Among natural polymers, starch and chitosan represent two biomolecules with proof of efficiency and low economic impact in various utilization fields. Therefore, herein, the features of hydrogel beads obtained from chitosan and three sorts of starch , grafted with acrylonitrile and then amidoximated, were deeply investigated for their use as sorbents for heavy metal ions and dyes. The hydrogel beads were prepared by ionotropic gelation/covalent cross-linking of chitosan and functionalized starches. The chemical structure of the hydrogel beads was analyzed by FT-IR spectroscopy; their morphology was revealed by optical and scanning electron microscopies, while the influence of the starch functionalization strategies on the crystallinity changes was evaluated by X-ray diffraction. Molecular dynamics simulations were used to reveal the influence of the grafting reactions and grafted structure on the starch conformation in solution and their interactions with chitosan. The sorption capacity of the hydrogel beads was tested in batch experiments, as a function of the beads’ features  and simulated polluted water, which included heavy metal ions (Cu In the last few years, extensive research has been undertaken to obtain specialized and selective sorbents containing natural polymers as a cheap and environmentally friendly solution for water cleaning. In this respect, polysaccharide-based hydrogels were studied, taking advantage of their low cost, availability, non-toxicity and biodegradability ,2. AmongA number of studies have shown that polymers containing amidoxime groups have high complex-forming capabilities with metal ions and can be successfully used in metal ion removal from aqueous solutions ,28,29,304+ ions [4+ ions as the initiator, was tested on three sorts of starch, namely PS, wheat (WS) and rice (RS) starches, followed by obtaining soluble derivatives by hydrolysis [Starch is an interesting bio-material, due to its abundance and low cost, but has poor mechanical properties and the fact that it is highly hydrophilic. To overcome these drawbacks, chemical modification of starch is usually applied, mainly by grafting reactions. A large range of polymers can be grafted on starch by ring-opening and radical polymerizations of various monomers in order to modulate the properties of the final products . For exa4+ ions  and then4+ ions . Ionic c4+ ions . Furtherdrolysis  The studdrolysis .2+, Co2+, Ni2+ and Zn2+ ions and for Direct Blue 15 and Congo red dyes was followed in batch experiments, as a function of the beads’ features , and contact time.Therefore, herein, the features of three sorts of starch  grafted with acrylonitrile  and then amidoximated  were deeply investigated, following the formation of composite hydrogel beads with CS. The current study starts with the premise that the use of starch from different botanical sources in grafting reactions can influence the properties of the obtained materials, and consequently their properties. Thus, we aimed to prepare hydrogel beads by ionotropic gelation and covalent cross-linking of CS and functionalized starches as SgAN or SgAx. The chemical structure of functionalized starches and the obtained hydrogel beads was followed by FT-IR spectroscopy; their morphology was revealed by optical and scanning electron microscopy, while the samples’ crystallinity changes in relation to the functionalization strategies was evaluated by X-ray diffraction. Molecular dynamics simulations were used to determine the influence of the grafting reactions and grafted structure on the starch conformation in solution and their interactions with chitosan. The sorption capacity of the beads for CuPrevious studies showed that the grafting reaction, in the current applied conditions, took place on the amylose part of starch  and the 4+ ions, which interacts with the -OH groups located at the C2 and C3 carbon atoms.The particle size analysis of native starches  simulations were performed in order to visualize the conformation modifications of the starch (amylose) backbone after AN grafting reaction and its further amidoxime functionalization. The different types of patterns observed in starch crystallization are determined mainly by the interaction between two starch molecules. This is why the three simulated systems contained two identical starch molecules  solvated in water , as is the case for the similar groups in CS; thus, a