title: A safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERS-CoV.
abstract: BACKGROUND
  Evidence points to the emergence of a novel human coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), which causes a severe acute respiratory syndrome (SARS)-like disease. In response, the development of effective vaccines and therapeutics remains a clinical priority. To accomplish this, it is necessary to evaluate neutralizing antibodies and screen for MERS-CoV entry inhibitors.
  
  
  METHODS
  In this study, we produced a pseudovirus bearing the full-length spike (S) protein of MERS-CoV in the Env-defective, luciferase-expressing HIV-1 backbone. We then established a pseudovirus-based inhibition assay to detect neutralizing antibodies and anti-MERS-CoV entry inhibitors.
  
  
  RESULTS
  Our results demonstrated that the generated MERS-CoV pseudovirus allows for single-cycle infection of a variety of cells expressing dipeptidyl peptidase-4 (DPP4), the confirmed receptor for MERS-CoV. Consistent with the results from a live MERS-CoV-based inhibition assay, the antisera of mice vaccinated with a recombinant protein containing receptor-binding domain (RBD, residues 377-662) of MERS-CoV S fused with Fc of human IgG exhibited neutralizing antibody response against infection of MERS-CoV pseudovirus. Furthermore, one small molecule HIV entry inhibitor targeting gp41 (ADS-J1) and the 3-hydroxyphthalic anhydride-modified human serum albumin (HP-HSA) could significantly inhibit MERS-CoV pseudovirus infection.
  
  
  CONCLUSIONS
  Taken together, the established MERS-CoV inhibition assay is a safe and convenient pseudovirus-based alternative to BSL-3 live-virus restrictions and can be used to rapidly screen MERS-CoV entry inhibitors, as well as evaluate vaccine-induced neutralizing antibodies against the highly pathogenic MERS-CoV.
authors: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.
authors: Guangyu
authors: G
authors: Zhao
authors: Lanying
authors: L
authors: Du
authors: Cuiqing
authors: C
authors: Ma
authors: Ye
authors: Y
authors: Li
authors: Lin
authors: L
authors: Li
authors: Vincent Km
authors: VK
authors: Poon
authors: Lili
authors: L
authors: Wang
authors: Fei
authors: F
authors: Yu
authors: Bo-Jian
authors: BJ
authors: Zheng
authors: Shibo
authors: S
authors: Jiang
authors: Yusen
authors: Y
authors: Zhou
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000302:isolation & purification*; D000914:Antibodies, Viral / Q000302:isolation & purification*; D000998:Antiviral Agents / Q000302:isolation & purification*; D002460:Cell Line; D017934:Coronavirus / Q000187:drug effects* / Q000276:immunology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D051379:Mice; D009500:Neutralization Tests / Q000379:methods*; D064370:Spike Glycoprotein, Coronavirus / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000998:Antiviral Agents; D064370:Spike Glycoprotein, Coronavirus; D008156:Luciferases
references: J Virol. 2004 Jun;78(11):5642-50
references: 15140961
references: N Engl J Med. 2013 Jun 27;368(26):2487-94
references: 23718156
references: Emerg Infect Dis. 2005 Mar;11(3):411-6
references: 15757556
references: J Antimicrob Chemother. 2013 Mar;68(3):573-6
references: 23221626
references: J Gen Virol. 2005 May;86(Pt 5):1435-1440
references: 15831955
references: J Virol. 2013 Aug;87(16):9379-83
references: 23785207
references: mBio. 2012 Nov 20;3(6):
references: 23170002
references: Retrovirology. 2010 Apr 26;7:37
references: 20420669
references: J Infect Dis. 2013 Jun 1;207(11):1743-52
references: 23532101
references: J Infect. 2012 Dec;65(6):477-89
references: 23072791
references: N Engl J Med. 2012 Nov 8;367(19):1814-20
references: 23075143
references: Nat Rev Microbiol. 2009 Mar;7(3):226-36
references: 19198616
references: Nature. 2003 Nov 27;426(6965):450-4
references: 14647384
references: Int J Infect Dis. 2013 Feb;17(2):e143-4
references: 23270612
references: J Med Chem. 1999 Aug 26;42(17):3203-9
references: 10464007
references: J Virol. 2013 Sep;87(17):9939-42
references: 23824801
references: Proc Natl Acad Sci U S A. 2004 Nov 2;101(44):15748-53
references: 15496474
references: Microbes Infect. 2013 Jun;15(6-7):506-10
references: 23518265
references: Nature. 2013 Mar 14;495(7440):251-4
references: 23486063
references: J Cell Biochem. 2008 Aug 15;104(6):2335-47
references: 18442051
references: PLoS One. 2013;8(1):e53568
references: 23320093
references: Virology. 2009 Oct 10;393(1):144-50
references: 19683779
references: J Gen Virol. 2004 Oct;85(Pt 10):3109-3113
references: 15448374
references: Biochem Biophys Res Commun. 2009 Jul 10;384(4):486-90
references: 19422787
references: J Biol Chem. 2007 Mar 30;282(13):9612-9620
references: 17276993
references: J Immunol. 2008 Jan 15;180(2):948-56
references: 18178835
references: J Virol. 2013 May;87(10):5502-11
references: 23468491
references: Biochem Biophys Res Commun. 2010 Jul 2;397(3):580-5
references: 20617558
references: Antimicrob Agents Chemother. 2009 Dec;53(12):4987-98
references: 19786602