Impact of a Functional Dairy Powder and Its Primary Component on the Growth of Pathogenic and Probiotic Gut Bacteria and Human Coronavirus 229E.

Milk boasts an array of potent bioactive compounds, such as lactoferrin (Lf), immunoglobulins, and functional proteins, all delivering substantial therapeutic benefits. In this study, Immune Powder (a functional dairy formulation) and its primary component called Fractionated Milk Protein (FMP) containing Lf, zinc, and immunoglobulins and formulated by Ausnutria Pty Ltd. were evaluated for their potential broad-spectrum pharmacological activity. In particular, this study investigated the antibacterial (against pathogenic Escherichia coli), prebiotic (promoting Lactobacillus delbrueckii growth), anti-inflammatory (inhibition of NO production in RAW264.7 macrophages), and antiviral (against human coronavirus 229E) effects of the samples. In addition, the impact of simulated gastric digestion on the efficacy of the samples was explored. LCMS-based proteomics was implemented to unveil cellular and molecular mechanisms underlying antiviral activity. The Immune Powder demonstrated antibacterial activity against E. coli (up to 99.74 ± 11.47% inhibition), coupled with prebiotic action (10.84 ± 2.2 viability fold-change), albeit these activities diminished post-digestion (p < 0.01). The Immune Powder effectively mitigated NO production in lipopolysaccharide-stimulated RAW264.7 macrophages, with declining efficacy post-digestion (p < 0.0001). The Immune Powder showed similar antiviral activity before and after digestion (p > 0.05) with up to 3-fold improvement. Likewise, FMP exhibited antibacterial potency pre-digestion at high concentrations (95.56 ± 1.23% inhibition at 125 mg/mL) and post-digestion at lower doses (61.82 ± 5.58% inhibition at 3906.25 µg/mL). FMP also showed enhanced prebiotic activity post-digestion (p < 0.0001), NO inhibition pre-digestion, and significant antiviral activity. The proteomics study suggested that the formulation and its primary component shared similar antiviral mechanisms by inhibiting scavenger receptor binding and extracellular matrix interaction.

Characterization of C-phycocyanin antioxidant, anti-inflammatory, anti-tumour, and anti-HCoV-229E activities and encapsulation for implementation in an innovative functional yogurt.

Functional bioactive ingredients isolated from microalgae as sustainable sources have become a new subject of pharmacology and functional foods. Thus, the work aims to produce crude phycocyanin (C-PC), define it, and investigate its pharmacological effects before warping it in a nanophytosome. Subsequently, the physicochemical properties of nanoparticles were evaluated. Both free and nanophytosomes of C-PC were incorporated into cow milk fermented with the probiotic Lactobacillus rhamnosus KU985435 to make functional yoghurt and the stability of C-PC of both phytosomes was assessed. The amino acid content of C-PC revealed the presence of eight of nine essential amino acids and eight of eleven non-essential amino acids. C-PC has a medium molecular weight (82.992 kDa). Some pharmacological effects like reducing inflammation (98.76 % ± 0.065), fighting free radicals (99.12 % ± 0.027), and being able to inhibit the human coronavirus 229 E with a selective index of 27.9 were observed. The maximum viral inhibitory activity was detected during the adsorption stage. Anti-human liver and colon carcinomas that exceeded Doxorubicin with very low cytotoxicity against normal cell lines were detected. C-PC is an unstable protein that could be degraded in the yoghurt during storage. Therefore, phytosome encapsulation can effectively stabilize C-PC (particle size 44.50 ± 12 nm and zeta-potential -32.4 ± 5 mV) and protect it from the acidic environment of the yoghurt. The produced yoghurt showed the desired physicochemical and functional properties and overall acceptance. The results prove that C-PC from spirulina algae is a renewable source of dyes. The encapsulation process using phytosomes gave it high stability against environmental influences, and therefore, it can be applied in the food and pharmaceutical industries in the future.

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.

Effect of a Total Extract and Saponins from Astragalus glycyphyllos L. on Human Coronavirus Replication In Vitro.

