Human coronavirus 229E inactivation on porous and nonporous materials using dielectric barrier discharge (DBD) plasma.

Viruses can spread through contaminated aerosols and contaminated surface materials, and effective disinfection techniques are essential for virus inactivation. Nonthermal plasma-generated reactive oxygen and nitrogen species can effectively inactivate the coronavirus. We aim to interpret the coronavirus inactivation level and mechanism of surface interaction with materials with and without dielectric barrier discharge (DBD) plasma treatment. Nonthermal plasma, particularly surface-type DBD plasma, can inactivate human coronavirus 229E (HCoV-229E) on porous (paper, wood, mask) and nonporous (plastic, stainless steel, glass, Cu) materials. Virus inactivation was analyzed using a 50% tissue culture infectivity dose (TCID50) using cell line, flow cytometry, and immunofluorescence. Surfaces contaminated with HCoV-229E were treated at different time intervals (0-5 h) with and without plasma exposure (natural decay in ambient air conditions). HCoV-229E persistence conformed to the following order: plastic > cover glass > stainless steel > mask > wood > paper > Cu with and without plasma exposure. HCoV-229E was more stable in plastic, cover glass, and stainless steel in 5 h, and the viable virus titer gradually decreased from its initial log10 order of 6.892 to 1.72, 1.53, and 1.32 TCID50/mL, respectively, under plasma exposure. No virus was observed in Cu after treatment for 5 h. The use of airflow, ambient nitrogen, and argon did not promote virus inactivation. Flow cytometry and immunofluorescence analysis demonstrated a low expression level of spike protein (fluorescence intensity) during plasma treatment and in E and M genes expression compared with the virus control.

Collection and transportation of SARS-CoV-2 and influenza A virus diagnostic samples: Optimizing the usage of guanidine-based chaotropic salts for enhanced biosafety and viral genome preservation.

Many virus lysis/transport buffers used in molecular diagnostics, including the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, contain guanidine-based chaotropic salts, primarily guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC). Although the virucidal effects of GuHCl and GITC alone against some enveloped viruses have been established, standardized data on their optimum virucidal concentrations against SARS-CoV-2 and effects on viral RNA stability are scarce. Thus, we aimed to determine the optimum virucidal concentrations of GuHCl and GITC against SARS-CoV-2 compared to influenza A virus (IAV), another enveloped respiratory virus. We also evaluated the effectiveness of viral RNA stabilization at the determined optimum virucidal concentrations under high-temperature conditions (35°C) using virus-specific real-time reverse transcription polymerase chain reaction. Both viruses were potently inactivated by 1.0 M GITC and 2.5 M GuHCl, but the GuHCl concentration for efficient SARS-CoV-2 inactivation was slightly higher than that for IAV inactivation. GITC showed better viral RNA stability than GuHCl at the optimum virucidal concentrations. An increased concentration of GuHCl or GITC increased viral RNA degradation at 35°C. Our findings highlight the need to standardize GuHCl and GITC concentrations in virus lysis/transport buffers and the potential application of these guanidine-based salts alone as virus inactivation solutions in SARS-CoV-2 and IAV molecular diagnostics.

Systematic investigation and modeling prediction of virus inactivation by ozone in wastewater: Decoupling the matrix effects.

