Tag Archives: epitopes

B-cell epitopes for the development of SARS-CoV-2 vaccine

J. Pharm. Pharmacogn. Res., vol. 10, no. 3, pp. 429-444, May-June 2022.

Original Article

Molecular characterization and prediction of B-cell epitopes for the development of SARS-CoV-2 vaccine through bioinformatics approach

[Caracterización molecular y predicción de epítopos de células B para el desarrollo de una vacuna contra el SARS-CoV-2 mediante un enfoque bioinformático]

Aamir Shehzad1, Martia Rani Tacharina1, Suryo Kuncorojakti2 , Hafiz Ishfaq Ahmad3, Rofiqul A’la1, Andi Yasmin Wijaya4, Wiwiek Tyasningsih5, Fedik Abdul Rantam1,4*

1Virology and Immunology Laboratory, Department of Microbiology, Faculty of Veterinary Medicine, Airlangga University, Surabaya, East Java, 60115, Indonesia.

2Division of Veterinary Anatomy, Department of Veterinary Science, Faculty of Veterinary Medicine Airlangga University, Surabaya, East Java, 60115, Indonesia.

3Department of Animal Breeding and Genetics, University of Veterinary and Animal Sciences, Lahore, Pakistan.

4Research Center for Vaccine Technology and Development, Institute of Tropical Disease, Universitas Airlangga, Surabaya, East Java, 60115 Indonesia.

5Bacteriology and Mycology Laboratory, Department of Microbiology, Faculty of Veterinary Medicine, Airlangga University, Surabaya, East Java, 60132, Indonesia.

*E-mail: fedik-a-r@fkh.unair.ac.id

Abstract

Context: The SARS-CoV-2 virus is the cause of the COVID-19 pandemic, which is a severe public health crisis worldwide.Aims: To analyze the SARS-CoV-2 isolates of Surabaya and predict ORF1ab polyprotein epitopes through the bioinformatics approach for vaccine candidate development.

Methods: Three genomic sequences of Surabaya isolates were obtained from the GISAID, NCBI and PDB Gen-bank databases and MEGA-11 software were used to understand the transformations in the isolates. The IEDB and VaxiJen, AllerTop, and ToxinPred web servers were used to predict B-cell epitopes and analyze their antigenicity, non-allergenicity, non-toxicity, respectively. Moreover, these epitopes were linked by EAAAK for 3D modeling, refinement, and validation through Swiss-Model, Galaxy Refine, and RAMPAGE web tools.

Results: The Surabaya isolates, RSDS-RCVTD-UNAIR-49-A, 54-A, and 42-A, had 10, 20, and 16 mutations in nucleotides and depicted a phylogenetically close relationship to isolates of Egypt, Pakistan, and Bangladesh, respectively. A total of 71 sequential Orf1ab B-cell epitopes were predicted, and only three peptides were found to be antigenic,  non-allergenic, and non-toxic. These epitopes were linked with the EAAAK linker to develop a 3D refined and validated structure. This construct was docked with TLR-3 receptor by the Cluspro webserver and found a high affinity of ORF1ab+TLR3 due to 15 hydrogen bonds. The construct demonstrated good humoral and cellular immune responses in the C-ImmSim server, and cloning in the expression vector pET28a (+) yielded a colon of 846bp.

Conclusions: ORF1ab B-cell epitopes could be useful for developing effective vaccines to combat SARS-CoV-2 infection.

Keywords: bioinformatics; epitopes; ORF1ab polyproteins; public health; Indonesia; SARS-CoV-2.

Resumen

Contexto: El virus SARS-CoV-2 es la causa de la pandemia de COVID-19, que es una grave crisis de salud pública a nivel mundial.Objetivos: Analizar los aislamientos de SARS-CoV-2 de Surabaya y predecir los epítopos de poliproteína ORF1ab mediante el enfoque bioinformático para el desarrollo de candidatos vacunales.

