Human‑to‑dog transmission of SARS‑CoV‑2, Colombia

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, has evolved to have a wide range of hosts, including non-human primates, wild and domestic animals. The ACE2 protein has a high level of conservation and is the common receptor inverte...

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Autores:
Rivero, Ricardo
Garay Montalvo, Evelin
Botero, Yesica
Serrano Coll, Héctor
Gastelbondo Pastrana, Bertha Irina
Muñoz, Marina
Ballesteros, Nathalia
Castañeda, Sergio
Patiño, Luz Helena
Ramírez, Juan David
Calderón Rangel, Alfonso
Guzmán Terán, Camilo Antonio
Martínez Bravo, Caty Milena
Alemán, Ader
Arrieta Bernate, German Javier
Mattar Velilla, Ameth Salim
Tipo de recurso:
Article of investigation
Fecha de publicación:
2022
Institución:
Corporación Universitaria del Caribe - CECAR
Repositorio:
Repositorio Digital CECAR
Idioma:
eng
OAI Identifier:
oai:repositorio.cecar.edu.co:cecar/10741
Acceso en línea:
https://repositorio.cecar.edu.co/handle/cecar/10741
Palabra clave:
SARS-CoV-2
transmission
Human‑to‑dog
Rights
openAccess
License
Derechos reservados - Corporación Universitaria de Caribe - CECAR
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network_acronym_str RepoCECAR2
network_name_str Repositorio Digital CECAR
repository_id_str
dc.title.eng.fl_str_mv Human‑to‑dog transmission of SARS‑CoV‑2, Colombia
title Human‑to‑dog transmission of SARS‑CoV‑2, Colombia
spellingShingle Human‑to‑dog transmission of SARS‑CoV‑2, Colombia
SARS-CoV-2
transmission
Human‑to‑dog
title_short Human‑to‑dog transmission of SARS‑CoV‑2, Colombia
title_full Human‑to‑dog transmission of SARS‑CoV‑2, Colombia
title_fullStr Human‑to‑dog transmission of SARS‑CoV‑2, Colombia
title_full_unstemmed Human‑to‑dog transmission of SARS‑CoV‑2, Colombia
title_sort Human‑to‑dog transmission of SARS‑CoV‑2, Colombia
dc.creator.fl_str_mv Rivero, Ricardo
Garay Montalvo, Evelin
Botero, Yesica
Serrano Coll, Héctor
Gastelbondo Pastrana, Bertha Irina
Muñoz, Marina
Ballesteros, Nathalia
Castañeda, Sergio
Patiño, Luz Helena
Ramírez, Juan David
Calderón Rangel, Alfonso
Guzmán Terán, Camilo Antonio
Martínez Bravo, Caty Milena
Alemán, Ader
Arrieta Bernate, German Javier
Mattar Velilla, Ameth Salim
dc.contributor.author.none.fl_str_mv Rivero, Ricardo
Garay Montalvo, Evelin
Botero, Yesica
Serrano Coll, Héctor
Gastelbondo Pastrana, Bertha Irina
Muñoz, Marina
Ballesteros, Nathalia
Castañeda, Sergio
Patiño, Luz Helena
Ramírez, Juan David
Calderón Rangel, Alfonso
Guzmán Terán, Camilo Antonio
Martínez Bravo, Caty Milena
Alemán, Ader
Arrieta Bernate, German Javier
Mattar Velilla, Ameth Salim
dc.contributor.corporatename.none.fl_str_mv Corporación Universitaria del caribe - CECAR
dc.contributor.researchgroup.none.fl_str_mv Salud Pública y Auditoría en Salud
dc.subject.proposal.none.fl_str_mv SARS-CoV-2
transmission
Human‑to‑dog
topic SARS-CoV-2
transmission
Human‑to‑dog
description Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, has evolved to have a wide range of hosts, including non-human primates, wild and domestic animals. The ACE2 protein has a high level of conservation and is the common receptor invertebrate species for a viral infection to occur; this receptor could give rise to anthroponotic events. This article describes the first event of symptomatic transmission in Latin America from a human to a dog by the B.1.625 lineage of SARS-CoV-2. We found 21 shared mutations in the complete genomes of viral sequences from owners and dogs. Further phylogenetic and molecular analysis showed that 100% co-localization of the clade helps to understand human-animal transmission. Prediction of the Spike protein structure of the sequenced virus and docking analyzes showed that the E484K mutation in the receptor-binding domain (RBD) could contribute to the viral affinity of dACE2. Therefore, close contact between SARS-CoV-2-infected humans and pets should be avoided to prevent the emergence of novel mutations of public health importance from anthroponotic events.