Water disinfection is undoubtedly regarded as a critical step in ensuring the water safety for human consumption, and ozone is widely used as a highly effective disinfectant for the control of pathogenic microorganisms in water. Although the diminished ozone efficiencies in complex water matrices have been widely reported, the specific extent to which individual components of matrix act on the virus inactivation by ozone remains unclear, and effective methodologies to predict the comprehensive effects of various factors are needed. In this study, the decoupled impact of the intricate water matrix on the ozone inactivation of viruses was systematically investigated and assessed from a simulative perspective. The concept of "equivalent ozone depletion rate constant" (k') was introduced to quantify the influence of different species, and a kinetic model was developed based on the k' values for simulating the ozone inactivation processes in complex matrix. The mechanisms through which diverse species influenced the ozone inactivation effectiveness were identified: 1) competition effects (k' = 105∼107 M-1s-1), including organic matters and reductive ions (SO32-, NO2-, and I-), which were the most influential species inhibiting the virus inactivation; 2) shielding effects (k' = 103∼104 M-1s-1), including Ca2+, Mg2+, and kaolin; 3) insignificant effects (k' = 0∼1 M-1s-1), including Cl-, SO42-, NO3-, NH4+, and Br-; 4) promotion effects (k' = ∼-103 M-1s-1), including CO32- and HCO3-. Prediction of ozone disinfection efficiency and evaluation of species contribution under complex aquatic matrices were successfully realized utilizing the model. The systematic understanding and methodologies developed in this research provide a reliable framework for predicting ozone inactivation efficiency under complex matrix, and a potential tool for accurate disinfectant dosage determination and interfering factors control in actual wastewater treatment processes.

Tenacity of Animal Disease Viruses on Wood Surfaces Relevant to Animal Husbandry.

The aim of this study was to analyse the hygienic suitability of wood often used in animal husbandry. To this end, the inactivation of viruses (Enterovirus E as a surrogate for non-enveloped viruses and Newcastle disease virus as a surrogate for enveloped viruses) on germ carriers consisting of various types of wood was studied over an extended period to assess the biosafety of wood as an agricultural building material. The study was designed to assess the intrinsic biocidal activity of the wood itself, without the use of a disinfectant. The laboratory tests were based on German test guidelines and current European standards. Five different types of wood germ carriers, i.e., spruce (Picea abies), pine (Pinus sylvestris), poplar (Populus sp.), beech (Fagus sylvatica) and Douglas fir (Pseudotsuga menziesii), as well as stainless-steel carriers, were inoculated with enveloped and non-enveloped viruses and stored for up to four months, and the remaining infectivity of the viruses was continuously assessed. The results showed that intact, finely sawn timber with a low depth of roughness had an inactivating effect on the viruses up to 7.5 decadal logarithmic levels. For the non-enveloped virus, inactivation was fastest on Douglas fir wood, with the target reduction for effective inactivation (reduction by factor 4.0 log10) being achieved after two weeks, and for the enveloped virus on pine wood, it was already achieved from the day of drying. The hygienic effects of the wood carriers may be due to their hygroscopic properties and wood constituents. These effects offer potential for further investigation, including tests with other wood species rich in extractives.

Detailed analysis of low temperature inactivation of respiratory syncytial virus.

Our previous findings indicated that many respiratory syncytial virus (RSV) isolates are unstable at 4 °C compared to 20 °C. Some of the strains completely lose infectivity after 24 h at 4 °C. This study analyzed the inactivation process at 4 °C using a representative strain, RSV/Sendai/851/13. After 24 h of storage at 4 °C, the virus was completely inactivated but retained its ability to attach to and to be taken into host cells. It suggested a reduced fusion ability between the viral and cellular membranes. During storage at 4 °C, the RSV fusion (F) protein underwent a conformational change and was no longer recognized by pre-fusion form-specific antibodies. When the RSV/Sendai/851/13 strain was passaged at 4 °C, a variant with an amino acid substitution, I148T, in the F protein fusion peptide was selected. Also, an amino acid change in G protein demonstrating stability at low temperatures was obtained. These results show that the inactivation of RSV at 4 °C is due to the loss of membrane fusion activity in the F protein, which cannot maintain its pre-fusion state at 4 °C.

Synchronously Sensitive Immunoassay and Efficient Inactivation of Living Zika Virus via DNAzyme Catalytic Amplification and In Situ Aggregation-Induced Emission Photosensitizer Generation.

