Micro-devices that use electric fields to trap, analyze and inactivate micro-organisms vary in concept, design and application. The application of electric fields to manipulate and inactivate bacteria and single-celled organisms has been described extensively in the literature. By contrast, the effect of such fields on viruses is not well understood. This review explores the possibility of using existing methods for manipulating and inactivating larger viruses and bacteria, for smaller viruses, such as SARS-CoV-2. It also provides an overview of the theoretical background. The findings may be used to implement new ideas and frame experimental parameters that optimize the manipulation, sampling and inactivation of SARS-CoV-2 electrically. Viruses, often debated to be non-living bodies, are complicated assemblies of proteins, nucleic acids and carbohydrates. Their incredibly small size 17–400 nm) makes it a challenge to manipulate them. Research on viruses becomes more urgent as health risks posed by viral pathogens become more frequent and deadly. Severe Acute Respiratory Syndrome Corona Virus 2 or SARS-CoV-2, a virus belonging to the corona virus family and the microbe responsible for the COVID-19 pandemic, has claimed over 6.7 million lives across the globe, as of December 2022 [7–400 nm Studying the mechanisms of virus deactivation could have huge benefits for battling pandemics such as COVID-19. Until now, various chemical , optical6 V/m [Nonuniform electric fields have been used before, albeit separately, to trap, analyze and inactivate single-celled organisms and bacteria. Viruses, on the other hand, are more difficult to manipulate and show resistance to inactivation treatments using electric fields as high as 2.9 × 106 V/m  due to t6 V/m .Previous reviews have only addressed the concentration of viruses using electric fields . This repp, E = 106 V/m) [pp and 1 MHz [A polarizable particle suspended in a liquid medium exposed to a nonuniform electric field experiences a force known as the dielectrophoretic force (DEP). When the medium is more polarizable than the particle, the particle will be repelled by the electrode, resulting in a negative DEP force (nDEP). Whereas when the particle is more polarizable, than the medium, the particle, will be attracted to the electrode, resulting in a positive DEP force (pDEP). Dielectrophoretic manipulation has been used to successfully trap and concentrate bacteria and viruses. Concentration of the viral particulates facilitates the formation of localized clusters that can be easily examined and inactivated. Morgan et al. successfully demonstrated the use of DEP in concentrating viruses . They co106 V/m) . The mannd 1 MHz .Recently, the use of nanofibers for trapping viruses was investigated . AlthougSmaller viruses were also shown to respond to the dielectrophoretic effect. Influenza (ϕ 80 nm) and Hepatitis virus (ϕ 32 nm) were manipulated and concentrated using negative dielectrophoresis (nDEP) on planar electrode arrays. Planar platforms and 3D structures that make use of quadrupole and octupole electrode cages establish points of confluence, where the virus can be trapped effectively . Cowpea A challenging problem is that with the increase in electrolyte conductivity, the ionic strength increases. This may result in more electrochemical reactions. Usually, virus samples require a relatively high ionic strength for their storage. Another challenge is that smaller viruses (<100 nm) show a random Brownian motion. Brownian motion is characterized by random movements that could hinder the trapping of viruses ,23. ThesInsulator-based DEP (iDEP) is a technique that uses insulating structures to concentrate the electric field. The technique seems to become more common for concentrating smaller viruses as well as larger viruses in recent years. For example, influenza virus (90 nm) , SindbisA summary of all the different aspects of the practical studies that concentrated viruses using DEP have been tabulated in The spherical SARS-CoV-2 virus is characterized by a lipid insulating membrane, and hence, the particle may not be easily polarizable. Tuning properties of the medium becomes instrumental if we desire to attract and concentrate the virus particles with pDEP. The Clausius–Mossotti factor of SARS-CoV-2 can be modelled by using a core-shell model, as depicted in ω. As a result, F will depend on ω as well. The fact that CM* are complex is indicated in Equations (1) and (2) by the asterisk in superscript. The symbol Note that the permittivities are complex quantities, the real and imaginary part of which usually will change with the applied radial frequency sγ is the conductivity of the solvent and oε the permittivity of vacuum oε = 8.854 × 10−12 F/m. In addition to ionic conduction, dipole relaxations may also occur in a dielectric medium. We assume that these are not strong in the present case and so they are neglected. This implies that If f, f = ω/(2π), will evidently derive from the conductivity and permittivity of the solvent, as well as from the conductivity and permittivity of the core and membrane of the particle.The change in the CM-factor with frequency For a layered sphere, the equation for which may also be written as:r and that of the outer layer is R. When we substitute Equation (5) in the CM factor given by Equation (2), we obtain:The core-shell structure of the virus is visualized in CM* given by Equation (7) will depend on the frequency. The same applies to the DEP force given by Equation (1), as we have already mentioned above.As indicated, the We should realize that v is the volume fraction of the virus particles. It is this Note that CM from Equations (5), (8) and (9) with a few lines of code using Matlab, Maple or Mathematica. The plots shown in We can compute sγ < pγ, a pDEP force on the corona virus can be produced at lower frequencies to effectively concentrate the virus. Unfortunately, studies have not delineated the electrical parameters of SARS-CoV-2. Nonetheless, for a KCl solution with a low conductivity, and as long as CM becomes positive. Selecting a non-aqueous medium, such as that of Micro-electrodes can be employed as sensors to quantify the virus present in a sample. The use of electric fields to count viruses dismisses the need for tagging and sample preparation. The usual method used for detecting and quantifying a specific virus is the Polymerase Chain Reaction (PCR), which is highly accurate, but not rapid. Significant work has been carried out on viruses where differences in the electrical parameters between the particle and the medium are utilized to give information about the quantity and type of virus present. Other techniques used to detect the presence and type of virus use antibodies coated on nanowires that record a change in conductance when a virus particle binds to the antibody . This me3 particles/mL. This technique can be easily integrated onto platforms that are designed to trap and inactivate viruses.Dielectrophoretic impedance measurement (DEPIM) is a technique that can quantify the trapped virus without the need for antibodies. The DEPIM technique is a combination of DEP manipulation and impedance measurement, which records the change in impedance as soon as a virus is trapped . NumerouAnother technique used to quantify and sample viruses treats the virus as an impurity and then measures the change in capacitance between the electrodes. The measured change in capacitance, as a result of the difference in permittivity of the virus particulate and the conducting medium, can then be empirically traced to the number of particles present in the medium by applying bioelectrochemical theory . The eleThis method utilizes the specificity of the virus’s composition, which is reflected by the effective permittivity of the membrane to identify the virus. An extension of the same method would be to measure the electrical capacitance per virus particle to find out the specific dielectric constant, and then subtract the contributions of the medium from the measured capacitance to identify the virus . The capAnother approach uses co-planar differential capacitive sensors refer to . Two capHowever, the calculations depend on the evaporation times of the media and a fast integrated analysis within the device itself may be a challenging goal.A list of practical studies that successfully sampled viruses using electric fields has been tabulated in The electrical inactivation of bacteria or viruses is a technique that refers to a process that invokes electric fields to render the organism incapable of replicating or infecting a host. Ions generated by electric activities, such as the corona discharge, have been proven to be effective in inactivating the virus ,41. This7 V/m. Lysis by irreversible electroporation demands a conductive medium. A higher conductivity of the medium leads to higher irreversible electroporation and, thus, a higher lysis rate [The electrical inactivation of bacteria is usually brought about by electrical lysis and irreversible electroporation. The lysis results from the creation of a transmembrane potential that is directed from the outside to the inside of the cell as a result of the accumulation of charges. When the transmembrane potential exceeds a certain threshold, electroporation occurs. With a further increase in the electric field, the electroporation is made permanent, which results in lysis. The transmembrane voltage to achieve cell lysis is around 1 V , which rsis rate . This re2 is useful to induce a strong DEP force in most dielectrophoretic studies, an increase in the field strength E leads to electroporation. We can optimize the geometry such that the divergence as well as the field strength are maximized to attract the particles at points of high electric field for an effective lysis.Many studies used pulsed excitation to kill bacteria ,50,51. WThe only paper that claims the inactivation of a virus by irreversible electroporation used AC to concentrate and lyse the virus. The study reported the electrical lysis of Vaccinia virus . The damSimilarly, a study in 2014 reported, although inadvertently, the lysis of Vaccinia virus on nanofiber probe arrays . Both stf is the conductivity factor, aE the applied field, R the radius of the virus, and θ the direction of aE. The factor f equals:r is the radius of the core. Note that we have modified Kotnik’s formula to the one given earlier by Neumann in 1989 [mV in response to a step voltage [mγ = 0. To produce a transmembrane potential of 1 V we would need a field of 8.3 V/μm. Grosse et al. extended the model to a spherical cell with two layers [ω.As iterated before, the membrane voltage to lyse cells is around 1 V. Ignoring the spikes, we may model SARS-CoV-2 as a core with one shell. The core-shell model is redrawn in ic essay . The conic essay ,61. They in 1989 . Schwan  voltage :(13)Vm≃3o layers . The expAnother lysis mechanism that evades the need for a conductive medium is the short pulse effect, which exploits the inhomogeneities in the cytoplasm of the bacterium. Pulse durations of an order smaller than the charging time of the bacterial cell membrane cause local heating near and around the inhomogeneities of the cellular material, causing cell lysis ,52. GeneIn 2021, a lot of studies focused on the inactivation of SARS-CoV-2 using agents other than the primary chemical and optical disinfectants. Chief among these studies was the simulation study carried out by Arbeitman et al.  that sug5−107 V/m stretch the dipoles found in the spike proteins of the virus and change the protein structure permanently. Irreversible structural changes especially on the receptor binding domain (RBD) of the spike protein, render the virus incapable of docking to the host cell’s receptors and thus inactivate the virus .Another study elucidated the effect of pulse electric fields on the conformation changes of the virus’ proteins . It suggUsing electric fields allows us to integrate concentration, quantification, and inactivation on a single microchip platform. For this, choosing the electrical parameters of the conducting medium is essential in trapping the virus at points of high electric field and achieving optimum quantification and inactivation. The electrode geometry is another crucial parameter. Geometries that maximize concentration as well as killing should be employed. A variety of customized configurations have been used to concentrate and inactivate bacteria and viruses. These configurations are either 3D or 2D. Of the two, 3D electrode configurations produce a DEP force with a larger divergence and, hence, have a greater penetrating power within the sample.For concentrating and killing smaller viruses, studies focus on 3D nano-electrode arrays with carbon nanotubes that create extremely high electric field strengths at the tips ,35,69. APlanar electrodes are very easy to fabricate; for example, interdigitated electrodes, castellated electrodes or matrix electrodes. Interdigitated or finger electrodes create a less nonuniform electric field with few high electric field points. These configurations are used mainly for dielectrophoresis ,22,74,75Using electric fields as a tool may allow us to integrate the trapping, sampling and inactivation of viruses on a single microchip platform. For this, optimizing the electrical parameters of the conducting medium is instrumental in facilitating a pDEP to trap the virus between the electrodes at points of high electric field to facilitate efficient sampling and inactivation. Sampling can be brought about by DEPIM measurements by measuring the change in capacitance between the planar electrodes right after the viruses are trapped. The electrode geometry is another sensitive parameter. Geometries that maximize both trapping and killing should be employed. The prime inactivation mechanism of SARS-CoV-2 is still unclear. Inactivation by irreversible electroporation and spike deactivation seem to be equally probable mechanisms that may take course when the virus is subjected to an electric field. Future experimental research should focus on exploring the inactivation mechanism of SARS-CoV-2 to confirm or dismiss the possibility of the inactivation of SARS-CoV-2 using moderate electric fields. Insight into the molecular interactions of the virus’s proteins and membrane lipids with electric fields and voltage potentials can help to establish a proof of concept. With a firm proof, products such as electric facemasks can be better designed, and a new generation of micro-fiber filters can be developed and deployed, which may help us ward off future pandemics.


