A sensor capable of quantifying avian influenza A virus (subtype H5) was developed by targeting hemagglutinin (HA) protein. This was accomplished by designing a solution-based sandwich assay that generated orthophosphate (PO43-) in proportion to the concentration of HA protein (and hence virus particles) present in a sample. Virus particles were captured and concentrated using an H5-specific primary antibody conjugated to magnetic microparticles. Following magnetic separation, an alkaline phosphatase (AP)-conjugated secondary antibody was introduced to bind to the captured virus. The resulting assembly was exposed to p-nitrophenyl phosphate (PNPP), producing PO43- in proportion to the amount of virus present. The generated phosphate was subsequently converted to glucose through the addition of maltose and maltose phosphorylase, and the resulting glucose was quantified amperometrically using an off-the-shelf commercial glucose test strip interfaced with a mini-potentiostat. Using this approach, limits of detection of 7.78 & times; 105 RNA copies per mL in binding buffer, 1.46 & times; 106 RNA copies per mL in artificial saliva, and 1.26 & times; 106 RNA copies per mL in tap water were obtained. Selectivity was demonstrated against respiratory pathogen pools, representative Gram-positive and Gram-negative bacteria, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This work presents a modular immunoassay-based strategy that leverages widely available glucose test strips for quantitative viral detection in a laboratory proof-of-concept format and may be adaptable to other targets through substitution of capture and detection antibodies.

The global impact of SARS-CoV-2 and the continued emergence of zoonotic coronaviruses underscore the urgent need for broad-spectrum antivirals for pandemic preparedness. Herein, we report AVI8122, a covalent pan-coronaviral inhibitor that targets 19 Mpros across the α, β, γ, and δ genera, encompassing bat, human, and other animal coronaviruses. AVI8122 exhibits low nanomolar potency and favorable pharmacokinetics in mice. Structural studies reveal that AVI8122 forms a covalent bond with the catalytic cysteine and maintains conserved interactions within the active sites of these Mpros. In cellulo, AVI8122 efficiently inhibited the replication of SARS-CoV-2 and its variants of concern as well as the activity of Mpros from all four genera. Furthermore, in mouse models, AVI8122 conferred dose-dependent protection against a lethal SARS-CoV-2 infection. Our findings position AVI8122 as an early lead compound and a tractable structural starting point for the development of broad-spectrum antivirals against future coronavirus spillover threats.

The main protease of SARS-CoV-2 is an essential enzyme required for polyprotein cleavage during viral replication and thus is an excellent target for development of direct-acting antiviral compounds. Continued research efforts have elucidated several peptidic small molecules like GC376, boceprevir, and nirmatrelvir with potent anticoronaviral activity bearing optimized amino acid side chain residues. To reduce synthetic complexity and cost, we used simple chemical surrogates that were commercially readily available to develop new inhibitors that mimic the potency of these drug compounds. We synthesized and tested several analogue chimeras of GC376 and boceprevir that have surrogate residues at the P1 and/or P2 position in order to further improve target binding. Both P1 variants with either a nonpolar cyclobutyl or polar thiazol-4-yl alanine resulted in low-micromolar to submicromolar Mpro inhibitors with strong antiviral activity in cell assays.

The vast array of immunoassay technologies used to assess protein interactions is costly or platform-specific. We present a label-free visual interference colour assay (VICA) that quantifies peptide and protein interactions by creating an iridescent surface allowing direct visualisation without spectrophotometric optics or microfluidics. A nanoporous aluminium oxide surface is tuned to match the refractive indices of the overlying protein layers to generate visual interference colours. To functionalise the surface, we created an affinity-capture system using a protein A-carboxyglutamic (GLA) construct that orients antibodies to enhance the signal. Using off-the-shelf antibodies, the platform can isolate analytes in buffer, whole blood, or serum. This surface generates a discernible colour change at concentrations as low as 50 femtomoles/mm2 and can monitor oligomer formation in sequential steps on the same slide. VICA provides comparable kinetic parameters to biolayer interferometry and traditional immunoassays while also allowing characterisation of proteins in large macromolecular complexes.

