1887

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is a high-priority pathogen in pandemic preparedness research. Reverse genetics systems are a valuable tool to study viral replication and pathogenesis, design attenuated vaccines and create defined viral assay systems for applications such as antiviral screening. Here we present a novel reverse genetics system for MERS-CoV that involves maintenance of the full-length viral genome as a cDNA copy inserted in a bacterial artificial chromosome amenable to manipulation by homologue recombination, based on the bacteriophage λ Red recombination system. Based on a full-length infectious MERS-CoV cDNA clone, optimal genomic insertion sites and expression strategies for GFP were identified and used to generate a reporter MERS-CoV expressing GFP in addition to the complete set of viral proteins. GFP was genetically fused to the N-terminal part of protein 4a, from which it is released during translation via porcine teschovirus 2A peptide activity. The resulting reporter virus achieved titres nearly identical to the wild-type virus 48 h after infection of Vero cells at m.o.i. 0.001 (1×10 p.f.u. ml and 3×10 p.f.u. ml, respectively), and allowed determination of the 50 % inhibitory concentration for the known MERS-CoV inhibitor cyclosporine A based on fluorescence readout. The resulting value was 2.41 µM, which corresponds to values based on wild-type virus. The reverse genetics system described herein can be efficiently mutated by Red-mediated recombination. The GFP-expressing reporter virus contains the full set of MERS-CoV proteins and achieves wild-type titres in cell culture.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.000919
2017-10-01
2020-01-26
Loading full text...

Full text loading...

/deliver/fulltext/jgv/98/10/2461.html?itemId=/content/journal/jgv/10.1099/jgv.0.000919&mimeType=html&fmt=ahah