Vulpes vulpes) that were submitted with neurological symptoms between December of 2021 and February of 2022. A histopathological analysis demonstrated that the virus was mainly present in the brain, with limited or no detection in the respiratory tract or other organs. Limited or no virus shedding was observed in throat and rectal swabs. A phylogenetic analysis showed that the three fox viruses were not closely related, but they were related to HPAI H5N1 clade 2.3.4.4b viruses that are found in wild birds. This suggests that the virus was not transmitted between the foxes. A genetic analysis demonstrated the presence of the mammalian adaptation E627K in the polymerase basic two (PB2) protein of the two fox viruses. In both foxes, the avian (PB2-627E) and the mammalian (PB2-627K) variants were present as a mixture in the virus population, which suggests that the mutation emerged in these specific animals. The two variant viruses were isolated, and virus replication and passaging experiments were performed. These experiments showed that the mutation PB2-627K increases the replication of the virus in mammalian cell lines, compared to the chicken cell line, and at the lower temperatures of the mammalian upper respiratory tract. This study showed that the HPAI H5N1 virus is capable of adaptation to mammals; however, more adaptive mutations are required to allow for efficient transmission between mammals. Therefore, surveillance in mammals should be expanded to closely monitor the emergence of zoonotic mutations for pandemic preparedness.During the 2020 to 2022 epizootic of highly pathogenic avian influenza virus (HPAI), several infections of mammalian species were reported in Europe. In the Netherlands, HPAI H5N1 virus infections were detected in three wild red foxes (IMPORTANCE Highly pathogenic avian influenza (HPAI) viruses caused high mortality among wild birds from 2021 to 2022 in the Netherlands. Recently, three wild foxes were found to be infected with HPAI H5N1 viruses, likely due to the foxes feeding on infected birds. Although HPAI is a respiratory virus, in these foxes, the viruses were mostly detected in the brain. Two viruses isolated from the foxes contained a mutation that is associated with adaptation to mammals. We show that the mutant virus replicates better in mammalian cells than in avian cells and at the lower body temperature of mammals. More mutations are required before viruses can transmit between mammals or can be transmitted to humans. However, infections in mammalian species should be closely monitored to swiftly detect mutations that may increase the zoonotic potential of HPAI H5N1 viruses, as these may threaten public health. Phoca vitulina), one juvenile gray seal , and a red fox (Vulpes vulpes) at a wildlife rehabilitation center in the United Kingdom was reported to be associated with HPAI H5N8 infections (Phoca vitulina) found at the German North Sea coast (Since the introduction of highly pathogenic avian influenza virus (HPAI) H5 clade 2.3.4.4b in 2016, this virus clade has caused repeated outbreaks in wild birds and poultry in Europe. Whereas low pathogenic avian influenza viruses replicate mostly in the digestive and respiratory epithelium with often mild disease in poultry and wild birds, these HPAI viruses can cause severe systemic disease, including viremia that leads to the diffuse infection of several internal organs. Infections with HPAI viruses of subtypes H5 and H7 are a notifiable disease in poultry, and after the detection of this virus, a poultry flock is generally culled to prevent further spread. The 2020 to 2021 epizootic was a devastating outbreak for poultry and was followed by the 2021 to 2022 epizootic, which has become the largest HPAI outbreak in terms of the number of culled animals that has ever occurred in Europe. In addition, high mortalities in an increasing number of species of wild birds were observed during both epizootics. Sporadically, HPAI H5 clade 2.3.4.4.b infections of free-living, wild carnivore species were reported in addition to the infections in wild birds and poultry. In late 2020, a disease and mortality event involving four juvenile common seals  airborne transmission of HPAI viruses between mammals: (i) the efficient attachment of the viral hemagglutinin (HA) glycoprotein to α2,6-linked sialic acid receptors. Mutations in HA are required to change the HA binding properties from the avian-type α2,3-linked sialic acid receptors to the mammalian-type α2,6-linked sialic acid receptor , 8. (ii) in mice –14. Furtin mice –, 15. Thein vitro in mammals, compared to birds.The previously described cases of HPAI H5 clade 2.3.4.4b infections in seals and foxes as well as the three infected foxes detected in the Netherlands between December of 2021 and February of 2022 suggest that the incidence of infections in mammals might be increasing. In this study, we analyzed virus localization in tissues from the three foxes with related histopathology and showed that the virus is mainly present in the brain, with limited detection in the respiratory tract. A phylogenetic analysis showed that the three fox viruses were not closely related, but they were related to viruses found in wild birds. A genetic analysis demonstrated the presence of mutation PB2-E627K in two out of three fox viruses. Two virus variants were isolated from one of these foxes, with one containing the avian PB2-627E variant and one containing the mammalian PB2-627K variant. The virus replication and stability of both isolates was studied over time and showed increased replication for the PB2-627K variant in human and dog cell lines, compared to the chicken cell line, and at the temperature of the mammalian upper respiratory tract, compared to the temperature of the avian respiratory tract, indicating more efficient replication Three foxes were found in the Netherlands at the municipalities Dorst, Heemskerk, and Oosterbeek, and they were observed to be showing abnormal behavior. The clinical signs in fox-Dorst were apparent blindness, head shaking, falling over, and opisthotonos. This animal was euthanized on 3/12/2021. Fox-Heemskerk showed convulsions, as reported, and was euthanized on 1/1/2022. Fox-Oosterbeek showed lethargy, crouching down with convex back and legs tucked under. The animal lacked fleeing behavior but was alert and did turn its head to look when approached. This fox was humanely dispatched on 7/2/2022. The distance between the different locations was 80 km to 105 km .The foxes were submitted for testing on avian influenza virus. Tracheal and rectal swabs were taken, and brain tissue was collected. Interestingly, the brain samples from two different locations  tested positive for avian influenza virus by a quantitative real-time RT-PCR targeting the matrix gene (M-PCR) with high virus genome loads, whereas no virus was detected in rectal swabs, and considerably lower virus loads were detected in the throat swabs of two foxes. Only for fox-Heemskerk were comparable virus loads detected in the brain samples and throat swab . The virAngiostrongylus vasorum) in two of the three foxes. These severe pulmonary changes and the moderate pulmonary changes in the third fox were not associated with virus protein expression (Capillaria spp.). In two of the three foxes, the nasal conchae was evaluated (Table S1). In fox-Heemskerk, virus protein expression was mainly observed in the olfactory epithelial cells with associated necrosis of these cells  was covered with mud. The most prominent gross finding was poorly collapsed lungs with a marbled red aspect, which was present with a slight variation in all three foxes. Histology revealed a subacute to chronic purulent granulomatous broncho-interstitial pneumonia with large numbers of parasitic structures . The viruses isolated from the foxes were not closely related, based on the phylogenetic analysis, and they differed from each other by between 127 and 176 nucleotides ; Fig. S1A genetic analysis was performed to investigate whether the mutations implicated in the adaptations to mammalian hosts occurred in the virus genomes. Mutation screening identified the previously described E627K mutation in the PB2 segment of the fox-Dorst virus. A minority variant analysis of the next-generation sequencing data from three samples of fox-Dorst showed a mixture of the avian (627E) and mammalian (627K) PB2 variants. The percentage of the 627K mutation varied between 37.4% of the virus population in the trachea swab, 46.1% in the amnion horn and medulla oblongata, and 19.5% in the cerebrum and cerebellum . Fox-HeeA limited dilution series of the amnion horn and medulla oblongata sample derived from fox-Dorst was inoculated into embryonated eggs for virus isolation. Full genome sequencing was performed for individual egg isolates. This showed that we isolated a HPAI H5N1 virus with the mammalian PB2-627K variant (100%), and a virus with the avian PB2-627E variant  that contained only one additional mutation (G485R) in the nucleoprotein (NP) (99%). To study the effect of PB2-E627K on virus replication, the virus titer was measured at specific time points after the infection of the mammalian A549 and MDCK cell lines as well as the avian DF-1 cell line.post hoc analyses. Due