Pathogen-Repellent Plastic Warp with Built-In Hierarchical Structuring Prevents the Contamination of Surfaces with Coronaviruses.

Amidst the COVID-19 pandemic, it is evident that viral spread is mediated through several different transmission pathways. Reduction of these transmission pathways is urgently needed to control the spread of viruses between infected and susceptible individuals. Herein, we report the use of pathogen-repellent plastic wraps (RepelWrap) with engineered surface structures at multiple length scales (nanoscale to microscale) as a means of reducing the indirect contact transmission of viruses through fomites. To quantify viral repellency, we developed a touch-based viral quantification assay to mimic the interaction of a contaminated human touch with a surface through the modification of traditional viral quantification methods (viral plaque and TCID50 assays). These studies demonstrate that RepelWrap reduced contamination with an enveloped DNA virus as well as the human coronavirus 229E (HuCoV-229E) by more than 4 log 10 (>99.99%) compared to a standard commercially available polyethylene plastic wrap. In addition, RepelWrap maintained its repellent properties after repeated 300 touches and did not show an accumulation in viral titer after multiple contacts with contaminated surfaces, while increases were seen on other commonly used surfaces. These findings show the potential use of repellent surfaces in reducing viral contamination on surfaces, which could, in turn, reduce the surface-based spread and transmission.

Establishment of Primary Transgenic Human Airway Epithelial Cell Cultures to Study Respiratory Virus-Host Interactions.

Primary human airway epithelial cell (hAEC) cultures represent a universal platform to propagate respiratory viruses and characterize their host interactions in authentic target cells. To further elucidate specific interactions between human respiratory viruses and important host factors in the airway epithelium, it is important to make hAEC cultures amenable to genetic modification. However, the short and finite lifespan of primary cells in cell culture creates a bottleneck for the genetic modification of these cultures. In the current study, we show that the incorporation of the Rho-associated protein kinase (ROCK) inhibitor (Y-27632) during cell propagation extends the life span of primary human cells in vitro and thereby facilitates the incorporation of lentivirus-based expression systems. Using fluorescent reporters for fluorescence-activated cell sorting (FACS)-based sorting, we generated homogenously fluorescent hAEC cultures that differentiate normally after lentiviral transduction. As a proof-of-principle, we demonstrate that host gene expression can be modulated post-differentiation via inducible short hairpin (sh)RNA-mediated knockdown. Importantly, functional characterization of these transgenic hAEC cultures with exogenous poly (I:C), as a proxy for virus infection, demonstrates that such modifications do not influence the host innate immune response. Moreover, the propagation kinetics of both human coronavirus 229E (HCoV-229E) and human respiratory syncytial virus (hRSV) were not affected. Combined, these results validate our newly established protocol for the genetic modification of hAEC cultures, thereby unlocking a unique potential for detailed molecular characterization of virus-host interactions in human respiratory epithelium.

Cyclosporin A inhibits the replication of diverse coronaviruses.

Low micromolar, non-cytotoxic concentrations of cyclosporin A (CsA) strongly affected the replication of severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus 229E and mouse hepatitis virus in cell culture, as was evident from the strong inhibition of GFP reporter gene expression and a reduction of up to 4 logs in progeny titres. Upon high-multiplicity infection, CsA treatment rendered SARS-CoV RNA and protein synthesis almost undetectable, suggesting an early block in replication. siRNA-mediated knockdown of the expression of the prominent CsA targets cyclophilin A and B did not affect SARS-CoV replication, suggesting either that these specific cyclophilin family members are dispensable or that the reduced expression levels suffice to support replication.

Antiseptic properties of two calix[4]arenes derivatives on the human coronavirus 229E.

Facing the lack in specific antiviral treatment, it is necessary to develop new means of prevention. In the case of the Coronaviridae this family is now recognized as including potent human pathogens causing upper and lower respiratory tract infections as well as nosocomial ones. Within the purpose of developing new antiseptics molecules, the antiseptic virucidal activity of two calix[4]arene derivatives, the tetra-para-sulfonato-calix[4]arene (C[4]S) and the 1,3-bis(bithiazolyl)-tetra-para-sulfonato-calix[4]arene (C[4]S-BTZ) were evaluated toward the human coronavirus 229E (HCoV 229E). Comparing these results with some obtained previously with chlorhexidine and hexamidine, (i) these two calixarenes did not show any cytotoxicity contrary to chlorhexidine and hexamidine, (ii) C[4]S showed as did hexamidine, a very weak activity against HCoV 229E, and (iii) the C[4]S-BTZ showed a stronger activity than chlorhexidine, i.e. 2.7 and 1.4log10 reduction in viral titer after 5min of contact with 10⁻³mol L⁻¹ solutions of C[4]S-BTZ and chlorhexidine, respectively. Thus, the C[4]S-BTZ appeared as a promising virucidal (antiseptic) molecule.

