A standardized procedure for quantitative evaluation of residual viral activity on antiviral treated textiles

The SARS-CoV-2 pandemic has increased the demand for antiviral technologies to mitigate or prevent the risk of viral transmission. Antiviral treated textiles have the potential to save lives, especially in healthcare settings that rely on reusable patient-care textiles and personal protective equipment. Currently, little is known about the role of textiles in cross-contamination and pathogen transmission, despite the wealth of information on hard surfaces and fomites harboring viruses that remain viable in certain circumstances. In addition, there is no international standard method for evaluating residual viral activity on textiles, which would allow a thorough investigation of the efficacy of antiviral textile products. Therefore, this pilot study aims to develop and refine a standardized protocol to quantitatively evaluate residual viral activity on antiviral textiles. Specifically, we focused on general textiles, such as bed linens, commonly used in healthcare settings for patient care. The Tissue Culture Infectious Dose 50 (TCID50) method is frequently used to quantitatively evaluate viral infectivity on textiles, but has not been established as a standard. This procedure involves observing the cytopathic effect of a given virus on cells grown in a 96-well plate after several days of incubation to determine the infectivity titer. We used HCoV-229E and Huh-7 human liver cancer cells for this investigation. We worked to improve the TCID50 method through variations of different steps within the protocol to attain reproducible results. Our proposed optimized hybrid protocol has shown evidence that the protocol is technically simpler and more efficient, and provides successful, consistent results. The analysis showed a significant difference between the treated fabric compared with controls.

Color-variable dual-dyed photodynamic antimicrobial polyethylene terephthalate (PET)/cotton blended fabrics.

The urgent demand for scalable, potent, color variable, and comfortable antimicrobial textiles as personal protection equipment (PPE) to help reduce infection transmission in hospitals and healthcare facilities has significantly increased since the start of the COVID-19 pandemic. Here, we explored photodynamic antimicrobial polyethylene terephthalate/cotton (TC) blended fabrics comprised of photosensitizer-conjugated cotton fibers and polyethylene terephthalate (PET) fibers dyed with disperse dyes. A small library of TC blended fabrics was constructed wherein the PET fibers were embedded with traditional disperse dyes dominating the fabric color, thereby enabling variable color expression, while the cotton fibers were covalently coupled with the photosensitizer thionine acetate as the microbicidal agent. Physical (SEM, CLSM, TGA, XPS and mechanical strength) and colorimetric (K/S and CIELab values) characterization methods were employed to investigate the resultant fabrics, and photooxidation studies with DPBF demonstrated the ability of these materials to generate reactive oxygen species (i.e., singlet oxygen) upon visible light illumination. The best results demonstrated a photodynamic inactivation of 99.985% (~ 3.82 log unit reduction, P = 0.0021) against Gram-positive S. aureus, and detection limit inactivation (99.99%, 4 log unit reduction, P ≤ 0.0001) against Gram-negative E. coli upon illumination with visible light (60 min; ~ 300 mW/cm2; λ ≥ 420 nm). Enveloped human coronavirus 229E showed a photodynamic susceptibility of ~ 99.99% inactivation after 60 min illumination (400-700 nm, 65 ± 5 mW/cm2). The presence of the disperse dyes on the fabrics showed no significant effects on the aPDI results, and furthermore, appeared to provide the photosensitizer with some measure of protection from photobleaching, thus improving the photostability of the dual-dyed fabrics. Taken together, these results suggest the feasibility of low cost, scalable and color variable thionine-conjugated TC blended fabrics as potent self-disinfecting textiles.

Vandetanib Blocks the Cytokine Storm in SARS-CoV-2-Infected Mice.

