This is a preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Short Report
The emergence of SARS-CoV-2 by an unusual genome reconstitution
Seong-Tshool Hong
1. Department of Biomedical Sciences and Institute for Medical Science, Jeonbuk National University Medical School, Jeonju, Jeonbuk 54907, South Korea.
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
SARS-CoV-2 has been spreading remarkedly fast around the world since its emergence while the origin of the virus remains ambiguous. Here, we constructed all of the original prototype genome sequences of SARS-CoV-2 by selecting the common nucleotide among the different virus strains with species. Phylogenetic analysis on the prototype sequences showed that SARS-CoV-2 was a direct descendant of Bat-CoV and was closely related to Pan-CoV, Bat-SL-CoV, and SARS-CoV. The pairwise comparison of SARS-CoV-2 with Bat-CoV showed an unusual replacement of the motif consisting of 7 amino acids within the spike protein of SARS-CoV-2. Database searches showed that the motif originated from a surface protein of Plasmodium malariae, suggesting that the SARS-CoV-2 was emerged after acquiring the motif of the malaria surface protein.
Keywords
SARS-CoV-2, prototype, genome, phylogenetic analysis.
Figure 1
Figure 2
Coronaviruses (CoVs) are prevalent causes of the common cold widely present in nature with a broad spectrum of hosts. Although they frequently infect humans, the natural host of CoVs are animals and thereby all of the CoVs for the common colds, HCoV–229E, HCoV-NL63, HCoV- OC43, and HCoV-HKU1, have zoonotic origins (1). The genomes of CoVs consist of a single- stranded, positive RNA of 26,000 to 32,000 base pairs and a variable number (from 6 to 11) of open reading frames (2). Because of their unusually large size with the characteristics of the RNA genomes, CoVs can frequently mutate to escape their natural hosts, causing serious diseases to humans. Outbreaks of SARS-CoV in 2003 and MERS-CoV in 2012 are well-known examples. Currently, another very serious pathogenic novel coronavirus, SARS-CoV–2, has emerged and caused a global pandemic.
The genome sequence of SARS-CoV–2 (3, 4) suggested that SARS-CoV–2 stemmed from coronaviruses of bat or pangolin (5, 6). Because of the ambiguity of the origin of SARS-CoV–2, we adopted a different approach to investigate the origin of SARS-CoV–2. First, we pooled out all of the available genome sequences of SARS-CoV–2 as well as its related viral species by the BLAST sequence similarity from all of the publicly available genome databases (Table S1). Since genetic mutation or recombination occurs on an individual basis and these events cannot occur at the same position repeatedly, the prototype sequence of the genome within a species is always the most prevalent base among the members. Therefore, we constructed the original prototype aa sequences of SARS-CoV–2 and its related species, Bat-CoV, Pan-CoV, Bat-SL-CoV, and SARS- CoV (Fig. S1 & Table S2). The prototype sequences for each viral species were determined by choosing the most prevalent nucleotide bases in each position within the same species from pairwise comparison. The prototype sequences of the five viral species were directly used for phylogenetic analysis by the neighbor-joining method using Unipro UGENE bioinformatics toolkits (7). As shown in Figure–1A, SARS-CoV–2 was a direct descendant of Bat-CoV and was closely related to Pan-CoV, Bat-SL-CoV, and SARS-CoV. The sequence alignment of the prototype amino acid sequences by using multiple sequence alignments (MSA) using MAFFT program (8) showed that the amino acid (aa) sequence of SARS-CoV–2 shared 98.67% sequence similarity with Bat-CoV while 94.51%, 94.35% and 82.93% of the sequences of SARS-CoV–2 were identical with that of Pan-CoV, Bat-SL-CoV, and SARS-CoV respectively (Table 1). The aa sequence similarities were well-matched with their phylogenetic distances. The zoonotic origin of SARS-CoV–2 is under debate to be either Bat-CoV or Pan-CoV. The current argument on the origin of SARS-CoV–2 would not be surprising since phylogenetic analyses can generate different results if individual genome sequences were used. Considering the sequence variability of RNA genomes as in the case of SARS-CoV–2, we believe that the prototype sequence analysis approach would generate a more precise result than conventional approaches in phylogenetic analysis for species consisting of RNA genome.
