Comparison of the Antiviral Activity of Remdesivir, Chloroquine, and Interferon-ß as Single or Dual Agents Against the Human Beta-Coronavirus OC43.

The human beta-coronavirus strain, OC43, provides a useful model for testing the antiviral activity of various agents. We compared the activity of several antiviral drugs against OC43, including remdesivir, chloroquine, interferon (IFN)-ß, IFN-λ1, and IFN-λ4, in two distinct cell types: human colorectal carcinoma cell line (HCT-8 cells) and normal human bronchial epithelial (NHBE) cells. We also tested whether these agents mediate additive, synergistic, or antagonistic activity against OC43 infection when used in combination. When used as single agents, remdesivir exhibited stronger antiviral activity than chloroquine, and IFN-ß exhibited stronger activity than IFN-λ1 or IFN-λ4 against OC43 in both HCT-8 and NHBE cells. Anakinra (IL-1 inhibitor) and tocilizumab (IL-6 inhibitor) did not mediate any antiviral activity. The combination of IFN-ß plus chloroquine or remdesivir resulted in higher synergy scores and higher expression of IFN-stimulated genes than did IFN-ß alone. In contrast, the combination of remdesivir plus chloroquine resulted in an antagonistic interaction in NHBE cells. Our findings indicate that the combined use of IFN-ß plus remdesivir or chloroquine induces maximal antiviral activity against human coronavirus strain OC43 in primary human respiratory epithelial cells. Furthermore, our experimental OC43 virus infection model provides an excellent method for evaluating the biological activity of antiviral drugs.

Nsp1 proteins of human coronaviruses HCoV-OC43 and SARS-CoV2 inhibit stress granule formation.

Stress granules (SGs) are cytoplasmic condensates that often form as part of the cellular antiviral response. Despite the growing interest in understanding the interplay between SGs and other biological condensates and viral replication, the role of SG formation during coronavirus infection remains poorly understood. Several proteins from different coronaviruses have been shown to suppress SG formation upon overexpression, but there are only a handful of studies analyzing SG formation in coronavirus-infected cells. To better understand SG inhibition by coronaviruses, we analyzed SG formation during infection with the human common cold coronavirus OC43 (HCoV-OC43) and the pandemic SARS-CoV2. We did not observe SG induction in infected cells and both viruses inhibited eukaryotic translation initiation factor 2α (eIF2α) phosphorylation and SG formation induced by exogenous stress. Furthermore, in SARS-CoV2 infected cells we observed a sharp decrease in the levels of SG-nucleating protein G3BP1. Ectopic overexpression of nucleocapsid (N) and non-structural protein 1 (Nsp1) from both HCoV-OC43 and SARS-CoV2 inhibited SG formation. The Nsp1 proteins of both viruses inhibited arsenite-induced eIF2α phosphorylation, and the Nsp1 of SARS-CoV2 alone was sufficient to cause a decrease in G3BP1 levels. This phenotype was dependent on the depletion of cytoplasmic mRNA mediated by Nsp1 and associated with nuclear accumulation of the SG-nucleating protein TIAR. To test the role of G3BP1 in coronavirus replication, we infected cells overexpressing EGFP-tagged G3BP1 with HCoV-OC43 and observed a significant decrease in virus replication compared to control cells expressing EGFP. The antiviral role of G3BP1 and the existence of multiple SG suppression mechanisms that are conserved between HCoV-OC43 and SARS-CoV2 suggest that SG formation may represent an important antiviral host defense that coronaviruses target to ensure efficient replication.

Antigenic structure of the human coronavirus OC43 spike reveals exposed and occluded neutralizing epitopes.

Human coronavirus OC43 is a globally circulating common cold virus sustained by recurrent reinfections. How it persists in the population and defies existing herd immunity is unknown. Here we focus on viral glycoprotein S, the target for neutralizing antibodies, and provide an in-depth analysis of its antigenic structure. Neutralizing antibodies are directed to the sialoglycan-receptor binding site in S1A domain, but, remarkably, also to S1B. The latter block infection yet do not prevent sialoglycan binding. While two distinct neutralizing S1B epitopes are readily accessible in the prefusion S trimer, other sites are occluded such that their accessibility must be subject to conformational changes in S during cell-entry. While non-neutralizing antibodies were broadly reactive against a collection of natural OC43 variants, neutralizing antibodies generally displayed restricted binding breadth. Our data provide a structure-based understanding of protective immunity and adaptive evolution for this endemic coronavirus which emerged in humans long before SARS-CoV-2.