to sample size limitations, three-way interactions containing time, virus, and cell type as variables could not be analyzed. However, the two-way interactions of time with virus and time with temperature could be assessed using the LMM. The differences between the two virus variants were statistically significant from 48 h p.i. onwards (P < 0.05) (Table S3). To assess the apparent absence of differences between the PB2-627E and PB2-627K virus replication curves on the DF-1 cells at 37°C, virus titer was compared by performing pairwise comparisons between both viruses at each time point and ignoring the data dependency (no random effects introduced in the model) due to replicated measures. These pairwise comparisons showed no significant differences (P > 0.05) between the two viruses from 48 h p.i. onwards. These results may indicate that PB2-E627K enhances virus replication in mammalian cells but not in avian cells at the temperature of the avian upper respiratory tract (37°C).The PB2-E627K mutation appears to increase virus replication from 48h postinfection (p.i.) onwards in mammalian cells  but not in avian cells (DF-1) at 37°C . The putP < 0.0001) and 48 h p.i. (P < 0.05) but significantly higher at 72 h p.i. (P < 0.05), indicating that the replication of both virus variants is delayed but not impaired . However, the passaging of the PB2-627E virus isolate, which contains 1.8% PB2-627K, showed that the 627K mutant has a replication benefit in all three cell lines at both temperatures tested . HoweverHere, we investigated three cases of HPAI H5N1 infections in wild foxes in the Netherlands to assess tissue tropism in wild mammals and to screen for adaptive mutations. All three foxes showed unusual tissue tropism, and evidence was found for mammalian adaptation. The full genome sequencing of the fox viruses, followed by a phylogenetic analysis, demonstrates that they belong to clade 2.3.4.4b and are related to viruses detected in wild birds. The fact that the three fox-viruses were not closely related as well as the large distance between the locations at which the three foxes were found suggest that these were separate virus introductions that likely originated from wild birds. Experimental infections via the consumption of infected bird carcasses have indicated that foxes are susceptible to avian influenza virus (AIV) infections, and this is also the most probable route of infection for free-living foxes . However16–In two of the three foxes, a minority population of viruses that contained the zoonotic mutation PB2-E627K was identified. The fact that this mutation was not detected in any of the wild bird sequences during the 2021 to 2022 epizootic in the Netherlands suggests that this mutation quickly arises upon the infection of mammals. Furthermore, as both the PB2-627E and PB2-627K variants were detected in these two foxes, it appears likely that this mammalian adaptation emerged within these specific animals. A similar event has occurred in two seals infected with the HPAI H5N8 virus in August of 2021 in Germany, and it indicates selection pressure for this adaptation in mammals . HistopaIt is currently unclear which factors have contributed to the increase of infections observed in red foxes in nature. The HPAI H5N1 clade 2.3.4.4b virus may be more capable of infecting mammals, the virus may be more infectious, or there may be a higher prevalence of the virus in wild birds during the 2021 to 2022 epizootic, compared to previous epizootics. Limited virus shedding and virus replication were observed in the respiratory systems and digestive tracts of the investigated foxes, which likely limits transmission between mammals at this state of the disease. Consistent with this, no evidence for transmission between foxes was found, based on the phylogenetic analysis of the viruses. The genetic analysis suggests that the zoonotic mutation PB2-E627K may arise in infected mammals. Although PB2-E627K is an important mammalian adaptation, previous research has indicated that several further adaptations are required for the efficient airborne transmission of AIV between mammals. For example, the PB2-E627K, HA-Q222L, and HA-G224S mammalian adaptations were required before 10 serial passages in ferrets to eventually produce airborne variants of HPAI  and for influenza A NP expression with IHC, as described previously . From alAll virus genome sequences were determined directly on the swab or tissue samples. The virus RNA was purified using a High Pure Viral RNA Kit , amplified using universal eight-segment primers, and directly sequenced, as described previously . PurifieIn addition to the virus sequences obtained from foxes and wild birds in the Netherlands in this study, the top five BLAST results for related sequences in Eurasia were included in the phylogenetic analysis. These H5N1 genome sequences were downloaded from the GISAID database . A phylo2. The cells were passaged when confluent using 0.05% Trypsin-EDTA .Madin-Darby canine kidney (MDCK) cells obtained from Philips-Duphar , chicken embryo fibroblasts (DF-1) , and human lung alveolar epithelial cells (A549)  were maintained in cell culture medium consisting of Dulbecco’s Modified Eagle Medium GlutaMAX (DMEM)  supplemented with 5% fetal calf serum  and 0.1% penicillin-streptomycin  at 37°C and 5% CO4 cells per well of a 96-well plate were seeded overnight in cell culture medium. Monolayers were infected with 10-fold serial dilutions in infection medium consisting of DMEM GlutaMAX supplemented with 0.1% penicillin-streptomycin and 0.3% bovine serum albumin. Each dilution was tested in eightfold, and each titration was diluted in triplicate. After 2 days of incubation at 37°C and 5% CO2, the monolayers were stained via immunoperoxidase monolayer assay. The monolayers were fixed with 10% neutral buffered formalin. The primary antibody was produced in-house from HB 65 mouse anti-NP and diluted 1:2,500. This was followed by HRP-conjugated rabbit anti-mouse that was diluted 1:500 . The titration was repeated on a different day, and the TCID50 titers were calculated using the Reed and Muench method  were incubated for 1 h with 1% penicillin and 1% gentamicin at room temperature. The homogenates were filtered and injected in nine-day-old specific-pathogen-free (SPF) embryonated chicken eggs (ECE), as described previously . Allantoh method .5 cells per well in cell culture medium. The next day, the cells were infected with 1.5 mL of virus diluted in infection medium at a multiplicity of infection of 0.01 (A549), 0.001 (MDCK), or 0.0005 (DF-1). The cells were incubated at 37°C and 5% CO2 or at 33°C and 5% CO2. At the indicated time points, 150 μL of medium was harvested and stored at –80°C. 150 μL of fresh infection medium was added after each harvest. The infectious virus titer of the collected medium was determined in triplo using the TCID50 protocol described above.The multiplicity of infection (MOI) was optimized for each cell line by selecting a dilution, which caused limited cell death but sufficient virus replication. MDCK, DF-1, and A549 cells were seeded in duplo on a 24-well plate at a density of 2.5 × 106 cells per T-25 flask. The next day, the cells were infected with diluted virus in 3 mL infection medium. Cells were incubated either at 37°C and 5% CO2 or at 33°C and 5% CO2 for 3 days before the medium was collected. The medium was diluted 1,000× and added to a fresh T-25 flask of A549, MDCK, or DF-1 cells. The remaining, undiluted medium was stored at –80°C for sequencing. The viral RNA was isolated using a Zymo Quick-RNA Viral 96 Kit . A region of 168 bp on the PB2 protein was amplified via RT-PCR using custom primers (Table S6)  and a Qiagen OneStep RT-PCR Kit . The amplicon length and concentration were analyzed using HS DNA 1000 with TapeStation 2200 (Agilent) as well as Quanti-IT (ThermoFisher) with ClarioStar (BMG). Region-specific amplicons were individually barcoded using Illumina’s Nextera XT Index V2 Kit with limit cycle PCR and PE150 sequenced on Illumina’s Miseq. The reads were mapped using the ViralProfiler-Workflow, an extension of the CLC Genomics Workbench . Consensus sequences were generated via a reference-based method, and minority variants were called using a cutoff of 1%. The minimal coverage at position PB2-627K was 2,000 reads.The stability of the virus isolates was determined by passaging 10 times on A549, MDCK, and DF-1 cells at identical temperatures and multiplicities of infection as the replication experiment described above. In short, the cells were seeded at a density of 2.5 × 10P < 0.05) kept in the final model were time:virus, time:temperature, and time:cell. Variable significance was assessed using the likelihood ratio test. This analysis was done using the statistical software R  in which this nested structure and the repeated measures were included as random effects. The virus titer was the response variable. The variables time, virus (two isolates: PB2-627E and PB2-627K), cell , and temperature 37°C or 33°C), as well as their interactions, were assessed to evaluate their significance. The significant interactions , A/red fox/Netherlands/22000097-005/2022 (fox-Heemskerk), and A/red fox/Netherlands/22002636-005/2022 (fox-Oosterbeek).