double crosslinked network is formed.The optical images in The samples’ porosity in the swollen state was estimated by measuring 50 pores in each SEM image from Thus, the obtained values of the aspect ratio are higher than 1.00 (circles), meaning that the pores have elongated shapes. In addition, the Feret diameter values showed that the larger (the higher diameter) and the more irregular shaped  pores were obtained when WS was used in hydrogel bead synthesis and were smaller with RS. The same trend was observed after bead amidoximation (SgAN-Ax samples). Larger amylose contents in RS (up to 30%) favor better grafting reactions and resulted in its better embedment into the hydrogel beads, leading to smaller pore sizes. PS and WS, with almost the same amylose content  formed hydrogel beads with larger pores. The amidoximated starch directly used in bead formation led to smaller size pores, as the starch had a very small influence on their porosity. In this synthesis route, the SgAx participation to crosslinking along with CS, and also the elongated form of SgAx both contributed to obtaining homogeneous beads with very close pore sizes, irrespective of the starch source. This observation is also supported by the MD simulations of the Ax-grafted starch that showed the side chains extended in the solvent, which were completely free to interact with ECH . To furtThe MD simulations showed that CS is able to interact with the SgAN molecules, coming into contact with both the AN side chains and the amylose backbone , 1591–1595 cm−1 to -NH2 groups on both CS and modified starch  and 1557–1559 cm−1 to vibrations of the protonated amine group. The same region in the FTIR spectra of the composite beads obtained by the amidoximation of the already formed CS/SgAN beads (namely CS/SgAN-Ax samples) demonstrated five individual peaks, with the peak ascribed to C=N in the oxime groups being shifted to about 1660 cm−1 and the new peak at about 1630 cm−1 being ascribed to the -OH groups bending. Furthermore, for these samples, the contribution of the peak at 1660 cm−1 diminished and the contribution of that at about 1597 cm−1 increased, suggesting a higher amount of free amino groups obtained by the post bead formation amidoximation reaction groups, which are evidently not involved in the crosslinking reactions with ECH.Thus, the region 1800–1500 cmThe modifications in the starch/CS crystallinity after hydrogel bead synthesis was followed by X-ray diffraction .The chitosan sample shows diffraction peaks at about 9° and 20° (2θ), which could be found in all the samples after hydrogel bead formation, as shown in The swelling behavior represents an important characteristic of a sorbent in its capacity to retain different ions or small molecules. Therefore, the swelling degree was evaluated as a function of time and pH . As show2) of chitosan can be protonated (–NH3+). The amidoximated starches show different points of zero charges, for example, at 4.6, 5 and 5.7 for RSgAx, WSgAx and PSgAx, respectively. Before these values, the primary amino groups of amidoximated starch and CS are protonated; therefore, below pH 4, the interpolymeric electrostatic repulsions in the composite beads and also the intramolecular repulsions between the ionized groups lead to an increase in the bead size; consequently, the equilibrium swelling ratio values increase with the pH decrease (swell between 70 and 90% at pH 2). On the contrary, at pH values up to the CS isoelectric point (6.5), ionization of the -OH groups in the amidoxime functional groups occurs, which could be associated with the protonated amino groups, decreasing the beads’ swelling capacity. Furthermore, the swelled beads are stable across the whole range of the tested pHs, suggesting that the crosslinking process was effective.Almost similar and constant values were obtained when the pH of the swelling media varied between 4 and 8 b, irresp2+, Co2+, Ni2+ and Zn2+ ions from aqueous solutions, in batch experiments. The influence of contact time on the hydrogel beads’ sorption capacity for metal ions is represented graphically in 2+. Furthermore, with some exceptions, the beads obtained with SgAx show better sorption capacities for the investigated ions, as compared to the beads post-amidoximation, following the same trend as that found for the beads’ swelling vs. time (2+ > Ni2+ > Zn2+ ≈ Co2+.Composite hydrogel beads with amidoximated