Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan

Jasper Fuk-Woo Chan, Kin-Hang Kok, Zheng Zhu, Hin Chu, Kelvin Kai-Wang To, Shuofeng Yuan, Kwok-Yung Yuen, Jasper Fuk-Woo Chan, Kin-Hang Kok, Zheng Zhu, Hin Chu, Kelvin Kai-Wang To, Shuofeng Yuan, Kwok-Yung Yuen

Abstract

A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus (2019-nCoV) was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike's receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet(s) containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.

Keywords: Coronavirus; SARS; Wuhan; bioinformatics; emerging; genome; respiratory; virus.

Figures

Figure 1.
Figure 1.
Betacoronavirus genome organization. The betacoronavirus genome comprises of the 5'-untranslated region (5'-UTR), open reading frame (orf) 1a/b (yellow box) encoding non-structural proteins (nsp) for replication, structural proteins including spike (blue box), envelop (orange box), membrane (red box), and nucleocapsid (cyan box) proteins, accessory proteins (purple boxes) such as orf 3, 6, 7a, 7b, 8 and 9b in the 2019-nCoV (HKU-SZ-005b) genome, and the 3'-untranslated region (3'-UTR). Examples of lineages A to D betacoronaviruses include human coronavirus (HCoV) HKU1 (lineage A), 2019-nCoV (HKU-SZ-005b) and SARS-CoV (lineage B), MERS-CoV and Tylonycteris bat CoV HKU4 (lineage C), and Rousettus bat CoV HKU9 (lineage D). The length of nsps and orfs are not drawn in scale.
Figure 2.
Figure 2.
Comparison of protein sequences of Spike stalk S2 subunit. Multiple alignment of Spike S2 amino acid sequences of 2019-nCoV HKU-SZ-005b (accession number MN975262), bat SARS-like coronavirus isolates bat-SL-CoVZXC21 and bat-SL-CoVZXC45 (accession number MG772934.1 and MG772933.1, respectively) and human SARS coronavirus (accession number NC004718) was performed and displayed using CLUSTAL 2.1 and BOXSHADE 3.21 respectively. The black boxes represent the identity while the grey boxes represent the similarity of the four amino acid sequences.
Figure 3.
Figure 3.
Comparison of protein sequences of A. Spike globular head S1, and B. S1 receptor-binding domain (RBD) subunit. Multiple alignment of Spike S1 amino acid sequences of 2019-nCoV HKU-SZ-005b (accession number MN975262), bat SARS-like coronavirus isolates bat-SL-CoVZXC21, bat-SL-CoVZXC45, bat-SL-CoV-YNLF_31C, bat-SL-CoV-YNLF_34C and bat SL-CoV HKU3-1 (accession number MG772934.1 and MG772933.1, KP886808, KP886809 and DQ022305, respectively), human SARS coronavirus GZ02 and Tor2 (accession number AY390556 and AY274119, respectively) and Paguma SARS-CoV (accession number AY515512) was performed and displayed using CLUSTAL 2.1 and BOXSHADE 3.21, respectively. The black background represents the identity while the grey background represents the similarity of the amino acid sequences. Orange box indicates the region of signal peptide, while green and blue boxes indicate the core domain and receptor binding domain respectively. Sequences of RBD, highlighted in (A) were used for comparison. External subdomain variable region of 2019-nCoV HKU-SZ-005b was predicted by comparison of amino acid similarity and published structural analysis [17]. Purple box indicates the external subdomain region.
Figure 3.
Figure 3.
Comparison of protein sequences of A. Spike globular head S1, and B. S1 receptor-binding domain (RBD) subunit. Multiple alignment of Spike S1 amino acid sequences of 2019-nCoV HKU-SZ-005b (accession number MN975262), bat SARS-like coronavirus isolates bat-SL-CoVZXC21, bat-SL-CoVZXC45, bat-SL-CoV-YNLF_31C, bat-SL-CoV-YNLF_34C and bat SL-CoV HKU3-1 (accession number MG772934.1 and MG772933.1, KP886808, KP886809 and DQ022305, respectively), human SARS coronavirus GZ02 and Tor2 (accession number AY390556 and AY274119, respectively) and Paguma SARS-CoV (accession number