To investigate how SARS-CoV–2 originated, we compared pairwise differences between the prototype aa sequences of SARS-CoV–2 and Bat-CoV. The pairwise aa sequence analysis showed that both of the two virus genomes encode 14 genes (Fig. 1B). The aa sequence mismatches were randomly distributed except for the consecutive 7 aa alteration (439 NNLDSKV 445) in the spike protein. Since genetic mutation is a random process, the mutated points are expected to be distributed randomly throughout the genome. Considering this nature of mutations, the consecutive 7-aa sequence difference in the spike protein was peculiar, indicating that an unusual event happened during the emergence of SARS-CoV–2 from Bat-CoV. Hence, 3D models of the spike proteins were made to analyze the consequence of alteration to the structure of the spike protein during the emergence of SARS-CoV–2. The spike protein of coronavirus (CoV) is the surface protein that binds to a receptor on the host cell surface. The spike protein consists of three large domains: a large ectodomain, a single-pass transmembrane anchor, and a short intracellular tail (9). The ectodomain is further divided into a receptor-binding subunit S1 and a membrane-fusion subunit S2. Coronavirus first binds to a receptor on the host cell surface through its S1 subunit and then fuses the viral and the host membranes through its S2 subunit, meaning that the S1 domain plays the most critical role for the virus’s invasion into its host (10, 11). As shown in Figure–1C, altering the consecutive 7-aa on the outer layer of the spike protein’s S1 domain did not affect the overall structural integrity of the spike protein. The 3D modeling also showed that the consecutive 7-aa alteration resulted in a new motif to occupy more space in the S1 domain than its original structure by transforming a partial α-helical structure to a random coil. Since the S1 domain acts as the binding domain for the entry of the virus, enlargement of its space as well as adaptation of different conformation in the outermost layer surface of the S1 domain would endow SARS-CoV–2 to expand its host range.
The unusual pattern of genetic alteration motivated us to investigate the origin of the 7-aa motif (439 NNLDSKV 445). To trace down its origin, we overlappingly defragmented the aa sequence of the spike protein of SARS-CoV–2 into 9 aa units. The fragmented aa sequence matched with all of the available protein sequences. All of the aa sequences matched only with the spike proteins of coronaviruses except for the 7-aa motif. Very surprisingly, the NNLDSKV motif perfectly matched only with a motif of a surface protein (GenBank Accession : XP_028861348.1) of Plasmodium malariae which causes malaria to humans. The NNLDSKV motif was located at aa 449–455 of a membrane-bound surface protein of P. malariae (Fig. 2).
The clinical presentation of COVID–19 very differs from that of other coronaviruses (12). The acquisition of the P. malariae gene in the spike protein seems to explain why. Plasmodium species, which is responsible for malaria, infect liver cells and erythrocytes to promote blood clotting and damages in the heart, liver, or kidney. Unlike other coronaviral infections, it is very interesting to note that symptoms similar to the case of the P. malariae infection were reported in COVID–19. Red cell distribution width (RDW) of erythrocytes has been reported to be enlarged after the malarial invasion because the growth of the malaria parasite causes the cells to be enlarged (13, 14). Foy et al., recently reported the observation of the same phenomenon in COVID–19 that elevated RDW is correlated with increased mortality risk in COVID–19 (15). Not only is the enlargement of erythrocytes in COVID–19 correlated with malaria infection, but also clinical presentations such as blood clotting and damages in the heart, liver, or kidney were also observed in COVID–19 (16–18). Furthermore, the acquisition of the P. malariae gene seems to explain why hydroxychloroquine, an anti-malaria drug for P. malariae infection, shows some efficacy in COVID–19 (18–20). Considering these facts, the investigation of different kinds of anti-malaria drugs for COVID–19 treatment needs to be pursued.
According to the current outbreak statistic of COVID–19 (https://www.worldometers. info/coronavirus/), a racial background affects the morbidity and mortality of COVID–19. It seems to be that the morbidity and mortality by COVID–19 are much more serious in the Western world than in Eastern countries. Multisystem inflammatory syndrome in children never reported and the morbidity and mortality by COVID–19 are much milder in Eastern countries. It would be very interesting to investigate the role of the NNLDSKV motif among different racial backgrounds including ACE–2 since the genetic polymorphism of ACE–2 is known to differ among different ethnic and racial groups (21).
- W. Ye, S. Yuan, K. S. Yuen, S. Y. Fung, C. P. Chan, D. Y. Jin, Zoonotic origins of human coronaviruses. Int. J. Biol. Sci. 16, 1686-1697 (2020).
- Song, Y. Xu, L. Bao, L. Zhang, P. Yu, Y. Qu, H. Zhu, W. Zhao, Y. Han, C. Qin, From SARS to MERS, thrusting coronaviruses into the spotlight. Viruses. 11 (2019), p. E59
- Wu, Y. Peng, B. Huang, X. Ding, X. Wang, P. Niu, J. Meng, Z. Zhu, Z. Zhang, J. Wang, J. Sheng, L. Quan, Z. Xia, W. Tan, G. Cheng, T. Jiang, Genome composition and divergence of the novel coronavirus (2019-nCoV) originating in China. Cell Host Microbe. 27, 325-328 (2020).