Members of the family Coronaviridae cause diseases in mammals, birds, and wildlife (bats), some of which may be transmissible to humans or specific to humans. In the human population, they can cause a wide range of diseases, mainly affecting the respiratory and digestive systems. In the scientific databases, there are huge numbers of research articles about the antiviral, antifungal, antibacterial, antiviral, and anthelmintic activities of medicinal herbs and crops with different ethnobotanical backgrounds. The subject of our research is the antiviral effect of isolated saponins, a purified saponin mixture, and a methanol extract of Astragalus glycyphyllos L. In the studies conducted for the cytotoxic effect of the substances, CC50 (cytotoxic concentration 50) and MTC (maximum tolerable concentration) were determined by the colorimetric method (MTT assay). The virus was cultured in the MDBK cell line. As a result of the experiments carried out on the influence of substances on viral replication (using MTT-based colorimetric assay for detection of human Coronavirus replication inhibition), it was found that the extract and the purified saponin mixture inhibited 100% viral replication. The calculated selective indices are about 13 and 18, respectively. The obtained results make them promising for a preparation with anti-Coronavirus action.

Enhanced broad spectrum in vitro antiviral efficacy of 3-F-4-MeO-Bn, 3-CN, and 4-CN derivatives of lipid remdesivir nucleoside monophosphate prodrugs.

Broad spectrum oral antivirals are urgently needed for the early treatment of many RNA viruses of clinical concern. We previously described the synthesis of 1-O-octadecyl-2-O-benzyl-glycero-3-phospho-RVn (V2043), an orally bioavailable lipid prodrug of remdesivir nucleoside (RVn, GS-441524) with broad spectrum antiviral activity against viruses with pandemic potential. Here we compared the relative activity of V2043 with new RVn lipid prodrugs containing sn-1 alkyl ether or sn-2 glycerol modifications. We found that 3-F-4-MeO-Bn, 3-CN-Bn, and 4-CN-Bn sn-2 glycerol modifications improved antiviral activity compared to V2043 when tested in vitro against clinically important RNA viruses from 5 virus families. These results support the continued development of V2043 and sn-2 glycerol modified RVn lipid prodrugs for the treatment of a broad range of RNA viruses for which there are limited therapies.

Dry-spun carbon nanotube ultrafiltration membranes tailored by anti-viral metal oxide coatings for human coronavirus 229E capture in water.

Although waterborne virus removal may be achieved using separation membrane technologies, such technologies remain largely inefficient at generating virus-free effluents due to the lack of anti-viral reactivity of conventional membrane materials required to deactivating viruses. Here, a stepwise approach towards simultaneous filtration and disinfection of Human Coronavirus 229E (HCoV-229E) in water effluents, is proposed by engineering dry-spun ultrafiltration carbon nanotube (CNT) membranes, coated with anti-viral SnO2 thin films via atomic layer deposition. The thickness and pore size of the engineered CNT membranes were fine-tuned by varying spinnable CNT sheets and their relative orientations on carbon nanofibre (CNF) porous supports to reach thicknesses less than 1 µm and pore size around 28 nm. The nanoscale SnO2 coatings were found to further reduce the pore size down to ∼21 nm and provide more functional groups on the membrane surface to capture the viruses via size exclusion and electrostatic attractions. The synthesized CNT and SnO2 coated CNT membranes were shown to attain a viral removal efficiency above 6.7 log10 against HCoV-229E virus with fast water permeance up to ∼4 × 103 and 3.5 × 103 L.m-2.h-1.bar-1, respectively. Such high performance was achieved by increasing the dry-spun CNT sheets up to 60 layers, orienting successive 30 CNT layers at 45°, and coating 40 nm SnO2 on the synthesized membranes. The current study provides an efficient scalable fabrication scheme to engineer flexible ultrafiltration CNT-based membranes for cost-effective filtration and inactivation of waterborne viruses to outperform the state-of-the-art ultrafiltration membranes.

