Development of Mouse Hepatitis Virus and SARS-CoV Infectious cDNA Constructs

  • R. S. Baric
  • A. C. Sims
Part of the Current Topics in Microbiology and Immunology book series (CT MICROBIOLOGY, volume 287)


The genomes of transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV) have been generated with a novel construction strategy that allows for the assembly of very large RNA and DNA genomes from a panel of contiguous cDNA subclones. Recombinant viruses generated from these methods contained the appropriate marker mutations and replicated as efficiently as wild-type virus. The MHV cloning strategy can also be used to generate recombinant viruses that contain foreign genes or mutations at virtually any given nucleotide. MHV molecular viruses were engineered to express green fluorescent protein (GFP), demonstrating the feasibility of the systematic assembly approach to create recombinant viruses expressing foreign genes. The systematic assembly approach was used to develop an infectious clone of the newly identified human coronavirus, the serve acute respiratory syndrome virus (SARS-CoV). Our cloning and assembly strategy generated an infectious clone within 2 months of identification of the causative agent of SARS, providing a critical tool to study coronavirus pathogenesis and replication. The availability of coronavirus infectious cDNAs heralds a new era in coronavirus genetics and genomic applications, especially within the replicase proteins whose functions in replication and pathogenesis are virtually unknown.


Recombinant Virus Infectious Bronchitis Virus Severe Acute Respiratory Syndrome Infectious Clone Mouse Hepatitis Virus 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. Almazán F, González JM, Pénzes Z, Izeta A, Calvo E, Plana-Durán J, Enjuanes L (2000) Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci USA 97:5516–5521Google Scholar
  2. Alonso S, Sola I, Teifke J, Reimann I, Izeta A, Balach M, Plana-Durán J, Moormann RJM, Enjuanes L (2002) In vitro and in vivo expression of foreign genes by transmissible gastroenteritis coronavirus-derived minigenomes. J Gen Virol 83:567–579PubMedGoogle Scholar
  3. Ballesteros ML, Sánchez CM, Enjuanes L (1997) Two amino acid changes at the N-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism. Virology 227:378–388CrossRefPubMedGoogle Scholar
  4. Bonilla PJ, Gorbalenya AE, Weiss SR (1994) Mouse hepatitis virus strain A59 RNA polymerase gene ORF 1a: heterogeneity among MHV strains. Virology 198:736–740CrossRefPubMedGoogle Scholar
  5. Boyer JC, Haenni AL (1994) Infectious transcripts and cDNA clones of RNA viruses. Virology 198:415–426CrossRefPubMedGoogle Scholar
  6. Casais R, Thiel V, Siddell SG, Cavanagh D, Britton P (2001) Reverse genetics system for the avian coronavirus infectious bronchitis virus. J Virol 75:12359–12369CrossRefPubMedGoogle Scholar
  7. Cavanagh D, Brian DA, Britton P, Enjuanes L, Horzinek MC, Lai MMC, Laude H, Plagemann PGW, Siddell S, Spaan W, Talbot PJ (1997) Nidovirales: a new order comprising Coronaviridae and Arteriviridae. Arch Virol 142:629–635Google Scholar
  8. Cho MK, Magnus D, Caplan AL, McGee D, Ethics of Genomics Group (1999) GENETICS:Ethical Considerations in Synthesizing a Minimal Genome. Science 286:2087–2090CrossRefPubMedGoogle Scholar
  9. Curtis KM, Yount B, Baric RS (2002) Heterologous gene expression from transmissible gastroenteritis virus replicon particles. J Virol 76:1422–1434PubMedGoogle Scholar
  10. Delmas B, Gelfi J, L'Haridon R, Vogel LK, Norén O, Laude H (1992) Aminopeptidase N is a major receptor for the enteropathogenic coronavirus TGEV. Nature 357:417–420CrossRefPubMedGoogle Scholar
  11. de Vries AAF, Horzinek MC, Rottier PJM, de Groot RJ (1997) The genome organization of the Nidovirales: similarities and differences between arteri-, toro-, and coronaviruses. Semin Virol 8:33–47CrossRefGoogle Scholar
  12. Drosten C, Günther S, Preiser W, van der Werf S, Brodt H-R, Becker S, Rabenau H, Panning M, Kolesnikova L, Fouchier RAM, Berger A, Burguiere A-M, Cinatl J, Eickmann M, Escriou N, Grywna K, Kramme S, Manuguerra J-C, Muller S, Rickerts W, Sturmer MV, S., Klenk H-D, Osterhaus ADME (2003) Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 348:1967–1976CrossRefPubMedGoogle Scholar
