Proceedings of the 2023 Viral Clearance Symposium, Session 4: Continuous Processing.

The continuous processing session at the 2023 Viral Clearance Symposium (VCS) focused on understanding how to effectively design viral clearance operations for use in continuous processes and methods to perform viral clearance studies. In this session, an approach to directly address control considerations with operating continuous-flow reactors for low pH viral inactivation was presented. Continuous-flow low pH incubation chamber design and implications for residence time determination were discussed. Additionally, viral clearance capability between batch operation and connected operation were demonstrated to be comparable for a connected bind-elute chromatography and flow-through chromatography step. Overall, this session provided additional scientific knowledge to support viral clearance strategies when implementing a continuous manufacturing process.

Proceedings of the 2023 Viral Clearance Symposium, Session 6: Viral Inactivation.

The use of detergents or low pH hold are commonly employed techniques in biologics downstream processing to inactivate enveloped viruses. These approaches have been demonstrated to be robust and are detailed in ASTM E2888 (low pH) and ASTM E3042-16 (Triton X-100), accordingly. One of the recent challenges is the need for a replacement of Triton X-100 with a more environmentally friendly detergent with similar log10 reduction value (LRV) achieved. The presentations in this session focused on a detailed assessment of a range of detergents. The most well characterized and potentially robust detergents identified were TDAO (n-Tetradecyl-N,N-dimethylamine-N-oxide) and Simulsol SL 11 W. Key performance factors assessed (in direct comparison with the industry standard Triton X-100) were viral inactivation kinetics (total elapsed time to achieve equilibrium), LRV achieved of enveloped viruses, toxicity, potential impact on product quality and process performance, clearance of residual detergent in subsequent downstream steps, assays to support assessment with appropriate limit of quantification, and commercial supply of detergent of the appropriate quality standard. Both TDAO and Simulsol SL11 had similar overall LRV as Triton-100. In addition, for the low pH viral inactivation, reduced LRV was observed at pH > 3.70 and low salt concentration (outside of the ASTM range), which is a cautionary note when applying low pH inactivation to labile proteins.

Determination of Optimal Antigen Yield and Virus Inactivation Conditions for the Production of the Candidate Foot-and-Mouth Disease Recombinant Vaccine Strain Asia1 Shamir-R in a Bioreactor.

Since the foot-and-mouth disease (FMD) outbreak in South Korea in 2010-2011, vaccination policies utilizing inactivated FMD vaccines composed of types O and A have been implemented nationwide. However, because type Asia1 occurred in North Korea in 2007 and intermittently in neighboring countries, the risk of type Asia1 introduction cannot be ruled out. This study evaluated the antigen yield and viral inactivation kinetics of the recombinant Asia1 Shamir vaccine strain (Asia1 Shamir-R). When Asia1 Shamir-R was proliferated in shaking flasks (1 L), a 2 L bioreactor (1 L), and a wave bioreactor (25 L), the antigen yields were 7.5 µg/mL, 5.2 µg/mL, and 3.8 µg/mL, respectively. The optimal FMDV inactivation conditions were 2 mM BEI at 26 °C and 1.0 mM BEI at 37 °C. There was no antigen loss due to BEI treatment, and only a decrease in antigen levels was observed during storage. The sera from pigs immunized with antigen derived from a bioreactor exhibited a neutralizing antibody titer of approximately 1/1000 against Asia1 Shamir and Asia1/MOG/05 viruses; therefore, Asia1 Shamir-R is expected to provide sufficient protection against both viruses. If an FMD vaccine production facility is established, this Asia1 Shamir-R can be employed for domestic antigen banks in South Korea.

Surface air gas discharge plasma: An ecofriendly virus inactivation approach to enhance CPRRs mediated antiviral genes expression against airborne bio-contaminant (human Coronavirus-229E).

