Deep mining of the Sequence Read Archive reveals major genetic innovations in coronaviruses and other nidoviruses of aquatic vertebrates.

Virus discovery by genomics and metagenomics empowered studies of viromes, facilitated characterization of pathogen epidemiology, and redefined our understanding of the natural genetic diversity of viruses with profound functional and structural implications. Here we employed a data-driven virus discovery approach that directly queries unprocessed sequencing data in a highly parallelized way and involves a targeted viral genome assembly strategy in a wide range of sequence similarity. By screening more than 269,000 datasets of numerous authors from the Sequence Read Archive and using two metrics that quantitatively assess assembly quality, we discovered 40 nidoviruses from six virus families whose members infect vertebrate hosts. They form 13 and 32 putative viral subfamilies and genera, respectively, and include 11 coronaviruses with bisegmented genomes from fishes and amphibians, a giant 36.1 kilobase coronavirus genome with a duplicated spike glycoprotein (S) gene, 11 tobaniviruses and 17 additional corona-, arteri-, cremega-, nanhypo- and nangoshaviruses. Genome segmentation emerged in a single evolutionary event in the monophyletic lineage encompassing the subfamily Pitovirinae. We recovered the bisegmented genome sequences of two coronaviruses from RNA samples of 69 infected fishes and validated the presence of poly(A) tails at both segments using 3'RACE PCR and subsequent Sanger sequencing. We report a genetic linkage between accessory and structural proteins whose phylogenetic relationships and evolutionary distances are incongruent with the phylogeny of replicase proteins. We rationalize these observations in a model of inter-family S recombination involving at least five ancestral corona- and tobaniviruses of aquatic hosts. In support of this model, we describe an individual fish co-infected with members from the families Coronaviridae and Tobaniviridae. Our results expand the scale of the known extraordinary evolutionary plasticity in nidoviral genome architecture and call for revisiting fundamentals of genome expression, virus particle biology, host range and ecology of vertebrate nidoviruses.

The harms of promoting the lab leak hypothesis for SARS-CoV-2 origins without evidence.

Science is humanity's best insurance against threats from nature, but it is a fragile enterprise that must be nourished and protected. The preponderance of scientific evidence indicates a natural origin for SARS-CoV-2. Yet, the theory that SARS-CoV-2 was engineered in and escaped from a lab dominates media attention, even in the absence of strong evidence. We discuss how the resulting anti-science movement puts the research community, scientific research, and pandemic preparedness at risk.

Validating the inactivation of viral pathogens with a focus on SARS-CoV-2 to safely transfer samples from high-containment laboratories.

Introduction: Pathogen leak from a high-containment laboratory seriously threatens human safety, animal welfare, and environmental security. Transportation of pathogens from a higher (BSL4 or BSL3) to a lower (BSL2) containment laboratory for downstream experimentation requires complete pathogen inactivation. Validation of pathogen inactivation is necessary to ensure safety during transportation. This study established a validation strategy for virus inactivation. Methods: SARS-CoV-2 wild type, delta, and omicron variants underwent heat treatment at 95°C for 10 minutes using either a hot water bath or a thermocycler. To validate the inactivation process, heat-treated viruses, and untreated control samples were incubated with A549-hACE2 and Vero E6-TMPRSS2-T2A-ACE2 cells. The cells were monitored for up to 72 hours for any cytopathic effects, visually and under a microscope, and for virus genome replication via RT-qPCR. The quality of post-treated samples was assessed for suitability in downstream molecular testing applications. Results: Heat treatment at 95°C for 10 minutes effectively inactivated SARS-CoV-2 variants. The absence of cytopathic effects, coupled with the inability of virus genome replication, validated the efficacy of the inactivation process. Furthermore, the heat-treated samples proved to be qualified for COVID-19 antigen testing, RT-qPCR, and whole-genome sequencing. Discussion: By ensuring the safety of sample transportation for downstream experimentation, this validation approach enhances biosecurity measures. Considerations for potential limitations, comparisons with existing inactivation methods, and broader implications of the findings are discussed.

A Novel Non-Destructive Rapid Tool for Estimating Amino Acid Composition and Secondary Structures of Proteins in Solution.

Amino-acid protein composition plays an important role in biology, medicine, and nutrition. Here, a groundbreaking protein analysis technique that quickly estimates amino acid composition and secondary structure across various protein sizes, while maintaining their natural states is introduced and validated. This method combines multivariate statistics and the thermostable Raman interaction profiling (TRIP) technique, eliminating the need for complex preparations. In order to validate the approach, the Raman spectra are constructed of seven proteins of varying sizes by utilizing their amino acid frequencies and the Raman spectra of individual amino acids. These constructed spectra exhibit a close resemblance to the actual measured Raman spectra. Specific vibrational modes tied to free amino and carboxyl termini of the amino acids disappear as signals linked to secondary structures emerged under TRIP conditions. Furthermore, the technique is used inversely to successfully estimate amino acid compositions and secondary structures of unknown proteins across a range of sizes, achieving impressive accuracy ranging between 1.47% and 5.77% of root mean square errors (RMSE). These results extend the uses for TRIP beyond interaction profiling, to probe amino acid composition and structure.

