The Novel A-Type Proanthocyanidin-Rich Phytocomplex SP4™ Acts as a Broad-Spectrum Antiviral Agent against Human Respiratory Viruses.

The appearance of new respiratory virus infections in humans with epidemic or pandemic potential has underscored the urgent need for effective broad-spectrum antivirals (BSAs). Bioactive compounds derived from plants may provide a natural source of new BSA candidates. Here, we investigated the novel phytocomplex formulation SP4™ as a candidate direct-acting BSA against major current human respiratory viruses, including coronaviruses and influenza viruses. SP4™ inhibited the in vitro replication of SARS-CoV-2, hCoV-OC43, hCoV-229E, Influenza A and B viruses, and respiratory syncytial virus in the low-microgram range. Using hCoV-OC43 as a representative respiratory virus, most of the antiviral activity of SP4™ was observed to stem primarily from its dimeric A-type proanthocyanidin (PAC-A) component. Further investigations of the mechanistic mode of action showed SP4™ and its PAC-A-rich fraction to prevent hCoV-OC43 from attaching to target cells and exert virucidal activity. This occurred through their interaction with the spike protein of hCoV-OC43 and SARS-CoV-2, thereby interfering with spike functions and leading to the loss of virion infectivity. Overall, these findings support the further development of SP4™ as a candidate BSA of a natural origin for the prevention of human respiratory virus infections.

Human Coronavirus 229E Infection Inactivates Pyroptosis Executioner Gasdermin D but Ultimately Leads to Lytic Cell Death Partly Mediated by Gasdermin E.

Human coronavirus 229E (HCoV-229E) is associated with upper respiratory tract infections and generally causes mild respiratory symptoms. HCoV-229E infection can cause cell death, but the molecular pathways that lead to virus-induced cell death as well as the interplay between viral proteins and cellular cell death effectors remain poorly characterized for HCoV-229E. Studying how HCoV-229E and other common cold coronaviruses interact with and affect cell death pathways may help to understand its pathogenesis and compare it to that of highly pathogenic coronaviruses. Here, we report that the main protease (Mpro) of HCoV-229E can cleave gasdermin D (GSDMD) at two different sites (Q29 and Q193) within its active N-terminal domain to generate fragments that are now unable to cause pyroptosis, a form of lytic cell death normally executed by this protein. Despite GSDMD cleavage by HCoV-229E Mpro, we show that HCoV-229E infection still leads to lytic cell death. We demonstrate that during virus infection caspase-3 cleaves and activates gasdermin E (GSDME), another key executioner of pyroptosis. Accordingly, GSDME knockout cells show a significant decrease in lytic cell death upon virus infection. Finally, we show that HCoV-229E infection leads to increased lytic cell death levels in cells expressing a GSDMD mutant uncleavable by Mpro (GSDMD Q29A+Q193A). We conclude that GSDMD is inactivated by Mpro during HCoV-229E infection, preventing GSDMD-mediated cell death, and point to the caspase-3/GSDME axis as an important player in the execution of virus-induced cell death. In the context of similar reported findings for highly pathogenic coronaviruses, our results suggest that these mechanisms do not contribute to differences in pathogenicity among coronaviruses. Nonetheless, understanding the interactions of common cold-associated coronaviruses and their proteins with the programmed cell death machineries may lead to new clues for coronavirus control strategies.

Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics.

The emergence of the COVID-19 pandemic prompted an increased interest in seasonal human coronaviruses. OC43, 229E, NL63, and HKU1 are endemic seasonal coronaviruses that cause the common cold and are associated with generally mild respiratory symptoms. In this study, we identified cell lines that exhibited cytopathic effects (CPE) upon infection by three of these coronaviruses and characterized their viral replication kinetics and the effect of infection on host surface receptor expression. We found that NL63 produced CPE in LLC-MK2 cells, while OC43 produced CPE in MRC-5, HCT-8, and WI-38 cell lines, while 229E produced CPE in MRC-5 and WI-38 by day 3 post-infection. We observed a sharp increase in nucleocapsid and spike viral RNA (vRNA) from day 3 to day 5 post-infection for all viruses; however, the abundance and the proportion of vRNA copies measured in the supernatants and cell lysates of infected cells varied considerably depending on the virus-host cell pair. Importantly, we observed modulation of coronavirus entry and attachment receptors upon infection. Infection with 229E and OC43 led to a downregulation of CD13 and GD3, respectively. In contrast, infection with NL63 and OC43 leads to an increase in ACE2 expression. Attempts to block entry of NL63 using either soluble ACE2 or anti-ACE2 monoclonal antibodies demonstrated the potential of these strategies to greatly reduce infection. Overall, our results enable a better understanding of seasonal coronaviruses infection kinetics in permissive cell lines and reveal entry receptor modulation that may have implications in facilitating co-infections with multiple coronaviruses in humans.IMPORTANCESeasonal human coronavirus is an important cause of the common cold associated with generally mild upper respiratory tract infections that can result in respiratory complications for some individuals. There are no vaccines available for these viruses, with only limited antiviral therapeutic options to treat the most severe cases. A better understanding of how these viruses interact with host cells is essential to identify new strategies to prevent infection-related complications. By analyzing viral replication kinetics in different permissive cell lines, we find that cell-dependent host factors influence how viral genes are expressed and virus particles released. We also analyzed entry receptor expression on infected cells and found that these can be up- or down-modulated depending on the infecting coronavirus. Our findings raise concerns over the possibility of infection enhancement upon co-infection by some coronaviruses, which may facilitate genetic recombination and the emergence of new variants and strains.