groups (CS/SgAN-Ax and CS/SgAx) were tested as supports for the uptake of Cuvs. time a. For ea2+ and Ni2+, tetrahedral by Zn2+ and square planar by Cu2+. Nevertheless, the found affinity for ion sorption in noncompetitive conditions towards the amidoximated starch-based beads of Cu2+ > Ni2+ > Zn2+ ≈ Co2+ suggests that most probably, sorption is favored by the lower ionic radius and by the structural arrangements that allow for the smallest energy consumption, with copper ions best fulfilling these conditions.The sorption capacity is usually influenced by the ions’ properties, such as ionic radii, hydrated radius, atomic weight, electronegativity, and others, as already observed in other studies ,43,44,45Another important parameter is the initial concentration, which herein was set to 300 mg/L and corresponds to different molar concentrations, i.e., Zn 4.587 mmol/L, Co 5.093 mmol/L, Cu 4.724 mmol/L, and Ni 5.11 mmol/L. Thus, the molar sorption capacity of the gel beads was calculated as the moles of metal ions sorbed after 360 min per moles of active sites in the hydrogel . Each amOrganic dyes are among the major concerns of water pollutants. Congo red (CR) and Direct Blue-15 (DB15) are organic dyes that can be easily dissolved in water, causing difficulties in their removal from contaminated water. Furthermore, they are toxic and carcinogenic, causing various diseases. Both dyes are secondary diazo dyes, with complex aromatic structures that make them non-biodegradable and quite stable. Moreover, they contain anionic sulphonic groups that can electrostatically interact with the protonated amino groups in the composite hydrogel beads. The sorption capacity of the hydrogel beads with amidoxime groups for both CR and DB15 is represented in Thus, PS-based hydrogel beads show the best affinity to CR, whereas for DB1, better results were obtained with RS-based hydrogel beads and lower values were found when hydrogel beads with WS functionalized starch were used, irrespective of the dye sorbed. Furthermore, better results were obtained with CS/SgAx than with CS/SgAN-Ax. This correlates with the chemical structure of the hydrogel beads and the amino groups of both CS and amidoximated starch could interact, in specific conditions, with the negatively charged dye molecules. The influence of the presence of ionic groups on the hydrogel matrix and their interaction with ionizable groups of dyes was also observed in other studies  The size2+, Co2+, Ni2+ and Zn2+) and two dyes (DB15 and CR). The MD simulations show that the AN-functionalized starch behaves in a similar manner to the native one, as the main starch chains wrapped around each other and the side chains were exposed to the exterior, whereas the Ax-functionalized starch molecules remained isolated for the entire simulation. The structures obtained in the MD simulation corroborate well with the XRD result; in the case of the SgAN, the side chains located at the exterior of the wrapped arrangement and the absence of amylopectin leads to a decrease in crystallinity, whereas for SgAx, the V-type pattern of single starch molecules in a coiled conformation is found. The comparison of the C/N and C/O atomic ratios at the surface and inside the beads revealed that there are some differences in the elemental composition and that inside the beads, the atomic ratios values are smaller than outside, but are the closest to the calculated values. FTIR spectra of the CS/SgAx samples were deconvoluted in four individual peaks, irrespective of starch nature, which can be ascribed to both components’ functional groups, since the CS/SgAN beads post-amidoximation shows five individual peaks. The new peak and also the variation in the others’ intensity suggested a high amount of free amino groups obtained by the post bead formation amidoximation reaction, groups which are obviously not involved in any crosslinked reactions with ECH. The hydrogel beads show good sorption capacities for Cu2+, Co2+, Ni2+ and Zn2+ ions and for Direct Blue 15 and Congo red dyes, with their performances being influenced by the synthesis protocol and starch sort. Future studies must continue with other sorption-related experiments as a function of different parameters  to elucidate the sorption mechanism for both inorganic and organic molecules.In this study, the features of three sorts of starch  grafted with acrylonitrile and then amidoximated were