Outbreaks of waterborne viruses pose a massive threatto humanhealth, claiming the lives of hundreds of thousands of people everyyear. Adsorption-based filtration offers a promising facile and environmentallyfriendly approach to help provide safe drinking water to a world populationof almost 8 billion people, particularly in communities that lackthe infrastructure for large-scale facilities. The search for a materialthat can effectively trap viruses has been mainly driven by a top-downapproach, in which old and new materials have been tested for thispurpose. Despite substantial advances, finding a material that achievesthis crucial goal and meets all associated challenges remains elusive.We suggest that the road forward should strongly rely on a complementarybottom-up approach based on our fundamental understanding of virusinteractions at interfaces. We review the state-of-the-art physicochemicalknowledge of the forces that drive the adsorption of viruses at solid–waterinterfaces. Compared to other nanometric colloids, viruses have heterogeneoussurface chemistry and diverse morphologies. We advocate that advancingour understanding of virus interactions would require describing theirphysicochemical properties using novel descriptors that reflect theirheterogeneity and diversity. Several other related topics are alsoaddressed, including the effect of coadsorbates on virus adsorption,virus inactivation at interfaces, and experimental considerationsto ensure well-grounded research results. We finally conclude withselected examples of materials that made notable advances in the field. Currently available approaches areeither of nonsufficient efficacy, come at high environmental and economiccosts, or require advanced infrastructure, and thus are not accessibleto large portions of the world population. Novel forward-looking approachesare required to meet the urgent need of providing safe drinking waterto a world population of almost 8 billion people, while simultaneouslyprotecting the environment from hazardous chemicals and greenhousegas emissions.Contaminated drinking water is responsiblefor more than 500 000deaths annually, mostly among children under 5 years old.17 Additionally, disinfection byproducts, especially when using chlorine-baseddisinfectants in the presence of natural organic matter (NOM), aretoxic to the environment and humans.19 Consequently,it is necessary to completely purify the water from NOMs to avoidthe production of these toxic byproducts, which in itself constitutesa major challenge. Moreover, the efficacy of disinfection approachesis largely compromised by the aggregation of viruses.24 Both chemical and UV disinfection come at high operational costsand expertise, making their accessibility limited to a small portionof the global population. Coagulation is driven by adding a chemical,i.e., the coagulant, that causes colloidal instability, forming largerparticles that can sediment faster. While being a relatively simpleprocess that can be utilized against a broad spectrum of colloids,its efficacy against viruses is considerably compromised due to boththe abundance of other colloids, such as NOM, and variations in waterchemistry and composition.26 The need to continuouslyfeed the system with coagulants, usually aluminum or iron salts, comesat a relatively high cost and is thus also inaccessible to a largeportion of the world population. Filtration by physical size exclusionis only possible using ultrafiltration or nanofiltration with poresizes smaller than 20 nm.27 These filtersrequire high overhead pressure, periodic back flushing, and chemicalcleaning, which all impose markedly high environmental and financialcosts.29 In the cases of coagulation and filtration,viruses are usually still infectious after treatment; they are retainedin the form of sediment or filter retentate, which, if not properlytreated, could pose a higher risk.30 Finally,none of these approaches is suitable for point-of-use (POU) applicationin developing communities. POU is the most promising water purificationstrategy in susceptible communities which lack the requisite infrastructureand expertise for large-scale water and wastewater treatment facilities,or where contamination occurs in the so-called last km, i.e., inthe water distribution systems shortly before it reaches the consumer.Traditionally, disinfection, coagulation, andultrafiltration arethe most commonly used approaches for water purification from viruses.Disinfection is typically conducted using chemical disinfectants orUV light. While regarded as one of the most effective approaches againstviruses, disinfection still shows varying efficiencies, even amongclosely related viruses with very subtle genetic and structural differences.31 Particularlyimportant is that such filters need to inactivate the adsorbed viruses,i.e., render them noninfectious, either directly upon adsorption orduring the regeneration processes.Adsorption-based filtration of viruses has emerged as an alternative,with the potential to overcome the limitations of the traditionallyused purification methods. Adsorption-based filtration is in particularcharacterized by the low energy and financial cost of operation, thelack of use of chemicals, the facile operational expertise, and thevariety of materials that could be used for building such filters.However, for it to achieve the needed global impact in fighting virusdissemination, adsorption-based filtration must fulfill many criteria:environmentally friendly, low cost, and wide availability; chemicaland mechanical stability; high adsorption capacity and efficiency;and simple regeneration and reuse.33 that could hinder bringing the viruses in close proximity to theadsorbing filter material, but primarily due to the lack of a materialthat can efficiently trap viruses. The latter is mainly attributedto the following reasons: (i) viruses cover a broad range of physicochemicalproperties; a material that would adsorb one virus might not adsorbthe other; (ii) there is an abundance of other contaminants in waterand wastewater, particularly NOM, which often compromises the efficacyof virus adsorption and retention; and (iii) the chemical propertiesof water  and the concentrationand type of other contaminants  are continuously changing, which may consequently affectthe efficacy of the clarification process.Fulfilling these criteriais a major challenge, partly due to theseveral physical and biological processesA large body of researchover the last two decades has been drivenby a top-down approach, in which various old and novel materials havebeen tested to adsorb viruses. These activities revealed several essentialmaterial properties that are necessary for high efficacy filtration.Still, the search for a material that fulfills the aforementionedcriteria remains elusive, resulting in very rare examples that madeit to real-life applications outside of the laboratory. In this work,we advocate that, for adsorption-based filtration to achieve its goal,it needs to be complemented by a bottom-up approach. Specifically,this approach must start with a fundamental understanding of the interactionsthat drive virus adsorption to solid–water interfaces, andhow this depends on the physicochemical properties of viruses andthe adsorbing surface. Such knowledge would dictate the designingprinciples for new materials to overcome the limitations of existingmaterials.This Perspective starts by providing a short introductionto waterborneviruses and how to address the challenge of extrapolating experimentalresults to the broad range of waterborne viruses. It then presentsa state-of-the-art understanding of virus interactions at solid–waterinterfaces, followed by a discussion on the existing knowledge gapsand how to address them. Afterward, a brief discussion is presentedon the potential mechanisms of virus inactivation at solid–waterinterfaces and what could still be explored to design a material thatnot only traps but also inactivates viruses. A few essential experimentalconsiderations to ensure nonambiguous interpretation of results forfuture studies are also identified. Finally, we conclude with selectedexamples of materials used for adsorption-based filtration of virusesthat made notable advances in the field.3536Viruses are infectious agents that use the cellularmachinery oftheir host, e.g., humans, animals, or bacteria, to make replicas ofthemselves. Viruses can be classified according to different criteria,including genome type, host, and structure. Here, we are particularlyconcerned with human waterborne viruses, which are mostly nonenveloped;i.e., they do not contain a lipid membrane around the proteinaceouscapsid and the encapsulated genome. The lack of an envelope contributesto their robustness, retaining their viability for extended periodsof time even under harsh environmental conditions outside of theirhost.39 For these reasons, and with only few exceptions, most virus filtrationstudies are conducted using bacteriophages,41 i.e., viruses that infect bacteria. Most bacteriophages pose nohealth risks to humans, they can be propagated to very high concentrations,and their infectivity can often be readily assessed within less than24 h; culturable human viruses, on the other hand, require between2–14 days to assess their infectivity.43Leviviridae family are the most commonlyused for filtration/inactivation research.41 The advantages offered by using bacteriophages must be consideredwith a caveat in mind: knowledge obtained using one virus is not directlytransferable to another virus; this does not only apply to knowledgetransfer from bacteriophages to human viruses, but also among bacteriophagesand human viruses themselves. Several studies have demonstrated thateven small differences between viruses, often from the same family,exhibit striking differences in their inactivation kinetics,17 interactions at interfaces,45 and purification efficiencies.47 Therefore, obtaining meaningful results without having to run virusfiltration/inactivation studies using all relevant human viruses,which is literally impossible, remains a challenge.Conducting studies with human viruses is not freefrom biosafetyrisks and is also very costly and time-consuming. In vitro propagationof human viruses to sufficiently high concentrations, relevant forfiltration studies, is in many cases not attainable within reasonabletime frames and costs. Some human viruses are not even culturablein vitro with no available infectivity assays, e.g., Noroviruses.48 While sometimes otherwiseclaimed, such reference organisms often do not represent the worst-casescenario and are usually chosen based on the availability and simplicityof propagation and quantification methods. Taking the diversity ofwaterborne viruses and the variety of filtration/inactivation mechanismsinto account, it is improbable to find one virus that represents theworst-case scenario for all of these processes. Therefore, using referenceorganisms might result in overestimating the filtration/inactivationefficiencies with adverse consequences when the tested technologiesare used out of the laboratory context. The other viable option isto study an array of viruses/bacteriophages to gain a mechanisticunderstanding of virus filtration/inactivation and correlating thesemechanisms to the physicochemical properties of the viruses. In thisway, one can use the physicochemical properties of any other virusto predict its filtration/inactivation efficiency. Indeed, severalresearch groups have followed this approach over the past decade.51 However, and despitethe advances achieved in offering a mechanistic understanding of variousvirus adsorption and inactivation processes, the predictive poweris still lacking.There aretwo options to address this challenge. The first is touse reference organisms. For example, the World Health Organization(WHO) recommends the use of Rotavirus as a reference organism forwaterborne viruses.n-octanol/water partition coefficient, kow,52 for organic moleculeshas been successfully used to predict their environmental fate, e.g.,bioaccumulation/bioconcentration,54 water solubility,56 soil/sediment attachment coefficients,57 and