Efficacy of Grignard Pure to Inactivate Airborne Phage MS2, a Common SARS-CoV-2 Surrogate.

Grignard Pure (GP) is a unique and proprietary blend of triethylene glycol (TEG) and inert ingredients designed for continuous antimicrobial treatment of air. TEG has been designated as a ″Safer Chemical" by the US EPA. GP has already received approval from the US EPA under its Section 18 Public Health Emergency Exemption program for use in seven states. This study characterizes the efficacy of GP for inactivating MS2 bacteriophage─a nonenveloped virus widely used as a surrogate for SARS-CoV-2. Experiments measured the decrease in airborne viable MS2 concentration in the presence of different concentrations of GP from 60 to 90 min, accounting for both natural die-off and settling of MS2. Experiments were conducted both by introducing GP aerosol into air containing MS2 and by introducing airborne MS2 into air containing GP aerosol. GP is consistently able to rapidly reduce viable MS2 bacteriophage concentration by 2-3 logs at GP concentrations of 0.04-0.5 mg/m3 (corresponding to TEG concentrations of 0.025 to 0.287 mg/m3). Related GP efficacy experiments by the US EPA, as well as GP (TEG) safety and toxicology, are also discussed.

Persistence of surrogates for high consequence viral and bacterial pathogens in a pilot-scale activated sludge treatment system.

The persistence of high consequence public health pathogens in a wastewater treatment system can significantly impact worker safety, as well as the public and downstream water bodies, particularly if the system is forced to shut down the treatment processes. This study utilizes organism viability to compare the persistence of three pathogen surrogates in wastewater using a pilot-scale activated sludge treatment (AST) system, operated to mimic treatment processes of large-scale plants. Bacillus globigii spores, surrogate for Bacillus anthracis, persisted in the AST system for at least a 50-day observation period leading to a possible steady condition far beyond the solid retention time for sludge particles. MS2 bacteriophage, surrogate for Poliovirus and other non-enveloped enteric viruses, was observed for up to 35 days after introduction, which largely and expectedly correlated to the measured solid retention time. Phi-6 bacteriophage, a surrogate for Ebola virus and other enveloped viruses, was detected for no more than 4 days after introduction, even though the AST system was operated to provide three times slower solids removal than for the other surrogates. This suggests Phi-6 is subject to inactivation under AST conditions rather than physical removal. These results may suggest similar persistence for the surrogated pathogens, leading to appropriate consequence management actions.

Immersive ultraviolet disinfection of E. coli and MS2 phage on woven cotton textiles.

Immersive ultraviolet disinfection provides a chemical-free technology for safer textiles, surfaces, and public spaces by inactivating communicable pathogens. This study examined immersive UV disinfection, using a disinfection cabinet, of E. coli and MS2 that was inoculated on white cotton T-shirts. The impact that porous materials have on UV disinfection is poorly understood with the majority of previous surface disinfection research focusing on hard, smooth surfaces. Several approaches were used in this study to characterize the light dynamics within the disinfection cabinet including colorimetric dosimetry coupons, biodosimetry, and spectroradiometry. Micro and macro geometry of porous surfaces are important factors to consider when using immersive UV technologies. The geometry of the cabinet impacted the distribution of emitted UV light within the disinfection cabinet and the physical properties of a porous material, such as the woven pattern of cotton, both contribute to UV disinfection efficiency. This work identified that light distribution is crucial for immersive UV technologies as the delivered fluence was highly variable within the disinfection cabinet and resulted in a difference of several logs of reduction for adjacent areas of T-shirt samples. Other inoculated areas achieved upwards of 1-log reductions values for MS2 and upwards of 2-log reductions for E. coli.

A simulation study to evaluate contamination during reuse of N95 respirators and effectiveness of interventions to reduce contamination.

