Influenza viruses are a major public health burden during seasonal epidemics and a continuous threat due to their potential to cause pandemics. Annual vaccination provides the best protection against the contagious respiratory illness caused by influenza viruses. However, the current production capacities for influenza vaccines are insufficient to meet the increasing demands. We explored the possibility to establish a continuous production process for influenza viruses using the duck-derived suspension cell line AGE1.CR. A two-stage bioreactor setup was designed in which cells were cultivated in a first stirred tank reactor where an almost constant cell concentration was maintained. Cells were then constantly fed to a second bioreactor where virus infection and replication took place. Using this two-stage reactor system, it was possible to continuously produce influenza viruses. Surprisingly, virus titers showed a periodic increase and decrease during the run-time of 17 days. These titer fluctuations were caused by the presence of defective interfering particles (DIPs), which we detected by PCR. Mathematical modeling confirmed this observation showing that constant virus titers can only emerge in the absence of DIPs. Even with very low amounts of DIPs in the seed virus and very low rates for de novo DIP generation, defective viruses rapidly accumulate and, therefore, represent a serious challenge for continuous vaccine production. Yet, the continuous replication of influenza virus using a two-stage bioreactor setup is a novel tool to study aspects of viral evolution and the impact of DIPs.

Influenza A viruses are respiratory pathogens that cause seasonal epidemics with up to 500,000 deaths each year. Yet there are currently only two classes of antivirals licensed for treatment and drug-resistant strains are on the rise. A major challenge for the discovery of new anti-influenza agents is the identification of drug targets that efficiently interfere with viral replication. To support this step, we developed a multiscale model of influenza A virus infection which comprises both the intracellular level where the virus synthesizes its proteins, replicates its genome, and assembles new virions and the extracellular level where it spreads to new host cells. This integrated modeling approach recapitulates a wide range of experimental data across both scales including the time course of all three viral RNA species inside an infected cell and the infection dynamics in a cell population. It also allowed us to systematically study how interfering with specific steps of the viral life cycle affects virus production. We find that inhibitors of viral transcription, replication, protein synthesis, nuclear export, and assembly/release are most effective in decreasing virus titers whereas targeting virus entry primarily delays infection. In addition, our results suggest that for some antivirals therapy success strongly depends on the lifespan of infected cells and, thus, on the dynamics of virus-induced apoptosis or the host's immune response. Hence, the proposed model provides a systems-level understanding of influenza A virus infection and therapy as well as an ideal platform to include further levels of complexity toward a comprehensive description of infectious diseases.

During the replication of influenza viruses, defective interfering particles (DIPs) can be generated. These are noninfectious deletion mutants that require coinfection with a wild-type virus but interfere with its helper virus replication. Consequently, coinfected cells mainly produce DIPs. Little is known about how such noninfectious virus particles affect the virus yield of cell culture-based influenza vaccine production. We compared infections of Madin-Darby canine kidney cells with two seed virus preparations of the influenza virus strain A/Puerto Rico/8/34 that contain different amounts of DIPs. A combination of conventional RT-PCR, RT-qPCR, and flow cytometry revealed that DI genomes indeed strongly accumulate in coinfected cells and impede the viral RNA synthesis. Additionally, cells infected at the higher DIP concentration showed a stronger antiviral response characterized by increased interferon-β expression and apoptosis induction. Furthermore, in the presence of DIPs, a significant fraction of cells did not show any productive accumulation of viral proteins at all. Together, these effects of DIPs significantly reduce the virus yield. Therefore, the accumulation of DIPs should be avoided during influenza vaccine production which can be achieved by quality controls of working seed viruses based on conventional RT-PCR. The strategy for the depletion of DIPs presented here can help to make cell culture-based vaccine production more reliable and robust.

With the aim to adapt high-yield adherent cell lines to suspension growth, Madin Darby canine kidney (MDCK) suspension cells were developed recently that achieved comparable influenza virus yields despite an early induction of apoptosis compared to the parental adherent cell line. For both cell lines, a comprehensive study under comparable infection conditions is performed comprising information on: time course of viral infection, antiviral state of cells, virus-induced apoptosis, and virus-induced cellular protein expression for early and late infection with influenza A/PuertoRico/8/34 H1N1. The proteomic analysis is performed with 2D differential gel electrophoreses followed by mass spectrometry. Based on flow cytometric data and on the differential expression of various stress and apoptosis-related proteins, the earlier onset of virus-induced apoptosis is confirmed for suspension cells. Surprisingly, the data indicated an increased virus release rate for suspension cells. These observations correlate with an increased expression of the apical marker protein ezrin, known to play a role in influenza-induced cytoskeletal rearrangement, and the differential expression of heterogeneous nuclear ribonucleoproteins, known to bind actively influenza viral proteins and play a central role in regulating gene expression. Based on these findings, additional studies towards the design of MDCK suspension cells with further increase in influenza virus yields will be performed.