The antiviral potential of the antiandrogen enzalutamide and the viral-androgen signaling interplay in seasonal coronaviruses.

The sex disparity in COVID-19 outcomes with males generally faring worse than females has been associated with the androgen-regulated expression of the protease TMPRSS2 and the cell receptor ACE2 in the lung and fueled interest in antiandrogens as potential antivirals. In this study, we explored enzalutamide, an antiandrogen used commonly to treat prostate cancer, as a potential antiviral against the human coronaviruses which cause seasonal respiratory infections (HCoV-NL63, -229E, and -OC43). Using lentivirus-pseudotyped and authentic HCoV, we report that enzalutamide reduced 229E and NL63 entry and infection in both TMPRSS2- and nonexpressing immortalized cells, suggesting a TMPRSS2-independent mechanism. However, no effect was observed against OC43. To decipher this distinction, we performed RNA-sequencing analysis on 229E- and OC43-infected primary human airway cells. Our results show a significant induction of androgen-responsive genes by 229E compared to OC43 at 24 and 72 h postinfection. The virus-mediated effect on AR-signaling was further confirmed with a consensus androgen response element-driven luciferase assay in androgen-depleted MRC-5 cells. Specifically, 229E induced luciferase-reporter activity in the presence and absence of the synthetic androgen mibolerone, while OC43 inhibited induction. These findings highlight a complex interplay between viral infections and androgen-signaling, offering insights for disparities in viral outcomes and antiviral interventions.

Antibacterial and anti-coronavirus investigation of selected Senegalese plant species according to an ethnobotanical survey.

ETHNOPHARMACOLOGICAL RELEVANCE: In Senegal, upper and lower respiratory tract infections constitute a real health problem. To manage these disorders, most people rely on the use of local medicinal plants. This is particularly the case for species belonging to the botanical families, Combretaceae, Fabaceae, Myrtaceae and Rubiaceae, which are widely used to treat various respiratory problems such as colds, flu, rhinitis, sinusitis, otitis, angina, bronchitis, bronchiolitis and also pneumonia. AIM OF THE STUDY: The aim of this study was to identify medicinal plants traditionally used for the management of infectious diseases, in particular those of the respiratory tract. On the basis of these ethnopharmacological uses, this study made it possible to highlight the antibacterial, antiviral and cytotoxic activities of selected plant species. MATERIALS AND METHODS: An ethnobotanical survey was conducted in Senegal among informants, including herbalists, traditional healers, and households, using medicinal plants in the management of infectious diseases, with a focus on respiratory tract infections. The most cited plant species were evaluated in vitro on a panel of 18 human pathogenic bacteria may be involved in respiratory infections and against the human coronavirus HCoV-229E in Huh-7 cells. The antiviral activity of the most active extracts against HCoV-229E was also evaluated on COVID-19 causing agent, SARS-CoV-2 in Vero-81 cells. In parallel, cytotoxic activities were evaluated on Huh-7 cells. RESULTS: A total of 127 informants, including 100 men (78.74%) and 27 women (21.26%) participated in this study. The ethnobotanical survey led to the inventory of 41 plant species belonging to 19 botanical families used by herbalists and/or traditional healers and some households to treat infectious diseases, with a specific focus on upper respiratory tract disorders. Among the 41 plant species, the most frequently mentioned in the survey were Guiera senegalensis J.F. Gmel. (95.2%), Combretum glutinosum Perr. Ex DC. (93.9%) and Eucalyptus spp. (82.8%). Combretaceae (30.2%) represented the most cited botanical family with six species, followed by Fabaceae (29.3%, 12 species). A total of 33 crude methanolic extracts of the 24 plant species selected for their number of citations were evaluated in vitro for their antimicrobial and cytotoxic activities. Guiera senegalensis, Combretum glutinosum, Vachellia nilotica subsp. tomentosa (Benth.) Kyal. & Boatwr, Eucalyptus camaldulensis Dehnh., and Terminalia avicennioides Guill. & Perr., showed antibacterial activities. The most active plants against HCoV-229E were: Ficus sycomorus L., Mitragyna inermis (Willd.) Kuntze, Pterocarpus erinaceus Poir., and Spermacoce verticillata L. One of these plants, Mitragyna inermis, was also active against SARS-CoV-2. CONCLUSION: This work confirmed the anti-infective properties of plant species traditionally used in Senegal. Overall, the most frequently cited plant species showed the best antibacterial activities. Moreover, some of the selected plant species could be considered as a potential source for the management of coronavirus infections. This new scientific data justified the use of these plants in the management of some infectious pathologies, especially those of the respiratory tract.