Sensitive identification and effective inactivation of the virus are paramount for the early diagnosis and treatment of viral infections to prevent the risk of secondary transmission of viruses in the environment. Herein, we developed a novel two-step fluorescence immunoassay using antibody/streptavidin dual-labeled polystyrene nanobeads and biotin-labeled G-quadruplex/hemin DNAzymes with peroxidase-mimicking activity for sensitive quantitation and efficient inactivation of living Zika virus (ZIKV). The dual-labeled nanobeads can specifically bind ZIKV through E protein targeting and simultaneously accumulate DNAzymes, leading to the catalytic oxidation of Amplex Red indicators and generation of intensified aggregation-induced emission fluorescence signals, with a detection limit down to 66.3 PFU/mL and 100% accuracy. Furthermore, robust reactive oxygen species generated in situ by oxidized Amplex Red upon irradiation can completely kill the virus. This sensitive and efficient detection-inactivation integrated system will expand the viral diagnostic tools and reduce the risk of virus transmission in the environment.

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.

High-Pressure Processing of Different Tissue Homogenates from Pigs Challenged with the African Swine Fever Virus.

African swine fever (ASF) is a disease that is a growing threat to the global swine industry. Regulations and restrictions are placed on swine movement to limit the spread of the virus. However, these are costly and time-consuming. Therefore, this study aimed to determine if high-pressure processing (HPP) sanitization techniques would be effective against the ASF virus. Here, it was hypothesized that HPP could inactivate or reduce ASF virus infectivity in tissue homogenates. To test this hypothesis, 30 aliquots of each homogenate (spleen, kidney, loin) were challenge-infected with the Turin/83 strain of ASF, at a 10 7.20 median hemadsorption dose (HAD)50/mL. Subsequently, eight aliquots of each homogenate were treated with 600 millipascal (600 MPa) HPP for 3, 5, and 7 min. Six untreated aliquots were used as the controls. Virological results showed a reduction in the viral titer of more than 7-log. These results support the validity of the study hypothesis since HPP treatment was effective in inactivating ASFV in artificially prepared samples. Overall, this study suggests the need for further investigation of other ASFV-contaminated meat products.

Effects of Strain Differences, Humidity Changes, and Saliva Contamination on the Inactivation of SARS-CoV-2 by Ion Irradiation.

One of the methods to inactivate viruses is to denature viral proteins using released ions. However, there have been no reports detailing the effects of changes in humidity or contamination with body fluids on the inactivation of viruses. This study investigated the effects of humidity changes and saliva contamination on the efficacy of SARS-CoV-2 inactivation with ions using multiple viral strains. Virus solutions with different infectious titers were dropped onto a circular nitrocellulose membrane and irradiated with ions from 10 cm above the membrane. After the irradiation of ions for 60, 90, and 120 min, changes in viral infectious titers were measured. The effect of ions on virus inactivation under different humidity conditions was also examined using virus solutions containing 90% mixtures of saliva collected from 10 people. A decrease in viral infectivity was observed over time for all strains, but ion irradiation further accelerated the decrease in viral infectivity. Ion irradiation can inactivate all viral strains, but at 80% humidity, the effect did not appear until 90 min after irradiation. The presence of saliva protected the virus from drying and maintained infectiousness for a longer period compared with no saliva. In particular, the Omicron strain retained its infectivity titer longer than the other strains. Ion irradiation demonstrated a consistent reduction in the number of infectious viruses when compared to the control across varying levels of humidity and irradiation periods. This underscores the notable effectiveness of irradiation, even when the reduction effect is as modest as 50%, thereby emphasizing its crucial role in mitigating the rapid dissemination of SARS-CoV-2.

Virus inactivation in low ozone exposure water reuse applications.