Discovery of First-in-Class PROTAC Degraders of SARS-CoV-2 Main Protease.

We have witnessed three coronavirus (CoV) outbreaks in the past two decades, including the COVID-19 pandemic caused by SARS-CoV-2. Main protease (MPro), a highly conserved protease among various CoVs, is essential for viral replication and pathogenesis, making it a prime target for antiviral drug development. Here, we leverage proteolysis targeting chimera (PROTAC) technology to develop a new class of small-molecule antivirals that induce the degradation of SARS-CoV-2 MPro. Among them, MPD2 was demonstrated to effectively reduce MPro protein levels in 293T cells, relying on a time-dependent, CRBN-mediated, and proteasome-driven mechanism. Furthermore, MPD2 exhibited remarkable efficacy in diminishing MPro protein levels in SARS-CoV-2-infected A549-ACE2 cells. MPD2 also displayed potent antiviral activity against various SARS-CoV-2 strains and exhibited enhanced potency against nirmatrelvir-resistant viruses. Overall, this proof-of-concept study highlights the potential of targeted protein degradation of MPro as an innovative approach for developing antivirals that could fight against drug-resistant viral variants.

Expression dynamics of the aplysia abyssovirus.

Two recent studies documented the genome of a novel, extremely large (35.9 kb), nidovirus in RNA sequence databases from the marine neural model Aplysia californica. The goal of the present study was to document the distribution and transcriptional dynamics of this virus, Aplysia abyssovirus 1 (AAbV), in maricultured and wild animals. We confirmed previous findings that AAbV RNA is widespread and reaches extraordinary levels in apparently healthy animals. Transmission electron microscopy identified viral replication factories in ciliated gill epithelial cells but not in neurons where viral RNA is most highly expressed. Viral transcripts do not exhibit evidence of discontinuous RNA synthesis as in coronaviruses but are consistent with production of a single leaderless subgenomic RNA, as in the Gill-associated virus of Penaeus monodon. Splicing patterns in chronically infected adults suggested high levels of defective genomes, possibly explaining the lack of obvious disease signs in high viral load animals.

SARS-CoV-2 Main Protease Inhibitors That Leverage Unique Interactions with the Solvent Exposed S3 Site of the Enzyme.

The main protease (MPro) of SARS-CoV-2 is crucial for the virus's replication and pathogenicity. Its active site is characterized by four distinct pockets (S1, S2, S4, and S1-3') and a solvent-exposed S3 site for accommodating a protein substrate. During X-ray crystallographic analyses of MPro bound with dipeptide inhibitors containing a flexible N-terminal group, we often observed an unexpected binding mode. Contrary to the anticipated engagement with the deeper S4 pocket, the N-terminal group frequently assumed a twisted conformation, positioning it for interactions with the S3 site and the inhibitor component bound at the S1 pocket. Capitalizing on this observation, we engineered novel inhibitors to engage both S3 and S4 sites or to adopt a rigid conformation for selective S3 site binding. Several new inhibitors demonstrated high efficacy in MPro inhibition. Our findings underscore the importance of the S3 site's unique interactions in the design of future MPro inhibitors as potential COVID-19 therapeutics.

Azapeptides with unique covalent warheads as SARS-CoV-2 main protease inhibitors.

The main protease (MPro) of SARS-CoV-2, the causative agent of COVID-19, is a pivotal nonstructural protein critical for viral replication and pathogenesis. Its protease function relies on three active site pockets for substrate recognition and a catalytic cysteine for enzymatic activity. To develop potential SARS-CoV-2 antivirals, we successfully synthesized a diverse range of azapeptide inhibitors with various covalent warheads to target MPro's catalytic cysteine. Our characterization identified potent MPro inhibitors, including MPI89 that features an aza-2,2-dichloroacetyl warhead with a remarkable EC50 value of 10 nM against SARS-CoV-2 infection in ACE2+ A549 cells and a selective index of 875. MPI89 is also remarkably selective and shows no potency against SARS-CoV-2 papain-like protease and several human proteases. Crystallography analyses demonstrated that these inhibitors covalently engaged the catalytic cysteine and used the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 stands as one of the most potent MPro inhibitors, suggesting the potential for further exploration of azapeptides and the aza-2,2-dichloroacetyl warhead for developing effective therapeutics against COVID-19.