Genetic homogeneity of a test population is essential to precisely associate a viral genome sequence and its phenotype at the nucleotide level. However, homogeneity is not easy to achieve for Marek's disease virus (MDV) due to its strictly cell-associated replication. To address this problem, two virulent infectious bacterial artificial chromosome (BAC) clones of MDV were generated from an MDV genome previously cloned as five overlapping cosmids. The Md5SN5BAC clone has the BAC vector inserted between the 3′ ends of and , such that no known ORFs should be disrupted. The BAC vector is flanked by P sites, so that it can be deleted from the viral genome by transfecting Md5SN5BAC into a newly developed chicken cell line that constitutively expresses Cre recombinase. The Md5B40BAC clone has the BAC vector replacing a portion of , a location similar to that used by other groups to construct MDV-BAC clones. Although both BACs were capable of producing infectious virulent MDV when inoculated into susceptible chickens, Md5B40BAC-derived viruses showed somewhat better replication and higher virulence. Removal of the BAC vector in Md5SN5BAC-derived viruses had no influence on virulence. Interestingly, when genetically homogeneous virulent MDV generated from Md5B40BAC was mixed with avirulent virus, the overall virulence of the mixed population was noticeably compromised, which emphasizes the importance of MDV population complexity in pathogenesis.


Article metrics loading...

Loading full text...

Full text loading...