OBJECTIVE: To assess the potential for contamination of personnel, patients, and the environment during use of contaminated N95 respirators and to compare the effectiveness of interventions to reduce contamination. DESIGN: Simulation study of patient care interactions using N95 respirators contaminated with a higher and lower inocula of the benign virus bacteriophage MS2. METHODS: In total, 12 healthcare personnel performed 3 standardized examinations of mannequins including (1) control with suboptimal respirator handling technique, (2) improved technique with glove change after each N95 contact, and (3) control with 1-minute ultraviolet-C light (UV-C) treatment prior to donning. The order of the examinations was randomized within each subject. The frequencies of contamination were compared among groups. Observations and simulations with fluorescent lotion were used to assess routes of transfer leading to contamination. RESULTS: With suboptimal respirator handling technique, bacteriophage MS2 was frequently transferred to the participants, mannequin, and environmental surfaces and fomites. Improved technique resulted in significantly reduced transfer of MS2 in the higher inoculum simulations (P < .01), whereas UV-C treatment reduced transfer in both the higher- and lower-inoculum simulations (P < .01). Observations and simulations with fluorescent lotion demonstrated multiple potential routes of transfer to participants, mannequin, and surfaces, including both direct contact with the contaminated respirator and indirect contact via contaminated gloves. CONCLUSION: Reuse of contaminated N95 respirators can result in contamination of personnel and the environment even when correct technique is used. Decontamination technologies, such as UV-C, could reduce the risk for transmission.

Evaluating the impact of ultraviolet C exposure conditions on coliphage MS2 inactivation on surfaces.

The COVID-19 pandemic has raised interest in using devices that generate ultraviolet C (UVC) radiation as an alternative approach for reducing or eliminating microorganisms on surfaces. Studies investigating the efficacy of UVC radiation against pathogens use a wide range of laboratory methods and experimental conditions that can make cross-comparison of results and extrapolation of findings to real-world settings difficult. Here, we use three different UVC-generating sources - a broad-spectrum pulsed xenon light, a continuous light-emitting diode (LED), and a low-pressure mercury vapour lamp - to evaluate the impact of different experimental conditions on UVC efficacy against the coliphage MS2 on surfaces. We find that a nonlinear dose-response relationship exists for all three light sources, meaning that linear extrapolation of doses resulting in a 1-log10 (90%) reduction does not accurately predict the dose required for higher (e.g. 3-log10 or 99.9%) log10 reductions. In addition, our results show that the inoculum characteristics and underlying substrate play an important role in determining UVC efficacy. Variations in microscopic surface topography may shield MS2 from UVC radiation to different degrees, which impacts UVC device efficacy. These findings are important to consider in comparing results from different UVC studies and in estimating device performance in field conditions.

Novel sustainable filter for virus filtration and inactivation.

The COVID-19 pandemic has caused a multi-scale impact on the world population that started from a nano-scale respiratory virus and led to the shutdown of macro-scale economies. Direct transmission of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) and its variants through aerosolized droplets is a major contributor towards increasing cases of this infection. To curb the spread, one of the best engineered solutions is the use of face masks to prevent the passage of infectious saliva micro-droplets from an infected person to a healthy person. The commercially available masks are single use, passive face-piece filters. These become difficult to breathe in during strenuous activities. Also, they need to be disposed regularly due to accumulation of unwanted particulate and pathogens over time. Frequent disposal of these masks is unsustainable for the environment. In this study, we have proposed a novel design for a filter for enhanced virus filtration, better breathability, and virus inactivation over time. The filter is called Hy-Cu named after its (Hy) drophobic properties and another significant layer comprises of copper (Cu). The breathability (pressure drop across filter) of Hy-Cu is tested and compared with widely used surgical masks and KN95 masks, both experimentally and numerically. The results show that the Hy-Cu filter offers at least 10% less air resistance as compared to commercially available masks. The experimental results on virus filtration and inactivation tests using MS2 bacteriophage (a similar protein structure as SARS-CoV-2) show that the novel filter has 90% filtering efficiency and 99% virus inactivation over a period of 2 h. This makes the Hy-Cu filter reusable and a judicious substitute to the single use masks.

Inactivation of MS2 bacteriophage on copper film deployed in high touch areas of a public transport system.

Although SARS-CoV-2 is primarily an airborne risk, the COVID-19 pandemic also highlighted the need for self-disinfection surfaces that could withstand the demand of high occupant densities characteristic of public transportation systems. The aim of this study was to evaluate the durability and antiviral activity of a copper film deployed for 90 days in two high touch locations within an active metropolitan bus and railcar. The antiviral efficacy of this copper film after being deployed in transit vehicles for 90 days (deployed copper film) was then compared to new (unused) copper film to determine if frequent touches and cleaning protocols could decrease the efficacy of the copper films. Deployed copper film, new copper film, and aluminium foil (positive control) coupons were inoculated with ~1 × 106 MS2 virus particles, allowed a contact time of either 5- or 10-min, and analysed for residual viral infectiousness. On both new and deployed copper films, MS2 was completely inactivated (≥5 log reduction) at both time points. These results suggest that the copper film may provide the durability demanded by high touch public spaces while maintaining the antiviral activity necessary to reduce exposure risk and viral transmission via surfaces in public transportation settings.