The clinical and virological features of two children's coinfections with human adenovirus type 7 and human coronavirus-229E virus.

Objective: Human adenovirus (HAdV) coinfection with other respiratory viruses is common, but adenovirus infection combined with human coronavirus-229E (HCoV-229E) is very rare. Study design and setting: Clinical manifestations, laboratory examinations, and disease severity were compared between three groups: one coinfected with HAdV-Ad7 and HCoV-229E, one infected only with adenovirus (mono-adenovirus), and one infected only with HCoV-229E (mono-HCoV-229E). Results: From July to August 2019, there were 24 hospitalized children: two were coinfected with HAdV-Ad7 and HCoV-229E, and 21 were infected with a single adenovirus infection. Finally, one 14-year-old boy presented with a high fever, but tested negative for HAdV-Ad7 and HCoV-229E. Additionally, three adult asymptotic cases with HCoV-229E were screened. No significant difference in age was found in the coinfection and mono-adenovirus groups (11 vs. 8 years, p = 0.332). Both groups had the same incubation period (2.5 vs. 3 days, p = 0.8302), fever duration (2.5 vs. 2.9 days, p = 0.5062), and length of hospital stay (7 vs. 6.76 days, p = 0.640). No obvious differences were found in viral loads between the coinfection and mono-adenovirus groups (25.4 vs. 23.7, p = 0.570), or in the coinfection and mono-HCoV-229E groups (32.9 vs. 30.06, p = 0.067). All cases recovered and were discharged from the hospital. Conclusion: HAdV-Ad7 and HCoV-229E coinfection in healthy children may not increase the clinical severity or prolong the clinical course. The specific interaction mechanism between the viruses requires further study.

Survival of human coronavirus 229E at different temperatures on various food-contact surfaces and food and under simulated digestive conditions.

The COVID-19 pandemic caused by SARS-CoV-2 has had a major impact on human health and the global economy. Various transmission possibilities of SARS-CoV-2 have been proposed, such as the surface of food in the cold chain and food packaging, as well as the fecal-oral route, although person-to-person contact via droplets and aerosols has been confirmed as the main route of transmission. This study evaluated the survivability of HCoV-229E, a SARS-CoV-2 surrogate, in suspension, on food-contact surfaces and on food at various temperatures, and in simulated digestive fluids by TCID50 assay. In suspension, HCoV-229E survived after 5 days at 20 °C with a 3.69 log reduction, after 28 days at 4 °C with a 3.07 log reduction, and after 12 weeks at -20 °C with a 1.18 log reduction. On food-contact surfaces, HCoV-229E was not detected on day 3 on stainless steel (SS), plastic (LDPE), and silicone rubber (SR) at 20 °C with a 3.28, 3.24 and 3.28 log reduction, respectively, and survived after 28 days on SS and LDPE at 4 °C with a 3.13 and 2.88 log reduction, respectively, and survived after 12 weeks on SS, LDPE, and SR at -20 °C with a 1.92, 1.32 and 1.99 log reduction, respectively. On food, HCoV-229E was not detected on day 3 on lettuce and day 4 on chicken breast and salmon at 20 °C with a 3.61, 3.26 and 3.08 log reduction, respectively, and on day 14 on lettuce and day 21 on chicken breast and salmon at 4 °C with a 3.88, 3.44 and 3.56 log reduction, respectively. The virus remained viable for 12 weeks in all foods at -20 °C with 2-2.47 log reduction. In addition, in simulated digestive fluid experiments, HCoV-229E was relatively resistant in simulated salivary fluid (SSF; pH 7, 5), fed state simulated gastric fluid (FeSSGF; pH 3, 5, 7), and fasted state simulated intestinal fluid (FaSSIF; pH 7). However, the virus was less tolerant in fasted state simulated gastric fluid (FaSSGF; pH 1.6) and fed state simulated intestinal fluid (FeSSIF; pH 5). Therefore, this study suggested that HCoV-229E remained infectious on various food-contact surfaces and foods; in particular, it survived longer at lower temperatures and survived depending on the pH of the simulated digestive fluid.

