Utilizing Rapid Hydrogen Peroxide Generation from 6-Hydroxycatechol to Design Moisture-Activated, Self-Disinfecting Coating.

A coating that can be activated by moisture found in respiratory droplets could be a convenient and effective way to control the spread of airborne pathogens and reduce fomite transmission. Here, the ability of a novel 6-hydroxycatechol-containing polymer to function as a self-disinfecting coating on the surface of polypropylene (PP) fabric was explored. Catechol is the main adhesive molecule found in mussel adhesive proteins. Molecular oxygen found in an aqueous solution can oxidize catechol and generate a known disinfectant, hydrogen peroxide (H2O2), as a byproduct. However, given the limited amount of moisture found in respiratory droplets, there is a need to enhance the rate of catechol autoxidation to generate antipathogenic levels of H2O2. 6-Hydroxycatechol contains an electron donating hydroxyl group on the 6-position of the benzene ring, which makes catechol more susceptible to autoxidation. 6-Hydroxycatechol-coated PP generated over 3000 µM of H2O2 within 1 h when hydrated with a small amount of aqueous solution (100 µL of PBS). The generated H2O2 was three orders of magnitude higher when compared to the amount generated by unmodified catechol. 6-Hydroxycatechol-containing coating demonstrated a more effective antimicrobial effect against both Gram-positive (Staphylococcus aureus and Staphylococcus epidermidis) and Gram-negative (Pseudomonas aeruginosa and Escherichia coli) bacteria when compared to unmodified catechol. Similarly, the self-disinfecting coating reduced the infectivity of both bovine viral diarrhea virus and human coronavirus 229E by as much as a 2.5 log reduction value (a 99.7% reduction in viral load). Coatings containing unmodified catechol did not generate sufficient H2O2 to demonstrate significant virucidal effects. 6-Hydroxycatechol-containing coating can potentially function as a self-disinfecting coating that can be activated by the moisture present in respiratory droplets to generate H2O2 for disinfecting a broad range of pathogens.

Pair of Functional Polyesters That Are Photo-Cross-Linkable and Electrospinnable to Engineer Elastomeric Scaffolds with Tunable Structure and Properties.

A pair of alkyne- and thiol-functionalized polyesters are designed to engineer elastomeric scaffolds with a wide range of tunable material properties (e.g., thermal, degradation, and mechanical properties) for different tissues, given their different host responses, mechanics, and regenerative capacities. The two prepolymers are quickly photo-cross-linkable through thiol-yne click chemistry to form robust elastomers with small permanent deformations. The elastic moduli can be easily tuned between 0.96 ± 0.18 and 7.5 ± 2.0 MPa, and in vitro degradation is mediated from hours up to days by adjusting the prepolymer weight ratios. These elastomers bear free hydroxyl and thiol groups with a water contact angle of less than 85.6 ± 3.58 degrees, indicating a hydrophilic nature. The elastomer is compatible with NIH/3T3 fibroblast cells with cell viability reaching 88 ± 8.7% relative to the TCPS control at 48 h incubation. Differing from prior soft elastomers, a mixture of the two prepolymers without a carrying polymer is electrospinnable and UV-cross-linkable to fabricate elastic fibrous scaffolds for soft tissues. The designed prepolymer pair can thus ease the fabrication of elastic fibrous conduits, leading to potential use as a resorbable synthetic graft. The elastomers could find use in other tissue engineering applications as well.

Economic simulation of batch and continuous aqueous two-phase purification for viral products.