Azvudine is a thymus-homing anti-SARS-CoV-2 drug effective in treating COVID-19 patients.

Azvudine (FNC) is a nucleoside analog that inhibits HIV-1 RNA-dependent RNA polymerase (RdRp). Recently, we discovered FNC an agent against SARS-CoV-2, and have taken it into Phase III trial for COVID-19 patients. FNC monophosphate analog inhibited SARS-CoV-2 and HCoV-OC43 coronavirus with an EC50 between 1.2 and 4.3 µM, depending on viruses or cells, and selective index (SI) in 15-83 range. Oral administration of FNC in rats revealed a substantial thymus-homing feature, with FNC triphosphate (the active form) concentrated in the thymus and peripheral blood mononuclear cells (PBMC). Treating SARS-CoV-2 infected rhesus macaques with FNC (0.07 mg/kg, qd, orally) reduced viral load, recuperated the thymus, improved lymphocyte profiles, alleviated inflammation and organ damage, and lessened ground-glass opacities in chest X-ray. Single-cell sequencing suggested the promotion of thymus function by FNC. A randomized, single-arm clinical trial of FNC on compassionate use (n = 31) showed that oral FNC (5 mg, qd) cured all COVID-19 patients, with 100% viral ribonucleic acid negative conversion in 3.29 ± 2.22 days (range: 1-9 days) and 100% hospital discharge rate in 9.00 ± 4.93 days (range: 2-25 days). The side-effect of FNC is minor and transient dizziness and nausea in 16.12% (5/31) patients. Thus, FNC might cure COVID-19 through its anti-SARS-CoV-2 activity concentrated in the thymus, followed by promoted immunity.

Comparative Host Interactomes of the SARS-CoV-2 Nonstructural Protein 3 and Human Coronavirus Homologs.

Human coronaviruses have become an increasing threat to global health; three highly pathogenic strains have emerged since the early 2000s, including most recently SARS-CoV-2, the cause of COVID-19. A better understanding of the molecular mechanisms of coronavirus pathogenesis is needed, including how these highly virulent strains differ from those that cause milder, common-cold-like disease. While significant progress has been made in understanding how SARS-CoV-2 proteins interact with the host cell, nonstructural protein 3 (nsp3) has largely been omitted from the analyses. Nsp3 is a viral protease with important roles in viral protein biogenesis, replication complex formation, and modulation of host ubiquitinylation and ISGylation. Herein, we use affinity purification-mass spectrometry to study the host-viral protein-protein interactome of nsp3 from five coronavirus strains: pathogenic strains SARS-CoV-2, SARS-CoV, and MERS-CoV; and endemic common-cold strains hCoV-229E and hCoV-OC43. We divide each nsp3 into three fragments and use tandem mass tag technology to directly compare the interactors across the five strains for each fragment. We find that few interactors are common across all variants for a particular fragment, but we identify shared patterns between select variants, such as ribosomal proteins enriched in the N-terminal fragment (nsp3.1) data set for SARS-CoV-2 and SARS-CoV. We also identify unique biological processes enriched for individual homologs, for instance, nuclear protein import for the middle fragment of hCoV-229E, as well as ribosome biogenesis of the MERS nsp3.2 homolog. Lastly, we further investigate the interaction of the SARS-CoV-2 nsp3 N-terminal fragment with ATF6, a regulator of the unfolded protein response. We show that SARS-CoV-2 nsp3.1 directly binds to ATF6 and can suppress the ATF6 stress response. Characterizing the host interactions of nsp3 widens our understanding of how coronaviruses co-opt cellular pathways and presents new avenues for host-targeted antiviral therapeutics.

A potential antiviral activity of Esculentoside A against binding interactions of SARS-COV-2 spike protein and angiotensin converting enzyme 2 (ACE2).

