Articles
Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan
Pages 221-236 | Received 16 Jan 2020, Accepted 17 Jan 2020, Published online: 28 Jan 2020
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, Wuhan, SARS, emerging, genome, respiratory, virus, bioinformatics
Introduction
Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales.There are four genera of CoVs, namely, Alphacoronavirus (αCoV), Betacoronavirus (βCoV), Deltacoronavirus (δCoV), and Gammacoronavirus (γCoV) [1].
Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs,
while avian species are the gene sources of most δCoVs and γCoVs.
CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV (SARS-CoV) which emerged in China in 2002–2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV (MERS-CoV) which has caused a persistent epidemic in the Arabian Peninsula since 2012 [2,3]. In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host [the Himalayan palm civet (Paguma larvata) for SARS-CoV and the dromedary camel (Camelus dromedarius) for MERS-CoV] before crossing species barriers to infect humans.
Prior to December 2019, 6 CoVs were known to infect human,
including 2 αCoV
(HCoV-229E and
HKU-NL63)
and
4 βCoV
(HCoV-OC43 [lineage A],
HCoV-HKU1 [lineage A]
SARS-CoV [lineage B] and
MERS-CoV [lineage C]).
The βCoV lineage A HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly [4].
In contrast, SARS-CoV (lineage B βCoV) and MERS-CoV (lineage C βCoV) may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively [5,6].
On 31 December 2019, the World Health Organization (WHO) was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China [7]. Subsequent virological testing showed that a novel CoV was detected in these patients.
As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan [8,9].
The earliest date of symptom onset was 1 December 2019 [10].
The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 (32%) required intensive care and 6 (15%) died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans [10].
We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan [11]. In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related β CoVs to provide insights into the potential source and control strategies.
Materials and methods
Viral sequences
The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank (accession no. MN975262) (Table 1). The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup.Table 1 of 3
| Accession number | Name displayed on the tree | Name of full-length genome | Year |
|---|---|---|---|
| AY274119 | Human SARS-CoV Tor2 2003 | SARS-related coronavirus isolate Tor2 | 2003 |
| AY278488 | Human SARS-CoV BJ01 2003 | SARS coronavirus BJ01 | 2003 |
| AY278491 | SARS coronavirus HKU-39849 2003 | SARS coronavirus HKU-39849 2003 | 2003 |
| AY390556 | Human SARS-CoV GZ02 2003 | SARS coronavirus GZ02 | 2003 |
| AY391777 | Human CoV OC43 2003 | Human coronavirus OC43 | 2003 |
| AY515512 | Paguma SARS CoV HC/SZ/61/03 2003 | SARS coronavirus HC/SZ/61/03 (paguma SARS) | 2018 |
| EF065513 | Bat CoV HKU9-1 2006 | Bat coronavirus HKU9-1 | 2006 |
| FJ588686 | Bat SL-CoV Rs672 2006 | Bat SARS CoV Rs672/2006 | 2006 |
| KC881005 | Bat SL-CoV RsSHC014 2013 | Bat SARS-like coronavirus RsSHC014 | 2013 |
| KC881006 | Bat SL-CoV Rs3367 2013 | Bat SARS-like coronavirus Rs3367 | 2013 |
| KY417146 | Bat SL-CoV Rs4231 2016 | Bat SARS-like coronavirus isolate Rs4231 | 2016 |
| KY417149 | Bat SL-CoV Rs4255 2016 | Bat SARS-like coronavirus isolate Rs4255 | 2016 |
| MG772933 | Bat SL-CoV ZC45 2018 | Bat SARS-like coronavirus isolate bat-SL-CoVZC45 | 2018 |
| MG772934 | Bat SL-CoV ZXC21 2018 | Bat SARS-like coronavirus isolate bat-SL-CoVZXC21 | 2018 |
| MK211377 | Bat CoV YN2018C 2018 | Coronavirus BtRs-BetaCoV/YN2018C | 2018 |
| MK211378 | Bat CoV YN2018D 2018 | Coronavirus BtRs-BetaCoV/YN2018Da | 2018 |
| MN975262 | HKU-SZ-005b | Human 2019-nCoV HKU-SZ-005b | 2020 |
| NC002645 | Human CoV 229E 2000 | Human coronavirus 229E | 2000 |
| NC006577 | Human CoV HKU1 2004 | Human coronavirus HKU1 | 2004 |
| NC009019 | Bat CoV HKU4-1 2006 | Bat coronavirus HKU4-1 | 2006 |
| NC009020 | Bat CoV HKU5-1 2006 | Bat coronavirus HKU5-1 | 2006 |
| NC014470 | Bat SARS-related CoV BM48-31 2009 | Bat coronavirus BM48-31/BGR/2008 | 2008 |
| NC019843 | Human MERS-CoV 2012 | Middle East respiratory syndrome coronavirus | 2012 |
aOne nucleotide was added within M gene to maintain the sequence in-frame.