deeply investigated, following the formation of composite hydrogel beads with CS, and tested as sorbents for four heavy metal ions , Direct Blue 15 (DB15) and Congo red (CR) were purchased from Sigma-Aldrich and were used as received. Acrylonitrile was distilled at about 77 °C and kept at a low temperature.CS powder, from Sigma-Aldrich, was used as received. The degree of acetylation of 15% and the average molar mass of 385 kDa were determined by the methods previously reported . PS (hum4)2 as the initiator in H2SO4 0.4 M, at 27 °C, under stirring for 1 h, and then separated in methanol. The polyamidoxime-grafted starch (SgAx) samples were obtained by analogous reactions of nitrile groups of SgAN, using hydroxylamine in an alkaline medium [v/v) solution. The same procedure was applied for all the types of starch. The AN average grafted length of three AN/starch structural units was determined from the 1H-NMR spectra of SgAN [The starches grafted with acrylonitrile (SgAN), obtained using potato (PSgAN), wheat (WSgAN), or rise (WSgAN) starch, were synthesized and characterized as described in , using Ce medium . SgAN wa of SgAN  as the rw/v), and then 2 mL ECH was added as the crosslinker. The obtained mixture was dripped  into an aqueous solution of sodium tripolyphosphate of 0.05 M, at room temperature, under gentle stirring (100 rpm). After 4 h, the formed beads were separated and transferred to an aqueous solution of 400 mL 0.1 M NaOH for 2 h, under slow stirring.The composite beads were obtained by two similar methods that have already been published , using CThe beads prepared using SgAN were subjected to amidoximation by analogous reactions, obtaining CS/SgAN-Ax beads; the CS/SgAN beads and 70 mL hydroxylamine solution were introduced into a two-necked flask equipped with stirrer and condenser, and the reaction was carried out under mild stirring for 5 h at 70 °C. Finally, the prepared microspheres (obtained by both methods) were washed with distilled water at a neutral pH, and dried at 104 °C .Information on the surface morphology was evaluated using the Various G4 UC scanning electron microscope , whereas the elemental composition with an energy dispersive X-ray spectroscopy analyzer  were analyzed by SEM. The investigations were carried out on samples sputtered with 10 nm platinum  in a high vacuum mode, using secondary electrons  and concentric backscattered detectors. The samples’ porosity in the swollen state was estimated by selecting 50 pores in each SEM image using ImageJ 1.48v analyzing software  [Optical images were obtained with a Nikon D3300 HDSLR camera, with an AF-P DX NIKKOR 18–55 mm f/3.5–5.6 G VR lens.−1, using 45 scans with a resolution of 4 cm−1 by the KBr pellet technique, using 2 mg of each sample. The spectra deconvolution was carried out using the Opus 4.7v software .FTIR analysis of the grafted samples was performed using a Bruker FT-IR spectrometer. Each spectrum was scanned in the range 400–4000 cmThe native starch, grafted starched (with AN and Ax) and CS-based composite beads were examined by X-ray diffraction by a Rigaku Miniflex 600 diffractometer , using CuKα-emissions in the angular range 3–60° (2θ), with a scanning step of 0.01° and a recording rate of 2°/min.The diameter  of native and AN and Ax-grafted starched was determined by a Morphologi G3SE particle characterization system . The samples were carefully spread out over a glass plate. To evaluate the hydrogel beads’ diameter, only full shaped particles, which were non-aggregated, were measured.Potentiometric titrations were carried out with a particle charge detector . The pH variation between 3 and 10 was achieved using 0.1 and 0.01 M solutions of NaOH and HCl, respectively. The potential measurements were carried out using 1 mg beads in 10 mL Millipore water, at room temperature.−5. Each simulation had a length of 200 ns, the composition of simulated systems being introduced in Three systems containing two starch molecules solvated in water and two systems containing two starch molecules and one chitosan molecule were built using the AmberTools 18 software  . Each st0 and Wt are the weights of the samples in the dried state and in the swollen state at time t, respectively.The swelling behavior of the composite beads was studied using the conventional gravimetric procedure, by immersing the dry samples for different time intervals in Millipore water with different pHs, at 25 °C. Swollen composite hydrogel beads were weighed at predetermined intervals, after wiping the excess surface liquid using filter paper. To calculate the swelling ratio, SR, the three measurements made for each sample and the mean data were used in Equation (1).