Influence of Different Inactivation Methods on Severe Acute Respiratory Syndrome Coronavirus 2 RNA Copy Number.

The outbreak of coronavirus disease 2019 (COVID-19) has spread across the world and was characterized as a pandemic. To protect medical laboratory personnel from infection, most laboratories inactivate the virus causing COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in clinical samples before testing. However, the effect of inactivation on the detection results remains unknown. Here, we used a digital PCR assay to determine the absolute SARS-CoV-2 RNA copy number in 63 nasopharyngeal swab samples and assess the effect of inactivation methods on viral RNA copy number. Viral inactivation was performed by three different methods: (i) incubation with the TRIzol LS reagent for 10 min at room temperature, (ii) heating in a water bath at 56°C for 30 min, and (iii) high-temperature treatment, including autoclaving at 121°C for 20 min, boiling at 100°C for 20 min, and heating at 80°C for 20 min. Compared to the amount of RNA in the original sample, TRIzol treatment destroyed 47.54% of the nucleocapsid protein (N) gene and 39.85% of open reading frame (ORF) 1ab. For samples treated at 56°C for 30 min, the copy number of the N gene and ORF 1ab was reduced by 48.55% and 56.40%, respectively. The viral RNA copy number dropped by 50 to 66% after heating at 80°C for 20 min. Nearly no viral RNA was detected after autoclaving at 121°C or boiling at 100°C for 20 min. These results indicate that inactivation reduced the quantity of detectable viral RNA and may cause false-negative results, especially in weakly positive cases. Thus, use of the TRIzol reagent rather than heat inactivation is recommended for sample inactivation, as the TRIzol reagent had the least effect on the RNA copy number among the tested methods.

Is the 2019 novel coronavirus related to a spike of cosmic rays?

WHO's pronouncement of the 2019 novel coronavirus outbreak as a pandemic disease came months after we published a warning that the present deepest minimum of the sunspot cycle would be likely to facilitate the onset of a viral pandemic. During a deep sunspot minimum (deepest in 100 years) such as we are now witnessing, two space related phenomena could have an effect on the disposition of viral disease and potential pandemics. With the weakening of the magnetic field in the Earth's vicinity, there would be a high flux of mutagenic cosmic rays. These processes would be likely to herald the onset of new pandemics. Neutron counts from Moscow Neutron Monitor show that the flux of cosmic rays reaching Earth in 2019 was indeed at a maximum over a timespan of half a century since 1962. It is of interest to note that immediately prior to the first recorded cases of the novel Corona virus in China a peak of cosmic rays was measured as is indicated by the Huon neutron monitor data. Recent research revealed that estimates of the timing of the most recent common ancestor of COVID-19 made with current sequence data point to emergence of the virus in late November 2019 to early December 2019, compatible with the earliest retrospectively confirmed cases and the cosmic ray spike in late November 2019. In our view, this strong cosmic ray spike was in some way connected with the onset of the outbreak.

UV-LED disinfection of Coronavirus: Wavelength effect.

UV light-emitting diodes (UV LEDs) are an emerging technology and a UV source for pathogen inactivation, however low UV-LED wavelengths are costly and have low fluence rate. Our results suggest that the sensitivity of human Coronavirus (HCoV-OC43 used as SARS-CoV-2 surrogate) was wavelength dependent with 267 nm ~ 279 nm > 286 nm > 297 nm. Other viruses showed similar results, suggesting UV LED with peak emission at ~286 nm could serve as an effective tool in the fight against human Coronaviruses.

Spectrum of virucidal activity from ultraviolet to infrared radiation.

The COVID-19 pandemic has sparked a demand for safe and highly effective decontamination techniques for both personal protective equipment (PPE) and hospital and operating rooms. The gradual lifting of lockdown restrictions warrants the expansion of these measures into the outpatient arena. Ultraviolet C (UVC) radiation has well-known germicidal properties and is among the most frequently reported decontamination techniques used today. However, there is evidence that wavelengths beyond the traditional 254 nm UVC - namely far UVC (222 nm), ultraviolet B, ultraviolet A, visible light, and infrared radiation - have germicidal properties as well. This review will cover current literature regarding the germicidal effects of wavelengths ranging from UVC through the infrared waveband with an emphasis on their activity against viruses, and their potential applicability in the healthcare setting for general decontamination during an infectious outbreak.