distribution in different cellular compartments.59 This was only possible due to the availabilityof experimentally determined kow for tensof organic molecules as well as data about their environmental fate.These data enabled the development and validation of computationalmodels that can estimate kow for new molecules,which is particularly useful for organic molecules that are challengingto investigate experimentally. Moreover, based on these data, it waspossible to establish and verify predictive models that can anticipatethe environmental fate of experimentally challenging and/or novelorganic molecules. The possibility to take most of the work from thelaboratory bench to the computer enables rapid and cost-effectivescreening of a large number of molecules with a fraction of the costand time required if this work was to be done experimentally. It isalso important to note that, for some organic molecules, experimentalassessment is practically impossible.To better identify the key requirements forachieving this predictivepower, we take inspiration from predicting the environmental fateof small organic molecules. For example, the development of the n-octanol/water partition coefficienthighlights the key hurdle to developing predictive models for viruses,i.e., the limited number of experimentally accessible viruses, whichimpedes the development and validation of broadly applicable models.With only few exceptions,61 most virus studies are conducted using oneto three viruses. These viruses are either selected to be as diverseas possible, e.g., different genome type, size, and morphology,60 or as similar as possible with systematic variation in their physicochemicalproperties.45 The former offers an overview of the diversity in virus interactionsbut can hardly yield any mechanistic understanding. The latter, dueto the similarity of the chosen viruses, could unintentionally resultin a biased mechanistic understanding of virus interactions.63 Additionally, even carefully selected viruses with systematic variationin their physicochemical properties still exhibit variations in multipleproperties simultaneously, e.g., charge and hydrophobicity.45 Selective variation of one property,e.g., charge, while maintaining all other properties fixed is a prerequisitefor attaining a detailed quantitative understanding of virus interactions.Unlike synthesized organic molecules, naturally existing viruses donot offer such versatility. Genetic engineering of viruses, particularlybacteriophages, has long been a difficult and labor-intensive process,65 hindering the production of bacteriophages with tailored propertiesfor virus studies. Recent advances in bacteriophage engineering streamlinedthis process, enabling targeted gene editing of bacteriophages withinless than a week.67 We envision future virus studies to be conducted using an arrayof genetically engineered bacteriophages to pinpoint the key mechanismsof interactions, as well as build predictive models that could beused to anticipate the interactions of human viruses.The example of the The designof genetically engineered bacteriophages has to be guidedby the aim of understanding how virus composition and structure relateto virus adsorption and inactivation. In the following section, wediscuss selected virus interactions that are crucial for virus adsorptionand inactivation at interfaces. We pay particular attention to knowledgegaps and how such gaps could potentially be filled by utilizing engineeredbacteriophages and/or other approaches.88 and the hydrophobic effect.95 Many of the adsorption-based filters intentionally or nonintentionallyexploit these two interactions to drive the adsorption of viruses.Most waterborne viruses are thought to carry a net negative chargeat circumneutral pH values.96 It is, therefore,logical to use positively charged material to trap the negativelycharged viruses. While this approach has shown promising results,74 noticeable variation in efficacy acrossdifferent viruses has been observed,79 raising doubts about its efficacy against the broadrange of waterborne viruses and under varying water chemistry. Inaddition, both positively charged and hydrophobic surfaces are susceptibleto competitive adsorption from other adsorbates, particularly NOM.97 More innovative approaches are needed in order to circumvent thesechallenges. For instance, it has been shown that some viruses stilladsorb to negatively charged surfaces under weak electrostatic repulsiveconditions,44 while no detectable adsorptionfor NOMs was observed under the same conditions.97 These results point to a potential approach for developingfiltration membranes that are repulsive to competitors, e.g., NOM,while still attractive to viruses. However, successful developmentof such a membrane or other ones would only be possible through aclear fundamental understanding of the effects of electrostatic interactions,the hydrophobic effect, water chemistry, and other adsorbates on virusattachment at solid–water interfaces. As previously mentioned,such understanding has to be complemented by reliable tools that canin-silico predict the interaction and thus filtration efficienciesof the broad range of human viruses based only on their structure.In the following subsections, we discuss these four key topics thatare relevant for virus adsorption, focusing on existing knowledgegaps and how to address them.Viruses are not motile; i.e., they are not capable of activelymoving by themselves. Their mobility outside and inside of their hostis solely driven by external forces and their surface interactions.From a physical chemistry point of view, they can be considered asabiotic colloids. A large body of literature suggests that virus interactionsto solid–water interfaces are primarily driven by electrostaticforces100 In comparison to synthetic nanoparticles, which frequently exhibituniform surface charge, viruses possess a rather complex charge character,104 are advantageous over methods that distinguish based oncharge only. The results of the latter are often compromised by impuritiesin virus solutions.106 But even when using the properexperimental approach, there are still two key challenges that hinderthe use of experimentally determined charges. The electric field thatdrives the electrophoretic mobility of viruses acts upon the wholevirus and thus reflects the sum of charges from different componentsof the virus (The isoelectric point (IEP) of viruses is oftenused as a physicochemicaldescriptor to rationalize and speculate about the electrostatic interactionsof viruses.he virus 2b. Convehe virus 2b. There80 This approach, however, showedlarge discrepancies compared to experimentally determined chargesand to virus surface interactions.80 For example, basedon such calculations, the bacteriophage MS2 is expected to carry anet positive charge at pH 6; however, at the samepH value, it shows strong repulsion from negatively charged surfaces,strong adsorption to positively charged surfaces, and electrophoreticmobilities indicative of net negative charge.44For these reasons, severalattempts have been made to find a calculabledescriptor for the charge of viruses. Inspired by globular proteins,initial attempts used the sequence of capsid proteins to calculatethe charge of ionizable amino acids at different pH values.107 This theory is experimentally supported by observations of differentelectrophoretic mobility between virus and virus-like particles (VLPs)without a genome.105 These results were,however, not consistent across literature, and some studies have alsoshown that VLPs had very similar adsorption behavior to that of genome-containingviruses.108 Despite the growing evidence that the genomecontribution toward electrostatic interactions might be limited ornegligible,62 whether this is also thecase for viruses of different genome sizes and types remains to beconfirmed. This is apropos because most of the aforementioned studieswere conducted using small ssRNA viruses from the Leviviridae family.Two main theories emerged to explain this inconsistency. Thefirstsuggests that there are contributions from the viral genome towardthe net charge of viruses.80 The use of this approach requiresknowledge of the 3D structure of the virus, as well as a method toidentify the amino acid residues that reside on the outer surfaceof the capsid. Due to advances in structural biology techniques, thenumber of high-resolution 3D structures of viruses is increasing exponentially.109 Surprisingly, identifying amino acid residueson the outer surface of the capsid turn out to be the main challengetoward applying this approach; computational sorting of amino acidresidues using tools, such as CapsidMap,110 does not agree with manual sorting using protein visualization softwares.44 This inconsistency calls for developing andverifying new computational approaches that can rapidly identify theposition of amino acid residues for the hundreds of available virusstructures without the need for tedious manual work. To circumventthe need for the 3D structures and identifying the position of aminoacids, Heffron and Mayer63 suggested usingthe capsid protein sequence after excluding the known and predictedgenome binding regions. While considering the 3D structure of virusesis inevitable, if we aim at obtaining a refined descriptor of viruscharge, the results of Heffron and Mayer highlighted a potential biasin previous literature, which mainly relied on Leviviridae viruses.63 Unlike many other viruses, thegenome of Leviviridae viruses binds extensively tothe inner surface of the capsid protein, which could have contributedto the success of using the charge from the outer surface of the capsidsin previous studies.80 For proper development of a chargedescriptor, future investigations need to consider a broader rangeof viruses.A second, more plausible, theory is that the chargecharacteristicof viruses is dominated by the ionizable amino acids on the outersurface of the capsid, with negligible contributions from the buriedamino acids, the ones on the inner side of the capsid, and the genome.Calculating viral charge based on the amino acids of the outer capsidshowed a very promising correlation with electrostatic virus interactionsat interfaces.The above-mentioned computational approaches haveconsidered theeffect of neither the spatial distribution of charges nor the surfacemorphology of viruses. Positively and negatively charged moietiescan have different spatial distributions on virus surfaces, e.g.,clustered or spread out. Such variation in distribution could occurin both single-protein capsids and multiprotein capsids. To the bestof the authors’ knowledge, the effect of the spatial distributionof the charge has not yet been investigated. In addition, virusesfeaturing uneven surfaces, such as Adenoviruses, Rotaviruses, andAstroviruses, present another challenge concerning how much thesefeatures contribute to surface interactions compared to the rest ofthe capsid proteins. Such relative contributions are also expectedto vary with the thickness of the electrical double layer, which itselfdepends on the water chemistry.Taken together, in order tomake significant advances in our understandingand prediction of electrostatic virus interactions, it is necessaryto develop and validate a calculable physicochemical descriptor forthe charge of viruses. This descriptor has to reflect the 3D distributionof the charged moieties, taking into account their radial position,spatial distribution, and virus morphology. Achieving this end goalis practically bound to engineering bacteriophages for which eachof the discussed parameters, e.g., spatial distribution of chargeand virus morphology, can be varied systematically while maintainingthe other parameters unchanged.44 Developing a quantitativeframework/expression to describe the contribution of the hydrophobiceffect to virus adsorption is, however, more challenging than in thecase of electrostatics. Electrostatic interactions are theoreticallywell-explained with expressions accurately describing the energy andforces of the electrostatic double layer for different geometries.111 Such quantitative theoretical description ofthe