Serum soluble Fas ligand is a severity and mortality prognostic marker for COVID-19 patients.

Finding cytokine storm initiator factors associated with uncontrolled inflammatory immune response is necessary in COVID-19 patients. The aim was the identification of Fas/Fas Ligand (FasL) role in lung involvement and mortality of COVID-19 patients. In this case-control study, mild (outpatient), moderate (hospitalized), and severe (ICU) COVID-19 patients and healthy subjects were investigated. RNA isolated from PBMCs for cDNA synthesis and expression of mFas/mFasL mRNA was evaluated by RT-PCR. Serum sFas/sFasL protein by ELISA and severity of lung involvement by CT-scan were evaluated. Also, we docked Fas and FasL via Bioinformatics software (in silico) to predict the best-fit Fas/FasL complex and performed molecular dynamics simulation (MDS) in hyponatremia and fever (COVID-19 patients), and healthy conditions. mFasL expression was increased in moderate and severe COVID-19 patients compared to the control group. Moreover, mFas expression showed an inverse correlation with myalgia symptom in COVID-19 patients. Elevation of sFasL protein in serum was associated with reduced lung injury and mortality. Bioinformatics analysis confirmed that blood profile alterations of COVID-19 patients, such as fever and hyponatremia could affect Fas/FasL complex interactions. Our translational findings showed that decreased sFasL is associated with lung involvement; severity and mortality in COVID-19 patients. We think that sFasL is a mediator of neutrophilia and lymphopenia in COVID-19. However, additional investigation is suggested. This is the first report describing that the serum sFasL protein is a severity and mortality prognostic marker for the clinical management of COVID-19 patients.

High temporal resolution Nanopore sequencing dataset of SARS-CoV-2 and host cell RNAs.

BACKGROUND: Recent studies have disclosed the genome, transcriptome, and epigenetic compositions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the effect of viral infection on gene expression of the host cells. It has been demonstrated that, besides the major canonical transcripts, the viral genome also codes for noncanonical RNA molecules. While the structural characterizations have revealed a detailed transcriptomic architecture of the virus, the kinetic studies provided poor and often misleading results on the dynamics of both the viral and host transcripts due to the low temporal resolution of the infection event and the low virus/cell ratio (multiplicity of infection [MOI] = 0.1) applied for the infection. It has never been tested whether the alteration in the host gene expressions is caused by aging of the cells or by the viral infection. FINDINGS: In this study, we used Oxford Nanopore's direct cDNA and direct RNA sequencing methods for the generation of a high-coverage, high temporal resolution transcriptomic dataset of SARS-CoV-2 and of the primate host cells, using a high infection titer (MOI = 5). Sixteen sampling time points ranging from 1 to 96 hours with a varying time resolution and 3 biological replicates were used in the experiment. In addition, for each infected sample, corresponding noninfected samples were employed. The raw reads were mapped to the viral and to the host reference genomes, resulting in 49,661,499 mapped reads (54,62 Gbs). The genome of the viral isolate was also sequenced and phylogenetically classified. CONCLUSIONS: This dataset can serve as a valuable resource for profiling the SARS-CoV-2 transcriptome dynamics, the virus-host interactions, and the RNA base modifications. Comparison of expression profiles of the host gene in the virally infected and in noninfected cells at different time points allows making a distinction between the effect of the aging of cells in culture and the viral infection. These data can provide useful information for potential novel gene annotations and can also be used for studying the currently available bioinformatics pipelines.

A flap endonuclease 1-assisted universal viral nucleic acid sensing system using surface-enhanced Raman scattering.

