F1000Research 2021, 9:52 Last updated: 07 JAN 2021 BRIEF REPORT Use of the informational spectrum methodology for rapid biological analysis of the novel coronavirus 2019-nCoV: prediction of potential receptor, natural reservoir, tropism and therapeutic/vaccine target [version 4; peer review: 2 approved] Veljko Veljkovic 1, Júlia Vergara-Alert2, Joaquim Segalés3,4, Slobodan Paessler5 1Biomed Protection, Galveston, TX, 77550, USA 2IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, Barcelona, 08193 Bellaterra, Spain 3Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona (UAB), Barcelona, 08193 Bellaterra, Spain 4UAB, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, Barcelona, 08193 Bellaterra, Spain 5Department of Pathology, Galveston National Laboratory, University of Texas Medical Branch, Galveston, TX, 77555, USA v4 First published: 27 Jan 2020, 9:52 Open Peer Review https://doi.org/10.12688/f1000research.22149.1 Second version: 31 Jan 2020, 9:52 https://doi.org/10.12688/f1000research.22149.2 Reviewer Status Third version: 27 Apr 2020, 9:52 https://doi.org/10.12688/f1000research.22149.3 Invited Reviewers Latest published: 06 Jan 2021, 9:52 https://doi.org/10.12688/f1000research.22149.4 1 2 version 4 Abstract (update) report A novel coronavirus recently identified in Wuhan, China (SARS-CoV-2) 06 Jan 2021 has expanded the number of highly pathogenic coronaviruses affecting humans. The SARS-CoV-2 represents a potential epidemic or version 3 pandemic threat, which requires a quick response for preparedness (revision) report against this infection. The present report uses the informational 27 Apr 2020 spectrum methodology to identify the possible origin and natural host of the new virus, as well as putative therapeutic and vaccine targets. version 2 The performed in silico analysis indicates that the newly emerging (update) report report SARS-CoV-2 is closely related to severe acute respiratory syndrome 31 Jan 2020 (SARS)-CoV and, to a lesser degree, Middle East respiratory syndrome (MERS)-CoV. Moreover, the well-known SARS-CoV receptor (ACE2) version 1 might be a putative receptor for the novel virus as well. Actin protein 27 Jan 2020 was also suggested as a host factor that participates in cell entry and pathogenesis of SARS-CoV-2; therefore, drugs modulating biological 1. Rita Casadio, University of Bologna, Bologna, activity of this protein (e.g. ibuprofen) were suggested as potential Italy candidates for treatment of this viral infection. Additional results indicated that civets and poultry are potential candidates for the Page 1 of 15 F1000Research 2021, 9:52 Last updated: 07 JAN 2021 natural reservoir of the SARS-CoV-2, and that domain 288-330 of S1 protein from the SARS-CoV-2 represents promising therapeutic and/or 2. Maria Salvato , University of Maryland, vaccine target. Baltimore, USA Keywords STARBIOS2 (EU-funded consortium), 2019-nCoV, Wuhan coronavirus, SARS, MERS Baltimore, USA Any reports and responses or comments on the article can be found at the end of the article. This article is included in the Disease Outbreaks gateway. This article is included in the Coronavirus collection. Corresponding author: Veljko Veljkovic (eveljkve@gmail.com) Author roles: Veljkovic V: Conceptualization, Formal Analysis, Methodology, Writing – Original Draft Preparation; Vergara-Alert J: Investigation, Writing – Original Draft Preparation; Segalés J: Investigation, Supervision, Writing – Original Draft Preparation; Paessler S: Investigation, Supervision, Writing – Original Draft Preparation Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2021 Veljkovic V et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite this article: Veljkovic V, Vergara-Alert J, Segalés J and Paessler S. Use of the informational spectrum methodology for rapid biological analysis of the novel coronavirus 2019-nCoV: prediction of potential receptor, natural reservoir, tropism and therapeutic/vaccine target [version 4; peer review: 2 approved] F1000Research 2021, 9:52 https://doi.org/10.12688/f1000research.22149.4 First published: 27 Jan 2020, 9:52 https://doi.org/10.12688/f1000research.22149.1 Page 2 of 15 F1000Research 2021, 9:52 Last updated: 07 JAN 2021 In the phylogenetic analysis, different amino acid sequences       UPDATE Updates from Version 3 of other coronaviruses were also included: (i) S1 proteins from In our article “Use of the informational spectrum methodology the following viruses: AVP78042, AVPvp78031, AY304486, for rapid biological analysis of the novel coronavirus 2019-nCoV: AY559093, JX163927, YN2018B, KY417146, used already prediction of potential receptor, natural reservoir, tropism and by other authors in the study of the