1887

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had spread to at least 215 countries, caused more than 8 000 000 confirmed infections and over 450 000 deaths, and overwhelmed healthcare systems worldwide. Like severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low-passage (P) strains of SARS-CoV-2 (Wash1 : P4 and Munich : P1) were cultured twice in Vero E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1 : P6 and minor variants in the Munich : P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated that the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies, we investigated the development of neutralizing antibody titres in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either or pathogenesis studies.

Funding
This study was supported by the:
  • Cynthia M McMillen , National Institutes of Health , (Award AI060525)
  • William B Klimstra , DSF Charitable Foundation
  • William B Klimstra , University of Pittsburgh Clinical and Translational Science Institute
  • William B Klimstra , Center for Vaccine Research, University of Pittsburgh
  • William B Klimstra , Dr. Patrick Gallagher, Chancellor of the University of Pittsburgh
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/content/journal/jgv/10.1099/jgv.0.001481
2020-08-21
2020-10-30
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References

  1. Patel A, Jernigan DB, nCo V. 2019-nCoV CDC Response Team Initial Public Health Response and Interim Clinical Guidance for the 2019 Novel Coronavirus Outbreak - United States, December 31, 2019-February 4, 2020. MMWR Morb Mortal Wkly Rep 2020; 69: 140 146 [CrossRef] [PubMed]
    [Google Scholar]
  2. Coronaviridae Study Group of the International Committee on Taxonomy of Viruses The species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 2020; 5: 536 544 [CrossRef] [PubMed]
    [Google Scholar]
  3. Zhu N, Zhang D, Wang W, Li X, Yang B et al. A novel coronavirus from patients with Pneumonia in China, 2019. N Engl J Med 2020; 382: 727 733 [CrossRef] [PubMed]
    [Google Scholar]
  4. Drosten C, Günther S, Preiser W, van der Werf S, Brodt H-R et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 2003; 348: 1967 1976 [CrossRef] [PubMed]
    [Google Scholar]
  5. Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T et al. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 2003; 348: 1953 1966 [CrossRef] [PubMed]
    [Google Scholar]
  6. Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus ADME, Fouchier RAM. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med 2012; 367: 1814 1820 [CrossRef] [PubMed]
    [Google Scholar]
  7. Ge X-Y, Li J-L, Yang X-L, Chmura AA, Zhu G et al. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature 2013; 503: 535 538 [CrossRef] [PubMed]
    [Google Scholar]
  8. Ithete NL, Stoffberg S, Corman VM, Cottontail VM, Richards LR et al. Close relative of human middle East respiratory syndrome coronavirus in bat, South Africa. Emerg Infect Dis 2013; 19: 1697 1699 [CrossRef] [PubMed]
    [Google Scholar]
  9. Wang L-F, Anderson DE. Viruses in bats and potential spillover to animals and humans. Curr Opin Virol 2019; 34: 79 89 [CrossRef] [PubMed]
    [Google Scholar]
  10. Zhou P, Yang X-L, Wang X-G, Hu B, Zhang L et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020; 579: 270 273 [CrossRef] [PubMed]
    [Google Scholar]
  11. McBride R, van Zyl M, Fielding BC. The coronavirus nucleocapsid is a multifunctional protein. Viruses 2014; 6: 2991 3018 [CrossRef] [PubMed]
    [Google Scholar]
  12. Siu YL, Teoh KT, Lo J, Chan CM, Kien F et al. The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles. J Virol 2008; 82: 11318 11330 [CrossRef] [PubMed]