Light-based technologies for management of COVID-19 pandemic crisis.

The global dissemination of the novel coronavirus disease (COVID-19) has accelerated the need for the implementation of effective antimicrobial strategies to target the causative agent SARS-CoV-2. Light-based technologies have a demonstrable broad range of activity over standard chemotherapeutic antimicrobials and conventional disinfectants, negligible emergence of resistance, and the capability to modulate the host immune response. This perspective article identifies the benefits, challenges, and pitfalls of repurposing light-based strategies to combat the emergence of COVID-19 pandemic.

Methods of Inactivation of SARS-CoV-2 for Downstream Biological Assays.

The scientific community has responded to the coronavirus disease 2019 (COVID-19) pandemic by rapidly undertaking research to find effective strategies to reduce the burden of this disease. Encouragingly, researchers from a diverse array of fields are collectively working towards this goal. Research with infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is undertaken in high-containment laboratories; however, it is often desirable to work with samples at lower-containment levels. To facilitate the transfer of infectious samples from high-containment laboratories, we have tested methods commonly used to inactivate virus and prepare the sample for additional experiments. Incubation at 80°C, a range of detergents, Trizol reagents, and UV energies were successful at inactivating a high titer of SARS-CoV-2. Methanol and paraformaldehyde incubation of infected cells also inactivated the virus. These protocols can provide a framework for in-house inactivation of SARS-CoV-2 in other laboratories, ensuring the safe use of samples in lower-containment levels.

Susceptibility of SARS-CoV-2 to UV irradiation.

The coronavirus SARS-CoV-2 pandemic became a global health burden. We determined the susceptibility of SARS-CoV-2 to irradiation with ultraviolet light. The virus was highly susceptible to ultraviolet light. A viral stock with a high infectious titer of 5 × 106 TCID50/mL was completely inactivated by UVC irradiation after nine minutes of exposure. The UVC dose required for complete inactivation was 1,048 mJ/cm2. UVA exposure demonstrated only a weak effect on virus inactivation over 15 minutes. Hence, inactivation of SARS-CoV-2 by UVC irradiation constitutes a reliable method for disinfection purposes in health care facilities and for preparing SARS-CoV-2 material for research purpose.

Rapid inactivation of SARS-CoV-2 with deep-UV LED irradiation.

The spread of novel coronavirus disease 2019 (COVID-19) infections worldwide has raised concerns about the prevention and control of SARS-CoV-2. Devices that rapidly inactivate viruses can reduce the chance of infection through aerosols and contact transmission. This in vitro study demonstrated that irradiation with a deep ultraviolet light-emitting diode (DUV-LED) of 280 ± 5 nm wavelength rapidly inactivates SARS-CoV-2 obtained from a COVID-19 patient. Development of devices equipped with DUV-LED is expected to prevent virus invasion through the air and after touching contaminated objects.

Far-UVC light (222 nm) efficiently and safely inactivates airborne human coronaviruses.

A direct approach to limit airborne viral transmissions is to inactivate them within a short time of their production. Germicidal ultraviolet light, typically at 254 nm, is effective in this context but, used directly, can be a health hazard to skin and eyes. By contrast, far-UVC light (207-222 nm) efficiently kills pathogens potentially without harm to exposed human tissues. We previously demonstrated that 222-nm far-UVC light efficiently kills airborne influenza virus and we extend those studies to explore far-UVC efficacy against airborne human coronaviruses alpha HCoV-229E and beta HCoV-OC43. Low doses of 1.7 and 1.2 mJ/cm2 inactivated 99.9% of aerosolized coronavirus 229E and OC43, respectively. As all human coronaviruses have similar genomic sizes, far-UVC light would be expected to show similar inactivation efficiency against other human coronaviruses including SARS-CoV-2. Based on the beta-HCoV-OC43 results, continuous far-UVC exposure in occupied public locations at the current regulatory exposure limit (~3 mJ/cm2/hour) would result in ~90% viral inactivation in ~8 minutes, 95% in ~11 minutes, 99% in ~16 minutes and 99.9% inactivation in ~25 minutes. Thus while staying within current regulatory dose limits, low-dose-rate far-UVC exposure can potentially safely provide a major reduction in the ambient level of airborne coronaviruses in occupied public locations.