hydrophobic effect is still missing; the available theoreticalformulations lack the broad consensus in the scientific community.Research over the last few decades, however, is slowly revealing thephysical origins of the hydrophobic effect.121 A quantitative description of the distance dependence of the hydrophobiceffect has largely been impeded by experimental challenges;120 e.g., reports about the effective range ofthe hydrophobic effect varied between approximately ten nanometers118 and a few micrometers.122 Currently, it is thought that the decay length of theseinteractions is in the range of ≤2 nm and its effective rangeis up to a few tens of nanometers.120 Equationsdescribing the distance-dependent hydrophobic interaction potentialshave been suggested for interacting planar surfaces and nanoparticles.125 These advances will hopefully lead to a unifying theoretical formulationto describe the hydrophobic effect. However, it is crucial to pointout that, like electrostatic interactions, these formulations arebased on homogeneous, smooth surfaces and nanoparticles. Therefore,in addition to the lack of a unifying theoretical formulation, theheterogeneous surface chemistry and morphology of viruses presentanother hurdle, as discussed for electrostatic interactions. Thesetwo challenges have several implications for assessing the contributionsfrom the hydrophobic effect toward virus interactions, which willbe discussed hereafter.The hydrophobic effect contributessignificantly to virus adsorption at interfaces, resulting in virusadsorption even under electrostatic repulsion between negatively chargedviruses and surfaces.126 With very rare exceptions,127 their application to study viruses is almostnonexistent, likely due to several experimental challenges. This ledto the development of new experimental approaches that are more suitablefor ranking viruses according to their hydrophobic character, e.g.,adsorption to hydrophobic surfaces89 and SDS-modifiedcapillary zone electrophoresis (CZE).103 Adsorption to hydrophobic surfaces also includes electrostatic contributions,because hydrophobic surfaces acquire a pH-dependent negative chargein aqueous environments,130 thus rendering ambiguous rankingthat reflects both the hydrophobic and electrostatic character ofthe viruses. SDS-modified CZE relies on the observation that the electrophoreticmobility of viruses shifts toward more negative mobility in the presenceof SDS,131 which is attributed to bindingof the SDS to hydrophobic patches on the surface of the viruses exposingits negatively charged polar end, thus increasing the negative chargeof the viruses. While offering a promising experimental tool to elucidatevirus hydrophobicity, further assessment and validation might be neededbefore being widely accepted by the research community. In particular,the potential bias if SDS binds with its negatively charged end tothe positively charged moieties on the surface of the virus, renderingthem neutral; this would erroneously attribute viruses with higherpositive-charge densities as more hydrophobic.Both experimental and computationalattempts to quantify the hydrophobic character of viruses yield anempirical qualitative ranking, rather than a quantitative descriptor.Experimental methods available to study hydrophobicity include microbialadhesion to hydrocarbons (MATH), hydrophobic interactions chromatography,reverse-phase chromatography, ANS fluorescence, and aqueous two-phasesystems (ATPS).134 and calculates a moving averagebased on the sequence of the capsid proteins.103 Such calculations can be readily performedusing the ProtScale application on the Expasy server.136 This approach yields what is known as the hydropathy index plots.These plots, however, offer no quantitative measure of the hydrophobicityof viruses, thus hindering direct and quantitative comparisons ofdifferent viruses. Another approach relies on calculating the ratioof apolar (hydrophobic) solvent accessible surface area (SASA) tothe total SASA of the virus capsid. When applied to four differentviruses of the Leviviridae family, this approachyielded almost identical values for the four viruses, despite theclear difference in their surface interaction with hydrophobic surfaces.44 Moreover, no improvement was observed when thisapproach was applied only to the amino acids on the outer side ofthe capsids.44 A more intricate approachwas achieved by applying a scoring system that exponentially increaseswith the area of each apolar patch on the outer surface of the viruscapsid,137 in which the total hydrophobicscore is equivalent to the sum of the scores of all hydrophobic patcheson the outer surface of the capsid. When applied to the same fourviruses, the calculated score could very well explain the hydrophobicinteractions observed in virus adsorption experiments.44 Further verification of this approach is stillneeded, ideally using engineered bacteriophages that have the sameamino acid residues on their surfaces but distributed differentlyto form hydrophobic patches of different sizes. Additionally, a representativephysicochemical descriptor for the hydrophobic character of viruseswould also need to reflect the surface morphology of the viruses.Even when usingsuch novel approaches, the challenges discussedwith experimental determination of virus charge also apply to hydrophobicity:(i) measured values reflect the hydrophobicity of the whole virus,while virus adsorption to surfaces might be dominated by single componentsof the virus or a small patch on the virus surface; and (ii) experimentalresults are unattainable for many human viruses. Three computationalapproaches have been proposed to assess the hydrophobic characterof viruses, the earliest of which assigns different scoring indicesfor each amino acid residue84 Changes invirus and surface charges with pH have a substantial effect on virusadsorption to interfaces;139 for example, while MS2 showedno detectable adsorption at pH > 6 to a negatively charged surfacecomposed of carboxyl-terminated self-assembled monolayers, it extensivelyadsorbed to the same surface at pH 5.44 Ionic strength also has a well-elucidated effect on electrostatics;the increase in ionic strength screens the electric double layer andthus shields electrostatics. This is particularly manifested in thecase of electrostatic repulsion, in which the increase in ionic strengthresults in shielding the repulsive forces, allowing viruses to attachto the surface via interactions, such as the hydrophobic effect.138 Changes in ionic compositionalso influence virus adsorption; arguably, the most striking changein virus interactions occurs when multivalent cations are added orremoved from solution.143 For instance, the addition of 1 mM Ca2+ increased theadsorption efficiency of MS2 to NOM by almost 2 orders of magnitude.140 Multivalent cations are orders of magnitudemore efficient than monovalent ones in reducing the electric potentialat interfaces; e.g., a CaCl2 electrolyte will be approximately100 times more effective in reducing the potential than a NaCl electrolytewith the same molar concentration.111 Notonly do multivalent cations affect the electric potential of chargedentities, but they can also bind to negatively charged moieties, thusaltering the surface charge and reducing the (negative) electric potentialfurther.111 This binding can mediate whatis known as cation bridging, in which a cation bridges two negativelycharged moieties, e.g., two carboxyl groups on the surface of a virusand an adsorbent. It has been speculated that cation bridging occursonly with specific negatively charged moieties, particularly carboxylgroups.84 For waterborne viruses, Mg2+ and Ca2+ are the two most relevant multivalentcations due to their abundance in surface water.144 Initial reports suggested that Ca2+ is moreefficient than Mg2+ for cation bridging.140 Using locally high concentrations of divalent cations atthe interface between the filtration material and water is a potential,but unexplored, approach to enhance adsorption-based filtration ofviruses. However, certain knowledge gaps about the role of divalentcations need to be addressed first. (i) The specificity of cationbridging to carboxyl groups needs to be verified. (ii) If cation bridgingis specific to carboxyl groups, does this imply that the higher thedensity of these groups, the larger the capacity of virus adsorption,or would electrostatic repulsion override the effect of the cations?(iii) What happens to the viruses under depletion conditions of thedivalent cations? Do they desorb or remain attached, and how is thisaffected by the physicochemical properties of the adsorbent? (iv)Is Ca2+ indeed more efficient than Mg2+? Ifyes, in which molarity range is this difference noticeable?The effect of water chemistry, i.e.,pH, ionic strength, and ionic composition, on electrostatic interactionsis well documented. The change in pH alters the protonation stateof ionizable moieties, thus changing the charge of both viruses andadsorbing surfaces; e.g., as pH decreases, more moieties are protonated,thus decreasing the net negative charge of a virus and/or a surface,provided that both are above their IEP.130 which is also reflected in virusadsorption to hydrophobic surfaces.44 Thisis worth noting because, taking into consideration the very diversescientific community that studies virus adsorption, this phenomenonmight go unnoticed.It is important to mention that solution chemistry affects not onlyelectrostatic interactions but also the hydrophobic effect. Hydrophobicsurfaces tend to acquire a net negative charge in aqueous environments.This charge depends on pH, ionic strength, and ionic composition,i.e., multivalent cations,148 NOM possesses a net negative charge at circumneutral pH values,149 thus likely exhibiting comparable electrostaticinteractions to many viruses. Several studies have demonstrated thatthe presence of NOM suppresses virus adsorption.153 This effect was, however, virus-dependent: for example, while MS2adsorption to soil minerals and sand was suppressed in the presenceof NOM, little to no effect was observed in the case of ΦX174.151 The mechanism by which coadsorbates affectvirus adsorption was neatly demonstrated using in situ adsorptionexperiments to positively charged surfaces of four different virusesthat exhibit different affinities to NOM.45 The results showed that viruses, e.g., MS2, that show net repulsiveinteractions with NOM exhibit markedly reduced adsorption in the presenceof NOM, whereas, viruses, e.g., Qβ, that show net attractiveinteractions with NOM are only minimally affected by the presenceof the NOM  model for a binary systemof adsorbates, the study also revealed the significant effect of minorinteraction details on the adsorption capacity of viruses, e.g., theminimum contact area required for virus attachment to the surfaceand the unfolding, i.e., spreading out, of NOM upon adsorption tosurfaces.In our discussion of the effectof coadsorbates on virus adsorption, we mainly consider NOM, whichis arguably the most abundant and ubiquitous coadsorbate in waterand wastewater. the NOM 3.45 As aThe suppressive effect of NOM on virus adsorptionpresents a massivechallenge for adsorption-based filtration. Novel and innovative ideasto overcome such challenge will likely emerge if a deeper understandingis obtained of multi-adsorbate systems in complex environments. Forexample, little is known about how controlling the size and patternof attractive patches on a surface would affect the adsorption kineticsand capacity of viruses in such systems.155 This is often mediated by chemical reactions withreactive oxygen species (ROS) that are present with locally high concentrationsin very close proximity to such surfaces and rapidly decay even atshort distances from the surface.155 However,the production of ROS often requires sunlight or the addition of chemicals,such as H2O2.158 The need for light exposure or the addition of chemicals complicatesthe use of adsorption-based filters and runs counter to the originalobjective of developing facile technologies that can be used for POUwater treatment. Some research results have indicated that stronginteractions