Vaccine manufacturing strategies that lower capital and production costs could improve vaccine access by reducing the cost per dose and encouraging localized manufacturing. Continuous processing is increasingly utilized to drive lower costs in biological manufacturing by requiring fewer capital and operating resources. Aqueous two-phase systems (ATPS) are a liquid-liquid extraction technique that enables continuous processing for viral vectors. To date, no economic comparison between viral vector purifications using traditional methods and ATPS has been published. In this work, economic simulations of traditional chromatography-based virus purification were compared to ATPS-based virus purification for the same product output in both batch and continuous modes. First, the modeling strategy was validated by re-creating a viral subunit manufacturing economic simulation. Then, ATPS capital and operating costs were compared to that of a traditional chromatography purification at multiple scales. At all scales, ATPS purification required less than 10% of the capital expenditure compared to chromatography-based purification. At an 11 kg per year production scale, the ATPS production costs were 50% less than purification with chromatography. Other chromatography configurations were explored, and may provide a production cost benefit to ATPS, but the purity and recovery were not experimentally verified. Batch and continuous ATPS were similar in capital and production costs. However, manual price adjustments suggest that continuous ATPS plant-building costs could be less than half that of batch ATPS at the 11 kg per year production scale. These simulations show the significant reduction in manufacturing costs that ATPS-based purification could deliver to the vaccine industry.

Design Rules for the Sequestration of Viruses into Polypeptide Complex Coacervates.

Encapsulation is a strategy that has been used to facilitate the delivery and increase the stability of proteins and viruses. Here, we investigate the encapsulation of viruses via complex coacervation, which is a liquid-liquid phase separation resulting from the complexation of oppositely charged polymers. In particular, we utilized polypeptide-based coacervates and explored the effects of peptide chemistry, chain length, charge patterning, and hydrophobicity to better understand the effects of the coacervating polypeptides on virus incorporation. Our study utilized two nonenveloped viruses, porcine parvovirus (PPV) and human rhinovirus (HRV). PPV has a higher charge density than HRV, and they both appear to be relatively hydrophobic. These viruses were compared to characterize how the charge, hydrophobicity, and patterning of chemistry on the surface of the virus capsid affects encapsulation. Consistent with the electrostatic nature of complex coacervation, our results suggest that electrostatic effects associated with the net charge of both the virus and polypeptide dominated the potential for incorporating the virus into a coacervate, with clustering of charges also playing a significant role. Additionally, the hydrophobicity of a virus appears to determine the degree to which increasing the hydrophobicity of the coacervating peptides can enhance virus uptake. Nonintuitive trends in uptake were observed with regard to both charge patterning and polypeptide chain length, with these parameters having a significant effect on the range of coacervate compositions over which virus incorporation was observed. These results provide insights into biophysical mechanisms, where sequence effects can control the uptake of proteins or viruses into biological condensates and provide insights for use in formulation strategies.

Empty and Full AAV Capsid Charge and Hydrophobicity Differences Measured with Single-Particle AFM.

Adeno-associated virus (AAV) is showing promise as a therapy for diseases that contain a single-gene deletion or mutation. One major scale-up challenge is the removal of empty or non-gene of interest containing AAV capsids. Analytically, the empty capsids can be separated from full capsids using anion exchange chromatography. However, when scaled up to manufacturing, the minute changes in conductivity are difficult to consistently obtain. To better understand the differences in the empty and full AAV capsids, we have developed a single-particle atomic force microscopy (AFM) method to measure the differences in the charge and hydrophobicity of AAV capsids at the single-particle level. The atomic force microscope tip was functionalized with either a charged or a hydrophobic molecule, and the adhesion force between the functionalized atomic force microscope tip and the virus was measured. We measured a change in the charge and hydrophobicity between empty and full AAV2 and AAV8 capsids. The charge and hydrophobicity differences between AAV2 and AAV8 are related to the distribution of charge on the surface and not the total charge. We propose that the presence of nucleic acids inside the capsid causes minor but measurable changes in the capsid structure that lead to measurable surface changes in charge and hydrophobicity.

Multimodal peptide ligand extracts parvovirus from interface in affinity aqueous two-phase system.