Genome characterization and phylogenetic analysis
Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees [12]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown next to the branches [13]. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site [14]. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Evolutionary analyses were conducted in MEGA X [15]. Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOXSHADE 3.21.Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction (PSIPRED) [16]. For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOXSHADE alignment.
Prediction of transmembrane domains was performed using the TMHMM 2.0 server (http://www.cbs.dtu.dk/services/TMHMM/). Secondary structure prediction in the 5′-untranslated region (UTR) and 3′-UTR was performed using the RNAfold WebServer (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) with minimum free energy (MFE) and partition function in Fold algorithms and basic options.
Results and discussion
Genome organization
The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other βCoVs, the 2019-nCoV genome contains two flanking untranslated regions (UTRs) and a single long open reading frame encoding a polyprotein.The 2019-nCoV genome is arranged in the order of 5′-replicase (orf1/ab)-structural proteins [Spike (S)-Envelope (E)-Membrane (M)-Nucleocapsid (N)]−3′ and lacks the hemagglutinin-esterase gene which is characteristically found in lineage A β-CoVs (Figure 1).
Figure 1.
Betacoronavirus genome organization.
Coronavirus genome comprises of 5′ untranslated region (5UTR) including 5′ leader sequence, open reading frame (orf) 1a/b (yellow box) encoding non-structural proteins (nsp) for replication, structural proteins including envelop (orange box), membrane (red) and nucleoprotein (cyan box), accessory proteins (purple boxes) such as orf 3, 6, 7a, 7b 8 and 9b of 2019-nCoV (HKU-SZ-005b) genome, and 3′ untranslated region (3UTR).
Examples of each betacoronavirus lineage are
human coronavirus (HCoV) HKU1 (lineage A),
2019-nCoV (HKU-SZ-005b) and
SARS-CoV (lineage B),
Human MERS-CoV and
bat CoV HKU9 (lineage C)
and Bat CoV HKU4 (lineage D).
The length of nsps and orfs are not drawn in scale.
The human SARS-CoV 5′- and 3′- UTR were used as references to adjust the prediction results.
Coronavirus genome comprises of 5′ untranslated region (5UTR) including 5′ leader sequence, open reading frame (orf) 1a/b (yellow box) encoding non-structural proteins (nsp) for replication, structural proteins including envelop (orange box), membrane (red) and nucleoprotein (cyan box), accessory proteins (purple boxes) such as orf 3, 6, 7a, 7b 8 and 9b of 2019-nCoV (HKU-SZ-005b) genome, and 3′ untranslated region (3UTR).
Examples of each betacoronavirus lineage are
human coronavirus (HCoV) HKU1 (lineage A),
2019-nCoV (HKU-SZ-005b) and
SARS-CoV (lineage B),
Human MERS-CoV and
bat CoV HKU9 (lineage C)
and Bat CoV HKU4 (lineage D).
The length of nsps and orfs are not drawn in scale.
The human SARS-CoV 5′- and 3′- UTR were used as references to adjust the prediction results.
The 5′- and 3′-UTRs are 265 and 358 nucleotides long, respectively.
The 5′- and 3 ′-UTR sequences of 2019-nCoV are similar to those of other βCoVs with nucleotide identities of ⩾83.6%.
The large replicase polyproteins pp1a and pp1ab encoded by the partially overlapping 5′-terminal orf1a/b within the 5′ two-thirds of the genome is proteolytic cleaved into 16 putative non-structural proteins (nsps). These putative nsps included two viral cysteine proteases, namely, nsp3 (papain-like protease) and nsp5 (chymotrypsin-like, 3C-like, or main protease), nsp12 (RNA-dependent RNA polymerase [RdRp]), nsp13 (helicase), and other nsps which are likely involved in the transcription and replication of the virus (Table 2).
( There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV (Table 3).
The major distinction between SARSr-CoV (SARS-related) and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots).