A sensor capable of quantifying both anti-SARS-CoV-2 spike receptor-binding domain (RBD) antibody levels and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in saliva and serum was developed. This was accomplished by exploiting the enzymatic reaction of maltose and orthophosphate (PO43-) in the presence of maltose phosphorylase to generate an equivalent amount of glucose that was detected using a commercial glucometer test strip and a potentiostat. Important for this approach is the ability to generate PO43- in an amount that is directly related to the concentration of the analytes. RBD-modified magnetic microparticles were used to capture anti-SARS-CoV-2 spike RBD antibodies, while particles modified with anti-SARS-CoV-2 nucleocapsid antibodies were used to capture SARS-CoV-2 nucleocapsid protein from inactivated virus samples. A magnet was used to isolate and purify the magnetic microparticles (with analyte attached), and alkaline phosphatase-conjugated secondary antibodies were bound to the analytes attached to the respective magnetic microparticles. Finally, through enzymatic reactions, specific amounts of PO43- (and subsequently glucose) were generated in proportion to the analyte concentration, which was then quantified using a commercial glucometer test strip. Utilizing glucose test strips makes the sensor relatively inexpensive, with a cost per test of ∼US $7 and ∼US $12 for quantifying anti-SARS-CoV-2 spike RBD antibody and SARS-CoV-2, respectively. Our sensor exhibited a limit of detection of 0.42 ng/mL for anti-SARS-CoV-2 spike RBD antibody, which is sensitive enough to quantify typical concentrations of antibodies in COVID-19-infected or vaccinated individuals (>1 μg/mL). The limit of detection for the SARS-CoV-2 virus is 300 pfu/mL (5.4 × 106 RNA copies/mL), which exceeds the performance recommended by the WHO (500 pfu/mL). In addition, the sensor exhibited good selectivity when challenged with competing analytes and could be used to quantify analytes in saliva and serum matrices with an accuracy of >94% compared to RT-qPCR.

This study explores the relationship between structural alterations of nirmatrelvir, such as homologation and deuteration, and metabolic stability of newly synthesized derivatives. We developed a reliable synthetic protocol toward dideutero-nirmatrelvir and its homologated analogues with high isotopic incorporation. Deuteration of the primary metabolic site of nirmatrelvir provides a 3-fold improvement of its human microsomal stability but is accompanied by an increased metabolism rate at secondary sites. Homologation of the lactam ring allows the capping group modification to decrease and delocalize the molecule's lipophilicity, reducing the metabolic rate at secondary sites. The effect of deuteration was less pronounced for the 6-membered lactam than for its 5-membered analogue in human microsomes, but the trend is reversed in the case of mouse microsomes. X-ray data revealed that the homologation of the lactam ring favors the orientation of the drug's nitrile warhead for interaction with the catalytic sulfur of the SARS-CoV-2 Mpro, improving its binding. Comparable potency against SARS-CoV-2 Mpro from several variants of concern and selectivity over human cysteine proteases cathepsin B, L, and S was observed for the novel deuterated/homologated derivative and nirmatrelvir. Synthesized compounds displayed a large interspecies variability in hamster, rat, and human hepatocyte stability assays. Overall, we aimed to apply a rational approach in changing the physicochemical properties of the drug to refine its biochemical and biological parameters.

Interferon regulatory factors (IRFs) are key elements of antiviral innate responses that regulate the transcription of interferons (IFNs) and IFN-stimulated genes (ISGs). While the sensitivity of human coronaviruses to IFNs has been characterized, antiviral roles of IRFs during human coronavirus infection are not fully understood. Type I or II IFN treatment protected MRC5 cells from human coronavirus 229E infection, but not OC43. Cells infected with 229E or OC43 upregulated ISGs, indicating that antiviral transcription is not suppressed. Antiviral IRFs, IRF1, IRF3 and IRF7, were activated in cells infected with 229E, OC43 or severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2). RNAi knockdown and overexpression of IRFs demonstrated that IRF1 and IRF3 have antiviral properties against OC43, while IRF3 and IRF7 are effective in restricting 229E infection. IRF3 activation effectively promotes transcription of antiviral genes during OC43 or 229E infection. Our study suggests that IRFs may be effective antiviral regulators against human coronavirus infection.

