The immunosensor was used for the determination of chlortoluron in natural groundwaters, showing the absence of matrix effects.Chlortoluron (3-(3-chloro-p-tolyl)-1,1-dimethyl urea) is an herbicide widely used in substitution to isoproturon to control grass weed in wheat and barley crops. Chlortoluron has been detected in groundwaters for more than 20 years; and dramatic increases in concentrations are observed after intense rain outbreaks. In this context; we developed an immunosensor for the determination of chlortoluron based on competitive binding of specific monoclonal antibodies on chlortoluron and immobilized biotinylated chlortoluron; followed by electrochemical detection on screen-printed carbon electrodes. The optimized immunosensor exhibited a logarithmic response in the range 0.01–10 µg·L The use of pesticides has exponentially increased since their creation to improve crop yields, in response to a constant increase in the world population ,2. Curre−1 were reported in surface waters close to oyster farms in the Pertuis Charentais (France) [−1 for individual pesticides and their relevant metabolites and 0.5 µg·L−1 for the total amount of pesticides [Chlortoluron has been detected in groundwaters for more than 20 years, and dramatic concentration increases have been observed during years with the most rainfall events . Recentl(France) . Many resticides ,15. Monisticides . Due to sticides ,18. Multsticides  or ultrasticides . These lsticides . These d−1 level [Chlorella vulgaris microalgae cells was described for the detection of simazine, atrazine, isoproturon, and diuron, but LOD values below the threshold value were observed for only diuron and isoproturon [Dictyosphaerium chlorelloides, Scenedesmus intermedius, and Scenedesmus sp.) entrapped in silicate sol-gel matrices were used for simazine, atrazine, propazine, terbuthylazine, and linuron with LOD values 10-fold higher than the legal limit [Chlamydomonas reinhardtii unicellular green alga, allowing the detection of three triazine  and two PUs (diuron and linuron) at the nM level (sub-µg·L−1) [−1, corresponding to the threshold value [−1 [−1 [Due to the mode of action of PU herbicides, most biosensors designed for their detection were based on the measurement of photosynthesis inhibition using appropriate biological elements like whole photosynthetic cells, chloroplasts, or thylakoids. For instance, an optical biosensor based on the measurement of the chlorophyll fluorescence of isolated chloroplasts was described for the detection of atrazine and diuron in drinking water at the sub-µg·L−1 level . A similproturon . Three oal limit . Site-di-µg·L−1) . Despiteld value . An elecld value . A similalue [−1 . An impe [−1 [−1 .2O2 and 3,3′,5,5′-tetramethylbenzidine (TMB). The formation of oxidized TMB was detected either by colorimetric or electrochemical methods.To the best of our knowledge, no immunosensor has been reported to date for the specific detection of chlortoluron, and only a colorimetric competitive ELISA was described showing a cross-reactivity for other PUs  . The aimAn indirect colorimetric immunoassay was first developed, and the method was transferred for electrochemical analysis using SPCE as the transducer. Chronoamperometry was used to investigate the response of the electrochemical immunosensor using the same labeled system. The proposed immunosensor was then used for the detection of chlortoluron in natural groundwater samples.−1 in methanol was purchased from Cluzeau Info Labo , and diluted solutions were prepared in PBS 1× buffer. pH 7.4. PBS 1× buffer was obtained by mixing 10 mmol·L−1 sodium hydrogen phosphate, 1.76 mmol·L−1 monopotassium phosphate, 137 mmol·L−1 sodium chloride, and 2.7 mmol·L−1 potassium chloride. Electrode treatment solution was composed of sulphuric acid 0.5 mol·L−1 and potassium chloride 0.1 mol·L−1. The “diazonium” solution was freshly prepared in 0.5 mol·L−1 hydrochloric acid solution by mixing 10 mmol·L−1 of 4-aminobenzoic acid and 10 mmol·L−1 of sodium nitrite. Different concentrations of streptavidin (from Streptomyces avidinii) were diluted in HEPES buffer . “TMB liquid substrate” was a ready-to-use solution containing H2O2 and 3,3′,5,5′-tetramethylbenzidine (TMB) HRP co-substrates. Blocking buffer was composed of either 1% or 3% bovine serum albumin (BSA) in PBS 1×. Microplate washing buffer was prepared with 0.1% Tween 20 in PBS 1×. All the aforementioned compounds were purchased from Sigma Aldrich . The activation solution was freshly prepared by mixing 0.1 mol·L−1 EDC and 25 mmol·L−1 NHS in MES buffer , purchased from Alfa Aesar.A standard solution of chlortoluron at 100 µg·L−1, pH 9 containing 0.1 mol·L−1 sodium bicarbonate and 0.01 mol·L−1 sodium carbonate.The synthesis of conjugated chlorotoluron-(PEG2-ethylamine)-biotin was performed in collaboration with Chimiothèque, UMR 5246-ICBMS, Université Claude Bernard Lyon 1 . Solutions of this compound were prepared in carbonate buffer 0.1 mol·LMouse monoclonal antibodies against chlortoluron (anti-ChlT-mAb) were especially produced for this study by Proteogenix . The secondary antibody was a peroxidase-bound anti-mouse IgG- antibody produced in rabbit (anti-IgG-HRP Ab) (Sigma Aldrich). Solutions of both antibodies were freshly prepared in PBS 1× containing 1% BSA (Sigma Aldrich).® membrane, hydrophilic polyethersulfone) and stored at 4 °C before use. Each sample was spiked with chlortoluron solution at 100 µg·L−1, and diluted successively with filtered water. Diluted samples were stocked at 4 °C in amber glass bottles.Groundwater samples were filtered through 0.2 µm syringe filters . One SPCE was composed of a three-electrode system including a graphite working electrode (4 mm diameter circle), a graphite auxiliary electrode (16 mm × 0.8 mm curved line), and an Ag/AgCl pseudo-reference electrode (5 mm × 1.5 mm straight line). The working and auxiliary electrodes were screen-printed using Electrodag 423 SS graphite paste  and the reference electrode using Ag/AgCl paste .Electrochemical treatments and measurements were performed using a MULTI AUTOLAB M204 potentiostat/galvanostat  controlled by NOVA 2.1 Software and a Multi Potentiostat µStat 8000P potentiostat/galvanostat  controlled by DropView 8400 Software.Colorimetric measurements were performed with an Epoch 2 Microplate Spectrophotometer . Nunc Maxisorp 96-well polystyrene microplates used for colorimetric assays were purchased from Thermofisher Scientific .Firstly, 100 µL of streptavidin solution were deposited in the microplate wells. After 90 min of incubation, the solution was removed, and 100 µL of biotinylated chlortoluron solution were incubated for 60 min at 37 °C. To avoid unspecific interactions, 300 µL of blocking buffer containing 3% BSA were added in each well and incubated for 120 min at room temperature. Then, 150 µL of anti-ChlT-mAb and 150 µL of chlortoluron solutions (assay) or PBS 1× (positive control) were mixed in a microtube and incubated for 30 min at 37 °C. Then, 100 µL of this mixture were added to each well and incubated for 60 min at 37 °C with immobilized chlortoluron. Next, 100 µL of anti-IgG-HRP Ab solution were allowed to react in each well for 30 min. Finally, the binding reaction was revealed using 100 µL of TMB liquid substrate. The absorbance of the oxidized TMB was read at 630 nm after 10 min of incubation at 37 °C. All the incubations were protected from light under orbital stirring at 350 rpm. Washing of microplate wells was performed between each step using 3 × 300 μL of washing buffer. All measurements were done in triplicate.−1. Then, 100 µL of “diazonium” solution were dropped on each electrode and left to react for 5 min at room temperature. Surface 4-carboxyphenyl groups were then generated by linear sweep voltammetry from +0.4 to −0.6 V vs. Ag/AgCl (scan rate 50 mV·s−1). In total, 50 µL of the activation solution containing EDC and NHS were then incubated for 60 min, and streptavidin immobilization was obtained after incubation of 20 µL of streptavidin solution for 60 min at 4 °C in the dark. After streptavidin immobilization, 20 µL of biotinylated chlortoluron solution were allowed to react for 60 min at 4 °C on the working electrode surface. In order to saturate unbound reactive groups, 50 µL of blocking buffer containing 1% BSA were incubated for 60 min. Then, 100 µL of anti-ChlT-mAb and 100 µL of chlortoluron solutions (assay) or PBS 1× (positive control) were mixed in a microtube and incubated for 30 min at 37 °C. Then, 50 µL of this mixture were dropped on the working electrode and incubated for 60 min. Next, 100 µL of anti-IgG-HRP Ab solution were allowed to react for 30 min, and 40 µL of TMB liquid substrate were added on the electrode. After 1 min of reaction, a potential of −0.2 V vs. Ag/AgCl was applied for 65 s and the current resulting from the reduction of oxidized TMB was concomitantly recorded. Washing of the electrode surface was performed between each step using 3 × 1 mL of PBS 1×. If not specified, incubations were done at room temperature. All assays were performed in triplicate.Electrode modification was adapted from the protocol described by Istamboulie et al. . Firstly0) was then calculated for each chlortoluron concentration by dividing the corrected signal value of the assay by the corrected value of the positive control.As described in the previous section, the signal values resulted either from colorimetric immunoassays or chronoamperometric immunosensors. Signal values were expressed as averages of triplicate assays. Three negative controls were performed by measuring the signal in the absence of streptavidin, immobilized chlortoluron, and primary antibodies, respectively. The positive control was performed in the absence of chlortoluron. For each optimization step, the ratio between positive control and negative control signal values was taken into account to determine the optimum conditions. The signal values of positive controls and assays were corrected from the mean value of negative controls. The percentage of binding . The limit of detection (LOD) was defined as the chlortoluron concentration inducing a binding decrease of 20%, with a maximum standard deviation of 7%.Calibration curves representing the %B/B−1, respectively  were obtained using biotinylated chlortoluron concentrations of 0.005 and 0.01 µg·L−1, respectively, but higher standard deviations were observed using a concentration of 0.01 µg·L−1. According to these results, subsequent competition tests were carried out using concentrations of 0.005, 0.16, and 1 µg·L−1 for biotinylated chlortoluron, monoclonal antibody, and secondary antibody, respectively.No significant differences were observed using the two concentrations of biotinylated chlortoluron tested. For a chlortoluron concentration of 1 µg·L−1 were tested to determine the sensitivity of the proposed immunoassay. The %B/B0 ratio was calculated based on the absorbance values measured for each chlortoluron concentration, as described in −1 . The calibration curve , and p is the slope at the inflexion point of the sigmoidal curve [A range of free chlortoluron concentrations between 0.05 and 500 µg·Lon curve  was drawal curve .