  1. Baigent, S. J., Petherbridge, L. J., Smith, L. P., Zhao, Y., Chesters, P. M. & Nair, V. K.(2006). Herpesvirus of turkey reconstituted from bacterial artificial chromosome clones induces protection against Marek's disease. J Gen Virol 87, 769–776.[CrossRef] [Google Scholar]
  2. Calnek, B. W.(2001). Pathogenesis of Marek's disease virus infection. In Marek's Disease (Current Topics in Microbiology and Immunology vol. 255), pp. 57–90. Edited by Hirai, K.. Berlin, Heidelberg, New York. : Springer-Verlag. [Google Scholar]
  3. Cho, B. R.(1978). An improved method for extracting cell-free herpesviruses of Marek's disease and turkeys from infected cell cultures. Avian Dis 22, 170–176.[CrossRef] [Google Scholar]
  4. Cox, E., Reddy, S., Iofin, I. & Cohen, J. I.(1998). Varicella-zoster virus ORF57, unlike its pseudorabies virus UL3.5 homolog, is dispensable for viral replication in cell culture. Virology 250, 205–209.[CrossRef] [Google Scholar]
  5. Cui, X., Lee, L. F., Reed, W. M., Kung, H. J. & Reddy, S. M.(2004). Marek's disease virus-encoded vIL-8 gene is involved in early cytolytic infection but dispensable for establishment of latency. J Virol 78, 4753–4760.[CrossRef] [Google Scholar]
  6. Dargan, D. J., Douglas, E., Cunningham, C., Jamieson, F., Stanton, R. J., Baluchova, K., McSharry, B. P., Tomasec, P., Emery, V. C. & other authors(2010). Sequential mutations associated with adaptation of human cytomegalovirus to growth in cell culture. J Gen Virol 91, 1535–1546.[CrossRef] [Google Scholar]
  7. Dean, H. J. & Cheung, A. K.(1993). A 3′ coterminal gene cluster in pseudorabies virus contains herpes simplex virus UL1, UL2, and UL3 gene homologs and a unique UL3.5 open reading frame. J Virol 67, 5955–5961. [Google Scholar]
  8. Drake, J. W. & Hwang, C. B.(2005). On the mutation rate of herpes simplex virus type 1. Genetics 170, 969–970.[CrossRef] [Google Scholar]
  9. Drake, J. W., Charlesworth, B., Charlesworth, D. & Crow, J. F.(1998). Rates of spontaneous mutation. Genetics 148, 1667–1686. [Google Scholar]
  10. Ferrin, L. J. & Camerini-Otero, R. D.(1991). Selective cleavage of human DNA: RecA-assisted restriction endonuclease (RARE) cleavage. Science 254, 1494–1497.[CrossRef] [Google Scholar]
  11. Fuchs, W. & Mettenleiter, T. C.(1996). DNA sequence and transcriptional analysis of the UL1 to UL5 gene cluster of infectious laryngotracheitis virus. J Gen Virol 77, 2221–2229.[CrossRef] [Google Scholar]
  12. Fuchs, W., Klupp, B. G., Granzow, H., Rziha, H. J. & Mettenleiter, T. C.(1996). Identification and characterization of the pseudorabies virus UL3.5 protein, which is involved in virus egress. J Virol 70, 3517–3527. [Google Scholar]
  13. Fuchs, W., Granzow, H., Klupp, B. G., Karger, A., Michael, K., Maresch, C., Klopfleisch, R. & Mettenleiter, T. C.(2007). Relevance of the interaction between alphaherpesvirus UL3.5 and UL48 proteins for virion maturation and neuroinvasion. J Virol 81, 9307–9318.[CrossRef] [Google Scholar]
  14. Gimeno, I. M., Witter, R. L., Hunt, H. D., Reddy, S. M., Lee, L. F. & Silva, R. F.(2005). The pp38 gene of Marek's disease virus (MDV) is necessary for cytolytic infection of B cells and maintenance of the transformed state but not for cytolytic infection of the feather follicle epithelium and horizontal spread of MDV. J Virol 79, 4545–4549.[CrossRef] [Google Scholar]
  15. Gimeno, I. M., Cortes, A. L. & Silva, R. F.(2008). Load of challenge Marek's disease virus DNA in blood as a criterion for early diagnosis of Marek's disease tumors. Avian Dis 52, 203–208.[CrossRef] [Google Scholar]
  16. Himly, M., Foster, D. N., Bottoli, I., Iacovoni, J. S. & Vogt, P. K.(1998). The DF-1 chicken fibroblast cell line: transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses. Virology 248, 295–304.[CrossRef] [Google Scholar]
  17. Jarosinski, K. W. & Osterrieder, N.(2010). Further analysis of Marek's disease virus horizontal transmission confirms that UL44 (gC) and UL13 protein kinase activity are essential, while US2 is nonessential. J Virol 84, 7911–7916.[CrossRef] [Google Scholar]
  18. Jarosinski, K. W., Margulis, N. G., Kamil, J. P., Spatz, S. J., Nair, V. K. & Osterrieder, N.(2007). Horizontal transmission of Marek's disease virus requires US2, the UL13 protein kinase, and gC. J Virol 81, 10575–10587.[CrossRef] [Google Scholar]
  19. Khattar, S. K., van Drunen Littel-van den Hurk, S., Babiuk, L. A. & Tikoo, S. K.(1995). Identification and transcriptional analysis of a 3′-coterminal gene cluster containing UL1, UL2, UL3, and UL3.5 open reading frames of bovine herpesvirus-1. Virology 213, 28–37.[CrossRef] [Google Scholar]