The targeted screening and sequencing approaches for COVID-19 surveillance need to be adjusted to fit the evolving surveillance objectives which necessarily change over time. We present the development of variant screening assays that can be applied to new targets in a timely manner and enable multiplexing of targets for efficient implementation in the laboratory. By targeting the HV69/70 deletion for Alpha, K417N for Beta, K417T for Gamma, and HV69/70 deletion plus K417N for sub-variants BA.1, BA.3, BA.4, and BA.5 of Omicron, we achieved simultaneous detection and differentiation of Alpha, Beta, Gamma, and Omicron in a single assay. Targeting both T478K and P681R mutations enabled specific detection of the Delta variant. The multiplex assays used in combination, targeting K417N and T478K, specifically detected the Omicron sub-variant BA.2. The limits of detection for the five variants of concern were 4-16 copies of the viral RNA per reaction. Both assays achieved 100% clinical sensitivity and 100% specificity. Analyses of 377 clinical samples and 24 wastewater samples revealed the Delta variant in 100 clinical samples (nasopharyngeal and throat swab) collected in November 2021. Omicron BA.1 was detected in 79 nasopharyngeal swab samples collected in January 2022. Alpha, Beta, and Gamma variants were detected in 24 wastewater samples collected in May-June 2021 from two major cities of Alberta (Canada), and the results were consistent with the clinical cases of multiple variants reported in the community.

An approach to assess severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (and past infection) was developed. For virus detection, the SARS-CoV-2 virus nucleocapsid protein (NP) was targeted. To detect the NP, antibodies were immobilized on magnetic beads to capture the NPs, which were subsequently detected using rabbit anti-SARS-CoV-2 nucleocapsid antibodies and alkaline phosphatase (AP)-conjugated anti-rabbit antibodies. A similar approach was used to assess SARS-CoV-2-neutralizing antibody levels by capturing spike receptor-binding domain (RBD)-specific antibodies utilizing RBD protein-modified magnetic beads and detecting them using AP-conjugated anti-human IgG antibodies. The sensing mechanism for both assays is based on cysteamine etching-induced fluorescence quenching of bovine serum albumin-protected gold nanoclusters where cysteamine is generated in proportion to the amount of either SARS-CoV-2 virus or anti-SARS-CoV-2 receptor-binding domain-specific immunoglobulin antibodies (anti-RBD IgG antibodies). High sensitivity can be achieved in 5 h 15 min for the anti-RBD IgG antibody detection and 6 h 15 min for virus detection, although the assay can be run in "rapid" mode, which takes 1 h 45 min for the anti-RBD IgG antibody detection and 3 h 15 min for the virus. By spiking the anti-RBD IgG antibodies and virus in serum and saliva, we demonstrate that the assay can detect the anti-RBD IgG antibodies with a limit of detection (LOD) of 4.0 and 2.0 ng/mL in serum and saliva, respectively. For the virus, we can achieve an LOD of 8.5 × 105 RNA copies/mL and 8.8 × 105 RNA copies/mL in serum and saliva, respectively. Interestingly, this assay can be easily modified to detect myriad analytes of interest.

The SARS-CoV-2 pandemic is an ongoing threat to global health, and wide-scale vaccination is an efficient method to reduce morbidity and mortality. We designed and evaluated two DNA plasmid vaccines, based on the pIDV-II system, expressing the SARS-CoV-2 spike gene, with or without an immunogenic peptide, in mice, and in a Syrian hamster model of infection. Both vaccines demonstrated robust immunogenicity in BALB/c and C57BL/6 mice. Additionally, the shedding of infectious virus and the viral burden in the lungs was reduced in immunized hamsters. Moreover, high-titers of neutralizing antibodies with activity against multiple SARS-CoV-2 variants were generated in immunized animals. Vaccination also protected animals from weight loss during infection. Additionally, both vaccines were effective at reducing both pulmonary and extrapulmonary pathology in vaccinated animals. These data show the potential of a DNA vaccine for SARS-CoV-2 and suggest further investigation in large animal and human studies could be pursued.

Recurring coronavirus outbreaks, such as the current COVID-19 pandemic, establish a necessity to develop direct-acting antivirals that can be readily administered and are active against a broad spectrum of coronaviruses. Described in this Article are novel α-acyloxymethylketone warhead peptidomimetic compounds with a six-membered lactam glutamine mimic in P1. Compounds with potent SARS-CoV-2 3CL protease and in vitro viral replication inhibition were identified with low cytotoxicity and good plasma and glutathione stability. Compounds 15e, 15h, and 15l displayed selectivity for SARS-CoV-2 3CL protease over CatB and CatS and superior in vitro SARS-CoV-2 antiviral replication inhibition compared with the reported peptidomimetic inhibitors with other warheads. The cocrystallization of 15l with SARS-CoV-2 3CL protease confirmed the formation of a covalent adduct. α-Acyloxymethylketone compounds also exhibited antiviral activity against an alphacoronavirus and non-SARS betacoronavirus strains with similar potency and a better selectivity index than remdesivir. These findings demonstrate the potential of the substituted heteroaromatic and aliphatic α-acyloxymethylketone warheads as coronavirus inhibitors, and the described results provide a basis for further optimization.

