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Original Articles

Genomic analysis and comparative multiple sequences of SARS-CoV2

Chang, Tai-Jaya,b,*; Yang, De-Mingc,d,e; Wang, Mong-Lienf,g; Liang, Kung-Howg,h; Tsai, Ping-Hsingi; Chiou, Shih-Hwai,j; Lin, Ta-Hsienk,l; Wang, Chin-Tienm,n

Author Information
Journal of the Chinese Medical Association: June 2020 - Volume 83 - Issue 6 - p 537-543
doi: 10.1097/JCMA.0000000000000335
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Abstract

1. INTRODUCTION

The severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV) transmitted from animals to humans have caused severe pneumonia in the world.1 SARS emerged in 2002 in Guangdong, China, and its subsequent global spread resulted in 8096 infected cases and 774 deaths.2 After China announced an outbreak of a new coronavirus in the city of Wuhan on December 31, 2019, lash to now, the world has become pandemic. Severe cases from the Huanan Seafood Wholesale market in Wuhan were confirmed pneumonia with the infection of a novel coronavirus (2019-nCoV),3 named SARS-CoV2 (International Committee on Taxonomy of Viruses).4,5

Classification of coronavirus divided into subfamily of the Coronaviridae: four genera (α, β, ρ and δ). Both SARS-CoV2 (2019) and SARS-CoV1 (2003) belong to coronavirus.6 Structural biology analysis of Wuhan coronavirus SARS-CoV2 divided it into ORF1ab, S, ORF3, E, M, ORF6, 7a, 8, and ORF10. The spike (S) protein allows the virus to attach the membrane of the host cell and the N (nucleocapsid) protein holds the virus RNA genome. The E (envelope) and M (membrane) alone with S protein form a viral envelope.7 The nonstructural RNA genome of ORF1ab, ORF3, ORF6, 7a, 8, and ORF10 contains highly conserved information for genome replication.8

The coronavirus transmission starts from attaching host cell membrane receptors before endocytosis to enter host cells. RNA gene1 of virus genome then begins its replication and synthesizes the subgenomic RNAs with new transcription afterward. Since then, N protein and new genomic RNA assemble to form helical nucleocapsids, which will interact with M protein inserted in endoplasmic reticulum and anchored in host Golgi. E and M proteins then begin to trigger budding processes. S together with helical N translates on membrane-bound polysomes rough endoplasmic reticulum and is transported to Golgi. Finally, virions are released by exocytosis to finish the life cycle and replication of the virus.9

SARS-CoV1 transmits possibly through bat and civet as intermediate hosts, and finally to human with the symptoms of severe respiratory impacts and 10% mortality rate. However, Wuhan SARS-CoV2 is suspected to be transmitted from bat (RaTG13) to pangolin as intermediate host before it is transmitted to humans by some unknown mechanisms with symptoms of severe respiratory impacts and unclear mortality rate.10 The genomic sequence of RaTG13 cited the 96% similarity with the Wuhan coronavirus.11 Although intermediate host is not clear, genomic sequence comparison obviously demonstrates S receptor-binding domain (RBD) of Wuhan SARS-CoV2 with the similarity in 90% homolog of pangolin. Then, pangolin might contribute the S protein region to be cross-transmitted to RATG13 to a recombinant new mutant Wuhan SARS-CoV2 to transmit to human finally.12

The S protein of SARS-CoV1 and SARS-CoV2 responsible for viral entry mediates the binding to host cell membrane of angiotensin-converting enzyme 2 (ACE2) through its RBD.13 The surface glycoprotein of spike of SARS-CoV comprises two components: S1 and S2. The S protein of SARS-CoV2 binds to the host receptor ACE2 through its S1 subunit, which contains RBD, followed by fusing the viral and host membranes through the S2 subunit, which contains the fusion peptide primed by host protease. To prime the fusion of viral membrane with host membrane, SARS-S is cleaved by a cellular protease called cathepsin L, thereby exposing the S2 domain of the S protein for membrane fusion, followed by endocytosis and forms low pH endosomes.14

Understanding the molecular mechanisms of genome selection and packaging is critical for developing antiviral strategies. Thus, in this report, we compared SARS-CoV2 sequences from different countries to analyze the genomic patterns of disease origin and evolution, providing genomic information for the development of new control methods against the worldwide SARS-CoV2 pandemic.