Development of novel antivrial agents that induce the degradation of the main protease of human-infecting coronaviruses.

The family of human-infecting coronaviruses (HCoVs) poses a serious threat to global health and includes several highly pathogenic strains that cause severe respiratory illnesses. It is essential that we develop effective broad-spectrum anti-HCoV agents to prepare for future outbreaks. In this study, we used PROteolysis TArgeting Chimera (PROTAC) technology focused on degradation of the HCoV main protease (Mpro), a conserved enzyme essential for viral replication and pathogenicity. By adapting the Mpro inhibitor GC376, we produced two novel PROTACs, P2 and P3, which showed relatively broad-spectrum activity against the human-infecting CoVs HCoV-229E, HCoV-OC43, and SARS-CoV-2. The concentrations of these PROTACs that reduced virus replication by 50 % ranged from 0.71 to 4.6 µM, and neither showed cytotoxicity at 100 µM. Furthermore, mechanistic binding studies demonstrated that P2 and P3 effectively targeted HCoV-229E, HCoV-OC43, and SARS-CoV-2 by degrading Mpro within cells in vitro. This study highlights the potential of PROTAC technology in the development of broad-spectrum anti-HCoVs agents, presenting a novel approach for dealing with future viral outbreaks, particularly those stemming from CoVs.

Human coronavirus 229E inactivation on porous and nonporous materials using dielectric barrier discharge (DBD) plasma.

Viruses can spread through contaminated aerosols and contaminated surface materials, and effective disinfection techniques are essential for virus inactivation. Nonthermal plasma-generated reactive oxygen and nitrogen species can effectively inactivate the coronavirus. We aim to interpret the coronavirus inactivation level and mechanism of surface interaction with materials with and without dielectric barrier discharge (DBD) plasma treatment. Nonthermal plasma, particularly surface-type DBD plasma, can inactivate human coronavirus 229E (HCoV-229E) on porous (paper, wood, mask) and nonporous (plastic, stainless steel, glass, Cu) materials. Virus inactivation was analyzed using a 50% tissue culture infectivity dose (TCID50) using cell line, flow cytometry, and immunofluorescence. Surfaces contaminated with HCoV-229E were treated at different time intervals (0-5 h) with and without plasma exposure (natural decay in ambient air conditions). HCoV-229E persistence conformed to the following order: plastic > cover glass > stainless steel > mask > wood > paper > Cu with and without plasma exposure. HCoV-229E was more stable in plastic, cover glass, and stainless steel in 5 h, and the viable virus titer gradually decreased from its initial log10 order of 6.892 to 1.72, 1.53, and 1.32 TCID50/mL, respectively, under plasma exposure. No virus was observed in Cu after treatment for 5 h. The use of airflow, ambient nitrogen, and argon did not promote virus inactivation. Flow cytometry and immunofluorescence analysis demonstrated a low expression level of spike protein (fluorescence intensity) during plasma treatment and in E and M genes expression compared with the virus control.

Interferon signaling in the nasal epithelium distinguishes among lethal and common cold coronaviruses and mediates viral clearance.

All respiratory viruses establish primary infections in the nasal epithelium, where efficient innate immune induction may prevent dissemination to the lower airway and thus minimize pathogenesis. Human coronaviruses (HCoVs) cause a range of pathologies, but the host and viral determinants of disease during common cold versus lethal HCoV infections are poorly understood. We model the initial site of infection using primary nasal epithelial cells cultured at an air-liquid interface (ALI). HCoV-229E, HCoV-NL63, and human rhinovirus-16 are common cold-associated viruses that exhibit unique features in this model: early induction of antiviral interferon (IFN) signaling, IFN-mediated viral clearance, and preferential replication at nasal airway temperature (33 °C) which confers muted host IFN responses. In contrast, lethal SARS-CoV-2 and MERS-CoV encode antagonist proteins that prevent IFN-mediated clearance in nasal cultures. Our study identifies features shared among common cold-associated viruses, highlighting nasal innate immune responses as predictive of infection outcomes and nasally directed IFNs as potential therapeutics.

Deciphering the similarities and disparities of molecular mechanisms behind respiratory epithelium response to HCoV-229E and SARS-CoV-2 and drug repurposing, a systems biology approach.