Samples of nasopharyngeal swabs (NPS) are commonly used for the detection of SARS-CoV-2 and diagnosis of COVID-19. As an alternative, self-collection of saliva and gargle samples minimizes transmission to healthcare workers and relieves the pressure of resources and healthcare personnel during the pandemic. This study aimed to develop an enhanced method enabling simultaneous viral inactivation and RNA preservation during on-site self-collection of saliva and gargle samples. Our method involves the addition of saliva or gargle samples to a newly formulated viral inactivation and RNA preservation (VIP) buffer, concentration of the viral RNA on magnetic beads, and detection of SARS-CoV-2 using reverse transcription quantitative polymerase chain reaction directly from the magnetic beads. This method has a limit of detection of 25 RNA copies per 200 μL of gargle or saliva sample and 9-111 times higher sensitivity than the viral RNA preparation kit recommended by the United States Centers for Disease Control and Prevention. The integrated method was successfully used to analyze more than 200 gargle and saliva samples, including the detection of SARS-CoV-2 in 123 gargle and saliva samples collected daily from two NPS-confirmed positive SARS-CoV-2 patients throughout the course of their infection and recovery. The VIP buffer is stable at room temperature for at least 6 months. SARS-CoV-2 RNA (65 copies/200 μL sample) is stable in the VIP buffer at room temperature for at least 3 weeks. The on-site inactivation of SARS-CoV-2 and preservation of the viral RNA enables self-collection of samples, reduces risks associated with SARS-CoV-2 transmission, and maintains the stability of the target analyte.

CRISPR-Cas systems integrated with nucleic acid amplification techniques improve both analytical specificity and sensitivity. We describe here issues and solutions for the successful integration of reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube under an isothermal condition (40 °C). Specific detection of a few copies of a viral DNA sequence was achieved in less than 20 min. However, the sensitivity was orders of magnitude lower for the detection of viral RNA due to the slow initiation of RPA when the complementary DNA (cDNA) template remained hybridized to RNA. During the delay of RPA, the crRNA-Cas12a ribonucleoprotein (RNP) gradually lost its activity in the RPA solution, and nonspecific amplification reactions consumed the RPA reagents. We overcame these problems by taking advantage of the endoribonuclease function of RNase H to remove RNA from the RNA-cDNA hybrids and free the cDNA as template for the RPA reaction. As a consequence, we significantly enhanced the overall reaction rate of an integrated assay using RT-RPA and CRISPR-Cas12a for the detection of RNA. We showed successful detection of 200 or more copies of the S gene sequence of SARS-CoV-2 RNA within 5-30 min. We applied our one-tube assay to 46 upper respiratory swab samples for COVID-19 diagnosis, and the results from both fluorescence intensity measurements and end-point visualization were consistent with those of RT-qPCR analysis. The strategy and technique improve the sensitivity and speed of RT-RPA and CRISPR-Cas12a assays, potentially useful for both semi-quantitative and point-of-care analyses of RNA molecules.

Tragically, the death toll from the COVID-19 pandemic continues to rise, and with variants being observed around the globe new therapeutics, particularly direct-acting antivirals that are easily administered, are desperately needed. Studies targeting the SARS-CoV-2 3CL protease, which is critical for viral replication, with different peptidomimetics and warheads is an active area of research for development of potential drugs. To date, however, only a few publications have evaluated the nitrile warhead as a viral 3CL protease inhibitor, with only modest activity reported. This article describes our investigation of P3 4-methoxyindole peptidomimetic analogs with select P1 and P2 groups with a nitrile warhead that are potent inhibitors of SARS-CoV-2 3CL protease and demonstrate in vitro SARS-CoV-2 antiviral activity. A selectivity for SARS-CoV-2 3CL protease over human cathepsins B, S and L was also observed with the nitrile warhead, which was superior to that with the aldehyde warhead. A co-crystal structure with SARS-CoV-2 3CL protease and a reversibility study indicate that a reversible, thioimidate adduct is formed when the catalytic sulfur forms a covalent bond with the carbon of the nitrile. This effort also identified efflux as a property limiting antiviral activity of these compounds, and together with the positive attributes described these results provide insight for further drug development of novel nitrile peptidomimetics targeting SARS-CoV-2 3CL protease.

