Virus Genes

, Volume 35, Issue 3, pp 753–766 | Cite as

Comparative sequence analysis of a highly oncogenic but horizontal spread-defective clone of Marek’s disease virus

  • Stephen J. SpatzEmail author
  • Yuguang Zhao
  • Lawrence Petherbridge
  • Lorraine P. Smith
  • Susan J. Baigent
  • Venugopal Nair
Original Paper


Marek’s disease virus (MDV) is a cell-associated alphaherpesvirus that induces rapid-onset T-cell lymphomas in poultry. MDV isolates vary greatly in pathogenicity. While some of the strains such as CVI988 are non-pathogenic and are used as vaccines, others such as RB-1B are highly oncogenic. Molecular determinants associated with differences in pathogenicity are not completely understood. Comparison of the genome sequences of phenotypically different strains could help to identify molecular determinants of pathogenicity. We have previously reported the construction of bacterial artificial chromosome (BAC) clones of RB-1B from which fully infectious viruses could be reconstituted upon DNA transfection into chicken cells. MDV reconstituted from one of these clones (pRB-1B-5) showed similar in vitro and in vivo replication kinetics and oncogenicity as the parental virus. However, unlike the parental RB-1B virus, the BAC-derived virus showed inability to spread between birds. In order to identify the unique determinants for oncogenicity and the ‘‘non-spreading phenotype’’ of MDV derived from this clone, we determined the full-length sequence of pRB-1B-5. Comparative sequence analysis with the published sequences of strains such as Md5, Md11, and CVI988 identified frameshift mutations in RLORF1, protein kinase (UL13), and glycoproteins C (UL44) and D (US6). Comparison of the sequences of these genes with the parental virus indicated that the RLORF1, UL44, and US6 mutations were also present in the parental RB-1B stock of the virus. However with regard to UL13 mutation, the parental RB-1B stock appeared to be a mixture of wild type and mutant viruses, indicating that the BAC cloning has selected a mutant clone. Although further studies are needed to evaluate the role of these genes in the horizontal-spreading defective phenotype, our data clearly indicate that mutations in these genes do not affect the oncogenicity of MDV.


Marek’s disease Gallid herpesvirus type 2 Oncogenicity Shedding-defective Genome Sequence 



We thank Jeremy Volkening for his excellent technical assistance and skills in bioinformatics and graphics. The authors would like to thank Dr. Laszlo Zsak for critical evaluation of this report. Part of this work was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and the Department of Environment Food and Rural Affairs (DEFRA), United Kingdom; and the United States Department of Agriculture CRIS program (Project Number 6612-32000-043).


