Biology subjects

Reichl, U.

Publications and source records attributed to Reichl, U..

8 recordsLinked to original sources

bioRxiv2021

Interferon induction and not replication interference mainly determines anti-influenza virus activity of defective interfering particles

Defective interfering (DI) RNAs arise during influenza virus replication, can be packaged into particles (DIPs) and suppress spread of wildtype (WT) virus. However, the molecular signatures of DI RNAs and the mechanism underlying antiviral activity are incompletely understood. Here, we show that any central deletion is sufficient to convert a viral RNA into a DI RNA and that antiviral activity of DIPs is inversely correlated with DI RNA length when induction of the interferon (IFN) system is disfavored. When induction of the IFN system was allowed, it was found to be the major contributor to DIP antiviral activity. Finally, while both DIPs and influenza virus triggered expression of IFN-stimulated genes (ISG) only virus stimulated robust expression of IFN. These results suggest a key role of innate immune activation in DIP antiviral activity and point towards previously unappreciated differences in DIP- and influenza virus-mediated activation of the effector functions of the IFN system. ImportanceDefective interfering (DI) RNAs naturally arise during RNA virus infection. They can be packaged into defective interfering particles (DIPs) and exert antiviral activity by suppressing viral genome replication and inducing the interferon (IFN) system. However, inhibition of influenza virus infection by DI RNAs has been incompletely understood. Here, we show that induction of the IFN system and not suppression of genome replication is the major determinant of DIP antiviral activity. Moreover, we demonstrate that DIPs induce IFN-stimulated genes (ISG) but not IFN with high efficiency. Our results reveal unexpected major differences in influenza virus and DIP activation of the IFN system, a key barrier against viral infection, and provide insights into how to design DIPs for antiviral therapy.

molecular biology↗

bioRxiv2021

MPA_Pathway_Tool: User-friendly, automatic assignment of microbial community data on metabolic pathways

MotivationTaxonomic and functional characterization of microbial communities from diverse environments such as the human gut or biogas plants by multi-omics methods plays an ever more important role. Researchers assign all identified genes, transcripts, or proteins to biological pathways to better understand the function of single species and microbial communities. However, due to the versatility of microbial metabolism and a still increasing number of new biological pathways, linkage to standard pathway maps such as the KEGG (Kyoto Encyclopedia of Genes and Genomes) central carbon metabolism is often problematic. ResultsWe successfully implemented and validated a new user-friendly, stand-alone web application, the MPA_Pathway_Tool. It consists of two parts, called Pathway-Creator and Pathway-Calculator. The Pathway-Creator enables an easy setup of user-defined pathways with specific taxonomic constraints. The Pathway-Calculator automatically maps microbial community data from multiple measurements on selected pathways and visualizes the results. Availability and ImplementationThe MPA_Pathway_Tool is implemented in Java and ReactJS. It is freely available on http://mpa-pathwaymapper.ovgu.de/. Further documentation and the complete source code are available on GitHub (https://github.com/danielwalke/MPA_Pathway_Tool). Contactdaniel.walke@ovgu.de, mailto:heyer@mpi-magdeburg.mpg.de heyer@mpi-magdeburg.mpg.de Supplementary InformationAdditional files and images are available at MDPI online. Highlightsuser-friendly generation of pathways, re-using of existent metabolic pathways, automated mapping of data

bioinformatics↗

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

Cell-free glycoengineering of the recombinant SARS-CoV-2 spike glycoprotein

The baculovirus-insect cell expression system is readily utilized to produce viral glycoproteins for research as well as for subunit vaccines and vaccine candidates, for instance against SARS-CoV-2 infections. However, the glycoforms of recombinant proteins derived from this expression system are inherently different from mammalian cell-derived glycoforms with mainly complex-type N-glycans attached, and the impact of these differences in protein glycosylation on the immunogenicity is severely underinvestigated. This applies also to the SARS-CoV-2 spike glycoprotein, which is the antigen target of all licensed vaccines and vaccine candidates including virus like particles and subunit vaccines that are variants of the spike protein. Here, we expressed the transmembrane-deleted human {beta}-1,2 N-acetlyglucosamintransferases I and II (MGAT1{triangleup}TM and MGAT2{triangleup}TM) and the {beta}-1,4-galactosyltransferase (GalT{triangleup}TM) in E. coli to in-vitro remodel the N-glycans of a recombinant SARS-CoV-2 spike glycoprotein derived from insect cells. In a cell-free sequential one-pot reaction, fucosylated and afucosylated paucimannose-type N-glycans were converted to complex-type galactosylated N-glycans. In the future, this in-vitro glycoengineering approach can be used to efficiently generate a wide range of N-glycans on antigens considered as vaccine candidates for animal trials and preclinical testing to better characterize the impact of N-glycosylation on immunity and to improve the efficacy of protein subunit vaccines.