- Wu, S. Zhao, B. Yu, Y. M. Chen, W. Wang, Z. G. Song, Y. Hu, Z. W. Tao, J. H. Tian, Y. Y. Pei, M. L. Yuan, Y. L. Zhang, F. H. Dai, Y, Liu, Q. M. Wang, J. J. Zheng, L. Xu, E. C. Holmes, Z. Zhang, Author Correction: A new coronavirus associated with human respiratory disease in China. Nature. 580 (7803):E7 (2020).
- G. Andersen, A. Rambaut, W. I. Lipkin, E. C. Holmes, Garry RF. The proximal origin of SARS-CoV-2. Nat. Med. 26, 450-452 (2020).
- T. Lam, M. H. Shum, H. C. Zhu, Y. G. Tong, X. B. Ni, Y. S. Liao, W. Wei, W. Y. Cheung, Li, L. F. Li, G. M. Leung, E. C. Holmes, Y. L. Hu, Y. Guan, Identifying SARS-CoV-2 related coronaviruses in Malayan pangolins. Nature (2020) DOI: 10.1038/s41586-020-2169- 0.
- Okonechnikov, O. Golosova, M. Fursov, the UGENE team. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics. 28: 1166-1167 (2012).
- Katoh, K. Misawa, K Kuma, T. Miyata, MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30, 3059–3066 (2002).
- Lan, J. Ge, J. Yu, S. Shan, H. Zhou, S. Fan, Q. Zhang, X. Shi, Q. Wang, L. Zhang, X. Wang, Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature doi: 10.1038/s41586-020-2180-5 (2020).
- Ou, Y. Liu, X. Lei, P. Li, D. Mi, L. Ren, L. Guo, R. Guo, T. Chen, J. Hu, Z. Xiang, Z. Mu, Chen, J. Chen, K. Hu, Q. Jin, J. Wang, Qian, Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat. Commun. 11, 1620 (2020).
- Zhang, Q. Wu, Z. Zhang, Probable Pangolin Origin of SARS-CoV-2 Associated with the COVID-19 Outbreak. Curr. Biol. 30, 1578 (2020).
- X. Lescure, L. Bouadma, D. Nguyen, M. Parisey, P. H. Wicky, S. Behillil, A. Gaymard, M. Bouscambert-Duchamp, F. Donati, Q. Le Hingrat, V. Enouf, N. Houhou-Fidouh, M. Valette, Mailles, J. C. Lucet, F. Mentre, X. Duval, D. Descamps, D. Malvy, J. F. Timsit, B. Lina, S. van-der-Werf, Y. Yazdanpanah, Clinical and virological data of the first cases of COVID-19 in Europe: a case series. Lancet Infect Dis. p: S1473-3099(20)30200-0 (2020).
- S. Jairajpuri, S. Rana, M. J. Hassan, F. Nabi, S. Jetley, An analysis of hematological parameters as a diagnostic test for malaria in patients with acute febrile illness: an institutional experience. Oman Med. J. 29, 12-17 (2014).
- S. Koltas, H. Demirhindi, S. Hazar, K. Ozcan, Supportive presumptive diagnosis of Plasmodium vivax malaria. Thrombocytopenia and red cell distribution width. Saudi Med. J. 28, 535-539 (2007).
- H. Foy et al., https://www.medrxiv.org/content/10.1101/2020.05.05.20091702v1.full.pdf (2020).
- Lodigiani, G. Iapichino, L. Carenzo, M. Cecconi, P. Ferrazzi, T. Sebastian, N. Kucher, J. D. Studt, C. Sacco, B. Alexia, M. T. Sandri, S. Barco; Humanitas COVID-19 task force, Venous and arterial thromboembolic complications in COVID-19 patients admitted to an academic hospital in Milan, Italy. Thromb. Res. 191, 9-14 (2020).
- Rismanbaf, S. Zarei .Liver and kidney injuries in COVID-19 and their effects on drug therapy; a letter to editor. Arch. Acad. Emerg. Med. 8, e17 (2020).
- J. Guzik, S. A. Mohiddin, A. Dimarco, V. Patel, K. Savvatis, F. M. Marelli-Berg, M. S. Madhur, M. Tomaszewski, P. Maffia, F. D'Acquisto, S. A. Nicklin, A. J. Marian, R. Nosalski, C. Murray, B. Guzik, C. Berry, R. M. Touyz, R. Kreutz, D. W. Wang, D. Bhella, O. Sagliocco, F. Crea, E. C. Thomson, I. B. McInnes, COVID-19 and the cardiovascular system: implications for risk assessment, diagnosis, and treatment options. Cardiovasc Res. Pii: cvaa 106. DOI: 10.1093/cvr/cvaa106 (2020).