Emerging bio-contaminants (airborne viruses) exploits and manipulate host (human) metabolism to produce new viral particles, evading the host's immune defences and leading to infections. Non-thermal plasma, operating at atmospheric pressure and ambient temperature, is explored for virus inactivation, generating RONS that interact and denatures viral proteins. However, various factors affecting virus survival influence the efficacy of non-thermal plasma. Glucose analogue 2-DG, a metabolic modifier used in this study, disrupts the glycolysis pathway viruses rely on, creating an unfavourable environment for replication. Here, airborne HCoV-229E bio-contaminant was treated with plasma for inactivation, and the presence of RONS was analysed. Metabolically altered lung cells were subsequently exposed to the treated airborne viruses. Cytopathic effect, spike protein, and cell death were evaluated via flow cytometry and confocal microscopy, and CPRRs mediated antiviral gene expression was evaluated using PCR. Gas plasma-treated viruses led to reduced virus proliferation in unaltered lung cells, although few virus particles survived the exposure, as confirmed by biological assessment (cytopathic effects and live/dead staining). A combination approach of gas plasma-treated viruses and altered lung cells displayed drastic virus reduction compared to the control group, established through confocal microscopy and flow cytometry. Furthermore, altered lung cell enhances gene transcription responsible for innate immunity when exposed to the gas plasma-treated virus, thereby impeding airborne virus propagation. This study demonstrates the significance of a surface air gas plasma and metabolic alteration approach in enhancing genes targeted towards antiviral innate immunity and tackling outbreaks of emerging bio-contaminants of concerns (airborne viruses).

Chemical inactivation strategies for SARS-CoV-2-infected cells and organoids.

Infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research, conducted in high-containment laboratories, requires transferring samples to lower containment labs for downstream applications, mandating sample inactivation. Here, we present a stepwise protocol for chemical inactivation of SARS-CoV-2 virus in culture supernatants or within infected cells and organoids, using eight chemical reagents validated via plaque assays. Additionally, we describe steps for troubleshooting virus inactivation, titer calculation, and log reduction. This protocol offers valuable resources for the COVID-19 research community, providing essential tools to advance research on this virus.

Viral inactivation for pH-sensitive antibody formats such as multi-specific antibodies.

Recently developed multi-specific antibody formats enable new therapeutic concepts. Conveniently, formats with an Fc domain allow purification in well-established mAb platform processes. However, due to the structural complexity of the formats, the assembled molecules may be sensitive to extreme pH commonly used for viral inactivation. An alternative to low pH incubation for virus inactivation is the use of a mixture of tri-n-butyl phosphate (TnBP, solvent) and Polysorbate 80 (PS80, detergent). While TnBP is toxic, this combination has a long history of use in the manufacturing of human plasma-derived products that are sensitive to low or high pH incubation. Data are provided demonstrating that the solvent/detergent (S/D) treatment using TnBP and PS80 can be successfully used for pH-sensitive, multi-specific antibody formats in the clarified cell culture fluid (CCCF). A different placement of the S/D within the purification process, namely during the capture by Protein A (PA), has been evaluated. This alternative placement allows effective viral inactivation by S/D while preserving the viral reduction and viral inactivation achieved through the PA step itself, enabling the cumulation of these effects. Furthermore, the process alternative simplifies the liquid handling by reducing the added volumes of the required S/D liquids, thus reducing the amount of toxic TnBP to a minimum. Data are shown demonstrating a complete removal of TnBP and PS80 in the process.

Human intestinal enteroids and predictive models validate the operational limits of sanitizers used for viral disinfection of vegetable process wash water.