Impact of SARS-CoV-2 Spike Mutations on Its Activation by TMPRSS2 and the Alternative TMPRSS13 Protease.

The continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) urges better understanding of the functional motifs in the spike (S) protein and their tolerance to mutations. Here, we focused on the S2' motif, which, during virus entry, requires cleavage by a host cell protease to release the fusion peptide. Though belonging to an immunogenic region, the SARS-CoV-2 S2' motif (811-KPSKR-815) has shown hardly any variation, with its three basic (K/R) residues being >99.99% conserved thus far. By creating a series of mutant pseudoviruses bearing the spikes of Wuhan-Hu-1, its G614 mutant or the Delta and Omicron variants, we show that residue K814 (preceding the scissile R815) is dispensable for TMPRSS2 yet favored by the alternative TMPRSS13 protease. Activation by TMPRSS13 was drastically reduced when the SARS-CoV-2 S2' motif was swapped with that of the low pathogenic 229E coronavirus (685-RVAGR-689), and also, the reverse effect was seen. This swap had no impact on recognition by TMPRSS2. In the Middle East respiratory syndrome coronavirus (MERS-CoV) spike, introducing a dibasic scissile motif was easily accepted by TMPRSS13 but less so by TMPRSS2, confirming that TMPRSS13 favors a sequence rich in K/R residues. Pseudovirus entry experiments in Calu-3 cells confirmed that the S2' mutations have minor impact on TMPRSS2. Our findings are the first to demonstrate which S2' residues are important for SARS-CoV-2 spike activation by these two airway proteases, with TMPRSS2 being more tolerant to variation than TMPRSS13. This preemptive insight will help to estimate the impact of S2' motif changes as they appear in new SARS-CoV-2 variants. IMPORTANCE Since its introduction in humans, SARS-CoV-2 is evolving with frequent appearance of new variants. The surveillance would benefit from proactive characterization of the functional motifs in the spike (S) protein, the most variable viral factor. This is linked to immune evasion but also influences spike functioning. Remarkably, though located in a strongly immunogenic region, the S2' cleavage motif has, thus far, remained highly conserved. This suggests that its sequence is critical for spike activation by airway proteases. To investigate this, we assessed how pseudovirus entry is affected by changes in the S2' motif. We demonstrate that TMPRSS2 readily accepts variations in this motif, whereas the alternative TMPRSS13 protease is more fastidious. The Wuhan-Hu-1, G614, Delta and Omicron spikes showed no difference in this regard. Being the first in its kind, our study will help to assess the impact of S2' variations as soon as they are detected during variant surveillance.

COVID-19 and the Importance of Being Prepared: A Multidisciplinary Strategy for the Discovery of Antivirals to Combat Pandemics.

During an emergency, such as a pandemic in which time and resources are extremely scarce, it is important to find effective and rapid solutions when searching for possible treatments. One possibility in this regard is the repurposing of available "on the market" drugs. This is a proof of the concept study showing the potential of a collaboration between two research groups, engaged in computer-aided drug design and control of viral infections, for the development of early strategies to combat future pandemics. We describe a QSAR (quantitative structure activity relationship) based repurposing study on molecular topology and molecular docking for identifying inhibitors of the main protease (Mpro) of SARS-CoV-2, the causative agent of COVID-19. The aim of this computational strategy was to create an agile, rapid, and efficient way to enable the selection of molecules capable of inhibiting SARS-CoV-2 protease. Molecules selected through in silico method were tested in vitro using human coronavirus 229E as a surrogate for SARS-CoV-2. Three strategies were used to screen the antiviral activity of these molecules against human coronavirus 229E in cell cultures, e.g., pre-treatment, co-treatment, and post-treatment. We found >99% of virus inhibition during pre-treatment and co-treatment and 90−99% inhibition when the molecules were applied post-treatment (after infection with the virus). From all tested compounds, Molport-046-067-769 and Molport-046-568-802 are here reported for the first time as potential anti-SARS-CoV-2 compounds.