Glucose-6-phosphate dehydrogenase deficiency enhances human coronavirus 229E infection.

The host cellular environment is a key determinant of pathogen infectivity. Viral gene expression and viral particle production of glucose-6-phosphate dehydrogenase (G6PD)-deficient and G6PD-knockdown cells were much higher than their counterparts when human coronavirus (HCoV) 229E was applied at 0.1 multiplicity of infection. These phenomena were correlated with increased oxidant production. Accordingly, ectopic expression of G6PD in G6PD-deficient cells or addition of antioxidant (such as alpha-lipoic acid) to G6PD-knockdown cells attenuated the increased susceptibility to HCoV 229E infection. All experimental data indicated that oxidative stress in host cells is an important factor in HCoV 229E infectivity.

Antiviral effects of saikosaponins on human coronavirus 229E in vitro.

1. Saikosaponins represent a group of oleanane derivatives, usually as glucosides, that are found in a number of plant families. Saikosaponins isolated from medicinal plants such as Bupleurum spp., Heteromorpha spp. and Scrophularia scorodonia have been reported to possess various biological activities, specifically antihepatitis, antinephritis, antihepatoma, anti-inflammation, immunomodulation and antibacterial effects. 2. The aim of the present study was to examine the anticoronaviral activity of saikosaponins (A, B2, C and D) and their mode of action. Using the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino) carbonyl-2H-tetrazolium hydroxide] (XTT) assay, results showed that all saikosaponins tested demonstrated antiviral activity at concentrations of 0.25-25 micromol/L, with the strongest activity being noted for saikosaponin B2 (IC50 = 1.7 +/- 0.1 micromol/L). Interestingly, both saikosaponins A (50% cellular cytotoxicity (CC50) concentration = 228.1 +/- 3.8 micromol/L; selectivity index (SI) = 26.6) and B2 (CC50 = 383.3 +/- 0.2 micromol/L; SI = 221.9) exhibited no cytotoxic effects on target cells at concentrations that achieved antiviral activity. In the time-of-addition studies, saikosaponin B2, at 6 micromol/L, significantly inhibited human coronavirus 229E infection following its addition at various time pre-infection (-4 to -1 h), coinfection (0 h) and post-infection (1-4 h). Furthermore, saikosaponin B2 also showed an inhibitory effect on viral attachment and penetration. 3. The present results indicate that saikosaponin B2 has potent anticoronaviral activity and that its mode of action possibly involves interference in the early stage of viral replication, such as absorption and penetration of the virus.

Stability and inactivation of SARS coronavirus.

The SARS-coronavirus (SARS-CoV) is a newly emerged, highly pathogenic agent that caused over 8,000 human infections with nearly 800 deaths between November 2002 and September 2003. While direct person-to-person transmission via respiratory droplets accounted for most cases, other modes have not been ruled out. Faecal shedding is common and prolonged and has caused an outbreak in Hong Kong. We studied the stability of SARS-CoV under different conditions, both in suspension and dried on surfaces, in comparison with other human-pathogenic viruses, including human coronavirus HCoV-229E. In suspension, HCoV-229E gradually lost its infectivity completely while SARS-CoV retained its infectivity for up to 9 days; in the dried state, survival times were 24 h versus 6 days. Thermal inactivation at 56 degrees C was highly effective in the absence of protein, reducing the virus titre to below detectability; however, the addition of 20% protein exerted a protective effect resulting in residual infectivity. If protein-containing solutions are to be inactivated, heat treatment at 60 degrees C for at least 30 min must be used. Different fixation procedures, e.g. for the preparation of immunofluorescence slides, as well as chemical means of virus inactivation commonly used in hospital and laboratory settings were generally found to be effective. Our investigations confirm that it is possible to care for SARS patients and to conduct laboratory scientific studies on SARS-CoV safely. Nevertheless, the agents tenacity is considerably higher than that of HCoV-229E, and should SARS re-emerge, increased efforts need to be devoted to questions of environmental hygiene.