Nanoparticle-Supported, Rapid, Digital Quantification of Neutralizing Antibodies Against SARS-CoV-2 Variants.

The measurement of neutralizing immune responses to viral infection is essential, given the heterogeneity of human immunity and the emergence of new virus strains. However, neutralizing antibody (nAb) assays often require high-level biosafety containment, sophisticated instrumentation, and long detection times. Here, as a proof-of-principle, we designed a nanoparticle-supported, rapid, electronic detection (NasRED) assay to assess the neutralizing potency of monoclonal antibodies (mAbs) against SARS-CoV-2.

Coronavirus M Protein Trafficking in Epithelial Cells Utilizes a Myosin Vb Splice Variant and Rab10.

The membrane (M) glycoprotein of coronaviruses (CoVs) serves as the nidus for virion assembly. Using a yeast two-hybrid screen, we identified the interaction of the cytosolic tail of Murine Hepatitis Virus (MHV-CoV) M protein with Myosin Vb (MYO5B), specifically with the alternative splice variant of cellular MYO5B including exon D (MYO5B+D), which mediates interaction with Rab10. When co-expressed in human lung epithelial A549 and canine kidney epithelial MDCK cells, MYO5B+D co-localized with the MHV-CoV M protein, as well as with the M proteins from Porcine Epidemic Diarrhea Virus (PEDV-CoV), Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome 2 (SARS-CoV-2).

Virology-the path forward.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases.

Viral anti-inflammatory serpin reduces immuno-coagulopathic pathology in SARS-CoV-2 mouse models of infection.

SARS-CoV-2 acute respiratory distress syndrome (ARDS) induces uncontrolled lung inflammation and coagulopathy with high mortality. Anti-viral drugs and monoclonal antibodies reduce early COVID-19 severity, but treatments for late-stage immuno-thrombotic syndromes and long COVID are limited. Serine protease inhibitors (SERPINS) regulate activated proteases.

Effect of exportin 1/XPO1 nuclear export pathway inhibition on coronavirus replication.

Nucleocytoplasmic transport of proteins using XPO1 (exportin 1) plays a vital role in cell proliferation and survival. Many viruses also exploit this pathway to promote infection and replication. Thus, inhibiting the XPO1-mediated nuclear export pathway with selective inhibitors has a diverse effect on virus replication by regulating antiviral, proviral, and anti-inflammatory pathways.

Intranasal Immunization with a Vaccinia Virus Vaccine Vector Expressing Pre-Fusion Stabilized SARS-CoV-2 Spike Fully Protected Mice against Lethal Challenge with the Heavily Mutated Mouse-Adapted SARS2-N501Y Strain of SARS-CoV-2.

The Omicron SARS-CoV-2 variant has been designated as a variant of concern because its spike protein is heavily mutated. In particular, the Omicron spike is mutated at five positions (K417, N440, E484, Q493, and N501) that have been associated with escape from neutralizing antibodies induced by either infection with or immunization against the early Washington strain of SARS-CoV-2. The mouse-adapted strain of SARS-CoV-2, SARS2-N501Y, contains a spike that is also heavily mutated, with mutations at four of the five positions in the Omicron spike associated with neutralizing antibody escape (K417, E484, Q493, and N501).

Erratum: An advanced workflow for single-particle imaging with the limited data at an X-ray free-electron laser. Corrigendum.

[This corrects the article DOI: 10.1107/S2052252520012798.].

Intranasal immunization with a vaccinia virus vaccine vector expressing pre-fusion stabilized SARS-CoV-2 spike fully protected mice against lethal challenge with the heavily mutated mouse-adapted SARS2-N501Y strain of SARS-CoV-2.

The Omicron SARS-CoV-2 variant has been designated a variant of concern because its spike protein is heavily mutated. In particular, Omicron spike is mutated at 5 positions (K417, N440, E484, Q493 and N501) that have been associated with escape from neutralizing antibodies induced by either infection with or immunization against the early Washington strain of SARS-CoV-2. The mouse-adapted strain of SARS-CoV-2, SARS2-N501Y , contains a spike that is also heavily mutated, with mutations at 4 of the 5 positions in Omicron spike associated with neutralizing antibody escape (K417, E484, Q493 and N501).

Diffraction data from aerosolized Coliphage PR772 virus particles imaged with the Linac Coherent Light Source.

Single Particle Imaging (SPI) with intense coherent X-ray pulses from X-ray free-electron lasers (XFELs) has the potential to produce molecular structures without the need for crystallization or freezing. Here we present a dataset of 285,944 diffraction patterns from aerosolized Coliphage PR772 virus particles injected into the femtosecond X-ray pulses of the Linac Coherent Light Source (LCLS). Additional exposures with background information are also deposited.