2. METHODS

2.1. Sequence resource

Studies focus on evolutionary and phylogenetic analyses have applied in disease progression for Wuhan lung pneumonia treatment. Herein, we apply genomic analysis to observe SARS-CoV2 sequences from GenBank (http://www.ncbi.nim.nih.gov/genebank/): MN 908947 (China, C1), MN985325 (USA: WA, UW), MN996527 (China, C2), MT007544 (Australia: Victoria, A1), MT027064 (USA: CA, UC), MT039890 (South Korea, K1), MT066175 (Taiwan, T1), MT066176 (Taiwan, T2), LC528232 (Japan, J1), and LC528233 (Japan, J2) for genomic sequence alignment analysis.

2.2. Method applied

Multiple Sequence Alignment by Clustalw (https://www.genome.jp/tools-bin/clustalw) web service is applied as our alignment tool.

3. RESULTS

3.1. E protein

The structures reveal that E has a short and hydrophilic N-amino terminus consisting of 7–12 amino acids, followed by a large hydrophobic transmembrane domain of 25 amino acids, and ends with a long, hydrophilic C-carboxyl terminus (C-terminal), which comprises the majority of the protein.15 By analyzing E protein alignment, one amino acid mutation “H” was observed from South Korea (K1) comparing the “L” from other nine sequences in E protein sequence alignment.

3.2.S protein

S protein mediates attachment of SARS-CoV1 to the host cell surface receptors and subsequent fusion between them to facilitate viral entry into the host cell.15 The expression of S protein at the cell membrane can mediate cell–cell fusion. This formation supports to offer a strategy to let spread the virus between cells to subvert function of virus-neutralizing antibodies mechanisms.

During analysis of S protein, one amino acid mutation at “W’ was observed from South Korea (K1) comparing “S” from other nine sequences. One amino acid mutation at “R” from Australia (A1) was observed comparing “S” from another nine sequences.

3.3. M and N proteins

The M protein is abundant which defines the shape of the viral envelope. N functions primarily to bind to RNA genome of SARS-CoV, making up the nucleocapsid.15 Although N protein is most involved in processes viral genome signaling, it is also involved RNA replication cycle with host cellular response to viral infection.

Although the sequence difference between SARS-CoV1 and SARS-CoV2 within M and N proteins, there is no SNP variant observed between M and N protein sequence alignments from different patients.

3.4. L and S subtypes

Possible subtypes suggested in reference to two subtypes were found in OFR8 with L and S subtypes. By alignment, leucine of “L” type appeared in UC, A1, K1, C1, T2, J2, and T1, while as, Serine of “S” type was observed in T1, C2, and UW.

Possible subtypes suggested in reference to two subtypes were found in OFR1ab with L and S subtypes. By alignment, RNA sequence “C” type appeared in J1, J2, K1, A1, C1, C2, and T2 “T” type was observed in T1, UW, and UC.

4. DISCUSSION

4.1. Point mutation

Although SARS-CoV1 and SARS-CoV2 share the sequence similarity with 80% homolog, their functions are various. In comparison to 10 strains from 10 patients in structural protein regions, one mutation is observed by analyzing E protein alignment, the amino acid mutation “H” was observed from South Korea (K1) comparing the “L” from other nine sequences in E protein sequence alignment.

