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Advances in manufacturing technology coupled with the increased potency of new biotherapeutic modalities have created an external environment where continuous manufacturing (CM) can address a growing need. Amgen has successfully implemented a hybrid CM process for a commercial lifecycle program. In this process, the bioreactor, harvest, capture column, and viral inactivation/depth filtration unit operations were integrated together in an automated, continuous module, while the remaining downstream unit operations took place in stand‐alone batch mode. CM operations are particularly suited for so‐called "high mix, low volume" manufacturing plants, where a variety of molecules are manufactured in relatively low volumes. The selected molecule fit this mold and was manufactured in a low‐capital micro‐footprint suite attached to an existing therapeutic production facility. Use of a hybrid process within an already operating facility required less capital and minimized complexity. To enable this hybrid CM process, an established fed‐batch process was converted to a perfusion process with continuous harvest. Development efforts included both process changes and the generation of a novel cell line adapted to long‐term perfusion. Chromatography resins were updated, and purification processes adapted to handle variable inputs due to the fluctuations in harvest titer from the lengthy production process. A novel automated single‐use (SU) viral inactivation (VI) skid was introduced, which entailed the development of a robust pH verification and alarm system, along with procedures for product isolation to allow discard of specific cycles. The CM process demonstrated consistent performance, meaning it met predefined performance criteria (including product quality attributes, or PQAs) when operated within established process parameters and manufactured according to applicable procedures. Using a 75% reduction in scale, it resulted in a five‐fold reduction in process media and buffer usage, a fifteen‐fold increase in mass per thaw, and an overall process productivity increase of 45‐fold (as measured by grams drug substance per liter per day.) The hybrid CM process also enabled increased material demand to be met with no change in cost of goods manufactured or plant capacity, due to the repurposing of existing facility space and the flexible duration of the hybrid CM harvest. Overall, the success of the hybrid CM platform represents an exciting opportunity to reduce costs and increase process efficiency in industry. [ABSTRACT FROM AUTHOR]

Recent studies offer valuable insights into viral inactivation for vaccine development. Schulze et al. have demonstrated the potential of heavy ion beam irradiation to create effective vaccines, which is particularly relevant in the context of airborne pandemics. Notably, the success in immunizing mice via intranasal administration with the inactivated influenza virus is encouraging, especially given the genetic similarities between influenza and SARS-CoV-2. However, the study raises important considerations. While heavy ion treatment shows advantages, there are concerns about viral inactivation completeness and the potential for surviving viruses, albeit at extremely low levels. Prolonged irradiation times and the risk of selective pressure leading to the evolution of resistant variants are highlighted. Biosafety concerns regarding accidental lab escape of resistant strains are crucial, emphasizing the need for caution during experiments. Moreover, limitations in Monte Carlo simulations of virus irradiation are discussed, pointing out the need for more comprehensive studies to assess the impact of secondary particles on virus inactivation under realistic irradiation conditions. Given these considerations, while the study presents a promising approach for vaccine development, further research is essential to address potential drawbacks and optimize the method for safe and effective application.

Recent studies offer valuable insights into viral inactivation for vaccine development. Schulze et al. have demonstrated the potential of heavy ion beam irradiation to create effective vaccines, which is particularly relevant in the context of airborne pandemics. Notably, the success in immunizing mice via intranasal administration with the inactivated influenza virus is encouraging, especially given the genetic similarities between influenza and SARS-CoV-2. However, the study raises important considerations. While heavy ion treatment shows advantages, there are concerns about viral inactivation completeness and the potential for surviving viruses, albeit at extremely low levels. Prolonged irradiation times and the risk of selective pressure leading to the evolution of resistant variants are highlighted. Biosafety concerns regarding accidental lab escape of resistant strains are crucial, emphasizing the need for caution during experiments. Moreover, limitations in Monte Carlo simulations of virus irradiation are discussed, pointing out the need for more comprehensive studies to assess the impact of secondary particles on virus inactivation under realistic irradiation conditions. Given these considerations, while the study presents a promising approach for vaccine development, further research is essential to address potential drawbacks and optimize the method for safe and effective application. [ABSTRACT FROM AUTHOR]

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. • S/D treatment allows efficient inactivation of enveloped viruses during the manufacturing process of pH sensitive multi-specific antibodies. • A novel placement of S/D incubation on the Protein A column reduces the amount of S/D required and simplifies the required liquid handling. • The novel placement ensures efficient virus inactivation, acceptable product quality and removal of S/D components without additional purification steps. [ABSTRACT FROM AUTHOR]

Quantifying viruses in wastewater via RT-qPCR provides total genomic data but does not indicate the virus capsid integrity or the potential risk for human infection. Assessing virus capsid integrity in sewage is important for wastewater-based surveillance, since discharged effluent may pose a public health hazard. While integrity assays using cell cultures can provide this information, they require specialised laboratories and expertise. One solution to overcome this limitation is the use of photo-reactive monoazide dyes (e.g., propidium monoazide [PMAxx]) in a capsid integrity-RT-qPCR assay (ci-RT-qPCR). In this study, we tested the efficiency of PMAxx dye at 50 μM and 100 μM concentrations on live and heat-inactivated model viruses commonly detected in wastewater, including adenovirus (AdV), hepatitis A (HAV), influenza A virus (IAV), and norovirus GI (NoV GI). The 100 μM PMAxx dye concentration effectively differentiated live from heat-inactivated viruses for all targets in buffer solution. This method was then applied to wastewater samples (n = 19) for the detection of encapsulated AdV, enterovirus (EV), HAV, IAV, influenza B virus (IBV), NoV GI, NoV GII, and SARS-CoV-2. Samples were negative for AdV, HAV, IAV, and IBV but positive for EV, NoV GI, NoV GII, and SARS-CoV-2. In the PMAxx-treated samples, EV, NoV GI, and NoV GII showed −0.52–1.15, 0.9–1.51, and 0.31–1.69 log reductions in capsid integrity, indicating a high degree of potentially infectious virus in wastewater. In contrast, SARS-CoV-2 was only detected using RT-qPCR but not after PMAxx treatment, indicating the absence of encapsulated and potentially infectious virus. In conclusion, this study demonstrates the utility of PMAxx dyes to evaluate capsid integrity across a diverse range of viruses commonly monitored in wastewater. [ABSTRACT FROM AUTHOR]

Introduction: Safe handling of biological samples sourced from wild ecosystems is a pressing concern for scientists in disparate fields, including ecology and evolution, OneHealth initiatives, bioresources, geography, veterinary medicine, conservation, and many others. This is especially relevant given the growing global research community and collaborative networks that often span international borders. Treatments to inactivate potential pathogens of concern during transportation and analysis of biospecimens while preserving molecular structures of interest are necessary. Objective: We provide a detailed resource on the effectiveness and limitations of TRIzol™ Reagent, a product commonly used in molecular biology to inactivate bacterial and viral pathogens found in wild animals. Methods: By literature review, we evaluate the mode of action of TRIzol Reagent and its main components on bacterial and viral structures. We also synthesize peer-reviewed literature on the effectiveness of TRIzol in inactivating a broad range of infectious bacteria and viruses. Key Findings: TRIzol Reagent inactivation is based on phenol, chaotropic salts, and sodium acetate. We find evidence of widespread efficacy in deactivating bacteria and a broad range of enveloped viruses. The efficacy against a subset of potential pathogens, including some nonenveloped viruses, remains uncertain. Conclusion: Available evidence suggests that TRIzol Reagent is effective in inactivating a broad spectrum of bacteria and viruses from cells, tissues, and liquids in biological samples when the matrices are exposed to at least 10 min at room temperature to the reagent. We highlight areas that require additional research and discuss implications for laboratory protocols. [ABSTRACT FROM AUTHOR]