  1. 1.
    A.S. Anderson, M.S. Parcells, R.W. Morgan, J. Virol. 72, 2548–2553 (1998)PubMedPubMedCentralGoogle Scholar
  2. 2.
    S.J. Baigent, L.J. Petherbridge, K. Howes, L.P. Smith, R.J. Currie, V.K. Nair, J. Virol. Methods 123, 53–64 (2005)CrossRefGoogle Scholar
  3. 3.
    A.D. Barrow, S.C. Burgess, S.J. Baigent, K. Howes, V.K. Nair, J. Gen. Virol. 84, 2635–2645 (2003)CrossRefGoogle Scholar
  4. 4.
    J. Besemer, M. Borodovsky, Nucleic Acids Res. 33, W451–W454 (2005)CrossRefGoogle Scholar
  5. 5.
    C. Blondeau, N. Chbab, C. Beaumont, K. Courvoisier, N. Osterrieder, J.-F. Vautherot, C. Denesvre, Vet. Res. 38, 419–433 (2007)CrossRefGoogle Scholar
  6. 6.
    A.C. Brown, S.J. Baigent, L.P. Smith, J.P. Chattoo, L.J. Petherbridge, P. Hawes, M.J. Allday, V. Nair, Proc. Natl. Acad. Sci. USA 103, 1687–1692 (2006)CrossRefGoogle Scholar
  7. 7.
    P. Brunovskis, L.F. Velicer, Virology 206, 324–338 (1995)CrossRefGoogle Scholar
  8. 8.
    B.W. Calnek, Crit. Rev. Microbiol. 12, 293–320 (1986)CrossRefGoogle Scholar
  9. 9.
    B.W. Calnek, in Curr Top Microbiol Immunol, vol. 255, ed. by K. Hirai (Springer-Verlag, Berlin, 2001) pp. 25–55Google Scholar
  10. 10.
    J.H. Chi, C.A. Harley, A. Mukhopadhyay, D.W. Wilson, J. Gen. Virol. 86, 253–261 (2005)CrossRefGoogle Scholar
  11. 11.
    F. Corpet, Nucleic Acids Res. 16, 10881–10890 (1988)CrossRefGoogle Scholar
  12. 12.
    P.M. Coussens, L.F. Velicer, J. Virol. 62, 2373–2379 (1988)PubMedPubMedCentralGoogle Scholar
  13. 13.
    F. Dorange, B.K. Tischer, J.F. Vautherot, N. Osterrieder, J. Virol. 76, 1959–1970 (2002)CrossRefGoogle Scholar
  14. 14.
    L. Fragnet, M.A. Blasco, W. Klapper, D. Rasschaert, J. Virol. 77, 5985–5996 (2003)CrossRefGoogle Scholar
  15. 15.
    W. Fuchs, B.G. Klupp, H. Granzow, C. Hengartner, A. Brack, A. Mundt, L.W. Enquist, T.C. Mettenleiter, J. Virol. 76, 8208–8217 (2002)CrossRefGoogle Scholar
  16. 16.
    H. Hu, J.I. Cohen, Virology 337, 304–311 (2005)CrossRefGoogle Scholar
  17. 17.
    Y. Izumiya, H.K. Jang, M. Sugawara, Y. Ikeda, R. Miura, Y. Nishimura, K. Nakamura, T. Miyazawa, C. Kai, T. Mikami, J. Gen. Virol. 80(Pt 9), 2417–2422 (1999)CrossRefGoogle Scholar
  18. 18.
    K.W. Jarosinski, N. Osterrieder, V.K. Nair, K.A. Schat, J. Virol. 79, 11647–11659 (2005)CrossRefGoogle Scholar
  19. 19.
    K.W. Jarosinski, K.A. Schat, Virus Genes 34, 9–22 (2007)CrossRefGoogle Scholar
  20. 20.
    D.C. Johnson, M.T. Huber, J. Virol. 76, 1–8 (2002)CrossRefGoogle Scholar
  21. 21.
    K. Katoh, K. Kuma, H. Toh, T. Miyata, Nucleic Acids Res. 33, 511–518 (2005)CrossRefGoogle Scholar
  22. 22.
    T.K. Kenyon, J.I. Cohen, C. Grose, J. Virol. 76, 10980–10993 (2002)CrossRefGoogle Scholar
  23. 23.
    B.G. Klupp, H. Granzow, G.M. Keil, T.C. Mettenleiter, J. Virol. 80, 6235–6246 (2006)CrossRefGoogle Scholar
  24. 24.
    M. Kozak, Nucleic Acids Res. 15, 8125–8148 (1987)CrossRefGoogle Scholar
  25. 25.
    S. Kumar, K. Tamura, M. Nei, Brief Bioinform. 5, 150–163 (2004)CrossRefGoogle Scholar