Inside the envelope, there is the nucleocapsid, which is formed from multiple copies of the nucleocapsid (N) protein, which are bound to the positive-sense single-stranded RNA genome in a continuous beads-on-a-string type conformation.16 The lipid bilayer envelope, membrane proteins, and nucleocapsid protect the virus when it is outside the host cell.17

Although the N protein holds the RNA genome, and M protein with E and S proteins together creates the viral envelope to protect the virus when it is outside the host cell, we do not find any point mutation of M and E proteins within 10 sequences.

The M protein is the most abundant structural protein and defines the shape of the viral envelope. Binding of M to N stabilizes the nucleocapsid (N protein–RNA complex), as well as the internal core of virions, and ultimately, promotes completion of viral assembly.18

During analysis of S protein, one amino acid mutation at “W” was observed from South Korea (K1) comparing “S” from other nine sequences. One amino acid mutation at “R” from Australia (A1) was observed comparing “S” from another nine sequences. Report19 mentioned a single amino acid reversion (L294-to-Q) in the S protein is sufficient to abrogate the phenotype and grows well at and below 32°C.

4.2. Spike protein and receptor (ACE)

A novel and pathogenic SARS-CoV2 was found in Wuhan, China in 2019, and its rapid national and international spread poses a global health emergency. The S protein mediates viral entry into host cells by first binding to a host receptor through the RBD in the S1 subunit and then fusing the viral and host membranes through the S2 subunit priming by host cell proteases.20–23 Unraveling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveals therapeutic targets. SARS-CoV and MERS-CoV RBDs recognize different receptors. SARS-CoV recognizes ACE2 as its receptor, whereas MERS-CoV recognizes dipeptidyl peptidase 4 as its receptor.13,24 Since SARS-CoV2 recognizes ACE2 as its host receptor binding to viral S protein,25 it is critical to define the RBD in SARS-CoV2 S protein in the most likely target for the mechanism of virus attachment such as new developing inhibitors, neutralizing antibodies, and vaccines.

Authors from Tai group26 demonstrate by characterizing SARS-CoV2 RBD to display a multiple sequence alignment of RBDs of SARS-CoV2, SARS-CoV, and MERS-CoV S proteins.

They identified the RBD in SARS-CoV2 S protein, and found that the RBD protein bound strongly to human and bat ACE2 receptors. SARS-CoV2 RBD displayed a significantly higher binding affinity to ACE2 receptor than SARS-CoV RBD. Subsequently, SARS-CoV RBD-specific antibodies could cross-react with SARS-CoV2 RBD protein. Meanwhile, SARS-CoV RBD-induced antisera which could cross-neutralize SARS-CoV2 suggests the potentials to develop SARS-CoV RBD-based vaccines for prevention of SARS-CoV2 and SARS-CoV infection.26

Hoffmann group mentions SARS-CoV1 and SARS-CoV2 share 76% amino acid identity in S protein region. By the amino acid alignment, they observe the receptor-binding motif of SARS-CoV1 corresponding to the sequences of bat-associated beta-coronavirus S proteins. Demonstration of high or low similarity by taking advantage of ACE2 as cellular receptor reveals that SARS-CoV2 possesses crucial amino acid residues for ACE2 binding.

They also found similarity signal to points out between SARS-CoV2 and SARS-CoV1 during transmitting host cells stage and then identified a potential target for antiviral intervention. Inspecting conserved amino acids within ACE2 domain, Hoffmann group performed SARS-CoV2 to transmit cell entry depends on ACE2 and transmembrane serine protease 2 two proteins and is blocked by applied clinically-proven protease inhibitor.27

4.3. SNP or subtype

We found that SNPs at locations 8782 (orf1ab: T8517C, synonymous) and 28 144 (ORF8: C251T, S84L) showed possible linkage in 10 sequences from different countries. As report “On the origin and continuing evolution of SARS-CoV-2” emphasized two subtypes of “L” and “S” from their data as they exhibited a “CT” haplotype (defined as “L” type because T28 144 is in the codon of leucine) and other “TC” haplotype (defined as “S” type because C28 144 is in the codon of serine) at these two sites.28 The authors show S is ancestral, related viruses like bat (RaTG13). They also depict that L is more prevalent with progressive, especially in Wuhan.29

However, according to data, it is too early to speculate on such consequences because there is no evidence whether it will affect some strategies such as vaccination. As mutation doesn’t occur within the S1 spike protein domain to influence the antigen targeting for vaccine production.