The recent emergence of the novel coronavirus (SARS-CoV-2) has resulted in a devastating pandemic with global concern. However, to date, there are no regimens to prevent and treat SARS-CoV-2 virus. There is an urgent need to identify novel leads with anti-viral properties that impede viral pathogenesis in the host system. Esculentoside A (EsA), a saponin isolated from the root of Phytolacca esculenta, is known to exhibit diverse pharmacological properties, especially anti-inflammatory activity. To our knowledge, SARS-CoV-2 uses angiotensin converting enzyme 2 (ACE2) to enter host cells. This is mediated through the proteins of SARS-CoV-2, especially the spike glycoprotein receptor binding domain. Thus, our primary goal is to prevent virus replication and binding to the host, which allows us to explore the efficiency of EsA on key surface drug target proteins using the computational biology paradigm approach. Here, the anti-coronavirus activity of EsA in vitro and its potential mode of inhibitory action on the S-protein of SARS-CoV-2 were investigated. We found that EsA inhibited the HCoV-OC43 coronavirus during the attachment and penetration stage. Molecular docking results showed that EsA had a strong binding affinity with the spike glycoprotein from SARS-CoV-2. The results of the molecular dynamics simulation revealed that EsA had higher stable binding with the spike protein. These results demonstrated that Esculentoside A can act as a spike protein blocker to inhibit SARS-CoV-2. Considering the poor bioavailability and low toxicity of EsA, it is suitable as novel lead for the inhibitor against binding interactions of SARS-CoV-2 of S-protein and ACE2.

Activation of STING Signaling Pathway Effectively Blocks Human Coronavirus Infection.

The COVID-19 pandemic poses a serious global health threat. The rapid global spread of SARS-CoV-2 highlights an urgent need to develop effective therapeutics for blocking SARS-CoV-2 infection and spread. Stimulator of Interferon Genes (STING) is a chief element in host antiviral defense pathways. In this study, we examined the impact of the STING signaling pathway on coronavirus infection using the human coronavirus OC43 (HCoV-OC43) model. We found that HCoV-OC43 infection did not stimulate the STING signaling pathway, but the activation of STING signaling effectively inhibits HCoV-OC43 infection to a much greater extent than that of type I interferons (IFNs). We also discovered that IRF3, the key STING downstream innate immune effector, is essential for this anticoronavirus activity. In addition, we found that the amidobenzimidazole (ABZI)-based human STING agonist diABZI robustly blocks the infection of not only HCoV-OC43 but also SARS-CoV-2. Therefore, our study identifies the STING signaling pathway as a potential therapeutic target that could be exploited for developing broad-spectrum antiviral therapeutics against multiple coronavirus strains in order to face the challenge of future coronavirus outbreaks.IMPORTANCE The highly infectious and lethal SARS-CoV-2 is posing an unprecedented threat to public health. Other coronaviruses are likely to jump from a nonhuman animal to humans in the future. Novel broad-spectrum antiviral therapeutics are therefore needed to control known pathogenic coronaviruses such as SARS-CoV-2 and its newly mutated variants, as well as future coronavirus outbreaks. STING signaling is a well-established host defense pathway, but its role in coronavirus infection remains unclear. In the present study, we found that activation of the STING signaling pathway robustly inhibits infection of HCoV-OC43 and SARS-CoV-2. These results identified the STING pathway as a novel target for controlling the spread of known pathogenic coronaviruses, as well as emerging coronavirus outbreaks.

AT-527, a Double Prodrug of a Guanosine Nucleotide Analog, Is a Potent Inhibitor of SARS-CoV-2 In Vitro and a Promising Oral Antiviral for Treatment of COVID-19.

