Dataset Information

Production of Defective Interfering Particles of Influenza A Virus in Parallel Continuous Cultures at Two Residence Times-Insights From qPCR Measurements and Viral Dynamics Modeling.

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 7.0·109 virions/mL and 8.4·109 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.

SUBMITTER: Tapia F

PROVIDER: S-EPMC6813217 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Production of Defective Interfering Particles of Influenza A Virus in Parallel Continuous Cultures at Two Residence Times-Insights From qPCR Measurements and Viral Dynamics Modeling.

Tapia Felipe F Laske Tanja T Wasik Milena A MA Rammhold Markus M Genzel Yvonne Y Reichl Udo U

Frontiers in bioengineering and biotechnology 20191018

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 on ...[more]

PMID: 31681751

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Project description:Defective interfering particles (DIPs) of influenza A virus (IAV) are suggested for use as broad-spectrum antivirals. We discovered a new type of IAV DIP named "OP7" that carries point mutations in its genome segment (Seg) 7 instead of a deletion as in conventional DIPs (cDIPs). Recently, using genetic engineering tools, we generated "OP7 chimera DIPs" that carry point mutations in Seg 7 plus a deletion in Seg 1. Together with cDIPs, OP7 chimera DIPs were produced in shake flasks in the absence of infectious standard virus (STV), rendering UV inactivation unnecessary. However, only part of the virions harvested were OP7 chimera DIPs (78.7%) and total virus titers were relatively low. Here, we describe the establishment of an OP7 chimera DIP production process applicable for large-scale production. To increase total virus titers, we reduced temperature from 37 to 32 °C during virus replication. Production of almost pure OP7 chimera DIP preparations (99.7%) was achieved with a high titer of 3.24 log10(HAU/100 µL). This corresponded to an 11-fold increase relative to the initial process. Next, this process was transferred to a stirred tank bioreactor resulting in comparable yields. Moreover, DIP harvests purified and concentrated by steric exclusion chromatography displayed an increased interfering efficacy in vitro. Finally, a perfusion process with perfusion rate control was established, resulting in a 79-fold increase in total virus yields compared to the original batch process in shake flasks. Again, a very high purity of OP7 chimera DIPs was obtained. This process could thus be an excellent starting point for good manufacturing practice production of DIPs for use as antivirals. KEY POINTS: • Scalable cell culture-based process for highly effective antiviral OP7 chimera DIPs • Production of almost pure OP7 chimera DIPs in the absence of infectious virus • Perfusion mode production and purification train results in very high titers.

Project description:Defective interfering particles (DIPs) of influenza A virus (IAV) are naturally occurring mutants that have an internal deletion in one of their eight viral RNA (vRNA) segments, rendering them propagation-incompetent. Upon coinfection with infectious standard virus (STV), DIPs interfere with STV replication through competitive inhibition. Thus, DIPs are proposed as potent antivirals for treatment of the influenza disease. To select corresponding candidates, we studied de novo generation of DIPs and propagation competition between different defective interfering (DI) vRNAs in an STV coinfection scenario in cell culture. A small-scale two-stage cultivation system that allows long-term semi-continuous propagation of IAV and its DIPs was used. Strong periodic oscillations in virus titers were observed due to the dynamic interaction of DIPs and STVs. Using next-generation sequencing, we detected a predominant formation and accumulation of DI vRNAs on the polymerase-encoding segments. Short DI vRNAs accumulated to higher fractions than longer ones, indicating a replication advantage, yet an optimum fragment length was observed. Some DI vRNAs showed breaking points in a specific part of their bundling signal (belonging to the packaging signal), suggesting its dispensability for DI vRNA propagation. Over a total cultivation time of 21 days, several individual DI vRNAs accumulated to high fractions, while others decreased. Using reverse genetics for IAV, purely clonal DIPs derived from highly replicating DI vRNAs were generated. We confirm that these DIPs exhibit a superior in vitro interfering efficacy compared to DIPs derived from lowly accumulated DI vRNAs and suggest promising candidates for efficacious antiviral treatment. IMPORTANCE Defective interfering particles (DIPs) emerge naturally during viral infection and typically show an internal deletion in the viral genome. Thus, DIPs are propagation-incompetent. Previous research suggests DIPs as potent antiviral compounds for many different virus families due to their ability to interfere with virus replication by competitive inhibition. For instance, the administration of influenza A virus (IAV) DIPs resulted in a rescue of mice from an otherwise lethal IAV dose. Moreover, no apparent toxic effects were observed when only DIPs were administered to mice and ferrets. IAV DIPs show antiviral activity against many different IAV strains, including pandemic and highly pathogenic avian strains, and even against nonhomologous viruses, such as SARS-CoV-2, by stimulation of innate immunity. Here, we used a cultivation/infection system, which exerted selection pressure toward accumulation of highly competitive IAV DIPs. These DIPs showed a superior interfering efficacy in vitro, and we suggest them for effective antiviral therapy.

Project description:A high particle to infectivity ratio is a feature common to many RNA viruses, with ~90-99% of particles unable to initiate a productive infection under low multiplicity conditions. A recent publication by Brooke et al. revealed that, for influenza A virus (IAV), a proportion of these seemingly non-infectious particles are in fact semi-infectious. Semi-infectious (SI) particles deliver an incomplete set of viral genes to the cell, and therefore cannot support a full cycle of replication unless complemented through co-infection. In addition to SI particles, IAV populations often contain defective-interfering (DI) particles, which actively interfere with production of infectious progeny. With the aim of understanding the significance to viral evolution of these incomplete particles, we tested the hypothesis that SI and DI particles promote diversification through reassortment. Our approach combined computational simulations with experimental determination of infection, co-infection and reassortment levels following co-inoculation of cultured cells with two distinct influenza A/Panama/2007/99 (H3N2)-based viruses. Computational results predicted enhanced reassortment at a given % infection or multiplicity of infection with increasing semi-infectious particle content. Comparison of experimental data to the model indicated that the likelihood that a given segment is missing varies among the segments and that most particles fail to deliver ≥1 segment. To verify the prediction that SI particles augment reassortment, we performed co-infections using viruses exposed to low dose UV. As expected, the introduction of semi-infectious particles with UV-induced lesions enhanced reassortment. In contrast to SI particles, inclusion of DI particles in modeled virus populations could not account for observed reassortment outcomes. DI particles were furthermore found experimentally to suppress detectable reassortment, relative to that seen with standard virus stocks, most likely by interfering with production of infectious progeny from co-infected cells. These data indicate that semi-infectious particles increase the rate of reassortment and may therefore accelerate adaptive evolution of IAV.

Dataset Information

Production of Defective Interfering Particles of Influenza A Virus in Parallel Continuous Cultures at Two Residence Times-Insights From qPCR Measurements and Viral Dynamics Modeling.

Publications

Production of Defective Interfering Particles of Influenza A Virus in Parallel Continuous Cultures at Two Residence Times-Insights From qPCR Measurements and Viral Dynamics Modeling.

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