Métodos: Se obtuvieron tres secuencias genómicas de aislamientos de Surabaya de las bases de datos GISAID, NCBI and PDB  Gen-bank y el software MEGA-11 para comprender las transformaciones en los aislamientos. Se utilizaron los servidores web IEDB y VaxiJen, AllerTop y ToxinPred para predecir epítopos de células B y analizar su antigenicidad, no alergenicidad y no toxicidad, respectivamente. Además, EAAAK vinculó estos epítopos para el modelado, el refinamiento y la validación en 3D a través de las herramientas web Swiss-Model, Galaxy Refine y RAMPAGE.

Resultados: Los aislamientos de Surabaya, RSDS-RCVTD-UNAIR-49-A, 54-A y 42-A, tenían 10, 20 y 16 mutaciones en nucleótidos y mostraban una relación filogenéticamente cercana con los aislamientos de Egipto, Pakistán y Bangladesh, respectivamente. Se predijeron un total de 71 epítopos de células B Orf1ab secuenciales, y solo tres péptidos resultaron ser antigénicos, no alergénicos y no tóxicos. Estos epítopos se vincularon con el enlazador EAAAK para desarrollar una estructura 3D refinada y validada. Esta construcción fue acoplada con el receptor TLR-3 por el servidor web Cluspro y encontró una alta afinidad de ORF1ab+TLR3 debido a 15 enlaces de hidrógeno. La construcción demostró buenas respuestas inmunitarias celulares y humorales en el servidor C-ImmSim, y la clonación en el vector de expresión pET28a (+) produjo un colon de 846 pb.

Conclusiones: Los epítopos de células B ORF1ab podrían ser útiles para desarrollar vacunas efectivas para combatir la infección por SARS-CoV-2.

Palabras Clave: bioinformática; epítopos; poliproteínas ORF1ab; Indonesia; salud pública; SARS-CoV-2.

This image has an empty alt attribute; its file name is jppres_pdf_free.png
Citation Format: Shehzad A, Kuncorojakti S, Tacharina MR, Ahmad HI, A'la R, Wijaya AY, Tyasningsih W, Rantam FA (2022) Molecular characterization and prediction of B-cell epitopes for the development of SARS-CoV-2 vaccine through bioinformatics approach. J Pharm Pharmacogn Res 10(3): 429–444.
References

Abraham Peele K, Srihansa T, Krupanidhi S, Ayyagari V S, Venkateswarulu TC (2021) Design of multi-epitope vaccine candidate against SARS-CoV-2: A in-silico study.J Biomol Struct Dyn 39: 3793–3801.

Adianingsih OR, Kharisma VD (2019) Study of B cell epitope conserved region of the Zika virus envelope glycoprotein to develop multi-strain vaccine. J App Pharm Sci 9: 098–103.

Ansori AN, Kusala MK, Normalina I, Indrasari S, Alamudi MY, Nidom RV, Nidom CA (2020) Immunoinformatic investigation of three structural protein genes in Indonesian SARS-CoV-2 isolates. Sys Rev Pharm 11: 422–434.

Ansori AN, Nidom RV, Kusala MK, Indrasari S, Normalina I, Nidom AN, Nidom CA (2021) Viroinformatics investigation of B-cell epitope conserved region in SARS-CoV-2 lineage B. 1.1. 7 isolates originated from Indonesia to develop vaccine candidate against COVID-19. J Pharm Pharmacogn Res 9: 766–779.

Biswas A, Bhattacharjee U, Chakrabarti AK, Tewari DN, Banu H, Dutta S (2020) Emergence of novel coronavirus and COVID-19: Whether to stay or die out? Crit Rev Microbiol 46: 182–193.

Bond CW, Leibowitz JL, Robb JA (1979) Pathogenic murine coronaviruses II. Characterization of virus-specific proteins of murine coronaviruses JHMV and A59V. Virology 94: 371–384.

Cai J, Sun W, Huang J, Gamber M, Wu J, He G (2020) Indirect virus transmission in cluster of COVID-19 cases, Wenzhou, China, 2020. Emerg Infect Dis 26: 1343–1345.

Callaway E (2020a) Making sense of coronavirus mutations. Nature 585: 174–177.

Callaway E (2020b) The coronavirus is mutating–does it matter? Nature 585: 174–178.