The impact of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19, is global and unprecedented. Although remdesivir has recently been approved by the FDA to treat SARS-CoV-2 infection, no oral antiviral is available for outpatient treatment. AT-527, an orally administered double prodrug of a guanosine nucleotide analog, was previously shown to be highly efficacious and well tolerated in hepatitis C virus (HCV)-infected subjects. Here, we report the potent in vitro activity of AT-511, the free base of AT-527, against several coronaviruses, including SARS-CoV-2. In normal human airway epithelial cells, the concentration of AT-511 required to inhibit replication of SARS-CoV-2 by 90% (EC90) was 0.47 µM, very similar to its EC90 against human coronavirus (HCoV)-229E, HCoV-OC43, and SARS-CoV in Huh-7 cells. Little to no cytotoxicity was observed for AT-511 at concentrations up to 100 µM. Substantial levels of the active triphosphate metabolite AT-9010 were formed in normal human bronchial and nasal epithelial cells incubated with 10 µM AT-511 (698 ± 15 and 236 ± 14 µM, respectively), with a half-life of at least 38 h. Results from steady-state pharmacokinetic and tissue distribution studies of nonhuman primates administered oral doses of AT-527, as well as pharmacokinetic data from subjects given daily oral doses of AT-527, predict that twice daily oral doses of 550 mg AT-527 will produce AT-9010 trough concentrations in human lung that exceed the EC90 observed for the prodrug against SARS-CoV-2 replication. This suggests that AT-527 may be an effective treatment option for COVID-19.

Human Milk Antibodies Against S1 and S2 Subunits from SARS-CoV-2, HCoV-OC43, and HCoV-229E in Mothers with A Confirmed COVID-19 PCR, Viral SYMPTOMS, and Unexposed Mothers.

BACKGROUND: Preexisting immunity to SARS-CoV-2 could be related to cross-reactive antibodies to common human-coronaviruses (HCoVs). This study aimed to evaluate whether human milk antibodies against to S1 and S2 subunits SARS-CoV-2 are cross-reactive to S1 and S2 subunits HCoV-OC43 and HCoV-229E in mothers with a confirmed COVID-19 PCR test, in mothers with previous viral symptoms during COVID-19 pandemic, and in unexposed mothers; Methods: The levels of secretory IgA (SIgA)/IgA, secretory IgM (SIgM)/IgM, and IgG specific to S1 and S2 SARS-CoV-2, and reactive to S1 + S2 HCoV-OC43, and HCoV-229E were measured in milk from 7 mothers with a confirmed COVID-19 PCR test, 20 mothers with viral symptoms, and unexposed mothers (6 Ctl1-2018 and 16 Ctl2-2018) using ELISA; Results: The S2 SARS-CoV-2 IgG levels were higher in the COVID-19 PCR (p = 0.014) and viral symptom (p = 0.040) groups than in the Ctl1-2018 group. We detected a higher number of positive correlations between the antigens and secretory antibodies in the COVID-19 PCR group than in the viral symptom and Ctl-2018 groups. S1 + S2 HCoV-OC43-reactive IgG was higher in the COVID-19 group than in the control group (p = 0.002) but did not differ for the other antibodies; Conclusions: Mothers with a confirmed COVID-19 PCR and mothers with previous viral symptoms had preexisting human milk antibodies against S2 subunit SARS-CoV-2. Human milk IgG were more specific to S2 subunit SARS-CoV-2 than other antibodies, whereas SIgA and SIgM were polyreactive and cross-reactive to S1 or S2 subunit SARS-CoV-2.

Prediction of Single Point Mutations in Human Coronavirus and Their Effects on Binding to 9-O-Acetylated Sialic Acid and Hidroxychloroquine.