−5 M. The glass bottles were placed on a slow-moving platform shaker  for 5–6 h. The concentrations of metal ions in the solution, before and after sorption, were analyzed using an atomic adsorption spectrometer (AAS) . The concentration of dyes, before and after sorption, was determined using a UV–Vis spectrophotometer , based on the calibration curves determined at the specific wavelength of 497 nm for CR and 597 nm for DR15. The sorption capacity (SC) of the hydrogel beads was calculated with Equations (2) and (3).Sorption experiments were conducted using glass bottles containing 0.1 g of the sorbents and 20 mL solution of metal ions or dyes, with an initial pH of about 6, initial concentration of metal ions of 300 mg/L and initial concentration of the dye solutions of 3 × 10


During a mouse plague in early 2021, a farmer from New South Wales, Australia, sought treatment for aseptic meningitis and was subsequently diagnosed with locally acquired lymphocytic choriomeningitis virus infection. Whole-genome sequencing identified a divergent and geographically distinct lymphocytic choriomeningitis virus strain compared with other published sequences. Mus musculus). In early 2021, a mouse plague started in western New South Wales, Australia, and spread to the adjoining jurisdictions of Queensland, Victoria, and South Australia, causing considerable losses to the Australia agricultural and grain industry. We report a case of acute LCMV infection in a male farmer from New South Wales.A member of the Arenaviridae family, lymphocytic choriomeningitis virus (LCMV) is an enveloped virus comprised of a bisegmented , negative-stranded RNA genome encoding 2 polypeptides per segment. First discovered in the 1930s during a study of epidemic encephalitis in St. Louis, Missouri, USA , we conducted PCR testing for LCMV on a sample of cerebrospinal fluid, which returned a positive result   . Our AusLCMV may have been introduced to Australia with the arrival of feral rodents brought by the Dutch and Spanish (17th century) or the French and British (18th century) (


Molecular iodine  with a film‐forming agent that extends the duration of the iodine's antimicrobial activity, for its ability to prevent influenza virus transmission between infected and susceptible animals in the guinea pig model of influenza virus transmission.We observed that a once‐daily topical application of this long‐lasting antiseptic to the nares of either the infected virus‐donor guinea pig or the susceptible virus‐recipient guinea pig, or to the nares of both animals, prior to virus inoculation effectively reduced transmission of a highly transmissible influenza A virus, even when the donor and recipient guinea pigs shared the same cage. Daily treatment of the recipient guinea pig starting 1 day after initial exposure to an infected donor guinea pig in the same cage was similarly effective in preventing detectable influenza virus infection in the recipient animal.We conclude that a daily application of this antiseptic formulation is efficacious in reducing the transmission of influenza A virus in the guinea pig model, and further study in this and other preclinical models is warranted. In the United States alone, 31.4 million outpatient visits,seasonal influenza has been vaccination. However, influenza vaccines are not optimally protective against influenza virus infection,The main strategy for prevention and control of The use of iodine as a topical antiseptic dates back nearly as far as its discovery in 18113−) that does not require additional iodine solubilizers and is less irritating to the skin than tincture of iodine while retaining its broad‐spectrum antimicrobial activity.Subsequently, povidone‐iodine (PVP‐I) was introduced in the 1950s as a novel iodine‐based antiseptic. PVP‐I is a complex of the polymer povidone (polyvinylpyrrolidone) and triiodide I3− that doStreptococcus pneumoniae,Haemophilus influenzae,Staphylococcus aureus,S. aureus. In addition, polymer and excipients in the Nasal Prep formulation protect PVP‐I from inactivation by nasal mucins and other organic compounds and increase its adhesion to mucosal