−1 and EC50 of 8.8 µg·L−1 were calculated as the chlortoluron concentrations leading to %B/B0 values of 80% and 50%, respectively, using the following equation:A LOD of 2.7 µg·L−1 to be reached, these first results validated the format of the competitive method proposed in this work.Even though the sensitivity of the immunoassay did not allow the critical value of 0.1 µg·L−1. −1). However, the risk of cross-reaction in real analysis is minimized since diuron was banned for agricultural use since 2003. Concerning the other molecules tested, a significant relative response was observed only when using metazachlor and dimethachlor at 5 µg·L−1. However, such a concentration is not commonly found in groundwaters.In order to assess its specificity, the optimized immunoassay was tested in the presence of several herbicides including another substituted phenylurea (diuron), a substituted urea (tebuthiuron), a sulfonylurea (triflusulfuron), three chloroacetamides , and a diazine (bentazone). The potential cross-response was tested using pesticide concentrations of 0.5 and 5 µg·L−1  was used to investigate the electrochemical characteristics of TMB on a SPCE, by scanning the potential between +1.0 and −1.0 V vs. Ag/AgCl at a scan rate of 50 mV·s−1 . As prev−1, respectively. Results showed that using a streptavidin concentration of 5 µg·L−1 was sufficient to obtain a correct electrode coating, in such a manner that the ratio between the positive and negative controls was higher than 5, whatever the concentration of biotinylated chlortoluron used , the concentration of antibody being set at 0.6 µg·L−1  of 63.5% and 23%, respectively. Based on these results, the next experiments were carried out with electrodes coated with 0.05 µg·L−1 of biotinylated chlortoluron, while competition was performed with a 0.6 µg·L−1 antibody solution.As shown in 6 µg·L−1 . While h−1. The chronoamperograms recorded for assays, positive controls, and negative controls are presented in −1 chlortoluron, it was −0.64 µA. Using a concentration of 0.1 µg·L−1, the measured current value was −1.17 µA, corresponding to a %B/B0 ratio of 71.6%. The average value of the the three negative controls was −0.33 µA. These data allowed a calibration curve representing the %B/B0 values versus chlortoluron concentrations to be drawn  and LOD (%B/B0 = 80%) values of 1.03 µg·L−1 and 22.4 ng·L−1, respectively. As expected, a highly sensitive response to chlortoluron was achieved with a detection limit approximately 120-fold lower than the colorimetric assay. These results confirmed the potential of the developed immunosensor for detecting chlortoluron at concentrations below the threshold value.The above equation allowed calculation of the EC50 (%B/BIn order to assess potential matrix effects, the developed immunosensor was tested with natural groundwaters. Groundwater samples were first filtered on 0.2 µm membrane and tested before and after being spiked with known concentrations of chlortoluron. The results were compared with those achieved using the buffer as solvent .0 decreased in a similar manner using either buffer or groundwaters as solvent (0 values obtained for 1 µg·L−1 chlortoluron were 71.3% and 58.2% for groundwaters 1 and 2, respectively, compared to 73.1% for the buffer. The result obtained for groundwater 2 spiked with 1 µg·L−1 chlortoluron could be explained by the presence of very low concentrations of chlortoluron in the original sample, in the µg·L−1 order, which could be responsible for the observed discrepancy.In the presence of increasing concentrations of chlortoluron, the value of %B/B solvent , showing−1, with an LOD of 22.4 ng·L−1. No matrix effects were observed when analyzing natural groundwaters spiked with fixed concentrations of chlortoluron. Considering that the maximal residue limit in drinking waters is 0.1 ng·L−1, and taking into account its good performance in terms of sensitivity, the developed biosensor appears to be a promising tool for in-field determination of chlortoluron herbicide.This work describes for the first time the development of an amperometric immunosensor for the sensitive detection of chlortoluron in waters intended for human consumption. New antibodies were successfully produced and used in combination with biotinylated chlortoluron for the development of a competitive immunoassay. The optimized immunosensor allowed the detection of chlortoluron at concentrations ranging from 0.01 to 10 µg·L


A transframe region within HIV-1 Gag-Pol (referred to as p6* or p6pol), directly linked to the protease (PR) N-terminus, plays a pivotal role in modulating PR activation. To identify specific p6* residues involved in PR activation, we created a series of p6* mutants by making substitutions for conserved p6* residues. Our results indicate that some p6* mutants were defective in terms of virus infectivity, despite displaying a wild-type virus particle processing pattern. Mutations at p6* F8 reduced virus infectivity associated with insufficient virus processing, due in part to impaired PR maturation and RT packaging. Our data strongly suggest that conserved Phe (F) residues at position 8 of p6* are involved in the PR maturation process. Due to id CA; p2, nucleocal p6gag , 6.in vitro [Since premature PR activation triggers premature Gag cleavage prior to Gag multimerization and virus particle formation (resulting in markedly reduced virus yields), temporal and spatial PR activation regulation is critical to virus assembly –9. Severin vitro , 15. Molin vitro –19. We pin vitro –22.Single amino acid substitutions of the four C-terminal p6* residues can significantly impair virus maturation, strongly suggesting that the C-terminal p6* tetrapeptide is important for PR activation regulation , 21. Dat5’-GGTACAGTCTCAATAGGGCTAATG-3’ (nt. 2577–51) or an upstream forward primer 5’-AATGATGCAGAGAGGCAAT-3’ (nt.1916-34). The amplified fragments were digested with BglII and BclI, and subcloned into a plasmid cassette pBRCla-Sal that contains HIV-1 coding sequence . Each mutation-containing pBRCla-Sal cassette was then digested with SpeI and SalI, and ligated into HIVgpt. The resultant constructs were confirmed by DNA sequencing.The parental HIV-1 proviral sequence in this study is HXB2 . All conHEK-293T or HeLa cells were maintained in DMEM supplemented with 10% fetal calf serum. Confluent HEK-293T cells were trypsinized, split 1:10 and seeded onto 10-cm plates 24 hours before transfections. For each construct, HEK-293T cells were transfected with 20 μg of plasmid DNA by the calcium phosphate precipitation method , with thgag monoclonal antibody (mouse hybridoma clone 183-H12-5C). For HIV-1 RT detection, the primary antibody is rabbit antiserum or a mouse anti-RT monoclonal antibody [Culture media from transfected HEK-293T cells were filtered (0.45-μm pores) and centrifuged through 2 ml 20% sucrose. Viral pellets and cell lysates mixed with sample buffer were subjected to 10% SDS-PAGE as described previously . HIV-1 Gantibody . MembranFor infections, 10 μg of wt or each of p6* mutant plasmid plus 5 μg of the VSV- G protein expression plasmid pHCMV-G  were co-https://www.hiv.lanl.gov/content/sequence/HIV/COMPENDIUM/2006_7/5.pdf). Primers used to generate mutants are shown in To identify specific p6* residues involved in PR activation, highly conserved residues believed to be replaceable without affecting encoded p6gag residues were altered using PCR-mediated site-directed mutagenesis. p6* sequences in published HIV-1 strains were aligned, with non-polymorphic residues treated as highly conserved residues  or Lys (K) replacement at F8 significantly reduced virus infectivity. Specifically, F8K exhibited a five-fold decrease in virus infectivity compared to the wt in parallel experiments. Western blot analysis data indicate that despite defective infectivity, most mutants displayed a virus particle processing profile similar to that of the wt . HoweverGiven the pivotal role of p6* in modulating the PR activation process, it is likely that the reduced infectivity observed in some p6* mutants may have been due to a defect in the PR activation process, leading to subsequent impairments in virus maturation. To test this possibility, all infectivity-defective mutants were subjected to immunoblot with an HIV-1 PR antiserum. Our data readily detected mature PR in all mutant particles. In addition to mature PR, bands corresponding to the PR precursor (p6*-PR) were observed for F8V and F8K, with substantial amounts of F8K PR present in precursor form . These din vitro [Since F8/L9 has been proposed as an internal p6* cleavage site , 33, 34,in vitro , 37. We Our results indicate that both F8I and F8K exhibited higher levels of virus-associated Pr55gag compared to the wt , lane 3.gag/Pr55gag ratios compared to the wt, suggesting a defect in virus maturation Click here for additional data file.


The COVID-19 pandemic has deeply influenced sanitization procedures, and high-level disinfection has been massively used to prevent SARS-CoV-2 spread, with potential negative impact on the environment and on the threat of antimicrobial resistance (AMR). Aiming to overcome these concerns, yet preserving the effectiveness of sanitization against enveloped viruses, we assessed the antiviral properties of the Probiotic Cleaning Hygiene System (PCHS), an eco-sustainable probiotic-based detergent previously proven to stably abate pathogen contamination and AMR. PCHS  was tested in comparison to common disinfectants (70% ethanol and 0.5% sodium hypochlorite), in suspension and carrier tests, according with the European UNI EN 14476:2019 and UNI EN 16777:2019 standards. Human alpha- and beta-coronaviruses hCoV-229E and SARS-CoV-2, human herpesvirus type 1, human and animal influenza viruses, and vaccinia virus were included in the study. The results showed that PCHS was able to inactivate 99.99% of all tested viruses within 1–2 h of contact, both in suspension and on surface. Notably, while control disinfectants became inactive within 2 h after application, the PCHS antiviral action persisted up to 24 h post-application, suggesting that its use may effectively allow a continuous prevention of virus spread via contaminated environment, without worsening environmental pollution and AMR concern.  The COVID-19 pandemic, caused by the new SARS-CoV-2 human coronavirus has deeply influenced the habits relative to hygiene and sanitization, shining light on the risk associated with environmental virus contamination, especially in the hospital environment. COVID-19 has in fact spread worldwide, causing at the moment over 244 million confirmed cases and 4.96 million deaths , suggestConsistently with these data, WHO has proposed preventive measures, and high-level virucidal chemical disinfectants have been mandatorily introduced by regulatory bodies for surface cleaning of indoor environments, including healthcare and non-healthcare settings, as interim recommendations to combat the COVID-19 health emergency ,15. Howereservoirs and transmission vectors to susceptible humans [Besides SARS-CoV-2, several enveloped viruses have been similarly shown to retain infectivity for long periods on hard surfaces, depending on virus type, surface characteristics, temperature, and humidity , includie humans ,31. Conse humans ,32,33, sAmong the viruses that might be transmitted by the contaminated environment, human coronaviruses , which can cause mild to severe respiratory infections , can per2O2), for sanitization [Based on the ability of many viruses to maintain infectivity on inanimate surfaces, the control of environmental viral contamination represents a key point to address. Current guidelines for COVID-19 management suggest using high level disinfectants, mostly 0.1–0.5% sodium hypochlorite (NaClO) and 1% hydrogen peroxide  [Aiming to obtain a long-term effective cleaning procedure, stably reducing viral contamination without impacting on environmental pollution and AMR, we tested the antiviral properties of an eco-sustainable probiotic-based sanitation system , that was previously shown to prevent pathogen recontamination by stably remodulating the hospital microbiome. Its action, based on the biological properties of selected probiotic s (−80%) ,46,47,48s (−80%) ,46, and s (−80%) , testinghttps://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:32010R0066, accessed on 23 October 2021) detergent containing 107 CFU/mL spores of selected probiotics belonging to the Bacillus genus . PCHS was tested at 1:10, 1:50, and 1:100 dilution in sterile distilled water.The Probiotic Cleaning Hygiene System (PCHS) used in all the assays was previously described . BrieflyThe enveloped viruses and appropriate target cells used for virus inocula preparation, virus titration, and standard inactivation assays, are summarized in 2 in the appropriate culture medium supplemented with 10% foetal bovine serum (FBS), 2 mM L-Glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (cell culture complete medium) .MRC-5 cells were cultured in Eagle Minimal Essential Medium Eagle (EMEM), whereas the other cell lines were grown in Dulbecco Minimal Essential Medium (DMEM). All the cell lines were expanded at 37 °C + 5% CO2 in culture medium additioned with 2% FBS. Infected cell cultures were incubated for different times until appearance of cytopathic effect (CPE) involving >80% of cultured cells, that corresponded to: 2 days for MVA and HSV-1, 5 days for SARS-CoV-2, 3 days for influenza viruses, and 7 days for hCoV-229E. At the end of the incubation time, cells and culture supernatants were collected. Cells were lysed by 3 cycles of rapid freezing/thawing in liquid nitrogen and 37 °C, interspersed with 30 s pulse-vortex. Cell lysate was then added to culture supernatant and viral particles were recovered by centrifugation at 20,000× g for 45 min at 4 °C. The virus pellets were suspended in 1 mL of PBS + 1% bovine serum albumin (BSA), then frozen and maintained at −80 °C until use. The virus stock titre was determined by infecting the