title: The effects of cell passages on the cell morphology and the outcome of herpes simplex virus type 1 infection.
abstract: Because cell cultures are essential in biological research which involves the analysis of virus morphogenesis, this study focused on examining the significance of cell passages. Human embryonic lung fibroblasts (MRC-5) at passage (P) 27 were seeded twice a week to P 32, P 40, and P 48, when just at confluence and then infected with herpes simplex virus type 1 (HSV-1). The structure of the non-virus-infected (MOCK) and HSV-1 infected cells, the amount of cellular infectious virus particles and the capability to express HSV-1 glycoproteins C (gC-1) and D (gD-1) were investigated by phase-contrast and immunofluorescence light microscopy, immunogold cryosection EM, plaque assays, immunoblots, and total protein assays. Modified cell structure including fragmentation of tubulin fibers were visible in MOCK from P 38 onwards. The quantity of vimentin remained unchanged while actin accumulated and beta-tubulin decreased in HSV-1 infected late P cells compared to early P cultures. Cells of high P counts contained significantly fewer infectious virus particles, very likely of lower virulence, and their expression of gC-1 and gD-1 were concordantly reduced. These observations indicate that the number of cell P must be considered in order to reproduce results of cell biology and viral morphogenesis. The MRC-5 cells ought not to be passaged more than ten times beyond P 27 in the laboratory.
authors.affiliation: The Protein Laboratory, Institute of Molecular Pathology, University of Copenhagen, The Panum Institute, Denmark.
authors.forename: H L
authors.initials: HL
authors.lastname: Jensen
authors.forename: B
authors.initials: B
authors.lastname: Norrild
mesh_terms: D018929:Cell Culture Techniques; D002460:Cell Line; D048429:Cell Size; D016922:Cellular Senescence; D003588:Cytopathogenic Effect, Viral; D003599:Cytoskeleton / Q000648:ultrastructure; D018259:Herpesvirus 1, Human / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008854:Microscopy, Electron / Q000379:methods; D008856:Microscopy, Fluorescence; D015388:Organelles / Q000648:ultrastructure; D014404:Tubulin / Q000032:analysis; D014759:Viral Envelope Proteins / Q000032:analysis; D010948:Viral Plaque Assay
chemical_list: D014404:Tubulin; D014759:Viral Envelope Proteins