synthetic biology↗

bioRxiv2021

Integrated cycles for urban biomass as a strategy to promote a CO2-neutral society - A feasibility study

Progressive global warming is one of the biggest challenges civilization is facing today. The establishment of a carbon dioxide (CO2)-neutral society based on sustainable value creation cycles is required to stop this development. The Integrated Cycles for Urban Biomass (ICU) concept is a new concept towards a CO2-neutral society. The integration of closed biomass cycles into residential buildings enable efficient resource utilization and avoid transport of biowaste. In this scenario, biowaste is degraded on-site into biogas that is converted into heat and electricity. The liquid fermentation residues are upgraded by nitrification processes (e.g., by a soiling(R)-process, EP3684909A1) to refined fertilizer, which can be used subsequently in house-internal gardens to produce fresh food for residents. Whereas this scenario sounds promising, comprehensive evaluations of produced amounts of biogas and food, saved CO2 and costs as well as social-cultural aspects are lacking. To assess these points, a feasibility study was performed, which estimated the material and energy flows based on simulations of the biogas process and food production. The calculations show that a residential complex with 100 persons can generate 21 % of the annual power (electrical and heat) consumption from the accumulated biowaste. The nitrogen (N) in the liquid fermentation residues enables the production of up to 6.3 t of fresh mass of lettuce per year in a 70 m2 professional hydroponic production area. The amount of produced lettuce corresponds to the amount of calories required to feed four persons for one year. Additionally, due to the reduction of biowaste transport and the in-house food and fertilizer production, 6 468 kg CO2-equivalent (CO2-eq) per year are saved compared to a conventional building. While the ICU concept is technically feasible, its costs are still 1.5 times higher than the revenues. However, the model predictions show that the ICU concept becomes economically feasible in case food prices further increase and ICU is implemented at larger scale, e.g.; at the district level. Finally, this study demonstrates that the ICU implementation can be a worthwile contribution towards a sustainable CO2-neutral society and enable to decrease the demand for agricultural land.

bioengineering↗

bioRxiv2021

Critical Assessment of Metaproteome Investigation (CAMPI): a Multi-Lab Comparison of Established Workflows

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carried out the first community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluated the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample. We observed that variability at the peptide level was predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappeared at the protein group level. While differences were observed for predicted community composition, similar functional profiles were obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.

microbiology↗

bioRxiv2021

Antiviral activity of influenza A virus defective interfering particles against SARS-CoV-2 replication in vitro through stimulation of innate immunity

SARS-CoV-2 causing COVID-19 emerged in late 2019 and resulted in a devastating pandemic. Although the first approved vaccines were already administered by the end of 2020, worldwide vaccine availability is still limited. Moreover, immune escape variants of the virus are emerging against which the current vaccines may confer only limited protection. Further, existing antivirals and treatment options against COVID-19 only show limited efficacy. Influenza A virus (IAV) defective interfering particles (DIPs) were previously proposed not only for antiviral treatment of the influenza disease but also for pan-specific treatment of interferon (IFN)-sensitive respiratory virus infections. To investigate the applicability of IAV DIPs as an antiviral for the treatment of COVID-19, we conducted in vitro co-infection experiments with cell culture-derived DIPs and the IFN-sensitive SARS-CoV-2 in human lung cells. We show that treatment with IAV DIPs leads to complete abrogation of SARS-CoV-2 replication. Moreover, this inhibitory effect was dependent on janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling. Further, our results suggest boosting of IFN-induced antiviral activity by IAV DIPs as a major contributor in suppressing SARS-CoV-2 replication. Thus, we propose IAV DIPs as an effective antiviral agent for treatment of COVID-19, and potentially also for suppressing the replication of new variants of SARS-CoV-2.

microbiology↗

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↗