Background and Objectives: Viral clearance studies are an essential part of a manufacturer's plan to ensure the safety of an injectable biologic product. In this way, viral safety is a critical quality attribute for biologics such as monoclonal antibodies (Mabs). Evaluation of virus purification by downstream processes is a key component of risk mitigation. In this study, the capability of continuous monoclonal antibody purification steps was evaluated in the process of instant monoclonal antibody purification in different stages of purification, and the amount of reduction or inactivation of each step was determined. Materials and Methods: Four enveloped and non-enveloped viral models VSV, Reovirus, EMCV, and HSV1 were used for spiking in selected samples in the designated tests, to have a comprehensive examination of the ability to clear the virus such as the type of genetic material, chemical resistance, and particle size. A TCID50 and qPCR methods were used to measure viral reduction. Two cell lines, Vero (African green monkey kidney) and L929 (Mouse fibroblast) were used for 4 model viruses propagation. The steps that were evaluated included 4 steps monoclonal antibody purification; cation exchange chromatography, acidic pH treatment, affinity chromatography, and nanofiltration. Results: The nano-filter stage showed the highest viral reduction and cation exchange chromatography showed the lowest reduction. The cumulative decrease using TCID50 is equal to 19.27 [log10] for all steps and for the qPCR method is equal to 12.47 [log10] in three steps of nano-filter, affinity chromatography, and ion exchange chromatography. Conclusion: The overall average reduction coefficient for all four model viruses is significantly high, which indicates the high capacity of the monoclonal antibody production process in inactivating and removing viruses leads to reducing the load of all four model viruses. [ABSTRACT FROM AUTHOR]

Background and Objectives: Viral clearance studies are an essential part of a manufacturer's plan to ensure the safety of an injectable biologic product. In this way, viral safety is a critical quality attribute for biologics such as monoclonal antibodies (Mabs). Evaluation of virus purification by downstream processes is a key component of risk mitigation. In this study, the capability of continuous monoclonal antibody purification steps was evaluated in the process of instant monoclonal antibody purification in different stages of purification, and the amount of reduction or inactivation of each step was determined. Materials and Methods: Four enveloped and non-enveloped viral models VSV, Reovirus, EMCV, and HSV1 were used for spiking in selected samples in the designated tests, to have a comprehensive examination of the ability to clear the virus such as the type of genetic material, chemical resistance, and particle size. A TCID50 and qPCR methods were used to measure viral reduction. Two cell lines, Vero (African green monkey kidney) and L929 (Mouse fibroblast) were used for 4 model viruses propagation. The steps that were evaluated included 4 steps monoclonal antibody purification; cation exchange chromatography, acidic pH treatment, affinity chromatography, and nanofiltration. Results: The nano-filter stage showed the highest viral reduction and cation exchange chromatography showed the lowest reduction. The cumulative decrease using TCID50 is equal to 19.27 [log10] for all steps and for the qPCR method is equal to 12.47 [log10] in three steps of nano-filter, affinity chromatography, and ion exchange chromatography. Conclusion: The overall average reduction coefficient for all four model viruses is significantly high, which indicates the high capacity of the monoclonal antibody production process in inactivating and removing viruses leads to reducing the load of all four model viruses.

BACKGROUND: Process integration of decellularization, antigen removal, viral inactivation and sterilization is essential for producing regenerative biomaterials that meet clinical requirements. OBJECTIVE: To systematically compare the effects of two combined processes of decellularization, antigen removal, virus inactivation and sterilization on the physicochemical properties of porcine dermal matrix. METHODS: Fresh porcine dermis was obtained and processed by two processes into acellular matrices. (1) Method A: 1% Triton X-100, DNase and RNase, and 1% tributyl phosphate were applied for decellularization, 1% peracetic acid and 25% ethanol for viral inactivation, and gamma-irradiation for terminal sterilization. (2) Method B: Dispase II, 0.5% Triton X-100 and DNase were used for decellularization, α-galactosidase for the antigen removal, 0.1% peracetic acid for viral inactivation and gamma-irradiation terminal sterilization. Porcine dermal matrices prepared by two methods were characterized. RESULTS AND CONCLUSION: (1) The collagen content of group A was lower than that of group B. The contents of elastin and sugar of group A were higher than those of group B. The material hardness value of group A was higher than that of group B. The drapability of the matrix material in group A was poor and the flexibility of the matrix material in group B was better. (2) The decellularization of group A did not affect the thermal stability of the matrix material. In group B, decellularization slightly reduced the thermal stability of the matrix material. After gamma ray sterilization, the thermal stability of the matrix material in group A decreased greatly and the thermal stability of the matrix material in group B decreased very little. (3) Compared with group B, the tensile strength and elasticity of the matrix material in group A were smaller. In vitro enzymatic degradation experiments showed that the matrix materials in group A had strong resistance to collagenase degradation, were sensitive to trypsin and were easily degraded; the matrix materials in group B were the opposite. (4) Scanning electron microscope showed that after sterilization by gamma rays, the three-dimensional structure of collagen could not be seen in the matrix material of group A; the collagen fibers were densely arranged, and there was a non-fibrous structural gel-like substance between the collagen fibers. In group B, the structure of collagen fibers in the matrix material was clear, and there was no non-fibrous structural glue-like substance between the collagen fibers. Hematoxylin-eosin staining and trichrome staining showed that after sterilization by gamma rays, a few nuclei were visible in the matrix material of group A; the collagen fibers were closely arranged, and the material was dense. The matrix material in group B had no nucleus and retained a good three-dimensional spatial structure of collagen fibers. (5) The results suggest that different processes have great influences on the physical and chemical properties of derived acellular matrices. Method B is effective in decellularizing and de-antigening and retains intact natural tissue matrix. Method A is more destructive to the material and results in lower collagen content, scratchability, thermal stability and poorer biomechanical properties compared with the Method B. [ABSTRACT FROM AUTHOR]

The pursuit of disinfecting porous materials or fomites to inactivate viral agents has special challenges. To address these challenges, a highly portable chlorine dioxide (ClO2) gas generation system was used to ascertain the ability of a gaseous preparation to inactivate a viral agent, the MS2 bacteriophage, when associated with potentially porous fomites of cloth, paper towel, and wood. The MS2 bacteriophage is increasingly used as a model to identify means of inactivating infectious viral agents of significance to humans. Studies showed that MS2 bacteriophage can be applied to and subsequently recovered from potential porous fomites such as cloth, paper towel, and wood. Paired with viral plaque assays, this provided a means for assessing the ability of gaseous ClO2 to inactivate bacteriophage associated with the porous materials. Notable results include 100% inactivation of 6 log bacteriophage after overnight exposure to 20 parts per million(ppm) ClO2. Reducing exposure time to 90 minutes and gas ppm to lower concentrations proved to remain effective in bacteriophage elimination in association with porous materials. Stepwise reduction in gas concentration from 76 ppm to 5 ppm consistently resulted in greater than 99.99% to 100% reduction of recoverable bacteriophage. This model suggests the potential of ClO2 gas deployment systems for use in the inactivation of viral agents associated with porous potential fomites. The ClO2 gas could prove especially helpful in disinfecting enclosed areas containing viral contaminated surfaces, rather than manually spraying and wiping them. [ABSTRACT FROM AUTHOR]