between viruses and surfaces could potentially resultin virus disintegration and thus inactivation.154 Based on experiments with isotopicallylabeled viruses, Murray and Laband100 postulatedthat van der Waals interactions between Poliovirus and CuO could resultin virus disintegration. Using a similar approach, Ryan et al.154 suggested that strong electrostatic interactionsof PRD1 and MS2 with iron oxides can cause virus disintegration. Gutierrezet al.79 recovered only 2% of infectionRotavirus after interaction with Hematite; in comparison, 64% of infectiousMS2 was recovered. TEM images suggested that Rotavirus might havebeen damaged by its interactions with Hematite. Conversely, in situadsorption experiments of MS2, fr, GA, and Qβ to positivelycharged surfaces, under conditions favoring very strong attractiveinteractions, have shown no signs of viral disintegration.44 Unequivocal evidence of virus disintegrationdue to physical interactions only is still missing, as well as themechanism behind such disintegration. Indeed, the search for a surfacethat can spontaneously inactivate nonenveloped viruses should be unrelentinglypursued. Future research should also investigate the potential synergeticeffect of various parameters, such as surface roughness, charge, andhydrophobicity, on virus inactivation. Identification of surface propertiesthat have virucidal effects would constitute a seminal advancementin our fight against waterborne viruses.Virus adsorption tocertain surfaces, such as iron (oxy)hydroxides,has been shown to accelerate virus inactivation.The scientific communitythat is interested in virus adsorptionto solid–water interfaces is a very broad one, including, amongothers, material scientists, environmental chemists, microbiologists,food scientists, virologists, and physicists. In this section, webriefly discuss a few experimental considerations to assist in avoidingmisinterpretation of experimental results.106 size distributions,106 and adsorption behavior44 of viruses.The effect of impurities on virus adsorption is mechanistically similarto the effect of coadsorbates depicted in 44High purity virus solutions area prerequisite for successful physicochemical characterization ofviruses and for mechanistic adsorption studies. It has been previouslyshown that different purification protocols result in different electrophoreticmobilities,46 While some studies claim that using the CsClgradient yields the highest purity,106 there arenot enough studies to reach a generic recommendation. Variations inthe rigor of experimental practice could also compromise the purityof the virus solution, even when using the same protocol. Furthermore,it has been reported that viruses might disintegrate over time, evenwhen stored in the dark at 4 °C, thus interfering with mechanisticadsorptions studies.44 Irrespective ofthe purification protocol, it is, therefore, recommended to periodicallyrun purity control experiments, e.g., adsorption experiments underconditions of well-known theoretical and experimental results.44Various protocolsexist for virus propagation, purification, and concentration, themost used of which are PEG precipitation, CsCl density gradient centrifugation,and buffer washing using dialysis or centrifugal ultrafiltration.Some protocols also comprise a combination of these methods.50). In the plaque assays, the virus-containingsolution is plated with the host cells in a Petri-dish; after thedue incubation period under optimized conditions for cell growth,the number of plaques, i.e., spots where cells were lysed by infectiousviruses, is counted. Each plaque represents one infectious virus,and the virus concentration is expressed as plaque forming units (PFU)/ml.A special variation of plaque assays is the focus forming assay (FFA),which is commonly used for viruses that do not lyse the host cellsand thus do not form plaques. FFA utilizes an immunostaining techniqueusing fluorescently labeled antibodies that are specific to one ofthe viral proteins. Infected cells will exhibit a high virus concentrationin their vicinity, forming clusters (foci). Virus concentrations arethus represented in focus forming units (FFU)/ml. TCID50 is an end point assay in which the end point reflects the dilutionthat results in killing 50% of the hosts. TCID50 is assessedby either monitoring the death of the host, e.g., laboratory rats,or monitoring the cytopathic effect of host cells; it is representedas TCID50/ml. Both plaque assays and TCID50 areperformed for a dilution series of the solution under consideration.The large majority of virus filtration/inactivation studies reportvirus infectivity only, either in PFU/ml or TCID50/ml,which is the most relevant parameter when assessing the efficiencyof the filtration/inactivation approach and the safety to consumethe effluent water. The efficiency of filtration is often reportedas log reduction values (LRV) in the virus concentration of the effluentover the influent concentration, where 1 LRV is equivalent to 90%filtration efficiency, 2 LRV ≡ 99% filtration efficiency, 3LRV ≡ 99.9% filtration efficiency, etc.Virussolutions contain several populations: infectious viruses, noninfectious/genome-containingviruses, and noninfectious/genome-free viruses 4a. The t46 As viruses get inactivated, the ratio ofinfectious viruses to the genome count decreases; this ratio is particularlyimportant when evaluating the efficiency of virus inactivation byany approach. Viruses could get inactivated while passing throughthe filter, e.g., due to interaction with dissolved ions. They couldalso get inactivated after attaching to the filter media. In orderto assess the inactivation of attached viruses, it would be necessaryto first elute them and assess their infectivity and genome count.Elution efficiency varies based on the virus and the adsorbent.160 Assuming that there is no preferential adsorption or elution ofinfectious or noninfectious viruses, the decreasein the ratio of the infectious viruses to genome count in the effluentor the eluent is a strong indicator of virus inactivation , inwhich a segment of the viral genome is replicated through PCR cyclestaking place in a thermocycler. The number of cycles required to achievea detectable concentration of the genome is then used to calculatethe genome count in the original sample based on a calibration curve.It is important to note that the genome count of lab-propagated virusesis often 1 to 3 orders of magnitude higher than the number of infectiousviruses.tivation 5.While these are the most common approaches to quantityvirus concentration,many other different techniques also exist, e.g., cryo-transmissionelectron microscopy (cryo-TEM) to count the number of virus particles,protein assays to determine the protein concentration of a virus,enzyme-linked immunosorbent assay (ELISA) to determine the concentrationof an antibody-binding virus protein, and tunable resistive pulsesensing (TRPS) for counting virus particles. Each of these approachesmeasures a different aspect of the virus concentration.40 Most of these filters take one of three forms:solid porous media,161 filtration columns,164 and thin membranes166 ,166 cellulose,73 glass fibers,79 or amyloid fibrils.46 The latter comprises the adsorption sites for virus attachment andis often made of metal oxide coating or nanoparticles, for example,magnesium oxyhydroxide,71 copper and copperoxides,166 and iron oxyhydroxide.46 In the following section, we will briefly discuss threerecent innovations which achieved notable advances in the field; acomprehensive literature review of adsorption-based filters has beenrecently covered by Sellaoui et al.40Numerousfilter materials have been tested to trap waterborne viruses.ranes166 6. Withv72 producednovel porouscarbon (NPC) from rice husk by silica removal, followed by steam activationand acid rinse. The material could be considered as a special formof activated carbon (AC). Conventionally produced AC often shows lowfiltration efficacy of viruses but is particularly effective againstsmall molecule contaminants. The results of Canh et al. showed a significantimprovement over previous attempts with AC, reducing the incubationtime from 8 h to 60 min while achieving approximately 5 LRV of MS2.The experiments were also unintentionally conducted in the presenceof 6.7 mg/L T0C; while being a very low concentration, it shows thatthe NPC could still be effective under minor competitive adsorptionconditions from dissolved carbon. Particularly important is that thematerial is produced from rice husk and thus is in full accordancewith the global need for waste valorization.Canh et al.46 produced a viral trapmade of amyloid fibrils and iron oxyhydroxide nanoparticles. The materialshowed more than 5 LRV of MS2 and approximately 0.5 LRV of Enterovirus71. The material seems to also have a partial inactivation effecton MS2; while the inactivation effect is not pronounced, it presentspromise for further optimization to reach higher efficiencies of inactivation.The material offers one of the most environmentally friendly approachesto water filtration. Its main component is β-lactoglobulin (BLG),which is derived from byproducts of the dairy industry. The secondcomponent of the material is iron oxyhydroxides, which is a naturallyexisting material with no toxicity to humans or the environment. Inaddition, the production of the material requires very little energyand no use of harsh chemicals or petrochemicals. A closely relatedmaterial, amyloid fibrils-carbon hybrid, has been previously usedto filter a broad range of contaminants, including heavy metal ions,metal cyanides, and nuclear waste,167 openingthe possibility for broader applications of a further developed versionof the material virus filtration and beyond.Palika et al.75 recently developeda granular material with a ceramic core coated with MgAl204. The material showed high efficiency in removal ofMS2 and fr, in which only 4 g of the material was enough to achieve>7 LRV of 1 L of virus-contaminated water. The efficiency remainedthe same for fr, even when filtering 2 L; for MS2, the efficiencywent down to approximately 4.5 LRV when filtering 2 L of virus-contaminatedwater. The granules rely on low-cost raw materials and are chemicallyand mechanically very stable. Their high stability enables regenerationof the material by backing at 400 °C. While this is a relativelyhigh temperature and the real extent of regeneration needs to be furtherassessed, the possibility to regenerate virus filters by applyingheat only, without the need for chemical cleaning, is a major steptoward sustainable filtration solutions.Yüzbasiet al.Adsorption-based filtrationoffers a propitious approach to addressthe challenge of providing safe drinking water for an enormous worldpopulation of almost 8 billion people, with minimal, if any, negativeimpact on the climate crisis and the environment. Despite the progressachieved through years of research and assessment of different materials,a filter that can meet all the expectations is still missing. We arguethat achieving substantial progress toward developing such filterswould be built on a better fundamental understanding of virus interactionsand inactivation at solid–water interfaces. Achieving suchan understanding entails adopting new descriptors for the physicochemicaldescriptors of viruses, advancing beyond traditionally used descriptorsthat were mainly developed to describe homogeneous, smooth, and sphericalnanoparticles. Viruses are, in contrast, chemically heterogeneouswith very diverse morphologies. To this end, it is necessary to harnessthe power of newly developed microbiological tools, in particularthe versatility offered by advances in targeted gene engineering ofbacteriophages. Additionally, the power of computational tools hasto be exploited in order to use such physicochemical descriptors topredict the efficacy of newly developed filters for the broad varietyof human waterborne viruses. Ultimately, top-down and bottom-up approachesfrom the molecular to the colloidal scale will have to be successfullycombined to meet all the challenges associated with the design ofefficient waterborne virus traps.


