Regulation of virion production by the ORF8 signal peptide across SARS-CoV-2 variants (preprint)

The open reading frame 8 (ORF8), an accessory protein of SARS-CoV-2, is prone to deletions and mutations across different viral variants, which was first described in several Singapore variants. The reason why viral evolution favors loss or inactivation of ORF8 is not fully understood, although the effects of ORF8 on inflammation, immune evasion, and disease severity have been described. Here we show using clinical ORF8 deficient viral isolates, virus like particles (VLPs) and viral replicons that ORF8 expression dampens viral particle production. ORF8 physically interacts with the viral Spike protein and induces Golgi fragmentation, overall contributing to less virus particle production. Using systematic ORF8 deletions, we mapped the particle reducing function to its N terminal signal peptide. Interestingly, this part of ORF8 is severely truncated in the recent XBB.1.5 variant, and when restored, suppresses viral particle production in the context of the entire viral genome. Collectively, our data support the model that evolutionary pressure exists to delete ORF8 sequence and expression across SARS-CoV-2 variants to fully enable viral particle production.

Assembly reactions of SARS-CoV-2 nucleocapsid protein with nucleic acid (preprint)

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-) protein into ribonucleoprotein particles (RNPs), 38{+/-}10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining mutant proteins in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multi-valent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N- protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

Rapid assembly of SARS-CoV-2 genomes reveals attenuation of the Omicron BA.1 variant through NSP6 (preprint)

Although the SARS-CoV-2 Omicron variant (BA.1) spread rapidly across the world and effectively evaded immune responses, its viral fitness in cell and animal models was reduced. The precise nature of this attenuation remains unknown as generating replication-competent viral genomes is challenging because of the length of the viral genome (30kb). Here, we designed a plasmid-based viral genome assembly and rescue strategy (pGLUE) that constructs complete infectious viruses or noninfectious subgenomic replicons in a single ligation reaction with >80% efficiency. Fully sequenced replicons and infectious viral stocks can be generated in 1 and 3 weeks, respectively. By testing a series of naturally occurring viruses as well as Delta-Omicron chimeric replicons, we show that Omicron nonstructural protein 6 harbors critical attenuating mutations, which dampen viral RNA replication and reduce lipid droplet consumption. Thus, pGLUE overcomes remaining barriers to broadly study SARS-CoV-2 replication and reveals deficits in nonstructural protein function underlying Omicron attenuation.

Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories (preprint)

A series of SARS-CoV-2 variants of concern (VOCs) have evolved in humans during the COVID-19 pandemic: Alpha, Beta, Gamma, Delta, and Omicron. Here, we used global proteomic and genomic analyses during infection to understand the molecular responses driving VOC evolution. We discovered VOC-specific differences in viral RNA and protein expression levels, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. For example, regulation of host translation was highly conserved, consistent with suppression of VOC replication in mice using the translation inhibitor plitidepsin. Conversely, modulation of the host inflammatory response was most divergent, where we found Alpha and Beta, but not Omicron BA.1, antagonized interferon stimulated genes (ISGs), a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated proinflammatory genes compared to other VOCs. Systematic comparison of Omicron subvariants revealed BA.5 to have evolved enhanced ISG and proinflammatory gene suppression that similarly correlated with Orf6 expression, effects not seen in BA.4 due to a mutation that disrupts the Orf6-nuclear pore interaction. Our findings describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans.

Omicron mutations enhance infectivity and reduce antibody neutralization of SARS-CoV-2 virus-like particles (preprint)

The Omicron SARS-CoV-2 virus contains extensive sequence changes relative to the earlier arising B.1, B.1.1 and Delta SARS-CoV-2 variants that have unknown effects on viral infectivity and response to existing vaccines. Using SARS-CoV-2 virus-like particles (SC2-VLPs), we examined mutations in all four structural proteins and found that Omicron showed increased infectivity relative to B.1, B.1.1 and similar to Delta, a property conferred by S and N protein mutations. Thirty-eight antisera samples from individuals vaccinated with tozinameran (Pfizer/BioNTech), elasomeran (Moderna), Johnson & Johnson vaccines and convalescent sera from unvaccinated COVID-19 survivors had moderately to dramatically reduced efficacy to prevent cell transduction by VLPs containing the Omicron mutations. The Pfizer/BioNTech and Moderna vaccine antisera showed strong neutralizing activity against VLPs possessing the ancestral spike protein (B.1, B.1.1), with 3-fold reduced efficacy against Delta and 15-fold lower neutralization against Omicron VLPs. Johnson & Johnson antisera showed minimal neutralization of any of the VLPs tested. Furthermore, the monoclonal antibody therapeutics Casirivimab and Imdevimab had robust neutralization activity against B.1, B.1.1 or Delta VLPs but no detectable neutralization of Omicron VLPs. Our results suggest that Omicron is at least as efficient at assembly and cell entry as Delta, and the antibody response triggered by existing vaccines or previous infection, at least prior to boost, will have limited ability to neutralize Omicron. In addition, some currently available monoclonal antibodies will not be useful in treating Omicron-infected patients.