Castiglione F, Mantile F, De Berardinis P, Prisco A (2012) How the interval between prime and boost injection affects the immune response in a computational model of the immune system. Comput Math Methods Med 2012: 842329.

Cevik M, Kuppalli K, Kindrachuk J, Peiris M (2020) Virology, transmission, and pathogenesis of SARS-CoV-2. BMJ 371: m3862.

Chan JF, Kok KH, Zhu Z, Chu H, To KK, Yuan S, Yuen KY (2020a) Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan. Emerg Microb Infect  9(1): 221-236.

Chan JFW, Zhang AJ, Yuan S, Poon VKM, Chan CCS, Lee ACY, Yuen KY (2020b) Simulation of the clinical and pathological manifestations of coronavirus disease 2019 (COVID-19) in a golden Syrian hamster model: implications for disease pathogenesis and transmissibility. Clin Infect Dis 71: 2428–2446.

Chukwudozie OS, Chukwuanukwu RC, Iroanya OO, Eze DM, Duru VC, Dele-Alimi TO, Okinedo EU (2020) Attenuated subcomponent vaccine design targeting the SARS-CoV-2 nucleocapsid phosphoprotein RNA binding domain: In silico analysis. J Immunol Res 2020: 2837670.

Cooper NR, Nemerow GR (1984) The role of antibody and complement in the control of viral infections. J Investig Dermatol 83: S121–S127.

Dandekar AA, Perlman S (2005) Immunopathogenesis of coronavirus infections: Implications for SARS. Nat Rev Immunol 5: 917–927.

Dhanda SK, Mahajan S, Paul S, Yan Z, Kim H, Jespersen MC, Peters B (2019) IEDB-AR: immune epitope database—analysis resource in 2019. Nucleic Acids Res 47: W502–W506.

Dong L, Tian J, He S, Zhu C, Wang J, Liu C, Yang J (2020) Possible vertical transmission of SARS-CoV-2 from an infected mother to her newborn. JAMA 323: 1846-1848.

Duffy S (2018) Why are RNA virus mutation rates so damn high? PLoS Biol16: e3000003.

Emameh RZ, Nosrati H, Taheri RA (2020) Combination of biodata mining and computational modelling in identification and characterization of ORF1ab polyprotein of SARS-CoV-2 isolated from oronasopharynx of an Iranian patient. Biol Proced Online 22: 8.

Falzone L, Musso N, Gattuso G, Bongiorno D, Palermo CI, Scalia G, Stefani S (2020) Sensitivity assessment of droplet digital PCR for SARS-CoV-2 detection. Int J Mol Med 46: 957–964.

Fitzsimmons WJ, Woods RJ, McCrone JT, Woodman A, Arnold JJ, Yennawar M, Lauring AS (2018) A speed–fidelity trade-off determines the mutation rate and virulence of an RNA virus. PLoS Biology 16: e2006459.

Fritz M, Rosolen B, Krafft E, Becquart P, Elguero E, Vratskikh O, Denolly S, Boson B, Vanhomwegen J, Gouilh MA, Kodjo A, Chirouze C, Rosolen SG, Legros V, Leroy EM (2020) High prevalence of SARS-CoV-2 antibodies in pets from COVID-19+ households. One Health 11: 100192.

Gangaev A, Ketelaars SL, Isaeva OI, Patiwael S, Dopler A, Hoefakker K, De Biasi S, Gibellini L, Mussini C, Guaraldi G, Girardis M (2020a) Identification and characterization of an immunodominant SARS-CoV-2-specific CD8 T cell response. Res Sq [Preprint] DOI: 10.21203/rs.3.rs-33197/v2

Gangaev A, Ketelaars SL, Patiwael S, Dopler A, Isaeva OI, Hoefakker K, Kvistborg P (2020b) Profound CD8 T-cell responses towards SARS-CoV-2 OFR1ab in COVID-19 patients. Res Sq [Preprint] DOI: 10.21203/rs.3.rs-33197/v1

Gao T, Gao Y, Liu X, Nie Z, Sun H, Lin K, Wang S (2021) Identification and functional analysis of the SARS-COV-2 nucleocapsid protein. BMC Microbiol 21: 58.

