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Abstract
Marek’s disease virus is the etiological agent of a major lymphoproliferative disorder in poultry and the prototype of the Mardivirus genus. Primary avian somatic cells are currently used for virus replication and vaccine production, but they are largely refractory to any genetic modification compatible with the preservation of intact viral susceptibility. We explored the concept of induction of viral replication permissiveness in an established pluripotent chicken embryonic stem cell-line (cES) in order to derive a new fully susceptible cell-line. Chicken ES cells were not permissive for Mardivirus infection, but as soon as differentiation was triggered, replication of Marek’s disease virus was detected. From a panel of cyto-differentiating agents, hexamethylene bis (acetamide) (HMBA) was found to be the most efficient regarding the induction of permissiveness. These initial findings prompted us to analyse the effect of HMBA on gene expression, to derive a new mesenchymal cell line, the so-called ESCDL-1, and monitor its susceptibility for Mardivirus replication. All Mardiviruses tested so far replicated equally well on primary embryonic skin cells and on ESCDL-1, and the latter showed no variation related to its passage number in its permissiveness for virus infection. Viral morphogenesis studies confirmed efficient multiplication with, as in other in vitro models, no extra-cellular virus production. We could show that ESCDL-1 can be transfected to express a transgene and subsequently cloned without any loss in permissiveness. Consequently, ESCDL-1 was genetically modified to complement viral gene deletions thus yielding stable trans-complementing cell lines. We herein claim that derivation of stable differentiated cell-lines from cES cell lines might be an alternative solution to the cultivation of primary cells for virology studies.
Highlights
Bearing in mind that hexamethylene bis(acetamide) (HMBA) had a favourable effect on virus multiplication either for GaHV-2 [9] or for VZV [45], we tested a panel of cyto-differentiating drugs on cES cells plated on gelatin-coated surfaces
Plating of a defined number of vBAC20EGFPVP22 infected sorted cells (13 000 cells per well in a 6-well plate) on either differentiated cells from cES or chicken embryo skin cell (CESC) showed that infection of the latter seemed to produce a higher number of plaques (Fig 1D), but the difference between cES differentiated cells and CESC was not significant when both cells were exposed to the same drug (Fig 1D)
We show that the acquisition of susceptibility to GaHV-2/Mardiviruses is dependent on the differentiation of cells derived from cES cells
Summary
When oriented toward a defined lineage, the differentiation of ES cells may lead to a unique permissiveness associated with a highly differentiated state as described for hepatocyte/hepatitis C virus interaction [18]. In this respect we have derived keratinocytes from cES cells [19] and shown that they were susceptible to GaHV-2 replication [20]. Mardiviruses of the GaHV-2 and MeHV-1 genotypes readily infected ESCDL-1 cells, and virus titres were in the range of those obtained on primary cells. The newly established ESCDL-1 cell line represents an attractive alternative to the tedious preparation of primary cells for research on Mardiviruses
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