- K. Singh, A. Singh, A. Shaikh, R. Singh, A. Misra, Chloroquine and hydroxychloroquine in the treatment of COVID-19 with or without diabetes: A systematic search and a narrative review with a special reference to India and other developing countries. Diabetes Metab. Syndr. 14, 241-246 (2020).
- L. M. Arnold, F. Buckner, Hydroxychloroquine for treatment of SARS-CoV-2 infection? Improving our confidence in a model-based approach to dose selection. Clin. Transl. Sci.(2020). DOI: 10.1111/cts.12797.
- Hussain, N. Jabeen, F. Raza, S. Shabbir, A. A. Baig, A. Amanullah, B. Aziz, Structural variations in human ACE2 may influence its binding with SARS-CoV-2 spike protein. J. Med. Virol. (2020). DOI: 10.1002/jmv.25832.
Acknowledgments
Funding: This work was supported by the funding from JINIS BDRD Research Institute, JINIS Biopharmaceuticals Inc. (Wanju, South Korea).
Author contributions: S.T.H. conceived and designed the study and wrote the paper. M.M.H. and S.S. performed the analysis of data. J.H. wrote the paper. H.J.K. and H.S.L discussed the result and further validated the data thoroughly. All authors participated in interpretation of data.
Competing interests: The authors declare no competing interesting.
Data availability: All data are available in the main manuscript and supplementary material.
Table 1. Amino acid sequence similarities among the 14 different ORFs of SARS-CoV-2 with Bat-CoV, Bat-SL-CoV, Pan-CoV and SARS-CoVs.
|
Gene |
Bat-CoV (%) |
Bat-SL-CoV (%) |
Pan-CoV (%) |
SARS-CoV (%) |
|
Whole genome |
98.67 |
94.35 |
94.51 |
82.93 |
|
ORF1a |
98.62 |
96.94 |
92.90 |
80.44 |
|
ORF1ab |
99 |
96.73 |
94.87 |
86.19 |
|
Spike (S) |
98.04 |
83.52 |
93.87 |
75.96 |
|
3a |
98.91 |
95.27 |
95.64 |
72.36 |
|
3b |
77.27 |
81.82 |
90.91 |
61.11 |
|
Envelope (E) |
100 |
100 |
100 |
94.74 |
|
Membrane (M) |
99.52 |
99.05 |
98.57 |
91.43 |
|
6 |
100 |
93.44 |
96.72 |
68.85 |
|
7a |
99.17 |
88.52 |
94.21 |
85.25 |
|
7b |
97.67 |
93.02 |
90.70 |
85.37 |
|
8 |
95.87 |
29.73 |
95.87 |
8a: 31.71; 8b: 40.48 |
|
Nucleocapsid (N) |
99.28 |
95.70 |
97.85 |
90.52 |
|
9b |
92.78 |
76.29 |
93.81 |
72.45 |
|
14 |
90.41 |
92.98 |
96.49 |
84.21 |
This is a list of supplementary files associated with this preprint. Click to download.
Andras Szilagyi
commented on 07 June, 2020
The conclusion of this article is false. The short sequence in question (NNLDSKV) in SARS-CoV-2 is actually identical to that in the pangolin coronavirus, and it also has 2 (3) identical residues in the RaTG13 coronavirus (RsSHC014 coronavirus), so it is not a 7-residue consecutive mutation. Too bad that the authors do not show the sequence alignments because then the reader would immediately see that the claim of the authors is false. Also, the NNLDSKV sequence can be found in at least 100 proteins from a wide range of organisms including bacteria, plants, and animals (this can be checked by e.g. the Peptide Match server of PIR), so contrary to the authors' claim it is not specific to P. malariae. In addition, (hydroxy)chloroquine is a wide-spectrum antiviral which is effective against several different viruses including HIV, flaviviruses and other coronaviruses, thus its effect is not specific to SARS-CoV-2, and has nothing to do with the presence of the NNLDSKV sequence. In summary, this article contains several serious errors and flaws, and its conclusions are invalid.
View 3 replies
Mary Olsen
commented on 07 June, 2020
You are right. The commenter did not understand the manuscript. Fig 1 clearly showed sequence alignments.
View 1 reply
Andras Szilagyi
replied on 08 June, 2020
Fig 1 does not contain a sequence alignment.