publishDate 2022
dc.date.issued.none.fl_str_mv 2022
dc.date.accessioned.none.fl_str_mv 2025-08-26T20:36:44Z
dc.type.none.fl_str_mv Artículo de revista
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dc.type.content.none.fl_str_mv Text
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dc.identifier.eissn.none.fl_str_mv 2045-2322
url https://repositorio.cecar.edu.co/handle/cecar/10741
identifier_str_mv 2045-2322
dc.language.iso.none.fl_str_mv eng
language eng
dc.relation.citationendpage.none.fl_str_mv 8
dc.relation.citationstartpage.none.fl_str_mv 1
dc.relation.citationvolume.none.fl_str_mv Volumen 12
dc.relation.ispartofjournal.none.fl_str_mv Scientific Reports
dc.relation.references.none.fl_str_mv Reina, J. E. SARS-CoV-2, una nueva zoonosis pandémica que amenaza al mundo. Vacunas 21, 17–22 (2020).
World Health Organization. WHO coronavirus (COVID-19) Dashboard. WHO coronavirus (COVID-19) dashboard with vaccination data. Who 1–5 (2021).
Sit, T. H. C. et al. Infection of dogs with SARS-CoV-2. Nature 586, 776–778 (2020).
Elbe, S. & Buckland-Merrett, G. Data, disease and diplomacy: GISAID’s innovative contribution to global health. Glob. Challenges 1, 33–46 (2017).
Premkumar, L. et al. The receptor-binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients. Sci. Immunol. 5, 1–9 (2020).
Gobeil, S.M.-C. et al. Effect of natural mutations of SARS-CoV-2 on spike structure, conformation and antigenicity. bioRxiv Prepr. Serv. Biol. https://doi.org/10.1101/2021.03.11.435037 (2021).
Goumenou, M., Spandidos, D. A. & Tsatsakis, A. Possibility of transmission through dogs being a contributing factor to the extreme COVID-19 outbreak in North Italy. Mol. Med. Rep. 21, 2293–2295 (2020).
Bosco-Lauth, A. M. et al. Experimental infection of domestic dogs and cats with SARS-CoV-2: Pathogenesis, transmission, and response to reexposure in cats. Proc. Natl. Acad. Sci. USA 117, 26382–26388 (2020).
Jangra, S. et al. SARS-CoV-2 spike E484K mutation reduces antibody neutralisation. Lancet Microbe. (2021).
Wibmer, C. K. et al. SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma. Nat. Med. https:// doi.org/10.1038/s41591-021-01285-x (2021).
Focosi, D. & Maggi, F. Neutralising antibody escape of SARS-CoV-2 spike protein: Risk assessment for antibody-based COVID-19 therapeutics and vaccines. Rev. Med. Virol. https://doi.org/10.1002/rmv.2231 (2021).
West, A. P., Barnes, C. O., Yang, Z. & Bjorkman, P. J. SARS-CoV-2 lineage B.1.526 emerging in the New York region detected by software utility created to query the spike mutational landscape. https://doi.org/10.1101/2021.02.14.431043.
Annavajhala, M. K. et al. A novel and expanding SARS-CoV-2 variant, B.1.526, identified in New York. https://doi.org/10.1101/ 2021.02.23.21252259.