Vegetables are globally associated with a considerable number of foodborne outbreaks caused by viral infections, specifically human norovirus. In fresh produce industry, washing represents a critical step for food safety as process wash water (PWW) needs to be maintained at appropriate microbial quality to prevent water-mediated cross-contamination. This study aimed to explore the disinfection efficacy of chlorine (free chlorine, FC), chlorine dioxide (ClO2) and peracetic acid (PAA) in PWW against infectious human norovirus and Tulane virus (TV). First, we tested the extent of TV inactivation in baby leaf, bell pepper, and vegetables mix PWW and monitored the viral decay by cell culture. Then, inactivation kinetics were defined for infectious human norovirus exposed to FC, ClO2 and PAA in baby leaves PWW using the human intestinal enteroids (HIE) system. Finally, kinetic inactivation models were fitted to TV reduction and decay of sanitizers to aid the implementation of disinfection strategies. Results showed that >8 log10 human norovirus and 3.9 log10 TV were inactivated by 20 ppm FC within 1 min; and by 3 ppm ClO2 in 1 min (TV) or 5 min (norovirus). PAA treatment at 80 ppm reduced ca. 2 log10 TV but not completely inactivated the virus even after 20 min exposure, while 5 min treatment prevented norovirus replication in HIE. TV inactivation in PWWs was described using an exponential decay model. Taking these data together, we demonstrated the value of applying the HIE model to validate current operational limits for the most commonly used sanitizers. The inactivation kinetics for human norovirus and TV, along with the predictive model described in this study expand the current knowledge to implement post-harvest produce safety procedures in industry settings.

Quantitative probability estimation of light-induced inactivation of SARS-CoV-2.

During the COVID pandemic caused by the SARS-CoV-2 virus, studies have shown the efficiency of deactivating this virus via ultraviolet light. The damage mechanism is well understood: UV light disturbs the integrity of the RNA chain at those locations where specific nucleotide neighbors occur. In this contribution, we present a model to address certain gaps in the description of the interaction between UV photons and the RNA sequence for virus inactivation. We begin by exploiting the available information on the pathogen's morphology, physical, and genomic characteristics, enabling us to estimate the average number of UV photons required to photochemically damage the virus's RNA. To generalize our results, we have numerically generated random RNA sequences and checked that the distribution of pairs of nucleotides susceptible of damage for the SARS-CoV-2 is within the expected values for a random-generated RNA chain. After determining the average number of photons reaching the RNA for a preset level of fluence (or photon density), we applied the binomial probability distribution to evaluate the damage of nucleotide pairs in the RNA chain due to UV radiation. Our results describe this interaction in terms of the probability of damaging a single pair of nucleotides, and the number of available photons. The cumulative probability exhibits a steep sigmoidal shape, implying that a relatively small change in the number of affected pairs may trigger the inactivation of the virus. Our light-RNA interaction model quantitatively describes how the fraction of affected pairs of nucleotides in the RNA sequence depends on the probability of damaging a single pair and the number of photons impinging on it. A better understanding of the underlying inactivation mechanism would help in the design of optimum experiments and UV sanitization methods. Although this paper focuses on SARS-CoV-2, these results can be adapted for any other type of pathogen susceptible of UV damage.

Heat inactivation of aqueous viable norovirus and MS2 bacteriophage.

AIMS: This study aimed to compare the heat inactivation kinetics of viable human norovirus with the surrogate, MS2 bacteriophage as well as assess the decay of the RNA signal. METHODS AND RESULTS: Human intestinal enteroids were used to analyze the heat inactivation kinetics of viable human norovirus compared to the surrogate MS2 bacteriophage, which was cultured using a plaque assay. Norovirus decay rates were 0.22 min-1, 0.68 min-1, and 1.11 min-1 for 50°C, 60°C, and 70°C, respectively, and MS2 bacteriophage decay rates were 0.0065 min-1, 0.045 min-1, and 0.16 min-1 for 50°C, 60°C, and 70°C, respectively. Norovirus had significantly higher decay rates than MS2 bacteriophage at all tested temperatures (P = .002-.007). No decrease of RNA titers as measured by reverse transcription-PCR for both human norovirus and MS2 bacteriophage over time was observed, indicating molecular methods do not accurately depict viable human norovirus after heat inactivation and treatment efficiency is underestimated. CONCLUSIONS: Overall, our data demonstrate that MS2 bacteriophage is a conservative surrogate to measure heat inactivation and potentially overestimates the infectious risk of norovirus. Furthermore, this study corroborates that measuring viral RNA titers, as evaluated by PCR methods, does not correlate with the persistence of viable norovirus under heat inactivation.