phylogenetic relation- therapeutic/vaccine target” [version 3; peer review: 2 approved] ship between SARS-CoV-2 and nearest bat and SARS-like we predicted actin protein as an important COVID-19 host factor. CoVs (GISAID database); and (ii) S1 proteins from three first This in silico prediction was a few months later experimentally isolated human MERS-CoV: AGG22542, AFS88936, AFY13307, confirmed. We extended our existing version of the article deposited in the GISAID database with these new data which point out actin as an important new therapeutic and preventing target for COVID-19 disease. The ISM Any further responses from the reviewers can be found at the end of the article Detailed description of the sequence analysis based on ISM has been published elsewhere2. According to this approach, sequences (protein or DNA) are transformed into signals by Introduction assignment of numerical values of each element (amino acid Fears are mounting worldwide over the cross-border spread of or nucleotide). These values correspond to electron-ion inter- the new coronavirus (denoted as SARS-CoV-2) that originated action potential6, determining electronic properties of amino in Wuhan, the largest city in central China, after its spread to acid/nucleotides, which are essential for their intermolecular many countries around the world. The newly emerging patho- interactions. The signal obtained is then decomposed in a peri- gen belongs to the same virus family as the deadly severe acute odical function by the Fourier transformation. The result is a respiratory syndrome and Middle East respiratory syndrome series of frequencies and their amplitudes. The obtained frequen- coronaviruses (SARS-CoV and MERS-CoV, respectively). cies correspond to the distribution of structural motifs (primary The World Health Organization (WHO) has recently published structure) with defined physico-chemical characteristics surveillance recommendations for a possible “large epidemic or responsible for the biological function of the putative protein even pandemic” (pandemic declared on March 11th, 2020) of the corresponding to the analyzed sequence. When comparing novel coronavirus and it has issued guidelines for hospitals across proteins that share same biological or biochemical function, the world. However, many questions about SARS-CoV-2 remain the technique allows detection of code/frequency pairs that are unanswered: (i) what is the origin and/or natural reservoir of the specific for their common biological properties. The method virus? (ii) is it easily transmitted from human to human? and is insensitive to the location of the motifs and, therefore, does (iii) what are the potential diagnostic, therapeutic and vaccine not require previous alignment of the sequences. In addition, targets? Currently, only nucleotide sequences of eight human this is the only method that allows immediate functional analysis. SARS-CoV-2 isolates are available without any additional infor- mation about biological properties of the virus, beyond the Phylogenetic analysis morphological confirmation of the virion using electronic micro- The phylogenetic tree of S1 proteins from coronaviruses was scopy. This is likely not enough information to answer the generated with the ISM-based phylogenetic algorithm ISTREE, important abovementioned questions. previously described in detail elsewhere7. In the presented analysis, we calculated the distance matrix with the amplitude on The informational spectrum method (ISM), a virtual spectroscopy the frequency F(0.257) as the distance measure between method for analysis of proteins, is based on the fundamen- sequences. tal electronic properties of amino acids and requires only nucleotide sequence availability to investigate proteins1. For this Results and discussion reason, ISM was previously used for analysis of novel viruses In order to compare informational similarity between for which little or no information were available2–5. Here, the SARS-CoV-2, SARS-CoV, MERS-CoV and Bat SARS-like CoV, SARS-CoV-2 was analyzed with ISM to identify its possible the cross-spectra (CS) of S1 proteins from these viruses were origin and natural host, as well as putative therapeutic and calculated. Figure 1a shows the CS of SARS-CoV-2, SARS-CoV vaccine targets. and MERS-CoV. These CS contain only one dominant peak corresponding to the frequency F(0.257). Figure 1b displays Methods the CS of S1 proteins from SARS-CoV-2 and Bat SARS-like Sequences CoV. Amplitudes in these latter CS are significantly lower than The S1 surface protein sequences from the first 8 human in those CS presented in Figure 1a. These results show that SARS-CoV-2, deposited in the publicly