Datensatz

Zusammenfassung

In cell culture-based influenza vaccine production significant efforts are directed towards virus seed optimization for maximum yields. Typically, high growth reassortants (HGR) containing backbones of six gene segments of e.g. influenza A/PR/8, are generated from wild type strains. Often, however, HA and TCID50 titres obtained do not meet expectations and further optimization measures are required. Flow cytometry is an invaluable tool to improve our understanding of mechanism related to progress of infection, virus-induced apoptosis, and cell-specific productivity. In this study, we performed infections with two influenza A/PR/8 variants (from NIBSC and RKI) and two A/PR/8-based HGRs (Wisconsin-like and Uruguay-like) to investigate virus replication, apoptosis and virus titres at different multiplicities of infection (MOI 0.0001, 0.1, 3). Flow cytometric analyses showed similar dynamics in the time course of infected and apoptotic cell populations for all four tested strains at MOI 0.0001. Interestingly, higher MOI resulted in an earlier increase of the populations of infected and apoptotic cells and showed strain-specific differences. Infections with A/PR/8 NIBSC resulted in an earlier increase in both cell populations compared to A/PR/8 RKI. The Uruguay-like reassortant showed the earliest increase in the concentration of infected cells and a late induction of apoptosis at all tested MOIs. In contrast, the Wisconsin-like reassortant showed strong apoptosis induction at high MOIs resulting in reduced titres compared to lower MOI. Maximum HA titres were unaffected by changes in the MOI for the two A/PR/8 and the Uruguay-like reassortant. Maximum TCID50 titres, however, decreased with increasing MOI for all strains. Overall, infections at very low MOI (0.0001) resulted not only in similar dynamics concerning progress of infection and induction of apoptosis but also in maximum virus yields. Highest HA titres were obtained for virus seed strains combining a fast progress in infection with a late onset of apoptosis. Therefore, both factors should be considered for the establishment of robust influenza vaccine production processes. Copyright © 2012 Elsevier Ltd. All rights reserved. [accessed November 2nd 2012]