The portfolio of SARS-CoV-2 small molecule drugs is currently limited to a handful that are either approved (remdesivir), emergency approved (dexamethasone, baricitinib, paxlovid, and molnupiravir), or in advanced clinical trials. Vandetanib is a kinase inhibitor which targets the vascular endothelial growth factor receptor (VEGFR), the epidermal growth factor receptor (EGFR), as well as the RET-tyrosine kinase. In the current study, it was tested in different cell lines and showed promising results on inhibition versus the toxic effect on A549-hACE2 cells (IC50 0.79 µM) while also showing a reduction of >3 log TCID50/mL for HCoV-229E. The in vivo efficacy of vandetanib was assessed in a mouse model of SARS-CoV-2 infection and statistically significantly reduced the levels of IL-6, IL-10, and TNF-α and mitigated inflammatory cell infiltrates in the lungs of infected animals but did not reduce viral load. Vandetanib also decreased CCL2, CCL3, and CCL4 compared to the infected animals. Vandetanib additionally rescued the decreased IFN-1ß caused by SARS-CoV-2 infection in mice to levels similar to that in uninfected animals. Our results indicate that the FDA-approved anticancer drug vandetanib is worthy of further assessment as a potential therapeutic candidate to block the COVID-19 cytokine storm.

Effect of BIO-PLYTM, a Platelet-Rich Plasma Derived Biologic on PRRSV-2-Infected Macrophages.

Porcine Reproductive and Respiratory Syndrome (PRRS) is the one of the most devastating diseases impacting the swine industry worldwide. Control and prevention methods rely on biosafety measures and vaccination. As an RNA virus with a high rate of mutation, vaccines are only partially effective against circulating and newly emerging strains. To reduce the burden of this disease, research on alternative control methods is needed. Here, we assess the in vitro antiviral effect of a novel platelet-rich plasma-derived biologic termed BIO-PLYTM (for the BIOactive fraction of Platelet-rich plasma LYsate) from both swine and equine origin. Our results show that BIO-PLYTM significantly reduces the amount of PRRSV viral load determined by RT-qPCR and the number of infectious viral particles measured by TCID50 in infected porcine alveolar and parenchymal macrophages. This study also showed limited toxicity of BIO-PLYTM in vitro and aspects of its immunomodulatory capacity evaluating the regulation of reactive oxygen species and cytokines production in infected cells. Finally, this study presents promising data on the effect of BIO-PLYTM on other RNA viruses such as human A influenza viruses and coronavirus.

A standardized procedure for quantitative evaluation of residual viral activity on antiviral treated textiles

The SARS-CoV-2 pandemic has increased the demand for antiviral technologies to mitigate or prevent the risk of viral transmission. Antiviral treated textiles have the potential to save lives, especially in healthcare settings that rely on reusable patient-care textiles and personal protective equipment. Currently, little is known about the role of textiles in cross-contamination and pathogen transmission, despite the wealth of information on hard surfaces and fomites harboring viruses that remain viable in certain circumstances. In addition, there is no international standard method for evaluating residual viral activity on textiles, which would allow a thorough investigation of the efficacy of antiviral textile products. Therefore, this pilot study aims to develop and refine a standardized protocol to quantitatively evaluate residual viral activity on antiviral textiles. Specifically, we focused on general textiles, such as bed linens, commonly used in healthcare settings for patient care. The Tissue Culture Infectious Dose 50 (TCID50) method is frequently used to quantitatively evaluate viral infectivity on textiles, but has not been established as a standard. This procedure involves observing the cytopathic effect of a given virus on cells grown in a 96-well plate after several days of incubation to determine the infectivity titer. We used HCoV-229E and Huh-7 human liver cancer cells for this investigation. We worked to improve the TCID50 method through variations of different steps within the protocol to attain reproducible results. Our proposed optimized hybrid protocol has shown evidence that the protocol is technically simpler and more efficient, and provides successful, consistent results. The analysis showed a significant difference between the treated fabric compared with controls.