References

  1. Sweileh WM. Global research trends of World Health Organization's top eight emerging pathogens. Global Health 2017;13:9 [CrossRef][PubMed]
    [Google Scholar]
  2. World Health Organization www.who.int/emergencies/mers-cov/en/ accessed 24 April 2017
  3. Bermingham A, Chand MA, Brown CS, Aarons E, Tong C et al. Severe respiratory illness caused by a novel coronavirus, in a patient transferred to the United Kingdom from the Middle East, September 2012. Euro Surveill 2012;17:20290[PubMed]
    [Google Scholar]
  4. Wernery U, Corman VM, Wong EY, Tsang AK, Muth D et al. Acute middle East respiratory syndrome coronavirus infection in livestock Dromedaries, Dubai, 2014. Emerg Infect Dis 2015;21:1019–1022 [CrossRef][PubMed]
    [Google Scholar]
  5. Alagaili AN, Briese T, Mishra N, Kapoor V, Sameroff SC et al. Middle East respiratory syndrome coronavirus infection in dromedary camels in Saudi Arabia. MBio 2014;5:e00884-14 [CrossRef][PubMed]
    [Google Scholar]
  6. Farag EA, Reusken CB, Haagmans BL, Mohran KA, Stalin Raj V, Raj S V et al. High proportion of MERS-CoV shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases, Qatar 2014. Infect Ecol Epidemiol 2015;5:28305 [CrossRef][PubMed]
    [Google Scholar]
  7. Corman VM, Jores J, Meyer B, Younan M, Liljander A et al. Antibodies against MERS coronavirus in dromedary camels, Kenya, 1992-2013. Emerg Infect Dis 2014;20:1319–1322 [CrossRef][PubMed]
    [Google Scholar]
  8. Müller MA, Corman VM, Jores J, Meyer B, Younan M et al. MERS coronavirus neutralizing antibodies in camels, Eastern Africa, 1983–1997. Emerg Infect Dis 2014;20:2093–2095 [CrossRef][PubMed]
    [Google Scholar]
  9. Breban R, Riou J, Fontanet A. Interhuman transmissibility of Middle East respiratory syndrome coronavirus: estimation of pandemic risk. Lancet 2013;382:694–699 [CrossRef][PubMed]
    [Google Scholar]
  10. Chowell G, Abdirizak F, Lee S, Lee J, Jung E et al. Transmission characteristics of MERS and SARS in the healthcare setting: a comparative study. BMC Med 2015;13:210 [CrossRef][PubMed]
    [Google Scholar]
  11. Drosten C, Meyer B, Müller MA, Corman VM, Al-Masri M et al. Transmission of MERS-coronavirus in household contacts. N Engl J Med 2014;371:828–835 [CrossRef][PubMed]
    [Google Scholar]
  12. Korea Centers for Disease Control and Prevention Middle East respiratory syndrome coronavirus outbreak in the Republic of Korea, 2015. Osong Public Health Res Perspect 2015;6:269–278 [CrossRef][PubMed]
    [Google Scholar]
  13. Assiri A, McGeer A, Perl TM, Price CS, Al Rabeeah AA et al. Hospital outbreak of Middle East respiratory syndrome coronavirus. N Engl J Med 2013;369:407–416 [CrossRef][PubMed]
    [Google Scholar]
  14. Oboho IK, Tomczyk SM, Al-Asmari AM, Banjar AA, Al-Mugti H et al. 2014 MERS-CoV outbreak in Jeddah-a link to health care facilities. N Engl J Med 2015;372:846–854 [CrossRef][PubMed]
    [Google Scholar]
  15. Mo Y, Fisher D. A review of treatment modalities for Middle East respiratory syndrome. J Antimicrob Chemother 2016;71:3340–3350 [CrossRef]
    [Google Scholar]
  16. Perlman S, Vijay R. Middle East respiratory syndrome vaccines. Int J Infect Dis 2016;47:23–28 [CrossRef][PubMed]
    [Google Scholar]
  17. Niemeyer D, Zillinger T, Muth D, Zielecki F, Horvath G et al. Middle East respiratory syndrome coronavirus accessory protein 4a is a type I interferon antagonist. J Virol 2013;87:12489–12495 [CrossRef][PubMed]
    [Google Scholar]
  18. Rabouw HH, Langereis MA, Knaap RCM, Dalebout TJ, Canton J et al. Middle East Respiratory coronavirus accessory protein 4a inhibits PKR-mediated antiviral stress responses. PLoS Pathog 2016;12:e1005982 [CrossRef]
    [Google Scholar]
  19. Thornbrough JM, Jha BK, Yount B, Goldstein SA, Li Y et al. Middle East respiratory syndrome coronavirus NS4b protein inhibits host rnase l activation. MBio 2016;7:e00258 [CrossRef]
    [Google Scholar]
  20. Matthews KL, Coleman CM, van der Meer Y, Snijder EJ, Frieman MB. The ORF4b-encoded accessory proteins of Middle East respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling. J Gen Virol 2014;95:874–882 [CrossRef]
    [Google Scholar]
  21. Yang Y, Ye F, Zhu N, Wang W, Deng Y et al. Middle East respiratory syndrome coronavirus ORF4b protein inhibits type I interferon production through both cytoplasmic and nuclear targets. Sci Rep 2015;5:17554 [CrossRef]
    [Google Scholar]
  22. Yang Y, Zhang L, Geng H, Deng Y, Huang B et al. The structural and accessory proteins M, ORF 4a, ORF 4b, and ORF 5 of Middle East respiratory syndrome coronavirus (MERS-CoV) are potent interferon antagonists. Protein Cell 2013;4:951–961 [CrossRef]
