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Abstract

Annual epidemic influenza virus infections cause up to 500.000 deaths and 3 to 5 million cases of severe illness with enormous healthcare expenses and non-productive time. Because of a high demand of vaccines effective production of virus material is inevitable. Since several years there are increasing efforts to develop mammalian cell culture-based production systems to overcome the limitations and drawbacks of the existing vaccine production in hen's eggs. To optimize these processes and to identify bottlenecks in virus replication we focus on analyzing cell physiology during both cell growth and virus infection. Here, we investigate the replication of equine influenza A virus in adherent Madin Darby canine kidney (MDCK) cells. Two methods, which are already established for virus detection are the hemagglutination assay (HA) for total virus particles and the tissue culture infectious dose (TCID50) determination for infectious virus concentrations. Both assays refer to virus particles in the culture supernatant. However, in order to directly correlate viral infection with other physiological states of the host cells a method based on a single-cell level is desireable. Flow cytometry is a suitable means to investigate the physiology of cell populations on this single-cell level. Therefore we established a sensitive immunofluorescent detection of both intracellular and surface-bound viral proteins using a combination of fluorochrome-labelled monoclonal antibodies. The method can be used for the quantitation of the population of infected cells in bioreactor samples. Especially at low multiplicities of infection or in early phases of viral infection additional information can be gained, as viral titers in the supernatant detectable by HA and TCID50 are still low because the release is a later event in viral replication. A diversification of the method for different influenza viruses and further host cells is subject to recent research.