Microwave-Generated Steam Decontamination of N95 Respirators Utilizing Universally Accessible Materials.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused a severe, international shortage of N95 respirators, which are essential to protect health care providers from infection. Given the contemporary limitations of the supply chain, it is imperative to identify effective means of decontaminating, reusing, and thereby conserving N95 respirator stockpiles. To be effective, decontamination must result in sterilization of the N95 respirator without impairment of respirator filtration or user fit. Although numerous methods of N95 decontamination exist, none are universally accessible. In this work, we describe a microwave-generated steam decontamination protocol for N95 respirators for use in health care systems of all sizes, geographies, and means. Using widely available glass containers, mesh from commercial produce bags, a rubber band, and a 1,100-W commercially available microwave, we constructed an effective, standardized, and reproducible means of decontaminating N95 respirators. Employing this methodology against MS2 phage, a highly conservative surrogate for SARS-CoV-2 contamination, we report an average 6-log10 plaque-forming unit (PFU) (99.9999%) and a minimum 5-log10 PFU (99.999%) reduction after a single 3-min microwave treatment. Notably, quantified respirator fit and function were preserved, even after 20 sequential cycles of microwave steam decontamination. This method provides a valuable means of effective decontamination and reuse of N95 respirators by frontline providers facing urgent need.IMPORTANCE Due to the rapid spread of coronavirus disease 2019 (COVID-19), there is an increasing shortage of protective gear necessary to keep health care providers safe from infection. As of 9 April 2020, the CDC reported 9,282 cumulative cases of COVID-19 among U.S. health care workers (CDC COVID-19 Response Team, MMWR Morb Mortal Wkly Rep 69:477-481, 2020, https://doi.org/10.15585/mmwr.mm6915e6). N95 respirators are recommended by the CDC as the ideal method of protection from COVID-19. Although N95 respirators are traditionally single use, the shortages have necessitated the need for reuse. Effective methods of N95 decontamination that do not affect the fit or filtration ability of N95 respirators are essential. Numerous methods of N95 decontamination exist; however, none are universally accessible. In this study, we describe an effective, standardized, and reproducible means of decontaminating N95 respirators using widely available materials. The N95 decontamination method described in this work will provide a valuable resource for hospitals, health care centers, and outpatient practices that are experiencing increasing shortages of N95 respirators due to the COVID-19 pandemic.

Airborne SARS-CoV-2 Is Rapidly Inactivated by Simulated Sunlight.

Aerosols represent a potential transmission route of COVID-19. This study examined effect of simulated sunlight, relative humidity, and suspension matrix on stability of SARS-CoV-2 in aerosols. Simulated sunlight and matrix significantly affected decay rate of the virus. Relative humidity alone did not affect the decay rate; however, minor interactions between relative humidity and other factors were observed. Mean decay rates (± SD) in simulated saliva, under simulated sunlight levels representative of late winter/early fall and summer were 0.121 ±â€…0.017 min-1 (90% loss, 19 minutes) and 0.306 ±â€…0.097 min-1 (90% loss, 8 minutes), respectively. Mean decay rate without simulated sunlight across all relative humidity levels was 0.008 ±â€…0.011 min-1 (90% loss, 286 minutes). These results suggest that the potential for aerosol transmission of SARS-CoV-2 may be dependent on environmental conditions, particularly sunlight. These data may be useful to inform mitigation strategies to minimize the potential for aerosol transmission.