surfaces,3M™ Skin and Nasal Antiseptic (PVP‐I solution 5% w/w [0.5% available iodine] USP) Patient Preoperative Skin Preparation Non‐Sterile Solution  is a safe and effective formulation of 5% PVP‐I with a proprietary film‐forming composition that extends the duration of the iodine's antimicrobial activity. It has broad‐spectrum microbiocidal activity against respiratory pathogens such as 22.12.Influenza A/Panama/2007/1999 (H3N2) virus (Pan/99) was originally derived from a 12‐plasmid reverse genetics system.2.2Female Hartley strain guinea pigs weighing 300–350 g were obtained from Charles River Laboratories. Animals were allowed free access to food and water and kept on a 12‐h light/dark cycle. Guinea pigs were anesthetized prior to intranasal inoculation and to nasal washing, using a mixture of ketamine (30 mg/kg) and xylazine (5 mg/kg), administered intramuscularly. All procedures were performed in accordance with the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee guidelines (protocol #IACUC‐2019‐0019) and were additionally approved by the US Army Medical Research and Development Command (USAMRDC) Animal Care and Use Review Office (ACURO) (protocol #DARPA‐6336). During guinea pig transmission experiments, strict measures were followed to prevent any cross‐contamination between animals, including handling recipient animals before donors and changing gloves between guinea pigs.2.3Nasal Prep , a commercially available 5% (w/w) PVP‐I solution, was used in these studies. The vehicle comparator  was produced by 3M specifically for these experiments, and the phosphate‐buffered saline (PBS) comparator was purchased (Gibco). All three treatments were applied to guinea pig nares in the same way: While guinea pigs were awake and in an upright position, we used a positive‐displacement pipette to deliver 50 μl to each naris . Awake administration of treatments was used instead of anesthetized administration after a comprehensive literature review2.44 plaque‐forming units (pfu) in 150 μl was instilled intranasally by applying 75 μl to each nostril. The inoculum dose was chosen to maximize the transmission rate of Pan/99 in our model. In our hands, peak nasal wash titers in donor guinea pigs reliably occur by Day 3 post inoculation at this dose (unpublished observations), and the time to peak nasal wash titer in donor guinea pigs has been shown by others to correlate inversely with transmission rate to recipients.On Day 0, Pan/99 stock virus was diluted in PBS supplemented with antibiotics  (PBS + P/S). An inoculum of 102.5BayesFactor (v.0.9.12–4.2)less likely than the null hypothesis.These exploratory experiments were performed in the absence of existing data to inform an estimate for effect size; thus, power calculations were not carried out a priori. Transmission experiments were performed in groups of four or eight transmission pairs (eight or 16 guinea pigs) in one or two transmission chambers at a time. Experiments were designed sequentially, with prior results informing the next experimental scheme to be tested, and the results are presented in the order in which experiments were conducted. Given the exploratory nature of these experiments, not every possible combination of intranasal treatment , treated animal , or transmission model (contact or airborne) was tested. Where possible, we minimized animal use by omitting groups that were unlikely to be substantively additionally informative, given prior results; the rationales for these omissions are noted in the text describing each experiment. Because power calculations were not performed and group sizes not established a priori, Bayesian methods3To assess the effect of topical Nasal Prep applied prophylactically in the nares of guinea pigs intranasally inoculated with influenza virus, we compared Nasal Prep to two controls, the Nasal Prep “vehicle”  or PBS. Four guinea pigs were treated intranasally with Nasal Prep, four with the vehicle, and two with PBS. Twenty‐four hours after treatment, all 10 guinea pigs were inoculated intranasally with influenza A/Panama/2007/1999 (H3N2) (“Pan/99”). All guinea pigs underwent nasal washes for virus titration every 2 days, starting on Day 1 post inoculation. The treatments  were also reapplied every 24 h; on nasal wash days, the treatment reapplication was done after nasal washing. As shown in Figure 4 pfu, the inoculum dose in the prior experiment. We1 pfu; thus, we