Mechanism of surfactant interactions with feline coronavirus: A physical chemistry perspective.

HYPOTHESIS: Surfactants are inexpensive chemicals with promising applications in virus inactivation, particularly for enveloped viruses. Yet, the detailed mechanisms by which surfactants deactivate coronaviruses remain underexplored. This study delves into the virucidal mechanisms of various surfactants on Feline Coronavirus (FCoV) and their potential applications against more pathogenic coronaviruses. EXPERIMENTS: By integrating virucidal activity assays with fluorescence spectroscopy, dynamic light scattering and laser Doppler electrophoresis, alongside liposome permeability experiments, we have analyzed the effects of non-ionic and ionic surfactants on viral activity. FINDINGS: The non-ionic surfactant octaethylene glycol monodecyl ether (C10EO8) inactivates the virus by disrupting the lipid envelope, whereas ionic surfactants like Sodium Dodecyl Sulfate and Cetylpyridinium Chloride predominantly affect the spike proteins, with their impact on the viral membrane being hampered by kinetic and thermodynamic constraints. FCoV served as a safe model for studying virucidal activity, offering a faster alternative to traditional virucidal assays. The study demonstrates that physicochemical techniques can expedite the screening of virucidal compounds, contributing to the design of effective disinfectant formulations. Our results not only highlight the critical role of surfactant-virus interactions but also contribute to strategic advancements in public health measures for future pandemic containment and the ongoing challenge of antimicrobial resistance.

Retention of Virus Versus Surrogate, by Ultrafiltration in Seawater: Case Study of Norovirus Versus Tulane.

In the field of chemical engineering and water treatment, the study of viruses, included surrogates, is well documented. Often, surrogates are used to study viruses and their behavior because they can be produced in larger quantities in safer conditions and are easier to handle. In fact, surrogates allow studying microorganisms which are non-infectious to humans but share some properties similar to pathogenic viruses: structure, composition, morphology, and size. Human noroviruses, recognized as the leading cause of epidemics and sporadic cases of gastroenteritis across all age groups, may be mimicked by the Tulane virus. The objectives of this work were to study (i) the ultrafiltration of Tulane virus and norovirus to validate that Tulane virus can be used as a surrogate for norovirus in water treatment process and (ii) the retention of norovirus and the surrogate as a function of water quality to better understand the use of the latter pathogenic viruses. Ultrafiltration tests showed significant logarithmic reduction values (LRV) in viral RNA: around 2.5 for global LRV (i.e., based on the initial and permeate average concentrations) and between 2 and 6 for average LRV (i.e., retention rate considering the increase of viral concentration in the retentate), both for norovirus and the surrogate Tulane virus. Higher reduction rates (from 2 to 6 log genome copies) are obtained for higher initial concentrations (from 101 to 107 genome copies per mL) due to virus aggregation in membrane lumen. Tulane virus appears to be a good surrogate for norovirus retention by membrane processes.

Heat stability of foodborne viruses - Findings, methodological challenges and current developments.

Heat treatment of food represents an important measure to prevent pathogen transmission. Thus far, evaluation of heat treatment processes is mainly based on data from bacteria. However, foodborne viruses have gained increasing attention during the last decades. Here, the published literature on heat stability and inactivation of human norovirus (NoV), hepatitis A virus (HAV) and hepatitis E virus (HEV) was reviewed. Data for surrogate viruses were not included. As stability assessment for foodborne viruses is often hampered by missing infectivity assays, an overview of applied methods is also presented. For NoV, molecular capsid integrity assays were mainly applied, but data from initial studies utilizing novel intestinal enteroid or zebrafish larvae assays are available now. However, these methods are still limited in applicability and sensitivity. For HAV, sufficient cell culture-based inactivation data are available, but almost exclusively for one single strain, thus limiting interpretation of the data for the wide range of field strains. For HEV, data are now available from studies using pig inoculation or cell culture. The results of the reviewed studies generally indicate that NoV, HAV and HEV possess a high heat stability. Heating at 70-72 °C for 2 min significantly reduces infectious titers, but often does not result in a >4 log10 decrease. However, heat stability greatly varied dependent on virus strain, matrix and heating regime. In addition, the applied method largely influenced the result, e.g. capsid integrity assays tend to result in higher measured stabilities than cell culture approaches. It can be concluded that the investigated foodborne viruses show a high heat stability, but can be inactivated by application of appropriate heating protocols. For HAV, suggestions for safe time/temperature combinations for specific foods can be derived from the published studies, with the limitation that they are mostly based on one strain only. Although significant improvement of infectivity assays for NoV and HEV have been made during the last years, further method development regarding sensitivity, robustness and broader applicability is important to generate more reliable heat inactivation data for these foodborne viruses in future.