Perez
commented on 08 June, 2020
They are right See &7 in perez, j., & Montagnier, L. (2020, April 25). COVID-19, SARS and Bats Coronaviruses Genomes Unexpected Exogeneous RNA Sequences. https://doi.org/10.31219/osf.io/d9e5g
Perez
commented on 08 June, 2020
This plasmodium inserts are trouve. Please see &7 in perez, j., & Montagnier, L. (2020, April 25). COVID-19, SARS and Bats Coronaviruses Genomes Unexpected Exogeneous RNA Sequences. https://doi.org/10.31219/osf.io/d9e5g
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Short Report
The emergence of SARS-CoV-2 by an unusual genome reconstitution
Seong-Tshool Hong
1. Department of Biomedical Sciences and Institute for Medical Science, Jeonbuk National University Medical School, Jeonju, Jeonbuk 54907, South Korea.
Corresponding Author
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SARS-CoV-2 has been spreading remarkedly fast around the world since its emergence while the origin of the virus remains ambiguous. Here, we constructed all of the original prototype genome sequences of SARS-CoV-2 by selecting the common nucleotide among the different virus strains with species. Phylogenetic analysis on the prototype sequences showed that SARS-CoV-2 was a direct descendant of Bat-CoV and was closely related to Pan-CoV, Bat-SL-CoV, and SARS-CoV. The pairwise comparison of SARS-CoV-2 with Bat-CoV showed an unusual replacement of the motif consisting of 7 amino acids within the spike protein of SARS-CoV-2. Database searches showed that the motif originated from a surface protein of Plasmodium malariae, suggesting that the SARS-CoV-2 was emerged after acquiring the motif of the malaria surface protein.
Figure 1
Figure 2
Coronaviruses (CoVs) are prevalent causes of the common cold widely present in nature with a broad spectrum of hosts. Although they frequently infect humans, the natural host of CoVs are animals and thereby all of the CoVs for the common colds, HCoV–229E, HCoV-NL63, HCoV- OC43, and HCoV-HKU1, have zoonotic origins (1). The genomes of CoVs consist of a single- stranded, positive RNA of 26,000 to 32,000 base pairs and a variable number (from 6 to 11) of open reading frames (2). Because of their unusually large size with the characteristics of the RNA genomes, CoVs can frequently mutate to escape their natural hosts, causing serious diseases to humans. Outbreaks of SARS-CoV in 2003 and MERS-CoV in 2012 are well-known examples. Currently, another very serious pathogenic novel coronavirus, SARS-CoV–2, has emerged and caused a global pandemic.
The genome sequence of SARS-CoV–2 (3, 4) suggested that SARS-CoV–2 stemmed from coronaviruses of bat or pangolin (5, 6). Because of the ambiguity of the origin of SARS-CoV–2, we adopted a different approach to investigate the origin of SARS-CoV–2. First, we pooled out all of the available genome sequences of SARS-CoV–2 as well as its related viral species by the BLAST sequence similarity from all of the publicly available genome databases (Table S1). Since genetic mutation or recombination occurs on an individual basis and these events cannot occur at the same position repeatedly, the prototype sequence of the genome within a species is always the most prevalent base among the members. Therefore, we constructed the original prototype aa sequences of SARS-CoV–2 and its related species, Bat-CoV, Pan-CoV, Bat-SL-CoV, and SARS- CoV (Fig. S1 & Table S2). The prototype sequences for each viral species were determined by choosing the most prevalent nucleotide bases in each position within the same species from pairwise comparison. The prototype sequences of the five viral species were directly used for phylogenetic analysis by the neighbor-joining method using Unipro UGENE bioinformatics toolkits (7). As shown in Figure–1A, SARS-CoV–2 was a direct descendant of Bat-CoV and was closely related to Pan-CoV, Bat-SL-CoV, and SARS-CoV. The sequence alignment of the prototype amino acid sequences by using multiple sequence alignments (MSA) using MAFFT program (8) showed that the amino acid (aa) sequence of SARS-CoV–2 shared 98.67% sequence similarity with Bat-CoV while 94.51%, 94.35% and 82.93% of the sequences of SARS-CoV–2 were identical with that of Pan-CoV, Bat-SL-CoV, and SARS-CoV respectively (Table 1). The aa sequence similarities were well-matched with their phylogenetic distances. The zoonotic origin of SARS-CoV–2 is under debate to be either Bat-CoV or Pan-CoV. The current argument on the origin of SARS-CoV–2 would not be surprising since phylogenetic analyses can generate different results if individual genome sequences were used. Considering the sequence variability of RNA genomes as in the case of SARS-CoV–2, we believe that the prototype sequence analysis approach would generate a more precise result than conventional approaches in phylogenetic analysis for species consisting of RNA genome.