The continued uncertainty of emerging infectious viral diseases has led to an extraordinary urgency to develop advanced molecular diagnostic tests that are faster, more reliable, simpler to use, and readily available than traditional methods. This study presents a system that can accurately and rapidly trace viral nucleic acids by employing flap endonuclease 1 (FEN1)-assisted specific DNA cleavage reactions and surface-enhanced Raman scattering (SERS)-based analysis. The designed Raman tag-labeled 5'- and 3'-flap provider DNA yielded structurally defined DNA substrates on magnetic nanoparticle surfaces when a target was present. The FEN1 enzyme subsequently processes the substrates formed via an invasive cleavage reaction, producing 5'-flap DNA products. Magnetic separation allows efficient purification of flap products from reaction mixtures. The isolated solution was directly applied onto high aspect-ratio plasmonic silver nanopillars serving as SERS-active substrates to induce amplified SERS signals. We verified the developed SERS-based sensing system using a synthetic target complementary to an avian influenza A (H9N2) virus gene and examined the detection performance of the system using complementary DNA (cDNA) derived from H9N2 viral RNA. As a result, we could detect a synthetic target with a detection limit of 41.1 fM with a single base-pair discrimination ability and achieved multiplexed detection capability for two targets. Using cDNA samples from H9N2 viruses, we observed a high concordance of R2 = 0.917 between the data obtained from SERS and the quantitative polymerase chain reaction. We anticipate that this enzyme-assisted SERS sensor may provide insights into the development of high-performance molecular diagnostic tools that can respond rapidly to viral pathogens.

Upregulation of hsa_circ_0004812 promotes COVID-19 cytokine storm via hsa-miR-1287-5p/IL6R, RIG-I axis.

BACKGROUND: SARS-CoV-2 is one of the most contagious viruses in the Coronaviridae (CoV) family, which has become a pandemic. The aim of this study is to understand more about the role of hsa_circ_0004812 in the SARS-CoV-2 related cytokine storm and its associated molecular mechanisms. MATERIALS AND METHODS: cDNA synthesis was performed after total RNA was extracted from the peripheral blood mononuclear cells (PBMC) of 46 patients with symptomatic COVID-19, 46 patients with asymptomatic COVID-19, and 46 healthy controls. The expression levels of hsa_circ_0004812, hsa-miR-1287-5p, IL6R, and RIG-I were determined using qRT-PCR, and the potential interaction between these molecules was confirmed by bioinformatics tools and correlation analysis. RESULTS: hsa_circ_0004812, IL6R, and RIG-I are expressed higher in the severe symptom group compared with the negative control group. Also, the relative expression of these genes in the asymptomatic group is lower than in the severe symptom group. The expression level of hsa-miR-1287-5p was positively correlated with symptoms in patients. The results of the bioinformatics analysis predicted the sponging effect of hsa_circ_0004812 as a competing endogenous RNA on hsa-miR-1287-5p. Moreover, there was a significant positive correlation between hsa_circ_0004812, RIG-I, and IL-6R expressions, and also a negative expression correlation between hsa_circ_0004812 and hsa-miR-1287-5p and between hsa-miR-1287-5p, RIG-I, and IL-6R. CONCLUSION: The results of this in-vitro and in silico study show that hsa_circ_0004812/hsa-miR-1287-5p/IL6R, RIG-I can play an important role in the outcome of COVID-19.

Spontaneous NLRP3 inflammasome-driven IL-1-ß secretion is induced in severe COVID-19 patients and responds to anakinra treatment.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may result in a severe pneumonia associated with elevation of blood inflammatory parameters, reminiscent of cytokine storm syndrome. Steroidal anti-inflammatory therapies have shown efficacy in reducing mortality in critically ill patients; however, the mechanisms by which SARS-CoV-2 triggers such an extensive inflammation remain unexplained. OBJECTIVES: To dissect the mechanisms underlying SARS-CoV-2-associated inflammation in patients with severe coronavirus disease 2019 (COVID-19), we studied the role of IL-1ß, a pivotal cytokine driving inflammatory phenotypes, whose maturation and secretion are regulated by inflammasomes. METHODS: We analyzed nod-like receptor protein 3 pathway activation by means of confocal microscopy, plasma cytokine measurement, cytokine secretion following in vitro stimulation of blood circulating monocytes, and whole-blood RNA sequencing. The role of open reading frame 3a SARS-CoV-2 protein was assessed by confocal microscopy analysis following nucleofection of a monocytic cell line. RESULTS: We found that circulating monocytes from patients with COVID-19 display ASC (adaptor molecule apoptotic speck like protein-containing a CARD) specks that colocalize with nod-like receptor protein 3 inflammasome and spontaneously secrete IL-1ß in vitro. This spontaneous activation reverts following patient's treatment with the IL-1 receptor antagonist anakinra. Transfection of a monocytic cell line with cDNA coding for the ORF3a SARS-CoV-2 protein resulted in ASC speck formation. CONCLUSIONS: These results provide further evidence that IL-1ß targeting could represent an effective strategy in this disease and suggest a mechanistic explanation for the strong inflammatory manifestations associated with COVID-19.