Purpose: We examined the inactivation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by a nitrogen-doped titanium dioxide (N-TiO2) visible-light photocatalyst that was activated via light irradiation in the natural environment and was safe for human use as a coating material. Methods: The photocatalytic activity of glass slides coated with three types of N-TiO2 without metal or loaded with copper or silver and copper was investigated by measuring acetaldehyde degradation. The titer levels of infectious SARS-CoV-2 were measured using cell culture after exposing photocatalytically active coated glass slides to visible light for up to 60 min. Results: N-TiO2 photoirradiation inactivated the SARS-CoV-2 Wuhan strain and this effect was enhanced by copper loading and further by the addition of silver. Hence, visible-light irradiation using silver and copper-loaded N-TiO2 inactivated the Delta, Omicron, and Wuhan strains. Conclusion: N-TiO2 could be used to inactivate SARS-CoV-2 variants, including emerging variants, in the environment. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 is a highly infectious virus and etiologic agent of COVID-19, which is spread by respiratory droplets, aerosols, and contaminated surfaces. Copper is a known antiviral agent, and has resulted in successful reduction of pathogens and infections by 83–99.9% when coated on surfaces in intensive care units. Additionally, copper has been shown to inactivate pathogens such as Coronavirus 226E, a close relative of SARS-CoV-2. Here, we examine the ability of two copper blends with differing compositions to inactivate SARS-CoV-2 virus at different time points. Copper Blend 2 (75.07% pure copper) was found to significantly reduce (over 50%) the viability of SARS-CoV-2 at 5 min of contact, with at least 98% reduction in recovered virus at 20 min (vs. plastic control). However, Copper Blend 1 (48.26% pure copper), was not found to significantly reduce viability of SARS-CoV-2 at any time point when compared to plastic. This may indicate that there is an important percentage of copper content in materials that is needed to effectively inactivate SARS-CoV-2. Overall, this study shows that over the course of 20 min, coatings made of copper materials can significantly reduce the recovery of infectious SARS-CoV-2 compared to uncoated controls, indicating the effective use of copper for viral inactivation on surfaces. Furthermore, it may suggest higher copper content has stronger antiviral properties. This could have important implications when short turnaround times are needed for cleaning and disinfecting rooms or equipment, especially in strained healthcare settings which are struggling to keep up with demand. [ABSTRACT FROM AUTHOR]

Viral inactivation (VI) is a process widely used across the pharmaceutical industry to eliminate the cytotoxicity resulting from trace levels of viruses introduced by adventitious agents. This process requires adding Triton X‐100, a non‐ionic detergent solution, to the protein solution and allowing sufficient time for this agent to inactivate the viruses. Differences in process parameters associated with vessel designs, aeration rate, and many other physical attributes can introduce variability in the process, thus making predicting the required blending time to achieve the desired homogeneity of Triton X‐100 more critical and complex. In this study we utilized a CFD model based on the lattice Boltzmann method (LBM) to predict the blend time to homogenize a Triton X‐100 solution added during a typical full‐scale commercial VI process in a vessel equipped with an HE‐3‐impeller for different modalities of the Triton X‐100 addition (batch vs. continuous). Although direct experimental progress of the blending process was not possible because of GMP restrictions, the degree of homogeneity measured at the end of the process confirmed that Triton X‐100 was appropriately dispersed, as required, and as computationally predicted here. The results obtained in this study were used to support actual production at the biomanufacturing site. [ABSTRACT FROM AUTHOR]

Quantifying viruses in wastewater via RT-qPCR provides total genomic data but does not indicate the virus capsid integrity or the potential risk for human infection. Assessing virus capsid integrity in sewage is important for wastewater-based surveillance, since discharged effluent may pose a public health hazard. While integrity assays using cell cultures can provide this information, they require specialised laboratories and expertise. One solution to overcome this limitation is the use of photo-reactive monoazide dyes (e.g., propidium monoazide [PMAxx]) in a capsid integrity-RT-qPCR assay (ci-RT-qPCR). In this study, we tested the efficiency of PMAxx dye at 50 μM and 100 μM concentrations on live and heat-inactivated model viruses commonly detected in wastewater, including adenovirus (AdV), hepatitis A (HAV), influenza A virus (IAV), and norovirus GI (NoV GI). The 100 μM PMAxx dye concentration effectively differentiated live from heat-inactivated viruses for all targets in buffer solution. This method was then applied to wastewater samples (n = 19) for the detection of encapsulated AdV, enterovirus (EV), HAV, IAV, influenza B virus (IBV), NoV GI, NoV GII, and SARS-CoV-2. Samples were negative for AdV, HAV, IAV, and IBV but positive for EV, NoV GI, NoV GII, and SARS-CoV-2. In the PMAxx-treated samples, EV, NoV GI, and NoV GII showed −0.52–1.15, 0.9–1.51, and 0.31–1.69 log reductions in capsid integrity, indicating a high degree of potentially infectious virus in wastewater. In contrast, SARS-CoV-2 was only detected using RT-qPCR but not after PMAxx treatment, indicating the absence of encapsulated and potentially infectious virus. In conclusion, this study demonstrates the utility of PMAxx dyes to evaluate capsid integrity across a diverse range of viruses commonly monitored in wastewater.

The laboratorial available methods applied in plasma disinfection can induce damage in other blood components. Antimicrobial photodynamic therapy (aPDT) represents a promising approach and is approved for plasma and platelet disinfection using non-porphyrinic photosensitizers (PSs), such as methylene blue (MB). In this study, the photodynamic action of three cationic porphyrins (Tri-Py(+)-Me, Tetra-Py(+)-Me and Tetra-S-Py(+)-Me) towards viruses was evaluated under white light irradiation at an irradiance of 25 and 150 mW·cm−2, and the results were compared with the efficacy of the approved MB. None of the PSs caused hemolysis at the isotonic conditions, using a T4-like phage as a model of mammalian viruses. All porphyrins were more effective than MB in the photoinactivation of the T4-like phage in plasma. Moreover, the most efficient PS promoted a moderate inactivation rate of the T4-like phage in whole blood. Nevertheless, these porphyrins, such as MB, can be considered promising and safe PSs to photoinactivate viruses in blood plasma. [ABSTRACT FROM AUTHOR]

A continuous viral inactivation (CVI) tubular reactor was designed for low pH viral inactivation within a continuous downstream system across multiple scales of operation. The reactors were designed to provide a minimum residence time of >60 min. The efficacy of this tubular reactor was tested with xenotropic murine leukemia virus (X‐MuLV) through pulse injection experiments. It was determined that the minimum residence time of the small‐scale reactor design, when operated at the target process flow rate, occurred between 63 and 67 min. Inactivation kinetics were compared between continuous operation and standard batch practices using three monoclonal antibodies. The quantification of the virus log reduction values (LRV) was similar between the two modes of operation and most of the acid‐treated samples had virus concentrations below the limit of detection. However, residual infectivity was still present in the endpoint batch samples of two experiments while the continuous samples always remained below the limit of detection. This provides the foundation for leveraging a standard batch‐based model to quantify the LRV for a CVI unit operation. [ABSTRACT FROM AUTHOR]