appropriate target cells seeded in 96-well plates, using the same culture conditions used for virus stock preparation, and evaluating the 50% tissue culture infectious dose (TCID50) per ml by the standard Spearman-Karber method, as previously described [Virus stocks were obtained by infecting specific 90% confluent target cells, subsequently incubated at the appropriate temperature  + 5% COescribed . Briefly8 TCID50/mL, as calculated by the Spearman-Karber method.All virus stocks contained around 105–107 TCID50, depending on virus type) were added to 90 µL of the appropriate dilution of PCHS , in the presence of 0.3% of BSA, to mimic the conditions that could be found on a hospital surface (“clean” conditions). Negative and positive controls were respectively represented by culture medium and 70% ethanol (EtOH). The suspension was incubated at room temperature for 1, 2, 4, 8, and 24 h; then it was collected, immediately diluted in 0.9 mL cold medium + 2% FBS , filtrated (0.45 µm) to remove the probiotic component, and then serially diluted (10-fold dilution) in cold medium + 2% FBS for titration of the residual virus amount by the Spearman-Karber method. Infected cells were then incubated at the appropriate temperature in the presence of 5% CO2 for the time needed to evidence virus CPE. Representative CPE pictures are shown in 50/mL. Each experimental condition was assessed in duplicate and the collected results represent the mean values of three independent assays. All the experiments with SARS-CoV-2 were performed in a BSL-III laboratory. Experimental controls and cytotoxicity evaluation of PCHS and standard disinfectants were performed in all cell types used in the assays, following the protocol indicated in the standard procedures.The antiviral activity of PCHS in suspension was assayed following the European standard procedure UNI EN 14476:2019, as indicated by the Technical Committee 216 (TC216) “Chemical disinfectants and antiseptics” of the European Committee for Standardization (CEN), which has been developing methods for testing the efficacy of disinfectants in Europe since 1989 ,52. Brie6 TCID50) were seeded on carrier surface with a micropipette, and the drop was spread on a surface area of about 1 cm diameter and left to dry at room temperature, then immediately covered with 100 µL of PCHS previously diluted 1:10, 1:50, and 1:100 in water. EtOH 70% and culture medium were used as positive and negative controls, respectively. After 1, 2, 4, 8, and 24h, 0.9 mL (10-fold dilution) of ice cold medium + 2% FCS were added to collect the residual virus. The collected medium was then filtered (0.45µm) to remove probiotic component and serially diluted (10-fold) in cold medium + 2% FCS. Each dilution was transferred to 96-well microplates (0.1 mL/well) containing a 90% confluent monolayer cells, which were then incubated at the appropriate conditions to evaluate the residual virus titre by the Spearman-Karber method. In the prevention assay, 100 µL of 1:10, 1:50, and 1:100 diluted PCHS were seeded on surface and left to dry at room temperature. EtOH 70% and culture medium were used as positive and negative controls, respectively. Immediately after drying, the surfaces were contaminated with 100 µL of virus inoculum (106 TCID50). After 1, 2, 4, 8, and 24h, samples were collected and titrated as described for the decontamination assay. The long-term ability of PCHS in preventing virus contamination was assayed by seeding 100 µL of 1:10, 1:50, and 1:100 diluted PCHS on surface and then contaminating the treated surface after 1, 2, 4, 8, and 24h with 100 µL of virus inoculum (106 TCID50). EtOH 70% and sodium hypochlorite (NaClO) 0.5% were used as positive controls; culture medium was used as a negative control. Virus inoculum was left in place for 2 h and then collected and titrated as described for the previous assays. Experimental controls and cytotoxicity evaluation of PCHS and standard disinfectants were performed on all cell types used in the assays, following that indicated in the standard procedures. Both EtOH and NaClO were cytotoxic at 10−1 dilution on all used cell lines, whereas PCHS did not show cell toxicity; direct comparison of virus titre was thus performed on the dilutions from 10−2 onwards.The antiviral ability of PCHS on hard non-porous surfaces was assessed following the European standard procedure UNI EN 16777:2019  and usinBacillus strains contained in the PCHS detergent , previously culturally isolated, were assessed by the API-ZYM system  for their ability to produce enzymes potentially useful to degrade virus components, following the manufacturer’s instructions.The https://www.r.project.org/, accessed on 10 May 2021) by Student’s t-test. A p-value ≤ 0.05 was considered significant.Statistical analyses were performed with Agilent GeneSpring GX v11.5 software  and R , showed that PCHS could efficiently inactivate all the tested viruses, regardless of the type of virus used , includiConsistently, PCHS exhibited a similar antiviral activity also against SARS-CoV-2 D, which Since in the suspension method the viruses come into contact with a large amount of the disinfectant, which may render them easier to inactivate, the antiviral activity of PCHS was also assessed in carrier tests, performed according to the UNI EN 16777:2019 standard European procedure. Based on the results obtained in suspension tests, only hCoV-229E and MVA viruses were used in the assays, to include a human coronavirus similar to SARS-CoV-2 and a highly resistant enveloped virus. Three different conditions were tested, to assess respectively: (1) the decontaminating ability of PCHS, (2) the PCHS ability to prevent virus contamination by PCHS, (3) the long-term stability of PCHS activity in preventing virus contamination.The results of decontamination-type assays showed that, similar to that observed in suspension tests, any dilution of PCHS could completely inactivate hCoV-229E (>4 Log decrease) within 1 h, whereas MVA was totally inactivated in 1 h by 1:10 and 1:50 dilutions and in 2 h by 1:100 diluted PCHS . The lowNext, the ability of PCHS-treated surfaces to prevent a subsequent virus contamination was assessed. To this purpose, PCHS was first applied on surface and left to dry, then the treated surface was contaminated with the virus inoculum, whose residual titre was evaluated after 1, 2, 4, 8, and 24 h of contact. The results, summarized in Lastly, the long-term ability of PCHS to prevent virus contamination was assessed on surfaces. PCHS  was applied on surfaces and left to dry, then virus inocula were added to treated surfaces after 1, 2, 4, 8, and 24 h. Viruses were left in place for 2 h and then collected to measure residual virus titre .Notably, PCHS-treated surfaces completely inactivated both viruses even 24 h after treatment, whereas EtOH- and NaClO-treated surfaces did not maintain their inactivating ability over time. EtOH lost its inactivating activity within 1 h and NaClO gradually lost its virucidal activity from 2 h on both MVA (−2.25 Logs at 2 h) and hCoV-229E (−3.4 Logs): at that time, in fact, the NaClO activity resulted below the threshold needed to define a compound as virucidal (−4 Logs). At longer times the antiviral activity of both chemical disinfectants disappeared completely.Bacillus strains were analyzed for their enzymatic activity, in order to highlight any eventual production of enzymes capable of degrading viral components. The three Bacillus strains included in the PCHS detergent  were individually tested by the API-ZYM system, allowing to identify and quantify simultaneously 19 different enzymatic activities. The results, summarized in B. pumilus and B. megaterium), α-chimotrypsin (B. pumilus and B. megaterium), naphtol-phosphohydrolase, α- and β-galactosidase (absent respectively in B. megaterium and B. subtilis), α- and β-glucosidase, N-acetyl-β-glucosamidase, and α-mannosidase (B. pumilus only). The presence of enzymes capable of processing lipids, proteins, and sugars, support the hypothesis that such probiotics could chemically degrade the outer components of enveloped viruses, achieving virus inactivation.To elucidate the possible contribution of probiotics in virus inactivation, PCHS-derived The COVID-19 pandemic, caused by SARS-CoV-2, has profoundly influenced sanitization procedures and the massive use of disinfectants has been indicated to prevent virus spread. On the other hand, contamination of viral origin has been reportedly evidenced for several viruses beside SARS-CoV-2 , possiblIn contrast, we recently reported that a sanitation approach based on the microbiome-balance principle , could stably and efficiently counteract pathogens compared to chemical disinfectants, through competitive exclusion mechanisms , decreas10 decrease of virus titre obtained within 1 h as the threshold to establish the antiviral activity of the product against a specific virus.Based on these premises, and in consideration of the current emergency linked to COVID-19, here we aimed to assess the antiviral potential of PCHS against the enveloped viruses potentially contaminating the healthcare setting, including SARS-CoV-2. PCHS was tested following suspension and surface procedures, according with the European standard UNI EN 14476:2019 and UNI EN 16777:2019. Indeed, both procedures prescribe the use of non-enveloped viruses and only MVA among the enveloped ones (since it is considered the most resistant enveloped virus); however, since our study aimed to analyze the enveloped viruses potentially able to persist on surfaces and similar to SARS-CoV-2, we included, beside MVA, the human coronaviruses hCoV-229E and SARS-CoV-2, the human herpesvirus HSV-1 and three human/animal type A influenza viruses. Three different concentrations of PCHS were tested, including a lower one that is used for routine cleaning (1:100 dilution) and two higher concentrations (1:10 and 1:50 dilutions), comparing their activity with that of 70% EtOH and 0.5% NaClO, two common disinfectants indicated for decontamination during the pandemic. Based on standard procedures evaluating the virucidal properties of a product, we considered a ≥4-LogThe results showed that PCHS was active on all enveloped viruses at all dilutions in a time-dependent manner. In particular, the 1:100 dilution completely inactivated within 1 h all tested viruses except for MVA, which was inactivated in 2 h. A variable situation was observed with influenza viruses, confirming that previously reported for chemical disinfectants, showing different activity depending on virus type and host origin . In factThe carrier/surface test results, performed on hCoV-229E and MVA, evidenced a high inactivation activity both in decontamination and prevention conditions. Interestingly, while 70% EtOH and 0.5% NaClO became inactive within 1 and 2 h, respectively, PCHS-treated surfaces maintained their antiviral properties even 24 h after PCHS application, suggesting that a daily PCHS sanitation  may assure a continuous stably decontaminated environment.Bacillus strains spread on the treated surface may be responsible for the continuous action. Accordingly, the analysis of the enzymes produced by the three PCHS-derived Bacillus species  revealed several enzymatic activities potentially able to degrade the virus shell components, including lipids, protein, and sugar residues. Consistent with this, the Bacillus genus is reportedly known as one of the most important bacterial sources of enzymes with remarkable properties, such as high resistance to extreme temperatures, pH, organic solvents, and oxidizing agents. Bacillus enzymes have been long used in medicine, biofilm destruction, animal feed, agriculture, degradation of feathers, wool, and hair [Bacillus genus possess sporulation capacity that renders them resistant to harsh environmental conditions and usable in concentrated detergents, which makes them interesting for medical and industrial purposes. Lastly, PCHS is a low-cost affordable system, which is also important from a cost-saving perspective [The surfactants contained in detergent are known to disrupt and damage the envelope of viruses ,68, and and hair ,70, thusand hair , suggestspective , and renspective . To thisspective . For nonIn light of its effective long-lasting antiviral activity and characteristics of sustainability, PCHS sanitation may be considered as a novel and safe perspective for the control and prevention of the spread of various enveloped viruses including SARS-CoV-2, also in light of the lack of negative side-effects on the environment health and on AMR concerns.