Electrochemical Biosensor for SARS-CoV-2 cDNA Detection Using AuPs-Modified 3D-Printed Graphene Electrodes.

A low-cost and disposable graphene polylactic (G-PLA) 3D-printed electrode modified with gold particles (AuPs) was explored to detect the cDNA of SARS-CoV-2 and creatinine, a potential biomarker for COVID-19. For that, a simple, non-enzymatic electrochemical sensor, based on a Au-modified G-PLA platform was applied. The AuPs deposited on the electrode were involved in a complexation reaction with creatinine, resulting in a decrease in the analytical response, and thus providing a fast and simple electroanalytical device. Physicochemical characterizations were performed by SEM, EIS, FTIR, and cyclic voltammetry. Square wave voltammetry was employed for the creatinine detection, and the sensor presented a linear response with a detection limit of 0.016 mmol L-1. Finally, a biosensor for the detection of SARS-CoV-2 was developed based on the immobilization of a capture sequence of the viral cDNA upon the Au-modified 3D-printed electrode. The concentration, immobilization time, and hybridization time were evaluated in presence of the DNA target, resulting in a biosensor with rapid and low-cost analysis, capable of sensing the cDNA of the virus with a good limit of detection (0.30 µmol L-1), and high sensitivity (0.583 µA µmol-1 L). Reproducible results were obtained (RSD = 1.14%, n = 3), attesting to the potentiality of 3D-printed platforms for the production of biosensors.

An electrochemical aptasensor with N protein binding aptamer-complementary oligonucleotide as probe for ultra-sensitive detection of COVID-19.

The emergence of the COVID-19 epidemic has affected the lives of hundreds of millions of people globally. There is no doubt that the development of fast and sensitive detection methods is crucial while the worldwide effective vaccination programs are miles away from actualization. In this study, we have reported an electrochemical N protein aptamer sensor with complementary oligonucleotide as probe for the specific detection of COVID-19. The electrochemical aptasensor was prepared by fixing the double-stranded DNA hybrid obtained by the hybridization of N protein aptamer and its Fc-labeled complementary strand on the surface of a gold electrode. After incubation with the target, the aptamer dissociated from the labeled complementary DNA oligonucleotide hybrid to preferentially bind with N protein in the solution. The concentration of N protein was measured by detecting the changes in electrochemical current signals induced by the conformational transformation of the complementary DNA oligonucleotide left on the electrode surface. The sensor had a linear relationship between the logarithm of the N protein concentration from 10 fM to 100 nM (ΔIp = 0.098 log CN protein/fM - 0.08433, R2 = 0.99), and the detection limitation was 1 fM (S/N = 3). The electrochemical aptamer sensor was applied to test the spiked concentrations of throat swabs and blood samples from three volunteers, and the obtained results proved that the sensor has great potentials for the early detection of COVID-19 in patients.

Infecting kidney organoids with a cDNA reporter clone of SARS-CoV-2.

