Semi-continuous propagation of influenza A virus and its defective interfering particles: analyzing the dynamic competition to select candidates for antiviral therapy

Pelz, Lars; Ruediger, Dylan; Alnaji, Fadi G.; Genzel, Yvonne; Brooke, Christopher B.; Kupke, Sascha Young; Reichl, Udo

doi:10.1101/2021.02.08.430251

Cited by 3 publications

(7 citation statements)

References 65 publications

(230 reference statements)

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“…This process was highly replicable between independently-generated libraries with inter-replicate Pearson correlation coefficients of ∼0.75 when comparing across the enrichment or depletion of individual junctions ( Figure 3 E). These maxima correlate well with previously-identified “sweet-spot” lengths from continuous culture propagation of influenza defective particles ( Pelz et al, 2021 ). Taken together, our data indicate that the length distribution of defective influenza segments can be explained by the competing processes of replication and packaging, regardless of their initial distribution upon formation.…”

Section: Resultssupporting

confidence: 85%

Section: Pb1 and Ha Exhibit Length-dependent Differences In Viral Incorporationsupporting

confidence: 85%

“…Interestingly, during replication and viral packaging our artificial libraries converge on a near-identical length as natural defective populations ( Pelz et al, 2021 ). This suggests that the contrasting pressures of replication speed and packaging are sufficient to shape defective influenza populations to their final, observed, lengths, from potentially disparate initial distributions.…”

Section: Discussionmentioning

confidence: 99%

“…For influenza A virus, a (-) sense, segmented, RNA virus, defective particles, interfering or otherwise, largely bear genomes with large internal deletions in the three polymerase-encoding segments ( Nayak et al, 1982 ; Nakajima et al, 1979 ; Akkina et al, 1984 ; Alnaji et al, 2019 ). These deletions generally reduce the size of each segment from ∼2.3kb to ∼400nt ( Saira et al, 2013 ; Pelz et al, 2021 ). It is unknown what drives this size and segment preference.…”

Section: Introductionmentioning

confidence: 99%

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Library-based analysis reveals segment and length dependent characteristics of defective influenza genomes

Mendes

Russell

2021

Preprint

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Parasitic elements of the viral population which are unable to replicate on their own yet rise to high frequencies, defective interfering particles are found in a variety of different viruses. Their presence is associated with a loss of population fitness, both through the depletion of key cellular resources and the stimulation of innate immunity. For influenza A virus, these particles contain large internal deletions in the genomic segments which encode components of the heterotrimeric polymerase. Using a library-based approach, we comprehensively profile the growth and replication of defective influenza species, demonstrating that they possess an advantage during genome replication, and that exclusion during packaging reshapes population composition in a manner consistent with their final, observed, distribution in natural populations. We find that an innate immune response is not linked to the size of a deletion; however, replication of defective segments can enhance their immunostimulatory properties. Overall, our results address several key questions in defective influenza A virus biology, and the methods we have developed to answer those questions may be broadly applied to other defective viruses.

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Section: Resultssupporting

confidence: 85%

Section: Pb1 and Ha Exhibit Length-dependent Differences In Viral Incorporationsupporting

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Library-based analysis reveals segment and length dependent characteristics of defective influenza genomes

Mendes

Russell

2021

Preprint

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“…Furthermore, model predictions suggest a more prominent replication advantage could also improve STV titer reduction in high virus concentration scenarios. A potential strategy to obtain DIPs with higher advantages over the STV is the selection of strongly accumulating DIPs from long-term continuous bioreactor cultivations [52]. Such DIPs consistently replicated at high levels indicating an increased advantage over their competition, i.e., other emerging DIPs.…”