As speculated, SARS-CoV2 RNA viruses cross species barriers into humans; they would not be well-adapted to host cells. They should modify and allow them to adapt and become able to replicate within, and broad transmit humans. However, we do not catch data about testing the relative replication cycle in human cells. It would be difficult to verify human interference with the impact of co-strains relatively. Thus, a balance between SARS-CoV2 virulence and patient’s genetics personal phenomena with environmental factors would be important to confirm the subtypes of origin and evolution.

Due to study limitations, we cannot handle the SARS-CoV2 biological study directly from patient specimens, which will not observe the correlation from clinical to laboratory analysis directly.

In conclusion, we analyzed 10 sequences from the NCBI database by genome alignment and found no difference in amino acid sequences within M and N proteins. There are two amino acid variances in S protein region. One mutation found from South Korea sequence is verified. Two possible “L” and “S” SNPs found in ORF1ab and ORF8 regions are detected. Since our data are limited to a small population, more studies about the biological symptoms of SARS-CoV2 in clinic animals and humans will manipulate an understanding on the origin of pandemic crisis.

Fig. 1
Fig. 1:
Genomic analysis of E protein amino acid sequence. We found one amino acid mutation at “H” from South Korea comparing the “L” from other nine sequences. Yellow line indicates the difference in 10 sequence alignments.
Fig. 2
Fig. 2:
Genomic analysis of M protein amino acid sequence. We do not observe any mutation in 10 sequences of M protein region.
Fig. 3
Fig. 3:
Genomic analysis of N protein amino acid sequence. We do not observe any mutation in 10 sequences of N protein region.
Fig. 4
Fig. 4:
Genomic analysis of S protein amino acid sequence. One amino acid mutation at “W” from South Korea comparing “S” in other nine sequences. One amino acid mutation at “R” from Australia was observed comparing “S” from another nine sequences. Two yellow lines indicate the difference in 10 sequence alignments.
Fig. 5
Fig. 5:
Genomic analysis of ORF8 protein amino acid sequence. Possible subtypes were found in OFR8 with L and S subtypes. “L” type appeared in UC, A1, K1, C1, T2, J2, and T1. “S” type was observed in T1, C2, and UW. Yellow line indicates the difference in 10 sequence alignments.
Fig. 6
Fig. 6:
Genomic analysis of orf1ab protein amino acid sequence. Genomic analysis regions of orf1ab RNA sequence. Yellow line indicates the difference in 10 RNA sequence alignment.

ACKNOWLEDGMENTS

This research was funded by Taipei Veterans General Hospital; grant numbers V107E-002-2, V108D46-004-MY2-1, V108E-006-4, 108E-006-5, and 109VACS-003.