Transfer Rate of Enveloped and Nonenveloped Viruses between Fingerpads and Surfaces.

Fomites can represent a reservoir for pathogens, which may be subsequently transferred from surfaces to skin. In this study, we aim to understand how different factors (including virus type, surface type, time since last hand wash, and direction of transfer) affect virus transfer rates, defined as the fraction of virus transferred, between fingerpads and fomites. To determine this, 360 transfer events were performed with 20 volunteers using Phi6 (a surrogate for enveloped viruses), MS2 (a surrogate for nonenveloped viruses), and three clean surfaces (stainless steel, painted wood, and plastic). Considering all transfer events (all surfaces and both transfer directions combined), the mean transfer rates of Phi6 and MS2 were 0.17 and 0.26, respectively. Transfer of MS2 was significantly higher than that of Phi6 (P < 0.05). Surface type was a significant factor that affected the transfer rate of Phi6: Phi6 is more easily transferred to and from stainless steel and plastic than to and from painted wood. Direction of transfer was a significant factor affecting MS2 transfer rates: MS2 is more easily transferred from surfaces to fingerpads than from fingerpads to surfaces. Data from these virus transfer events, and subsequent transfer rate distributions, provide information that can be used to refine quantitative microbial risk assessments. This study provides a large-scale data set of transfer events with a surrogate for enveloped viruses, which extends the reach of the study to the role of fomites in the transmission of human enveloped viruses like influenza and SARS-CoV-2. IMPORTANCE This study created a large-scale data set for the transfer of enveloped viruses between skin and surfaces. The data set produced by this study provides information on modeling the distribution of enveloped and nonenveloped virus transfer rates, which can aid in the implementation of risk assessment models in the future. Additionally, enveloped and nonenveloped viruses were applied to experimental surfaces in an equivalent matrix to avoid matrix effects, so results between different viral species can be directly compared without confounding effects of different matrices. Our results indicating how virus type, surface type, time since last hand wash, and direction of transfer affect virus transfer rates can be used in decision-making processes to lower the risk of viral infection from transmission through fomites.

Effectiveness of the Nanosilver/TiO2-Chitosan Antiviral Filter on the Removal of Viral Aerosols.

Background: The precaution of airborne transmission of viruses, such as influenza, SARS, MERS, and COVID-19, is essential for reducing infection. In this study, we applied a zero-valent nanosilver/titania-chitosan (nano-Ag0/TiO2-CS) filter bed, whose broad-spectrum antimicrobial efficacy has been proven previously, for the removal of viral aerosols to minimize the risk of airborne transmission. Methods: The photochemical deposition method was used to synthesize the nano-Ag0/TiO2-CS antiviral material. The surface morphology, elemental composition, and microstructure of the nano-Ag0/TiO2-CS were analyzed by a scanning electron microscopy/energy dispersive X-ray spectroscopy and a transmission electron microscopy, respectively. The MS2 bacteriophages were used as surrogate viral aerosols. The antiviral efficacy of nano-Ag0/TiO2-CS was evaluated by the MS2 plaque reduction assay (PRA) and filtration experiments. In the filtration experiments, the MS2 aerosols passed through the nano-Ag0/TiO2-CS filter, and the MS2 aerosol removal efficiency was evaluated by an optical particle counter and culture method. Results and Conclusions: In the MS2 PRA, 3 g of nano-Ag0/TiO2-CS inactivated 97% of MS2 bacteriophages in 20 mL liquid culture (2 ± 0.5 × 1016 PFU/mL) within 2 hours. The removal efficiency of nano-Ag0/TiO2-CS filter (thickness: 6 cm) for MS2 aerosols reached up to 93%. Over 95% of MS2 bacteriophages on the surface of the nano-Ag0/TiO2-CS filter were inactivated within 20 minutes. The Wells-Riley model predicted that when the nano-Ag0/TiO2-CS filter was used in the ventilation system, airborne infection probability would reduce from 99% to 34.6%. The nano-Ag0/TiO2-CS filter could remain at 50% of its original antiviral efficiency after continuous operation for 1 week, indicating its feasibility for the control of the airborne transmission.