  13. Eleouet JF, Rasschaert D, Lambert P, Levy L, Vende P, Laude H (1995) Complete sequence (20 kilobasee not been fully characterized. The structure and function of the ∼20-kb MHV replicase domain will likely remain a fertile area of research for the next decade and reveal novel protein functions that participate and regulate discontinuous transcription and high-frequency RNA recombination. Although large panels of reagents are available for analyzing replicase protein expression, processing, and subcellular localization, a spectrum of genetically informative mutations have not been systematically targeted to any of these replicase proteins. Given the complexity and size of the coronavirus replicase gene, the number of potential mutants that can be generated is enormous and will likely require bioinformatic approaches for building and testing specific hypotheses. For example, the ORF1a C-terminal MHV p15 protein is highly conserved among group I through III coronaviruses and contains a large number of conserved cysteine residues and predicted phosphorylation, myristylation, and glycosylation sites (prosite, spect of coronavirus transcription. J Virol 71:5148–5160Google Scholar
  14. Fu K, Baric RS (1994) Map locations of mouse hepatitis virus temperature-sensitive mutants: confirmation of variable rates of recombination. J Virol 68:7458–7466PubMedGoogle Scholar
  15. Fu KS, Baric RS (1992) Evidence for variable rates of recombination in the MHV genome. Virology 189:88–102CrossRefPubMedGoogle Scholar
  16. Grimes B, Cooke H (1998) Engineering mammalian chromosomes. Hum Mol Genet 7:1635–1640CrossRefPubMedGoogle Scholar
  17. Hsue B, Masters PS (1999) Insertion of a new transcriptional unit into the genome of mouse hepatitis virus. J Virol 73:6128–6135PubMedGoogle Scholar
  18. Hutchison CA III, Peterson SN, Gill SR, Cline RT, White O, Fraser CM, Smith HO, Venter JC (1999) Global transposon mutagenesis and a minimal mycoplasma genome. Science 286:2165–2169CrossRefPubMedGoogle Scholar
  19. Izeta A, Smerdou C, Alonso S, Penzes Z, Méndez A, Plana-Durán J, Enjuanes L (1999) Replication and packaging of transmissible gastroenteritis coronavirus-derived synthetic minigenomes. J Virol 73:1535–1545PubMedGoogle Scholar
  20. Ksiazek TG, Erdman D, Goldsmith C, Zaki S, Peret T, Emery S, Tong S, Urbani C, Comer JA, Lim W, Rollin PE, Dowell S, Ling A-E, Humphrey C, Shieh W-J, Guarner J, Paddock CD, Rota P, Fields B, DeRisi J, Yang J-Y, Cox N, Hughes J, LeDuc JW, Bellini WJ, Anderson LJ (2003) A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 348:1953–1966CrossRefPubMedGoogle Scholar
  21. Kuo L, Godeke G-J, Raamsman MJB, Masters PS, Rottier PJM (2000) Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier. J Virol 74:1393–1406CrossRefPubMedGoogle Scholar
  22. Lai MMC, Cavanagh D (1997) The molecular biology of coronaviruses. Adv Virus Res 48:1–100CrossRefPubMedGoogle Scholar
  23. Laude H, Rasschaert D, Delmas B, Godet M, Gelfi J, Bernard C (1990) Molecular biology of transmissible gastroenteritis virus. Vet Microbiol 23:147–154CrossRefPubMedGoogle Scholar
  24. Lee HJ, Shieh CK, Gorbalenya AE, Koonin EV, Lamonica N, Tuler J, Bagdzhadzhyan A, Lai MMC (1991) The complete sequence (22 kilobases) of murine coronavirus gene-1 encoding the putative proteases and RNA polymerase. Virology 180:567–582CrossRefPubMedGoogle Scholar
  25. Leparc-Goffart I, Hingley ST, Chua MM, Phillips J, Lavi E, Weiss SR (1998) Targeted recombination within the spike gene of murine coronavirus mouse hepatitis virus-A59: Q159 is a determinant of hepatotropism. J Virol 72:9628–9636PubMedGoogle Scholar
  26. Masters PS (1999) Reverse genetics of the largest RNA viruses. Adv Virus Res 53:245–264PubMedGoogle Scholar
  27. McGoldrick A, Lowings JP, Paton DJ (1999) Characterisation of a recent virulent transmissible gastroenteritis virus from Britain with a deleted ORF 3a. Arch Virol 144:763–770CrossRefPubMedGoogle Scholar
  28. Narayanan K, Makino S (2001) Cooperation of an RNA packaging signal and a viral envelope protein in coronavirus RNA packaging. J Virol 75:9059–9067CrossRefPubMedGoogle Scholar
  29. Ng LFP, Liu DX (2002) Membrane association and dimerization of a cysteine-rich, 16-kilodalton polypeptide released from the C-terminal region of the coronavirus infectious bronchitis virus 1a polyprotein. J Virol 76:6257–6267CrossRefPubMedGoogle Scholar