AY515512) was performed and displayed using CLUSTAL 2.1 and BOXSHADE 3.21, respectively. The black background represents the identity while the grey background represents the similarity of the amino acid sequences. Orange box indicates the region of signal peptide, while green and blue boxes indicate the core domain and receptor binding domain respectively. Sequences of RBD, highlighted in (A) were used for comparison. External subdomain variable region of 2019-nCoV HKU-SZ-005b was predicted by comparison of amino acid similarity and published structural analysis [17]. Purple box indicates the external subdomain region.
Figure 4.
Figure 4.
Analysis of orf3b. A. Multiple alignment of orf3b protein sequence between 2019-nCoV (HKU-SZ-005b), SARS-CoV and SARS-related CoV. B. A novel putative short protein found in orf3b.
Figure 5.
Figure 5.
Analysis of orf8 to show novel putative protein. (A) Phylogenetic analysis of orf8 amino acid sequences of 2019-nCoV HKU-SZ-005b (accession number MN975262), bat SARS-like coronavirus isolates bat-SL-CoVZXC21 and bat-SL-CoVZXC45 (accession number MG772934.1 and MG772933.1, respectively) and human SARS coronavirus (accession number AY274119) was performed using the neighbour-joining method with bootstrap 1000. The evolutionary distances were calculated using the JTT matrix-based method. (B) Multiple alignment was performed and displayed using CLUSTAL 2.1 and BOXSHADE 3.21, respectively. The black background represents the identity while the grey background represents the similarity of the amino acid sequences. (C) Structural analysis of Orf8 was performed using PSI-blast-based secondary structure PREDiction (PSIPRED). Predicted helix structure (h) and strand (s) were boxed with red and yellow respectively.
Figure 6.
Figure 6.
Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees using amino acid sequences of (A) orf1ab polypeptide; (B) Spike glycoprotein; (C) Envelope protein; (D) Membrane protein; (E) Nucleoprotein.
Figure 6.
Figure 6.
Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees using amino acid sequences of (A) orf1ab polypeptide; (B) Spike glycoprotein; (C) Envelope protein; (D) Membrane protein; (E) Nucleoprotein.
Figure 6.
Figure 6.
Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees using amino acid sequences of (A) orf1ab polypeptide; (B) Spike glycoprotein; (C) Envelope protein; (D) Membrane protein; (E) Nucleoprotein.
Figure 6.
Figure 6.
Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees using amino acid sequences of (A) orf1ab polypeptide; (B) Spike glycoprotein; (C) Envelope protein; (D) Membrane protein; (E) Nucleoprotein.
Figure 6.
Figure 6.
Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees using amino acid sequences of (A) orf1ab polypeptide; (B) Spike glycoprotein; (C) Envelope protein; (D) Membrane protein; (E) Nucleoprotein.
Figure 7.
Figure 7.
Secondary structure prediction and comparison in the 5′-untranslated region (UTR) and 3′-UTR using the RNAfold WebServer (with minimum free energy and partition function in Fold algorithms and basic options. The SARS 5′- and 3′- UTR was used as a reference to adjust the prediction results.(A) SARS-CoV 5'-UTR; (B) 2019-nCoV (HKU-SZ-005b) 5'-UTR; (C) ZC45 5'-UTR; (D) SARS-CoV 3'-UTR; (E) 2019-nCoV (HKU-SZ-005b) 3'-UTR; (F) ZC45 3'-UTR.
Figure 7.
Figure 7.
Secondary structure prediction and comparison in the 5′-untranslated region (UTR) and 3′-UTR using the RNAfold WebServer (with minimum free energy and partition function in Fold algorithms and basic options. The SARS 5′- and 3′- UTR was used as a reference to adjust the prediction results.(A) SARS-CoV 5'-UTR; (B) 2019-nCoV (HKU-SZ-005b) 5'-UTR; (C) ZC45 5'-UTR; (D) SARS-CoV 3'-UTR; (E) 2019-nCoV (HKU-SZ-005b) 3'-UTR; (F) ZC45 3'-UTR.

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