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High-resolution optical fluorescence microscopies and, in particular, super-resolution fluorescence microscopy, are rapidly adopting highly sensitive cameras as their preferred photodetectors. Camera-based parallel detection facilitates high-speed live cell imaging with the highest spatial resolution. Here, we show that the drive to use ever more sensitive, photon-counting image sensors in cameras can, however, have detrimental effects on the spatial resolution of the resulting images. This is particularly noticeable in applications that demand a high space-bandwidth product, where the image magnification is close to the Nyquist sampling limit of the sensor. Most scientists will often select image sensors based on parameters such as pixel size, quantum efficiency, signal-to-noise performance, dynamic range, and frame rate of the sensor. A parameter that is, however, typically overlooked is the sensor's modulation transfer function (MTF). We have determined the wavelength-specific MTF of front- and back-illuminated image sensors and evaluated how it affects the spatial resolution that can be achieved in high-resolution fluorescence microscopy modalities. We find significant differences in image sensor performance that cause the resulting spatial resolution to vary by up to 28%. This result shows that the choice of image sensor has a significant impact on the imaging performance of all camera-based optical microscopy modalities., (© 2024. The Author(s).)

Vaccine development is essential for pandemic preparedness. We previously conducted a Phase 1 clinical trial of the vector vaccine candidate MVA-MERS-S against the Middle East respiratory syndrome coronavirus (MERS-CoV), expressing its full spike glycoprotein (MERS-CoV-S), as a homologous two-dose regimen (Days 0 and 28). Here, we evaluate the safety (primary objective) and immunogenicity (secondary and exploratory objectives: magnitude and characterization of vaccine-induced humoral responses) of a third vaccination with MVA-MERS-S in a subgroup of trial participants one year after primary immunization. MVA-MERS-S booster vaccination is safe and well-tolerated. Both binding and neutralizing anti-MERS-CoV antibody titers increase substantially in all participants and exceed maximum titers observed after primary immunization more than 10-fold. We identify four immunogenic IgG epitopes, located in the receptor-binding domain (RBD, n = 1) and the S2 subunit (n = 3) of MERS-CoV-S. The level of baseline anti-human coronavirus antibody titers does not impact the generation of anti-MERS-CoV antibody responses. Our data support the rationale of a booster vaccination with MVA-MERS-S and encourage further investigation in larger trials. Trial registration: Clinicaltrials.gov NCT03615911., (© 2022. The Author(s).)

Middle East respiratory syndrome coronavirus (MERS-CoV) is enzootic in dromedary camels across the Middle East and Africa. Virus-induced pneumonia in humans results from animal contact, with a potential for limited onward transmission. Phenotypic changes have been suspected after a novel recombinant clade (lineage 5) caused large nosocomial outbreaks in Saudi Arabia and South Korea in 2016. However, there has been no functional assessment. Here we perform a comprehensive in vitro and ex vivo comparison of viruses from parental and recombinant virus lineages (lineage 3, n = 7; lineage 4, n = 8; lineage 5, n = 9 viruses) from Saudi Arabia, isolated immediately before and after the shift toward lineage 5. Replication of lineage 5 viruses is significantly increased. Transcriptional profiling finds reduced induction of immune genes IFNB1, CCL5, and IFNL1 in lung cells infected with lineage 5 strains. Phenotypic differences may be determined by IFN antagonism based on experiments using IFN receptor knock out and signaling inhibition. Additionally, lineage 5 is more resilient against IFN pre-treatment of Calu-3 cells (ca. 10-fold difference in replication). This phenotypic change associated with lineage 5 has remained undiscovered by viral sequence surveillance, but may be a relevant indicator of pandemic potential., (© 2021. The Author(s).)