Preparing for another Ebola Outbreak: The impact of viral inactivation methods on commonly measured biochemistry analytes in plasma and urine.

INTRODUCTION: Infectious specimens containing viruses like Ebola require sample manipulation to ensure the safety of laboratory staff, which may negatively impact biochemistry test results. We evaluated the impact of viral inactivation methods on 25 biochemistry analytes in plasma, and seven biochemistry analytes in urine. METHODS: Fifteen lithium heparinized plasma specimens with and without gel underwent the following viral inactivation methods: 1) untreated, 2) Triton X-100 treatment, 2) heated for 60 min then Triton X-100 treatment, 3) heated for 60 min, 4) heated for 75 min, and 5) heated for 90 min. Electrolytes, protein, enzymes, glucose, as well as hepatic and renal markers were measured on the Roche Cobas e601, c502 or c702. Urinalysis analytes were measured on the Siemens CLINITEK. Acceptable recovery was based on Institute for Quality Management in Healthcare 2021 guidelines or ± 1 for urinalysis. RESULTS: Potassium and lactate dehydrogenase were impacted by the presence of gel. Viral inactivation with Triton X-100 had minimal impact on the biochemistry results. Heat inactivation resulted in significant negative bias in alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, creatinine, total protein, amylase, lactate dehydrogenase and creatine kinase. Positive bias in phosphate, aspartate transaminase, total bilirubin, and uric acid were observed after heat inactivation. CONCLUSION: Reliable results for commonly measured electrolytes, enzymes and proteins can be obtained after viral inactivation by Triton X-100 treatment at room temperature. However, heat inactivation has significant negative impact on routine biochemistry enzymes and alternative testing processes should be explored.

Ozone and photodynamic inactivation of norovirus surrogate bacteriophage MS2 in fresh Brazilian berries and surfaces.

This study assessed the efficacy of ozone (bubble diffusion in water; 6.25 ppm) and photodynamic inactivation (PDT) using curcumin (75 µM) as photosensitizer (LED emission 430-470 nm; 33.6 mW/cm2 irradiance; 16.1, 20.2, and 24.2 J/cm2 light dose) against the Norovirus surrogate bacteriophage MS2 in Brazilian berries (black mulberry and pitanga) and surfaces (glass and stainless steel). Contaminated berries and surfaces were immersed in ozonized water or exposed to PDT-curcumin for different time intervals. Transmission electron microscopy was used to assess the effects of the treatments on MS2 viral particles. The MS2 inactivation by ozone and PDT-curcumin varied with the fruit and the surface tested. Ozone reduced the MS2 titer up to 3.6 log PFU/g in black mulberry and 4.1 log PFU/g in pitanga. On surfaces, the MS2 reduction by ozone reached 3.6 and 4.8 log PFU/cm2 on glass and stainless steel, respectively. PDT-curcumin reduced the MS2 3.2 and 4.8 log PFU/g in black mulberry and pitanga and 2.7 and 3.3 log PFU/cm2 on glass and stainless steel, respectively. MS2 particles were disintegrated by exposure of MS2 to ozone and PDT-curcumin on pitanga. Results can contribute to establishing effective practices for controlling NoV in fruits and surfaces, estimated based on MS2 bacteriophage behavior.