Geourjon C, Deleage G (1995) SOPMA: Significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Bioinformatics 11: 681–684.

Ghoshal B, Ghoshal B, Swift S, Tucker A (2021) Uncertainty estimation in SARS-CoV-2 B-cell epitope prediction for vaccine development. In: International Conference on Artificial Intelligence in Medicine. Springer, Cham, p. 361–366.

GISAD (2021) GISAID Database. Available from https://www.epicov.org/epi3/frontend#6d403 [Accessed July 27, 2021].

Graham RL, Sparks JS., Eckerle LD, Sims AC, Denison MR (2008) SARS coronavirus replicase proteins in pathogenesis. Virus Res 133: 88–100.

Grote A, Hiller K, Scheer M, Münch R, Nörtemann B, Hempel DC, Jahn D (2005) JCat: A novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res 33: W526–W531.

Grubaugh ND, Hanage WP, Rasmussen AL (2020) Making sense of mutation: What D614G means for the COVID-19 pandemic remains unclear. Cell 182: 794–795.

Gupta S, Kapoor P, Chaudhary K, Gautam A, Kumar R, Open Source Drug Discovery Consortium, Raghava GP (2013) In silico approach for predicting toxicity of peptides and proteins. PloS One 8: e73957.

Hachim A, Kavian N, Cohen CA, Chin AW, Chu DK, Mok CK, Tsang OT, Yeung YC, Perera RA, Poon LL, Peiris JM (2020) ORF8 and ORF3b antibodies are accurate serological markers of early and late SARS-CoV-2 infection. Nat Immunol 21: 1293–1301.

Han H, Ma Q, Li C, Liu R, Zhao L, Wang W, Xia Y (2020) Profiling serum cytokines in COVID-19 patients reveals IL-6 and IL-10 are disease severity predictors. Emerg Microbes Infect 9: 1123–1130.

Harcourt J, Tamin A, Lu X, Kamili S, Sakthivel SK, Murray J, Thornburg N J (2020) Isolation and characterization of SARS-CoV-2 from the first US COVID-19 patient. BioRxiv [Preprint] DOI: 10.1101/2020.03.02.972935

Hernández‐Huerta MT, Pérez‐Campos Mayoral L, Romero Díaz C, Martínez Cruz M, Mayoral‐Andrade G, Sanchez Navarro LM, Matias‐Cervantes CA (2021) Analysis of SARS‐CoV‐2 mutations in Mexico, Belize, and isolated regions of Guatemala and its implication in the diagnosis. Med Virol 93: 2099–2114.

Horiike T (2016) An introduction to molecular phylogenetic analysis. Rev Agri Sci 4: 36-45.

Hu J, He CL, Gao Q, Zhang GJ, Cao XX, Long QX, Huang AL (2020) The D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity. BioRxiv [Preprint] DOI: 10.1101/2020.06.20.161323

Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, Cao B (2020a) Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395: 497–506.

Huang Y, Yang C, Xu XF, Xu W, Liu SW (2020b) Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19. Acta Pharmacol Sin 41: 1141–1149.

Kathwate GH (2022) In silico design and characterization of multi-epitopes vaccine for SARS-CoV2 from its spike proteins. Int J Pept Res Ther 28: 37.

Khailany RA, Safdar M, Ozaslan M (2020) Genomic characterization of a novel SARS-CoV-2. Gene Reports 19: 100682.

Khan MK, Zaman S, Chakraborty S, Chakravorty R, Alam MM, Bhuiyan TR, Seraj ZI (2014) In silico predicted mycobacterial epitope elicits in vitro T-cell responses. Mo.l Immunol 61: 16–22.

Khatoon N, Pandey RK, Prajapati VK (2017) Exploring leishmania secretory proteins to design B and T cell multi-epitope subunit vaccine using immunoinformatics approach. Sci Rep 7: 8285.

Kim JI (2020) New potential for healing the trauma of Maori from Brain Education. [Interview] Dr. Lily George, Director of Education, New Zealand Headquarters of ECO. IBREA Report 12: 3–7.