Aqueous two-phase systems (ATPS) have found various applications in bioseparations and microencapsulation. The primary goal of this technique is to partition target biomolecules in a preferred phase, rich in one of the phase-forming components. However, there is a lack of understanding of biomolecule behavior at the interface between the two phases. Biomolecule partitioning behavior is studied using tie-lines (TL), where each TL is a group of systems at thermodynamic equilibrium. Across a TL, a system can either have a bulk PEG-rich phase with citrate-rich droplets, or the opposite can occur. We found that porcine parvovirus (PPV) was recovered at a higher amount when PEG was the bulk phase and citrate was in droplets and that the salt and PEG concentrations are high. To improve the recovery, A PEG 10 kDa-peptide conjugate was formed using the multimodal WRW ligand. When WRW was present, less PPV was caught at the interface of the two-phase system, and more was recovered in the PEG-rich phase. While WRW did not significantly increase the PPV recovery in the high TL system, which was found earlier to be optimal for PPV recovery, the peptide did greatly enhance recovery at a lower TL. This lower TL has a lower viscosity and overall system PEG and citrate concentration. The results provide both a method to increase virus recovery in a lower viscosity system, as well as provide interesting thoughts into the interfacial phenomenon and how to recover virus in a phase and not at the interface.

Virucidal N95 Respirator Face Masks via Ultrathin Surface-Grafted Quaternary Ammonium Polymer Coatings.

N95 respirator face masks serve as effective physical barriers against airborne virus transmission, especially in a hospital setting. However, conventional filtration materials, such as nonwoven polypropylene fibers, have no inherent virucidal activity, and thus, the risk of surface contamination increases with wear time. The ability of face masks to protect against infection can be likely improved by incorporating components that deactivate viruses on contact. We present a facile method for covalently attaching antiviral quaternary ammonium polymers to the fiber surfaces of nonwoven polypropylene fabrics that are commonly used as filtration materials in N95 respirators via ultraviolet (UV)-initiated grafting of biocidal agents. Here, C12-quaternized benzophenone is simultaneously polymerized and grafted onto melt-blown or spunbond polypropylene fabric using 254 nm UV light. This grafting method generated ultrathin polymer coatings which imparted a permanent cationic charge without grossly changing fiber morphology or air resistance across the filter. For melt-blown polypropylene, which comprises the active filtration layer of N95 respirator masks, filtration efficiency was negatively impacted from 72.5 to 51.3% for uncoated and coated single-ply samples, respectively. Similarly, directly applying the antiviral polymer to full N95 masks decreased the filtration efficiency from 90.4 to 79.8%. This effect was due to the exposure of melt-blown polypropylene to organic solvents used in the coating process. However, N95-level filtration efficiency could be achieved by wearing coated spunbond polypropylene over an N95 mask or by fabricating N95 masks with coated spunbond as the exterior layer. Coated materials demonstrated broad-spectrum antimicrobial activity against several lipid-enveloped viruses, as well as Staphylococcus aureus and Escherichia coli bacteria. For example, a 4.3-log reduction in infectious MHV-A59 virus and a 3.3-log reduction in infectious SuHV-1 virus after contact with coated filters were observed, although the level of viral deactivation varied significantly depending on the virus strain and protocol for assaying infectivity.

Temporal Changes in Extracellular Vesicle Hemostatic Protein Composition Predict Favourable Left Ventricular Remodeling after Acute Myocardial Infarction.

The subset of plasma extracellular vesicles (EVs) that coprecipitate with low-density lipoprotein (LDL-EVs) carry coagulation and fibrinolysis pathway proteins as cargo. We investigated the association between LDL-EV hemostatic/fibrinolysis protein ratios and post-acute myocardial infarction (post-AMI) left ventricular (LV) remodeling which precedes heart failure. Protein concentrations of von Willebrand factor (VWF), SerpinC1 and plasminogen were determined in LDL-EVs extracted from plasma samples obtained at baseline (within 72 h post-AMI), 1 month and 6 months post-AMI from 198 patients. Patients were categorized as exhibiting adverse (n = 98) or reverse (n = 100) LV remodeling based on changes in LV end-systolic volume (increased or decreased ≥15) over a 6-month period. Multiple level longitudinal data analysis with structural equation (ML-SEM) model was used to assess predictive value for LV remodeling independent of baseline differences. At baseline, protein levels of VWF, SerpinC1 and plasminogen in LDL-EVs did not differ between patients with adverse versus reverse LV remodeling. At 1 month post-AMI, protein levels of VWF and SerpinC1 decreased whilst plasminogen increased in patients with adverse LV remodeling. In contrast, VWF and plasminogen decreased whilst SerpinC1 remained unchanged in patients with reverse LV remodeling. Overall, compared with patients with adverse LV remodeling, higher levels of SerpinC1 and VWF but lower levels of plasminogen resulted in higher ratios of VWF:Plasminogen and SerpinC1:Plasminogen at both 1 month and 6 months post-AMI in patients with reverse LV remodeling. More importantly, ratios VWF:Plasminogen (AUC = 0.674) and SerpinC1:Plasminogen (AUC = 0.712) displayed markedly better prognostic power than NT-proBNP (AUC = 0.384), troponin-I (AUC = 0.467) or troponin-T (AUC = 0.389) (p < 0.001) to predict reverse LV remodeling post-AMI. Temporal changes in the ratios of coagulation to fibrinolysis pathway proteins in LDL-EVs outperform current standard plasma biomarkers in predicting post-AMI reverse LV remodeling. Our findings may provide clinical cues to uncover the cellular mechanisms underpinning post-AMI reverse LV remodeling.