available (i) S1 proteins from SARS-CoV-2, SARS-CoV, MERS-CoV GISAID database (assessed on January 19, 2020), were ana- and Bat SARS-like CoV encode common information, which lyzed by ISM. The studied sequences were BetaCoV/Wuhan/ is represented with the frequency F(0.257), and (ii) S1 proteins IVDC-HB-04/2020, BetaCoV/Wuhan/IVDC-HB-01/2019, Beta- from SARS-CoV-2 are remarkable more informationally CoV/Wuhan/IVDC-HB-05/2019, BetaCoV/Wuhan/IPBCAMS- similar with S1 from SARS-CoV and MERS-CoV than with WH-01/2019, BetaCoV/Wuhan/WIV04/2019, BetaCoV/ S1 from Bat SARS-like CoV. This suggests that biological Wuhan-Hu-1/2019, BetaCoV/Nonthaburi/61/2020, and BetaCoV/ properties of SARS-CoV-2 are apparently more similar to Nonthaburi/74/2020. SARS-CoV and MERS-CoV than to Bat SARS-like CoV. Page 3 of 15 F1000Research 2021, 9:52 Last updated: 07 JAN 2021 Figure 1. Cross-spectrum (CS) of S1 proteins. (a) CS of S1 from SARS-CoV, MERS-CoV and SARS-CoV-2; (b) CS of Bat SARS-like CoV and SARS-CoV-2. The abscissa represents the frequencies from the Fourier transform of the sequence of electron-ion interaction potential corresponding to the amino-acid sequence of proteins. The lowest frequency is 0.0 and the highest is 0.5. The ordinate represents the signal-to-noise ratio (the ratio between signal intensity at one particular IS frequency and the main value of the whole spectrum, S/N). To confirm this conclusion, the ISM-base phylogenetic tree by functional studies in vitro12. Calculation of the CS for S1 for S1 proteins was calculated (Figure 2). In this calcula- protein from the SARS-CoV-2 and all ACE2 sequences avail- tion the amplitude on the frequency F(0.257) was used as able at the UniProt database revealed that the highest amplitudes the distance measure. As observed in Figure 2, all analyzed on the frequency F(0.257) correspond to ACE2 from civet SARS-CoV-2 S1 amino acid sequences are grouped with and chicken. This result indicates that these species can be SARS-CoV and MERS-CoV and separated from Bat SARS-like included as potential candidates for the natural reservoir of the CoV. This indicates that SARS-CoV-2 are more phylogenetically SARS-CoV-2. However, it is possible that SARS-CoV-2 viruses similar to SARS-CoV and MERS-CoV than to Bat SARS-like use very different receptors in the natural host(s) and not only CoV. This result differs from those obtained with the homology- the ACE2 as it is the putative case in humans. An experimen- based phylogenetic analysis, which showed that SARS-CoV-2 tal study performed on chicken, however, indicated lack of are closely related to Bat SARS-like CoV (https://platform.gisaid. susceptibility of this species to the novel virus13; civets so org/epi3/frontend#lightbox1296857287). far have not been tested, but the indicated study confirmed susceptibility of domestic cat to SARS-CoV-2. It has been previously shown that the dominant frequency in the informational spectrum of viral envelope proteins corresponds Finally, the S1 amino acid sequence from the SARS-CoV-2 to interaction between the virus and its receptor2,3,7,8. The ISM was scanned to look for the domain that gives the highest contri- analysis showed that the frequency component F(0.257) is bution to the information represented by the frequency F(0.257) present in the CS of S1 SARS-CoV and its receptor angi- (Figure 4a). This analysis revealed domain 266–330 (number- otensin converting enzyme 2 (ACE2)9, but not in the CS of S1 ing concerns the maturated protein) is essential for interaction MERS-CoV and its main receptor dipeptidyl peptidase 4 of SARS-CoV-2 with ACE2. Of note is the striking homology (DPP4)10. Of note is that both receptors ACE2 and DPP4 are between these domains of S1 proteins from SARS-CoV-2 expressed in airway epithelia. Presence of F(0.257) in the and SARS-CoV, but not from MERS-CoV for which ACE2 is informational spectrum of MERS-CoV (Figure 1) suggests not the main receptor (Figure 4b). also possible interaction between this virus and the ACE2. The dominant peak on the frequency F(0.257) in the CS of S1 from Further, S1 spike proteins from SARS-CoV (Table 1) and SARS-CoV and MERS-CoV and ACE2 supports this possibil- SARS-CoV-2 (Table 2) were compared. The CS of S1 proteins ity (Figure 3), although this has not been formally proved for from SARS-CoV (Figure 5a) and SARS-CoV-2 (Figure 5b) MERS-CoV11. were assessed. Principal information encoded in S1 proteins from SARS-CoV and SARS-CoV-2 is represented with two As it is shown in Figure 1a, the frequency F(0.257) is also different frequencies F(0.222) and F(0.478), respectively. This present in the informational spectrum of the SARS-CoV-2, result indicates some potential