Advertisement

Use of Diploid Human Fibroblasts as a Model System to Culture, Grow, and Study Human Cytomegalovirus Infection

  • Elizabeth A. FortunatoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1119)

Abstract

Primary human diploid fibroblasts are used routinely to study host/pathogen interactions of human cytomegalovirus (HCMV). Fibroblasts’ ease of culture and tremendous permissiveness for infection allow the study of all facets of infection, an abbreviated list of which includes ligand/receptor interactions, activation of cell signaling responses, and dysregulation of the cell cycle and DNA repair processes. Another advantage to fibroblasts’ permissiveness for HCMV is the capability to grow high titer stocks of virus in them. This chapter will discuss the production of viral stocks of HCMV in primary human fibroblasts, commencing with culturing and infection of cells and continuing through harvest, titration (determining the infectious capacity of a particular virus preparation), and storage of viral stocks for use in downstream experiments.

Key words

Human cytomegalovirus Culture of primary fibroblasts Preparation and storage of virus stocks Titrating viral stocks Growth of virus in culture 

References

  1. 1.
    Sinzger C, Jahn G (1996) Human cytomegalovirus cell tropism and pathogenesis. Intervirology 39:302–319PubMedGoogle Scholar
  2. 2.
    Casavant NC, Luo MH, Rosenke K, Winegardner T, Zurawska A, Fortunato EA (2006) Potential role for p53 in the permissive life cycle of human cytomegalovirus. J Virol 80:8390–8401PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Fortunato EA, Spector DH (1998) p53 and RPA are sequestered in viral replication centers in the nuclei of cells infected with human cytomegalovirus. J Virol 72:2033–2039PubMedCentralPubMedGoogle Scholar
  4. 4.
    Fortunato EA, Sanchez V, Yen JY, Spector DH (2002) Infection of cells with human cytomegalovirus during S phase results in a blockade to immediate early gene expression that can be overcome by inhibition of the proteasome. J Virol 76(11):5369–5379Google Scholar
  5. 5.
    Salvant BS, Fortunato EA, Spector DH (1998) Cell cycle dysregulation by human cytomegalovirus: influence of the cell cycle phase at the time of infection and effects on cyclin transcription. J Virol 72:3729–3741PubMedCentralPubMedGoogle Scholar
  6. 6.
    Irmiere A, Gibson W (1983) Isolation and characterization of a noninfectious virion-like particle released from cells infected with human strains of cytomegalovirus. Virology 130:118–133PubMedCrossRefGoogle Scholar
  7. 7.
    Benyesh-Melnick M, Probstmeyer F, McCombs R, Brunschwig JP, Vonka V (1966) Correlation between infectivity and physical virus particles in human cytomegalovirus. J Bacteriol 92:1555–1561PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Biological SciencesUniversity of IdahoMoscowUSA

Personalised recommendations