proceeded to explore whether Nasal Prep could reduce influenza virus transmission in the guinea pig model.Even though the use of Nasal Prep did not result in consistent reduction of viral titers in the nasal washes of directly inoculated guinea pigs, the infectious dose required to infect a virus‐naïve, susceptible guinea pig (the “virus recipient”) via animal‐to‐animal transmission from an inoculated guinea pig (the “virus donor”) is likely to be orders of magnitude lower than 10In the guinea pig influenza virus transmission model, guinea pigs can transmit virus between one another while occupying the same or separate cages. In the contact transmission model Figure , both viWe first tested the efficacy of Nasal Prep in preventing airborne transmission of influenza virus in the guinea pig model. Twenty‐four hours before infecting the donor guinea pigs, we applied Nasal Prep or PBS to all animals' nares, both donors and recipients. The next day, donor guinea pigs were inoculated with Pan/99, and then, 3 h later, the inoculated virus‐donor animals and susceptible virus‐recipient animals were retreated with Nasal Prep or PBS and then paired in cages that only permit virus transmission via the airborne route , or PBS, starting 24 h prior to virus inoculation of untreated donor guinea pigs. This experiment was performed using the airborne transmission model in the same way as the prior one Figure . As expeBecause daily intranasal Nasal Prep efficiently prevented influenza virus transmission to treated recipient guinea pigs in the airborne model, we next evaluated its efficacy in the contact model , with treatment application starting 1 day after the recipient guinea pigs had already been cocaged with untreated, infected donor guinea pigs. In this experiment, donor guinea pigs were intranasally inoculated with influenza virus under anesthesia. Three hours later, after the donor animals had fully recovered from anesthesia, each donor was paired with an untreated recipient guinea pig in the same cage  is insufficient to establish that Nasal Prep can be used on a daily basis for longer periods of time, such as during a respiratory virus epidemic or pandemic. Systemic absorption of elemental iodine from mucous membranes is approximately 5–10 times greater than from the intact skin.Dr. Bouvier reports that she is an employee of the Icahn School of Medicine at Mount Sinai, which received funding from 3M Company during the conduct of the study. Dr. Landgrebe, Dr. Parthasarathy, and Dr. Wlashin report that they are employees of 3M, which received funding from DARPA during the conduct of the study. 3M employees conceived of the study and contributed to experimental design but had no direct participation in the conduct of the experiments or the collection or analysis of the data. Dr. Landgrebe, Dr. Parthasarathy, and Dr. Wlashin also report three patents pending that are relevant to this work, entitled “Treatment and Prophylaxis of Viral Infections,” “Transmission Prevention of Viruses with Application of Antiseptic Composition,” and “Virus Transmission with Application of a Composition.” Dr. Gaaloul and Mr. Komen report having nothing to disclose.Nassima Gaaloul ben Hnia: Investigation; methodology. Mathew Kipkemboi Komen: Investigation; methodology. Katie F. Wlaschin: Conceptualization; funding acquisition; resources. Ranjani V. Parthasarathy: Conceptualization; funding acquisition; resources. Kevin D. Landgrebe: Conceptualization; funding acquisition; resources.https://publons.com/publon/10.1111/irv.13035.The peer review history for this article is available at Figure S1. Comparison of nasal wash titers from Nasal Prep‐treated donor guinea pigs presented in Figure 7 to those presented in Figure 1 and Figure 3. The geometric mean (brown closed symbols and solid lines) and standard deviation (brown dashed lines) of the nasal wash virus titers from the 12 Nasal Prep‐treated donor guinea pigs shown in Figures 1 and 3A are presented with the individual nasal wash titers of the 4 Nasal Prep‐treated guinea pigs shown in Figure 7A (red open symbols and dotted lines). The horizontal black dotted line represents the limit of detection (LOD) of the plaque assay; points plotted below the LOD are graphed at y = 1 to allow presentation on a log‐scale axis.Text S1. R Script and Console Output for Bayes Factor and Posterior Probability CalculationsClick here for additional data file.