PURPOSE: Identifying the molecular mechanisms behind SARS-CoV-2 disparities and similarities will help find new treatments. The present study determines networks' shared and non-shared (specific) crucial elements in response to HCoV-229E and SARS-CoV-2 viruses to recommend candidate medications. METHODS: We retrieved the omics data on respiratory cells infected with HCoV-229E and SARS-CoV-2, constructed PPIN and GRN, and detected clusters and motifs. Using a drug-gene interaction network, we determined the similarities and disparities of mechanisms behind their host response and drug-repurposed. RESULTS: CXCL1, KLHL21, SMAD3, HIF1A, and STAT1 were the shared DEGs between both viruses' protein-protein interaction network (PPIN) and gene regulatory network (GRN). The NPM1 was a specific critical node for HCoV-229E and was a Hub-Bottleneck shared between PPI and GRN in HCoV-229E. The HLA-F, ADCY5, TRIM14, RPF1, and FGA were the seed proteins in subnetworks of the SARS-CoV-2 PPI network, and HSPA1A and RPL26 proteins were the seed in subnetworks of the PPI network of HCOV-229E. TRIM14, STAT2, and HLA-F played the same role for SARS-CoV-2. Top enriched KEGG pathways included cell cycle and proteasome in HCoV-229E and RIG-I-like receptor, Chemokine, Cytokine-cytokine, NOD-like receptor, and TNF signaling pathways in SARS-CoV-2. We suggest some candidate medications for COVID-19 patient lungs, including Noscapine, Isoetharine mesylate, Cycloserine, Ethamsylate, Cetylpyridinium, Tretinoin, Ixazomib, Vorinostat, Venetoclax, Vorinostat, Ixazomib, Venetoclax, and epoetin alfa for further in-vitro and in-vivo investigations. CONCLUSION: We suggested CXCL1, KLHL21, SMAD3, HIF1A, and STAT1, ADCY5, TRIM14, RPF1, and FGA, STAT2, and HLA-F as critical genes and Cetylpyridinium, Cycloserine, Noscapine, Ethamsylate, Epoetin alfa, Isoetharine mesylate, Ribavirin, and Tretinoin drugs to study further their importance in treating COVID-19 lung complications.

Design, synthesis, docking, and antiviral evaluation of some novel pyrimidinone-based α-aminophosphonates as potent H1N1 and HCoV-229E inhibitors.

Dialkyl/aryl aminophosphonates, 3a-g and 4a-e were synthesized using the LiClO4 catalyzed Kabachnic Fields-type reaction straightforwardly and efficiently. The synthesized phosphonates structures were characterized using elemental analyses, FT-IR, 1H NMR, 13C NMR, and MS spectroscopy. The new compounds were subjected to in-silico molecular docking simulations to evaluate their potential inhibition against Influenza A Neuraminidase and RNA-dependent RNA polymerase of human coronavirus 229E. Subsequently, the compounds were further tested in vitro using a cytopathic inhibition assay to assess their antiviral activity against both human Influenza (H1N1) and human coronavirus (HCoV-229E). Diphenyl ((2-(5-cyano-6-oxo-4-phenyl-1,6-dihydropyrimidin-2-yl) hydrazinyl) (furan-2-yl) methyl) phosphonate (3f) and diethyl ((2-(5-cyano-6-oxo-4-phenyl-1,6-dihydropyrimidin-2-yl) hydrazinyl) (1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl) methyl) phosphonate (4e) were demonstrated direct inhibition activity against Influenza A Neuraminidase and RNA-dependent RNA polymerase. This was supported by their highly favorable binding energies in-silico, with top-ranked values of -12.5 kcal/mol and -14.2 kcal/mol for compound (3f), and -13.5 kcal/mol and -9.89 kcal/mol for compound (4e). Moreover, they also displayed notable antiviral efficacy in vitro against both viruses. These compounds demonstrated significant antiviral activity, as evidenced by selectivity indices (SI) of 101.7 and 51.8, respectively against H1N1, and 24.5 and 5.1 against HCoV-229E, respectively.

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).

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.

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.

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.

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.

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.

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.

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.

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).

In vitro assessment of antibacterial and antiviral activity of three copper products after 200 rounds of simulated use.

Copper has well-documented antibacterial effects but few have evaluated it after prolonged use and against bacteria and viruses. Coupons from three copper formulations (solid, thermal coating, and decal applications) and carbon steel controls were subjected to 200 rounds simulated cleaning using a Wiperator™ and either an accelerated hydrogen peroxide, quaternary ammonium, or artificial sweat products. Antibacterial activity against S. aureus and P. aeruginosa was then evaluated using a modified Environmental Protection Agency protocol. Antiviral activity against coronavirus (229E) and norovirus (MNV-1) surrogates was assessed using the TCID50 method. Results were compared to untreated control coupons. One hour after inoculation, S. aureus exhibited a difference in log kill of 1.16 to 4.87 and P. aeruginosa a log kill difference of 3.39-5.23 (dependent upon copper product and disinfectant) compared to carbon steel. MNV-1 demonstrated an 87-99% reduction on each copper surfaces at 1 h and 99% reduction at 2 h compared to carbon steel. Similarly, coronavirus 229E exhibited a 97-99% reduction after 1 h and 90-99% after 2 h. Simulated use with artificial sweat did not hinder the antiviral nor the antibacterial activity of Cu surfaces. Self-sanitizing copper surfaces maintained antibacterial and antiviral activity after 200 rounds of simulated cleaning.

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.