To investigate how SARS-CoV–2 originated, we compared pairwise differences between the prototype aa sequences of SARS-CoV–2 and Bat-CoV. The pairwise aa sequence analysis showed that both of the two virus genomes encode 14 genes (Fig. 1B). The aa sequence mismatches were randomly distributed except for the consecutive 7 aa alteration (439 NNLDSKV 445) in the spike protein. Since genetic mutation is a random process, the mutated points are expected to be distributed randomly throughout the genome. Considering this nature of mutations, the consecutive 7-aa sequence difference in the spike protein was peculiar, indicating that an unusual event happened during the emergence of SARS-CoV–2 from Bat-CoV. Hence, 3D models of the spike proteins were made to analyze the consequence of alteration to the structure of the spike protein during the emergence of SARS-CoV–2. The spike protein of coronavirus (CoV) is the surface protein that binds to a receptor on the host cell surface. The spike protein consists of three large domains: a large ectodomain, a single-pass transmembrane anchor, and a short intracellular tail (9). The ectodomain is further divided into a receptor-binding subunit S1 and a membrane-fusion subunit S2. Coronavirus first binds to a receptor on the host cell surface through its S1 subunit and then fuses the viral and the host membranes through its S2 subunit, meaning that the S1 domain plays the most critical role for the virus’s invasion into its host (10, 11). As shown in Figure–1C, altering the consecutive 7-aa on the outer layer of the spike protein’s S1 domain did not affect the overall structural integrity of the spike protein. The 3D modeling also showed that the consecutive 7-aa alteration resulted in a new motif to occupy more space in the S1 domain than its original structure by transforming a partial α-helical structure to a random coil. Since the S1 domain acts as the binding domain for the entry of the virus, enlargement of its space as well as adaptation of different conformation in the outermost layer surface of the S1 domain would endow SARS-CoV–2 to expand its host range.
The unusual pattern of genetic alteration motivated us to investigate the origin of the 7-aa motif (439 NNLDSKV 445). To trace down its origin, we overlappingly defragmented the aa sequence of the spike protein of SARS-CoV–2 into 9 aa units. The fragmented aa sequence matched with all of the available protein sequences. All of the aa sequences matched only with the spike proteins of coronaviruses except for the 7-aa motif. Very surprisingly, the NNLDSKV motif perfectly matched only with a motif of a surface protein (GenBank Accession : XP_028861348.1) of Plasmodium malariae which causes malaria to humans. The NNLDSKV motif was located at aa 449–455 of a membrane-bound surface protein of P. malariae (Fig. 2).
The clinical presentation of COVID–19 very differs from that of other coronaviruses (12). The acquisition of the P. malariae gene in the spike protein seems to explain why. Plasmodium species, which is responsible for malaria, infect liver cells and erythrocytes to promote blood clotting and damages in the heart, liver, or kidney. Unlike other coronaviral infections, it is very interesting to note that symptoms similar to the case of the P. malariae infection were reported in COVID–19. Red cell distribution width (RDW) of erythrocytes has been reported to be enlarged after the malarial invasion because the growth of the malaria parasite causes the cells to be enlarged (13, 14). Foy et al., recently reported the observation of the same phenomenon in COVID–19 that elevated RDW is correlated with increased mortality risk in COVID–19 (15). Not only is the enlargement of erythrocytes in COVID–19 correlated with malaria infection, but also clinical presentations such as blood clotting and damages in the heart, liver, or kidney were also observed in COVID–19 (16–18). Furthermore, the acquisition of the P. malariae gene seems to explain why hydroxychloroquine, an anti-malaria drug for P. malariae infection, shows some efficacy in COVID–19 (18–20). Considering these facts, the investigation of different kinds of anti-malaria drugs for COVID–19 treatment needs to be pursued.
According to the current outbreak statistic of COVID–19 (https://www.worldometers. info/coronavirus/), a racial background affects the morbidity and mortality of COVID–19. It seems to be that the morbidity and mortality by COVID–19 are much more serious in the Western world than in Eastern countries. Multisystem inflammatory syndrome in children never reported and the morbidity and mortality by COVID–19 are much milder in Eastern countries. It would be very interesting to investigate the role of the NNLDSKV motif among different racial backgrounds including ACE–2 since the genetic polymorphism of ACE–2 is known to differ among different ethnic and racial groups (21).
- W. Ye, S. Yuan, K. S. Yuen, S. Y. Fung, C. P. Chan, D. Y. Jin, Zoonotic origins of human coronaviruses. Int. J. Biol. Sci. 16, 1686-1697 (2020).
- Song, Y. Xu, L. Bao, L. Zhang, P. Yu, Y. Qu, H. Zhu, W. Zhao, Y. Han, C. Qin, From SARS to MERS, thrusting coronaviruses into the spotlight. Viruses. 11 (2019), p. E59
- Wu, Y. Peng, B. Huang, X. Ding, X. Wang, P. Niu, J. Meng, Z. Zhu, Z. Zhang, J. Wang, J. Sheng, L. Quan, Z. Xia, W. Tan, G. Cheng, T. Jiang, Genome composition and divergence of the novel coronavirus (2019-nCoV) originating in China. Cell Host Microbe. 27, 325-328 (2020).