Cryptococcus, a notorious pathogen since the advent of the HIV/AIDS-pandemic. A cryptococcal infection usually manifests itself as a pneumonia and/or meningitis. The genus includes ten species, most of them belonging to the C. gattii/C. neoformans species complexes, and some non-pathogenic species  [Naganishia albida, Naganishia diffluens and Papilliotrema laurentii) [Cryptococcosis is the collective heading of infections caused by members of the basidiomycetous yeast genus  luteus) . Previouurentii) , 2.Cryptococcus neoformans sensu stricto (previously C. neoformans variety grubii) is globally the major cause of systemic cryptococcosis among immunocompromised individuals. It is strongly associated with bird excreta, especially pigeon droppings [Cryptococcus deneoformans (formerly C. neoformans variety neoformans) is less common but remain a major cause of cryptococcosis in Europe [C. deneoformans × C. neoformans, which occurs predominantly in Mediterranean Europe [C. neoformans and C. deneoformans is that the latter is associated with skin-infections and more often seen in elderly patients [roppings . Cryptocn Europe . This apn Europe . Relevanpatients , 4.Cryptococcus gattii sensu lato (formerly C. neoformans variety gattii) became notorious due to the unprecedented outbreak on Vancouver Island . Until then, C. gattii was recognized as a pathogen exclusive to tropical and subtropical regions [C. gattii is –like C. neoformans– a species complex, it comprises six lineages of which five are recognized as species while the sixth needs to be named [C. gattii VGII-outbreak lineage’ is now called C. deuterogattii. The environmental niches are trees and plants, and it is globally distributed like its sibling C. gattii sensu stricto. As both species occur in the environment of temperate climate zones it is obvious that they cannot longer be regarded as strict (sub)tropical pathogens. The remaining three pathogenic species have a predilection for the immunocompromised host. C. bacillisporus and C. decagattii infections are mainly reported from the America’s, with some inexplicable cases outside these continents [Cryptococcus tetragattii is, like C. neoformans, a frequent cause of cryptococcal meningitis among HIV-positive patients from sub-Saharan African and the Indian subcontinent  species-level identification is of importance and could contribute to adequate treatment.PCR-fingerprinting, AFLP genotyping, microsatellite typing, multi-locus sequencing typing and whole genome sequencing are widely used to investigate the molecular epidemiology , 6–9 Fi. The bipC. gattii sensu lato infections need to be more aggressively treated than C. neoformans sensu lato, as the former produce cryptococcoma’s that are difficult to eradicate [Recommended treatment is a 2-weeks induction therapy with amphotericin B plus 5-fluorocytosine, followed by fluconazole as suppressive therapy . C. gattradicate . Antifunradicate .


Li+/Li). The Li+ diffusion coefficient of Co2VO4 is evaluated by theoretical calculation to be as high as 3.15 × 10–10 cm2 s−1, proving Co2VO4 a promising anode in fast-charging LIBs. A hexagonal porous Co2VO4 nanodisk (PCVO ND) structure is designed accordingly, featuring a high specific surface area of 74.57 m2 g−1 and numerous pores with a pore size of 14 nm. This unique structure succeeds in enhancing Li+ and electron transfer, leading to superior fast-charging performance than current commercial anodes. As a result, the PCVO ND shows a high initial reversible capacity of 911.0 mAh g−1 at 0.4 C, excellent fast-charging capacity (344.3 mAh g−1 at 10 C for 1000 cycles), outstanding long-term cycling stability (only 0.024% capacity loss per cycle at 10 C for 1000 cycles), confirming the commercial feasibility of PCVO ND in fast-charging LIBs.High-energy–density lithium-ion batteries (LIBs) that can be safely fast-charged are desirable for electric vehicles. However, sub-optimal lithiation potential and low capacity of commonly used LIBs anode cause safety issues and low energy density. Here we hypothesize that a cobalt vanadate oxide, CoThe online version contains supplementary material available at 10.1007/s40820-021-00758-5. Lithium-ion batteries (LIBs) are widely used for portable electronic devices in the past decade because of their high-energy density, long cycling life, low self-discharge, the absence of memory effect, and low environmental impact , 2. In r−1 at a current density of > 10 A g−1 . In. In−1 be+/Li). Figure −1, with an initial coulombic efficiency of 76.9%. The high discharge capacity in the first cycle is due to the formation of the SEI film. Figure S4 shows the cycling performance of PCVO ND at 0.4 C. PCVO ND displayed a high initial reversible capacity of 911.0 mAh g−1. The capacity showed a slight decrease before the 50th cycle and then increased slightly in the subsequent cycles. The capacity was recovered to 817.9 mAh g−1 after 100 cycles, corresponding to a high capacity retention of 89.8%, suggesting high cycling stability. Similar results have also been reported for the electrochemical performance of transition metal oxides in the literature [+ ion accessibility with cycling. The exceptional electrochemical performance of PCVO ND electrode at 0.4 C was verified by electrochemical impedance spectroscopy (EIS) test. Figure S5a, b showed Nyquist plots and equivalent circuit model of PCVO ND electrode before cycling and after 1, 10, 50, and 100 cycles. R1 and R2 represent internal resistance, including electrolyte resistance, the internal resistance of active material, and contact resistance between the current collector and active material, while R3 represents the charge transfer resistance. The internal resistance of the PCVO ND electrode before cycling is much larger than that for the following cycles, mainly due to the much-enhanced ion and electron transfer in the electrode. Subsequently, there was little change in the impedance of the PCVO ND electrode, indicating low transfer resistance and good stability of PCVO ND.The PCVO ND was measured at 0.4 C within the voltage range of 0.01–3.0 V  and outstanding long-term cycling stability (only 0.024% capacity loss per cycle at 10 C for 1000 cycles). A LIB with such PCVO ND anode can be fully charged within 5 min. Thus our PCVO ND met the requirement of anodes for fast-charging LIBs in EVs by the US Department of Energy and showed better fast-charging performance than previously reported fast-charging anodes, summarized in Fig. + diffusion coefficient of Co2VO4 can facilitate fast electrochemical reactions, and the porous nanodisk structural design can reduce the electron and ion diffusion length, further enhancing the electrochemical kinetics; (iii) Numerous internal pores of PCVO ND and uniform nanoscale pore size buffer the volume change, and the close contact between electrode and electrolyte by PCVO ND' large specific surface area stabilize the SEI film, leading to superior long-term cycling stability of PCVO ND at a high rate.Above good electrochemical performances  along with high Liles Fig. d. Notabl2VO4 were absent in the electrode after 1000 cycles. Meanwhile, two new peaks at 44.3° and 65.7° corresponded to the (015) and (110) diffraction planes of LiVO2, respectively [2 (lattice spacing of 0.201 nm). And the lattice spacing of 0.151 nm corresponded to the (220) of CoO [xVO2 + (y + 2) Li+  + (y + 2) e− ⟷ Co + Li2O + Lix+yVO2 after 1000 cycles, further confirming the stability of PCVO ND cycled at a high rate.To further investigate PCVO ND's stability, the morphologies of PCVO ND before and after 1000 cycles were examined. Figure S6a–d showed the SEM images of the PCVO ND electrode before and after 1000 cycles at 10 C. The PCVO ND exhibits a high structural stability and still maintains a hexagonal structure after 1000 cycles , confirming the excellent long-term cycle stability of PCVO ND. The structure of the PCVO ND after 1000 cycles was also investigated to conform the stability of PCVO ND. The XRD pattern of PCVO ND after 1000 cycles at 10 C is shown in Fig. S6e. Compared to the pristine PCVO ND, the peaks of Coectively . The HRT) of CoO . The XRD−1  and scan rate (v) which can be described by the following equation:To get deep insight into the excellent lithium storage kinetics of PCVO ND, CV measurements were carried at different scan rates ranging from 0.2 to 1.0 mV s−1 Fig. f. The shi) versus log (v). As shown in Fig. 1v) and diffusion-controlled storage contribution (k2v1/2) using the following formula:In this equation, a and b are adjustable parameters. The value of b ranges from 0.5 to 1.0 . Thus, Si anode was not further studied in this paper. Figure 4Ti5O12 at 400 mA g−1. Li4Ti5O12 displayed the lowest discharge capacity of 164.1 mAh g−1 and intercalated Li+ at the highest potential (~ 1.55 V vs. Li+/Li). Such low capacity, along with high operation voltage, sacrificed the cell voltage and cell energy seriously. In contrast, PCVO ND showed the highest discharge capacity of 910.9 mAh g−1. And the insertion of Li+ into PCVO ND occurred at a lower potential (~ 0.65 V vs. Li+/Li). The energy density formula in Fig. E is the energy density relative to the LTO anode, Cn is the specific capacity of different anodes, Vn is the voltage difference between discharge platform and commercial 4 V cathode materials  [In order to evaluate the practical application, the electrochemical performances of PCVO ND were compared with that of anodes in commercial LIBs. Figure  to CVO) .Fig. 5a 4Ti5O12 if the two are coupled with a typical 4 V cathode  was 15 orders of magnitude higher than that of Li+ at the lowest voltage (~ 0.1 V vs. Li+/Li) close to that of the Li-plating, which resulted in a safety risk due to high surface Li-plating . In order to compare the PCVO ND's safety with graphite at high rates, the PCVO ND and graphite anodes were charged and discharged at 4000 mA g−1 for 1000 cycles. Both PCVO ND and graphite anodes showed high cycling stability , safe lithiation potential (~ 0.65 V vs. Li+/Li), high Li+ diffusion coefficient (6.95 × 10–10 cm2 s−1), and high cycling stability. In order to further enhance Li+ and electrons transfer, we designed a hexagonal porous Co2VO4 nanodisk (PCVO ND) structure, which features a high specific surface area of 74.57 m2 g−1 and numerous pores with a uniform pore size of 14 nm in PCVO ND. The porous nanodisk structural design can reduce the electron and ion diffusion length at the electrode level, further enhancing the electrochemical kinetics. Moreover, the close contact between electrode and electrolyte by the large specific surface area of PCVO ND stabilizes the SEI film, leading to superior fast-charging performance of PCVO ND. As a result, the PCVO ND showed a high initial reversible capacity of 911.0 mAh g−1 at 0.4 C, excellent fast-charging capacity (average capacity of 579.2 mAh g−1 at 4 C and 344.3 mAh g−1 at 10 C for 1000 cycles), outstanding long-term cycling stability (only 0.024% capacity loss per cycle at 10 C for 1000 cycles). Due to its superior fast-charging performance and simple preparation, the PCVO ND has high commercial feasibility in high-energy–density fast-charging LIBs.Our studies showed that CoSupplementary file1 (PDF 694 KB)Below is the link to the electronic supplementary material.