In drinking water applications, an ozone exposure (Ct) based framework has been historically used to validate ozone disinfection. However, significant viral inactivation can be achieved with little to no measurable ozone exposure. Additionally, ozone exposure depends on multiple water quality variables as well as the calculation/ozone measurement method used. In this study, we evaluated alternative ozone monitoring frameworks as well as the impact of water quality variables on ozone decay kinetics and virus/coliform inactivation. Here we show that both change in UV254 absorbance and applied O3:TOC were well correlated with viral inactivation and these frameworks were resilient to changes in water quality. Both increasing temperature (12-30 °C) and pH (5.5-8.4) was shown to significantly increase the ozone decay rate and decreased the resulting ozone exposure by as much as ∼90% in the case of pH. However, due to the increased reaction rate of ozone with viruses at elevated temperature and pH, there was only a minor impact (∼20% in the case of pH) in overall disinfection performance for a given O3:TOC. These frameworks were also considered for variable source water with TOC (5-11 mg/L) and TSS (1.2-5.8 mg/L). Change in UV254 absorbance or applied ozone dose (mg/L) were the strongest indicators of disinfection performance for source waters of variable TOC, however site-specific testing may be needed to apply this framework. Challenge testing with influent nitrite indicated that ozone disinfection performance is significantly impacted (>50% reduction in inactivation) in the presence of nitrite thus enforcing the importance of accounting for this value in the applied ozone dose. Multi-point ozone dissolution was investigated as an alternative ozone application method that may present a benefit with respect to overall disinfection performance especially if nitrite was present. Developing and validating these alternative monitoring frameworks and ozone application methods is imperative in water reuse applications where unnecessary elevated ozone exposure may lead to harmful byproduct formation.

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.

The effect of enzymatic and viability dye treatment in combination with long-range PCR on assessing Tulane virus infectivity.

Human norovirus (HuNoV) is regularly involved in food-borne infections. To detect infectious HuNoV in food, RT-qPCR remains state of the art but also amplifies non-infectious virus. The present study combines pre-treatments, RNase and propidium monoazide, with three molecular analyses, including long-range PCR, to predominantly detect infectious Tulane virus (TuV), a culturable HuNoV surrogate. TuV was exposed to inactivating conditions to assess which molecular method most closely approximates the reduction in infectious virus determined by cell culture (TCID50). After thermal treatments (56 °C/5 min, 70 °C/5 min, 72 °C/20 min), TCID50 reductions of 0.3, 4.4 and 5.9 log10 were observed. UV exposure (40/100/1000 mJ/cm2) resulted in 1.1, 2.5 and 5.9 log10 reductions. Chlorine (45/100 mg/L for 1 h) reduced infectious TuV by 2.0 and 3.0 log10. After thermal inactivation standard RT-qPCR, especially with pre-treatments, showed the smallest deviation from TCID50. On average, RT-qPCR with pre-treatments deviated by 1.1-1.3 log10 from TCID50. For UV light, long-range PCR was closest to TCID50 results. Long-range reductions deviated from TCID50 by ≤0.1 log10 for mild and medium UV-conditions. However, long-range analyses often resulted in qPCR non-detects. At higher UV doses, RT-qPCR with pre-treatments differed by ≤1.0 log10 from TCID50. After chlorination the molecular methods repeatedly deviated from TCID50 by >1.0 log10, Overall, each method needs to be further optimized for the individual types of inactivation treatment.

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).

Virucidal activity of a plant-oil-based oral rinse against respiratory viruses.

BACKGROUND: Respiratory viruses have been reported to infect the salivary glands and the throat, which are potential reservoirs for virus replication and transmission. Therefore, strategies to reduce the amount of infective virus particles in the oral mucous membranes could lower the risk of transmission. METHODS: The viral inactivation capacity of a plant-oil-based oral rinse (Salviathymol®) was evaluated in comparison with chlorhexidine (Chlorhexamed® FORTE) using a quantitative suspension test according to EN 14476. FINDINGS: Salviathymol efficiently inactivated severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), respiratory syncytial virus (RSV) and two influenza strains to undetectable levels. CONCLUSION: Salviathymol has potential as preventive measure to lower transmission of respiratory viruses.

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.

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.

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.