The benefits of continuous processing over batch manufacturing are widely acknowledged across the biopharmaceutical industry, primary of which are higher productivity and greater consistency in product quality. Furthermore, the reduced equipment and facility footprint lead to significantly lower capital costs. Technology enablers have a major role in this migration from batch to continuous processing. In this review, we highlight the various enablers that are facilitating adoption of continuous upstream and downstream bioprocessing. This includes new bioreactors and cell retention devices for upstream operations, and on-column and continuous flow refolding, novel continuous chromatography, and single-pass filtration systems for downstream processes. We also elucidate the significant roles of process integration and control as well as of data analytics in these processes. Technology enablers have a major role in migration from batch to continuous processing. Perfusion bioreactors are used for continuous cell culture and product formation, with continuous addition of fresh media and removal of spent media and product while simultaneously retaining the cells by various devices, including membranes, filtration, or centrifugation. Protein refolding, chromatography, and single-pass filtration systems are key steps. Microbial-based proteins are expressed in inclusion bodies and refolded into active conformations before further purification by chromatography; they are then formulated using single-pass tangential flow filtration technologies. Mammalian monoclonal antibody biotherapeutics are subjected to chromatography and viral inactivation before being formulated to produce the drug substance. Process integration and control, and data analytics, additionally have significant role. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is evolving, with emergence of mutational variants due to the error-prone replication process of RNA viruses, in general. More recently, the Delta and Omicron variants (including sub-variants BA.1–5) predominate globally, and a Delta–Omicron recombinant termed Deltacron has emerged. The emergence of variants of concern (VOC) demonstrating immune evasion and potentially greater transmissibility and virulence naturally raises concern in both the infection control communities and the public at large, as to the continued suitability of interventions intended to mitigate the risk of viral dissemination and acquisition of the associated disease COVID-19. We evaluated the virucidal efficacy of targeted surface hygiene products (an ethanol/quaternary ammonium compound (QAC)-containing disinfectant spray, a QAC disinfectant wipe, a lactic acid disinfectant wipe, and a citric acid disinfectant wipe) through both theoretical arguments and empirical testing using international standard methodologies (ASTM E1053-20 hard surface test and EN14476:2013+A2:2019 suspension test) in the presence of soil loads simulating patients' bodily secretions/excretions containing shed virus. The results demonstrate, as expected, complete infectious viral inactivation (≥3.0 to ≥4.7 log10 reduction in infectious virus titer after as little as 15 s contact time at room temperature) by these surface hygiene agents of the original SARS-CoV-2 isolate and its Beta and Delta VOC. Through appropriate practices of targeted surface hygiene, it is expected that irrespective of the SARS-CoV-2 VOC encountered as the current pandemic unfolds (and, for that matter, any emerging and/or re-emerging enveloped virus), the chain of infection from virus-contaminated fomites to the hand and mucous membranes of a susceptible person may be disrupted. [ABSTRACT FROM AUTHOR]

Aims: Enteric viruses are recognized as a major concern in health care and in the food sector in Canada. Novel clean-label strategies for controlling enteric viruses are sought in the food industry. In this study, we examined the antiviral potential of plant extracts and essential oils on murine norovirus 1 (MNV-1), hepatitis A virus (HAV) and herpes simplex 1 (HSV-1)., Methods and Results: Inactivation of the viruses by grape seed, blueberry, green tea, and cranberry extracts and by rosemary and thyme essential oils was measured using plaque formation assay. Concentrations ranging from 50 to 200,000 ppm with a contact time of 90 min were tested. Grape seed extract at 10,000 ppm was the most effective (p < 0.05) at reducing MNV-1 and HAV infectious titers, respectively by 2.85 ± 0.44 log10 and 1.94 ± 0.17 log10. HSV-1 titer was reduced by 3.81 ± 0.40 log10 at 1000 ppm grape seed extract., Conclusions: Among the plant products tested, grape seed extract was found the most effective at reducing the infectious titers of MNV-1, HAV and HSV., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Inactivation of SARS-CoV-2 virus is necessary to mitigate risk but may interfere with diagnostic assay performance. We examined the effect of heat inactivation on a prototype SARS-CoV-2 antigen immunoassay run on the ARCHITECT automated analyzer. Recombinant full-length SARS-CoV-2 nucleocapsid protein and virus lysate detection was reduced by 66 and 31%, respectively. Several nonionic detergents were assessed as inactivation alternatives based on infectivity in cultured Vero CCL81 cells. Incubation of SARS-CoV-2 in 0.1% Tergitol 15-S-9 for 10 min significantly reduced infectivity and increased the immunoassay signal for cultured lysate and patient specimens. Tergitol 15-S-9 can inactivate SARS-CoV-2 while preserving epitopes on the nucleocapsid protein for enhanced detection by immunoassay antibodies.

Water contamination is a global health problem, and the need for safe water is ever-growing due to the public health implications of unsafe water. Contaminated water could contain pathogenic bacteria, protozoa, and viruses that are implicated in several debilitating human diseases. The prevalence and survival of waterborne viruses differ from bacteria and other waterborne microorganisms. In addition, viruses are responsible for more severe waterborne diseases such as gastroenteritis, myocarditis, and encephalitis among others, hence the need for dedicated attention to viral inactivation. Disinfection is vital to water treatment because it removes pathogens, including viruses. The commonly used methods and techniques of disinfection for viral inactivation in water comprise physical disinfection such as membrane filtration, ultraviolet (UV) irradiation, and conventional chemical processes such as chlorine, monochloramine, chlorine dioxide, and ozone among others. However, the production of disinfection by-products (DBPs) that accompanies chemical methods of disinfection is an issue of great concern due to the increase in the risks of harm to humans, for example, the development of cancer of the bladder and adverse reproductive outcomes. Therefore, this review examines the conventional disinfection approaches alongside emerging disinfection technologies, such as photocatalytic disinfection, cavitation, and electrochemical disinfection. Moreover, the merits, limitations, and log reduction values (LRVs) of the different disinfection methods discussed were compared concerning virus removal efficiency. Future research needs to merge single disinfection techniques into one to achieve improved viral disinfection, and the development of medicinal plant-based materials as disinfectants due to their antimicrobial and safety benefits to avoid toxicity is also highlighted.

Adeno-associated virus (AAV) is an essential instrument in the neuroscientist’s toolkit, which allows delivery of DNA to provide labeling with fluorescent proteins or genetic instructions to regulate gene expression. In the field of neural regeneration, the transduction of neurons enables the observation and regulation of axon growth and regeneration, and in the future will likely be a mechanism for delivering molecular therapies to promote sprouting and regeneration after central nervous system injury. Traditional formulations of AAV preparations permit efficient viral transduction under physiologic conditions, but an improved understanding of the mechanistic limitations of AAV transduction may facilitate production of more resilient AAV strains for investigative and therapeutic purposes. We studied AAV transduction in the context of prior exposure of AAV serotype 8 (AAV8) to environmental pH within the range encountered during endosomal endocytosis (pH 7.4 to pH 4.4), during which low pH-triggered structural and autoproteolytic changes to the viral capsid are believed to be necessary for endosome escape and virus uncoating. Due to the fundamental nature of these processes, we hypothesized that premature exposure of AAV8 particles to acidic pH would decrease viral transduction of HT1080 cells in vitro, as measured by fluorescent reporter gene expression using high-content imaging analysis. We found that increasingly acidic incubation conditions were associated with concomitant reductions in transduction efficiency, and that quantitative levels of reporter gene expression in transduced cells were similarly decreased. The biggest decrease in transduction occurred between pH 7.4 and pH 6.4, suggesting the possible co-occurrence of a pH-associated event and viral inactivation within that range. Taken together, these findings indicate that exposure of AAV8 to acidic pH for as little as 1 hour is deleterious to transduction ability. Future studies are necessary to understand the pH-associated causative mechanisms involved. This study was approved by the University of Miami Institutional Animal Care and Use Committee, USA (Protocol #18-108-LF) on July 12, 2018.