    [Google Scholar]
  13. J Alsaadi EA, Jones IM. Membrane binding proteins of coronaviruses. Future Virol 2019; 14: 275 286 [CrossRef] [PubMed]
    [Google Scholar]
  14. Letko M, Marzi A, Munster V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat Microbiol 2020; 5: 562 569 [CrossRef] [PubMed]
    [Google Scholar]
  15. Bosch BJ, van der Zee R, de Haan CAM, Rottier PJM. The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex. J Virol 2003; 77: 8801 8811 [CrossRef] [PubMed]
    [Google Scholar]
  16. Heald-Sargent T, Gallagher T, Ready GT. Ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence. Viruses 2012; 4: 557 580 [CrossRef] [PubMed]
    [Google Scholar]
  17. Belouzard S, Chu VC, Whittaker GR. Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites. Proc Natl Acad Sci U S A 2009; 106: 5871 5876 [CrossRef] [PubMed]
    [Google Scholar]
  18. Burkard C, Verheije MH, Wicht O, van Kasteren SI, van Kuppeveld FJ et al. Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner. PLoS Pathog 2014; 10: e1004502 [CrossRef] [PubMed]
    [Google Scholar]
  19. Millet JK, Whittaker GR. Host cell proteases: critical determinants of coronavirus tropism and pathogenesis. Virus Res 2015; 202: 120 134 [CrossRef] [PubMed]
    [Google Scholar]
  20. Li F, Structure LF. Structure, function, and evolution of coronavirus spike proteins. Annu Rev Virol 2016; 3: 237 261 [CrossRef] [PubMed]
    [Google Scholar]
  21. Coutard B, Valle C, de Lamballerie X, Canard B, Seidah NG et al. The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade. Antiviral Res 2020; 176: 104742 [CrossRef] [PubMed]
    [Google Scholar]
  22. Shang J, Wan Y, Luo C, Ye G, Geng Q et al. Cell entry mechanisms of SARS-CoV-2. Proc Natl Acad Sci U S A 2020; 117: 11727 11734 [CrossRef] [PubMed]
    [Google Scholar]
  23. Shang J, Ye G, Shi K, Wan Y, Luo C et al. Structural basis of receptor recognition by SARS-CoV-2. Nature 2020; 581: 221 224 [CrossRef] [PubMed]
    [Google Scholar]
  24. Hoffmann M, Kleine-Weber H, Pöhlmann S. A multibasic cleavage site in the spike protein of SARS-CoV-2 is essential for infection of human lung cells. Mol Cell 2020; 78: 779 784 [CrossRef] [PubMed]
    [Google Scholar]
  25. Grifoni A, Weiskopf D, Ramirez SI, Mateus J, Dan JM et al. Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals. Cell 2020; 181: 1489 1501 [CrossRef] [PubMed]
    [Google Scholar]
  26. Guo L, Ren L, Yang S, Xiao M, Chang YF et al. Profiling early humoral response to diagnose novel coronavirus disease (COVID-19). Clin Infect Dis 2020
    [Google Scholar]
  27. Lv H, Wu NC, Tak-Yin Tsang O, Yuan M, Perera R et al. Cross-Reactive antibody response between SARS-CoV-2 and SARS-CoV infections. Cell Rep 2020; 107725:
    [Google Scholar]
  28. Wu F, Wang A, Liu M, Wang Q, Chen J et al. Neutralizing antibody responses to SARS-CoV-2 in a COVID-19 recovered patient cohort and their implications.. The Lancet. 2020; in press:
    [Google Scholar]
  29. Liu L, To KK-W, Chan K-H, Wong Y-C, Zhou R et al. High neutralizing antibody titer in intensive care unit patients with COVID-19. Emerg Microbes Infect 2020; 9: 1 30 [CrossRef] [PubMed]
    [Google Scholar]
  30. Poh CM, Carissimo G, Wang B, Amrun SN, Lee CY-P et al. Two linear epitopes on the SARS-CoV-2 spike protein that elicit neutralising antibodies in COVID-19 patients. Nat Commun 2020; 11: 2806 [CrossRef] [PubMed]
    [Google Scholar]
  31. Harcourt J, Tamin A, Lu X, Kamili S, Sakthivel SK et al. Severe acute respiratory syndrome coronavirus 2 from patient with coronavirus disease, United States. Emerg Infect Dis 2020; 26: 1266 1273 [CrossRef] [PubMed]