SARS-CoV-2 infection is associated with lower blood oxygen levels, even in patients without hypoxia requiring hospitalization. This discordance illustrates the need for a more unifying explanation as to whether SARS-CoV-2 directly or indirectly affects erythropoiesis. Here, we show significantly enriched CD71+ erythroid precursors/progenitors in the blood circulation of COVID-19 patients. We found that these cells have distinctive immunosuppressive properties. In agreement, we observed a strong negative correlation between the frequency of these cells with T and B cell proportions in COVID-19 patients. The expansion of these CD71+ erythroid precursors/progenitors was negatively correlated with the hemoglobin levels. A subpopulation of abundant erythroid cells, CD45+ CD71+ cells, co-express ACE2, TMPRSS2, CD147, and CD26, and these can be infected with SARS-CoV-2. In turn, pre-treatment of erythroid cells with dexamethasone significantly diminished ACE2/TMPRSS2 expression and subsequently reduced their infectivity with SARS-CoV-2. This provides a novel insight into the impact of SARS-CoV-2 on erythropoiesis and hypoxia seen in COVID-19 patients.

An estimated two billion people worldwide have been infected with hepatitis B virus (HBV). Despite the high infectivity of HBV in vivo, a lack of easily infectable in vitro culture systems hinders studies of HBV. Overexpression of the sodium taurocholate co-transporting polypeptide (NTCP) bile acid transporter in hepatoma cells improved infection efficiency. We report here a hepatoma cell culture system that does not require dimethyl sulfoxide (DMSO) for HBV infection. We overexpressed NTCP in Huh7.5 cells and allowed these cells to differentiate in a medium supplemented with human serum (HS) instead of fetal bovine serum (FBS). We show that human serum culture enhanced HBV infection in Huh7.5-NTCP cells, e.g., in HS cultures, HBV pgRNA levels were increased by as much as 200-fold in comparison with FBS cultures and 19-fold in comparison with FBS+DMSO cultures. Human serum culture increased levels of hepatocyte differentiation markers, such as albumin secretion, in Huh7.5-NTCP cells to similar levels found in primary human hepatocytes. N-glycosylation of NTCP induced by culture in human serum may contribute to viral entry. Our study demonstrates an in vitro HBV infection of Huh7.5-NTCP cells without the use of potentially toxic DMSO.

SARS-CoV-2 is the etiological agent of COVID19. There are currently several licensed vaccines approved for human use and most of them target the spike protein in the virion envelope to induce protective immunity. Recently, variants that spread more quickly have emerged. There is evidence that some of these variants are less sensitive to neutralization in vitro, but it is not clear whether they can evade vaccine induced protection. In this study, we tested SARS-CoV-2 spike RBD as a vaccine antigen and explored the effect of formulation with Alum/MPLA or AddaS03 adjuvants. Our results show that RBD induces high titers of neutralizing antibodies and activates strong cellular immune responses. There is also significant cross-neutralization of variants B.1.1.7 and B.1.351 and to a lesser extent, SARS-CoV-1. These results indicate that recombinant RBD can be a viable candidate as a stand-alone vaccine or as a booster shot to diversify our strategy for COVID19 protection.

Replication of SARS-CoV-2, the coronavirus causing COVID-19, requires a main protease (Mpro) to cleave viral proteins. Consequently, Mpro is a target for antiviral agents. We and others previously demonstrated that GC376, a bisulfite prodrug with efficacy as an anti-coronaviral agent in animals, is an effective inhibitor of Mpro in SARS-CoV-2. Here, we report structure-activity studies of improved GC376 derivatives with nanomolar affinities and therapeutic indices >200. Crystallographic structures of inhibitor-Mpro complexes reveal that an alternative binding pocket in Mpro, S4, accommodates the P3 position. Alternative binding is induced by polar P3 groups or a nearby methyl. NMR and solubility studies with GC376 show that it exists as a mixture of stereoisomers and forms colloids in aqueous media at higher concentrations, a property not previously reported. Replacement of its Na+ counter ion with choline greatly increases solubility. The physical, biochemical, crystallographic, and cellular data reveal new avenues for Mpro inhibitor design.