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

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

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. Furthermore, 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 points • An automated perfusion process for production of IAV DIPs was established. • DIP titers of 7.40E + 9 plaque forming units per mL were reached. • A novel tubular cell retention device enabled continuous virus harvesting. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00253-021-11561-y

Determination of the bendability of ductile materials

Understanding fracture in the bending of metal sheet is important but difficult especially for ductile materials were fracture is hard to quantify. New bend tests allow fracture analysis at higher bending strains, however, most of them are impractical for industrial application. This work investigates the link between material failure in bending of brittle and highly ductile materials and local tensile ductility which can be measured in a simple tensile test. For this, a 3-point bend test is developed based on the widely accepted VDA238-100, enabling the fracture of highly ductile aluminium alloys. The failure strain of brittle and ductile aluminium sheets in bending is determined. Uni-axial tensile tests are performed in combination with a digital image correlation (DIC) strain measurement system. Using the surface strain data, obtained from the DIC system during tensile testing and bend testing, a correlation between the local ductility in tensile testing and the fracture strain in bending was identified

Single-Cell Analysis Uncovers a Vast Diversity in Intracellular Viral Defective Interfering RNA Content Affecting the Large Cell-to-Cell Heterogeneity in Influenza A Virus Replication

Virus replication displays a large cell-to-cell heterogeneity; yet, not all sources of this variability are known. Here, we study the effect of defective interfering (DI) particle (DIP) co-infection on cell-to-cell variability in influenza A virus (IAV) replication. DIPs contain a large internal deletion in one of their eight viral RNAs (vRNA) and are, thus, defective in virus replication. Moreover, they interfere with virus replication. Using single-cell isolation and reverse transcription polymerase chain reaction, we uncovered a large between-cell heterogeneity in the DI vRNA content of infected cells, which was confirmed for DI mRNAs by single-cell RNA sequencing. A high load of intracellular DI vRNAs and DI mRNAs was found in low-productive cells, indicating their contribution to the large cell-to-cell variability in virus release. Furthermore, we show that the magnitude of host cell mRNA expression (some factors may inhibit virus replication), but not the ribosome content, may further affect the strength of single-cell virus replication. Finally, we show that the load of viral mRNAs (facilitating viral protein production) and the DI mRNA content are, independently from one another, connected with single-cell virus production. Together, these insights advance single-cell virology research toward the elucidation of the complex multi-parametric origin of the large cell-to-cell heterogeneity in virus infections

Antiviral Activity of Influenza A Virus Defective Interfering Particles against SARS-CoV-2 Replication In Vitro through Stimulation of Innate Immunity

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (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 show only 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

Influenza A virus OP7 defective interfering particles: Cell culture-based production and antiviral efficacy in vivo

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Review on quality assurance along the CFRP value chain - Non-destructive testing of fabrics, preforms and CFRP by HF radio wave techniques

Eddy current testing is well established for non-destructive testing of electrical conductive materials [1]. The development of radio frequency (RF) eddy current technology with frequency ranges up to 100 MHz made it possible to extend the classical fields of application even towards less conductive materials like CFRP [2][3](Table 2). It turns out that RF eddy current technology on CFRP generates a growing number of valuable information for comprehensive material diagnostic. Both permittivity and conductivity of CFRP influence the complex impedance measured with RF eddy current devices. The electrical conductivity contains information about fiber texture like orientations, gaps or undulations in a multilayered material. The permittivity characterization influenced by dielectric properties allows the determination of local curing defects on CFRP e.g. hot spots, thermal impacts or polymer degradation. An explanation for that effect is seen in the measurement frequency range and the capacitive structure of the carbon rovings. Using radio wave frequencies for testing, the effect of displacement currents cannot be neglected anymore. The capacitive structures formed by the carbon rovings is supposed to further strengthen the dielectric influences on eddy current measurement signal [3]. This report gives an overview of several realized applications and should be understood as a general introduction of CFRP testing by HF Radio Wave techniques

Economic precariousness: A new channel in the housing market cycle

Abstract: Demographic and institutional elements, as important drivers of the housing market, should not be neglected since it is not only financial and monetary elements that matter in the case of the housing market. In this context, one relationship, which still remains unclear, is the relationship between the housing and the labour markets. Some research has been undertaken to support the hypothesis that high rates of homeownership lead to high unemployment via increases in the reservation wage. However, further research is needed to address the possible implications of the institutional settings of the labour market in the dynamics of the housing market. The aim of this paper is to bring some light on the link between both markets. In particular, this contribution explains how the housing cycle could be ‘amplified’ via a new channel, i.e. economic precariousness, which is closely related to job insecurity. Subsequently, we provide evidence in the case of five developed economies, Ireland, the Netherlands, Spain, the United Kingdom and the United States, over the period 1985-2013.Not appicabl