    [Google Scholar]
  32. Wölfel R, Corman VM, Guggemos W, Seilmaier M, Zange S et al. Virological assessment of hospitalized patients with COVID-2019. Nature 2020; 581: 465 469 [CrossRef] [PubMed]
    [Google Scholar]
  33. Alfson KJ, Avena LE, Beadles MW, Staples H, Nunneley JW et al. Particle-to-PFU ratio of Ebola virus influences disease course and survival in cynomolgus macaques. J Virol 2015; 89: 6773 6781 [CrossRef] [PubMed]
    [Google Scholar]
  34. Klimstra WB, Ryman KD, Johnston RE. Adaptation of Sindbis virus to BHK cells selects for use of heparan sulfate as an attachment receptor. J Virol 1998; 72: 7357 7366 [CrossRef] [PubMed]
    [Google Scholar]
  35. Amarasinghe SL, Su S, Dong X, Zappia L, Ritchie ME et al. Opportunities and challenges in long-read sequencing data analysis. Genome Biol 2020; 21: 30 [CrossRef] [PubMed]
    [Google Scholar]
  36. Soneson C, Yao Y, Bratus-Neuenschwander A, Patrignani A, Robinson MD et al. A comprehensive examination of nanopore native RNA sequencing for characterization of complex transcriptomes. Nat Commun 2019; 10: 3359 [CrossRef] [PubMed]
    [Google Scholar]
  37. Ogando NS, Dalebout TJ, Zevenhoven-Dobbe JC, Limpens RWAL, van der Meer Y et al. SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology. J Gen Virol 2020 [CrossRef] [PubMed]
    [Google Scholar]
  38. Anderson DE, Tan CW, Chia WN, Young BE, Linster M et al. Lack of cross-neutralization by SARS patient sera towards SARS-CoV-2. Emerg Microbes Infect 2020; 9: 900 902 [CrossRef] [PubMed]
    [Google Scholar]
  39. Haveri A, Smura T, Kuivanen S, Österlund P, Hepojoki J et al. Serological and molecular findings during SARS-CoV-2 infection: the first case study in Finland, January to February 2020. Euro Surveill 2020; 25: [CrossRef] [PubMed]
    [Google Scholar]
  40. Okba NMA, Müller MA, Li W, Wang C, GeurtsvanKessel CH et al. Severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease patients. Emerg Infect Dis 2020; 26: 1478 1488 [CrossRef] [PubMed]
    [Google Scholar]
  41. Perera RA, Mok CK, Tsang OT, Lv H, Ko RL et al. Serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), March 2020. Euro Surveill 2020; 25: [CrossRef] [PubMed]
    [Google Scholar]
  42. To KK-W, Tsang OT-Y, Leung W-S, Tam AR, Wu T-C et al. Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study. Lancet Infect Dis 2020; 20: 565 574 [CrossRef] [PubMed]
    [Google Scholar]
  43. Hartman AL, Nambulli S, McMillen CM, White AG, Tilston-Lunel NL. SARS-CoV-2 infection of African green monkeys results in mild respiratory disease discernible by PET/CT imaging and prolonged shedding of infectious virus from both respiratory and gastrointestinal tracts. bioRxiv 2020 137687 20.06.2020
    [Google Scholar]
  44. Lau S-Y, Wang P, Mok BW-Y, Zhang AJ, Chu H et al. Attenuated SARS-CoV-2 variants with deletions at the S1/S2 junction. Emerg Microbes Infect 2020; 9: 837 842 [CrossRef] [PubMed]
    [Google Scholar]
  45. Davidson AD, Williamson MK, Lewis S, Shoemark D, Carroll MW et al. Characterisation of the transcriptome and proteome of SARS-CoV-2 using direct RNA sequencing and tandem mass spectrometry reveals evidence for a cell passage induced in-frame deletion in the spike glycoprotein that removes the furin-like cleavage site. bioRxiv 2020
    [Google Scholar]
  46. Liu Z, Zheng H, Lin H, Li M, Yuan R et al. Identification of common deletions in the spike protein of SARS-CoV-2. J Virol 2020 [CrossRef] [PubMed]
    [Google Scholar]
  47. Walls AC, Park Y-J, Tortorici MA, Wall A, McGuire AT et al. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell 2020; 181: 281 292 [CrossRef] [PubMed]