Surface air gas discharge plasma: An ecofriendly virus inactivation approach to enhance CPRRs mediated antiviral genes expression against airborne bio-contaminant (human Coronavirus-229E).

Emerging bio-contaminants (airborne viruses) exploits and manipulate host (human) metabolism to produce new viral particles, evading the host's immune defences and leading to infections. Non-thermal plasma, operating at atmospheric pressure and ambient temperature, is explored for virus inactivation, generating RONS that interact and denatures viral proteins. However, various factors affecting virus survival influence the efficacy of non-thermal plasma. Glucose analogue 2-DG, a metabolic modifier used in this study, disrupts the glycolysis pathway viruses rely on, creating an unfavourable environment for replication. Here, airborne HCoV-229E bio-contaminant was treated with plasma for inactivation, and the presence of RONS was analysed. Metabolically altered lung cells were subsequently exposed to the treated airborne viruses. Cytopathic effect, spike protein, and cell death were evaluated via flow cytometry and confocal microscopy, and CPRRs mediated antiviral gene expression was evaluated using PCR. Gas plasma-treated viruses led to reduced virus proliferation in unaltered lung cells, although few virus particles survived the exposure, as confirmed by biological assessment (cytopathic effects and live/dead staining). A combination approach of gas plasma-treated viruses and altered lung cells displayed drastic virus reduction compared to the control group, established through confocal microscopy and flow cytometry. Furthermore, altered lung cell enhances gene transcription responsible for innate immunity when exposed to the gas plasma-treated virus, thereby impeding airborne virus propagation. This study demonstrates the significance of a surface air gas plasma and metabolic alteration approach in enhancing genes targeted towards antiviral innate immunity and tackling outbreaks of emerging bio-contaminants of concerns (airborne viruses).

Tertiary folds of the SL5 RNA from the 5' proximal region of SARS-CoV-2 and related coronaviruses.

Coronavirus genomes sequester their start codons within stem-loop 5 (SL5), a structured, 5' genomic RNA element. In most alpha- and betacoronaviruses, the secondary structure of SL5 is predicted to contain a four-way junction of helical stems, some of which are capped with UUYYGU hexaloops. Here, using cryogenic electron microscopy (cryo-EM) and computational modeling with biochemically determined secondary structures, we present three-dimensional structures of SL5 from six coronaviruses. The SL5 domain of betacoronavirus severe-acute-respiratory-syndrome-related coronavirus 2 (SARS-CoV-2), resolved at 4.7 Å resolution, exhibits a T-shaped structure, with its UUYYGU hexaloops at opposing ends of a coaxial stack, the T's "arms." Further analysis of SL5 domains from SARS-CoV-1 and MERS (7.1 and 6.4 to 6.9 Å resolution, respectively) indicate that the junction geometry and inter-hexaloop distances are conserved features across these human-infecting betacoronaviruses. The MERS SL5 domain displays an additional tertiary interaction, which is also observed in the non-human-infecting betacoronavirus BtCoV-HKU5 (5.9 to 8.0 Å resolution). SL5s from human-infecting alphacoronaviruses, HCoV-229E and HCoV-NL63 (6.5 and 8.4 to 9.0 Å resolution, respectively), exhibit the same coaxial stacks, including the UUYYGU-capped arms, but with a phylogenetically distinct crossing angle, an X-shape. As such, all SL5 domains studied herein fold into stable tertiary structures with cross-genus similarities and notable differences, with implications for potential protein-binding modes and therapeutic targets.

Open-source milligram-scale, four channel, automated protein purification system.

Liquid chromatography purification of multiple recombinant proteins, in parallel, could catalyze research and discovery if the processes are fast and approach the robustness of traditional, "one-protein-at-a-time" purification. Here, we report an automated, four channel chromatography platform that we have designed and validated for parallelized protein purification at milligram scales. The device can purify up to four proteins (each with its own single column), has inputs for up to eight buffers or solvents that can be directed to any of the four columns via a network of software-driven valves, and includes an automated fraction collector with ten positions for 1.5 or 5.0 mL collection tubes and four positions for 50 mL collection tubes for each column output. The control software can be accessed either via Python scripting, giving users full access to all steps of the purification process, or via a simple-to-navigate touch screen graphical user interface that does not require knowledge of the command line or any programming language. Using our instrument, we report milligram-scale, parallelized, single-column purification of a panel of mammalian cell expressed coronavirus (SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-229E) trimeric Spike and monomeric Receptor Binding Domain (RBD) antigens, and monoclonal antibodies targeting SARS-CoV-2 Spike (S) and Influenza Hemagglutinin (HA). We include a detailed hardware build guide, and have made the controlling software open source, to allow others to build and customize their own protein purifier systems.