The 2022 global spread of Monkeypox Virus (MPXV) underlined the need to investigate safe-handling procedures of clinical and research samples. Here we evaluated the efficiency in reducing MPXV infectious titer of Triton X-100 (0.1 and 0.2%), UV-C irradiation (15 or 30 min), and heat (56 °C 30 min or 70 °C 5 min). The treatment of MPXV at 70 °C resulted in the strongest decrease of MPXV infectious titer (5.4 Log TCID50/mL), 56 °C and UV-C had a lighter impact (3.9 and 4.3Log), Triton X-100 was less efficient (1.8–2.5Log). Notably, SARS-CoV-2 was much more susceptible to Triton X-100 (4.0 Log decrease). UV-C had the highest impact on MPXV DNA detection by PCR (2.2–4.3 Ct value increase); protein detection by ELISA was dramatically impaired by heating. Overall, UV-C and heating were more effective in lowering MPXV infectious titer but their impact on nucleic acids or protein detection assays must be considered. • MPXV titer is highly reduced at 70 °C, less by UV-C or 56 °C, poorly by Triton X-100. • Treatment with Triton X-100 is more effective in inactivating SARS-CoV-2 than MPXV. • MPXV DNA detection is partially impaired by UV-C exposition. • Protein detection is dramatically affected by heat inactivation protocols. [ABSTRACT FROM AUTHOR]

Different light-based strategies have been investigated to inactivate viruses. Herein, we developed an HIV-based pseudotyped model of SARS-CoV-2 (SC2) to study the mechanisms of virus inactivation by using two different strategies; photoinactivation (PI) by UV-C light and photodynamic inactivation (PDI) by Photodithazine photosensitizer (PDZ). We used two pseudoviral particles harboring the Luciferase-IRES-ZsGreen reporter gene with either a SC2 spike on the membrane or without a spike as a naked control pseudovirus. The mechanism of viral inactivation by UV-C and PDZ-based PDI were studied via biochemical characterizations and quantitative PCR on four levels; free-cell viral damage; viral cell entry; DNA integration; and expression of reporter genes. Both UV-C and PDZ treatments could destroy single stranded RNA (ssRNA) and the spike protein of the virus, with different ratios. However, the virus was still capable of binding and entering into the HEK 293T cells expressing angiotensin-converting enzyme 2 (ACE-2). A dose-dependent manner of UV-C irradiation mostly damages the ssRNA, while PDZ-based PDI mostly destroys the spike and viral membrane in concentration and dose-dependent manners. We observed that the cells infected by the virus and treated with either UV-C or PDZ-based PDI could not express the luciferase reporter gene, signifying the viral inactivation, despite the presence of RNA and DNA intact genes. [ABSTRACT FROM AUTHOR]

Photocatalysis has attracted great attention because of its direct utilisation of sunlight to obtain various chemical reactions, causing water splitting, organic pollutant degradation, and water disinfection. Nanophotocatalysts can be employed for various applications, including hydrogen storage, green diesel production, fuel cell applications, industrial manufacturing methods, pharmaceutical industries, and catalytic degradation of contaminants/hazardous materials. Photocatalytic inactivation and removal of pathogenic viruses, antibiotic-resistant bacteria and antibiotic resistance genes can be considered as simple and effective technique with low-cost, eco-friendliness, and low energy consumption features. The high specific surface areas, abundant functional groups, large amounts of active sites are some advantages of the nanostructures for photocatalytic activity with high efficiency. However, some important limitations/drawbacks of nanophotocatalysts for industrial and commercial applications such as the low selectivity, aggregation/sedimentation, difficult separation, low-usage of visible light, fast charge recombination, and low migration potential of photogenerated electrons/holes are need to be comprehensively and analytically investigated and addressed by researchers. This critical review highlighted the recent advancements in photocatalytic disinfection of pathogenic viruses and antibiotic-resistant bacteria, focussing on the development of highly efficient nanophotocatalysts and their underlying mechanisms of inactivation/removal of these pathogens. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 (HSV-1) is a ubiquitous and widespread human pathogen, which gives rise to a range of diseases, including cold sores, corneal blindness, and encephalitis. Currently, the use of nucleoside analogs, such as acyclovir and penciclovir, in treating HSV-1 infection often presents limitation due to their side effects and low efficacy for drug-resistance strains. Therefore, new anti-herpetic drugs and strategies should be urgently developed. Here, we reported that baicalein, a naturally derived compound widely used in Asian countries, strongly inhibited HSV-1 replication in several models. Baicalein was effective against the replication of both HSV-1/F and HSV-1/Blue (an acyclovir-resistant strain) in vitro. In the ocular inoculation mice model, baicalein markedly reduced in vivo HSV-1/F replication, receded inflammatory storm and attenuated histological changes in the cornea. Consistently, baicalein was found to reduce the mortality of mice, viral loads both in nose and trigeminal ganglia in HSV-1 intranasal infection model. Moreover, an ex vivo HSV-1-EGFP infection model established in isolated murine epidermal sheets confirmed that baicalein suppressed HSV-1 replication. Further investigations unraveled that dual mechanisms, inactivating viral particles and inhibiting IκB kinase beta (IKK-β) phosphorylation, were involved in the anti-HSV-1 effect of baicalein. Collectively, our findings identified baicalein as a promising therapy candidate against the infection of HSV-1, especially acyclovir-resistant strain.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has raised the need of versatile means for virus decontamination. Millimeter waves are used in biochemical research in dynamic nuclear polarization enhanced nuclear magnetic resonance (DNP/NMR) spectroscopy. However, their efficiency in object decontamination for viruses has not been tested yet. Here we report the high efficiency of 95 GHz waves in killing both coronavirus 229E and poliovirus. An exposure of 2 s to 95 GHz waves reduced the titer of these viruses by 99.98% and 99.375%, respectively, and formed holes in the envelope of 229E virions as detected by scanning electron microscopy (SEM) analysis. The ability of 95 GHz waves to reduce the coronavirus titer to a range of limited infective dose of SARS-CoV-2 for humans and animal models along with precise focusing capabilities for these waves suggest 95 GHz waves as an effective way to decontaminate objects. [ABSTRACT FROM AUTHOR]

ABSTRACT The global emergence of novel pathogenic viruses presents an important challenge for research, as high biosafety levels are required to process samples. While inactivation of infectious agents facilitates the use of less stringent safety conditions, its effect on other biological entities of interest present in the sample is generally unknown. Here, we analyzed the effect of five inactivation methods (heat, ethanol, formaldehyde, psoralen, and TRIzol) on microbiome composition and diversity in samples collected from four different body sites (gut, nasal, oral, and skin) and compared them against untreated samples from the same tissues. We performed 16S rRNA gene sequencing and estimated abundance and diversity of bacterial taxa present in all samples. Nasal and skin samples were the most affected by inactivation, with ethanol and TRIzol inducing the largest changes in composition, and heat, formaldehyde, TRIzol, and psoralen inducing the largest changes in diversity. Oral and stool microbiomes were more robust to inactivation, with no significant changes in diversity and only moderate changes in composition. Firmicutes was the taxonomic group least affected by inactivation, while Bacteroidetes had a notable enrichment in nasal samples and moderate enrichment in fecal and oral samples. Actinobacteria were more notably depleted in fecal and skin samples, and Proteobacteria exhibited a more variable behavior depending on sample type and inactivation method. Overall, our results demonstrate that inactivation methods can alter the microbiome in a tissue-specific manner and that careful consideration should be given to the choice of method based on the sample type under study. IMPORTANCE Understanding how viral infections impact and are modulated by the microbiome is an important problem in basic research but is also of high clinical relevance under the current pandemic. To facilitate the study of interactions between microbial communities and pathogenic viruses under safe conditions, the infectious agent is generally inactivated prior to processing samples. The effect of this inactivation process in the microbiome is, however, unknown. Further, it is unclear whether biases introduced by inactivation methods are dependent on the sample type under study. Estimating the magnitude and nature of the changes induced by different methods in samples collected from various body sites thus provides important information for current and future studies that require inactivation of pathogenic agents.