Survival of the enveloped bacteriophage Phi6 (a surrogate for SARS-CoV-2) in evaporated saliva microdroplets deposited on glass surfaces.

Survival of respiratory viral pathogens in expelled saliva microdroplets is central to their transmission, yet the factors that determine survival in such microdroplets are not well understood. Here we combine microscopy imaging with virus viability assays to study survival of three bacteriophages suggested as good models for respiratory pathogens: the enveloped Phi6 (a surrogate for SARS-CoV-2), and the non-enveloped PhiX174 and MS2. We measured virus viability in human saliva microdroplets, SM buffer, and water following deposition on glass surfaces at various relative humidities (RH). Saliva and water microdroplets dried out rapidly, within minutes, at all tested RH levels (23%, 43%, 57%, and 78%), while SM microdroplets remained hydrated at RH ≥ 57%. Generally, the survival of all three viruses in dry saliva microdroplets was significantly greater than those in SM buffer and water under all RH (except PhiX174 in water under 57% RH survived the best among 3 media). Thus, atmosphere RH and microdroplet hydration state are not sufficient to explain virus survival, indicating that the virus-suspended medium, and association with saliva components in particular, likely play a role in virus survival. Uncovering the exact properties and components that make saliva a favorable environment for the survival of viruses, in particular enveloped ones like Phi6, is thus of great importance for reducing transmission of viral respiratory pathogens including SARS-CoV-2.

Multivalent nanoparticle-based vaccines protect hamsters against SARS-CoV-2 after a single immunization.

The COVID-19 pandemic continues to wreak havoc as worldwide SARS-CoV-2 infection, hospitalization, and death rates climb unabated. Effective vaccines remain the most promising approach to counter SARS-CoV-2. Yet, while promising results are emerging from COVID-19 vaccine trials, the need for multiple doses and the challenges associated with the widespread distribution and administration of vaccines remain concerns. Here, we engineered the coat protein of the MS2 bacteriophage and generated nanoparticles displaying multiple copies of the SARS-CoV-2 spike (S) protein. The use of these nanoparticles as vaccines generated high neutralizing antibody titers and protected Syrian hamsters from a challenge with SARS-CoV-2 after a single immunization with no infectious virus detected in the lungs. This nanoparticle-based vaccine platform thus provides protection after a single immunization and may be broadly applicable for protecting against SARS-CoV-2 and future pathogens with pandemic potential.

Scalable in-hospital decontamination of N95 filtering face-piece respirator with a peracetic acid room disinfection system.

BACKGROUND: Critical shortages of personal protective equipment, especially N95 respirators, during the coronavirus disease 2019 (COVID-19) pandemic continues to be a source of concern. Novel methods of N95 filtering face-piece respirator decontamination that can be scaled-up for in-hospital use can help address this concern and keep healthcare workers (HCWs) safe. METHODS: A multidisciplinary pragmatic study was conducted to evaluate the use of an ultrasonic room high-level disinfection system (HLDS) that generates aerosolized peracetic acid (PAA) and hydrogen peroxide for decontamination of large numbers of N95 respirators. A cycle duration that consistently achieved disinfection of N95 respirators (defined as ≥6 log10 reductions in bacteriophage MS2 and Geobacillus stearothermophilus spores inoculated onto respirators) was identified. The treated masks were assessed for changes to their hydrophobicity, material structure, strap elasticity, and filtration efficiency. PAA and hydrogen peroxide off-gassing from treated masks were also assessed. RESULTS: The PAA room HLDS was effective for disinfection of bacteriophage MS2 and G. stearothermophilus spores on respirators in a 2,447 cubic-foot (69.6 cubic-meter) room with an aerosol deployment time of 16 minutes and a dwell time of 32 minutes. The total cycle time was 1 hour and 16 minutes. After 5 treatment cycles, no adverse effects were detected on filtration efficiency, structural integrity, or strap elasticity. There was no detectable off-gassing of PAA and hydrogen peroxide from the treated masks at 20 and 60 minutes after the disinfection cycle, respectively. CONCLUSION: The PAA room disinfection system provides a rapidly scalable solution for in-hospital decontamination of large numbers of N95 respirators during the COVID-19 pandemic.