difference(s) in the virus-host suggesting that ACE2 might be the receptor for this novel interaction of these two viruses although they apparently use the coronavirus too. This prediction was subsequently confirmed same receptor ACE2. Page 4 of 15 F1000Research 2021, 9:52 Last updated: 07 JAN 2021 Figure 2. Informational spectrum method-based phylogenetic tree for S1 proteins from SARS-CoV, MERS-CoV, Bat SARS-like CoV and SARS-CoV-2. The frequency F(0.257) as the distance measure was used. Figure 3. Cross-spectrum of ACE2 and S1 proteins from SARS-CoV and MERS-CoV. The abscissa and the ordinate are as described in Figure 1. To identify the host proteins involved in the attachment and/or plays an important role in the early entry events during human internalization of the SARS-CoV-2, the UniProt database coronavirus infections14. Actin proteins were selected as the (https://www.uniprot.org) was screened by ISM for human best candidate interactors for the SARS-CoV-2 among the proteins with the dominant peak on the frequency F(0.478). The host proteins that are characterized with frequency F(0.478). list of human proteins that have a dominant peak in IS at the Figure 5c shows that CS of actins from different mammalian frequency F(0.478) are given in Table 3. According to the IS species (Table 4) contains the dominant peak on F(0.478), sug- criterion, these proteins are potential candidate interactors with gesting that actin probably encodes the conserved information the SARS-CoV-2 S1 protein. Further, literature data mining important for their biological function. was performed to identify which proteins presented in Table 3 might be involved in the processes of infection with human The data mining of the PubMed database (www.ncbi.nlm.nih. coronaviruses. This analysis revealed that the actin protein gov/pubmed/) also showed that actin protein plays an important Page 5 of 15 F1000Research 2021, 9:52 Last updated: 07 JAN 2021 Figure 4. Domain of S1 protein which is important for SARS-CoV-2/ACE2 interaction. (a) Mapping of the domain of S1 protein from SARS-CoV-2 (BetaCoV/Wuhan/IVDC-HB-01/2019) which gives the dominant contribution to the information represented with the frequency F(0.257). (b) Sequence homology between domains of S1 proteins from SARS-CoV and SARS-CoV-2 with essential contribution to the information corresponding to the frequency F(0.257). Table 1. S1 proteins from SARS_CoV (uniprot.org). SPIKE_CVHSA Spike glycoprotein OS=Human SARS coronavirus Q19QX0_CVHSA Spike glycoprotein OS=Human SARS coronavirus A7J8L4_CVHSA Spike glycoprotein OS=Human SARS coronavirus J9SFL2_CVHSA Spike glycoprotein OS=Human SARS coronavirus J9TDZ0_CVHSA Spike glycoprotein OS=Human SARS coronavirus Q202F4_CVHSA Spike glycoprotein OS=Human SARS coronavirus Q202E5_CVHSA Spike glycoprotein OS=Human SARS coronavirus Q202E9_CVHSA Spike glycoprotein OS=Human SARS coronavirus Q202F5_CVHSA Spike glycoprotein OS=Human SARS coronavirus Q202E6_CVHSA Spike glycoprotein OS=Human SARS coronavirus Q202H5_CVHSA Spike glycoprotein OS=Human SARS coronavirus Q202F2_CVHSA Spike glycoprotein OS=Human SARS coronavirus Q202F9_CVHSA Spike glycoprotein OS=Human SARS coronavirus Q202G8_CVHSA Spike glycoprotein OS=Human SARS coronavirus Q202G3_CVHSA Spike glycoprotein OS=Human SARS coronavirus Q202H8_CVHSA Spike glycoprotein OS=Human SARS coronavirus Q202G5_CVHSA Spike glycoprotein OS=Human SARS coronavirus Page 6 of 15 F1000Research 2021, 9:52 Last updated: 07 JAN 2021 role in the rapid virus cell-to-cell spread and dissemination Table 2. S1 proteins from SARS-CoV-2 (GISAID). of infection15. Additionally, the actin filament reorganization is a key step in lung inflammation induced by systemic inflam- matory responses caused by infectious agents16. These findings BetaCoV/Wuhan/IVDC-HB-04/2020 indicate that interaction between actin proteins and the S1 could BetaCoV/Wuhan/IVDC-HB-01/2019 be involved in the infection and pathogenesis of SARS-CoV-2. BetaCoV/Wuhan/IVDC-HB-05/2019 In consequence, the possibility to interfere on this interaction might represent a valid hypothesis for development of promising BetaCoV/Wuhan/IPBCAMS-WH-01/2019 prevention and therapeutic strategies. BetaCoV/Wuhan/WIV04/2019 BetaCoV/Wuhan-Hu-1/2019 The here suggested role of actin in SARS-CoV-2 infection and pathogenesis was experimentally confirmed few months after BetaCoV/Nonthaburi/61/2020 submission of this manuscript. The ultrastructural analysis BetaCoV/Nonthaburi/74/2020 of SARS-CoV-2 interactions with the host cell via high reso- BetaCoV/Wuhan/WIV07/2019 lution scanning electron microscopy revealed the cell-to-cell transmission of virus which is mediated by actin17. The recent