Biology subjects

Genzel, Y.

Publications and source records attributed to Genzel, Y..

4 recordsLinked to original sources

bioRxiv2025

Quantitative analysis of proteomic changes in monoclonal MDCK cell lines infected with human influenza A virus

AbstractSuspension MDCK cells are a substrate for producing influenza A virus (IAV) and typically show very high virus yields compared to other animal cells. Due to the significant heterogeneity within cell populations, studying and comparing clonal cell lines with regard to specific properties, such as superior growth or higher productivity, could facilitate process optimization. In this study, we analyzed the expressed proteins of clonal cell lines to identify intrinsic characteristics of effective IAV producers. We compared proteome changes in two IAV-infected monoclonal suspension MDCK cell lines: C59, a low-yield IAV producer with fast cell growth and small cell diameter, and C113, a high-yield IAV producer with average cell growth and large cell diameter. We examined growth rate, size, metabolism and IAV production. A total of 5177 host cell proteins were detected in both cell lines using DIA-PASEF mode with a TimsTOFpro mass spectrometer. Analysis of the differentially expressed proteins revealed that fatty acid oxidation and branched-chain amino acid degradation were upregulated in highly productive cells. In contrast, steroid biosynthesis and DNA replication were more active in faster-growing cells. Following infection, 122 proteins were significantly upregulated (p<0.05, log2-fold change [≥]1) in the high-producing cell line. These proteins were associated with membrane trafficking, interactions with the IAV-NS1 protein and virus production. Additionally, 98 proteins associated with antiviral pathways such as the proto-oncogenic receptor tyrosine kinase MET and tumor necrosis factor (TNF) signaling were downregulated (p<0.05, log2-fold change [≤]1). In the cell line that produced lower IAV titers, 77 proteins were downregulated and 57 were upregulated after infection. RNA metabolism appeared to be downregulated, while the tricarboxylic acid (TCA) cycle and the stress response were both upregulated. In the high-yield C113 clone, only proteins associated with apoptosis and the target of rapamycin kinase (TOR) were expressed following infection. This may indicate a more effective release of virus particles. A comparison of intracellular IAV protein levels demonstrated that M1 and NA levels were 4-fold and 8-fold higher, respectively, for the high-yield C113 cell line. These findings again suggest better virus release.

molecular biology↗

bioRxiv2023

Generation of OP7 chimera defective interfering particle preparations free of infectious influenza A virus that shows antiviral efficacy in mice

Influenza A virus (IAV) defective interfering particles (DIPs) are considered as new promising antiviral agents. Conventional DIPs (cDIPs) contain a deletion in the genome and can only replicate upon co-infection with infectious standard virus (STV), during which they suppress STV replication. We previously discovered a new type of IAV DIP "OP7" that entails genomic point mutations and displays higher antiviral efficacy than cDIPs. To avoid safety concerns for the medical use of OP7 preparations, we developed a production system that does not depend on infectious IAV. We reconstituted a mixture of DIPs consisting of cDIPs and OP7 chimera DIPs, in which both harbor a deletion in their genome. To complement the defect, the deleted viral protein is expressed by the suspension cell line used for production in shake flasks. Here, DIP preparations harvested are not contaminated with infectious virions, and the fraction of OP7 chimera DIPs depended on the multiplicity of infection. Intranasal administration of OP7 chimera DIP material was well tolerated. A rescue from an otherwise lethal IAV infection and no signs of disease upon OP7 chimera DIP co-infection demonstrated the remarkable antiviral efficacy. The clinical development of this new class of broad-spectrum antiviral may contribute to pandemic preparedness.

molecular biology↗

bioRxiv2021

Cell culture-based production of defective interfering influenza A virus particles in perfusion mode using an alternating tangential flow filtration system

Respiratory diseases including influenza A virus (IAV) infections represent a major threat to human health. While the development of a vaccine requires a lot of time, a fast countermeasure could be the use of defective interfering particles (DIPs) for antiviral therapy. IAV DIPs are usually characterized by a large internal deletion in one viral RNA segment. Consequentially, DIPs can only propagate in presence of infectious standard viruses (STVs), compensating the missing gene function. Here, they interfere with and suppress the STV replication and might act "universally" against many IAV subtypes. We recently reported a production system for purely clonal DIPs utilizing genetically modified cells. In the present study, we established an automated perfusion process for production of a DIP, called DI244, using an alternating tangential flow filtration (ATF) system for cell retention. Viable cell concentrations and DIP titers more than 10-times higher than for a previously reported batch cultivation were observed. Further, we investigated a novel tubular cell retention device for its potential for continuous virus harvesting into the permeate. Very comparable performances to typically used hollow fiber membranes were found during the cell growth phase. During the virus replication phase the tubular membrane, in contrast to the hollow fiber membrane, allowed 100% of the produced virus particles to pass through. To our knowledge, this is the first time a continuous virus harvest was shown for a membrane-based perfusion process. Overall, the process established offers interesting possibilities for advanced process integration strategies for next-generation virus particle and virus vector manufacturing. Key pointsO_LIAn automated perfusion process for production of IAV DIPs was established C_LIO_LIDIP titers of 7.40E+9 plaque forming units per mL were reached C_LIO_LIA novel tubular cell retention device enabled continuous virus harvesting C_LI

bioengineering↗

bioRxiv2021

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

Defective interfering particles (DIPs) of influenza A virus (IAV) are naturally occurring mutants that comprise an internal deletion in one of their eight viral RNA (vRNA) segments, rendering them propagation-incompetent. Upon co-infection 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 a STV co-infection 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, a sweet spot of 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 than DIPs derived from lowly accumulated DI vRNAs and suggest promising candidates for efficacious antiviral treatment. ImportanceDefective 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 non-homologous viruses, like 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.

evolutionary biology↗