Virus inactivation at moderately low pH varies with virus and buffer properties.

BACKGROUND: Virus inactivation is a critical operation in therapeutic protein manufacturing. Low pH buffers are a widely used strategy to ensure robust enveloped virus clearance. However, the choice of model virus can give varying results in viral clearance studies. Pseudorabies virus (SuHV) or herpes simplex virus-1 (HSV-1) are frequently chosen as model viruses to demonstrate the inactivation for the herpes family. RESULTS: In this study, SuHV, HSV-1, and equine arteritis virus (EAV) were used to compare the inactivation susceptibility at pH 4.0 and 4°C. SuHV and HSV-1 are from the same family, and EAV was chosen as a small, enveloped virus. Glycine, acetate, and citrate buffers at pH 4.0 and varying buffer strengths were studied. The inactivation susceptibility was found to be in the order of SuHV > HSV > EAV. The buffer effectiveness was found to be in the order of citrate > acetate > glycine. The smaller virus, EAV, remained stable and infectious in all the buffer types and compositions studied. CONCLUSION: The variation in inactivation susceptibility of herpes viruses indicated that SuHV and HSV cannot be interchangeably used as a virus model for inactivation studies. Smaller viruses might remain adventitiously infective at moderately low pH.

Single-Particle Characterization of SARS-CoV-2 Isoelectric Point and Comparison to Variants of Interest.

SARS-CoV-2, the cause of COVID-19, is a new, highly pathogenic coronavirus, which is the third coronavirus to emerge in the past 2 decades and the first to become a global pandemic. The virus has demonstrated itself to be extremely transmissible and deadly. Recent data suggest that a targeted approach is key to mitigating infectivity. Due to the proliferation of cataloged protein and nucleic acid sequences in databases, the function of the nucleic acid, and genetic encoded proteins, we make predictions by simply aligning sequences and exploring their homology. Thus, similar amino acid sequences in a protein usually confer similar biochemical function, even from distal or unrelated organisms. To understand viral transmission and adhesion, it is key to elucidate the structural, surface, and functional properties of each viral protein. This is typically first modeled in highly pathogenic species by exploring folding, hydrophobicity, and isoelectric point (IEP). Recent evidence from viral RNA sequence modeling and protein crystals have been inadequate, which prevent full understanding of the IEP and other viral properties of SARS-CoV-2. We have thus experimentally determined the IEP of SARS-CoV-2. Our findings suggest that for enveloped viruses, such as SARS-CoV-2, estimates of IEP by the amino acid sequence alone may be unreliable. We compared the experimental IEP of SARS-CoV-2 to variants of interest (VOIs) using their amino acid sequence, thus providing a qualitative comparison of the IEP of VOIs.

Osmolyte enhanced aqueous two-phase system for virus purification.