Causes and Consequences of Coronavirus Spike Protein Variability.

Coronaviruses are a large family of enveloped RNA viruses found in numerous animal species. They are well known for their ability to cross species barriers and have been transmitted from bats or intermediate hosts to humans on several occasions. Four of the seven human coronaviruses (hCoVs) are responsible for approximately 20% of common colds (hCoV-229E, -NL63, -OC43, -HKU1). Two others (SARS-CoV-1 and MERS-CoV) cause severe and frequently lethal respiratory syndromes but have only spread to very limited extents in the human population. In contrast the most recent human hCoV, SARS-CoV-2, while exhibiting intermediate pathogenicity, has a profound impact on public health due to its enormous spread. In this review, we discuss which initial features of the SARS-CoV-2 Spike protein and subsequent adaptations to the new human host may have helped this pathogen to cause the COVID-19 pandemic. Our focus is on host forces driving changes in the Spike protein and their consequences for virus infectivity, pathogenicity, immune evasion and resistance to preventive or therapeutic agents. In addition, we briefly address the significance and perspectives of broad-spectrum therapeutics and vaccines.

Influenza-like illness symptoms due to endemic human coronavirus reinfections are not influenced by the length of the interval separating reinfections.

After 3 years of its introduction to humans, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been declared as endemic. Little is known about the severity of the disease manifestation that future infections may cause, especially when reinfections occur after humoral immunity from a previous infection or vaccination has waned. Such knowledge could inform policymakers regarding the frequency of vaccination. Reinfections by endemic human coronaviruses (HCoVs) can serve as a model system for SARS-CoV-2 endemicity. We monitored 44 immunocompetent male adults with blood sampling every 6 months (for 17 years), for the frequency of HCoV (re-)infections, using rises in N-antibodies of HCoV-NL63, HCoV-29E, HCoV-OC43, and HCoV-HKU1 as markers of infection. Disease associations during (re-)infections were examined by comparison of self-reporting records of influenza-like illness (ILI) symptoms, every 6 months, by all participants. During 8,549 follow-up months, we found 364 infections by any HCoV with a median of eight infections per person. Symptoms more frequently reported during HCoV infection were cough, sore throat, and myalgia. Two hundred fifty-one of the 364 infections were species-specific HCoV-reinfections, with a median interval of 3.58 (interquartile range 1.92-5.67) years. The length of the interval between reinfections-being either short or long-had no influence on the frequency of reporting ILI symptoms. All HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1 (re-)infections are associated with the reporting of ILIs. Importantly, in immunocompetent males, these symptoms are not influenced by the length of the interval between reinfections. IMPORTANCE: Little is known about the disease following human coronavirus (HCoV) reinfection occurring years after the previous infection, once humoral immunity has waned. We monitored endemic HCoV reinfection in immunocompetent male adults for up to 17 years. We found no influence of reinfection interval length in the disease manifestation, suggesting that immunocompetent male adults are adequately protected against future HCoV infections.

Repurposing screen identifies novel candidates for broad-spectrum coronavirus antivirals and druggable host targets.

Libraries composed of licensed drugs represent a vast repertoire of molecules modulating physiological processes in humans, providing unique opportunities for the discovery of host-targeting antivirals. We screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) repurposing library with approximately 12,000 molecules for broad-spectrum coronavirus antivirals and discovered 134 compounds inhibiting an alphacoronavirus and mapping to 58 molecular target categories. Dominant targets included the 5-hydroxytryptamine receptor, the dopamine receptor, and cyclin-dependent kinases. Gene knock-out of the drugs' host targets including cathepsin B and L (CTSB/L; VBY-825), the aryl hydrocarbon receptor (AHR; Phortress), the farnesyl-diphosphate farnesyltransferase 1 (FDFT1; P-3622), and the kelch-like ECH-associated protein 1 (KEAP1; Omaveloxolone), significantly modulated HCoV-229E infection, providing evidence that these compounds inhibited the virus through acting on their respective host targets. Counter-screening of all 134 primary compound candidates with SARS-CoV-2 and validation in primary cells identified Phortress, an AHR activating ligand, P-3622-targeting FDFT1, and Omaveloxolone, which activates the NFE2-like bZIP transcription factor 2 (NFE2L2) by liberating it from its endogenous inhibitor KEAP1, as antiviral candidates for both an Alpha- and a Betacoronavirus. This study provides an overview of HCoV-229E repurposing candidates and reveals novel potentially druggable viral host dependency factors hijacked by diverse coronaviruses.