Super-resolution structured illumination microscopy (SIM) has become a widely used method for biological imaging. Standard reconstruction algorithms, however, are prone to generate noise-specific artifacts that limit their applicability for lower signal-to-noise data. Here we present a physically realistic noise model that explains the structured noise artifact, which we then use to motivate new complementary reconstruction approaches. True-Wiener-filtered SIM optimizes contrast given the available signal-to-noise ratio, and flat-noise SIM fully overcomes the structured noise artifact while maintaining resolving power. Both methods eliminate ad hoc user-adjustable reconstruction parameters in favor of physical parameters, enhancing objectivity. The new reconstructions point to a trade-off between contrast and a natural noise appearance. This trade-off can be partly overcome by further notch filtering but at the expense of a decrease in signal-to-noise ratio. The benefits of the proposed approaches are demonstrated on focal adhesion and tubulin samples in two and three dimensions, and on nanofabricated fluorescent test patterns.

Viruses manipulate cellular metabolism and macromolecule recycling processes like autophagy. Dysregulated metabolism might lead to excessive inflammatory and autoimmune responses as observed in severe and long COVID-19 patients. Here we show that SARS-CoV-2 modulates cellular metabolism and reduces autophagy. Accordingly, compound-driven induction of autophagy limits SARS-CoV-2 propagation. In detail, SARS-CoV-2-infected cells show accumulation of key metabolites, activation of autophagy inhibitors (AKT1, SKP2) and reduction of proteins responsible for autophagy initiation (AMPK, TSC2, ULK1), membrane nucleation, and phagophore formation (BECN1, VPS34, ATG14), as well as autophagosome-lysosome fusion (BECN1, ATG14 oligomers). Consequently, phagophore-incorporated autophagy markers LC3B-II and P62 accumulate, which we confirm in a hamster model and lung samples of COVID-19 patients. Single-nucleus and single-cell sequencing of patient-derived lung and mucosal samples show differential transcriptional regulation of autophagy and immune genes depending on cell type, disease duration, and SARS-CoV-2 replication levels. Targeting of autophagic pathways by exogenous administration of the polyamines spermidine and spermine, the selective AKT1 inhibitor MK-2206, and the BECN1-stabilizing anthelmintic drug niclosamide inhibit SARS-CoV-2 propagation in vitro with IC 50 values of 136.7, 7.67, 0.11, and 0.13 μM, respectively. Autophagy-inducing compounds reduce SARS-CoV-2 propagation in primary human lung cells and intestinal organoids emphasizing their potential as treatment options against COVID-19.

To estimate the seroprevalence and temporal course of SARS-CoV-2 neutralizing antibodies, we embedded a multi-tiered seroprevalence survey within an ongoing community-based cohort study in Bonn, Germany. We first assessed anti-SARS-CoV-2 immunoglobulin G levels with an immunoassay, followed by confirmatory testing of borderline and positive test results with a recombinant spike-based immunofluorescence assay and a plaque reduction neutralization test (PRNT). Those with a borderline or positive immunoassay result were retested after 4 to 5 months. At baseline, 4771 persons participated (88% response rate). Between April 24 th and June 30 th , 2020, seroprevalence was 0.97% (95% CI: 0.72-1.30) by immunoassay and 0.36% (95% CI: 0.21-0.61) when considering only those with two additional positive confirmatory tests. Importantly, about 20% of PRNT+ individuals lost their neutralizing antibodies within five months. Here, we show that neutralizing antibodies are detectable in only one third of those with a positive immunoassay result, and wane relatively quickly.

Autophagy is an essential cellular process affecting virus infections and other diseases and Beclin1 (BECN1) is one of its key regulators. Here, we identified S-phase kinase-associated protein 2 (SKP2) as E3 ligase that executes lysine-48-linked poly-ubiquitination of BECN1, thus promoting its proteasomal degradation. SKP2 activity is regulated by phosphorylation in a hetero-complex involving FKBP51, PHLPP, AKT1, and BECN1. Genetic or pharmacological inhibition of SKP2 decreases BECN1 ubiquitination, decreases BECN1 degradation and enhances autophagic flux. Middle East respiratory syndrome coronavirus (MERS-CoV) multiplication results in reduced BECN1 levels and blocks the fusion of autophagosomes and lysosomes. Inhibitors of SKP2 not only enhance autophagy but also reduce the replication of MERS-CoV up to 28,000-fold. The SKP2-BECN1 link constitutes a promising target for host-directed antiviral drugs and possibly other autophagy-sensitive conditions.