External Quality Assessment for Molecular Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Clinical Laboratories.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a huge threat to public health. Viral nucleic acid testing is the diagnostic gold standard and can play an important role in the prevention and control of this infection. In this study, bacteriophage MS2 virus-like particles encapsulating specific RNA sequences of SARS-CoV-2 and other coronaviruses were prepared by genetic engineering. The assessment panel, consisting of four positive samples with concentrations of 2.8, 3.5, 4.2, and 4.9 log10 copies/mL and five negative samples with other human coronaviruses, was prepared and distributed to evaluate the accuracy of routine viral RNA detection. Results of 931 panels from 844 laboratories were collected. The overall percentage agreement, positive percentage agreement (PPA), and negative percentage agreement, defined as the percentage of agreement between the correct results and total results submitted for all, positive, and negative samples were 96.8% (8109/8379), 93.9% (3497/3724), and 99.1% (4612/4655), respectively. For samples with concentrations of 4.9 and 4.2 log10 copies/mL, the PPAs were >95%. However, for 3.5 and 2.8 log10 copies/mL, the PPAs were 94.6% (881/931) and 84.9% (790/931), respectively. For all negative samples, the negative percentage agreement values were >95%. Thus, most laboratories can reliably detect SARS-CoV-2. However, further improvement and optimization are required to ensure the accuracy of detection in panel members with lower concentrations of viral RNA.

Automated SARS-COV-2 RNA extraction from patient nasopharyngeal samples using a modified DNA extraction kit for high throughput testing.

BACKGROUND: The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) has prompted a need for mass testing to identify patients with viral infection. The high demand has created a global bottleneck in testing capacity, which prompted us to modify available resources to extract viral RNA and perform reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-COV-2. OBJECTIVES: Report on the use of a DNA extraction kit, after modifications, to extract viral RNA that could then be detected using an FDA-approved SARS-COV-2 RT-qPCR assay. MATERIALS AND METHODS: Initially, automated RNA extraction was performed using a modified DNA kit on samples from control subjects, a bacteriophage, and an RNA virus. We then verified the automated extraction using the modified kit to detect in-lab propagated SARSCOV-2 titrations using an FDA approved commercial kit (S, N, and ORF1b genes) and an in-house primer-probe based assay (E, RdRp2 and RdRp4 genes). RESULTS: Automated RNA extraction on serial dilutions SARS-COV-2 achieved successful one-step RT-qPCR detection down to 60 copies using the commercial kit assay and less than 30 copies using the in-house primer-probe assay. Moreover, RT-qPCR detection was successful after automated RNA extraction using this modified protocol on 12 patient samples of SARS-COV-2 collected by nasopharyngeal swabs and stored in viral transport media. CONCLUSIONS: We demonstrated the capacity of a modified DNA extraction kit for automated viral RNA extraction and detection using a platform that is suitable for mass testing. LIMITATIONS: Small patient sample size. CONFLICT OF INTEREST: None.

Nanoscale Structural Characterization of Individual Viral Particles Using Atomic Force Microscopy Infrared Spectroscopy (AFM-IR) and Tip-Enhanced Raman Spectroscopy (TERS).

Viruses are infections species that infect a large spectrum of living systems. Although displaying a wide variety of shapes and sizes, they are all composed of nucleic acid encapsulated into a protein capsid. After virions enter the host cell, they replicate to produce multiple copies of themselves. They then lyse the host, releasing virions to infect new cells. The high proliferation rate of viruses is the underlying cause of their fast transmission among living species. Although many viruses are harmless, some of them are responsible for severe diseases such as AIDS, viral hepatitis, and flu. Traditionally, electron microscopy is used to identify and characterize viruses. This approach is time- and labor-consuming, which is problematic upon pandemic proliferation of previously unknown viruses, such as H1N1 and COVID-19. Herein, we demonstrate a novel diagnosis approach for label-free identification and structural characterization of individual viruses that is based on a combination of nanoscale Raman and infrared spectroscopy. Using atomic force microscopy-infrared (AFM-IR) spectroscopy, we were able to probe structural organization of the virions of Herpes Simplex Type 1 viruses and bacteriophage MS2. We also showed that tip-enhanced Raman spectroscopy (TERS) could be used to reveal protein secondary structure and amino acid composition of the virus surface. Our results show that AFM-IR and TERS provide different but complementary information about the structure of complex biological specimens. This structural information can be used for fast and reliable identification of viruses. This nanoscale bimodal imaging approach can be also used to investigate the origin of viral polymorphism and study mechanisms of virion assembly.