Korber B, Fischer WM, Gnanakaran S, Yoon H, Theiler J, Abfalterer W, Montefiori DC (2020) Tracking changes in SARS-CoV-2 spike: Evidence that D614G increases infectivity of the COVID-19 virus. Cell 182: 812–827.

Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33: 1870-1874.

Lau SY, Wang P, Mok BWY, Zhang AJ, Chu H, Lee ACY, Chen H (2020) Attenuated SARS-CoV-2 variants with deletions at the S1/S2 junction. EmergMicrobes Infect 9: 837–842.

Lewis DM, Leibrand S, Leibrand H (2020) A test-based strategy for safely shortening quarantine for COVID-19. MedRxiv [Preprint] DOI:10.1101/2020.11.24.20238287

Li G, Fan Y, Lai Y, Han T, Li Z, Zhou P, Wu J (2020a) Coronavirus infections and immune responses. JMed Virol 92: 424–432.

Li J, Wang H, Mao L, Yu H, Yu X, Sun Z, Wang X (2020b) Rapid genomic characterization of SARS-CoV-2 viruses from clinical specimens using nanopore sequencing. Sci Rep 10: 17492.

Liu K, Pan X, Li L, Yu F, Zheng A, Du P, Han P, Meng Y, Zhang Y, Wu L, Chen Q (2021) Binding and molecular basis of the bat coronavirus RaTG13 virus to ACE2 in humans and other species. Cell 184(13): 3438–3451.

Mapleson D, Drou N, Swarbreck D (2015) RAMPART: A workflow management system for de novo genome assembly. Bioinformatics 31: 1824–1826.

Martin A, Nateqi J, Gruarin S, Munsch N, Abdarahmane I, Zobel M, Knapp B (2020) An artificial intelligence-based first-line defence against COVID-19: Digitally screening citizens for risks via a chatbot. Sci Rep 10: 19012.

Meng XY, Zhang HX, Mezei M, Cui M (2011) Molecular docking: a powerful approach for structure-based drug discovery. Curr Comput Aided Drug Des 7: 146–157.

Naveed M, Tehreem S, Arshad S, Bukhari SA, Shabbir MA, Essa R, Khan I (2021) Design of a novel multiple epitope-based vaccine: An immunoinformatics approach to combat SARS-CoV-2 strains. J Infect Public Health 14: 938–946.

Nemati AS, Tafrihi M, Sheikhi F, Tabari AR, Haditabar A (2021) Designing a new multi epitope-based vaccine against COVID-19 disease: An immunoinformatic study based on reverse vaccinology approach. Res Sq [Preprint] DOI: 10.21203/rs.3.rs-206270/v1

Oany AR, Emran AA, Jyoti TP (2014) Design of an epitope-based peptide vaccine against spike protein of human coronavirus: an in silico approach. Drug Des Devel Ther 8: 1139.

Ozono S, Zhang Y, Ode H, Seng TT, Imai K, Miyoshi K, Tokunaga K (2021) Naturally mutated spike proteins of SARS-CoV-2 variants show differential levels of cell entry. Nat Commun 12:848.

Pandey RK, Bhatt TK, Prajapati VK (2018) Novel immunoinformatics approaches to design multi-epitope subunit vaccine for malaria by investigating anopheles salivary protein. Sci Rep 8: 1125.

Park WB, Kwon NJ, Choi SJ, Kang CK, Choe PG, Kim JY, Oh MD (2020) Virus isolation from the first patient with SARS-CoV-2 in Korea. J Korean Med Sci 35: 10–14.

Rapin N, Lund O, Bernaschi M, Castiglione F (2010) Computational immunology meets bioinformatics: the use of prediction tools for molecular binding in the simulation of the immune system. PloS One 5: e9862.

Safavi A, Kefayat A, Mahdevar E, Abiri A, Ghahremani F (2020) Exploring the out of sight antigens of SARS-CoV-2 to design a candidate multi-epitope vaccine by utilizing immunoinformatics approaches. Vaccine 38: 7612–7628.