- Wu, S. Zhao, B. Yu, Y. M. Chen, W. Wang, Z. G. Song, Y. Hu, Z. W. Tao, J. H. Tian, Y. Y. Pei, M. L. Yuan, Y. L. Zhang, F. H. Dai, Y, Liu, Q. M. Wang, J. J. Zheng, L. Xu, E. C. Holmes, Z. Zhang, Author Correction: A new coronavirus associated with human respiratory disease in China. Nature. 580 (7803):E7 (2020).
- G. Andersen, A. Rambaut, W. I. Lipkin, E. C. Holmes, Garry RF. The proximal origin of SARS-CoV-2. Nat. Med. 26, 450-452 (2020).
- T. Lam, M. H. Shum, H. C. Zhu, Y. G. Tong, X. B. Ni, Y. S. Liao, W. Wei, W. Y. Cheung, Li, L. F. Li, G. M. Leung, E. C. Holmes, Y. L. Hu, Y. Guan, Identifying SARS-CoV-2 related coronaviruses in Malayan pangolins. Nature (2020) DOI: 10.1038/s41586-020-2169- 0.
- Okonechnikov, O. Golosova, M. Fursov, the UGENE team. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics. 28: 1166-1167 (2012).
- Katoh, K. Misawa, K Kuma, T. Miyata, MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30, 3059–3066 (2002).
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Acknowledgments
Funding: This work was supported by the funding from JINIS BDRD Research Institute, JINIS Biopharmaceuticals Inc. (Wanju, South Korea).
Author contributions: S.T.H. conceived and designed the study and wrote the paper. M.M.H. and S.S. performed the analysis of data. J.H. wrote the paper. H.J.K. and H.S.L discussed the result and further validated the data thoroughly. All authors participated in interpretation of data.
Competing interests: The authors declare no competing interesting.
Data availability: All data are available in the main manuscript and supplementary material.
Table 1. Amino acid sequence similarities among the 14 different ORFs of SARS-CoV-2 with Bat-CoV, Bat-SL-CoV, Pan-CoV and SARS-CoVs.
|
Gene |
Bat-CoV (%) |
Bat-SL-CoV (%) |
Pan-CoV (%) |
SARS-CoV (%) |
|
Whole genome |
98.67 |
94.35 |
94.51 |
82.93 |
|
ORF1a |
98.62 |
96.94 |
92.90 |
80.44 |
|
ORF1ab |
99 |
96.73 |
94.87 |
86.19 |
|
Spike (S) |
98.04 |
83.52 |
93.87 |
75.96 |
|
3a |
98.91 |
95.27 |
95.64 |
72.36 |
|
3b |
77.27 |
81.82 |
90.91 |
61.11 |
|
Envelope (E) |
100 |
100 |
100 |
94.74 |
|
Membrane (M) |
99.52 |
99.05 |
98.57 |
91.43 |
|
6 |
100 |
93.44 |
96.72 |
68.85 |
|
7a |
99.17 |
88.52 |
94.21 |
85.25 |
|
7b |
97.67 |
93.02 |
90.70 |
85.37 |
|
8 |
95.87 |
29.73 |
95.87 |
8a: 31.71; 8b: 40.48 |
|
Nucleocapsid (N) |
99.28 |
95.70 |
97.85 |
90.52 |
|
9b |
92.78 |
76.29 |
93.81 |
72.45 |
|
14 |
90.41 |
92.98 |
96.49 |
84.21 |
Andras Szilagyi
commented on 07 June, 2020
The conclusion of this article is false. The short sequence in question (NNLDSKV) in SARS-CoV-2 is actually identical to that in the pangolin coronavirus, and it also has 2 (3) identical residues in the RaTG13 coronavirus (RsSHC014 coronavirus), so it is not a 7-residue consecutive mutation. Too bad that the authors do not show the sequence alignments because then the reader would immediately see that the claim of the authors is false. Also, the NNLDSKV sequence can be found in at least 100 proteins from a wide range of organisms including bacteria, plants, and animals (this can be checked by e.g. the Peptide Match server of PIR), so contrary to the authors' claim it is not specific to P. malariae. In addition, (hydroxy)chloroquine is a wide-spectrum antiviral which is effective against several different viruses including HIV, flaviviruses and other coronaviruses, thus its effect is not specific to SARS-CoV-2, and has nothing to do with the presence of the NNLDSKV sequence. In summary, this article contains several serious errors and flaws, and its conclusions are invalid.