    [Google Scholar]
  48. Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 2020; 181: 271 280 [CrossRef] [PubMed]
    [Google Scholar]
  49. Bertram S, Glowacka I, Blazejewska P, Soilleux E, Allen P et al. TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells. J Virol 2010; 84: 10016 10025 [CrossRef] [PubMed]
    [Google Scholar]
  50. Ou X, Liu Y, Lei X, Li P, Mi D et al. Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat Commun 2020; 11: 1620 [CrossRef] [PubMed]
    [Google Scholar]
  51. Kim S-J, Nguyen V-G, Park Y-H, Park B-K, Chung H-C. A novel synonymous mutation of SARS-CoV-2: is this possible to affect their antigenicity and immunogenicity?. Vaccines 2020; 8: E220 220 [CrossRef] [PubMed]
    [Google Scholar]
  52. Koyama T, Weeraratne D, Snowdon JL, Parida L. Emergence of drift variants that may affect COVID-19 vaccine development and antibody treatment. Pathogens 2020; 9: 324 [CrossRef] [PubMed]
    [Google Scholar]
  53. Korber B, Fischer WM, Gnanakaran S, Yoon H, Theiler J et al. Tracking changes in SARS-CoV-2 spike: evidence that D614G increases infectivity of the COVID-19 virus. Cell 2020 [CrossRef] [PubMed]
    [Google Scholar]
  54. Zhang L, Jackson CB, Mou H, Ojha A, Rangarajan ES et al. The D614G mutation in the SARS-CoV-2 spike protein reduces S1 shedding and increases infectivity. bioRxiv 2020 2020.06.12.148726 [CrossRef] [PubMed]
    [Google Scholar]
  55. Andersen KG, Rambaut A, Lipkin WI, Holmes EC, Garry RF. The proximal origin of SARS-CoV-2. Nat Med 2020; 26: 450 452 [CrossRef] [PubMed]
    [Google Scholar]
  56. Heidner HW, McKnight KL, Davis NL, Johnston RE. Lethality of PE2 incorporation into Sindbis virus can be suppressed by second-site mutations in E3 and E2. J Virol 1994; 68: 2683 2692 [CrossRef] [PubMed]
    [Google Scholar]
  57. Cavallo L, Oliva R. D936Y and other mutations in the fusion core of the SARS-Cov-2 spike protein heptad repeat 1 undermine the Post-Fusion assembly. bioRxiv 2020
    [Google Scholar]
  58. Case JB, Rothlauf PW, Chen RE, Liu Z, Zhao H et al. Neutralizing antibody and soluble ACE2 inhibition of a replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2. eCell Host and Microb 2020; in press:
    [Google Scholar]
  59. Ryman KD, Gardner CL, Burke CW, Meier KC, Thompson JM et al. Heparan sulfate binding can contribute to the neurovirulence of neuroadapted and nonneuroadapted Sindbis viruses. J Virol 2007; 81: 3563 3573 [CrossRef] [PubMed]
    [Google Scholar]
  60. Bernard KA, Klimstra WB, Johnston RE. Mutations in the E2 glycoprotein of Venezuelan equine encephalitis virus confer heparan sulfate interaction, low morbidity, and rapid clearance from blood of mice. Virology 2000; 276: 93 103 [CrossRef] [PubMed]
    [Google Scholar]
  61. Smit JM, Waarts B-L, Kimata K, Klimstra WB, Bittman R et al. Adaptation of alphaviruses to heparan sulfate: interaction of Sindbis and Semliki forest viruses with liposomes containing lipid-conjugated heparin. J Virol 2002; 76: 10128 10137 [CrossRef] [PubMed]
    [Google Scholar]
  62. Klimstra WB, Ryman KD, Bernard KA, Nguyen KB, Biron CA et al. Infection of neonatal mice with Sindbis virus results in a systemic inflammatory response syndrome. J Virol 1999; 73: 10387 10398 [CrossRef] [PubMed]
    [Google Scholar]
  63. Gardner CL, Hritz J, Sun C, Vanlandingham DL, Song TY et al. Deliberate attenuation of Chikungunya virus by adaptation to heparan sulfate-dependent infectivity: a model for rational arboviral vaccine design. PLoS Negl Trop Dis 2014; 8: e2719 [CrossRef] [PubMed]
    [Google Scholar]
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