Rampant pathogenesis induced by communicable microbes has necessitated development of technologies for rapid and sustained disinfection of surfaces. Copper nanoparticles (CuNPs) have been widely reported for their antimicrobial properties. However, nanostructured copper is prone to oxidative dissolution in the oil phase limiting its sustained use on surfaces and coatings. The current study reports a systematic investigation of a simple synthesis protocol using fatty acid stabilizers (particularly essential oils) for synthesis of copper nanoparticles in the oil phase. Of the various formulations synthesized, rosemary oil stabilized copper nanoparticles (RMO CuNPs) were noted to have the best inactivation kinetics and were also most stable. Upon morphological characterization by TEM and EELS, these were found to be monodispersed ( φ 5-8 nm) with copper coexisting in all three oxidation states on the surface of the nanoparticles. The nanoparticles were drop cast on woven fabric of around 500 threads per inch and exposed to gram positive bacteria ( Staphylococcus aureus ), gram negative bacteria ( Escherichia coli and Pseudomonas aeruginosa ), enveloped RNA virus (phi6), non-enveloped RNA virus (MS2) and non-enveloped DNA virus (T4) to encompass the commonly encountered groups of pathogens. It was possible to completely disinfect 10 7 copies of all microorganisms within 40 min of exposure. Further, this formulation was incorporated with polyurethane as thinners and used to coat non-woven fabrics. These also exhibited antimicrobial properties. Sustained disinfection with less than 9% cumulative copper loss for upto 14 washes with soap water was observed while the antioxidant activity was also preserved. Based on the studies conducted, RMO CuNP in oil phase was found to have excellent potential of integration on surface coatings, paints and polymers for rapid and sustained disinfection of microbes on surfaces., (Creative Commons Attribution license.)

As part of a viral mitigating strategy for continuous bioprocessing, that utilizes a plug flow reactor (PFR) for continuous viral inactivation (CVI), understanding the minimum residence time as a function of reactor scale and operational conditions is critical. An empirical‐based model was utilized to calculate the minimum duration a virus particle experiences within a plug flow reactor as a function of reactor design and operational conditions. This empirical model's calculations were challenged by pulse injecting the bacteriophage ΦX‐174 in non‐inactivating conditions and monitoring the discharge of the PFR with infectivity assays. The initial proposed empirical model, with the constraint of requiring an operational Dean number of >100, proved to be effective at calculating first breakthrough of ΦX‐174 but only for the appropriate Dean number conditions. With the knowledge gained from the first empirical model, a second was generated to eliminate the Dean number constraint. This second modified empirical model proved to be successful at calculating the first breakthrough at all Dean number's tested, however CVI operation at the lower Dean's number will lead to an increased asymmetry (i.e., increased tailing) in the residence time distribution. [ABSTRACT FROM AUTHOR]

Batch low‐pH hold is a common processing step to inactivate enveloped viruses for biologics derived from mammalian sources. Increased interest in the transition of biopharmaceutical manufacturing from batch to continuous operation resulted in numerous attempts to adapt batch low‐pH hold to continuous processing. However, control challenges with operating this system have not been directly addressed. This article describes a low‐cost, column‐based continuous viral inactivation system constructed with off‐the‐shelf components. Model‐based, reaction‐invariant pH controller is implemented to account for the nonlinearities with Bayesian estimation addressing variations in the operation. The residence time distribution is modeled as a plug flow reactor with axial dispersion in series with a continuously stirred tank reactor, and is periodically estimated during operation through inverse tracer experiments. The estimated residence time distribution quantifies the minimum residence time, which is used to adjust feed flow rates. Controller validation experiments demonstrate that pH and minimum residence time setpoint tracking and disturbance rejection are achieved with fast and accurate response and no instability. Viral inactivation testing demonstrates tight control of logarithmic reduction values over extended operation. This study provides tools for the design and operation of continuous viral inactivation systems in service of increasing productivity, improving product quality, and enhancing patient safety. [ABSTRACT FROM AUTHOR]

One of the main concerns in human milk banks (HMB) is the transmission of human cytomegalovirus (HCMV) that could be present in the milk of infected women. There are consistent data showing that this virus is destroyed by Holder pasteurization (62.5°C for 30 min), but there is a lack of information about the response of the virus to the treatment at lower temperatures in strict HMB conditions. In order to analyze the effectiveness of different temperatures of pasteurization to eliminate HCMV in human milk, a preliminary assay was performed incubating HCMV-spiked raw milk samples from donor mothers at tested temperatures in a PCR thermocycler and the viral infectivity was assayed on cell cultures. No signs of viral replication were observed after treatments at temperatures equal or >53°C for 30, 20, and 10 min, 58°C for 5 min, 59°C for 2 min, and 60°C for 1 min. These data were confirmed in a pasteurizer-like model introducing HCMV-spiked milk in disposable baby bottles. No viral infectivity was detected on cell cultures after heating treatment of milk for 30 min at temperatures from 56 to 60°C. Thus, our results show that by using conventional pasteurization conditions, temperatures in the range of 56–60°C are enough to inactivate HCMV. Consequently, we consider that, in order to provide a higher quality product, the current recommendation to pasteurize both mother's own milk and donated milk at 62.5°C must be re-evaluated.

The laboratorial available methods applied in plasma disinfection can induce damage in other blood components. Antimicrobial photodynamic therapy (aPDT) represents a promising approach and is approved for plasma and platelet disinfection using non-porphyrinic photosensitizers (PSs), such as methylene blue (MB). In this study, the photodynamic action of three cationic porphyrins (Tri-Py(+)-Me, Tetra-Py(+)-Me and Tetra-S-Py(+)-Me) towards viruses was evaluated under white light irradiation at an irradiance of 25 and 150 mW·cm−2, and the results were compared with the efficacy of the approved MB. None of the PSs caused hemolysis at the isotonic conditions, using a T4-like phage as a model of mammalian viruses. All porphyrins were more effective than MB in the photoinactivation of the T4-like phage in plasma. Moreover, the most efficient PS promoted a moderate inactivation rate of the T4-like phage in whole blood. Nevertheless, these porphyrins, such as MB, can be considered promising and safe PSs to photoinactivate viruses in blood plasma.

In the absence of a vaccine, preventing the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the primary means to reduce the impact of the 2019 coronavirus disease (COVID-19). Multiple studies have reported the presence of SARS-CoV-2 genetic material on surfaces suggesting that fomite transmission of SARS-CoV-2 is feasible. High temperature inactivation of virus has been previously suggested, but not shown. In the present study, we investigated the environmental stability of SARS-CoV-2 in a clinically relevant matrix dried onto stainless steel at a high temperature. The results show that at 54.5 °C, the virus half-life was 10.8 ± 3.0 min and the time for a 90% decrease in infectivity was 35.4 ± 9.0 min. These findings suggest that in instances where the environment can reach temperatures of at least 54.5 °C, such as in vehicle interior cabins when parked in warmer ambient air, that the potential for exposure to infectious virus on surfaces could be decreased substantially in under an hour. [ABSTRACT FROM AUTHOR]

• Triton X-100 did not interfere with electrolytes, enzymes, and proteins in plasma. • Heat exposure negatively impacted assessment of enzymes in plasma. • Triton X-100 negatively impacted leukocyte esterase analysis in urine. 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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

Background and Aim We need the next-generation of whole-inactivated influenza vaccines to create stronger cross-protection against different influenza subtypes. This research aimed to apply the inactivation process of the influenza virus through gamma radiation as a candidate for the development of whole-inactivated vaccines. Methods and Materials The influenza virus strain A/PR/8/34 (A/Puerto Rico/8/34 [H1N1]) was propagated in Madin-Darby Canine Kidney (MDCK) epithelial cells. After ultrafiltration, the virus infectivity titer was calculated by 50% Tissue Culture Infectious Dose (TCID 50%) method based on the Karber formula. Alternatively, the gamma cell-220 was applied for virus inactivation via gamma rays. The D10 value factor and optimum dose of virus inactivation were calculated based on the dose/survival curve and the initial viral titer. In addition, antigenic properties of irradiated viruses compared to un-irradiated viruses and complete inactivation of the irradiated samples with optimum dose were also evaluated by hemagglutination assay and safety test, respectively. Ethical Considerations The Research Ethics Committee of Islamic Azad University, Tehran Medical Branch, Iran approved this study (Code: IR.IAU.TMU.REC.1397.309). Results According to the concentrated virus titer (TCID50: 105.75/ml) and dose/survival curve, the D10 value factor and optimum dose of virus inactivation were calculated at 4.878 and 28.048 kGy, respectively. On the other hand, owing to the results obtained from the safety test and hemagglutination assay, the optimum dose of virus inactivation was determined to be 28 kGy by maintaining the antigenic properties. Conclusion Gamma radiation appears to be a good candidate for vaccine development through maintaining the antigenic structures.