Challenges for estimating human norovirus infectivity by viability RT-qPCR as compared to replication in human intestinal enteroids.

Viability RT-qPCR, a molecular detection method combining viability marker pre-treatment with RT-qPCR, has been proposed to infer infectivity of viruses which is particularly relevant for non-culturable viruses or sophisticated cell culture systems. Being human noroviruses (HuNoV) most frequently associated with foodborne outbreaks, this study compared different viability techniques and infectivity in human intestinal enteroids (HIE) to ultimately determine whether the molecular approaches could serve as rapid assays to predict HuNoV inactivation in high-risk food. To this end, the performance of three viability RT-qPCR assays with different intercalating markers ((Viability PCR Crosslinker Kit (CL), propidium monoazide (PMAxx™), and platinum chloride (PtCl4)) in estimating survival of HuNoV exposed to thermal and high pressure (HPP) treatments was compared to replication tested in the HIE cell culture model. A nearly full-length genomic molecular assay coupled with PMAxx™ to infer HuNoV thermal inactivation was also assessed. The experimental design included HuNoV genogroup I.3 [P13], GII.4 Sydney [P16], GII.6 [P7], along with Tulane virus (TV) serving as surrogate. Finally, viability RT-qPCR was tested in HPP-treated strawberry puree, selected as a food matrix with high viral contamination risk. PMAxx™ and CL performed evenly, while PtCl4 affected HuNoV infectivity. Taking all experimental data together, viability RT-qPCR was demonstrated to be an improved method over direct RT-qPCR to estimate viral inactivation at extreme thermal (95 °C) and HPP (450 MPa) exposures, but not under milder conditions as amplification signals were detected. Despite its complexity and limitations, the HIE demonstrated a more robust model than viability RT-qPCR to assess HuNoV infectivity.

SARS-CoV-2 inactivation in laboratory animal tissues with 4% formaldehyde or 5% glutaraldehyde for transfer to biosafety level 1 laboratories.

The SARS-CoV-2 pandemic required the immediate need to transfer inactivated tissue from biosafety level (BSL)-3 to BSL-1 areas to enable downstream analytical methods. No validated SARS-CoV-2 inactivation protocols were available for either formaldehyde (FA)-fixed or glutaraldehyde (GA)-fixed tissues. Therefore, representative tissue from ferrets and hamsters was spiked with 2.2 × 106 tissue culture infectious dose 50% per ml (TCID50/ml) SARS-CoV-2 or were obtained from mice experimentally infected with SARS-CoV-2. SARS-CoV-2 inactivation was demonstrated with 4% FA or 5% GA at room temperature for 72 hours by a titer reduction of up to 103.8 TCID50/ml in different animal tissues with a maximum protein content of 100 µg/mg and a thickness of up to 10 mm for FA and 8 mm for GA. Our protocols can be easily adapted for validating the inactivation of other pathogens to allow for the transfer of biological samples from BSL-3 areas to BSL-1 laboratories.

Sunlight Inactivation of Enveloped Viruses in Clear Water.

Enveloped virus fate in the environment is not well understood; there are no quantitative data on sunlight inactivation of enveloped viruses in water. Herein, we measured the sunlight inactivation of two enveloped viruses (Phi6 and murine hepatitis virus, MHV) and a nonenveloped virus (MS2) over time in clear water with simulated sunlight exposure. We attenuated UV sunlight wavelengths using long-pass 50% cutoff filters at 280, 305, and 320 nm. With the lowest UV attenuation tested, all decay rate constants (corrected for UV light screening, k̂) were significantly different from dark controls; the MS2 k̂ was equal to 4.5 m2/MJ, compared to 16 m2/MJ for Phi6 and 52 m2/MJ for MHV. With the highest UV attenuation tested, only k̂ for MHV (6.1 m2/MJ) was different from the dark control. Results indicate that the two enveloped viruses decay faster than the nonenveloped virus studied, and k̂ are significantly impacted by UV attenuation. Differences in k̂ may be due to the presence of viral envelopes but may also be related to other differing intrinsic properties of the viruses, including genome length and composition. Reported k̂ values can inform strategies to reduce the risk from exposure to enveloped viruses in the environment.

