S1D). Regarding the spike gene, the 2019-nCoV is closest to the bat
CoVs, while the 3a and 8b accessory genes are both closest to the SARSCoVs. Although phylogenetic analyses for the whole genome and individual genes clearly show that the 2019-nCoV is most closely related to SARS-like bat Cell Host & Microbe 27, March 11, 2020 3
Please cite this article in press as: Wu et al., Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.02.001
Cell Host & Microbe
Commentary viruses (Figures 1B and 1C), we did not find a single strain of a SARS-like bat virus that harbors all proteins with the most similarity to counterparts of the 2019nCoV (Figures 1B and 1C). Given the close relationship between 2019-nCoV and SARS-CoVs or SARSlike bat CoVs (Figures 1B and 1C), an examination of the amino acid substitutions in different proteins could shed light into how 2019-nCoV differs structurally and functionally from SARS-CoVs. In total, there were 380 amino acid substitutions between the amino acid sequences of 2019-nCoV (HB01) and the corresponding consensus sequences of SARS and SARS-like viruses (Figure 2 and Tables S1E and S1F). No amino acid substitutions occurred in nonstructural protein 7 (nsp7), nsp13, envelope, matrix, or accessory proteins p6 and 8b (Table S1F). Respectively, 102 and 61 amino acid substitutions are located in nsp3 and nsp2. In addition, 27 amino acid substitutions were found in the spike protein with a length of 1,273 amino acids, including six substitutions in the RBD at amino acid region 357-528 and six substitutions in the underpinning subdomain (SD) at amino acid region 569-655. Moreover, four substitutions (Q560L, S570A, F572T, and S575A) in the C-terminal of the receptor-binding subunit S1 domain (Figure 2) are situated in two peptides previously reported to be antigens for SARSCoV (Guo et al., 2004). Due to very limited knowledge of this novel virus, we are unable to give reasonable explanations for the significant number of amino acid substitutions between the 2019-nCoV and SARS or SARS-like CoVs. For example, no amino acid substitutions were present in the receptor-bind-
4 Cell Host & Microbe 27, March 11, 2020
ing motifs that directly interact with human receptor ACE2 protein in SARS-CoV (Ge et al., 2013), but six mutations occurred in the other region of the RBD. Whether these differences could affect the host tropism and transmission property of the 2019-nCoV compared to SARS-CoV is worthy of future investigation. SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j.chom.2020.02.001. ACKNOWLEDGMENTS This work was supported by the National Key Plan for Scientific Research and Development of China (2016YFD0500301 and 2016YFC1200200), CAMS Initiative for Innovative Medicine (CAMS-I2M and 2016-I2M-1-005), the National Natural Science Foundation of China (U1603126), the Central Public-Interest Scientific Institution Basal Research Fund (2016ZX310195, 2017PT31026, and 2018PT31016), and NIH R01AI069120 (United States). REFERENCES China CDC (2020). Tracking the Epidemic. http:// weekly.chinacdc.cn/news/TrackingtheEpidemic. htm?from=timeline#Beijing%20Municipality% 20Update. Cui, J., Li, F., and Shi, Z.L. (2019). Origin and evolution of pathogenic coronaviruses. Nat. Rev. Microbiol. 17, 181–192. Drosten, C., Kellam, P., and Memish, Z.A. (2014). Evidence for camel-to-human transmission of MERS coronavirus. N. Engl. J. Med. 371, 1359–1360. Ge, X.Y., Li, J.L., Yang, X.L., Chmura, A.A., Zhu, G., Epstein, J.H., Mazet, J.K., Hu, B., Zhang, W., Peng, C., et al. (2013). Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature 503, 535–538. Guan, Y., Zheng, B.J., He, Y.Q., Liu, X.L., Zhuang, Z.X., Cheung, C.L., Luo, S.W., Li, P.H., Zhang, L.J.,
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