    [Google Scholar]
  23. Pfefferle S, Krähling V, Ditt V, Grywna K, Mühlberger E et al. Reverse genetic characterization of the natural genomic deletion in SARS-Coronavirus strain Frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in vitro and in vivo. Virol J 2009;6:131 [CrossRef]
    [Google Scholar]
  24. Tischer BK, von Einem J, Kaufer B, Osterrieder N. Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. BioTechniques 2006;40:191–197
    [Google Scholar]
  25. Yount B, Curtis KM, Fritz EA, Hensley LE, Jahrling PB et al. Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus. Proc Natl Acad Sci USA 2003;100:12995–13000 [CrossRef][PubMed]
    [Google Scholar]
  26. Corman VM, Eckerle I, Bleicker T, Zaki A, Landt O et al. Detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction. Euro Surveill 2012;17:pii=20285[PubMed]
    [Google Scholar]
  27. Scobey T, Yount BL, Sims AC, Donaldson EF, Agnihothram SS et al. Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus. Proc Natl Acad Sci USA 2013;110:16157–16162 [CrossRef]
    [Google Scholar]
  28. de Felipe P. Skipping the co-expression problem: the new 2A "CHYSEL" technology. Genet Vaccines Ther 2004;2:13 [CrossRef]
    [Google Scholar]
  29. Van Boheemen S, de Graaf M, Lauber C, Bestebroer TM, Raj VS et al. Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans. MBio 2012;3:e00473-12 [CrossRef][PubMed]
    [Google Scholar]
  30. Pfefferle S, Schöpf J, Kögl M, Friedel CC, Müller MA et al. The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors. PLoS Pathog 2011;7:e1002331 [CrossRef][PubMed]
    [Google Scholar]
  31. de Wilde AH, Raj VS, Oudshoorn D, Bestebroer TM, van Nieuwkoop S et al. MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment. J Gen Virol 2013;94:1749–1760 [CrossRef][PubMed]
    [Google Scholar]
  32. Almazán F, Dediego ML, Sola I, Zuñiga S, Nieto-Torres JL et al. Engineering a replication-competent, propagation-defective Middle East respiratory syndrome coronavirus as a vaccine candidate. MBio 2013;4:e00650-13 [CrossRef][PubMed]
    [Google Scholar]
  33. Weiss SR, Leibowitz JL. Coronavirus pathogenesis. Adv Virus Res 2011;81:85–164 [CrossRef][PubMed]
    [Google Scholar]
  34. Weiss SR, Navas-Martin S. Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. Microbiol Mol Biol Rev 2005;69:635–664 [CrossRef][PubMed]
    [Google Scholar]
  35. Kim JH, Lee SR, Li LH, Park HJ, Park JH et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 2011;6:e18556 [CrossRef][PubMed]
    [Google Scholar]
  36. Leardkamolkarn V, Sirigulpanit W. Establishment of a stable cell line coexpressing dengue virus-2 and green fluorescent protein for screening of antiviral compounds. J Biomol Screen 2012;17:283–292 [CrossRef][PubMed]
    [Google Scholar]
  37. Poirier JT, Reddy PS, Idamakanti N, Li SS, Stump KL et al. Characterization of a full-length infectious cDNA clone and a GFP reporter derivative of the oncolytic picornavirus SVV-001. J Gen Virol 2012;93:2606–2613 [CrossRef]
    [Google Scholar]
  38. Thomas JM, Klimstra WB, Ryman KD, Heidner HW. Sindbis virus vectors designed to express a foreign protein as a cleavable component of the viral structural polyprotein. J Virol 2003;77:5598–5606 [CrossRef][PubMed]
    [Google Scholar]
  39. Mateos-Gomez PA, Morales L, Zuñiga S, Enjuanes L, Sola I. Long-distance RNA-RNA interactions in the coronavirus genome form high-order structures promoting discontinuous RNA synthesis during transcription. J Virol 2013;87:177–186 [CrossRef][PubMed]
    [Google Scholar]
  40. Sola I, Moreno JL, Zúñiga S, Alonso S, Enjuanes L. Role of nucleotides immediately flanking the transcription-regulating sequence core in coronavirus subgenomic mRNA synthesis. J Virol 2005;79:2506–2516 [CrossRef][PubMed]
    [Google Scholar]
  41. Dediego ML, Alvarez E, Almazán F, Rejas MT, Lamirande E et al. A severe acute respiratory syndrome coronavirus that lacks the E gene is attenuated in vitro and in vivo. J Virol 2007;81:1701–1713 [CrossRef][PubMed]
    [Google Scholar]
  42. Ortego J, Ceriani JE, Patiño C, Plana J, Enjuanes L. Absence of E protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway. Virology 2007;368:296–308 [CrossRef]
    [Google Scholar]
  43. Matrosovich M, Matrosovich T, Garten W, Klenk H-D. New low-viscosity overlay medium for viral plaque assays. Virol J 2006;3:63 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.000919
Loading
/content/journal/jgv/10.1099/jgv.0.000919
Loading

Data & Media loading...

Most cited articles

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error