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Transcriptomic Characterization Reveals Attributes of High Influenza Virus Productivity in MDCK Cells.

ABSTRACT: The Madin-Darby Canine Kidney (MDCK) cell line is among the most commonly used cell lines for the production of influenza virus vaccines. As cell culture-based manufacturing is poised to replace egg-based processes, increasing virus production is of paramount importance. To shed light on factors affecting virus productivity, we isolated a subline, H1, which had twice the influenza virus A (IAV) productivity of the parent (P) through cell cloning, and characterized H1 and P in detail on both physical and molecular levels. Transcriptome analysis revealed that within a few hours after IAV infection, viral mRNAs constituted over one fifth of total mRNA, with several viral genes more highly expressed in H1 than P. Functional analysis of the transcriptome dynamics showed that H1 and P responded similarly to IAV infection, and were both subjected to host shutoff and inflammatory responses. Importantly, H1 was more active in translation and RNA processing intrinsically and after infection. Furthermore, H1 had more subdued inflammatory and antiviral responses. Taken together, we postulate that the high productivity of IAV hinges on the balance between suppression of host functions to divert cellular resources and the sustaining of sufficient activities for virus replication. Mechanistic insights into virus productivity can facilitate the process optimization and cell line engineering for advancing influenza vaccine manufacturing.

SUBMITTER: Ye Q

PROVIDER: S-EPMC8620111 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Similar to the recent COVID-19 pandemic, influenza A virus poses a constant threat to the global community. For the treatment of flu disease, both antivirals and vaccines are available with vaccines the most effective and safest approach. In order to overcome limitations in egg-based vaccine manufacturing, cell culture-based processes have been established. While this production method avoids egg-associated risks in face of pandemics, process intensification using animal suspension cells in high cell density perfusion cultures should allow to further increase manufacturing capacities worldwide. In this work, we demonstrate the development of a perfusion process using Madin-Darby canine kidney (MDCK) suspension cells for influenza A (H1N1) virus production from scale-down shake flask cultivations to laboratory scale stirred tank bioreactors. Shake flask cultivations using semi-perfusion mode enabled high-yield virus harvests (4.25 log10(HAU/100 μL)) from MDCK cells grown up to 41 × 106 cells/mL. Scale-up to bioreactors with an alternating tangential flow (ATF) perfusion system required optimization of pH control and implementation of a temperature shift during the infection phase. Use of a capacitance probe for on-line perfusion control allowed to minimize medium consumption. This contributed to a better process control and a more economical performance while maintaining a maximum virus titer of 4.37 log10(HAU/100 μL) and an infectious virus titer of 1.83 × 1010 virions/mL. Overall, this study clearly demonstrates recent advances in cell culture-based perfusion processes for next-generation high-yield influenza vaccine manufacturing for pandemic preparedness. KEY POINTS: • First MDCK suspension cell-based perfusion process for IAV produciton was established. • "Cell density effect" was overcome and process was intensified by reduction of medium use and automated process control. • The process achieved cell density over 40 × 106 cells/mL and virus yield over 4.37 log10(HAU/100 μL).

Project description:BackgroundThe MDCK cell line is the primary cell line used for influenza vaccine production. Using genetic engineering technology to change the expression and activity of genes that regulate virus proliferation to obtain high-yield vaccine cell lines has attracted increasing attention. A comprehensive understanding of the key genes, targets, and molecular mechanisms of viral regulation in cells is critical to achieving this goal, yet the post-transcriptional regulation mechanism involved in virus proliferation-particularly the effect of lncRNA on influenza virus proliferation-is still poorly understood. Therefore, this study used high-throughput RNA-seq technology to identify H1N1 infection-induced lncRNA and mRNA expression changes in MDCK cells and explore the regulatory relationship between these crucial lncRNAs and their target genes.ResultsIn response to H1N1 infection in MDCK cells 16 h post-infection (hpi) relative to uninfected controls, we used multiple gene function annotation databases and initially identified 31,501 significantly differentially expressed (DE) genes and 39,920 DE lncRNAs (|log2FC| > 1, p < 0.05). Among these, 102 lncRNAs and 577 mRNAs exhibited predicted correlations with viral response mechanisms. Based on the magnitude of significant expression differences, related research, and RT-qPCR expression validation at the transcriptional level, we further focused on 18 DE mRNAs and 32 DE lncRNAs. Among these, the differential expression of the genes RSAD2, CLDN1, HCLS1, and IFIT5 in response to influenza virus infection was further verified at the protein level using Western blot technology, which showed results consistent with the RNA-seq and RT-qPCR findings. We then developed a potential molecular regulatory network between these four genes and their six predicted lncRNAs.ConclusionsThe results of this study will contribute to a more comprehensive understanding of the molecular mechanism of host cell non-coding RNA-mediated regulation of influenza virus replication. These results may also identify methods for screening target genes in the development of genetically engineered cell lines capable of high-yield artificial vaccine production.

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Transcriptomic Characterization Reveals Attributes of High Influenza Virus Productivity in MDCK Cells.

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