Induced pluripotent stem cell (iPSC)-derived kidney organoids can be used for disease modeling and drug testing. Here, we describe a protocol to prepare stocks of an infectious clone of SARS-CoV-2 expressing a stable mNeonGreen reporter (icSARS-CoV-2-mNG). We demonstrate the infection of kidney organoids, primarily at the proximal tubular cells, with icSARS-CoV-2-mNG. Using a TCID50 (tissue culture infectious dose 50) assay and confocal microscopy, we show the quantification of SARS-CoV-2-mNG signal in proximal tubular cells of the kidney organoids. For complete details on the use and execution of this protocol, please refer to Rahmani et al. (2022).

A Nanopore Sensor for Multiplexed Detection of Short Polynucleotides Based on Length-Variable, Poly-Arginine-Conjugated Peptide Nucleic Acids.

Real-time and easy-to-use detection of nucleic acids is crucial for many applications, including medical diagnostics, genetic screening, forensic science, or monitoring the onset and progression of various diseases. Herein, an exploratory single-molecule approach for multiplexed discrimination among similar-sized single-stranded DNAs (ssDNA) is presented. The underlying strategy combined (i) a method based on length-variable, short arginine (poly-Arg) tags appended to peptide nucleic acid (PNA) probes, designed to hybridize with selected regions from complementary ssDNA targets (cDNA) in solution and (ii) formation and subsequent detection with the α-hemolysin nanopore of (poly-Arg)-PNA-cDNA duplexes containing two overhangs associated with the poly-Arg tail and the non-hybridized segment from ssDNA. We discovered that the length-variable poly-Arg tail marked distinctly the molecular processes associated with the nanopore-mediated duplexes capture, trapping and unzipping. This enabled the detection of ssDNA targets via the signatures of (poly-Arg)-PNA-cDNA blockade events, rendered most efficient from the ß-barrel entrance of the nanopore, and scaled proportional in efficacy with a larger poly-Arg moiety. We illustrate the approach by sensing synthetic ssDNAs designed to emulate fragments from two regions of SARS-CoV-2 nucleocapsid phosphoprotein N-gene.

High-resolution melting analysis after nested PCR for the detection of SARS-CoV-2 spike protein G339D and D796Y variations.

High-resolution melting (HRM) analysis was performed to detect G339D and D796Y variations in the SARS-CoV-2 Omicron variant spike protein. We employed two-step PCR consisting of the first RT-PCR and the second nested PCR to prepare the amplicon for HRM analysis. The melting temperatures (Tm) of the amplicon from the cDNA of the Omicron variant receptor binding domain (RBD) were 73.1 °C (G339D variation) and 75.1 °C (D796Y variation), respectively. These Tm values were clearly distinct from those of SARS-CoV-2 isolate Wuhan-Hu-1. HRM analysis after the two-step PCR was conducted on Omicron variant-positive specimens. The HRM curve and Tm value obtained with the Omicron variant-positive specimen were coincident with those of the amplicon from cDNA of the Omicron variant RBD. Our study demonstrates the utility of HRM analysis after two-step PCR for the detection of mutations in SARS-CoV-2 gene.

Rotational diffusometric sensor with isothermal amplification for ultra-sensitive and rapid detection of SARS-CoV-2 nsp2 cDNA.

In the wake of a pandemic, the development of rapid, simple, and accurate molecular diagnostic tests can significantly aid in reducing the spread of infections. By combining particle imaging with molecular assays, a quick and highly sensitive biosensor can readily identify a pathogen at low concentrations. Here, we implement functionalized particle-enabled rotational diffusometry in combination with loop-mediated isothermal amplification for the rapid detection of the SARS-CoV-2 nsp2 gene in the recombinant plasmid as a proof of concept for COVID-19 diagnostics. By analyzing the images of blinking signals generated by these modified particles, the change in micro-level viscosity due to nucleic acid amplification was measured. The high sensitivity of rotational diffusometry enabled facile detection within 10 min, with a limit of detection of 70 ag/µL and a sample volume of 2 µL. Tenfold higher detection sensitivity was observed for rotational diffusometry in comparison with real-time PCR. In addition, the system stability and the effect of temperature on rotational diffusometric measurements were studied and reported. These results demonstrated the utility of a rotational diffusometric platform for the rapid and sensitive detection of SARS-CoV-2 cDNA fragments.