Federation Drug American (FDA). Fact sheet for health care providers emergency use authorization of bamlanivimab and etesevimab. 1–36 (2020).
O’Toole, Á. et al. pangolin: Lineage assignment in an emerging pandemic as an epidemiological tool. in prep. https://doi.org/10. 1093/ve/veab064/6315289. (2021).
Rambaut, A. et al. A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology. Nat. Microbiol. 5, 1403–1407 (2020).
Stevanovic, V. et al. Seroprevalence of SARS-CoV-2 infection among pet animals in Croatia and potential public health impact. Transbound. Emerg. Dis. 00, 1–7 (2020).
Perisé-Barrios, A. J. et al. Humoral responses to SARS-CoV-2 by healthy and sick dogs during the COVID-19 pandemic in Spain. Vet. Res. 52, 1–11 (2021).
Jangra, S. et al. SARS-CoV-2 spike E484K mutation reduces antibody neutralisation. Lancet Microbe 2, e283–e284 (2021).
Cele, S. et al. Escape of SARS-CoV-2 501YV2 from neutralization by convalescent plasma. Nature 593, 142–146 (2021).
Andreano, E. et al. SARS-CoV-2 escape in vitro from a highly neutralizing COVID-19 convalescent plasma. bioRxiv Prepr. Serv. Biol. https://doi.org/10.1101/2020.12.28.424451 (2020).
Fernández, A. Structural impact of mutation D614G in SARS-CoV-2 spike protein: Enhanced infectivity and therapeutic opportunity. ACS Med. Chem. Lett. 11, 1667–1670 (2020).
Zhang, L. et al. SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity. Nat. Commun. 11, 1–9 (2020).
Zhang, Z. et al. The molecular basis for SARS-CoV-2 binding to dog ACE2. Nat. Commun. 12, 4195 (2021).
Naveca, F. G. et al. COVID-19 in Amazonas, Brazil, was driven by the persistence of endemic lineages and P.1 emergence. Nat. Med. 27, 1230–1238 (2021).
Faria, N. R. et al. Genomics and epidemiology of the P.1 SARS-CoV-2 lineage in Manaus, Brazil. Science (80-). 372, 815–821 (2021).
du Plessis, L. et al. Establishment and lineage dynamics of the SARS-CoV-2 epidemic in the UK. Science (80-). 371, 708–712 (2021).
Early introductions and transmission of SARS-CoV-2 variant B.1.1.7 in the United States Graphical abstract. https://doi.org/10. 1016/j.cell.2021.03.061.
Mallapaty, S. COVID mink analysis shows mutations are not dangerous—Yet. Nature 587, 340–341 (2020).
Oude Munnink, B. B. et al. Transmission of SARS-CoV-2 on mink farms between humans and mink and back to humans.
World Health Organization. SARS-CoV-2 mink-associated variant strain—Denmark. https://www.who.int/emergencies/disea se-outbreak-news/item/2020-DON301. (Accesed 31 July 2021)
Luan, J., Lu, Y., Jin, X. & Zhang, L. Spike protein recognition of mammalian ACE2 predicts the host range and an optimized ACE2 for SARS-CoV-2 infection. (2020). https://doi.org/10.1016/j.bbrc.2020.03.047.
Corman, V. et al. Diagnostic detection of 2019-nCoV by real-time RT-PCR. https://virologie-ccm.charite.de/en/ (2020). (Accesed 16 Apr 2021)
Miller, J. M. et al. Guidelines for Safe work practices in human and animal medical diagnostic laboratories recommendations of a CDC-convened, Biosafety Blue Ribbon Panel Centers for Disease Control and Prevention MMWR Editorial and Production Staff MMWR Editorial Board. Centers Dis. Control Prev. Morb. Mortal. Wkly. Rep. 61, 105 (2012).
World Health Organization. Laboratory Biosafety Manual 3rd edn. (World Health Organization, 2004)
du Sert, N. P. et al. The arrive guidelines 2.0: Updated guidelines for reporting animal research. PLoS Biol. 18, e3000410 (2020).