Estimated Inactivation of Coronaviruses by Solar Radiation With Special Reference to COVID-19.

Using a model developed for estimating solar inactivation of viruses of biodefense concerns, we calculated the expected inactivation of SARS-CoV-2 virus, cause of COVID-19 pandemic, by artificial UVC and by solar ultraviolet radiation in several cities of the world during different times of the year. The UV sensitivity estimated here for SARS-CoV-2 is compared with those reported for other ssRNA viruses, including influenza A virus. The results indicate that SARS-CoV-2 aerosolized from infected patients and deposited on surfaces could remain infectious outdoors for considerable time during the winter in many temperate-zone cities, with continued risk for re-aerosolization and human infection. Conversely, the presented data indicate that SARS-CoV-2 should be inactivated relatively fast (faster than influenza A) during summer in many populous cities of the world, indicating that sunlight should have a role in the occurrence, spread rate and duration of coronavirus pandemics.

Experimental aerosol survival of SARS-CoV-2 in artificial saliva and tissue culture media at medium and high humidity.

SARS-CoV-2, the causative agent of the COVID-19 pandemic, may be transmitted via airborne droplets or contact with surfaces onto which droplets have deposited. In this study, the ability of SARS-CoV-2 to survive in the dark, at two different relative humidity values and within artificial saliva, a clinically relevant matrix, was investigated. SARS-CoV-2 was found to be stable, in the dark, in a dynamic small particle aerosol under the four experimental conditions we tested and viable virus could still be detected after 90 minutes. The decay rate and half-life was determined and decay rates ranged from 0.4 to 2.27 % per minute and the half lives ranged from 30 to 177 minutes for the different conditions. This information can be used for advice and modelling and potential mitigation strategies.

Shining a light on the pathogenicity of health care providers' mobile phones: Use of a novel ultraviolet-C wave disinfection device.

BACKGROUND: Mobile phones are known to carry pathogenic bacteria and viruses on their surfaces, posing a risk to healthcare providers (HCPs) and hospital infection prevention efforts. We utilize an Ultraviolet-C (UV-C) device to provide an effective method for mobile phone disinfection and survey HCPs about infection risk. METHODS: Environmental swabs were used to culture HCPs' personal mobile phone surfaces. Four cultures were obtained per phone: before and after the UV-C device's 30-second disinfecting cycle, at the beginning and end of a 12-hour shift. Surveys were administered to participants pre- and poststudy. RESULTS: Total bacterial colony forming units were reduced by 90.5% (P = .006) after one UV-C disinfection cycle, and by 99.9% (P = .004) after 2 cycles. Total pathogenic bacterial colony forming units were decreased by 98.2% (P = .038) after one and >99.99% (P = .037) after 2 disinfection cycles. All survey respondents were willing to use the UV-C device daily to weekly, finding it convenient and beneficial. DISCUSSION: This novel UV-C disinfecting device is effective in reducing pathogenic bacteria on mobile phones. HCPs would frequently use a phone disinfecting device to reduce infection risk. CONCLUSIONS: In light of the ongoing coronavirus (COVID-19) pandemic, a standardized approach to phone disinfection may be valuable in preventing healthcare-associated infections.

Physicochemical properties of SARS-CoV-2 for drug targeting, virus inactivation and attenuation, vaccine formulation and quality control.

The material properties of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its proteins are discussed. We review the viral structure, size, rigidity, lipophilicity, isoelectric point, buoyant density and centrifugation conditions, stability against pH, temperature, UV light, gamma radiation, and susceptibility to various chemical agents including solvents and detergents. Possible inactivation, downstream, and formulation conditions are given including suitable buffers and some first ideas for quality-control methods. This information supports vaccine development and discussion with competent authorities during vaccine approval and is certainly related to drug-targeting strategies and hygienics. Several instructive tables are given, including the pI and grand average of hydropathicity (GRAVY) of SARS-CoV-1 and -2 proteins in comparison. SARS-CoV-1 and SARS-CoV-2 are similar in many regards, so information can often be derived. Both are unusually stable, but sensitive at their lipophilic membranes. However, since seemingly small differences can have strong effects, for example, on immunologically relevant epitope settings, unevaluated knowledge transfer from SARS-CoV-1 to SARS-CoV-2 cannot be advised. Published knowledge regarding downstream processes, formulations and quality assuring methods is, as yet, limited. However, standard approaches employed for other viruses and vaccines seem to be feasible including virus inactivation, centrifugation conditions, and the use of adjuvants.