An advanced workflow for single-particle imaging with the limited data at an X-ray free-electron laser.

An improved analysis for single-particle imaging (SPI) experiments, using the limited data, is presented here. Results are based on a study of bacteriophage PR772 performed at the Atomic, Molecular and Optical Science instrument at the Linac Coherent Light Source as part of the SPI initiative. Existing methods were modified to cope with the shortcomings of the experimental data: inaccessibility of information from half of the detector and a small fraction of single hits.

Low-signal limit of X-ray single particle diffractive imaging.

An outstanding question in X-ray single particle imaging experiments has been the feasibility of imaging sub 10-nm-sized biomolecules under realistic experimental conditions where very few photons are expected to be measured in a single snapshot and instrument background may be significant relative to particle scattering. While analyses of simulated data have shown that the determination of an average image should be feasible using Bayesian methods such as the EMC algorithm, this has yet to be demonstrated using experimental data containing realistic non-isotropic instrument background, sample variability and other experimental factors. In this work, we show that the orientation and phase retrieval steps work at photon counts diluted to the signal levels one expects from smaller molecules or with weaker pulses, using data from experimental measurements of 60-nm PR772 viruses.

Evaluation of the performance of classification algorithms for XFEL single-particle imaging data.

Using X-ray free-electron lasers (XFELs), it is possible to determine three-dimensional structures of nanoscale particles using single-particle imaging methods. Classification algorithms are needed to sort out the single-particle diffraction patterns from the large amount of XFEL experimental data. However, different methods often yield inconsistent results.

Single-particle imaging without symmetry constraints at an X-ray free-electron laser.

The analysis of a single-particle imaging (SPI) experiment performed at the AMO beamline at LCLS as part of the SPI initiative is presented here. A workflow for the three-dimensional virus reconstruction of the PR772 bacteriophage from measured single-particle data is developed. It consists of several well defined steps including single-hit diffraction data classification, refined filtering of the classified data, reconstruction of three-dimensional scattered intensity from the experimental diffraction patterns by orientation determination and a final three-dimensional reconstruction of the virus electron density without symmetry constraints.

Rapid sample delivery for megahertz serial crystallography at X-ray FELs.

Liquid microjets are a common means of delivering protein crystals to the focus of X-ray free-electron lasers (FELs) for serial femtosecond crystallography measurements. The high X-ray intensity in the focus initiates an explosion of the microjet and sample. With the advent of X-ray FELs with megahertz rates, the typical velocities of these jets must be increased significantly in order to replenish the damaged material in time for the subsequent measurement with the next X-ray pulse.

Author Correction: Supersaturation-controlled microcrystallization and visualization analysis for serial femtosecond crystallography.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Supersaturation-controlled microcrystallization and visualization analysis for serial femtosecond crystallography.

Time-resolved serial femtosecond crystallography with X-ray free electron laser (XFEL) holds the potential to view fast reactions occurring at near-physiological temperature. However, production and characterization of homogeneous micron-sized protein crystals at high density remain a bottleneck, due to the lack of the necessary equipments in ordinary laboratories. We describe here supersaturation-controlled microcrystallization and visualization and analysis tools that can be easily used in any laboratory.

Merging single-shot XFEL diffraction data from inorganic nanoparticles: a new approach to size and orientation determination.

X-ray free-electron lasers (XFELs) provide new opportunities for structure determination of biomolecules, viruses and nanomaterials. With unprecedented peak brilliance and ultra-short pulse duration, XFELs can tolerate higher X-ray doses by exploiting the femtosecond-scale exposure time, and can thus go beyond the resolution limits achieved with conventional X-ray diffraction imaging techniques. Using XFELs, it is possible to collect scattering information from single particles at high resolution, however particle heterogeneity and unknown orientations complicate data merging in three-dimensional space.

Corrigendum: Diffraction data of core-shell nanoparticles from an X-ray free electron laser.

This corrects the article DOI: 10.1038/sdata.2017.

Conformational landscape of a virus by single-particle X-ray scattering.

Using a manifold-based analysis of experimental diffraction snapshots from an X-ray free electron laser, we determine the three-dimensional structure and conformational landscape of the PR772 virus to a detector-limited resolution of 9 nm. Our results indicate that a single conformational coordinate controls reorganization of the genome, growth of a tubular structure from a portal vertex and release of the genome. These results demonstrate that single-particle X-ray scattering has the potential to shed light on key biological processes.