Rapid and Repetitive Inactivation of SARS‐CoV‐2 and Human Coronavirus on Self‐Disinfecting Anionic Polymers

While the ongoing COVID‐19 pandemic affirms an urgent global need for effective vaccines as second and third infection waves are spreading worldwide and generating new mutant virus strains, it has also revealed the importance of mitigating the transmission of SARS‐CoV‐2 through the introduction of restrictive social practices. Here, it is demonstrated that an architecturally‐ and chemically‐diverse family of nanostructured anionic polymers yield a rapid and continuous disinfecting alternative to inactivate coronaviruses and prevent their transmission from contact with contaminated surfaces. Operating on a dramatic pH‐drop mechanism along the polymer/pathogen interface, polymers of this archetype inactivate the SARS‐CoV‐2 virus, as well as a human coronavirus surrogate (HCoV‐229E), to the minimum detection limit within minutes. Application of these anionic polymers to frequently touched surfaces in medical, educational, and public‐transportation facilities, or personal protection equipment, can provide rapid and repetitive protection without detrimental health or environmental complications.

Flavonols and dihydroflavonols inhibit the main protease activity of SARS-CoV-2 and the replication of human coronavirus 229E.

Since December 2019, the deadly novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the current COVID-19 pandemic. To date, vaccines are available in the developed countries to prevent the infection of this virus; however, medicines are necessary to help control COVID-19. Human coronavirus 229E (HCoV-229E) causes the common cold. The main protease (Mpro) is an essential enzyme required for the multiplication of these two viruses in the host cells, and thus is an appropriate candidate to screen potential medicinal compounds. Flavonols and dihydroflavonols are two groups of plant flavonoids. In this study, we report docking simulation with two Mpro enzymes and five flavonols and three dihydroflavonols, in vitro inhibition of the SARS-CoV-2 Mpro, and in vitro inhibition of the HCoV 229E replication. The docking simulation results predicted that (+)-dihydrokaempferol, (+)- dihydroquercetin, (+)-dihydromyricetin, kaempferol, quercetin, myricentin, isoquercitrin, and rutin could bind to at least two subsites (S1, S1', S2, and S4) in the binding pocket and inhibit the activity of SARS-CoV-2 Mpro. Their affinity scores ranged from -8.8 to -7.4 (kcal/mol). Likewise, these compounds were predicted to bind and inhibit the HCoV-229E Mpro activity with affinity scores ranging from -7.1 to -7.8 (kcal/mol). In vitro inhibition assays showed that seven available compounds effectively inhibited the SARS-CoV-2 Mpro activity and their IC50 values ranged from 0.125 to 12.9 µM. Five compounds inhibited the replication of HCoV-229E in Huh-7 cells. These findings indicate that these antioxidative flavonols and dihydroflavonols are promising candidates for curbing the two viruses.

Toward Universal Photodynamic Coatings for Infection Control.

The dual threats posed by the COVID-19 pandemic and hospital-acquired infections (HAIs) have emphasized the urgent need for self-disinfecting materials for infection control. Despite their highly potent antimicrobial activity, the adoption of photoactive materials to reduce infection transmission in hospitals and related healthcare facilities has been severely hampered by the lack of scalable and cost-effective manufacturing, in which case high-volume production methods for fabricating aPDI-based materials are needed. To address this issue here, we examined the antimicrobial efficacy of a simple bicomponent spray coating composed of the commercially-available UV-photocrosslinkable polymer N-methyl-4(4'-formyl-styryl)pyridinium methosulfate acetal poly(vinyl alcohol) (SbQ-PVA) and one of three aPDI photosensitizers (PSs): zinc-tetra(4-N-methylpyridyl)porphine (ZnTMPyP4+), methylene blue (MB), and Rose Bengal (RB). We applied these photodynamic coatings, collectively termed SbQ-PVA/PS, to a variety of commercially available materials. Scanning electron microscopy (SEM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) confirmed the successful application of the coatings, while inductively coupled plasma-optical emission spectroscopy (ICP-OES) revealed a photosensitizer loading of 0.09-0.78 nmol PS/mg material. The antimicrobial efficacy of the coated materials was evaluated against methicillin-susceptible Staphylococcus aureus ATCC-29213 and human coronavirus strain HCoV-229E. Upon illumination with visible light (60 min, 400-700 nm, 65 ± 5 mW/cm2), the coated materials inactivated S. aureus by 97-99.999% and HCoV-229E by 92-99.999%, depending on the material and PS employed. Photobleaching studies employing HCoV-229E demonstrated detection limit inactivation (99.999%) even after exposure for 4 weeks to indoor ambient room lighting. Taken together, these results demonstrate the potential for photodynamic SbQ-PVA/PS coatings to be universally applied to a wide range of materials for effectively reducing pathogen transmission.

