Abstract

Defective interfering particles (DIPs) are a natural byproduct of influenza A virus (IAV) replication. DIPs interfere with the propagation and spread of infectious standard virus (STV), reduce virus yields by competing for viral and cellular resources, and induce antiviral responses. These properties open exciting possibilities for the development of DIP-based antivirals. Exploring options for cell culture-based DIP production, we have established a fully continuous cultivation process, where one bioreactor is used to grow cells that are fed to two bioreactors operated in parallel for virus production. This system allows head-to-head comparisons of STV and DIP replication dynamics over extended time periods. Cultivations were performed at two residence times (RT, 22 and 36 h) using MDCK suspension cells grown in a fully defined medium. For infection, we used a virus seed generated by reverse genetics containing STVs and a known DIP carrying a deletion in segment 1 (delS1(1)). Four days post infection, DIPs achieved maximum concentrations of 7.0 center dot 10(9) virions/mL and 8.4 center dot 10(9) virions/mL for RTs of 22 and 36 h, respectively. Furthermore, oscillations in virus titers with two to three maxima were found for DIP accumulation at 36 and 22 h RT, respectively. To complement the study, a basic mathematical model using simple kinetics and a reasonable number of parameters to describe DIP-propagation in continuous cultures was established. Upon fitting the model individually to each of the two data sets, oscillations in the viral dynamics and the cell population dynamics were described well. Modeling suggests that both STV inactivation and virus degradation have to be taken into account to achieve good agreement of simulations and experimental data for longer RTs. Together, the high DIP titers obtained, and the successful simulation of the experimental data showed that the combination of continuous bioreactors and mathematical models can enable studies regarding DIP dynamics over extended time periods and allow large scale manufacturing of DIP-based antivirals.