Does the exponential Wells-Riley model provide a good fit for human coronavirus and rhinovirus? A comparison of four dose-response models based on human challenge data.

The risk assessments during the COVID-19 pandemic were primarily based on dose-response models derived from the pooled datasets for infection of animals susceptible to SARS-CoV. Despite similarities, differences in susceptibility between animals and humans exist for respiratory viruses. The two most commonly used dose-response models for calculating the infection risk of respiratory viruses are the exponential and the Stirling approximated ß-Poisson (BP) models. The modified version of the one-parameter exponential model or the Wells-Riley model was almost solely used for infection risk assessments during the pandemic. Still, the two-parameter (α and ß) Stirling approximated BP model is often recommended compared to the exponential dose-response model due to its flexibility. However, the Stirling approximation restricts this model to the general rules of ߠ≫ 1 and α â‰ª ß, and these conditions are very often violated. To refrain from these requirements, we tested a novel BP model by using the Laplace approximation of the Kummer hypergeometric function instead of the conservative Stirling approximation. The datasets of human respiratory airborne viruses available in the literature for human coronavirus (HCoV-229E) and human rhinovirus (HRV-16 and HRV-39) are used to compare the four dose-response models. Based on goodness-of-fit criteria, the exponential model was the best fitting model for the HCoV-229E (k = 0.054) and for HRV-39 datasets (k = 1.0), whereas the Laplace approximated BP model followed by the exact and Stirling approximated BP models are preferred for both the HRV-16 (α = 0.152 and ß = 0.021 for Laplace BP) and the HRV-16 and HRV-39 pooled datasets (α = 0.2247 and ß = 0.0215 for Laplace BP).

Exploration of 1,2,3-triazolo fused triterpenoids as inhibitors of human coronavirus 229E targeting the viral nsp15 protein.

The coronavirus disease-19 (COVID-19) pandemic has raised major interest in innovative drug concepts to suppress human coronavirus (HCoV) infections. We previously reported on a class of 1,2,3-triazolo fused betulonic acid derivatives causing strong inhibition of HCoV-229E replication via the viral nsp15 protein, which is proposedly related to compound binding at an intermonomer interface in hexameric nsp15. In the present study, we further explored the structure-activity relationship (SAR), by varying the substituent at the 1,2,3-triazolo ring as well as the triterpenoid skeleton. The 1,2,3-triazolo fused triterpenoids were synthesized by a multicomponent triazolization reaction, which has been developed in-house. Several analogs possessing a betulin, oleanolic acid, or ursolic acid core displayed favorable activity and selectivity (EC50 values for HCoV-229E: 1.6-3.5 µM), but neither of them proved as effective as the lead compound containing betulonic acid. The 18ß-glycyrrhetinic acid-containing analogs had low selectivity. The antiviral findings were rationalized by in silico docking in the available structure of the HCoV-229E nsp15 protein. The new SAR insights will aid the further development of these 1,2,3-triazolo fused triterpenoid compounds as a unique type of coronavirus inhibitors.

Enhanced enrichment of collected airborne coronavirus and influenza virus samples via a ConA-coated microfluidic chip for PCR detection.

The severe acute respiratory syndrome (SARS-CoV-2) outbreak triggered global concern and emphasized the importance of virus monitoring. During a seasonal influenza A outbreak, relatively low concentrations of 103-104 viral genome copies are available per 1 m3 of air, which makes detection and monitoring very challenging because the limit of detection of most polymerase chain reaction (PCR) devices is approximately 103 viral genome copies/mL. In response to the urgent need for the rapid detection of airborne coronaviruses and influenza viruses, an electrostatic aerosol-to-hydrosol (ATH) sampler was combined with a concanavalin A (ConA)-coated high-throughput microfluidic chip. The samples were then used for PCR detection. The results revealed that the enrichment capacity of the ATH sampler was 30,000-fold for both HCoV-229E and H1N1 influenza virus, whereas the enrichment capacities provided by the ConA-coated microfluidic chip were 8-fold and 16-fold for HCoV-229E and H1N1 virus, respectively. Thus, the total enrichment capacities of our combined ATH sampler and ConA-coated microfluidic chip were 2.4 × 105-fold and 4.8 × 105-fold for HCoV-229E and H1N1 virus, respectively. This methodology significantly improves PCR detection by providing a higher concentration of viable samples.