Due to the current spreading of the new disease CoViD-19, the World Health Organization formally declared a world pandemic on March 11, 2020. The present trends indicate that the pandemic will have an enormous clinical and economic impact on population health. Infections are initiated by the transmembrane spike (S) glycoproteins of human coronavirus (hCoV) binding to host receptors. Ongoing research and therapeutic product development are of vital importance for the successful treatment of CoViD-19. To contribute somewhat to the overall effort, herein, single point mutations (SPMs) of the binding site residues in hCoV-OC43 S that recognizes cellular surface components containing 9-O-acetylated sialic acid (9-O-Ac-Sia) are explored using an in silico protein engineering approach, while their effects on the binding of 9-O-Ac-Sia and Hidroxychloroquine (Hcq) are evaluated using molecular docking simulations. Thr31Met and Val84Arg are predicted to be the critical - most likely SPMs in hCoV-OC43 S for the binding of 9-O-Ac-Sia and Hcq, respectively, even though Thr31Met is a very likely SPM in the case of Hcq too. The corresponding modes of interaction indicate a comparable strength of the Thr31Met/9-O-Ac-Sia and Val84Arg/Hcq (or Thr31Met/Hcq) complexes. Given that the binding site is conserved in all CoV S glycoproteins that associate with 9-O-acetyl-sialoglycans, the high hydrophobic affinity of Hcq to hCoV-OC43 S speaks in favor of its ability to competitively inhibit rapid S-mediated virion attachment in high-density receptor environments, but its considerably low specificity to hCoV-OC43 S may be one of the key obstacles in considering the potential of Hcq to become a drug candidate.

Climate and Human coronaviruses 229E and Human coronaviruses OC43 Infections: Respiratory Viral Infections Prevalence in Hospitalized Children in Cheonan, Korea.

The study of climate and respiratory viral infections using big data may enable the recognition and interpretation of relationships between disease occurrence and climatic variables. In this study, realtime reverse transcription quantitative PCR (qPCR) methods were used to identify Human respiratory coronaviruses (HCoV). infections in patients below 10 years of age with respiratory infections who visited Dankook University Hospital in Cheonan, South Korea, from January 1, 2012, to December 31, 2018. Out of the 9010 patients who underwent respiratory virus real-time reverse transcription qPCR test, 364 tested positive for HCoV infections. Among these 364 patients, 72.8% (n = 265) were below 10 years of age. Data regarding the frequency of infections was used to uncover the seasonal pattern of the two viral strains, which was then compared with local meteorological data for the same time period. HCoV-229E and HCoV-OC43 showed high infection rates in patients below 10 years of age. There was a negative relationship between HCoV-229E and HCoV-OC43 infections with air temperature and wind-chill temperatures. Both HCoV-229E and HCoV-OC43 rates of infection were positively related to atmospheric pressure, while HCoV-229E was also positively associated with particulate matter concentrations. Our results suggest that climatic variables affect the rate in which children below 10 years of age are infected with HCoV. These findings may help to predict when prevention strategies may be most effective.

Coronavirus hemagglutinin-esterase and spike proteins coevolve for functional balance and optimal virion avidity.

Human coronaviruses OC43 and HKU1 are respiratory pathogens of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spillover. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase (HE) acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity toward clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations led to cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and coevolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses. Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.

Ancestral origin, antigenic resemblance and epidemiological insights of novel coronavirus (SARS-CoV-2): Global burden and Bangladesh perspective.

SARS-CoV-2, a new coronavirus strain responsible for COVID-19, has emerged in Wuhan City, China, and continuing its global pandemic nature. The availability of the complete gene sequences of the virus helps to know about the origin and molecular characteristics of this virus. In the present study, we performed bioinformatic analysis of the available gene sequence data of SARS-CoV-2 for the understanding of evolution and molecular characteristics and immunogenic resemblance of the circulating viruses. Phylogenetic analysis was performed for four types of representative viral proteins (spike, membrane, envelope and nucleoprotein) of SARS-CoV-2, HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HKU1, MERS-CoV, HKU4, HKU5 and BufCoV-HKU26. The findings demonstrated that SARS-CoV-2 exhibited convergent evolutionary relation with previously reported SARS-CoV. It was also depicted that SARS-CoV-2 proteins were highly similar and identical to SARS-CoV proteins, though proteins from other coronaviruses showed a lower level of resemblance. The cross-checked conservancy analysis of SARS-CoV-2 antigenic epitopes showed significant conservancy with antigenic epitopes derived from SARS-CoV. Descriptive epidemiological analysis on several epidemiological indices was performed on available epidemiological outbreak information from several open databases on COVID-19 (SARS-CoV-2). Satellite-derived imaging data have been employed to understand the role of temperature in the environmental persistence of the virus. Findings of the descriptive analysis were used to describe the global impact of newly emerged SARS-CoV-2, and the risk of an epidemic in Bangladesh.