Target DNA- and pH-responsive DNA hydrogel-based capillary assay for the optical detection of short SARS-CoV-2 cDNA.

DNA is recognized as a powerful biomarker for clinical diagnostics because its specific sequences are closely related to the cause and development of diseases. However, achieving rapid, low-cost, and sensitive detection of short-length target DNA still remains a considerable challenge. Herein, we successfully combine the catalytic hairpin assembly (CHA) technique with capillary action to develop a new and cost-effective method, a target DNA- and pH-responsive DNA hydrogel-based capillary assay, for the naked eye detection of 24 nt short single-stranded target DNA. Upon contact of target DNA, three individual hairpin DNAs hybridize with each other to sufficiently amplify Y-shaped DNA nanostructures (Y-DNA) until they are completely consumed via CHA cycling reactions. Each arm of the resultant Y-DNA contains sticky ends with i-motif DNA structure-forming sequences that can be self-assembled in an acidic environment (pH 5.0) to form target DNA- and pH-responsive DNA hydrogels by means of i-motif DNA-driven crosslinking. When inserting a capillary tube in the resultant solution, the liquid level inside clearly reduces due to the decrease in capillary force induced by the gels. In this way, the developed assay demonstrates sensitive and quantitative detection, with a detection limit of approximately 10 pM of 24 nt short complementary DNA (cDNA) targeting SARS-CoV-2 RNA genes at room temperature within 1 h. The assay is further shown to successfully detect target cDNA in serum, and it is also applied to detect several types of target sequences. Requiring no analytic equipment, precise temperature control, or enzymatic reactions, the developed DNA hydrogel-based capillary assay has potential as a promising naked eye detection platform for target DNA in resource-limited clinical settings.

Reverse Genetics with a Full-Length Infectious cDNA Clone of Bovine Torovirus.

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified in the new family Tobaniviridae. While reverse genetics systems have been established for various CoVs, none exist for ToVs. Here, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and hemagglutinin (HA)-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated a recombinant virus (rEGFP) wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. rEGFP expressed EGFP in infected cells but showed significantly lower levels of viral growth than wtBToV. Moreover, rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in nonstructural proteins (NSPs) that emerged during passage exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a phenotype similar to that of these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. IMPORTANCE ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae. Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, pathogenesis, and BToV vaccine development.

High Induction of IL-6 Secretion From hUCMSCs Optimize the Potential of hUCMSCs and TCZ as Therapy for COVID-19-Related ARDS.

Biological and cellular interleukin-6 (IL-6)-related therapies have been used to treat severe COVID-19 pneumonia with hyperinflammatory syndrome and acute respiratory failure, which prompted further exploration of the role of IL-6 in human umbilical cord mesenchymal stem cell (hUCMSC) therapy. Peripheral blood mononuclear cells (PBMCs) were responders cocultured with hUCMSCs or exogenous IL-6. A PBMC suppression assay was used to analyze the anti-inflammatory effects via MTT assay. The IL-6 concentration in the supernatant was measured using ELISA. The correlation between the anti-inflammatory effect of hUCMSCs and IL-6 levels and the relevant roles of IL-6 and IL-6 mRNA expression was analyzed using the MetaCore functional network constructed from gene microarray data. The location of IL-6 and IL-6 receptor (IL-6R) expression was further evaluated. We reported that hUCMSCs did not initially exert any inhibitory effect on PHA-stimulated proliferation; however, a potent inhibitory effect on PHA-stimulated proliferation was observed, and the IL-6 concentration reached approximately 1000 ng/mL after 72 hours. Exogenous 1000 ng/mL IL-6 inhibited PHA-stimulated inflammation but less so than hUCMSCs. The inhibitory effects of hUCMSCs on PHA-stimulated PBMCs disappeared after adding an IL-6 neutralizing antibody or pretreatment with tocilizumab (TCZ), an IL-6R antagonist. hUCMSCs exert excellent anti-inflammatory effects by inducing higher IL-6 levels, which is different from TCZ. High concentration of IL-6 cytokine secretion plays an important role in the anti-inflammatory effect of hUCMSC therapy. Initial hUCMSC therapy, followed by TCZ, seems to optimize the therapeutic potential to treat COVID-19-related acute respiratory distress syndrome (ARDS).