Due to the high variation in viral surface properties, a platform method for virus purification is still lacking. A potential alternative to the high-cost conventional methods is aqueous two-phase systems (ATPSs). However, optimizing virus purification in ATPS requires a large experimental design space, and the optimized systems are generally found to operate at high ATPS component concentrations. The high concentrations capitalize on hydrophobic and electrostatic interactions to obtain high viral particle yields. This study investigated using osmolytes as driving force enhancers to reduce the high concentration of ATPS components while maintaining high yields. The partitioning behavior of porcine parvovirus (PPV), a nonenveloped mammalian virus, and human immunodeficiency virus-like particle (HIV-VLP), a yeast-expressed enveloped VLP, were studied in a polyethylene glycol (PEG) 12 kDa-citrate system. The partitioning of the virus modalities was enhanced by osmoprotectants glycine and betaine, while trimethylamine N-oxide was ineffective for PPV. The increased partitioning to the PEG-rich phase pertained only to viruses, resulting in high virus purification. Recoveries were 100% for infectious PPV and 92% for the HIV-VLP, with high removal of the contaminant proteins and more than 60% DNA removal when glycine was added. The osmolyte-induced ATPS demonstrated a versatile method for virus purification, irrespective of the expression system.

Physiochemical properties of enveloped viruses and arginine dictate inactivation.

BACKGROUND: Therapeutic protein manufacturing would benefit by having an arsenal of ways to inactivate viruses. There have been many publications on the virus inactivation ability of arginine at pH 4.0, but the mechanism of this inactivation is unknown. This study explored how virus structure and solution conditions enhance virus inactivation by arginine and leads to a better understanding of the mechanism of virus inactivation by arginine. RESULTS: Large diameter viruses from the Herpesviridae family (SuHV-1, HSV-1) with loosely packed lipids were highly inactivated by arginine, whereas small diameter, enveloped viruses (equine arteritis virus (EAV) and bovine viral diarrhea virus (BVDV)) with tightly packed lipids were negligibly inactivated by arginine. To increase the inactivation of viruses resistant to arginine, arginine-derivatives and arginine peptides were tested. Derivates and peptides demonstrated that a greater capacity for clustering and added hydrophobicity enhanced virus inactivation. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) detected increases in virus size after arginine exposure, supporting the mechanism of lipid expansion. CONCLUSIONS: Arginine most likely interacts with the lipid membrane to cause inactivation. This is shown by larger viruses being more sensitive to inactivation and expansion of the viral size. The enhancement of arginine inactivation when increased hydrophobic molecules are present or arginine is clustered demonstrates a potential mechanism of how arginine interacts with the lipid membrane.

Single-particle chemical force microscopy to characterize virus surface chemistry.

Two important viral surface characteristics are the hydrophobicity and surface charge, which determine the viral colloidal behavior and mobility. Chemical force microscopy allows the detection of viral surface chemistry in liquid samples with small amounts of virus sample. This single-particle method requires the functionalization of an atomic force microscope (AFM) probe and covalent bonding of viruses to a surface. A hydrophobic methyl-modified AFM probe was used to study the viral surface hydrophobicity, and an AFM probe terminated with either negatively charged carboxyl acid or positively charged quaternary amine was used to study the viral surface charge. With an understanding of viral surface properties, the way in which viruses interact with the environment can be better predicted.

Thermostabilization of viruses via complex coacervation.

Widespread vaccine coverage for viral diseases could save the lives of millions of people each year. For viral vaccines to be effective, they must be transported and stored in a narrow temperature range of 2-8 °C. If temperatures are not maintained, the vaccine may lose its potency and would no longer be effective in fighting disease; this is called the cold storage problem. Finding a way to thermally stabilize a virus and end the need to transport and store vaccines at refrigeration temperatures will increase access to life-saving vaccines. We explore the use of polymer-rich complex coacervates to stabilize viruses. We have developed a method of encapsulating virus particles in liquid complex coacervates that relies on the electrostatic interaction of viruses with polypeptides. In particular, we tested the incorporation of two model viruses; a non-enveloped porcine parvovirus (PPV) and an enveloped bovine viral diarrhea virus (BVDV) into coacervates formed from poly(lysine) and poly(glutamate). We identified optimal conditions (i.e., the relative amount of the two polypeptides) for virus encapsulation, and trends in this composition matched differences in the isoelectric point of the two viruses. Furthermore, we were able to achieve a ∼103-104-fold concentration of virus into the coacervate phase, such that the level of virus remaining in the bulk solution approached our limit of detection. Lastly, we demonstrated a significant enhancement of the stability of non-enveloped PPV during an accelerated aging study at 60 °C over the course of a week. Our results suggest the potential for using coacervation to aid in the purification and formulation of both enveloped and non-enveloped viruses, and that coacervate-based formulations could help limit the need for cold storage throughout the transportation and storage of vaccines based on non-enveloped viruses.