Zika virus diagnostic testing and laboratory research increased considerably when Zika virus began spreading through the Americas in 2015, increasing the risk for potential Zika virus exposure of laboratory workers and biomedical researchers. We report 4 cases of laboratory-associated Zika virus disease in the United States during 2016–2019. Of these, 2 were associated with needlestick injuries; for the other 2 cases, the route of transmission was undetermined. In laboratories in which work with Zika virus is performed, good laboratory biosafety practices must be implemented and practiced to reduce the risk for infection among laboratory personnel. Zika virus is a flavivirus that was first isolated in 1947 from a rhesus macaque in the Zika Forest in Uganda. Zika virus is primarily transmitted to humans by infected mosquitoes, but other confirmed transmission modes include intrauterine, sexual, and intrapartum transmission, and probable modes include transmission through blood transfusion and breastfeeding  3 microbiology laboratory sustained a needlestick injury with a bifurcated needle; information on whether the skin was punctured was not available. The incident occurred during in vitro inoculation of human skin cells with wild-type Zika virus for vaccine research purposes. She was wearing 2 pairs of nitrile gloves and working in a biosafety cabinet. She immediately used a surgical sponge and chlorohexidine to scrub the wound for 15 minutes, then washed her hands with soap and water. After 9 days, she experienced a low-grade fever, generalized maculopapular rash, headache, myalgia, and fatigue; mild unilateral conjunctivitis occurred the next day. She did not live in an area with local Zika virus transmission, and in the month before illness onset she had no other risk factors for acquisition of Zika virus infection . She reported full resolution of her symptoms within 5 days. Zika virus infection was confirmed through the detection of Zika virus RNA in serum and urine and Zika virus IgM and neutralizing antibodies in serum .7 PFU/mL  of headache, arthralgia, myalgia, fatigue, and a rash that initially appeared on his face and spread to his whole body during the next 2 days. The arthralgia and myalgia became progressively more severe and debilitating through day 5, but recovery occurred by day 13. Zika virus infection was confirmed through detection of Zika virus RNA in serum and semen and with serologic methods . He had The patient reported that he typically worked with large quantities (4–100 L) of Zika virus in the laboratory but did not recall any specific exposure or incident of concern within the 2 weeks before illness onset. His activities included clarifying Zika virus materials through filters, performing pump-driven chromatography, using buffers to dilute concentrated Zika virus, and adding formaldehyde to initiate Zika virus inactivation. The recommended personal protective equipment (PPE) he routinely wore included a first PPE layer, donned in an external area, of disposable laboratory coat or coverall, booties, a hairnet, goggles, and 1 pair of gloves and a second PPE layer of a second coverall, hairnet, pair of gloves, and disposable face shield donned once inside the laboratory; no mask was used. He performed his work inside a biosafety cabinet when possible but could not do so when using larger containers . The liquid could sometimes potentially splash. On 1 occasion during the probable exposure period, while he was working in a biosafety cabinet, a large droplet of live virus dripped onto his glove; he immediately changed the outer glove. He reported it was possible he might have rubbed his face with the back of a gloved hand; however, no confirmed mucus membrane exposure could be identified. An additional 12 employees working with Zika virus in the same laboratory were subsequently tested and showed no serologic evidence of recent or past Zika virus infection.In October 2019, a male researcher in a vaccine research laboratory experienced fever, rash, arthralgia, and conjunctival injection. His laboratory activities sometimes involved working with Zika virus, including performing serum neutralization testing, and he had worked with Zika virus 8 and 10 days before symptom onset. He routinely wore gloves in the laboratory, but more detailed PPE information was unavailable. An investigation did not identify any specific exposure or reported breach in biosafety procedures, and no sharps were used in the laboratory. He did not live in an area with a history of Zika virus transmission and he had no other risk factors for Zika virus infection. Confirmation of infection was by detection of Zika virus RNA in urine and by serologic methods . SymptomDuring the 4-year period from 2016–2019, 4 cases of laboratory-acquired Zika virus infection were reported in the United States: 2 associated with needlestick injuries and 2 in which the means of exposure was undetermined. In laboratories where work with Zika virus is performed, good laboratory safety practices are critical to reduce the risk to personnel of Zika virus exposure and disease.Many factors affect the likelihood of Zika virus infection following exposure, including the type and severity of any injury or exposure, route of exposure, viral concentration and dose, transmissibility of the strain, immediate management of any recognized exposure, and the worker’s health status. At least 3 other potential occupational exposures to Zika virus have occurred among researchers without subsequent Zika virus infection: a bite from an infected mouse that punctured the skin of a gloved researcher’s finger . Therefore, the infections were likely laboratory-acquired.The Biosafety in Microbiological and Biomedical Laboratories guidelines recommend BSL-2 practices, safety equipment, and facilities for working with Zika virus (Laboratory personnel should have appropriate training regarding precautions to prevent exposures associated with the tasks they perform (>2 weeks postexposure. This serum sample should be tested for Zika virus IgM and if positive, tested by plaque-reduction neutralization test, and results compared with those from the baseline sample to assess for asymptomatic infection. Similarly, if a person is symptomatic within 2 weeks of exposure and test results on collected samples are negative, indicating the illness is unrelated to Zika virus infection, consideration should be given to obtaining an additional serum sample at >2 weeks postexposure and similarly evaluating for asymptomatic infection.Appropriate evaluation and management of occupational Zika virus exposures is crucial. If an incident occurs, established workplace procedures for initial wound management or mucous membrane exposures should be followed and the event immediately reported to a supervisor. No specific Zika virus post-exposure prophylaxis exists; however, as soon as possible after the incident, a baseline serum sample should be obtained and stored in case comparison with a convalescent serum sample is needed. Persons should be advised to take steps to prevent potential sexual transmission of Zika virus and to avoid mosquito bites if in a geographic area with risk for mosquito-borne transmission of Zika virus. These measures should be continued until laboratory testing excludes infection; if Zika virus infection is confirmed, additional counseling should be provided. If symptoms consistent with Zika virus disease occur within 2 weeks of the exposure, serum and urine should be collected and tested by using appropriate molecular and serologic methods. For an exposed person who remains asymptomatic, a serum sample should be obtained Although Zika virus transmission has declined substantially in recent years, research using Zika virus is ongoing. Exposure and infection are occupational risks for laboratory and biomedical research workers who work with live virus. Strong infection prevention practices are essential for reducing this risk (


Equus caballus in Japan, 1978, with a (nt) identity of at least 97%. The newly isolated virus CHIKV-China/YN2018-1 became more infectious after passage of the BHK-21 cell line to the Vero cell line. The newly identified RRV gene had the highest homology with the 2006 RRV isolate from Australia, with a (nt) identity of at least 94%. In addition, numerous known and unknown viruses have also been detected in mosquitoes from Yunnan province, China, and propagation tests will be carried out.We collected 5,500 mosquitoes belonging to six species in three locations in China. Their viromes were tested using metagenomic sequencing and bioinformatic analysis. The affluent viral sequences that were detected and annotated belong to 22 viral taxonomic families. Then, PCR was performed to confirm the results, followed by phylogenetic analysis. Herein, part of mosquito virome was identified, including chikungunya virus (CHIKV), Getah virus (GETV), and Ross river virus (RRV). After metagenomic analysis, seven CHIKV sequences were verified by PCR amplification, among which CHIKV-China/YN2018-1 had the highest homology with the CHIKV isolated in Senegal, 1983, with a nucleotide (nt) identity of at least 81%, belonging to genotype West Africa viral genes. Five GETV sequences were identified, which had a high homology with the GETV sequences isolated from  Mosquitoes are important biological vectors of many infectious viruses, including Getah virus (GETV), Ross River virus (RRV), chikungunya virus (CHIKV), Zika virus (ZIKV), and dengue virus (DENV), which were a menace to global public health and security . MosquitThe aim of this study was to establish an efficient monitoring method for mosquito-borne viral distribution in Yunnan Province, China, and provide valuable information in the field of viral isolation, prevention, and control. Through metagenomic sequencing and PCR, the diversity and abundant viromes of mosquito-borne viruses in Yunnan province were obtained. The existence of RRV and novel GETV were verified, and CHIKV was isolated. Our initial study on mosquito-borne virus metagenomics provides important technical support for the distribution, diversity, and monitoring of mosquito-borne viruses in China and other countries.Armigeres obturbans (Ar. obturbans), Anopheles sinensis (An. sinensis), Culex quinquefasciatus (C. quinquefasciatus), Culex tritaeniorhynchus (C. tritaeniorhynchus), Aedes albopictus , and Aedes aegypti (Ae. aegypti). After identifying the mosquito species, we put every 100 mosquitoes of the same species in a cryopreservation tube  The E value ≤10 e-5 in BLAST hits was considered statistically significant. Sequences of bacterial and eukaryotes were deleted, and virus-like sequences were analyzed.The sequences were aligned with non-redundant databases and virus reference databases using BLASTx and BLASTn in GenBank.  supplemented with 10% fetal bovine serum  and 1% penicillin and streptomycin ; cells were maintained in humidified 5% COMosquito samples verified to be positive by PCR were used for virus isolation. In short, the mosquito sample solution was mixed 1:6 with DMEM containing 2% FBS, followed by inoculation on a monolayer of BHK-21 cells and culture together for 5–6 days. We checked the culture daily for virus-induced cytopathic effect (CPE). Cultures without a CPE were blind passaged 3 times.After isolation, the obtained virus CHIKV-China/YN2018-1 was first identified by PCR technology. Based on the envelope 1 (E1) protein gene Table 3,g for 15 min, followed by centrifugation at 60,000 × g for 4 h. We got the virus precipitate by gently removing and discarding the supernatant, and equal proportion mixture of 6.1% (V/V) glutaraldehyde fixative (pH 7.2) and DMEM was used to resuspend the precipitate, and 25 μl was added to the copper grid, followed by the addition of one drop of 3% phosphotungstic acid for negative staining and observation using a microscope.After verification by PCR and Western blotting, the newly isolated CHIKV was inoculated on a monolayer of BHK-21 cells to produce CPE. Then, the cells were collected and repeatedly frozen and thawed three times. After that, the virus suspension was harvested by centrifuging at 10,000 × 50 (50% tissue culture infective dose) was acquired based on the Reed-Muench method . After passage in the Vero cell line from the BHK-21 cell line of CHIKV (P5 and P10), the purified RNA was used as template for cDNA synthesis using a SuperScript™ III first-strand synthesis system (Invitrogen) following the manufacturer’s instructions. Subsequently, we amplified the E1 genes of passages 5 and 10 of CHIKV and sequenced them. The four E1 genes were aligned with MEGA 7.0.t-test was applied in the comparison between groups. Each experiment was set in triplicate, and each independent experiment was repeated three times. It was considered statistically significant when the value of p < 0.05.All data were input in SPSS 17.0 for statistical analysis. We have deposited the metagenomic sequencing data in the GenBank Sequence Reads Archive. The obtained accession numbers were SRR15058239 to SRR15058241. We deposited the amplified sequences in the GenBank, whose accession numbers were as follows: CHIKV-China/YN2018E1-1/2/3/4 (MZ494489–MZ494492), CHIKV-China/YN2018NS3-1/2/3/4/5 (MZ494493–MZ494497), GETV-China/YN2018E2-1/2/3 (MZ494498–MZ494500), GETV-China/YN2018NS3-1/2 (MZ494501–MZ494502), and RRV-China/YN2018C-1/2 (MZ494503–MZ494504), respectively.Togaviridae and Bunyaviridae.In order to acquire clean data of mosquito viruses, we eliminated host sequences and barcode DNA. A total of 25,768,166 reads were obtained by Illumina sequencing, and the mean read length was 184.13 nt Table 1.SOAPdenovo was performed to assemble virus sequences into contigs, and 14,127 viral contigs were obtained, of which many virus-like contigs were remarkably similar, such as 63 CHIKV-like contigs with a read coverage of 37× (292–906 nt), 126 GETV-like contigs with a read coverage of 54× (226–872 nt), and 35 RRV-like contigs with a read coverage of 12× (235–843 nt). The CHIKV-like contigs have 