Natural and Recombinant SARS-CoV-2 Isolates Rapidly Evolve In Vitro to Higher Infectivity through More Efficient Binding to Heparan Sulfate and Reduced S1/S2 Cleavage.

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include (i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increases the positive charge of the surface of this domain, (ii) insertions into the NTD of heterologous peptides containing positively charged amino acids, and (iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide, and makes viruses less capable of syncytium formation. These viral adaptations result in higher affinity of viral particles to heparin, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers, and 2 orders of magnitude higher infectivity. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA-positive (RNA+) viruses, evolution to HS binding may result in virus attenuation in vivo. IMPORTANCE The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to the ACE2 receptor and, later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the NTD and in the P1' position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells, and decrease the genome equivalent to PFU (GE/PFU) ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high-affinity interaction of the spikes with the ACE2 receptor.

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants.

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called 'Tiled-ClickSeq', which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5'UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

Rescue of Infectious Sindbis Virus by Yeast Spheroplast-Mammalian Cell Fusion.

Sindbis virus (SINV), a positive-sense single stranded RNA virus that causes mild symptoms in humans, is transmitted by mosquito bites. SINV reverse genetics have many implications, not only in understanding alphavirus transmission, replication cycle, and virus-host interactions, but also in biotechnology and biomedical applications. The rescue of SINV infectious particles is usually achieved by transfecting susceptible cells (BHK-21) with SINV-infectious mRNA genomes generated from cDNA constructed via in vitro translation (IVT). That procedure is time consuming, costly, and relies heavily on reagent quality. Here, we constructed a novel infectious SINV cDNA construct that expresses its genomic RNA in yeast cells controlled by galactose induction. Using spheroplasts made from this yeast, we established a robust polyethylene glycol-mediated yeast: BHK-21 fusion protocol to rescue infectious SINV particles. Our approach is timesaving and utilizes common lab reagents for SINV rescue. It could be a useful tool for the rescue of large single strand RNA viruses, such as SARS-CoV-2.

Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals.

The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity among samples. Mixed allelic frequencies along the 20kb ORF1ab gene in one sample, suggested the presence of a defective viral RNA species subpopulation maintained in mixture with functional RNA in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.

Whole-Genome Sequencing of Pathogens in Saliva : A Target-Enrichment Approach for SARS-CoV-2.

Outbreak analysis and transmission surveillance of viruses can be performed via whole-genome sequencing after viral isolation. Such techniques have recently been applied to characterize and monitor SARS-CoV-2 , the etiological agent of the COVID-19 pandemic. However, the isolation and culture of SARS-CoV-2 is time consuming and requires biosafety level 3 containment, which is not ideal for many resource-constrained settings. An alternate method, bait capture allows target enrichment and sequencing of the entire SARS-CoV-2 genome eliminating the need for viral culture. This method uses a set of hybridization probes known as "baits" that span the genome and provide sensitive, accurate, and minimal off-target hybridization. Baits can be designed to detect any known virus or bacteria in a wide variety of specimen types, including oral secretions. The bait capture method presented herein allows the whole genome of SARS-CoV-2 in saliva to be sequenced without the need to culture and provides an outline of bait design and bioinformatic analysis to guide a bioinformatician.