  26. 26.
    L.F. Lee, P. Wu, D. Sui, D. Ren, J. Kamil, H.J. Kung, R.L. Witter, Proc. Natl. Acad. Sci. USA 97, 6091–6096 (2000)CrossRefGoogle Scholar
  27. 27.
    H.C. Liu, E.J. Soderblom, M.B. Goshe, J. Virol. Methods 135, 66–75 (2006)CrossRefGoogle Scholar
  28. 28.
    K. Maotani, A. Kanamori, K. Ikuta, S. Ueda, S. Kato, K. Hirai, J. Virol. 58, 657–660 (1986)PubMedPubMedCentralGoogle Scholar
  29. 29.
    T.C. Mettenleiter, J. Virol. 76, 1537–1547 (2002)CrossRefGoogle Scholar
  30. 30.
    T.C. Mettenleiter, Vet. Microbiol. 113, 163–169 (2006)CrossRefGoogle Scholar
  31. 31.
    J.F. Moffat, L. Zerboni, P.R. Kinchington, C. Grose, H. Kaneshima, A.M. Arvin, J. Virol. 72, 965–974 (1998)PubMedPubMedCentralGoogle Scholar
  32. 32.
    J.F. Moffat, L. Zerboni, M.H. Sommer, T.C. Heineman, J.I. Cohen, H. Kaneshima, A.M. Arvin, Proc. Natl. Acad. Sci. USA 95, 11969–11974 (1998)CrossRefGoogle Scholar
  33. 33.
    R.W. Morgan, A. Anderson, J. Kent, M.S. Parcells, in Current Research on Marek’s Disease, ed. by R.F. Silva, H.H. Cheng, P.M. Coussens (American Association of Avian Pathologists, Kennett Square, Pennsylvania, 1996), pp. 207–212Google Scholar
  34. 34.
    M. Niikura, J. Dodgson, H. Cheng, Arch. Virol. 151, 537–549 (2005)CrossRefGoogle Scholar
  35. 35.
    M. Niikura, H.C. Liu, J.B. Dodgson, H.H. Cheng, Poult. Sci. 83, 1117–1123 (2004)CrossRefGoogle Scholar
  36. 36.
    M. Niikura, R.L. Witter, H.K. Jang, M. Ono, T. Mikami, R.F. Silva, Acta Virol. 43, 159–163 (1999)PubMedGoogle Scholar
  37. 37.
    Y. Nishiyama, Rev. Med. Virol. 14, 33–46 (2004)CrossRefGoogle Scholar
  38. 38.
    K.J. O’Regan, M.A. Bucks, M.A. Murphy, J.W. Wills, R.J. Courtney, Virology 358, 192–200 (2007)CrossRefGoogle Scholar
  39. 39.
    K. Ohashi, P.H. O’Connell, K.A. Schat, Virology 199, 275–283 (1994)CrossRefGoogle Scholar
  40. 40.
    N. Osterrieder, Virus Res. 59, 165–177 (1999)CrossRefGoogle Scholar
  41. 41.
    N. Osterrieder, J.P. Kamil, D. Schumacher, B.K. Tischer, S. Trapp, Nat. Rev. Microbiol. 4, 283–294 (2006)CrossRefGoogle Scholar
  42. 42.
    M.S. Parcells, A.S. Anderson, R.W. Morgan, Virus Genes 9, 5–13 (1994)CrossRefGoogle Scholar
  43. 43.
    L. Petherbridge, A.C. Brown, S.J. Baigent, K. Howes, M.A. Sacco, N. Osterrieder, V.K. Nair, J. Virol. 78, 13376–13380 (2004)CrossRefGoogle Scholar
  44. 44.
    S.M. Reddy, D. Sui, P. Wu, L. Lee, Acta Virol. 43, 174–180 (1999)PubMedGoogle Scholar
  45. 45.
    F.A. Rey, EMBO Rep. 7, 1000–1005 (2006)CrossRefGoogle Scholar
  46. 46.
    B.J. Ryckman, R.J. Roller, J. Virol. 78, 399–412 (2004)CrossRefGoogle Scholar
  47. 47.
    A.A. Schaffer, L. Aravind, T.L. Madden, S. Shavirin, J.L. Spouge, Y.I. Wolf, E.V. Koonin, S.F. Altschul, Nucleic Acids Res. 29, 2994–3005 (2001)CrossRefGoogle Scholar
  48. 48.
    K.A. Schat, Cancer Surv. 6, 1–37 (1987)PubMedGoogle Scholar
  49. 49.
    K.A. Schat, B.W. Calnek, J. Fabricant, Avian Pathol. 11, 593–605 (1982)CrossRefGoogle Scholar