Super-resolved structured illumination microscopy (SR-SIM) is among the fastest fluorescence microscopy techniques capable of surpassing the optical diffraction limit. Current custom-build instruments are able to deliver two-fold resolution enhancement with high acquisition speed. SR-SIM is usually a two-step process, with raw-data acquisition and subsequent, time-consuming post-processing for image reconstruction. In contrast, wide-field and (multi-spot) confocal techniques produce high-resolution images instantly. Such immediacy is also possible with SR-SIM, by tight integration of a video-rate capable SIM with fast reconstruction software. Here we present instant SR-SIM by VIGOR (Video-rate Immediate GPU-accelerated Open-Source Reconstruction). We demonstrate multi-color SR-SIM at video frame-rates, with less than 250 ms delay between measurement and reconstructed image display. This is achieved by modifying and extending high-speed SR-SIM image acquisition with a new, GPU-enhanced, network-enabled image-reconstruction software. We demonstrate high-speed surveying of biological samples in multiple colors and live imaging of moving mitochondria as an example of intracellular dynamics.

Linear 2D- or 3D-structured illumination microscopy (SIM or3D-SIM, respectively) enables multicolor volumetric imaging of fixed and live specimens with subdiffraction resolution in all spatial dimensions. However, the reliance of SIM on algorithmic post-processing renders it particularly sensitive to artifacts that may reduce resolution, compromise data and its interpretations, and drain resources in terms of money and time spent. Here we present a protocol that allows users to generate high-quality SIM data while accounting and correcting for common artifacts. The protocol details preparation of calibration bead slides designed for SIM-based experiments, the acquisition of calibration data, the documentation of typically encountered SIM artifacts and corrective measures that should be taken to reduce them. It also includes a conceptual overview and checklist for experimental design and calibration decisions, and is applicable to any commercially available or custom platform. This protocol, plus accompanying guidelines, allows researchers from students to imaging professionals to create an optimal SIM imaging environment regardless of specimen type or structure of interest. The calibration sample preparation and system calibration protocol can be executed within 1-2 d.

Super-resolved structured illumination microscopy (SR-SIM) is an important tool for fluorescence microscopy. SR-SIM microscopes perform multiple image acquisitions with varying illumination patterns, and reconstruct them to a super-resolved image. In its most frequent, linear implementation, SR-SIM doubles the spatial resolution. The reconstruction is performed numerically on the acquired wide-field image data, and thus relies on a software implementation of specific SR-SIM image reconstruction algorithms. We present fairSIM, an easy-to-use plugin that provides SR-SIM reconstructions for a wide range of SR-SIM platforms directly within ImageJ. For research groups developing their own implementations of super-resolution structured illumination microscopy, fairSIM takes away the hurdle of generating yet another implementation of the reconstruction algorithm. For users of commercial microscopes, it offers an additional, in-depth analysis option for their data independent of specific operating systems. As a modular, open-source solution, fairSIM can easily be adapted, automated and extended as the field of SR-SIM progresses.

The large virus family Paramyxoviridae includes some of the most significant human and livestock viruses, such as measles-, distemper-, mumps-, parainfluenza-, Newcastle disease-, respiratory syncytial virus and metapneumoviruses. Here we identify an estimated 66 new paramyxoviruses in a worldwide sample of 119 bat and rodent species (9,278 individuals). Major discoveries include evidence of an origin of Hendra- and Nipah virus in Africa, identification of a bat virus conspecific with the human mumps virus, detection of close relatives of respiratory syncytial virus, mouse pneumonia- and canine distemper virus in bats, as well as direct evidence of Sendai virus in rodents. Phylogenetic reconstruction of host associations suggests a predominance of host switches from bats to other mammals and birds. Hypothesis tests in a maximum likelihood framework permit the phylogenetic placement of bats as tentative hosts at ancestral nodes to both the major Paramyxoviridae subfamilies (Paramyxovirinae and Pneumovirinae). Future attempts to predict the emergence of novel paramyxoviruses in humans and livestock will have to rely fundamentally on these data.