Section: Discussionmentioning

confidence: 99%

Multiscale model of defective interfering particle replication for influenza A virus infection in animal cell culture

et al. 2021

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Cell culture-derived defective interfering particles (DIPs) are considered for antiviral therapy due to their ability to inhibit influenza A virus (IAV) production. DIPs contain a large internal deletion in one of their eight viral RNAs (vRNAs) rendering them replication-incompetent. However, they can propagate alongside their homologous standard virus (STV) during infection in a competition for cellular and viral resources. So far, experimental and modeling studies for IAV have focused on either the intracellular or the cell population level when investigating the interaction of STVs and DIPs. To examine these levels simultaneously, we conducted a series of experiments using highly different multiplicities of infections for STVs and DIPs to characterize virus replication in Madin-Darby Canine Kidney suspension cells. At several time points post infection, we quantified virus titers, viable cell concentration, virus-induced apoptosis using imaging flow cytometry, and intracellular levels of vRNA and viral mRNA using real-time reverse transcription qPCR. Based on the obtained data, we developed a mathematical multiscale model of STV and DIP co-infection that describes dynamics closely for all scenarios with a single set of parameters. We show that applying high DIP concentrations can shut down STV propagation completely and prevent virus-induced apoptosis. Interestingly, the three observed viral mRNAs (full-length segment 1 and 5, defective interfering segment 1) accumulated to vastly different levels suggesting the interplay between an internal regulation mechanism and a growth advantage for shorter viral RNAs. Furthermore, model simulations predict that the concentration of DIPs should be at least 10000 times higher than that of STVs to prevent the spread of IAV. Ultimately, the model presented here supports a comprehensive understanding of the interactions between STVs and DIPs during co-infection providing an ideal platform for the prediction and optimization of vaccine manufacturing as well as DIP production for therapeutic use.

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Accumulation of copy-back viral genomes during respiratory syncytial virus infection is preceded by diversification of the copy-back viral genome population followed by selection

Felt¹,

Achouri²,

Faber³

et al. 2022

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RNA viruses generate non-standard viral genomes during their replication, including viral genomes of the copy-back (cbVG) type that cannot replicate in the absence of a standard virus. cbVGs play a crucial role in shaping virus infection outcomes due to their ability to interfere with virus replication and induce strong immune responses. However, despite their critical role during infection, the principles that drive the selection and evolution of cbVGs within a virus population are poorly understood. As cbVGs are dependent on the virus replication machinery to be generated and replicated, we hypothesized that host factors that affect virus replication exert selective pressure on cbVGs and drive their evolution within a virus population. To test this hypothesis, we used respiratory syncytial virus (RSV) as model and took an experimental evolution approach by serially passaging RSV in immune competent A549 control and immune deficient A549 STAT1 KO cells which allow higher levels of virus replication. As predicted, we observed that virus populations accumulated higher amounts of cbVGs in the more permissive A549 STAT1 KO cells over time but, unexpectedly, the predominant cbVG species after passages in the two conditions were different. While A549 STAT1 KO cells accumulated relatively short cbVGs, A549 control cells mainly contained cbVGs of much longer predicted size that have not been described previously. These long cbVGs were the first species generated in both cell lines in vitro and the predominant ones observed in samples from RSV infected patients. Although high replication levels are associated with cbVG generation and accumulation, our data show that high levels of virus replication are critical for cbVG population diversification, a process that preceded the generation of shorter cbVGs that selectively accumulated over time. Taken together, we show that selection and evolution of cbVGs within a virus population is shaped by how resistant (low replication) or permissive (high replication) a host is to RSV.

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Semi-continuous propagation of influenza A virus and its defective interfering particles: analyzing the dynamic competition to select candidates for antiviral therapy

Cited by 3 publications

References 65 publications

Library-based analysis reveals segment and length dependent characteristics of defective influenza genomes

Library-based analysis reveals segment and length dependent characteristics of defective influenza genomes

Multiscale model of defective interfering particle replication for influenza A virus infection in animal cell culture

Accumulation of copy-back viral genomes during respiratory syncytial virus infection is preceded by diversification of the copy-back viral genome population followed by selection

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