Katoh, K. & Frith, M. C. Adding unaligned sequences into an existing alignment using MAFFT and LAST. Bioinformatics 28, 3144–3146 (2012).
Minh, B. Q. et al. IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 37, 1530–1534 (2020).
Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A. & Jermiin, L. S. ModelFinder: Fast model selection for accurate phylogenetic estimates. Nat. Methods. 14, 587–589 (2017).
Hoang, D. T., Chernomor, O., von Haeseler, A., Minh, B. Q. & Vinh, L. S. UFBoot2: Improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35, 518–522 (2018).
Rambaut, A. FigTree. http://tree.bio.ed.ac.uk/software/figtree/. (Accesed 11 July 2021)
Letunic, I. & Bork, P. Interactive Tree Of Life (iTOL) v5: An online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 49, W293–W296 (2021).
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 2021, 1–11. https://doi.org/10.1038/s41586- 021-03819-2 (2021).
Mirdita, M., Steinegger, M. & Söding, J. MMseqs2 desktop and local web server app for fast, interactive sequence searches. Bioinformatics 35, 2856–2858 (2019).
Lan, J. et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581, 215–220 (2020).
Du, X. et al. Insights into protein–ligand interactions: Mechanisms, models, and methods. Int. J. Mol. Sci. 17, 1–34 (2016).
Dolinsky, T. J., Nielsen, J. E., McCammon, J. A. & Baker, N. A. PDB2PQR: An automated pipeline for the setup of PoissonBoltzmann electrostatics calculations. Nucleic Acids Res. 32, W665–W667 (2004).
dc.rights.none.fl_str_mv Derechos reservados - Corporación Universitaria de Caribe - CECAR
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spelling Rivero, RicardoGaray Montalvo, EvelinBotero, YesicaSerrano Coll, HéctorGastelbondo Pastrana, Bertha IrinaMuñoz, MarinaBallesteros, NathaliaCastañeda, SergioPatiño, Luz HelenaRamírez, Juan DavidCalderón Rangel, AlfonsoGuzmán Terán, Camilo AntonioMartínez Bravo, Caty MilenaAlemán, AderArrieta Bernate, German JavierMattar Velilla, Ameth SalimCorporación Universitaria del caribe - CECARSalud Pública y Auditoría en Salud2025-08-26T20:36:44Z2022https://repositorio.cecar.edu.co/handle/cecar/107412045-2322Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, has evolved to have a wide range of hosts, including non-human primates, wild and domestic animals. The ACE2 protein has a high level of conservation and is the common receptor invertebrate species for a viral infection to occur; this receptor could give rise to anthroponotic events. This article describes the first event of symptomatic transmission in Latin America from a human to a dog by the B.1.625 lineage of SARS-CoV-2. We found 21 shared mutations in the complete genomes of viral sequences from owners and dogs. Further phylogenetic and molecular analysis showed that 100% co-localization of the clade helps to understand human-animal transmission. Prediction of the Spike protein structure of the sequenced virus and docking analyzes showed that the E484K mutation in the receptor-binding domain (RBD) could contribute to the viral affinity of dACE2. Therefore, close contact between SARS-CoV-2-infected humans and pets should be avoided to prevent the emergence of novel mutations of public health importance from anthroponotic events.Auditoría en saludSalud Pública8 páginasapplication/pdfengDerechos reservados - Corporación Universitaria de Caribe - CECARhttps://creativecommons.org/licenses/by-nc/4.0/Atribución-NoComercial 4.0 Internacional (CC BY-NC 4.0)info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2https://doi.org/10.1038/s41598-022-11847-9Human‑to‑dog transmission