Broad spectrum post-entry inhibitors of coronavirus replication: Cardiotonic steroids and monensin.

A small molecule screen identified several cardiotonic steroids (digitoxin and ouabain) and the ionophore monensin as potent inhibitors of HCoV-229E, HCoV-OC43, and SARS-CoV-2 replication with EC50s in the low nM range. Subsequent tests confirmed antiviral activity in primary cell models including human nasal epithelial cells and lung organoids. Addition of digitoxin, ouabain, or monensin strongly reduced viral gene expression as measured by both viral protein and RNA accumulation. Furthermore, the compounds acted post virus entry. While the antiviral activity of digitoxin was dependent upon activation of the MEK and JNK signaling pathways but not signaling through GPCRs, the antiviral effect of monensin was reversed upon inhibition of several signaling pathways. Together, the data demonstrates the potent anti-coronavirus properties of two classes of FDA approved drugs that function by altering the properties of the infected cell, rendering it unable to support virus replication.

Discovery of Anti-Coronavirus Cinnamoyl Triterpenoids Isolated from Hippophae rhamnoides during a Screening of Halophytes from the North Sea and Channel Coasts in Northern France.

The limited availability of antiviral therapy for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spurred the search for novel antiviral drugs. Here, we investigated the potential antiviral properties of plants adapted to high-salt environments collected in the north of France. Twenty-five crude methanolic extracts obtained from twenty-two plant species were evaluated for their cytotoxicity and antiviral effectiveness against coronaviruses HCoV-229E and SARS-CoV-2. Then, a bioguided fractionation approach was employed. The most active crude methanolic extracts were partitioned into three different sub-extracts. Notably, the dichloromethane sub-extract of the whole plant Hippophae rhamnoides L. demonstrated the highest antiviral activity against both viruses. Its chemical composition was evaluated by ultra-high performance liquid chromatography (UHPLC) coupled with mass spectrometry (MS) and then it was fractionated by centrifugal partition chromatography (CPC). Six cinnamoyl triterpenoid compounds were isolated from the three most active fractions by preparative high-performance liquid chromatography (HPLC) and identified by high resolution MS (HR-MS) and mono- and bi-dimensional nuclear magnetic resonance (NMR). Specifically, these compounds were identified as 2-O-trans-p-coumaroyl-maslinic acid, 3ß-hydroxy-2α-trans-p-coumaryloxy-urs-12-en-28-oic acid, 3ß-hydroxy-2α-cis-p-coumaryloxy-urs-12-en-28-oic acid, 3-O-trans-caffeoyl oleanolic acid, a mixture of 3-O-trans-caffeoyl oleanolic acid/3-O-cis-caffeoyl oleanolic acid (70/30), and 3-O-trans-p-coumaroyl oleanolic acid. Infection tests demonstrated a dose-dependent inhibition of these triterpenes against HCoV-229E and SARS-CoV-2. Notably, cinnamoyl oleanolic acids displayed activity against both SARS-CoV-2 and HCoV-229E. Our findings suggest that Hippophae rhamnoides could represent a source of potential antiviral agents against coronaviruses.

Remdesivir Derivative VV116 Is a Potential Broad-Spectrum Inhibitor of Both Human and Animal Coronaviruses.

Coronaviruses represent a significant threat to both human and animal health, encompassing a range of pathogenic strains responsible for illnesses, from the common cold to more severe diseases. VV116 is a deuterated derivative of Remdesivir with oral bioavailability that was found to potently inhibit SARS-CoV-2. In this work, we investigated the broad-spectrum antiviral activity of VV116 against a variety of human and animal coronaviruses. We examined the inhibitory effects of VV116 on the replication of the human coronaviruses HCoV-NL63, HCoV-229E, and HCoV-OC43, as well as the animal coronaviruses MHV, FIPV, FECV, and CCoV. The findings reveal that VV116 effectively inhibits viral replication across these strains without exhibiting cytotoxicity, indicating its potential for safe therapeutic use. Based on the results of a time-of-addition assay and an rNTP competitive inhibition assay, it is speculated that the inhibitory mechanism of VV116 against HCoV-NL63 is consistent with its inhibition of SARS-CoV-2. Our work presents VV116 as a promising candidate for broad-spectrum anti-coronavirus therapy, with implications for both human and animal health, and supports the expansion of its therapeutic applications as backed by detailed experimental data.