Sensitive and multiplexed RNA detection with Cas13 droplets and kinetic barcoding (preprint)

Rapid and sensitive quantification of RNA is critical for detecting infectious diseases and identifying disease biomarkers. Recent direct detection assays based on CRISPR-Cas13a1-4 avoid reverse transcription and DNA amplification required of gold-standard PCR assays5, but these assays have not yet achieved the sensitivity of PCR and are not easily multiplexed to detect multiple viruses or variants. Here we show that Cas13a acting on single target RNAs loaded into droplets exhibits stochastic nuclease activity that can be used to enable sensitive, rapid, and multiplexed virus quantification. Using SARS-CoV-2 RNA as the target and combinations of CRISPR RNA (crRNA) that recognize different parts of the viral genome, we demonstrate that reactions confined to small volumes can rapidly achieve PCR-level sensitivity. By tracking nuclease activity within individual droplets over time, we find that Cas13a exhibits rich kinetic behavior that depends on both the target RNA and crRNA. We demonstrate that these kinetic signatures can be harnessed to differentiate between different human coronavirus species as well as SARS-CoV-2 variants within a single droplet. The combination of high sensitivity, short reaction times, and multiplexing makes this droplet-based Cas13a assay with kinetic barcoding a promising strategy for direct RNA identification and quantification.

Rapid assessment of SARS-CoV-2 evolved variants using virus-like particles (preprint)

Newly evolved SARS-CoV-2 variants are driving ongoing outbreaks of COVID-19 around the world. Efforts to determine why these viral variants have improved fitness are limited to mutations in the viral spike (S) protein and viral entry steps using non-SARS-CoV-2 viral particles engineered to display S. Here we show that SARS-CoV-2 virus-like particles can package and deliver exogenous transcripts, enabling analysis of mutations within all structural proteins and rapid dissection of multiple steps in the viral life cycle. Identification of an RNA packaging sequence was critical for engineered transcripts to assemble together with SARS-CoV-2 structural proteins S, nucleocapsid (N), membrane (M) and envelope (E) into non-replicative SARS-CoV-2 virus-like particles (SC2-VLPs) that deliver these transcripts to ACE2- and TMPRSS2-expressing cells. Using SC2-VLPs, we tested the effect of 30 individual mutations within the S and N proteins on particle assembly and entry. While S mutations unexpectedly did not affect these steps, SC2-VLPs bearing any one of four N mutations found universally in more-transmissible viral variants (P199L, S202R, R203M and R203K) showed increased particle production and up to 10-fold more reporter transcript expression in receiver cells. Our study provides a platform for rapid testing of viral variants outside a biosafety level 3 setting and identifies viral N mutations and viral particle assembly as mechanisms to explain the increased spread of current viral variants, including Delta (N:R203M). One-Sentence SummaryR203M substitution within SARS-CoV-2 N, found in delta variant, improves RNA packaging into virus-like particles by 10-fold.

Accelerated RNA detection using tandem CRISPR nucleases (preprint)

Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided recognition of RNA that triggers cleavage and release of a fluorescent reporter molecule1,2, but long reaction times hamper sensitivity and speed when applied to point-of-care testing. Here we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 RNA copies/microliter in 20 minutes. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that detected SARS-CoV-2 RNA from nasopharyngeal samples with PCR-derived Ct values up to 29 in microfluidic chips, using a compact imaging system. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables direct RNA detection in a format amenable to point-of-care infection diagnosis, as well as to a wide range of other diagnostic or research applications.

Launching a saliva-based SARS-CoV-2 surveillance testing program on a university campus (preprint)

Regular surveillance testing of asymptomatic individuals for SARS-CoV-2 has played a vital role in SARS-CoV-2 outbreak prevention on college and university campuses. Here we describe the voluntary saliva testing program instituted at the University of California, Berkeley during an early period of the SARS-CoV-2 pandemic in 2020. The program was administered as a research study ahead of clinical implementation, enabling us to launch surveillance testing while continuing to optimize the assay. Results of both the testing protocol itself and the study participants experience show how the program succeeded in providing routine, robust testing capable of contributing to outbreak prevention within a campus community and offer strategies for encouraging participation and a sense of civic responsibility.