BetaCoV/Wuhan/WIV06/2019 analysis of the SARS-CoV-2 RNA–protein interactome in BetaCoV/Wuhan/WIV05/2019 infected human cells confirmed this finding18. It was also BetaCoV/Wuhan/WIV02/2019 demonstrated that spike protein of SARS-CoV-2 targets to the BetaCoV/Wuhan/HBCDC-HB-01/2019 dendritic spines of neurons, which are F-actin-enriched struc- tures [X]. This result suggests that modulation of the neural BetaCoV/Zhejiang/WZ-01/2020 morphology by control of F-actin dynamics or rearrangement Figure 5. CS of S1 proteins from SARS-CoV and SARS-CoV-2 and actin proteins. (a) CS of S1 proteins from human SARS-CoV; (b) CS of S1 proteins from SARS-CoV-2; (c) CS of mammalian actin proteins. The abscissa and the ordinate are as described in Figure 1. Page 7 of 15 F1000Research 2021, 9:52 Last updated: 07 JAN 2021 Table 3. Human proteins (uniprot.org) with the dominant peak on the frequency F(0.478) in the informational spectrum. ABCB8_HUMAN ATP-binding cassette sub-family B member 8, (Human) ACTB_HUMAN Actin, cytoplasmic 1 (Human) ACTC_HUMAN Actin, alpha cardiac muscle 1 (Human) ACTK_HUMAN Kappa-actin (Human) ACTS_HUMAN Actin, alpha skeletal muscle (Human) ATL3_HUMAN ADAMTS-like protein 3 (Human) AUP1_HUMAN Ancient ubiquitous protein 1 (Human) CA064_HUMAN Putative uncharacterized protein C1orf64 (Human) CETN2_HUMAN Centrin-2 (Human) CPNE1_HUMAN Copine-1 (Human) CR034_HUMAN Uncharacterized protein C18orf34 (Human) CSEN_HUMAN Calsenilin (Human) EXOC4_HUMAN Exocyst complex component 4 (Human) F108B_HUMAN Abhydrolase domain-containing protein FAM108B1 (Human) FGF13_HUMAN Fibroblast growth factor 13 (Human) FRMD1_HUMAN FERM domain-containing protein 1 (Human) GCDH_HUMAN Glutaryl-CoA dehydrogenase, mitochondrial (Human) GKN2_HUMAN Gastrokine-2 (Human) GPDA_HUMAN Glycerol-3-phosphate dehydrogenase [NAD+] (Human) HPS1_HUMAN Hermansky-Pudlak syndrome 1 protein (Human) HXK3_HUMAN Hexokinase-3 (Human) IL23R_HUMAN Interleukin-23 receptor (Human) KAD3_HUMAN GTP:AMP phosphotransferase mitochondrial (Human) KRA71_HUMAN Keratin-associated protein 7-1 (Human) LGMN_HUMAN Legumain (Human) MYOG_HUMAN Myogenin (Human) NTR2_HUMAN Neurotensin receptor type 2 (Human) RD3_HUMAN Protein RD3 (Human) S2543_HUMAN Solute carrier family 25 member 43 (Human) SMDF_HUMAN Neuregulin-1, sensory and motor neuron-derived factor (Human) SOX17_HUMAN Transcription factor SOX-17 (Human) THOC4_HUMAN THO complex subunit 4 (Human) TXND1_HUMAN Thioredoxin domain-containing protein 1 (Human) VATE2_HUMAN Vacuolar ATP synthase subunit E 2 (Human) ZN516_HUMAN Zinc finger protein 516 (Human) Page 8 of 15 F1000Research 2021, 9:52 Last updated: 07 JAN 2021 Table 4. Mammalian actin proteins (uniprot.org). ACTB_HUMAN Actin cytoplasmic 1 OS=Homo sapiens ACTB_MOUSE Actin cytoplasmic 1 OS=Mus musculus ACTC_HUMAN Actin alpha cardiac muscle 1 OS=Homo sapiens ACTB_CAVPO Actin cytoplasmic 1 OS=Cavia porcellus ACTS_MOUSE Actin alpha skeletal muscle OS=Mus musculus ACTB_PONAB Actin cytoplasmic 1 OS=Pongo abelii ACTA_MOUSE Actin aortic smooth muscle OS=Mus musculus ACTH_MOUSE Actin gamma-enteric smooth muscle OS=Mus musculus ACTG_MOUSE Actin cytoplasmic 2 OS=Mus musculus ACTB_MESAU Actin cytoplasmic 1 OS=Mesocricetus auratus ACTG_HUMAN Actin cytoplasmic 2 OS=Homo sapiens ACTS_RABIT Actin alpha skeletal muscle OS=Oryctolagus cuniculus ACTH_HUMAN Actin gamma-enteric smooth muscle OS=Homo sapiens ACTC_MOUSE Actin alpha cardiac muscle 1 OS=Mus musculus ACTC_RAT Actin alpha cardiac muscle 1 OS=Rattus norvegicus ACTA_HUMAN Actin aortic smooth muscle OS=Homo sapiens ACTG_RAT Actin cytoplasmic 2 OS=Rattus norvegicus ACTB_RAT Actin cytoplasmic 1 OS=Rattus norvegicus ACTB_BOVIN Actin cytoplasmic 1 OS=Bos taurus ACTS_HUMAN Actin alpha skeletal muscle OS=Homo sapiens is responsible for the neurological manifestations observed In silico methods are considered very important tools to gener- in COVID-19 patients19. ate first hypotheses and identify first drug candidates against newly discovered agents, like in the case of SARS-CoV-2, Interestingly, further data mining revealed that ibuprofen especially in the short-term. ISM, a technology based on elec- (FDA approved drug with excellent safety record) attenuates tronic biology, allowed identifying potential importance of interleukin-1β-induced inflammation as well as actin human actin proteins for viral infection/dissemination as reorganization20. Actin was also found to be the primary well as one FDA approved drug that may have an indirect component by which ibuprofen can bind to the tissue in antiviral activity within weeks of the initial outbreak. However, different organs21. This suggests that ibuprofen might impact additional experiments are required to confirm our initial