REFERENCES

1. Fehr AR, Channappanavar R, Perlman S. Middle East respiratory syndrome: emergence of a pathogenic human coronavirus. Annu Rev Med 2017;68:387–99.
2. de Wit E, van Doremalen N, Falzarano D, Munster VJ. SARS and MERS: recent insights into emerging coronaviruses. Nat Rev Microbiol 2016;14:523–34.
3. WHO (2020). Novel Coronavirus (2019-nCoV) Situation Report 23. Available at https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200212-sitrep-23-ncov.pdf?sfvrsn=41e9fb78_4
4. “Coronavirus Disease 2019”. World Health Organization. Retrieved 15 March 2020. 2020. Available at https://www.who.int/emergencies/diseases/novel-coronavirus-2019/events-as-they-happen
5. Naming the 2019 Coronavirus Feb 5, 2020. Available at https://talk.ictvonline.org
6. Marra MA, Jones SJ, Astell CR, Holt RA, Brooks-Wilson A, Butterfield YS, et al. The genome sequence of the SARS-associated coronavirus. Science 2003; 5624:1399–404.
7. Rota PA, Oberste MS, Monroe SS, Nix WA, Campagnoli R, Icenogle JP, et al. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 2003;300:1394–9.
8. Anderson KG, Rambaut A, Lipkin WI, Holes EC, Garry RF. The proximal origin of SARS-CoV-2. Nature Medicine 2020 17 March.
9. Fehr AR, Perlman S. Coronaviruses: an overview of their replication and pathogenesis. Methods Mol Biol 2015;1282:1–23.
10. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020;395:497–506.
11. Chan JF, Yuan S, Kok KH, To KK, Chu H, Yang J, et al. A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. Lancet 2020;395:514–23.
12. Chan JF, Kok KH, Zhu Z, Chu H, To KK, Yuan S, et al. Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan. Emerg Microbes Infect 2020;9:221–36.
13. Li W, Moore MJ, Vasilieva N, Sui J, Wong SK, Berne MA, et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 2003;426:450–4.
14. Zhao Y, Zhao Z, Wang Y, Zhou Y, Ma Y, Zuo W. Single-cell RNA expression profiling of ACE2, the putative receptor of Wuhan 2019-nCov. Available at https://doi.org/10.1101/2020.01.26.919985.
15. Schoeman D, Fielding BC. Coronavirus envelope protein: current knowledge. Virol J 2019;16:69.
16. Chang CK, Hou MH, Chang CF, Hsiao CD, Huang TH. The SARS coronavirus nucleocapsid protein – forms and functions. Antiviral Res 2014;103:39–50.
17. Neuman BW, Kiss G, Kunding AH, Bhella D, Baksh MF, Connelly S, et al. A structural analysis of M protein in coronavirus assembly and morphology. Journal Struct Biol 2011;17411–22.
18. Escors D, Ortego J, Laude H, Enjuanes L. The membrane M protein carboxy terminus binds to transmissible gastroenteritis coronavirus core and contributes to core stability. J Virol 2001;75:1312–24.
19. Shen S, Law YC, Liu DX. A single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature. Virology 2004;326:288–98.
20. Liu S, Xiao G, Chen Y, He Y, Niu J, Escalante CR, et al. Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors. Lancet 2004;363:938–47.
21. Wang Q, Wong G, Lu G, Yan J, Gao GF. MERS-CoV spike protein: Targets for vaccines and therapeutics. Antiviral Res 2016;133:165–77.
22. Li F, Li W, Farzan M, Harrison SC. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 2005;309:1864–8.
23. Lu G, Hu Y, Wang Q, Qi J, Gao F, Li Y, et al. Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26. Nature 2013;500:227–31.
24. Raj VS, Mou H, Smits SL, Dekkers DH, Müller MA, Dijkman R, et al. Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature 2013;495:251–4.
25. Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020;579:270–3.
26. Tai W, He L, Zhang X, Pu J, Voronin D, Jiang S, et al. Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine Cell mole Immunol 2020 March 19. doi.org/10.1038/s41423-020-0400-4.
27. Hoffmann M, Kleine-Weber H, Schroeder S, Kruger N, Herrler TErichsen S, et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 2020 Mar 4. pii: S0092–8674(20)30229-4
28. Lam TTY, Shum MHH, Zhu HC, Tong YG, Ni XB, Liao YS, et al. Identification of 2019-nCoV related coronaviruses in Malayan pangolins in Southern China. Nature 2020. Doi: 10.1038/s41586-020-2169-0
29. Tang X, Wu C, Li X, Song Y, Yao X, Wu X, et al. On the origin and continuing evolution of SARS-CoV-2 NSR 2020 nwaa036, https://doi.org/10.1093/nsr/nwaa036
Keywords:

Genomic analysis; Multiple sequences; Severe acute respiratory syndrome coronavirus 2

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