92.6%–95.1% nt homology with the known CHIKV sequences. The GETV-like contigs have 83.7%–91.3% nt homology with the known GETV sequences. The RRV-like contigs have 81.6%–90.5% nt homology with the known RRV sequences. Next, we designed and synthesized specific primers to amplify the identified viruses, to further confirm the results of metavirome sequencing Table 3.Alphavirus of Togaviridae. The results were verified by RT-PCR. Four 1,317-nucleotide-long segments (CHIKV-China/YN2018E1-1/2/3/4) and five 1,068-nucleotide-long segments (CHIKV-China/YN2018NS3-1/2/3/4/5) were amplified, and shared ~81%–95% and ~94%–98% nucleotide (nt) identity and ~83%–88% and ~86%–95% amino acid (aa) with each other, respectively. We aligned these segments using BLASTN against the GenBank, and identified that they shared high identity with the E1 gene and the non-structural protein 3 (NS3) of CHIKV from Senegal isolated in 1983, with at least 81% and 80% nt identity and 83% and 83% aa identity, respectively, suggesting that these viral genes from a CHIKV variant  and two 1,572-nucleotide-long segments (GETV-China/YN2018NS3-1/2) were amplified, and shared ~96%–97% and ~97% nt identity and ~91%–93% and ~93% aa identity with each other, respectively. We aligned these segments using BLASTN against the GenBank, and identified that they shared high identity with the envelope 2 (E2) gene and the NS3 gene of GETV sequence from Equus caballus isolated in Japan, 1978, with at least 97% and 97% nt identity and 93% and 93% aa identity, respectively, suggesting they were from a GETV variant  were amplified, and shared ~94% nt identity and ~87% aa identity with each other, respectively. We aligned these segments using BLASTN against the GenBank, and identified that they shared high identity with the capsid protein (C) gene of RRV, with at least 94% nt identity and 89% aa identity, respectively, with the RRV sequence from human isolated in Australia, 2006 . As predicted, E1 gene/protein was positively expressed in CHIKV-infected cells rather than mock-infected cells. In order to further verify the isolation of CHIKV, we observed the virus particles through negative-stain electron microscopy, which were shown rounded with approximately 70 nm diameter and tiny protrusions on the surface, which presented CHIKV-like particles  in comparison with detection in BHK-21 cells.After sequencing, MegAlign was utilized to align the CHIKV-China/YN2018-1 E genes of P5 and P10. Nucleotide identity analysis showed that E1 gene of P5 shared 99.2% nucleotide identity with that of P10. Then, follow-up infection was performed on Vero cell lines, and the 5th and 10th generations of CHIKV-China/YN2018-1 E genes were sequenced. Nucleotide comparison analysis showed that 9 nucleotide sites of P5 and 14 nucleotide sites of P10 of E genes presented polymorphism. Alignment of the amino acid sequence showed that one vital amino acid site (position: 226 aa) of E1 protein in P5 and P10 was mutated, with amino acid A  changed to V . Moreover, after detecting the viral titers in Vero cells, the P5 and P10 of CHIKV-China/YN2018-1 were 1.67 × 10Ae. aegypti/Ae. albopictus mosquito species  of E1 proteins in P5 and P10 both mutated, with amino acid A  changed to V . It has been reported that E1-A226V mutation at the position of 226 in E1 protein can significantly improve the adaptability of CHIKV to bopictus . Then, wIt was obvious that our results indicated that the distribution of viruses detected in mosquitoes obtained from Yunnan province depends on the geographical location of mosquitoes. However, our current research explored only a small part of the mosquito viromes in this area; further research is needed in more regions or countries to explore the abundance and diversity of the mosquito viromes. In general, our research has brought useful insights for virus isolation and identification of CHIKV and new GETV, which potentially will be used in the field of viral epidemiology.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/PX and NL conceived and designed the experiments. GF, JZ, CL, YZ, and DZ performed the experiments. PX and YL analyzed the data. HZ contributed reagents/materials/analysis tools. PX and NL wrote the paper. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China [grant number 32002312] and the Wenzhou Basic Scientific Research Project [grant number Y2020103].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.


Extensive RVFV replication was detected in many tissues of the mosquito with the notable exception of the interior of ovarian follicles.Rift Valley fever virus (RVFV) causes a zoonotic mosquito-borne haemorrhagic disease that emerges to produce rapid large-scale outbreaks in livestock within sub-Saharan Africa. A range of mosquito species in Africa have been shown to transmit RVFV, and recent studies have assessed whether temperate mosquito species are also capable of transmission. In order to support vector competence studies, the ability to visualize virus localization in mosquito cells and tissue would enhance the understanding of the infection process within the mosquito body. Here, the application of in situ hybridization utilizing RNAscope Phenuiviridae and genus Phlebovirus, which has a tri-segmented genome consisting of a small (S) segment encoding the virus nucleoprotein and a non-structural protein, a medium (M) segment encoding two glycoproteins, Gn and Gc, and a large (L) segment encoding the virus RNA-dependent RNA polymerase. Rift Valley fever virus is transmitted to and between mammals by mosquitoes, although human infections usually result from close contact with contaminated carcasses. Abortions in ruminants are often observed within livestock following infection with RVFV. In addition, the movement of infected livestock has been implicated in the establishment of RVFV in areas outside of sub-Saharan Africa, such as the Arabian Peninsula [Rift Valley fever is an economically significant zoonotic disease of livestock in sub-Saharan Africa. Outbreaks can occur suddenly, often driven by the onset of heavy rainfall, resulting in significant morbidity and mortality amongst livestock and humans . The caueninsula  and islaeninsula ,4.Aedes and Culex, reviewed in [®, a form of in situ hybridization using RNA probes, was developed to detect RNA within formalin-fixed, paraffin-embedded tissue [® for detection of RVFV within mammalian liver tissue [A range of mosquito species have been associated with transmission of RVFV, particularly those within the genera iewed in ,6. Estabiewed in ,8. Formad tissue . A recenr tissue , a key tGAPDH) gene confirmed the methodology was effective for insect tissue. Staining with RVFV probes demonstrated extensive RVFV RNA labelling in key structures such as the basal layer of the mosquito midgut and the proventriculus, suggesting points of entry for the virus following consumption of a bloodmeal.In this study, we have adapted a similar approach, but using probes designed against the virus nucleoprotein gene on the S segment, to investigate the presence and distribution of RVFV RNA within an infected mosquito. Control probes targeting mosquito glyceraldehyde-3-dehydrogenase /mL. Mosquito infection with RVFV was conducted as previously described [A colonized, indigenous mosquito strain of Caldbeck , was oriescribed . A secon® (Advanced Cell Diagnostic ACD) is a commercially available RNA in situ hybridization (RNA-ISH) technology, applied here to study RVFV replication and localization with light microscopy of the whole mosquito following oral infection. Novel probes targeting the negative sense nucleoprotein-coding gene of RVFV strain ZH501  and mosquito GAPDH gene from Culex quinquifasciatus, Say 1823,  were designed and produced by Advanced Cell Diagnostics. Mosquito sections were selected based on preliminary images of uninfected Culex pipiens stained with haematoxylin and eosin. A negative control probe, supplied with the RNAscope® 2.5 High Definition (HD)–Red Assay kit , was performed on parallel sections targeting the bacterial dihydrodipicolinate reductase (DapB) gene that was not expected to be present in the sample; any observed staining to this negative probe was due to non-specific binding. RNAscope® staining procedures were conducted following the manufacturer’s instructions; all steps were performed at room temperatures with reagents supplied with the kit, unless stated. Slides were dewaxed for 5 min in xylene, immersed in 100% alcohol for 2 min, then dried. Slides were treated with hydrogen peroxide for 10 min, washed in water and placed into the target retrieval solution (supplied with the kit) for 15 min at 98–100 °C, then washed immediately with distilled water, followed by 100% alcohol and dried. A hydrophobic barrier was drawn around the section using the pen and templates provided and dried for 20 min or overnight. Sections were treated with Protease Plus (ACD) at 40 °C for 15 min.RNAscopeGAPDH, DapB) for 2 h at 40 °C and then washed in 1× wash buffer (twice for 2 min). After each of the subsequent hybridization steps, the sections were washed in 1× wash buffer (twice for 2 min). Solution AMP 1 (supplied by the manufacturer ACD) was applied for 30 min, followed by AMP 2 for 15 min, AMP 3 for 30 min and AMP 4 for 15 min, all at 40 °C. Buffers AMP 5 and 6 were incubated at room temperature for 30 and 15 min, respectively. Fast Red was then used to detect the signal by incubating at room temperature for 10 min. Slides were washed with water and counterstained with Gills 1 haematoxylin for 2 min and dried, either for 1 h at 60 °C or overnight at room temperature. A coverslip was applied using EcoMount . Images were captured with a Pannoramic 250 Flash II slide scanner , and image selection was performed on digitized images in a Pannoramic Viewer .Prepared sections were washed with water before applying the probes  were fed a blood meal containing RVFV strain ZH501 and maintained for 7 days at 25 °C. Each mosquito was then fixed in formalin, and preliminary sections were stained with haematoxylin and eosin (To visualize virus infection within the tissues of nd eosin  to ident® procedure and detect virus distribution, RNA probes were designed to detect a mosquito host target and RVFV virus genome. Sections were counterstained to highlight tissue structures. The negative control probe based on the DapB gene (To validate the RNAscopeSpecific staining with the RVFV probe was detected in 20% of infected mosquitoes, similar to that observed in previous vector competence studies . Whole mAt higher magnification, the extent of RVFV replication could be assessed within individual tissues . Images Midgut (Legs (Thorax (Head (Johnston’s organ (Ommatidia (Ovarian follicles (Culex pipiens to support virus escape from the midgut and replication at secondary sites, as recently reported by [® in mosquito infection studies, demonstrating a novel highly sensitive method to detect virus RNA localization within the mosquito vector.The results reported within this study complement those reported by , where Lorted by . These d


Ivermectin is widely used in both animals and humans as an FDA-approved parasiticide. Ivermectin has also been reported to have antiviral activity against several viruses including coronaviruses. There are reports that indicate ivermectin may have some role in diminishing the disease caused by SARS-CoV-2, but the evidence is inconclusive. The objective of this study was to determine if ivermectin was efficacious in inhibiting avian infectious bronchitis virus  replication in chicken embryos. Briefly, our approach was to use the Massachusetts vaccine strain of IBV in combination with various doses of ivermectin and then inoculate these preparations into chicken embryos to determine if IBV replication was inhibited. The embryos were examined for IBV lesions and samples of chorioallantoic fluid were collected for IBV RT-PCR analysis. Several trials were performed, and the results of our study indicate that ivermectin did not inhibit IBV replication in chicken embryos. These reports piqued our interest in determining the potential of using ivermectin to combat coronaviral diseases of poultry. Therefore, the objective of this study was to determine if ivermectin had antiviral efficacy against the poultry coronavirus, avian infectious bronchitis virus, using a chicken embryo model.During the first quarter of 2020 the COVID-19 pandemic was spreading across the United States at an alarming rate with many afflicted people being hospitalized. The medical community developed extreme interest in searching for drugs to combat the disease. Many drugs received consideration. Although these drugs were FDA approved, they were approved for disease conditions and indications other than COVID-19. Some of these drugs received much attention in the popular press and included antiviral, anti-inflammatory and anti-parasitic drugs. Drugs that were evaluated included remdesivir, baricitinib, choloroquine/hydroxychloroquine, famotidine, ivermectin and others . Heidary2.2.1.This study was conducted at the University of Nebraska – Lincoln. The protocols were approved by the Institutional Animal Care and Use Committee (Project ID 2114).2.2.50) was determined by a previously published method . SARS-CoV-2 does not infect chickens or chicken embryos [in vitro cell culture system [in vitro cell culture system with an avian influenza virus [Although SARS-CoV-2 and IBV are both coronaviruses we recognize there are important differences between the two. One report states that the genomes of the two viruses have only 43% identity . SARS-Co embryos ,13 and Ie system . Antivirza virus . There aza virus . PerhapsNegative research results are often deemed not worthy of reporting. However, because of the public health concerns and information being distributed about ivermectin, we believe the results of this study are germane. Unfortunately, there have been those that have consumed veterinary formulations of ivermectin in an apparent attempt to prevent or treat COVID-19 resulting in hospitalization .