Inactivation of human coronaviruses using an automated room disinfection device.

The emergence of more virulent and epidemic strains of viruses, especially in the context of COVID-19, makes it more important than ever to improve methods of decontamination. The objective of this study was to evaluate the potential of on-demand production of chlorine species to inactivate human coronaviruses. The commercial prototype disinfection unit was provided by Unipolar Water Technologies. The Unipolar device generates active chlorine species using an electrochemical reaction and dispenses the disinfectant vapour onto surfaces with an aspirator. The minimum effective concentration and exposure time of disinfectant were evaluated on human hepatoma (Huh7) cells using 50% tissue culture infectious dose (TCID50) assay and human coronavirus 229E (HCoV-229E), a surrogate for pathogenic human coronaviruses. We showed that chlorine species generated in the Unipolar device inactivate HCoV-229E on glass surfaces at ≥ 400 parts per million active chlorine concentration with a 5 min exposure time. Here, inactivation refers to the inability of the virus to infect the Huh7 cells. Importantly, no toxic effect was observed on Huh7 cells for any of the active chlorine concentrations and contact times tested.

Induction of antiviral gene expression by cyclosporine A, but not inhibition of cyclophilin A or B, contributes to its restriction of human coronavirus 229E infection in a lung epithelial cell line.

The development of antivirals with an extended spectrum of activity is an attractive possibility to protect against future emerging coronaviruses (CoVs). Cyclosporine A (CsA), a clinically approved immunosuppressive drug, has established antiviral activity against diverse unrelated viruses, including several CoVs. However, its antiviral mechanisms of action against CoV infection have remained elusive, precluding the rational design of non-immunosuppressive derivatives with improved antiviral activities. In this study, we evaluated the mechanisms of CsA against HCoV-229E infection in a human lung epithelial cell line. We demonstrate that the antiviral activity of CsA against HCoV-229E is independent of classical CsA target proteins, cyclophilin A or B, which are not required host factors for HCoV-229E in A549 cells. Instead, CsA treatment induces expression of antiviral genes in a manner dependent on interferon regulatory factor 1, but independent of classical interferon responses, which contributes to its inhibitory effect against HCoV-229E infection. Our results also point to a role for the HCoV-229E nucleoprotein in antagonizing activation of type I interferon, but we show that CsA treatment does not affect evasion of innate immune signalling pathways by HCoV-229E. Overall, our findings further the understanding of the antiviral mechanisms of CsA against CoV infection and highlight a novel immunomodulatory strategy to inhibit CoV infection that may inform future drug development efforts.

Superior anti-pulmonary viral potential of Natrialba sp. M6-producing surfactin and C50 carotenoid pigment with unveiling its action modes.

BACKGROUND: Respiratory viruses, particularly adenoviruses (ADV), influenza A virus (e.g., H1N1), and coronaviruses (e.g., HCoV-229E and SARS-CoV-2) pose a global public health problem. Therefore, developing natural wide-spectrum antiviral compounds for disrupting the viral life cycle with antioxidant activity provides an efficient treatment approach. Herein, biosurfactant (Sur) and C50 carotenoid pigment (Pig) of haloalkaliphilic archaeon Natrialba sp. M6 which exhibited potent efficacy against hepatitis and anti-herpes simplex viruses, were investigated against pulmonary viruses. METHODS: The cytotoxicity of the extracted Sur and Pig was examined on susceptible cell lines for ADV, HIN1, HCoV-229E, and SARS-CoV-2. Their potential against the cytopathic activity of these viruses was detected with investigating the action modes (including, virucidal, anti-adsorption, and anti-replication), unveiling the main mechanisms, and using molecular docking analysis. Radical scavenging activity was determined and HPLC analysis for potent extract (Sur) was performed. RESULTS: All current investigations stated higher anti-pulmonary viruses of Sur than Pig via mainly virucidal and/or anti-replicative modes. Moreover, Sur had stronger ADV's capsid protein binding, ADV's DNA polymerase inhibition, suppressing hemagglutinin and neuraminidase of H1N1, and inhibiting chymotrypsin-like (3CL) protease of SARS-CoV-2, supporting with in-silico analysis, as well as radical scavenging activity than Pig. HPLC analysis of Sur confirmed the predominate presence of surfactin in it. CONCLUSION: This study declared the promising efficacy of Sur as an efficient pharmacological treatment option for these pulmonary viruses and considered as guide for further in vivo research.