of SARS‑CoV‑2, ColombiaArtículo de revistahttp://purl.org/coar/resource_type/c_2df8fbb1Textinfo:eu-repo/semantics/articlehttp://purl.org/redcol/resource_type/ARTinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/version/c_970fb48d4fbd8a85Colombia81Volumen 12Scientific ReportsReina, J. E. SARS-CoV-2, una nueva zoonosis pandémica que amenaza al mundo. Vacunas 21, 17–22 (2020).World Health Organization. WHO coronavirus (COVID-19) Dashboard. WHO coronavirus (COVID-19) dashboard with vaccination data. Who 1–5 (2021).Sit, T. H. C. et al. Infection of dogs with SARS-CoV-2. Nature 586, 776–778 (2020).Elbe, S. & Buckland-Merrett, G. Data, disease and diplomacy: GISAID’s innovative contribution to global health. Glob. Challenges 1, 33–46 (2017).Premkumar, L. et al. The receptor-binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients. Sci. Immunol. 5, 1–9 (2020).Gobeil, S.M.-C. et al. Effect of natural mutations of SARS-CoV-2 on spike structure, conformation and antigenicity. bioRxiv Prepr. Serv. Biol. https://doi.org/10.1101/2021.03.11.435037 (2021).Goumenou, M., Spandidos, D. A. & Tsatsakis, A. Possibility of transmission through dogs being a contributing factor to the extreme COVID-19 outbreak in North Italy. Mol. Med. Rep. 21, 2293–2295 (2020).Bosco-Lauth, A. M. et al. Experimental infection of domestic dogs and cats with SARS-CoV-2: Pathogenesis, transmission, and response to reexposure in cats. Proc. Natl. Acad. Sci. USA 117, 26382–26388 (2020).Jangra, S. et al. SARS-CoV-2 spike E484K mutation reduces antibody neutralisation. Lancet Microbe. (2021).Wibmer, C. K. et al. SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma. Nat. Med. https:// doi.org/10.1038/s41591-021-01285-x (2021).Focosi, D. & Maggi, F. Neutralising antibody escape of SARS-CoV-2 spike protein: Risk assessment for antibody-based COVID-19 therapeutics and vaccines. Rev. Med. Virol. https://doi.org/10.1002/rmv.2231 (2021).West, A. P., Barnes, C. O., Yang, Z. & Bjorkman, P. J. SARS-CoV-2 lineage B.1.526 emerging in the New York region detected by software utility created to query the spike mutational landscape. https://doi.org/10.1101/2021.02.14.431043.Annavajhala, M. K. et al. A novel and expanding SARS-CoV-2 variant, B.1.526, identified in New York. https://doi.org/10.1101/ 2021.02.23.21252259.Federation Drug American (FDA). Fact sheet for health care providers emergency use authorization of bamlanivimab and etesevimab. 1–36 (2020).O’Toole, Á. et al. pangolin: Lineage assignment in an emerging pandemic as an epidemiological tool. in prep. https://doi.org/10. 1093/ve/veab064/6315289. (2021).Rambaut, A. et al. A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology. Nat. Microbiol. 5, 1403–1407 (2020).Stevanovic, V. et al. Seroprevalence of SARS-CoV-2 infection among pet animals in Croatia and potential public health impact. Transbound. Emerg. Dis. 00, 1–7 (2020).Perisé-Barrios, A. J. et al. Humoral responses to SARS-CoV-2 by healthy and sick dogs during the COVID-19 pandemic in Spain. Vet. Res. 52, 1–11 (2021).Jangra, S. et al. SARS-CoV-2 spike E484K mutation reduces antibody neutralisation. Lancet Microbe 2, e283–e284 (2021).Cele, S. et al. Escape of SARS-CoV-2 501YV2 from neutralization by convalescent plasma. Nature 593, 142–146 (2021).Andreano, E. et al. SARS-CoV-2 escape in vitro from a highly neutralizing COVID-19 convalescent plasma. bioRxiv Prepr. Serv. Biol. https://doi.org/10.1101/2020.12.28.424451 (2020).Fernández, A. Structural impact of mutation D614G in SARS-CoV-2 spike protein: Enhanced infectivity and therapeutic opportunity. ACS Med. Chem. Lett. 11, 1667–1670 (2020).Zhang, L. et al. SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity. Nat. Commun. 