We describe two neurological cases of Oropouche virus infection in northern Brazil, where the virus is endemic but neglected as a pathogen. This study reiterates the necessity of developing protocols for diagnosing infections and training medical personnel to recognize the pathogenicity of Oropouche virus in neurological infections. West Nile virus (WNV), Zika virus (ZIKV), dengue virus (DENV), Saint Louis encephalitis virus (SLEV), and chikungunya virus (CHIKV), can cause infection or impairment to the CNS , are less known to be associated with CNS infections.The emergence of encephalitic arboviruses in Brazil requires the attention of health and surveillance professionals to investigate neurological impairment as a possible symptom of arbovirus infections , and is transmitted via the urban cycle to humans by Culicoides paraensis. The virus circulates in Central America (Panama and Trinidad and Tobago) and South America (Brazil and Peru), causing more than 30 epidemics and over half a million clinical cases of Oropouche fever. The disease caused by OROV is characterized by acute febrile illness, accompanied by headache, arthralgia, myalgia, photophobia, and other systemic manifestations. Some patients exhibit neurological manifestations, such as aseptic meningitis or even meningoencephalitis, but this is rare or underdetected. In some cases, the symptoms of Oropouche fever commonly reappear a few days after the end of the first febrile episode; however, the symptoms are usually less severe, and patients generally recover completely without sequelae, even in severe cases . Computed tomography (CT) of the head and magnetic resonance imaging of the brain showed no alterations; however, the electroencephalogram identified episodes of slow waves in the temporal lobe. The patient was discharged from the hospital 48 h after admission, without convulsions and in a generally good condition. The next day, she experienced vomiting, diarrhea, fever (38 °C), and a new convulsive episode. She was clinically re-evaluated at the hospital, and while normotensive, a sample of clear CSF was collected 4 days after the onset of symptoms that tested negative for bacterial and fungal growth.In the Brazilian National Reference Laboratory for arboviruses, quantitative reverse-transcription polymerase chain reaction (RT-qPCR) for CHIKV , but the majority of OROV human infections are reported in Brazil, with a cumulative estimate of over 450 million infections since the first OROV epidemic in 1961  in the state of Pará, Brazil  currently circulating in the south and central Americas (Travassos da Rosa et al. These two cases stress the necessity of training health workers, especially medical staff, to recognize the pathogenicity of OROV in neurological infections and amplifying the diagnostic capacity for OROV. Additionally, OROV should be recognized as a potential pathogen in cases of fever associated with neurological symptoms, regardless of meningitis and meningoencephalitis diagnosis, especially in patients with a history of exposure to arboviruses.The poor epidemiological and pathological OROV scenario, regarding knowledge of the scientific community, especially in northern Brazil where the virus is endemic, reflects how OROV is neglected by the health professionals working in this region. Indeed, OROV is usually not considered in the investigation of etiological agents of neurological infections, including meningitis and encephalitis, indicating that studies are needed to improve the understanding of neurological OROV infections and amplify the laboratorial capacity for OROV diagnosis. We believe that such studies may lead to the eventual inclusion of OROV infections within the scope of clinical neurological diseases and in the improvement of epidemiological data of OROV in Brazil.


Batch adsorption experiments of alginate/clay hybrid composite beads investigated the effect of metal concentration in the range of 1–4 mg L−1 on Cu(II) removal, adsorption kinetic for maximum 240 min, and Langmuir and Freundlich adsorption isotherms by using atomic absorption spectrometry. The pseudo-second-order kinetic model best fitted the adsorption for alginate/montmorillonite beads (R2 = 0.994), while the diffusion process was predominant for montmorillonite/kaolin beads (R2 = 0.985). The alginate/clay hybrid materials best fitted the Langmuir isotherm model.Massive amounts of industrial and agricultural water around the world are polluted by various types of contaminants that harm the environment and affect human health. Alginic acid is a very versatile green polymer used for heavy metal adsorption due to its availability, biocompatibility, low cost, and non-toxic characteristics. The aim of this paper was to prepare new low-cost hybrid composite beads using sodium alginate with treated montmorillonite and kaolin for the adsorption of copper (Cu) cations. Modified and unmodified clays were investigated by studying their morphology and elemental composition, functional groups, and mean particle size and particle size distribution. The characterization of alginate/clay hybrid composite beads was carried out by evaluating surface morphology , crystallinity , and point of zero charge (pH Massive amounts of industrial and agricultural water around the world are polluted by various types of contaminants that harm the environment and affect human health. Removing contaminants from effluents with sustainable adsorbent materials made from abundant and inexpensive cellulose, alginate, starch, chitosan is a feasible approach to deal with this problem ,4,5,6,7.−1 of copper. Other authors reported that a solution with a concentration of 1 g L−1 acidified alginic acid was able to sequester 54.11% of Cu(II) [Among these molecules, alginic acid is a very versatile green polymer used for heavy metal adsorption due to its availability, biocompatibility, low cost, and non-toxic characteristics. Alginates are anionic polysaccharides obtained from seaweed, consisting of a linear chain of 1–4) β-D-mannuronic acid (M) and residues of α-L-guluronic acid (G) arranged in irregular blocks, having applications in interdisciplinary fields such as medicine , drug def Cu(II)  or starcf Cu(II)  and tern β-D-mann−1 of Cd ions [Clay is a mixture of aluminosilicates that contains silicon, aluminum, manganese, titanium, potassium, calcium, and sodium ,16,17. T Cd ions . Besides Cd ions  and for  Cd ions . In this−1 [−1, most of the aquatic species are sensitive to the Cu(II) [Copper (Cu) is a heavy metal occurring in geological deposits, sites of volcanic activity, and following weathering and erosion of rocks and soils . Anthrop−1 . Howevere Cu(II) . This hie Cu(II) ,40, chele Cu(II) , and elee Cu(II)  are succ2+ ions from synthetic waters by batch adsorption. We hypothesized that the carboxyl (–COO–) and hydroxyl (–OH) functional groups present in alginate and the reactive –OH groups present in mineral clays would act as adsorption sites, facilitating the successful removal of Cu2+ from synthetic water.The objectives of this study were: (i) to improve by chemical treatment the affinity of montmorillonite and kaolin for copper cations; (ii) to reinforce sodium alginate with modified clays, thus obtaining new hybrid composite materials; (iii) to use the new hybrid composite materials as potential adsorbents for removing Cu6H7O6Na)n, 90.8% purity, characterized by a molecular mass in the range of 10,000–600,000 g/mol, with max. Pb content of 0.002%, mass loss at drying of max. 15%, and viscosity (1% at 20 °C) of 350–550 mPa·s was used as a polymeric matrix. We prepared a 1% (w/v) sodium alginate solution by dissolving sodium alginate into 100 mL of distilled water at a temperature of 90 °C, stirring at 600 rpm for 2 h.Sodium alginate powder , (C− ions (tested with 0.1 M AgNO3 solution). Finally, the samples were dried in an oven at a temperature of 90 °C for 6 h and ground before use. A schematic flow chart illustrating the steps for the chemical modification of clays is shown in 2 × 2H2O), analytical-grade, was used as the crosslinking agent.Montmorillonite and kaolin clays were acquired from Siceram S.A. . In order to increase the cationic exchange capacity, the starting clays were modified according to procedure described by Taleb et al.  with litw/v, were added into the calcium alginate solution under magnetic agitation at a temperature of 90 °C, stirring at 600 rpm for 1 h. In order to eliminate the air bubbles, the obtained solution was sonicated in an ultrasonic bath for 30 min. Calcium alginate/montmorillonite and calcium alginate/kaolin hybrid composite beads were obtained by pouring the previously obtained solutions dropwise into a 1 M CaCl2 solution under stirring at 200 rpm, by means of a peristaltic pump. The obtained beads were collected and washed with distilled water and dried in an oven at a temperature of 30 °C for 72 h. Calcium alginate microbeads were prepared in the same conditions, as a reference material. Weight reductions about 7 times and 10 times for alginate/clays beads and calcium alginate beads, respectively, compared with the wet beads, were recorded after drying.Modified montmorillonite and kaolin, respectively, 2% Modified and unmodified clays were investigated by studying their morphology and elemental composition, functional groups, and particle size distribution.The characterization of alginate/clay hybrid composite beads was carried out by evaluating their surface morphology, chemical structure, mean size and particle size distribution, and crystallinity. −1. Twenty scans were collected for each sample before and after Cu ions adsorption at a spectral resolution of 2 cm−1. Point of zero charge (pHpzc) measurements for clays particles in suspension were carried out by help of a Zetasizer Nano , at the pH values ranging from 2 to 13 adjusted with 1 mM NaCl or 1 mM HCl. The pHpzc value was determined from a plot of initial pH versus the pH of the samples. X-ray diffraction (XRD) analysis was performed using a PANalytical X’Pert PRO MPD spectrometer  with a Cu anode.A scanning electron microscopy (SEM) with field emission using a QUANTA 450 FEG model  coupled with an energy dispersive X-Ray analysis detector (EDX) and a gun at a 1.2 nm resolution, with a resolution of 133 eV was used to characterize the morphology of the materials. Microscopy analysis was performed in low-vacuum mode with a vacuum of 50 Pa. Attenuated total reflectance–Fourier transform infrared spectroscopy (ATR–FT-IR) was performed with an Interspec 200-X