Virus and chlorine adsorption onto guanidine modified cellulose nanofibers using covalent and hydrogen bonding.

Unsafe drinking water leads to millions of human deaths each year, while contaminated wastewater discharges are a significant threat to aquatic life. To relieve the burden of unsafe water, we are in search of an inexpensive material that can adsorb pathogenic viruses from drinking water and adsorb toxic residual chlorine from wastewater. To impart virus and chlorine removal abilities to cellulosic materials, we modified the primary hydroxyl group with a positively charged guanidine group, to yield guanidine modified cellulose derivatives. Microcrystalline cellulose (MC) bearing covalently bonded guanidine hydrochloride (MC-GC) and hydrogen-bonded guanidine hydrochloride (MC-GH) were synthesized, and electrospun into nanofibers after blending with the non-ionogenic polyvinyl alcohol (PVA), to produce large pore sized, high surface area membranes. The MC-GC/PVA and MC-GH/PVA nanofibers were stabilized against water dissolution by crosslinking with glutaraldehyde vapor. The water-stable MC-GC/PVA mats were able to remove more than 4 logs of non-enveloped porcine parvovirus (PPV) and enveloped Sindbis virus and reached 58% of chlorine removal. The MC-GC/PVA nanofibers demonstrated better performance for pathogen removal and dechlorination than MC-GH/PVA nanofibers. This first study of MC-GC/PVA electrospun mats for virus removal shows they are highly effective and merit additional research for virus removal.

Surface Chemistry Can Unlock Drivers of Surface Stability of SARS-CoV-2 in a Variety of Environmental Conditions.

The surface stability and resulting transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), specifically in indoor environments, have been identified as a potential pandemic challenge requiring investigation. This novel virus can be found on various surfaces in contaminated sites such as clinical places; however, the behavior and molecular interactions of the virus with respect to the surfaces are poorly understood. Regarding this, the virus adsorption onto solid surfaces can play a critical role in transmission and survival in various environments. In this article, we first give an overview of existing knowledge concerning viral spread, molecular structure of SARS-CoV-2, and the virus surface stability is presented. Then, we highlight potential drivers of the SARS-CoV-2 surface adsorption and stability in various environmental conditions. This theoretical analysis shows that different surface and environmental conditions including temperature, humidity, and pH are crucial considerations in building fundamental understanding of the virus transmission and thereby improving safety practices.

Changes in Membrane Dielectric Properties of Porcine Kidney Cells Provide Insight into the Antiviral Activity of Glycine.

The ability to monitor the status and progression of viral infections is important for development and screening of new antiviral drugs. Previous research illustrated that the osmolyte glycine (Gly) reduced porcine parvovirus (PPV) infection in porcine kidney (PK-13) cells by stabilizing the capsid protein and preventing virus capsid assembly into viable virus particles. Dielectrophoresis (DEP) was examined herein as a noninvasive, electric field- and frequency-dependent tool for real-time monitoring of PK-13 cell responses to obtain information about membrane barrier functionality and polarization. DEP responses of PK-13 cells were compared to those of PPV-infected cells in the absence and presence of the osmolyte glycine. With infection progression, PK-13 DEP spectra shifted toward lower frequencies, reducing crossover frequencies (fCO). The spherical single-shell model was used to extract PK-13 cell dielectric properties. Upon PPV infection, specific membrane capacitance increased over the time progression of virus attachment, penetration, and capsid protein production and assembly. Following glycine treatment, the DEP spectra displayed attenuated fCO and specific membrane capacitance values shifted back toward uninfected PK-13 cell values. These results suggest that DEP can be used to noninvasively monitor the viral infection cycle and screen antiviral compounds. DEP can augment traditional tools by elucidating membrane polarization changes related to drug mechanisms that interrupt the virus infection cycle.