  20. Kishi, M., Bradley, G., Jessip, J., Tanaka, A. & Nonoyama, M.(1991). Inverted repeat regions of Marek's disease virus DNA possess a structure similar to that of the a sequence of herpes simplex virus DNA and contain host cell telomere sequences. J Virol 65, 2791–2797. [Google Scholar]
  21. Lee, L. F., Lupiani, B., Silva, R. F., Kung, H. J. & Reddy, S. M.(2008). Recombinant Marek's disease virus (MDV) lacking the Meq oncogene confers protection against challenge with a very virulent plus strain of MDV. Vaccine 26, 1887–1892.[CrossRef] [Google Scholar]
  22. Lupiani, B., Lee, L. F., Cui, X., Gimeno, I., Anderson, A., Morgan, R. W., Silva, R. F., Witter, R. L., Kung, H. J. & Reddy, S. M.(2004). Marek's disease virus-encoded Meq gene is involved in transformation of lymphocytes but is dispensable for replication. Proc Natl Acad Sci U S A 101, 11815–11820.[CrossRef] [Google Scholar]
  23. Mao, W., Niikura, M., Silva, R. F. & Cheng, H. H.(2008). Quantitative evaluation of viral fitness due to a single nucleotide polymorphism in the Marek's disease virus UL41 gene via an in vitro competition assay. J Virol Methods 148, 125–131.[CrossRef] [Google Scholar]
  24. Montgomery, A. & Centifanto, Y.(1989). Heterogeneity within an HSV-1 wild-type strain and its importance in pathogenesis. Proc Soc Exp Biol Med 191, 362–369.[CrossRef] [Google Scholar]
  25. Niikura, M., Dodgson, J. & Cheng, H.(2006). Direct evidence of host genome acquisition by the alphaherpesvirus Marek's disease virus. Arch Virol 151, 537–549.[CrossRef] [Google Scholar]
  26. Osterrieder, N., Kamil, J. P., Schumacher, D., Tischer, B. K. & Trapp, S.(2006). Marek's disease virus: from miasma to model. Nat Rev Microbiol 4, 283–294.[CrossRef] [Google Scholar]
  27. Petherbridge, L., Brown, A. C., Baigent, S. J., Howes, K., Sacco, M. A., Osterrieder, N. & Nair, V. K.(2004). Oncogenicity of virulent Marek's disease virus cloned as bacterial artificial chromosomes. J Virol 78, 13376–13380.[CrossRef] [Google Scholar]
  28. Petherbridge, L., Xu, H., Zhao, Y., Smith, L. P., Simpson, J., Baigent, S. & Nair, V.(2009). Cloning of Gallid herpesvirus 3 (Marek's disease virus serotype-2) genome as infectious bacterial artificial chromosomes for analysis of viral gene functions. J Virol Methods 158, 11–17.[CrossRef] [Google Scholar]
  29. Reddy, S. M., Lupiani, B., Gimeno, I. M., Silva, R. F., Lee, L. F. & Witter, R. L.(2002). Rescue of a pathogenic Marek's disease virus with overlapping cosmid DNAs: use of a pp38 mutant to validate the technology for the study of gene function. Proc Natl Acad Sci U S A 99, 7054–7059.[CrossRef] [Google Scholar]
  30. Sambrook, J., Fritsch, E. F. & Maniatis, T.(1989).Molecular Cloning, 2nd edn. Cold Spring Harbor, NY. : Cold Spring Harbor Laboratory. [Google Scholar]
  31. Schumacher, D., Tischer, B. K., Fuchs, W. & Osterrieder, N.(2000). Reconstitution of Marek's disease virus serotype 1 (MDV-1) from DNA cloned as a bacterial artificial chromosome and characterization of a glycoprotein B-negative MDV-1 mutant. J Virol 74, 11088–11098.[CrossRef] [Google Scholar]
  32. Silva, R. F., Reddy, S. M. & Lupiani, B.(2004). Expansion of a unique region in the Marek's disease virus genome occurs concomitantly with attenuation but is not sufficient to cause attenuation. J Virol 78, 733–740.[CrossRef] [Google Scholar]
  33. Spatz, S. J.(2010). Accumulation of attenuating mutations in varying proportions within a high passage very virulent plus strain of Gallid herpesvirus type 2. Virus Res 149, 135–142.[CrossRef] [Google Scholar]
  34. Tulman, E. R., Afonso, C. L., Lu, Z., Zsak, L., Rock, D. L. & Kutish, G. F.(2000). The genome of a very virulent Marek's disease virus. J Virol 74, 7980–7988.[CrossRef] [Google Scholar]
  35. Witter, R. L.(2001). Protective efficacy of Marek's disease vaccine. In Marek's Disease (Current Topics in Microbiology and Immunology vol. 255), pp. 57–90. Edited by Hirai, K.. Berlin, Heidelberg, New York. : Springer-Verlag. [Google Scholar]
  36. Yamamoto, H., Hattori, M., Ohashi, K., Sugimoto, C. & Onuma, M.(1995). Kinetic analysis of T cells and antibody production in chickens infected with Marek's disease virus. J Vet Med Sci 57, 945–946.[CrossRef] [Google Scholar]
  37. Zhao, Y., Petherbridge, L., Smith, L. P., Baigent, S. & Nair, V.(2008). Self-excision of the BAC sequences from the recombinant Marek's disease virus genome increases replication and pathogenicity. Virol J 5, 19.[CrossRef] [Google Scholar]

Data & Media loading...

Most cited this month Most Cited RSS feed

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