the SARS-CoV-2-induced disease by indirect interaction findings. with actin proteins. Previously, ibuprofen was predicted as a candidate entry inhibitor for Ebola virus using the same in silico approach22, and this prediction was confirmed experi- In conclusion, results of the presented in silico analysis mentally at a later time point23,24. Actin also represents the suggest the following: (i) the newly emerging SARS-CoV-2 is quercetin-binding protein, suggesting that this natural compound highly related to SARS-CoV and, to a lesser degree, MERS- could be use in prevention and treatment of COVID-1925. The CoV, and ACE2 is a likely receptor of it; (ii) civets and poul- recent study performed on 113 healthcare workers working in try are potential candidates for the natural reservoir of the areas with a high risk of COVID-19 demonstrated the SARS-CoV-2, (iii) human actin proteins possibly participate in synergic effect of quercetin and vitamin C against COVID-1926. attachment/internalisation of SARS-CoV-2, (iv) drugs which These results prompt the possibility to experimentally test the interact with actin proteins (e.g. ibuprofen, quercetin) should be effects of ibuprofen and quercetin on SARS-CoV-2 infection investigated as possible therapeutics for treatment of SARS-CoV-2 under in vitro and in vivo conditions. infection, and (v) domain 266-330 of S1 protein from the Page 9 of 15 F1000Research 2021, 9:52 Last updated: 07 JAN 2021 SARS-CoV-2 represents promising therapeutic and/or vaccine Data availability target. Further research on these issues are needed, includ- Underlying data ing the development of reverse genetics and animal models to Sequence data of the viruses were obtained from the GISAID study the biology of SARS-CoV-2. Due to the fast evolving EpiFlu™ Database. To access the database each individual user of scientific knowledge on SARS-CoV-2, the first prediction should complete the “Registration Form For Individual Users”, has been already confirmed, while the chicken as potential which is available alongside detailed instructions. After submis- candidate as intermediate host has not been supported. sion of the Registration form, the user will receive a password. Importantly, link between ibuprofen/actin interactions and There are not any other restrictions for the access to GISAID. viral entry remains an exciting path for future therapeutic Conditions of access to, and use of, the GISAID EpiFlu™ investigations. Database and Data are defined by the Terms of Use. References 1. Veljković V, Cosić I, Dimitrijević B, et al.: Is it possible to analyze DNA and 14. Owczarek K, Szczepanski A, Milewska A, et al.: Early events during human protein sequences by the methods of digital signal processing? IEEE Trans coronavirus OC43 entry to the cell. Sci Rep. 2018; 8(1): 7124. Biomed Eng. 1985; 32(5): 337–41. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text 15. Doceul V, Hollinshead M, van der Linden L, et al.: Repulsion of superinfecting 2. Veljkovic V, Niman HL, Glisic S, et al.: Identification of hemagglutinin virions: a mechanism for rapid virus spread. Science. 2010; 327(5967): structural domain and polymorphisms which may modulate swine H1N1 873–876. interactions with human receptor. BMC Struct Biol. 2009; 9: 62 . PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text | Free Full Text 16. Du L, Zhou J, Zhang J, et al.: Actin filament reorganization is a key step in 3. Veljkovic V, Glisic S, Muller CP, et al.: In silico analysis suggests interaction lung inflammation induced by systemic inflammatory response syndrome. between Ebola virus and the extracellular matrix. Front Microbiol. 2015; 6: Am J Respir Cell Mol Biol. 2012; 47(5): 597–603. 135. PubMed Abstract | Publisher Full Text PubMed Abstract | Publisher Full Text | Free Full Text 17. Caldas LA, Carneiro FA, Higa LM, et al.: Ultrastructural analysis of 4. Paessler S, Veljkovic V: Prediction of influenza vaccine effectiveness for the SARS-CoV-2 interactions with the host cell via high resolution scanning influenza season 2017/18 in the US [version 1; peer review: 2 approved]. electron microscopy. Sci Rep. 2020; 10(1): 16099. F1000Res. 2017; 6: 2067. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text | Free Full Text 18. Schmidt N, Lareau CA, Keshishian H, et al.: The SARS-CoV-2 RNA–protein 5. Koma T, Veljkovic V, Anderson DE, et al.: Zika virus infection elicits auto- interactome in infected human cells. Nat Microbiol. 2020. antibodies to C1q. Sci Rep. 2018; 8(1): 1882. PubMed Abstract | Publisher Full Text PubMed Abstract | Publisher Full Text | Free Full Text 19. Chen CY, Chou YC, Hsueh YP: SARS-CoV-2 D614 and G614 spike variants 6. Veljković V, Slavić I: Simple general-model pseudopotential. Phys Rev Let. impair neuronal synapses and exhibit differential fusion ability. bioRxiv. 