Cold plasma within a stable supercavitation bubble - A breakthrough technology for efficient inactivation of viruses in water.

Water scarcity, one of the most pressing challenges we face today, has developed for many reasons, including the increasing number of waterborne pollutants that affect the safety of the water environment. Waterborne human, animal and plant viruses represent huge health, environmental, and financial burden and thus it is important to efficiently inactivate them. Therefore, the main objective of this study was to construct a unique device combining plasma with supercavitation and to evaluate its efficiency for water decontamination with the emphasis on inactivation of viruses. High inactivation (>5 log10 PFU/mL) of bacteriophage MS2, a human enteric virus surrogate, was achieved after treatment of 0.43 L of recirculating water for up to 4 min. The key factors in the inactivation were short-lived reactive plasma species that damaged viral RNA. Water treated with plasma for a short time required for successful virus inactivation did not cause cytotoxic effects in the in vitro HepG2 cell model system or adverse effects on potato plant physiology. Therefore, the combined plasma-supercavitation device represents an environmentally-friendly technology that could provide contamination-free and safe water.

Virus Inactivation by Formaldehyde and Common Lysis Buffers.

Numerous mammalian viruses are routinely analyzed in clinical diagnostic laboratories around the globe or serve as indispensable model systems in viral research. Potentially infectious viral entities are handled as blood, biopsies, or cell and tissue culture samples. Countless protocols describe methods for virus fixation and inactivation, yet for many, a formal proof of safety and completeness of inactivation remains to be shown. While modern nucleic acid extraction methods work quite effectively, data are largely lacking on possible residual viral infectivity, e.g., when assessed after extended culture times, which maximizes the sensitivity for low levels of residual infectiousness. Therefore, we examined the potency and completeness of inactivation procedures on virus-containing specimens when applying commonly used fixatives like formaldehyde or nucleic acid extraction/lysis buffers. Typical representatives of different virus classes, including RNA and DNA viruses, enveloped and non-enveloped, such as adenovirus, enterovirus, lentivirus, and coronavirus, were used, and the reduction in the in vitro infectiousness was assessed for standard protocols. Overall, a 30-minute incubation with formaldehyde at room temperature effectively inactivated all tested enveloped and non-enveloped viruses. Full inactivation of HIV-1 and ECHO-11 was also achieved with all buffers in the test, whereas for SARS-CoV-2 and AdV-5, only five of the seven lysis buffers were fully effective under the tested conditions.

Ultrafast inactivation of SARS-CoV-2 by 254-nm UV-C irradiation on porous and non-porous media of medical interest using an omnidirectional chamber.

Covid-19 has spurred a renewed interest in decontamination techniques for air, objects and surfaces. Beginning in 2020, urgent effort was done to permit the reuse of UV-C for inactivating SARS-CoV-2. However, those studies diverged widely on the dose necessary to reach this goal; until today, the real value of the sensitivity of the virus to a 254-nm illumination is not known precisely. In this study, decontamination was performed in an original UV-C large decontamination chamber (UVCab, ON-LIGHT, France) delivering an omnidirectional irradiation with an average dose of 50 mJ/cm2 in 60 s. Viral inactivation was checked by both cell culture and PCR test. SARS-CoV-2 was inactivated by UV-C light within 3 s on both porous (disposable gown) and non-porous (stainless steel and apron) surfaces. For the porous surface, an irradiation of 5 min was needed to achieve a completely negative PCR signal. The Z value estimating the sensitivity of SARS-CoV-2 to UV-C in the experimental conditions of our cabinet was shown to be > 0.5820 m2/J. These results illustrate the ability of this apparatus to inactivate rapidly and definitively high loads of SARS-CoV-2 deposited on porous or non-porous supports and opens new perspectives on material decontamination using UV-C.