INFLUENCE OF HOST CELL DEFENCE DURING INFLUENZA VACCINE PRODUCTION IN MDCK CELLS

For cell culture-based influenza vaccine production virus yield optimisation is of crucial importance. In particular, with the recent threat of the new H1N1 pandemic, not only seasonal vaccines but also pre-/pandemic vaccines have to be supplied in large quantities. In vivo influenza replication is limited by the immune system, but for production cell lines the impact of cellular defence mechanisms on virus yield is unknown. In influenza-infected adherent Madin-Darby canine kidney (MDCK) cells the interferon (IFN) response and subsequent induction of the antiviral state was monitored. Virus yield and host cell signalling intensity were strain-dependent. By over-expression of viral antagonists IFN-signalling could be reduced up to 90%. However, maximum virus titre determined by real-time PCR and HA-assay was not altered significantly. Stimulation of the antiviral state by conditioned medium led to enhanced IFN-signalling, which initially slowed down virus replication but had only minor effects on final virus titres. Interestingly, minireplicon assays revealed that canine Mx proteins are lacking the antiviral activity against influenza of their human or mouse counterparts. In summary, for MDCK cell culture-based influenza virus production host cell defence mechanisms seem to play only a minor role for final virus yields. Antiviral mechanisms of these epithelial cells may slow down influenza replication, which in vivo gains time for the immune system to be activated, but do not reduce maximum virus titres obtained in the bioprocess

Influenza A virus propagation in MDCK: Intracellular virus replication, virus release and cell-cycle preferential infection analysis

Cell culture-based processes for vaccine manufacturing offer advantages over egg-based processes in terms of product uniformity and sterility, production time and scaling up capacity1,2. Regarding influenza vaccines, MDCK cells are one of the host cell lines currently used to manufacture licensed products; however, virus titers remain lower compared to those obtained in eggs and further increase of specific and volumetric yields is required. To identify bottlenecks in influenza A virus (IAV) production, we thoroughly studied IAV replication in MDCK cells. For this, we analyzed different features of the infection process such as viral RNA replication, intracellular localization of viral components, virus release and morphology of the particles, and the preferential infection in different cell-cycle phases. Using synchronous infections, we found that production of infectious particles dropped much earlier than the production of total particles. Furthermore, we found that the maximum virus release rate was reached when all viral RNA species attained their maximum intracellular concentration. Using qPCR we determined that the vRNA maximum concentration per cell was 10‑fold higher than the specific viral titers obtained, indicating that vRNA concentration does not limit IAV particle assembly. When we evaluated the morphology of particles released using electron microscopy, we observed that a higher fraction of the viral particles produced at late times possess an abnormal morphology, concurring with the increased production of non-infectious viruses. Using imaging flow cytometry, we determined that the export of influenza viral genome segments (ribonucleoprotein complexes, vRNPs) from the nucleus to the cytoplasm strongly correlated with the onset of virus release. However, our results also suggest that the induction of apoptosis caused that virus assembly became deficient producing more non-infectious particles at late infection times. Lastly, using low MOI infections and imaging flow cytometry, we found that -in contrast to previous publications- IAV did not preferentially infect a specific cell cycle phase and no cell cycle arrest induction was observed during the time frame of the experiment (9 hpi). In summary, the data presented here offers a comprehensive overview of the dynamics of IAV infection in MDCK and might contribute to the development of molecular or cell culture-based strategies to improve IAV production in MDCK cells. 1 Gallo-Ramírez et al, 2015. Exp. Rev. Vaccines 14 (9). 2 Pardue et al. 2011. Exp. Rev. Vaccines 10 (8)

Multiscale Modeling of Influenza A Virus Infection Supports the Development of Direct-Acting Antivirals

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

Modeling the intracellular dynamics of influenza virus replication to understand the control of viral RNA synthesis

Influenza viruses transcribe and replicate their negative-sense RNA genome inside the nucleus of host cells via three viral RNA species. In the course of an infection, these RNAs show distinct dynamics, suggesting that differential regulation takes place. To investigate this regulation in a systematic way, we developed a mathematical model of influenza virus infection at the level of a single mammalian cell. It accounts for key steps of the viral life cycle, from virus entry to progeny virion release, while focusing in particular on the molecular mechanisms that control viral transcription and replication. We therefore explicitly consider the nuclear export of viral genome copies (vRNPs) and a recent hypothesis proposing that replicative intermediates (cRNA) are stabilized by the viral polymerase complex and the nucleoprotein (NP). Together, both mechanisms allow the model to capture a variety of published data sets at an unprecedented level of detail. Our findings provide theoretical support for an early regulation of replication by cRNA stabilization. However, they also suggest that the matrix protein 1 (M1) controls viral RNA levels in the late phase of infection as part of its role during the nuclear export of viral genome copies. Moreover, simulations show an accumulation of viral proteins and RNA toward the end of infection, indicating that transport processes or budding limits virion release. Thus, our mathematical model provides an ideal platform for a systematic and quantitative evaluation of influenza virus replication and its complex regulation. Copyright © 2012 by the American Society for Microbiology. [accessed November 2nd 2012