Different light-based strategies have been investigated to inactivate viruses. Herein, we developed an HIV-based pseudotyped model of SARS-CoV-2 (SC2) to study the mechanisms of virus inactivation by using two different strategies; photoinactivation (PI) by UV-C light and photodynamic inactivation (PDI) by Photodithazine photosensitizer (PDZ). We used two pseudoviral particles harboring the Luciferase-IRES-ZsGreen reporter gene with either a SC2 spike on the membrane or without a spike as a naked control pseudovirus. The mechanism of viral inactivation by UV-C and PDZ-based PDI were studied via biochemical characterizations and quantitative PCR on four levels; free-cell viral damage; viral cell entry; DNA integration; and expression of reporter genes. Both UV-C and PDZ treatments could destroy single stranded RNA (ssRNA) and the spike protein of the virus, with different ratios. However, the virus was still capable of binding and entering into the HEK 293T cells expressing angiotensin-converting enzyme 2 (ACE-2). A dose-dependent manner of UV-C irradiation mostly damages the ssRNA, while PDZ-based PDI mostly destroys the spike and viral membrane in concentration and dose-dependent manners. We observed that the cells infected by the virus and treated with either UV-C or PDZ-based PDI could not express the luciferase reporter gene, signifying the viral inactivation, despite the presence of RNA and DNA intact genes.

Copper (Cu) is an effective antimicrobial material; however, its activity is inhibited by oxidation. Titanium dioxide (TiO 2 ) photocatalysis prevents Cu oxidation and improves its antimicrobial activity and stability. In this study, the virucidal efficacy of Cu-doped TiO 2 nanoparticles (Cu-TiO 2 ) with three different oxidation states of the Cu dopant (i.e., zero-valent Cu (Cu 0 ), cuprous (Cu I ), and cupric (Cu II ) oxides) was evaluated for the phiX174 bacteriophage under visible light illumination (Vis/Cu-TiO 2 ). Cu I -TiO 2 exhibited superior virucidal activity (5 log inactivation in 30 min) and reusability (only 11 % loss of activity in the fifth cycle) compared to Cu 0 -TiO 2 and Cu II -TiO 2 . Photoluminescence spectroscopy and photocurrent measurements showed that Cu I -TiO 2 exhibited the highest charge separation efficiency and photocurrent density (approximately 0.24 μA/cm 2 ) among the three materials, resulting in the most active redox reactions of Cu. Viral inactivation tests under different additives and viral particle integrity analyses (i.e., protein oxidation and DNA damage analyses) revealed that different virucidal species played key roles in the three Vis/Cu-TiO 2 systems; Cu(III) was responsible for the viral inactivation by Vis/Cu I -TiO 2 . The Vis/Cu I -TiO 2 system exhibited substantial virucidal performance for different viral species and in different water matrices, demonstrating its potential practical applications. The findings of this study offer valuable insights into the design of effective and sustainable antiviral photocatalysts for disinfection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Herpes simplex virus type 1 (HSV-1) is a ubiquitous and widespread human pathogen, which gives rise to a range of diseases, including cold sores, corneal blindness, and encephalitis. Currently, the use of nucleoside analogs, such as acyclovir and penciclovir, in treating HSV-1 infection often presents limitation due to their side effects and low efficacy for drug-resistance strains. Therefore, new anti-herpetic drugs and strategies should be urgently developed. Here, we reported that baicalein, a naturally derived compound widely used in Asian countries, strongly inhibited HSV-1 replication in several models. Baicalein was effective against the replication of both HSV-1/F and HSV-1/Blue (an acyclovir-resistant strain) in vitro. In the ocular inoculation mice model, baicalein markedly reduced in vivo HSV-1/F replication, receded inflammatory storm and attenuated histological changes in the cornea. Consistently, baicalein was found to reduce the mortality of mice, viral loads both in nose and trigeminal ganglia in HSV-1 intranasal infection model. Moreover, an ex vivo HSV-1-EGFP infection model established in isolated murine epidermal sheets confirmed that baicalein suppressed HSV-1 replication. Further investigations unraveled that dual mechanisms, inactivating viral particles and inhibiting I κ B kinase beta (IKK- β) phosphorylation, were involved in the anti-HSV-1 effect of baicalein. Collectively, our findings identified baicalein as a promising therapy candidate against the infection of HSV-1, especially acyclovir-resistant strain. Image 1 Baicalein exerts potent ability against HSV-1 infection and dual mechanisms were disclosed. • Baicalein is highly effective against HSV-1infection ex vivo and in vivo. • Inactivation of viral particles and suppression of NF- κ B activation were involved in the anti-viral effect of baicalein. • Hence, our work offers experimental basis for baicalein as a potential drug in treating HSV-1 associated diseases. [ABSTRACT FROM AUTHOR]

Outside the host, viruses will eventually lose their ability to infect cells due to conformational changes that occur to proteins on the viral capsid. In order to undergo a conformational change, these proteins require energy to activate the chemical reaction that leads to the conformational change. In this study, data from the literature is used to calculate the energy required for viral inactivation for a variety of different viruses by means of the Arrhenius equation. We find that some viruses (rhinovirus, poliovirus, human immunodeficiency virus, Alkhumra hemorrhagic fever virus, and hepatitis A virus) have high inactivation energies, indicative of breaking of a chemical double bond. We also find that several viruses (respiratory syncytial virus, poliovirus, and norovirus) have nonlinear Arrhenius plots, suggesting that there is more than a single pathway for inactivation of these viruses. [ABSTRACT FROM AUTHOR]

Aims: Develop an effective laboratory method to consistently recover viral loads from porous concrete coupons sufficient for disinfectant efficacy testing. Investigate the role of concrete matrix pH on the recovery of foot-and-mouth disease virus (FMDV) and African Swine Fever virus (ASFV) from porous concrete. Compare parameters off FMDV and ASFV inactivation on porous and nonporous surfaces in quantitative carrier tests of a liquid chemical disinfectant. Methods and results: Concrete test coupons were fabricated from commercial and industrial sources and carbonated by exposure to 5% CO2 in a humidified incubator, lowering the matrix pH. Neither dried FMDV nor ASFV were recovered from high-pH concrete control coupons. Recovery of infectious virus from lower pH carbonated concrete was similar to stainless steel coupon controls. Exposure to the liquid disinfectant VirkonTM S inactivated FMDV and ASFV on porous concrete. Conclusions: Concrete matrix pH had a greater impact than surface porosity on the ability to recover viable virus from unsealed concrete. Significance and Impact of the Study: Concrete is commonly found in environments where virus decontamination is required. This study demonstrates a reproducible method to recover sufficient viral loads from porous concrete coupons to facilitate quantitative carrier testing. This method provides a basis for evidence-based validation testing of chemical disinfectants to inactivate pH-sensitive viruses on unsealed concrete. [ABSTRACT FROM AUTHOR]

The current global debacle of COVID-19, spelled by SARS-CoV-2 needs no elaboration. With incessant and constantly clambering number of deaths across various nations, the need of the hour is to develop readily deployable, fast, affordable detection assays and kits, yielding precise and consistent results as well as timely availability of efficacious anti-SARS-CoV-2 strategies to contain it. Conventionally employed real time PCR based technique for detection of the virus suffers from a couple of handicaps. Amongst other approaches, CRISPR based technology has ushered in new hopes. Recent efforts have been directed toward developing CRISPR/Cas based low-cost, rapid detection methods as well as development of one-pot assay platforms. The plausible application of CRISPR-Cas system to counteract the viral assault has also been assessed. The write up in this article mirrors the current status, the prospects and the practical snags of CRISPR/Cas technology for the detection and inactivation of the novel corona virus, SARS-CoV-2.

