Mycoplasma contamination and viral immunomodulatory activity: Dendritic cells

skychen

During in vitro investigations on the interaction of classical swine fever virus (CSFV) – an immunosuppressive viral pathogen – with monocytederived
dendritic cells (MoDC) a soluble factor with a strong anti-proliferative activity for T lymphocyteswas found. This activity, with an inhibitory
dilution 50% (ID50) of 103–107, was induced after virus infection of monocytes differentiating into DC. UV-inactivation of the supernatants and
blocking experiments with a monoclonal antibody against the E2 envelope protein of CSFV initially indicated a virus-dependency. However, further
investigations including filtration and centrifugation experiments as well as antibiotic treatment demonstrated the involvement of mycoplasma. This
was confirmed by a Hoechst 33258 staining, PCR and mycoplasma cultures—Mycoplasma hyorhinis was identified as the contaminant. Elucidation
of a mycoplasma presence occurred under conditions in which the original virus stocks prepared in SK6 cells were negative for mycoplasma using
the above tests. Moreover, conventional passage of the virus on the SK6 cells used for this purpose did not reveal any mycoplasma. It was the
passage of virus in MoDC rather than SK6 cells that was required to expose the contamination. Three passages of the anti-proliferative supernatants
on MoDC cultures increased the ID50 103-fold; only when these MoDC-derived supernatants were employed was the mycoplasma contaminant
also detectable on SK6 cells. In conclusion, these data demonstrate that regular testing of cell lines and virus stocks for mycoplasma does not
necessarily identify their presence, and that application of passage in MoDC cultures could prove an aid for identifying initially undetectable levels
of mycoplasma contamination.


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