Saitou N, Nei M (1987) The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406–425.

Sen Gupta PS, Biswal S, Singha D, Rana MK (2021) Binding insight of clinically oriented drug famotidine with the identified potential target of SARS-CoV-2. J Biomol Struct Dyn 39: 5327–5333.

Siegrist CA (2008) Vaccine Immunology. Vaccines. SaundersElsevier, p. 17–36.

Singh A, Thakur M, Sharma LK, Chandra K (2020) Designing a multi-epitope peptide based vaccine against SARS-CoV-2. Sci Rep 10: 16219.

Tahir ul Qamar M, Shahid F, Aslam S, Ashfaq UA, Aslam S, Fatima I, Chen LL (2020) Reverse vaccinology assisted designing of multiepitope-based subunit vaccine against SARS-CoV-2. Infect DisPoverty 9: 132.

Tamura K, Nei M, Kumar S (2004) Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci U S A 101: 11030–11035.

Tang T, Bidon M, Jaimes JA, Whittaker GR, Daniel S (2020) Coronavirus membrane fusion mechanism offers a potential target for antiviral development. Antivir Res 178: 104792.

Valencia I, Peiró C, Lorenzo Ó, Sánchez-Ferrer CF, Eckel J, Romacho T (2020) DPP4 and ACE2 in diabetes and COVID-19: therapeutic targets for cardiovascular complications? Front Pharmacol 11: 1161.

Voloch CM, da Silva Francisco Jr R, de Almeida LG, Cardoso CC, Brustolini OJ, Gerber AL, de Vasconcelos ATR (2021) Genomic characterization of a novel SARS-CoV-2 lineage from Rio de Janeiro, Brazil. Virol J 95: e00119-21.

Watanabe Y, Allen JD, Wrapp D, McLellan JS, Crispin M (2020) Site-specific glycan analysis of the SARS-CoV-2 spike. Science 369: 330–333.

Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Schwede T (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46: W296–W303.

Weisblum Y, Schmidt F, Zhang F, DaSilva J, Poston D, Lorenzi JC, Bieniasz PD (2020) Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants. Elife 9: e61312.

Weiss SR, Navas-Martin S (2005) Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. Microbiol Mol Biol Rev 69: 635–664.

WHO (2020a) World Health Organization. Naming the coronavirus disease (COVID-19) and the virus that causes it. Available at: https://www. who.int/emergencies/diseases/novel-coronavirus-2019/technicalguidance/naming-the-coronavirus-disease-(covid-2019)-and-thevirus-that-causes-it. [Accessed 2 March, 2020].

WHO (2020b) World Health Organization. Coronavirus disease 2019 (COVID-19): situation report, 32. Available at: https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200221-sitrep-32-covid-19.pdf [Accessed January 05, 2022].

WHO (2021) World Health Organization. Coronavirus disease. Available from https://covid19.who.int/ [Accessed August 27, 2021].

Yadav V, Rajput M, Diwakar RP, Kumar R (2020) An Overview on transmission of diseases in special reference to COVID-19 and potential Tar-gets to control this pandemic. J Adv Microbiol Res 4: 015.

Zhang J, Zeng H, Gu J, Li H, Zheng L, Zou Q (2020) Progress and prospects on vaccine development against SARS-CoV-2. Vaccines 8: 153.

Zhang Y, Chen Y, Li Y, Huang F, Luo B, Yuan Y, Xia B, Ma X, Yang T, Yu F, Liu J (2021) The ORF8 protein of SARS-CoV-2 mediates immune evasion through down-regulating MHC-Ι. Proc Natl Acad Sci U S A 118: e2024202118.

Zheng YY, Ma YT, Zhang JY, Xie X (2020) COVID-19 and the cardiovascular system. Nat Rev Cardiol 17: 259–260.

Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, Si HR, Zhu Y, Li B, Huang CL, Chen HD (2020) A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579: 270–273.

Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, Zhao X, Huang B, Shi W, Lu R, Niu P (2020) A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med 382: 727–733.

© 2022 Journal of Pharmacy & Pharmacognosy Research (JPPRes)