  30. Penzes Z, González JM, Calvo E, Izeta A, Smerdou C, Mendez A, Sánchez CM, Sola I, Almazán F, Enjuanes L (2001) Complete genome sequence of transmissible gastroenteritis coronavirus PUR46-MAD clone and evolution of the Purdue virus cluster. Virus Genes 23:105–118CrossRefPubMedGoogle Scholar
  31. Peters CJ, Sanchez A, Rollin PE, Ksiazek TG, Murphy FA (1996) Filoviridae: Marburg and Ebola Viruses. In: Fields BN, Knipe DM, Howley PM, Chanock RM, Melnick JL, Monath TP, Roizman B and Straus SE (eds) Field's Virology. Lippincott Williams and Wilkens, Philadelphia, pp 1161–1176Google Scholar
  32. Pingoud A, Jeltsch A (2001) Structure and function of type II restriction endonucleases. Nucl Acids Res 29:3705–3727CrossRefPubMedGoogle Scholar
  33. Repass JF, Makino S (1998) Importance of the positive-strand RNA secondary structure of a murine coronavirus defective interfering RNA internal replication signal in positive-strand RNA synthesis. J Virol 72:7926–7933PubMedGoogle Scholar
  34. Rice CM, Grakoui A, Galler R, Chambers TJ (1989) Transcription of infectious yellow fever RNA from full-length cDNA templates produced by in vitro ligation. New Biol 1:285–296PubMedGoogle Scholar
  35. Risco C, Antón IM, Enjuanes L, Carrascosa JL (1996) The transmissible gastroenteritis coronavirus contains a spherical core shell consisting of M and N proteins. J Virol 70:4773–4777PubMedGoogle Scholar
  36. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  37. Sánchez CM, Gebauer F, Suñé C, Méndez A, Dopazo J, Enjuanes L (1992) Genetic evolution and tropism of transmissible gastroenteritis coronaviruses. Virology 190:92–105CrossRefPubMedGoogle Scholar
  38. Sánchez CM, Izeta A, Sánchez-Morgado JM, Alonso S, Sola I, Balasch M, Plana-Durán J, Enjuanes L (1999) Targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence. J Virol 73:7607–7618PubMedGoogle Scholar
  39. Schaad M, Baric RS (1994) Genetics of mouse hepatitis virus transcription: evidence that subgenomic negative strands are functional templates. J Virol 68:8169–8179PubMedGoogle Scholar
  40. Siddell SG (1995) The Coronaviridae: an introduction. In: Siddell SG (ed) The Coronaviridae. Plenum Press, New York The Viruses, pp 1–10Google Scholar
  41. Siddell SG, Sawicki D, Meyer Y, Thiel V, Sawicki S (2001) Identification of the mutations responsible for the phenotype of three MHV RNA-negative ts mutants. Adv Exp Med Biol 494:453–458PubMedGoogle Scholar
  42. Smith GA, Enquist LW (2000) A self-recombining bacterial artificial chromosome and its application for analysis of herpesvirus pathogenesis. Proc Natl Acad Sci USA 97:4873–4878CrossRefPubMedGoogle Scholar
  43. Stalcup RP, Baric RS, Leibowitz JL (1998) Genetic complementation among three panels of mouse hepatitis virus gene 1 mutants. Virology 241:112–121CrossRefPubMedGoogle Scholar
  44. Thiel V, Herold J, Schelle B, Siddell SG (2001) Viral replicase gene products suffice for coronavirus discontinuous transcription. J Virol 75:6676–6681CrossRefPubMedGoogle Scholar
  45. Tresnan DB, Levis R, Holmes KV (1996) Feline aminopeptidase N serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup I. J Virol 70:8669–8674PubMedGoogle Scholar
  46. van Zijl M, Quint W, Briaire J, de Rover T, Gielkens A, Berns A (1988) Regeneration of herpesviruses from molecularly cloned subgenomic fragments. J Virol 62:2191–2195PubMedGoogle Scholar
  47. Wesley RD, Woods RD, Cheung AK (1991) Genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus. J Virol 65:3369–3373PubMedGoogle Scholar
  48. Williams GD, Chang R-Y, Brian DA (1999) A phylogenetically conserved hairpin-type 3′ untranslated region pseudoknot functions in coronavirus RNA replication. J Virol 73:8349–8355PubMedGoogle Scholar
  49. Yount B, Curtis KM, Baric RS (2000) Strategy for systematic assembly of large RNA and DNA genomes: the transmissible gastroenteritis virus model. J Virol 74:10600–10611CrossRefPubMedGoogle Scholar
  50. Yount B, Denison MR, Weiss SR, Baric RS (2002) Systematic assembly of a full length infectious cDNA of mouse hepatitis virus stain A59. J Virol 76:11065–11078CrossRefPubMedGoogle Scholar
  51. Yount B, Curtis KM, Fritz EA, Hensley LE, Jahrling PB, Prentice E, Denison MR, Geisbert TW, Baric RS (2003) Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus. Proc Natl Acad Sci USA 100:12995–13000CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • R. S. Baric
    • 1
  • A. C. Sims
    • 1
  1. 1.Department of EpidemiologyUniversity of North CarolinaChapel HillUSA

Personalised recommendations