Integrated Laboratory Demonstrations of Multi-Object Adaptive Optics on a Simulated 10-Meter Telescope at Visible Wavelengths

One important frontier for astronomical adaptive optics (AO) involves methods such as Multi-Object AO and Multi-Conjugate AO that have the potential to give a significantly larger field of view than conventional AO techniques. A second key emphasis over the next decade will be to push astronomical AO to visible wavelengths. We have conducted the first laboratory simulations of wide-field, laser guide star adaptive optics at visible wavelengths on a 10-meter-class telescope. These experiments, utilizing the UCO/Lick Observatory's Multi-Object / Laser Tomography Adaptive Optics (MOAO/LTAO) testbed, demonstrate new techniques in wavefront sensing and control that are crucial to future on-sky MOAO systems. We (1) test and confirm the feasibility of highly accurate atmospheric tomography with laser guide stars, (2) demonstrate key innovations allowing open-loop operation of Shack-Hartmann wavefront sensors (with errors of ~30 nm) as will be needed for MOAO, and (3) build a complete error budget model describing system performance. The AO system maintains a performance of 32.4% Strehl on-axis, with 24.5% and 22.6% at 10" and 15", respectively, at a science wavelength of 710 nm (R-band) over the equivalent of 0.8 seconds of simulation. The MOAO-corrected field of view is ~25 times larger in area than that limited by anisoplanatism at R-band. Our error budget is composed of terms verified through independent, empirical experiments. Error terms arising from calibration inaccuracies and optical drift are comparable in magnitude to traditional terms like fitting error and tomographic error. This makes a strong case for implementing additional calibration facilities in future AO systems, including accelerometers on powered optics, 3D turbulators, telescope and LGS simulators, and external calibration ports for deformable mirrors.Comment: 29 pages, 11 figures, submitted to PAS

Influenza A virus propagation in MDCK: Intracellular virus replication, virus release and cell-cycle preferential infection analysis

Cell culture-based processes for vaccine manufacturing offer advantages over egg-based processes in terms of product uniformity and sterility, production time and scaling up capacity1,2. Regarding influenza vaccines, MDCK cells are one of the host cell lines currently used to manufacture licensed products; however, virus titers remain lower compared to those obtained in eggs and further increase of specific and volumetric yields is required. To identify bottlenecks in influenza A virus (IAV) production, we thoroughly studied IAV replication in MDCK cells. For this, we analyzed different features of the infection process such as viral RNA replication, intracellular localization of viral components, virus release and morphology of the particles, and the preferential infection in different cell-cycle phases. Using synchronous infections, we found that production of infectious particles dropped much earlier than the production of total particles. Furthermore, we found that the maximum virus release rate was reached when all viral RNA species attained their maximum intracellular concentration. Using qPCR we determined that the vRNA maximum concentration per cell was 10‑fold higher than the specific viral titers obtained, indicating that vRNA concentration does not limit IAV particle assembly. When we evaluated the morphology of particles released using electron microscopy, we observed that a higher fraction of the viral particles produced at late times possess an abnormal morphology, concurring with the increased production of non-infectious viruses. Using imaging flow cytometry, we determined that the export of influenza viral genome segments (ribonucleoprotein complexes, vRNPs) from the nucleus to the cytoplasm strongly correlated with the onset of virus release. However, our results also suggest that the induction of apoptosis caused that virus assembly became deficient producing more non-infectious particles at late infection times. Lastly, using low MOI infections and imaging flow cytometry, we found that -in contrast to previous publications- IAV did not preferentially infect a specific cell cycle phase and no cell cycle arrest induction was observed during the time frame of the experiment (9 hpi). In summary, the data presented here offers a comprehensive overview of the dynamics of IAV infection in MDCK and might contribute to the development of molecular or cell culture-based strategies to improve IAV production in MDCK cells. 1 Gallo-Ramírez et al, 2015. Exp. Rev. Vaccines 14 (9). 2 Pardue et al. 2011. Exp. Rev. Vaccines 10 (8)

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

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

Finitely generated free Heyting algebras via Birkhoff duality and coalgebra

Algebras axiomatized entirely by rank 1 axioms are algebras for a functor and thus the free algebras can be obtained by a direct limit process. Dually, the final coalgebras can be obtained by an inverse limit process. In order to explore the limits of this method we look at Heyting algebras which have mixed rank 0-1 axiomatizations. We will see that Heyting algebras are special in that they are almost rank 1 axiomatized and can be handled by a slight variant of the rank 1 coalgebraic methods

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