View 3 replies
Seong-Tshool Hong
replied on 07 June, 2020
The 7-aa motif (NNLDSKV ) is not present in pangolin CoV and Plant and not in 100 proteins. I am not quite sure whether you searched database correctly. However, the same motif is present in some bacteria. Also we did not say that the motif is specific to P. malariae. All we said was the motif is also present in P. malariae. I think you over-and mis-interpreted the paper.
View 1 reply
Andras Szilagyi
replied on 08 June, 2020
Yes, the pangolin CoV does contain the NNLDSKV motif. See Fig. 5 in https://pubs.acs.org/doi/10.1021/acs.jproteome.0c00129 or Fig. 3 in https://www.sciencedirect.com/science/article/pii/S0960982220303602 or Fig. 3 in https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1008421 (published articles). Like I indicated, I ran the Peptide Match server at https://research.bioinformatics.udel.edu/peptidematch/index.jsp with the NNLDSKV sequence, and it hits 101 proteins, 63 from bacteria, 11 from plants, 9 from animals, etc.
Oluvan
replied on 09 June, 2020
The “ pangolin” sequence came from samples with human derived contamination in it. Up to 17% on Trace. Look up the SRA and see if you can correlate the virus reads to the first animal traced on the result.
View 1 reply
Andras Szilagyi
replied on 09 June, 2020
Too little information. Please provide links to database records and analyses to back up your claim.
View 1 reply
Oluvan
replied on 08 July, 2020
https://www.ncbi.nlm.nih.gov/bioproject/PRJNA573298 Which was where the raw sequences came from. See it with your own eyes.
Shannon
replied on 08 August, 2020
The preponderance of evidence is that an ancestral virus jumped from bats to pangolin and it is an offshoot otherwise unrelated to SARS-2. The exploitation of furin cleavage is the hot-spot to research right now. No other β-CoV has this feature. Mouse Hepatitis and Infectious Bronchitis virus are the closest ones.
Mary Olsen
commented on 07 June, 2020
You are right. The commenter did not understand the manuscript. Fig 1 clearly showed sequence alignments.
View 1 reply
Andras Szilagyi
replied on 08 June, 2020
Fig 1 does not contain a sequence alignment.
Perez
commented on 08 June, 2020
They are right See &7 in perez, j., & Montagnier, L. (2020, April 25). COVID-19, SARS and Bats Coronaviruses Genomes Unexpected Exogeneous RNA Sequences. https://doi.org/10.31219/osf.io/d9e5g
Perez
commented on 08 June, 2020
This plasmodium inserts are trouve. Please see &7 in perez, j., & Montagnier, L. (2020, April 25). COVID-19, SARS and Bats Coronaviruses Genomes Unexpected Exogeneous RNA Sequences. https://doi.org/10.31219/osf.io/d9e5g
Seong-Tshool Hong
replied on 07 June, 2020
The 7-aa motif (NNLDSKV ) is not present in pangolin CoV and Plant and not in 100 proteins. I am not quite sure whether you searched database correctly. However, the same motif is present in some bacteria. Also we did not say that the motif is specific to P. malariae. All we said was the motif is also present in P. malariae. I think you over-and mis-interpreted the paper.
View 1 reply
Andras Szilagyi
replied on 08 June, 2020
Yes, the pangolin CoV does contain the NNLDSKV motif. See Fig. 5 in https://pubs.acs.org/doi/10.1021/acs.jproteome.0c00129 or Fig. 3 in https://www.sciencedirect.com/science/article/pii/S0960982220303602 or Fig. 3 in https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1008421 (published articles). Like I indicated, I ran the Peptide Match server at https://research.bioinformatics.udel.edu/peptidematch/index.jsp with the NNLDSKV sequence, and it hits 101 proteins, 63 from bacteria, 11 from plants, 9 from animals, etc.
Oluvan
replied on 09 June, 2020
The “ pangolin” sequence came from samples with human derived contamination in it. Up to 17% on Trace. Look up the SRA and see if you can correlate the virus reads to the first animal traced on the result.
View 1 reply
Andras Szilagyi
replied on 09 June, 2020
Too little information. Please provide links to database records and analyses to back up your claim.
View 1 reply
Oluvan
replied on 08 July, 2020
https://www.ncbi.nlm.nih.gov/bioproject/PRJNA573298 Which was where the raw sequences came from. See it with your own eyes.
Shannon
replied on 08 August, 2020
The preponderance of evidence is that an ancestral virus jumped from bats to pangolin and it is an offshoot otherwise unrelated to SARS-2. The exploitation of furin cleavage is the hot-spot to research right now. No other β-CoV has this feature. Mouse Hepatitis and Infectious Bronchitis virus are the closest ones.