Improved Culture Methods for Human Coronaviruses HCoV-OC43, HCoV-229E, and HCoV-NL63.

HCoV-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1 are four of the seven known human coronaviruses (HCoVs) and, unlike the highly pathogenic SARS-CoV, MERS-CoV, and SARS-CoV-2, these four so-called seasonal HCoVs generally cause mild upper-respiratory-tract illness. As Biosafety Level 2 (BSL-2) pathogens, the seasonal HCoVs are more accessible and can be used as surrogates for studying the highly pathogenic HCoVs. However, scientists have for many years found these difficult to study because of the lack of a universal culture system and the inability of typical culture methods to yield high-titer infectious stocks. We have developed assays to grow and quantify infectious virus and viral RNA for HCoV-OC43, -229E, and -NL63. We identified which immortalized cell lines should be used to optimize the replication of HCoV-OC43, -229E, and -NL63 in order to generate high titers (Vero E6, Huh-7, and LLC-MK2 cells, respectively). Here we present protocols for improved propagation and quantification of each seasonal HCoV. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Growth of HCoVs Basic Protocol 2: Quantification of HCoV by plaque assay Basic Protocol 3: Quantification of HCoV RNA products of replication Basic Protocol 4: Concentrating HCoVs via ultracentrifugation.

An engineered A549 cell line expressing CD13 and TMPRSS2 is permissive to clinical isolate of human coronavirus 229E.

The lack of suitable in vitro culture model has hampered research on wild-type (WT) human coronaviruses. While 3D tissue or organ cultures have been instrumental for this purpose, such models are challenging, time-consuming, expensive and require extensive cell culture adaptation and directed evolution. Consequently, high-throughput applications are beyond reach in most cases. Here we developed a robust A549 cell line permissive to a human coronavirus 229E (HCoV-229E) clinical isolate by transducing CD13 and transmembrane serine protease 2 (TMPRSS2), henceforth referred to as A549++ cells. This modification allowed for productive infection, and a more detailed analysis showed that the virus might use the TMPRSS2-dependent pathway but can still bypass this pathway using cathepsin-mediated endocytosis. Overall, our data showed that A549++ cells are permissive to HCoV-229E clinical isolate, and applicable for further studies on HCoV-229E infectiology. Moreover, this line constitutes a uniform platform for studies on multiple members of the Coronaviridae family.

Evaluation of in vitro antiviral activity of SARS-CoV-2 Mpro inhibitor pomotrelvir and cross-resistance to nirmatrelvir resistance substitutions.

The unprecedented scale of the COVID-19 pandemic and the rapid evolution of SARS-CoV-2 variants underscore the need for broadly active inhibitors with a high barrier to resistance. The coronavirus main protease (Mpro) is an essential cysteine protease required for viral polyprotein processing and is highly conserved across human coronaviruses. Pomotrelvir is a novel Mpro inhibitor that has recently completed a phase 2 clinical trial. In this report, we demonstrated that pomotrelvir is a potent competitive inhibitor of SARS-CoV-2 Mpro with high selectivity against human proteases. In the enzyme assay, pomotrelvir is also active against Mpro proteins derived from human coronaviruses CoV-229E, CoV-OC43, CoV-HKU1, CoV-NL63, MERS, and SARS-CoV. In cell-based SARS-CoV-2 replicon and SARS-CoV-2 infection assays, pomotrelvir has shown potent inhibitory activity and is broadly active against SARS-CoV-2 clinical isolates including Omicron variants. Many resistance substitutions of the Mpro inhibitor nirmatrelvir confer cross-resistance to pomotrelvir, consistent with the finding from our enzymatic analysis that pomotrelvir and nirmatrelvir compete for the same binding site. In a SARS-CoV-2 infection assay, pomotrelvir is additive when combined with remdesivir or molnupiravir, two nucleoside analogs targeting viral RNA synthesis. In conclusion, our results from the in vitro characterization of pomotrelvir antiviral activity support its further clinical development as an alternative COVID-19 therapeutic option.