Hydroxyl Radical Generation Through the Fenton-Like Reaction of Hematin- and Catechol-Functionalized Microgels.

Hydroxyl radical (•OH) is a potent reactive oxygen species with the ability to degrade hazardous organic compounds, kill bacteria, and inactivate viruses. However, an off-the-shelf, portable, and easily activated biomaterial for generating •OH does not exist. Here, microgels were functionalized with catechol, an adhesive moiety found in mussel adhesive proteins, and hematin (HEM), a hydroxylated Fe3+ ion-containing porphyrin derivative. When the microgel was hydrated in an aqueous solution with physiological pH, molecular oxygen in the solution oxidized catechol to generate H2O2, which was further converted to •OH by HEM. The generated •OH was able to degrade organic dyes, including orange II and malachite green. Additionally, the generated •OH was antimicrobial against both gram-negative (Escherichia coli) and gram-positive (Staphylococcus epidermidis) bacteria with the initial concentration of 106-107 CFU/mL. These microgels also reduced the infectivity of a non-enveloped porcine parvovirus and an enveloped bovine viral diarrhea virus by 3.5 and 4.5 log reduction values, respectively (99.97-99.997% reduction in infectivity). These microgels were also functionalized with positively charged [2-(methacryloyloxy)ethyl] trimethylammonium chloride (METAC), which significantly enhanced the antibacterial and antiviral activities through electrostatic interaction between the negatively charged pathogens and the microgel. These microgels can potentially serve as a lightweight and portable source of disinfectant, for an on-demand generation of •OH with a wide range of applications.

Virus Isoelectric Point Determination Using Single-Particle Chemical Force Microscopy.

Virus colloidal behavior is governed by the interaction of the viral surface and the surrounding environment. One method to characterize the virus surface charge is the isoelectric point (pI). Traditional determination of virus pI has focused on the bulk characterization of a viral solution. However, virus capsids are extremely heterogeneous, and a single-particle method may give more information on the range of surface charge observed across a population. One method to measure the virus pI is chemical force microscopy (CFM). CFM is a single-particle technique that measures the adhesion force of a functionalized atomic force microscope (AFM) probe and, in this case, a virus covalently bound to a surface. Non-enveloped porcine parvovirus (PPV) and enveloped bovine viral diarrhea virus (BVDV) were used to demonstrate the use of CFM for viral particles with different surface properties. We have validated the CFM to determine the pI of PPV to be 4.8-5.1, which has a known pI value of 5.0 in the literature, and to predict the unknown pI of BVDV to be 4.3-4.5. Bulk measurements, ζ-potential, and aqueous two-phase system (ATPS) cross-partitioning methods were also used to validate the new CFM method for the virus pI. Most methods were in good agreement. CFM can detect the surface charge of viral capsids at a single-particle level and enable the comparison of surface charge between different types of viruses.

Arginine-enveloped virus inactivation and potential mechanisms.

Arginine synergistically inactivates enveloped viruses at a pH or temperature that does little harm to proteins, making it a desired process for therapeutic protein manufacturing. However, the mechanisms and optimal conditions for inactivation are not fully understood, and therefore, arginine viral inactivation is not used industrially. Optimal solution conditions for arginine viral inactivation found in the literature are high arginine concentrations (0.7-1 M), a time of 60 min, and a synergistic factor of high temperature (≥40°C), low pH (≤pH 4), or Tris buffer (5 mM). However, at optimal conditions full inactivation does not occur over all enveloped viruses. Enveloped viruses that are resistant to arginine often have increased protein stability or membrane stabilizing matrix proteins. Since arginine can interact with both proteins and lipids, interaction with either entity may be key to understanding the inactivation mechanism. Here, we propose three hypotheses for the mechanisms of arginine induced inactivation. Hypothesis 1 describes arginine-induced viral inactivation through inhibition of vital protein function. Hypothesis 2 describes how arginine destabilizes the viral membrane. Hypothesis 3 describes arginine forming pores in the virus membrane, accompanied by further viral damage from the synergistic factor. Once the mechanisms of arginine viral inactivation are understood, further enhancement by the addition of functional groups, charges, or additives may allow the inactivation of all enveloped viruses in mild conditions.