Table 2. Putative functions and proteolytic cleavage sites of 16 nonstructural proteins(nsps) in orf1a/b as predicted by bioinformatics.
| NSP | Putative function/domain | Amino acid position | Putative cleave site |
|---|---|---|---|
| nsp1 | suppress antiviral host response | M1 – G180 | (LNGG'AYTR) |
| nsp2 | unknown | A181 – G818 | (LKGG'APTK) |
| nsp3 | putative PL-pro domain | A819 – G2763 | (LKGG'KIVN) |
| nsp4 | complex with nsp3 and 6: DMV formation | K2764 – Q3263 | (AVLQ'SGFR) |
| nsp5 | 3CL-pro domain | S3264 – Q3569 | (VTFQ'SAVK) |
| nsp6 | complex with nsp3 and 4: DMV formation | S3570 – Q3859 | (ATVQ'SKMS) |
| nsp7 | complex with nsp8: primase | S3860 – Q3942 | (ATLQ'AIAS) |
| nsp8 | complex with nsp7: primase | A3943 – Q4140 | (VKLQ'NNEL) |
| nsp9 | RNA/DNA binding activity | N4141 – Q4253 | (VRLQ'AGNA) |
| nsp10 | complex with nsp14: replication fidelity | A4254 – Q4392 | (PMLQ'SADA) |
| nsp11 | short peptide at the end of orf1a | S4393 – V4405 | (end of orf1a) |
| nsp12 | RNA-dependent RNA polymerase | S4393 – Q5324 | (TVLQ'AVGA) |
| nsp13 | helicase | A5325 – Q5925 | (ATLQ'AENV) |
| nsp14 | ExoN: 3′–5′ exonuclease | A5926 – Q6452 | (TRLQ'SLEN) |
| nsp15 | XendoU: poly(U)-specific endoribonuclease | S6453 – Q6798 | (PKLQ'SSQA) |
| nsp16 | 2'-O-MT: 2'-O-ribose methyltransferase | S6799 – N7096 | (end of orf1b) |
Table 3. Amino acid identity between the 2019 novel coronavirus and bat SARS-like coronavirus or human SARS-CoV.
| Amino acid identity (%) | 2019-nCoV | 2019-nCoV |
|---|---|---|
| vs. bat-SL-CoVZXC21 | vs. SARS-CoV | |
| NSP1 | 96 | 84 |
| NSP2 | 96 | 68 |
| NSP3 | 93 | 76 |
| NSP4 | 96 | 80 |
| NSP5 | 99 | 96 |
| NSP6 | 98 | 88 |
| NSP7 | 99 | 99 |
| NSP8 | 96 | 97 |
| NSP9 | 96 | 97 |
| NSP10 | 98 | 97 |
| NSP11 | 85 | 85 |
| NSP12 | 96 | 96 |
| NSP13 | 99 | 100 |
| NSP14 | 95 | 95 |
| NSP15 | 88 | 89 |
| NSP16 | 98 | 93 |
| Spike | 80 | 76 |
| Orf3a | 92 | 72 |
| Orf3b | 32 | 32 |
| Envelope | 100 | 95 |
| Membrane | 99 | 91 |
| Orf6 | 94 | 69 |
| Orf7a | 89 | 85 |
| Orf7b | 93 | 81 |
| Orf8/Orf8b | 94 | 40 |
| Nucleoprotein | 94 | 94 |
| Orf9b | 73 | 73 |
There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV (Table 3).
The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots.
Spike
(1-13 figures!)
https://www.tandfonline.com/doi/full/10.1080/22221751.2020.1719902
Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain (NTD) and receptor-binding domain (RBD),while the S2 subunit contains conserved fusion peptide (FP), heptad repeat (HR) 1 and 2, transmembrane domain (TM), and cytoplasmic domain (CP). We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs (SL-CoV ZXC21 and ZC45) and human SARS-CoV (Figure 2). Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials [18]. Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV (Figure 3(A)), the core domain of RBD (excluding the external subdomain) are highly conserved (Figure 3(B)). Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain (RBD) when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity.
Spike 1 subunit:
https://www.tandfonline.com/doi/full/10.1080/22221751.2020.1719902
Spik2 2 subunit: ( link) 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.
RBD
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.
RBD:
ORF3
Orf3b
A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b (Figure 4). It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wild-type virus in several cell types [19], suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points [20]. Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling [21]. Subsequently, orf3b homologues identified from three bat SARS-related-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV [22]. IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study.
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.
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