Repurposing the Ebola and Marburg Virus Inhibitors Tilorone, Quinacrine, and Pyronaridine: In Vitro Activity against SARS-CoV-2 and Potential Mechanisms.

Severe acute respiratory coronavirus 2 (SARS-CoV-2) is a newly identified virus that has resulted in over 2.5 million deaths globally and over 116 million cases globally in March, 2021. Small-molecule inhibitors that reverse disease severity have proven difficult to discover. One of the key approaches that has been widely applied in an effort to speed up the translation of drugs is drug repurposing. A few drugs have shown in vitro activity against Ebola viruses and demonstrated activity against SARS-CoV-2 in vivo. Most notably, the RNA polymerase targeting remdesivir demonstrated activity in vitro and efficacy in the early stage of the disease in humans. Testing other small-molecule drugs that are active against Ebola viruses (EBOVs) would appear a reasonable strategy to evaluate their potential for SARS-CoV-2. We have previously repurposed pyronaridine, tilorone, and quinacrine (from malaria, influenza, and antiprotozoal uses, respectively) as inhibitors of Ebola and Marburg viruses in vitro in HeLa cells and mouse-adapted EBOV in mice in vivo. We have now tested these three drugs in various cell lines (VeroE6, Vero76, Caco-2, Calu-3, A549-ACE2, HUH-7, and monocytes) infected with SARS-CoV-2 as well as other viruses (including MHV and HCoV 229E). The compilation of these results indicated considerable variability in antiviral activity observed across cell lines. We found that tilorone and pyronaridine inhibited the virus replication in A549-ACE2 cells with IC50 values of 180 nM and IC50 198 nM, respectively. We used microscale thermophoresis to test the binding of these molecules to the spike protein, and tilorone and pyronaridine bind to the spike receptor binding domain protein with K d values of 339 and 647 nM, respectively. Human Cmax for pyronaridine and quinacrine is greater than the IC50 observed in A549-ACE2 cells. We also provide novel insights into the mechanism of these compounds which is likely lysosomotropic.

Photodynamic Coatings on Polymer Microfibers for Pathogen Inactivation: Effects of Application Method and Composition.

A substantial increase in the risk of hospital-acquired infections (HAIs) has greatly impacted the global healthcare industry. Harmful pathogens adhere to a variety of surfaces and infect personnel on contact, thereby promoting transmission to new hosts. This is particularly worrisome in the case of antibiotic-resistant pathogens, which constitute a growing threat to human health worldwide and require new preventative routes of disinfection. In this study, we have incorporated different loading levels of a porphyrin photosensitizer capable of generating reactive singlet oxygen in the presence of O2 and visible light in a water-soluble, photo-cross-linkable polymer coating, which was subsequently deposited on polymer microfibers. Two different application methods are considered, and the morphological and chemical characteristics of these coated fibers are analyzed to detect the presence of the coating and photosensitizer. To discern the efficacy of the fibers against pathogenic bacteria, photodynamic inactivation has been performed on two different bacterial strains, Staphylococcus aureus and antibiotic-resistant Escherichia coli, with population reductions of >99.9999 and 99.6%, respectively, after exposure to visible light for 1 h. In response to the current COVID-19 pandemic, we also confirm that these coated fibers can inactivate a human common cold coronavirus serving as a surrogate for the SARS-CoV-2 virus.