The purpose of the study was to assess the effectiveness of the removal of nucleic acids (NA), Hepatitis B (HBV) and C (HCV) viruses and parvovirus B19 (B19V) from blood plasma fractions obtained by alcohol separation. The studies were performed in a model experiment by fractionation normal pool plasma donation contaminated with virus-containing samples in the laboratory. It was found that the multi-step process to extract a fraction II, designed to provide the immunoglobulin G, provided the elimination of NA HBV and HCV to undetectable levels by PCR, and the total reduction factor compared to the original composition of the plasma HBV DNA >5.35 log, HCV RNA >4.84 log. Factor DNA V19V reduction was at the level of 4.56 log(CI 4.49-4.63). Upon receipt of the fraction V (for albumin) reduction factor of the test virus was >3.0 log wich in combination with additional purification step provides a reliable level of safety. The selection fraction I resulted in effectiveness of the elimination of HBV DNA and V19V DNA less than 2 orders, HCV RNA - less than 3 orders of magnitude, it is not enough to recognize the stage reliable. Experiments confirmed that the high risk of contamination of production pools parvovirus B19 additional safety measures are necessary, including mandatory investigation of plasma DNA V19V. In general the development of a full cycle of drug from blood plasma from donors, basic process alcohol fractionation steps necessary to supplement validation virus elimination and inactivation considering residual risk of contamination with pathogenic agents feedstock.

Due to rising consumer preference for natural remedies, the search for natural antiviral agents has accelerated considerably in recent years. Among the natural sources of compounds with potential antiviral proprieties, berries are interesting candidates, due to their association with health-promoting properties, including antioxidant, antimutagenic, anticancer, antimicrobial, anti-inflammatory, and neuroprotective properties. The past two decades have witnessed a flurry of new findings. Studies suggest promising antiviral proprieties against enveloped and non-enveloped viruses, particularly of cranberries, blueberries, blackcurrants, black raspberries, and pomegranates. The aim of this review is to assemble these findings, to list the implied mechanisms of action, and thereby point out promising subjects for research in this field, in the hope that compounds obtainable from natural sources such as berries may be used someday to treat, or even prevent, viral infections.

Blue LED light has proven to have a powerful bacteria-killing ability; however, little is known about its mechanism of virucidal activity. Therefore, we analyzed the effect of blue light on different respiratory viruses, such as adenovirus, respiratory syncytial virus and SARS-CoV-2. The exposure of samples to a blue LED light with a wavelength of 420 nm (i.e., in the visible range) at 20 mW/cm2 of irradiance for 15 min appeared optimal and resulted in the complete inactivation of the viral load. These results were similar for all the three viruses, demonstrating that both enveloped and naked viruses could be efficiently inactivated with blue LED light, regardless of the presence of envelope and of the viral genome nature (DNA or RNA). Moreover, we provided some explanations to the mechanisms by which the blue LED light could exert its antiviral activity. The development of such safe and low-cost light-based devices appears to be of fundamental utility for limiting viral spread and for sanitizing small environments, objects and surfaces, especially in the pandemic era.

The prevalence of lethal viral infections necessitates the innovation of novel disinfection techniques for contaminated surfaces, air, and wastewater as significant transmission media of disease. The instigated research has led to the development of photocatalysis as an effective renewable solar-driven technology relying on the reactive oxidative species, mainly hydroxyl (OH●) and superoxide (O2●−) radicals, for rupturing the capsid shell of the virus and loss of pathogenicity. Metal-free graphitic carbon nitride (g-C3N4), which possesses a visible light active bandgap structure, low toxicity, and high thermal stability, has recently attracted attention for viral inactivation. In addition, g-C3N4-based photocatalysts have also experienced a renaissance in many domains, including environment, energy conversion, and biomedical applications. Herein, we discuss the three aspects of the antiviral mechanism, intending to highlight the advantages of photocatalysis over traditional viral disinfection techniques. The sole agenda of the review is to summarize the significant research on g-C3N4-based photocatalysts for viral inactivation by reactive oxidative species generation. An evaluation of the photocatalysis operational parameters affecting viral inactivation kinetics is presented. An overview of the prevailing challenges and sustainable solutions is presented to fill in the existing knowledge gaps. Given the merits of graphitic carbon nitride and the heterogeneous photocatalytic viral inactivation mechanism, we hope that further research will contribute to preventing the ongoing Coronavirus pandemic and future calamities.

Continuous production of monoclonal antibodies is gaining more and more importance. To ensure continuous flow through the entire process as well as viral safety, continuous viral clearance needs to be investigated as well. This study focuses on low pH viral inactivation inside a coiled flow inverter (CFI). Computational fluid dynamics (CFD) simulation is used to gain further insight into the inactivation process inside the apparatus. The influence of viruses in comparison to different tracer elements on the residence time distribution (RTD) behavior is investigated. Finally, the viral inactivation kinetics are implemented into the CFD simulation and real process conditions are simulated. These are compared to experimental results. To the authors' knowledge, this study represents the first successful simulation of continuous viral inactivation inside a CFI. It allows the detailed analysis of processes inside the apparatus and the prediction of experimental virus study results and will therefore contribute to the effective planning of future validation studies. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) and related recombinant proteins continue to gain importance in the treatment of a great variety of diseases. Despite significant advances, their manufacturing can still present challenges owing to their molecular complexity and stringent regulations with respect to product purity, stability, safety, and so forth. In this context, protein aggregates are of particular concern due to their immunogenic potential. During manufacturing, mAbs routinely undergo acidic treatment to inactivate viral contamination, which can lead to their aggregation and thereby to product loss. To better understand the underlying mechanism so as to propose strategies to mitigate the issue, we systematically investigated the denaturation and aggregation of two mAbs at low pH as well as after neutralization. We observed that at low pH and low ionic strength, mAb surface hydrophobicity increased whereas molecular size remained constant. After neutralization of acidic mAb solutions, the fraction of monomeric mAb started to decrease accompanied by an increase on average mAb size. This indicates that electrostatic repulsion prevents denatured mAb molecules from aggregation under acidic pH and low ionic strength, whereas neutralization reduces this repulsion and coagulation initiates. Limiting denaturation at low pH by d‐sorbitol addition or temperature reduction effectively improved monomer recovery after neutralization. Our findings might be used to develop innovative viral inactivation procedures during mAb manufacturing that result in higher product yields. [ABSTRACT FROM AUTHOR]

A continuous viral inactivation (CVI) chamber has been designed to operate with acceptable residence time distribution (RTD) characteristics. However, altering the CVI's geometry and operation to accommodate the scale was not obvious. In this work, we elucidate the influence of Dean vortices and leverage the transition into the weak turbulent regime to establish relationships between input variables and process outputs. This study was targeted to understand and quantify the impact of viscosity, Dean number, internal diameter, and path length on the RTD. When the Dean number exceeds 70, radial mixing generated by the Dean vortices began to consistently alter the axial dispersive effects experienced by the pulse injection. Increasing to a Dean number of >100, the axial dispersive effects were dominated by the Dean vortices which allowed the calculation of the minimum and maximum residence time to be generated. This work provides a method to calculate operational solutions for a tubular incubation reactor in terms of path length, internal diameter, flow rate, and target minimum and maximum residence time specifications that assures both viral residence times while also establishing criteria to maximize product quality during continuous operation. [ABSTRACT FROM AUTHOR]