The landscape of human brain immune response in patients with severe COVID-19 (preprint)

In coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the relationship between brain tropism, neuroinflammation and host immune response has not been well characterized. We analyzed 68,557 single-nucleus transcriptomes from three brain regions (dorsolateral prefrontal cortex, medulla oblongata and choroid plexus) and identified an increased proportion of stromal cells and monocytes in the choroid plexus of COVID-19 patients. Differential gene expression, pseudo-temporal trajectory and gene regulatory network analyses revealed microglial transcriptome perturbations, mediating a range of biological processes, including cellular activation, mobility and phagocytosis. Quantification of viral spike S1 protein and SARS-CoV-2 transcripts did not support the notion of brain tropism. Overall, our findings suggest extensive neuroinflammation in patients with acute COVID-19.

Immunological and cardio-vascular pathologies associated with SARS-CoV-2 infection in golden syrian hamster (preprint)

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection in golden Syrian hamster (GSH) causes lungs pathology and resembles to human corona virus disease (Covid-19). Extra-pulmonary pathologies and immunological parameters of SARS-CoV-2 infection remained undefined in GSH. Using in silico modelling, we identified the similarities between human and hamster angiotensin-converting enzyme-2 (ACE-2), neuropilin-1 (NRP-1) that bind to receptor-binding domain (RBD) and S1 fragment of spike protein of SARS-CoV-2. SARS-CoV-2 infection led to lung pathologies, and cardiovascular complications (CVC) marked by interstitial coronary fibrosis and acute inflammatory response. Serum lipidomic and metabolomic profile of SARS-CoV-2-infected GSH revealed changes in serum triglycerides (TG) and low-density lipoprotein (LDL), and alterations in metabolites that correlated with Covid19. Together, we propose GSH as an animal model to study SARS-CoV-2 infection and its therapy associated with pulmonary and extra-pulmonary pathologies.

Hypoxia reduces cell attachment of SARS-CoV-2 spike protein by modulating the expression of ACE2 and heparan sulfate (preprint)

A main clinical parameter of Covid-19 pathophysiology is hypoxia. Here we show that hypoxia decreases the attachment of the receptor binding domain (RBD) and the S1 subunit (S1) of the spike protein to epithelial cells. In Vero E6 cells, hypoxia reduces the protein levels of ACE2, which might in part explain the observed reduction of the infection rate. However, hypoxia also inhibits the binding of the spike to human lung epithelial cells lacking ACE2 expression, indicating that hypoxia modulates the expression of additional binding partners of SARS-CoV-2. We show that hypoxia also decreases the total cell surface levels of heparan sulfate, a known attachment receptor of SARS-CoV-2, by reducing the expression of syndecan-1 and syndecan3, the main proteoglycans containing heparan sulfate. Our study indicates that hypoxia acts to prevent SARS-CoV-2 infection, suggesting that the hypoxia signaling pathway might offer therapeutic opportunities for the treatment of Covid-19.

Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples (preprint)

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI-FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against the gold standard, nasopharyngeal swab specimens. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

Rapid detection of SARS-CoV-2 with Cas13 (preprint)

To combat disease outbreaks such as the COVID-19 pandemic, flexible diagnostics for rapid viral detection are greatly needed. We report a nucleic acid test that integrates distinct mechanisms of DNA and RNA amplification optimized for high sensitivity and rapid kinetics, linked to Cas13 detection for specificity. We paired this workflow, termed Diagnostics with Coronavirus Enzymatic Reporting (DISCoVER), with extraction-free sample lysis using shelf-stable reagents that are widely available at low cost. DISCoVER has been validated on saliva samples to incentivize frequent testing for more widespread community surveillance and robustly detected attomolar levels of SARS-CoV-2 within 30 minutes, while avoiding false positives in virus-negative saliva. Furthermore, DISCoVER is compatible with multiplexed CRISPR probes to enable simultaneous detection of a human gene control or alternative pathogens.