11, 1–9 (2020).Zhang, Z. et al. The molecular basis for SARS-CoV-2 binding to dog ACE2. Nat. Commun. 12, 4195 (2021).Naveca, F. G. et al. COVID-19 in Amazonas, Brazil, was driven by the persistence of endemic lineages and P.1 emergence. Nat. Med. 27, 1230–1238 (2021).Faria, N. R. et al. Genomics and epidemiology of the P.1 SARS-CoV-2 lineage in Manaus, Brazil. Science (80-). 372, 815–821 (2021).du Plessis, L. et al. Establishment and lineage dynamics of the SARS-CoV-2 epidemic in the UK. Science (80-). 371, 708–712 (2021).Early introductions and transmission of SARS-CoV-2 variant B.1.1.7 in the United States Graphical abstract. https://doi.org/10. 1016/j.cell.2021.03.061.Mallapaty, S. COVID mink analysis shows mutations are not dangerous—Yet. Nature 587, 340–341 (2020).Oude Munnink, B. B. et al. Transmission of SARS-CoV-2 on mink farms between humans and mink and back to humans.World Health Organization. SARS-CoV-2 mink-associated variant strain—Denmark. https://www.who.int/emergencies/disea se-outbreak-news/item/2020-DON301. (Accesed 31 July 2021)Luan, J., Lu, Y., Jin, X. & Zhang, L. Spike protein recognition of mammalian ACE2 predicts the host range and an optimized ACE2 for SARS-CoV-2 infection. (2020). https://doi.org/10.1016/j.bbrc.2020.03.047.Corman, V. et al. Diagnostic detection of 2019-nCoV by real-time RT-PCR. https://virologie-ccm.charite.de/en/ (2020). (Accesed 16 Apr 2021)Miller, J. M. et al. Guidelines for Safe work practices in human and animal medical diagnostic laboratories recommendations of a CDC-convened, Biosafety Blue Ribbon Panel Centers for Disease Control and Prevention MMWR Editorial and Production Staff MMWR Editorial Board. Centers Dis. Control Prev. Morb. Mortal. Wkly. Rep. 61, 105 (2012).World Health Organization. Laboratory Biosafety Manual 3rd edn. (World Health Organization, 2004)du Sert, N. P. et al. The arrive guidelines 2.0: Updated guidelines for reporting animal research. PLoS Biol. 18, e3000410 (2020).Katoh, K. & Frith, M. C. Adding unaligned sequences into an existing alignment using MAFFT and LAST. Bioinformatics 28, 3144–3146 (2012).Minh, B. Q. et al. IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 37, 1530–1534 (2020).Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A. & Jermiin, L. S. ModelFinder: Fast model selection for accurate phylogenetic estimates. Nat. Methods. 14, 587–589 (2017).Hoang, D. T., Chernomor, O., von Haeseler, A., Minh, B. Q. & Vinh, L. S. UFBoot2: Improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35, 518–522 (2018).Rambaut, A. FigTree. http://tree.bio.ed.ac.uk/software/figtree/. (Accesed 11 July 2021)Letunic, I. & Bork, P. Interactive Tree Of Life (iTOL) v5: An online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 49, W293–W296 (2021).Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 2021, 1–11. https://doi.org/10.1038/s41586- 021-03819-2 (2021).Mirdita, M., Steinegger, M. & Söding, J. MMseqs2 desktop and local web server app for fast, interactive sequence searches. Bioinformatics 35, 2856–2858 (2019).Lan, J. et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581, 215–220 (2020).Du, X. et al. Insights into protein–ligand interactions: Mechanisms, models, and methods. Int. J. Mol. Sci. 17, 1–34 (2016).Dolinsky, T. J., Nielsen, J. E., McCammon, J. A. & Baker, N. A. PDB2PQR: An automated pipeline for the setup of PoissonBoltzmann electrostatics calculations. 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