Pathogen reduction of SARS-CoV-2 virus in plasma and whole blood using riboflavin and UV light.

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has recently been identified as the causative agent for Coronavirus Disease 2019 (COVID-19). The ability of this agent to be transmitted by blood transfusion has not been documented, although viral RNA has been detected in serum. Exposure to treatment with riboflavin and ultraviolet light (R + UV) reduces blood-borne pathogens while maintaining blood product quality. Here, we report on the efficacy of R + UV in reducing SARS-CoV-2 infectivity when tested in human plasma and whole blood products. STUDY DESIGN AND METHODS: SARS-CoV-2 (isolate USA-WA1/2020) was used to inoculate plasma and whole blood units that then underwent treatment with riboflavin and UV light (Mirasol Pathogen Reduction Technology System, Terumo BCT, Lakewood, CO). The infectious titers of SARS-CoV-2 in the samples before and after R + UV treatment were determined by plaque assay on Vero E6 cells. Each plasma pool (n = 9) underwent R + UV treatment performed in triplicate using individual units of plasma and then repeated using individual whole blood donations (n = 3). RESULTS: Riboflavin and UV light reduced the infectious titer of SARS-CoV-2 below the limit of detection for plasma products at 60-100% of the recommended energy dose. At the UV light dose recommended by the manufacturer, the mean log reductions in the viral titers were ≥ 4.79 ± 0.15 Logs in plasma and 3.30 ± 0.26 in whole blood units. CONCLUSION: Riboflavin and UV light effectively reduced the titer of SARS-CoV-2 to the limit of detection in human plasma and by 3.30 ± 0.26 on average in whole blood. Two clades of SARS-CoV-2 have been described and questions remain about whether exposure to one strain confers strong immunity to the other. Pathogen-reduced blood products may be a safer option for critically ill patients with COVID-19, particularly those in high-risk categories.

Inactivation of severe acute respiratory syndrome coronavirus 2 in plasma and platelet products using a riboflavin and ultraviolet light-based photochemical treatment.

BACKGROUND AND OBJECTIVE: Severe acute respiratory distress syndrome coronavirus-2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is a member of the coronavirus family. Coronavirus infections in humans are typically associated with respiratory illnesses; however, viral RNA has been isolated in serum from infected patients. Coronaviruses have been identified as a potential low-risk threat to blood safety. The Mirasol Pathogen Reduction Technology (PRT) System utilizes riboflavin and ultraviolet (UV) light to render blood-borne pathogens noninfectious, while maintaining blood product quality. Here, we report on the efficacy of riboflavin and UV light against the pandemic virus SARS-CoV-2 when tested in both plasma and platelets units. MATERIALS AND METHODS: Stock SARS-CoV-2 was grown in Vero cells and inoculated into either plasma or platelet units. Those units were then treated with riboflavin and UV light. The infectious titres of SARS-CoV-2 were determined by plaque assay using Vero cells. A total of five (n = 5) plasma and three (n = 3) platelet products were evaluated in this study. RESULTS: In both experiments, the measured titre of SARS-CoV-2 was below the limit of detection following treatment with riboflavin and UV light. The mean log reductions in the viral titres were ≥3·40 and ≥4·53 for the plasma units and platelet units, respectively. CONCLUSION: Riboflavin and UV light effectively reduced the titre of SARS-CoV-2 in both plasma and platelet products to below the limit of detection in tissue culture. The data suggest that the process would be effective in reducing the theoretical risk of transfusion transmitted SARS-CoV-2.