1972; 29: 105. 2020. Publisher Full Text Publisher Full Text 7. Perovic VR, Muller CP, Niman HL, et al.: Novel phylogenetic algorithm to 20. Li R, Song X, Li G, et al.: Ibuprofen attenuates interleukin-1β-induced monitor human tropism in Egyptian H5N1-HPAIV reveals evolution toward inflammation and actin reorganization via modulation of RhoA signaling efficient human-to-human transmission. PLoS One. 2013; 8(4): e61572. in rabbit chondrocytes. Acta Biochim Biophys Sin (Shanghai). 2019; 51(10): PubMed Abstract | Publisher Full Text | Free Full Text 1026–1033. 8. Schmier S, Mostafa A, Haarmann T, et al.: In Silico Prediction and PubMed Abstract | Publisher Full Text Experimental Confirmation of HA Residues Conferring Enhanced Human 21. Kunsman GW, Rohrig TP: Tissue distribution of ibuprofen in a fatal Receptor Specificity of H5N1 Influenza A Viruses. Sci Rep. 2015; 5: 11434. overdose. Am J Forensic Med Pathol. 1993; 14(1): 48–50. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text 9. Li W, Moore MJ, Vasilieva N, et al.: Angiotensin-converting enzyme 2 is a 22. Veljkovic V, Goeijenbier M, Glisic S, et al.: In silico analysis suggests functional receptor for the SARS coronavirus. Nature. 2003; 426(6965): repurposing of ibuprofen for prevention and treatment of EBOLA virus 450–4. disease [version 1; peer review: 2 approved]. F1000Res. 2015; 4: 104. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text | Free Full Text 10. Raj VS, Mou H, Smits SL, et al.: Dipeptidyl peptidase 4 is a functional receptor 23. Zhao Y, Ren J, Harlos K, et al.: Toremifene interacts with and destabilizes the for the emerging human coronavirus-EMC. Nature. 2013; 495(7440): 251–4. Ebola virus glycoprotein. Nature. 2016; 535(7610): 169–172. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text | Free Full Text 11. Müller MA, Raj VS, Muth D, et al.: Human coronavirus EMC does not require 24. Paessler S, Huang C, Sencanski M, et al.: Ibuprofen as a template molecule the SARS-coronavirus receptor and maintains broad replicative capability for drug design against Ebola virus. Front Biosci (Landmark Ed). 2018; 23: in mammalian cell lines. mBio. 2012; 3(6): pii: e00515-12. 947–953. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text 12. Letko M, Marzi A, Munster V: Functional assessment of cell entry and 25. Böhl M, Czupalla C, Tokalov SV, et al.: Identification of actin as quercetin- receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat binding protein: an approach to identify target molecules for specific Microbiol. 2020; 5(4): 562–569. ligands. Anal Biochem. 2005; 346(2): 295–9. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text 13. Shi J, Wen Z, Zhong G, et al.: Susceptibility of ferrets, cats, dogs, and other 26. Effect of Quercetin on Prophylaxis and Treatment of COVID-19. Clinical trial domesticated animals to SARS-coronavirus 2. Science. 2020; pii: eabb7015. registration NCT04377789. PubMed Abstract | Publisher Full Text | Free Full Text Reference Source Page 10 of 15 F1000Research 2021, 9:52 Last updated: 07 JAN 2021 Open Peer Review Current Peer Review Status: Version 4 Reviewer Report 07 January 2021 https://doi.org/10.5256/f1000research.34828.r76710 © 2021 Salvato M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Maria Salvato 1 Institute of Human Virology, University of Maryland, Baltimore, MD, USA 2 STARBIOS2 (EU-funded consortium), Baltimore, USA It is unclear which new data demonstrate that actin is an important new therapeutic and preventing target for COVID-19 disease. Is it the data referred from other authors? The cross- spectra frequency of .478 of Mammalian Actin and SARS-CoV-2 are the same, but does this vary depending on whether the actin species and the SARS-CoV host species match? In other words, does a human SARS-CoV2 have the same frequency as a bat actin? The figure legend should specify which mammalian host the "mammalian" actins represent. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Immunology, viral pathogenesis, viral genetics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Version 3 Reviewer Report 05 May 2020 https://doi.org/10.5256/f1000research.26010.r62772 © 2020 Salvato M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Maria Salvato Page 11 of 15 F1000Research 2021, 9:52 Last updated: 07 JAN 2021 1 Institute of Human Virology, University of Maryland, Baltimore, MD, USA 2 STARBIOS2 (EU-funded consortium), Baltimore, USA The authors have made the minor corrections requested and the manuscript looks like a valuable contribution to the literature. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Immunology, viral pathogenesis, viral genetics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Version 2 Reviewer Report 16 April 2020 https://doi.org/10.5256/f1000research.24526.r61263 © 2020 Salvato M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Maria Salvato 1 Institute of Human Virology, University of Maryland, Baltimore, MD, USA 2 STARBIOS2 (EU-funded consortium), Baltimore, USA This manuscript presents a machine-learning analysis of the published sequences of the novel 2019-nCoV. The authors use the Informational Spectrum Method (ISM), a virtual spectroscopy method for protein analysis based on the electronic properties of each amino acid. Their goal is to identify sites on the virus most likely to interact with other molecules like drugs, antibodies or viral receptors. Due to the rapidity of this field and the time elapsed since the manuscript was submitted (Jan 27), most of their conclusions are no longer new: nCoV is most related to SARS-CoV and less to MERS- CoV, ACE2 is a likely receptor, the natural reservoir might be civets and poultry, human actin proteins participate in internalization, ibuprofen that interacts with actin proteins should be investigated as a therapeutic, and finally that domain 266-330 of the S1 protein should be targeted by drugs or vaccines. It is a nice piece of work. The conclusions could be updated, for example they could say that the first of these predictions are supported by recent publications, that bat CoVs now appear to be the most closely related and bats are more likely to be the natural reservoir, and the link between ibuprofen/actin interactions and viral entry remains an exciting path for future therapeutics. Page 12 of 15 F1000Research 2021, 9:52 Last updated: 07 JAN 2021 Minor corrections ○In the first sentence of “Update” the authors refer to ‘these proteins’…do they mean Actin and SARS-CoV proteins? If they only mean actin they should say so, and then the sentence would read: “…actin protein is suggested as a host factor that participates in infection and pathogenesis of 2019-nCoV. Drugs modulating the biological activity of actin (e.g., ibuprofen) were suggested as candidates that should be investigated for the treatment of 2019-nCoV infection.“ ○ In the UPDATE, last sentences should say "...which are presented in Figure 5." ○ Introduction pg 3 “Fears are mounting worldwide over the cross-border spread of the new strain of coronavirus (denoted as 2019-nCoV) originated in Wuhan,….” Instead say “…that originated in Wuhan…” ○ Introduction pg 3 “…eight human 2019-nCoV isolates are available without any additional information about biological properties of the virus, beyond the morphological confirmation…” Replace “morphology” with “morphological”, it is an adjective. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Immunology, viral pathogenesis, viral genetics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Reviewer Report 18 March 2020 https://doi.org/10.5256/f1000research.24526.r60149 Page 13 of 15 F1000Research 2021, 9:52 Last updated: 07 JAN 2021 © 2020 Casadio R. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Rita Casadio Biocomputing Group, CIG, Interdepartmental Center «Luigi Galvani» for Integrated Studies of Bioinformatics, Biophysics and Biocomplexity, University of Bologna, Bologna, Italy Possibly the ISM method invented by the authors has high potentiality. Here, apparently, the method is applied to answer urgent questions in relation to sequence analysis of the novel coronavirus 2019-nCoV. It is interesting to verify that different papers at the moment seem to have reached the same conclusions, although in my opinion a comparison with other methods which are already published would add to the paper. On top, R.Yan et al,1 Science 2020 4 March, beautifully detailed the putative region of the virus/ACE2 interaction. I recommend the authors quote this finding as well and if possible add the reference. This would help in validating a quite interesting method of sequence analysis. References 1. Yan R, Zhang Y, Li Y, Xia L, et al.: Structural basis for the recognition of the SARS-CoV-2 by full- length human ACE2.Science. 2020. PubMed Abstract | Publisher Full Text Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Computational Biology, Structural bioinformatics, Functional annotation, Machine and deep learning I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Page 14 of 15 F1000Research 2021, 9:52 Last updated: 07 JAN 2021 The benefits of publishing with F1000Research: • Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage For pre-submission enquiries, contact research@f1000.com Page 15 of 15