  50. 50.
    D. Schumacher, B.K. Tischer, W. Fuchs, N. Osterrieder, J. Virol. 74, 11088–11098 (2000)CrossRefGoogle Scholar
  51. 51.
    D. Schumacher, B.K. Tischer, S.M. Reddy, N. Osterrieder, J. Virol. 75, 11307–11318 (2001)CrossRefGoogle Scholar
  52. 52.
    D. Schumacher, B.K. Tischer, S. Trapp, N. Osterrieder, J. Virol. 79, 3987–3997 (2005)CrossRefGoogle Scholar
  53. 53.
    B. Silva, L.F. Lee, M. Heidari, I. Gimeno, S. Spatz, J. Zelepsky, in 4th International Workshop on the Molecular Pathogenesis of Marek’s Disease Virus, ed. by M.S. Parcells, R.W. Morgan, J. Burnside, C.J. Schmidt, E.L. Bernberg, C. Kenny, E. Eppler (University of Delaware, Newark, Delaware, 2006), p. 28Google Scholar
  54. 54.
    S.J. Spatz, L. Petherbridge, Y. Zhao, V. Nair, J. Gen. Virol. 88, 1080–1096 (2007)CrossRefGoogle Scholar
  55. 55.
    S.J. Spatz, R.F. Silva, Virus Genes 35, 41–53 (2006)CrossRefGoogle Scholar
  56. 56.
    R. Staden, K.F. Beal, J.K. Bonfield, Methods Mol. Biol. 132, 115–130 (2000)PubMedGoogle Scholar
  57. 57.
    B.K. Tischer, D. Schumacher, D. Chabanne-Vautherot, V. Zelnik, J.F. Vautherot, N. Osterrieder, J. Virol. 79, 5889–5899 (2005)CrossRefGoogle Scholar
  58. 58.
    B.K. Tischer, D. Schumacher, M. Messerle, M. Wagner, N. Osterrieder, J. Gen. Virol. 83, 997–1003 (2002)CrossRefGoogle Scholar
  59. 59.
    S. Trapp, M.S. Parcells, J.P. Kamil, D. Schumacher, B.K. Tischer, P.M. Kumar, V.K. Nair, N. Osterrieder, J. Exp. Med. 203, 1307–1317 (2006)CrossRefGoogle Scholar
  60. 60.
    E.R. Tulman, C.L. Afonso, Z. Lu, L. Zsak, D.L. Rock, G.F. Kutish, J. Virol. 74, 7980–7988 (2000)CrossRefGoogle Scholar
  61. 61.
    K. Venugopal, Res. Vet. Sci. 69, 17–23 (2000)CrossRefGoogle Scholar
  62. 62.
    V. Vittone, E. Diefenbach, D. Triffett, M.W. Douglas, A.L. Cunningham, R.J. Diefenbach, J. Virol. 79, 9566–9571 (2005)CrossRefGoogle Scholar
  63. 63.
    F. Wagenaar, J.M. Pol, B. Peeters, A.L. Gielkens, N. de Wind, T.G. Kimman, J. Gen. Virol. 76(Pt 7), 1851–1859 (1995)CrossRefGoogle Scholar
  64. 64.
    M. Wagner, Z. Ruzsics, U.H. Koszinowski, Trends Microbiol. 10, 318–324 (2002)CrossRefGoogle Scholar
  65. 65.
    H. Yamada, Y.M. Jiang, S. Oshima, T. Daikoku, Y. Yamashita, T. Tsurumi, Y. Nishiyama, J. Gen. Virol. 79(Pt 8), 1989–1995 (1998)CrossRefGoogle Scholar
  66. 66.
    V. Zelnik, V. Majerciak, D. Szabova, H. Geerligs, J. Kopacek, L.J. Ross, J. Pastorek, Acta Virol. 43, 164–168 (1999)PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Stephen J. Spatz
    • 1
    Email author
  • Yuguang Zhao
    • 2
  • Lawrence Petherbridge
    • 2
  • Lorraine P. Smith
    • 2
  • Susan J. Baigent
    • 2
  • Venugopal Nair
    • 2
  1. 1.Southeast Poultry Research Laboratory, Agricultural Research Service, United States Department of AgricultureAthensUSA
  2. 2.Institute for Animal Health, Compton LaboratoryComptonUK

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