Duplex formation between the template and the nascent strand in the transcription-regulating sequences determines the site of template switching in SARS - CoV-2 (preprint)

Recently published transcriptomic data of the SARS-CoV-2 coronavirus show that there is a large variation in the frequency and steady state levels of subgenomic mRNA sequences. This variation is derived from discontinuous subgenomic RNA synthesis where the polymerase switches template from a 3 proximal genome body sequence to a 5 untranslated leader sequence. This leads to a fusion between the common 5 leader sequence and a 3 proximal body sequence in the RNA product. This process revolves around a common core sequence (CS) that is present at both the template sites that make up the fusion junction. Base-pairing between the leader CS and the nascent complementary minus strand body CS, and flanking regions (together called the transcription regulating sequence, TRS) is vital for this template switching event. However, various factors can influence the site of template switching within the same TRS duplex. Here, we model the duplexes formed between the leader and complementary body TRS regions, hypothesising the role of the stability of the TRS duplex in determining the major sites of template switching for the most abundant mRNAs. We indicate that the stability of secondary structures and the speed of transcription play key roles in determining the probability of template switching in the production of subgenomic RNAs.

EVs analysis in the COVID-19 era: insights on serum inactivation protocols towards downstream isolation and analysis (preprint)

Since the outbreak of COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2 positive individuals demanded the use of inactivation protocols to ensure laboratory operators safety. While not standardized, these practices can be roughly divided in two categories, namely heat inactivation and solvent-detergent treatments. As such, these routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the EVs community, yet deep investigations in this direction havent been reported so far. In the attempt of sparking interest on this highly relevant topic, we here provide preliminary insights on SARS-CoV-2 inactivation practices to be adopted prior serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entailed the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat-treatment, however accompanied by a marked enrichment in EVs-associated contaminants. On the contrary, solvent/detergent treatment is promising for small EVs (< 150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.

IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2 (preprint)

Commonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on detection of a single viral target, adding time and cost or risk of false-negative results. Currently, no test combines detection of widely used SARS-CoV-2 E- and N-gene targets and a sample control in a single, multiplexed reaction. We developed the IGI-LuNER RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (NER). This combined, cost-effective test can be performed in 384-well plates with detection sensitivity suitable for clinical reporting, and will aid in future sample pooling efforts, thus improving throughput of SARS-CoV-2 detection. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/20247338v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@74929corg.highwire.dtl.DTLVardef@1457971org.highwire.dtl.DTLVardef@2825ddorg.highwire.dtl.DTLVardef@1cde2b6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Optimizing COVID-19 control with asymptomatic surveillance testing in a university environment (preprint)

The high proportion of transmission events derived from asymptomatic or presymptomatic infections make SARS-CoV-2, the causative agent in COVID-19, difficult to control through the traditional non-pharmaceutical interventions (NPIs) of symptom-based isolation and contact tracing. As a consequence, many US universities are developing asymptomatic surveillance testing labs, to augment existing NPIs and control outbreaks on campus. We built a stochastic branching process model of COVID-19 dynamics at UC Berkeley to advise optimal control strategies in a university environment. Our model combines behavioral interventions in the form of group size limits to deter superspreading, symptom-based isolation, and contact tracing, with asymptomatic surveillance testing. We find that behavioral interventions offer a cost-effective means of epidemic control: group size limits of twelve or fewer greatly reduce superspreading, and rapid isolation of symptomatic infections can halt rising epidemics, depending on the frequency of asymptomatic transmission in the population. Surveillance testing can overcome uncertainty surrounding asymptomatic infections, with the most effective approaches prioritizing frequent testing with rapid turnaround time to isolation over test sensitivity. Importantly, contact tracing amplifies population-level impacts of all infection isolations, making even delayed interventions effective. Combination of behavior-based NPIs and asymptomatic surveillance also reduces variation in daily case counts to produce more predictable epidemics. Furthermore, targeted, intensive testing of a minority of high transmission risk individuals can effectively control the COVID-19 epidemic for the surrounding population. We offer this blueprint and easy-to-implement modeling tool to other academic or professional communities navigating optimal return-to-work strategies for the 2021 year.

Direct detection of SARS-CoV-2 using CRISPR-Cas13a and a mobile phone (preprint)

The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic and pre- symptomatic carriers of the virus. CRISPR-based diagnostics that utilize RNA and DNA-targeting enzymes can augment gold-standard PCR-based testing if they can be made rapid, portable and accurate. Here we report the development of an amplification-free CRISPR-Cas13a-based mobile phone assay for direct detection of SARS-CoV-2 from nasal swab RNA extracts. The assay achieved ~100 copies/L sensitivity in under 30 minutes and accurately detected a set of positive clinical samples in under 5 minutes. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity, and we directly quantified viral load using enzyme kinetics. Combined with mobile phone-based quantification, this assay can provide rapid, low-cost, point-of-care screening to aid in the control of SARS-CoV-2.