Spectrophotometer  in the spectral region of 4000–750 cm−1. A solution of copper in HNO3 at a concentration of 1000 mg L−1, the ICP multielement standard solution IV , was used as the calibration standard.The batch adsorption experiments of alginate/clay hybrid composite beads were performed to evaluate the Cu(II)removal, adsorption kinetics for maximum 240 min, and Langmuir and Freundlich adsorption isotherms with the help of ContrAA 800 atomic absorption spectrometer  with the flame technique, using solutions with metal concentrations in the range of 1–4 mg LC0 is the initial adsorbate concentration (mg L−1)and Ce is the adsorbate concentration (mg L−1) at the liquid–phase equilibrium.Cu(II) adsorption and the equilibrium adsorption capacity were calculated using Equations (1) and (2).eq) was calculated using Equation (2):V represents the volume of adsorbate (L), and m is the mass of the adsorbent used .Data in The size distribution of the clay particles versus the relative intensity of light scattered by the particles is shown in As shown in The FTIR spectra of the original and treated clays are shown in −1, attributed to the Si–O stretching of montmorillonite. The band located about 915 cm−1 was ascribed to the presence of Al2OH species that are characteristic of dioctahedral smectites [−1 was attributed to the Si2OH bending mode. The band located at 840 cm−1 was assigned to the AlMgOH bending mode. All absorption spectra shown in −1, attributed to the tensile vibration of the Si–O bond. The absorption peak at 910 cm−1 in the kaolin structure was due to the presence of Al2OH, while the absorption band at 788 cm−1 occurred due to the bending vibration of the Si2OH group. The absorption spectrum of kaolin after treatment showed two localized peaks at 3689 cm−1 and 3619 cm−1 attributed to the stretching vibration of Al(OH)Al [−1 in the case of montmorillonite was attributed to the bending vibration of the H–O–H group. After treating the clays, the absorption bands of the functional groups increased in intensity.All spectra exhibited a main band located at 1035 cmmectites , while tAl(OH)Al . This coThe characterization of clays performed by SEM/EDAX and FT-IR analyses revealed that the treatment of clays with HCl and NaCl improved the surface properties, allowing the clays to retain heavy metal pollutants.The surface morphology of alginate/clay composite beads was studied by SEM analysis, which is illustrated in SEM analysis revealed that the Ca alginate beads had a smoot surface, while the alginate loaded with montmorillonite/kaolin beads presented irregularities that allow more effective contact with the environment to which they are exposed due to their specific surface . This leThe crystallinity of the prepared beads in comparison with that of the original and treated clays is presented in 2 and sodium calcium aluminum silicate, which are the main components of the base material, could be observed at their specific 2θ angles and Miller indices of 26.62 ((0 1 1) for SiO2 and (−1 1 4) for the aluminosilicate); 50.1 ((1 1 2) for SiO2 and (0 −4 6) for the aluminosilicate), and 20.84 ((1 0 0) for SiO2). Kaolin showed specific peaks for SiO2 at 26.62 (0 1 1), 20.84 (1 0 0), and 50.1 (1 1 2), for Al2Na2O6Si at 27.77 (0 4 0), 22.01 (2 0 −2), and 28.07 (0 0 −4), and for TiO2 at 24.95 (1 0 1), 47.17 (2 0 0), and 38.01 (0 0 4). By comparing the results in visible peak intensity between the various stages of processing that the base material went through, it is clearly visible that by modifying montmorillonite and kaolin, a more refined structure emerged that displayed clearer crystallinity, which can be associated with a more homogeneous structure of the material.The XRD analysis of the samples showed differences in peak intensities for each processing step which the materials underwent. In the case of montmorillonite, the specific peaks for SiOThe addition of alginate, however, reduced the peak intensity due to its organic nature, as it covered the base materials and diluted the X-ray intensity during the analysis. This is also an indicator that the two materials were combined efficiently, which increased the homogeneity of the final product.pzc value, the surface of the hybrid composites became negative, which facilitated the adsorption of positive ions [The alginate/montmorillonite and alginate/kaolin particles found in suspension in an NaCl solution exhibited almost similar zeta potentials and point of zero charge at pH 2.7 ± 0.01 for the alginate/montmorillonite beads and 2.4 ± 0.01 for the alginate/kaolin beads. At pH values greater than the pHive ions . The adsive ions . In Figu−1, 2 mg L−1, 3 mg L−1, and 4 mg L−1 pollutant concentrations using an amount of 250 mg of adsorbent.The effect of Cu(II) concentration on time was assessed at 1 mg L−1 to 4 mg L−1, the percentage of removed Cu(II) decreased from 76.1 ± 1.6% to 52.4 ± 0.7% in the case of the alginate/montmorillonite beads (−1 [te beads A and frote beads B. This beads (−1 . As it ieq and tq are the adsorption capacities at equilibrium and time t (mg g−1), and k1, K2, and iK are the rate of pseudo-first-order adsorption (l min−1), pseudo-second-order adsorption (g/mg min), and intradiffusion, respectively; iC is an arbitrary constant representing the boundary layer thickness.The dynamic of the adsorption process was investigated by using pseudo-first-order, pseudo-second-order, and Weber’s intraparticle diffusion kinetic models, according to the Equations (3)–(5):The pseudo-first-order kinetic model is based on the hypothesis that the rate of change of solute uptake with time is directly proportional to the difference in saturation concentration and the amount of solid uptake with time. The pseudo-second-order kinetic model states that the rate-limiting step is the chemical sorption or chemisorption and predicts the behavior over the whole range of adsorption. The intraparticle diffusion model assumes that the diffusion of the adsorbate controls the adsorption process.e–qtq) versus contact time, tt/q versus contact time, and adsorbate uptake versus the square root of time (t0.5) calculated according to Equations (3)–(5), respectively, are shown in eq (mg/g) were evaluated from the slope and intercept of the regression lines.A plot of Log. A diffusion process was predominant for montmorillonite/kaolin beads (R2 = 0.985). It was also observed that the rate constant for montmorillonite beads was in the order diffusion > pseudo-second-order model > pseudo-first-order model. In the case of the intraparticle diffusion model, the adsorbate faster diffused through the solution into the pores of alginate/montmorillonite beads (Ki = 0.027 ± 0.0014 mg/g min0.5). This behavior was correlated with the pore dimensions of montmorillonite powder  did not significantly differ when using pseudo-first-order and pseudo-second-order models. The qmax was 0.54 ± 0.040 mg g−1 for the alginate/montmorillonite beads and 0.341 ± 0.023 mg g−1 for the alginate/kaolin beads. The experimental adsorption capacity of the alginate/montmorillonite and alginate/kaolin beads was 0.398 mg g−1 and 0.202 mg g−1, respectively. The adsorption kinetic parameters obtained by the pseudo-second-order model best provided a correlation with the experimental data. A similar heterogeneous process was reported for the removal of aqueous Ba2+ by natural and Fe(III) oxide-modified allophane, beidellite, and zeolite adsorbents [The results in e powder . For thesorbents . The psesorbents , and of sorbents .−1 copper ion concentration. The Langmuir adsorption isotherm quantitatively describes the formation of a monolayer of adsorbate on the outer surface of the adsorbent, after which, no further adsorption takes place.Langmuir and Freundlich adsorption models were used to obtain the adsorption isotherms. The isotherms were obtained at 3 mg LThe parameters of the Langmuir and Freundlich adsorption isotherms are reported in L) was between 0 and 1. For the alginate/montmorillonite and alginate/kaolin beads, the maximum monolayer adsorption capacity (qmax) was close to the experimental value (eq) for Cu(II) removal, indicating values of 0.6802 ± 0.02 mg g−1 and 0.3389 ± 0.03 mg g−1, respectively. The Langmuir model showed higher R2 than that the Freundlich model for all prepared alginate/clay beads, indicating the monolayer adsorption capacity of the alginate/clay hybrid composite materials. All alginate/clay beads showed a value of inverse adsorption intensity of the process (1/n) below 1, which indicates large Cu(II) adsorption. The KF values for alginate/montmorillonite and alginate/kaolin beads were higher , indicating a great removal capacity for Cu(II) as a pollutant from synthetic waters. The phenomenon is in accordance with other studies regarding heavy metals adsorption on different activated adsorbents [The equilibrium adsorption process was favorable for both adsorbents, because the dimensionless separation factor  retained on the alginate/clay hybrid beads, FTIR analysis was performed .−1 (-OH) and 2928–2850 cm−1 (C-H groups) decreased in intensity, indicating the formation of hydrogen bonds between the pollutant and the hydroxyl groups of alginate and clay. Similar chemical interactions were found in the case of a cellulose/clay composite hydrogel developed for the adsorption of dye [As it is observed in n of dye .Alginate/montmorillonite and alginate/kaolin beads were prepared by the ionic gelation method. Activated clays showed improved surface properties, nanometric size, and polydispersity, and were able to retain heavy metal pollutants. The SEM and FTIR results highlighted the interactions between the alginate and the clays, leading to the formation of hybrid composite beads with better adsorption properties for Cu(II) than the original clays.−1 for Cu(II) removal. This study demonstrates that the low-cost hybrid material beads based on alginate, montmorillonite, and kaolin can be recommended as promising adsorbents for Cu(II) removal from wastewaters, with improved adsorption properties.The optimal contact time for Cu(II) removal from an aqueous solution was 210 min for the alginate/montmorillonite beads and 135 min for the alginate/kaolin beads. The adsorption processes followed a pseudo-second-order kinetic for the alginate/montmorillonite beads and an intraparticle diffusion model for the alginate/kaolin beads. The alginate/montmorillonite and alginate/kaolin beads well fitted the Langmuir isotherm model and exhibited an adsorption capacity between 0.33 and 0.68 mg g