Bunyavirales and Mononegavirales) containing nine families . Among these TBVs, some are very pathogenic, causing huge mortality, and hence, deserve to be covered under the umbrella of one health. About 38 viral species are being transmitted by <10% of the tick species of the families Ixodidae and Argasidae. All TBVs are RNA viruses except for the African swine fever virus from the family Asfarviridae. Tick-borne viral diseases have also been classified as an emerging threat to public health and animals, especially in resource-poor communities of the developing world. Tick-host interaction plays an important role in the successful transmission of pathogens. The ticks' salivary glands are the main cellular machinery involved in the uptake, settlement, and multiplication of viruses, which are required for successful transmission into the final host. Furthermore, tick saliva also participates as an augmenting tool during the physiological process of transmission. Tick saliva is an important key element in the successful transmission of pathogens and contains different antimicrobial proteins, e.g., defensin, serine, proteases, and cement protein, which are key players in tick-virus interaction. While tick-virus interaction is a crucial factor in the propagation of tick-borne viral diseases, other factors  are also involved. Some immunological factors, e.g., toll-like receptors, scavenger receptors, Janus-kinase (JAK-STAT) pathway, and immunodeficiency (IMD) pathway are involved in tick-virus interaction by helping in virus assembly and acting to increase transmission. Ticks also harbor some endogenous viruses as internal microbial faunas, which also play a significant role in tick-virus interaction. Studies focusing on tick saliva and its role in pathogen transmission, tick feeding, and control of ticks using functional genomics all point toward solutions to this emerging threat. Information regarding tick-virus interaction is somewhat lacking; however, this information is necessary for a complete understanding of transmission TBVs and their persistence in nature. This review encompasses insight into the ecology and vectorial capacity of tick vectors, as well as our current understanding of the predisposing, enabling, precipitating, and reinforcing factors that influence TBV epidemics. The review explores the cellular, biochemical, and immunological tools which ensure and augment successful evading of the ticks' defense systems and transmission of the viruses to the final hosts at the virus-vector interface. The role of functional genomics, proteomics, and metabolomics in profiling tick-virus interaction is also discussed. This review is an initial attempt to comprehensively elaborate on the epidemiological determinants of TBVs with a focus on intra-vector physiological processes involved in the successful execution of the docking, uptake, settlement, replication, and transmission processes of arboviruses. This adds valuable data to the existing bank of knowledge for global stakeholders, policymakers, and the scientific community working to devise appropriate strategies to control ticks and TBVs.Ticks  are the second most important vector for transmission of pathogens to humans, livestock, and wildlife. Ticks as vectors for viruses have been reported many times over the last 100 years. Tick-borne viruses (TBVs) belong to two orders ( Pests are causing damage to our lives. Several organisms act as vectors and transmit or cause diseases in the agriculture sector, humans, wildlife, and livestock. Ticks (Acari: Ixodida) are blood-sucking ectoparasites and act as vectors of pathogens infecting livestock, wildlife, and humans across the world , bacterial , viral , and rickettsial  diseases and Lyme disease , also known as tibo viruses, constitute various viruses that are transmitted successfully between two different environments. These are the host environment, the stable one, while the other is the opposite of stable, i.e., tick internal environment  and eight RNA virus families    and tick saliva pass through a common buccal canal. Tick saliva originates from branched and paired alveolar salivary glands, which are present anterolaterally and extend into the posterior sides of the body. From each salivary gland, a duct originates that enters a broad shallow tube, i.e., salivarium. This tube lies above the pharynx in the pharyngeal region valve and opens into the food canal at the anterior opening of the pharynx  holocyclus, type 2 female alveoli contain only two granular cell types , along with some granular cells. Type 3 alveoli are located in the posterior and peripheral regions of the glands  savignyi  spinigera. Type 4 alveoli are not distinguishable in larval stages  americanum , and size of salivary glands takes place  at the feeding site and enhances the tick feeding process  samguneus, I. scapularis, R. microplus, I. ricinus, and A. americanum , and D2; among these, A2/B2 is considered to be derived from PGE2 because of the alkaline nature of tick saliva  in the Ixodid species, and is involved in change in exosomal origin   in I. scapularis  asiaticum  in A. americanum  exhibit biological transmission: they enter into the vector, infect, and replicate before reaching the vertebrate host. Following the entry of a virus into the midgut of ticks, it must escape from the midgut and reach the tick salivary glands from which it will be transmitted to the vertebrate host  cell infection and replication and (ii) virus release. Although there are some records of virus detection in saliva, little is known about this critical stage of tick-borne virus transmission Nuttall, . ExperimIxodes persulcatus  does not follow these dynamics  . It is now recognized that tick-borne viruses can be transmitted effectively even when an infectious virus is not detectable in the blood Turell, .via the lymphatics to the thoracic duct and into the bloodstream. Primary viremia seeds are extraneural tissues that support further virus replication and shedding of the virus into circulation. In studies on mosquito-borne viruses, the threshold level of viremia was defined as the lowest amount of virus capable of causing an infection in around 1 to 5% of the vector population feeding on the viraemic host  infected by the bite of a tick infected with tick-borne encephalitis virus, the virus replicates first in the skin site of tick feeding and in lymph nodes that drain the site. Neutrophils, monocytes/macrophages, and Langerhans cells attracted to the tick feeding site become infected  were considered not to play a significant role in the transmission cycle of louping ill virus  and bank voles (Myodesglareolus) demonstrated efficient transmission in the absence of viremia or at comparatively low viremia levels. In contrast, pine voles (Pitymyssubterraneus), which developed high levels of viremia, produced only a few infected ticks . Uninfected I. ricinus nymphs became infected with the virus when co-feeding with infected ticks, while the hares showed only low or undetectable levels of viremia  from mothers as transovarial transmission was <5 percent  of ticks . The biocontrol potential of entomopathogenic fungi for tick control has been examined in various laboratory bioassays (Ebani and Mancianti, Natural enemies of ticks include parasitoid wasps, insectivorous birds, nematodes, R microplus. Similarly, M. anisoplae can also aid in the management of the tick population with relatively fewer adverse effects on the environment. Moreover, it has been reported as potentially effective against a wide range of arthropods and, thus, can cause the death of non-target species (Azagi et al., Ixodiphagus hookeri wasps can parasitize various forms of ticks, such as larvae and nymphs. It has been suggested that the odor from tick host animals attracts parasitoid wasps (Sormunen et al., Various species of fungi were reported as pathogenic to a wide range of tick species and cause high mortality in susceptible species, such as B. thuringiensis, exhibit mortality in ticks, specifically the larval form of ticks, and higher mortality rates were recorded with an increase in spore concentration. This control strategy offers potential for the control of ectoparasites (Ebani and Mancianti, Some entomopathogenic bacteria, such as RNA interference is an extensively used gene silencing technique for the genetic manipulation of ticks. It has been proved as an effective tool to identify and characterize tick-pathogen interference, tick protective antigens, and screening. It is a nucleic acid-based reverse genetic method used to determine gene function and its possible effects on the metabolic pathway by disrupting gene expression. Four methods, namely, injection, soaking, feeding, and virus production, of dsRNA have been implied to deliver dsRNA for RNA interference in ticks (Niu et al., Through this technique, a large number of genes can be recognized as potent candidates for a vaccine. This approach is relatively cheap and requires minimum use of laboratory animals. Selected antigens, after characterization and evaluation, can be produced as recombinant proteins that can be used for vaccine trials (Sudhakar et al., Based on the above-mentioned discussion, it can be concluded that tick-borne viruses are a major threat to public health, and tick-virus interaction is the key point of the spread of these infections. Different factors both from the side of ticks and viruses are involved in virus replication and blockage in tick saliva and midgut. By controlling/ modifying the proteomics of tick saliva, transmission routes, and vector control strategies, the damage caused by tick-borne viruses can be minimized. We are hopeful that this review will enhance the public perception regarding these viruses and tick-virus interaction and will provide insight into future investigations regarding their control using the factors involved at the tick-virus interaction level.All authors equally contributed in manuscript writing and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.