We describe the successful detection of human, porcine and canine picornaviruses (CanPV) in sewage sludge (at each stage of treatment) from Louisville, Kentucky, USA, using Pan-enterovirus amplicon-based long-read Illumina sequencing. Based on publicly available sequence data in GenBank, this is the first detection of CanPV in the USA and the first detection globally using wastewater-based epidemiology. Our findings also suggest there might be clusters of endemic porcine enterovirus (which have been shown capable of causing systemic infection in porcine) circulation in the USA that have not been sampled for around two decades. Our findings highlight the value of WBE coupled with amplicon based long-read Illumina sequencing for virus surveillance and demonstrates this approach can provide an avenue that supports a “One Health” model to virus surveillance. Finally, we describe a new CanPV assay targeting the capsid protein gene region that can be used globally, especially in resource limited settings for its detection and molecular epidemiology. To the Editor: In situations where most pathogenic, human-infecting virus infections do not result in clinical manifestations, such as with Enteroviruses (EVs)  were collected (three per week), over three weeks a from thAssay 1 amplicons from these five confirmed EV positive samples were subjected to assay 3 and Long-read Illumina sequencing (LRIS). Seventy-three long-read contigs were recovered from the five EV positive samples (Table S2). Though more variants were recovered using LRIS, both SS and LRIS were congruent with respect to the EV types detected in four  of the five samples (Tables S1 and S2). SS showed multiple peaks in the fifth sample  while LRIS delineated the different virus types   does noter year] ). A simiPhylogenetic analysis of the two CanPV contigs  showed that they belong to a group of unclassified canine picornaviruses that (based on publicly available sequence data in GenBank) have not been previously described in the USA. They have however been described in dogs in the United Arab Emirates (UAE), China and Hong Kong for over a decade (2008 to 2019)  and moreOne-Health framework [Our findings show that sludge from different stages  of conventional wastewater treatment can be used for virus surveillance. We recovered porcine (EV-G), canine (CanPV) and human (EV-A and EV-C) picornaviruses demonstrating this approach provides an avenue that facilitates surveillance of both human viruses and animal viruses and a ramework . In addiClick here for additional data file.


Virus mutation is one of the most important research issues which plays a critical role in disease progression and has prompted substantial scientific publications. Mutation extraction from published literature has become an increasingly important task, benefiting many downstream applications such as vaccine design and drug usage. However, most existing approaches have low performances in extracting virus mutation due to both lack of precise virus mutation information and their development based on human gene mutations.We developed ViMRT, a text-mining tool and search engine for automated virus mutation recognition using natural language processing. ViMRT mainly developed 8 optimized rules and 12 regular expressions based on a development dataset comprising 830 papers of 5 human severe disease-related viruses. It achieved higher performance than other tools in a test dataset  and has been applied well to two other viruses, influenza virus and severe acute respiratory syndrome coronavirus-2 . These results indicate that ViMRT is a high-performance method for the extraction of virus mutation from the biomedical literature. Besides, we present a search engine for researchers to quickly find and accurately search virus mutation-related information including virus genes and related diseases.http://bmtongji.cn:1225/mutation/index.ViMRT software is freely available at  Viruses cause many human diseases including cancers by persistent chronic infection and induce public health insecurity . For exaHowever, those unstructured texts are limited by the insufficient feasibility of traditional manual curation in utilization, as it cannot be carried out on all of the available literature. Furthermore, due to the inconsistency of virus mutations and the lack of standard mutation nomenclature, the conventional ‘Keyword Search’ may be difficult to quickly and non-redundantly get desired mutation information from literature in PubMed. As such, extracting, mining and integrating virus mutation-related information from those extremely large amounts of literature has become an increasingly important task in many downstream applications, such as virus mutation database development , semi-standard mutation (SSM: written form of mutation containing punctuation) and natural language mutation  and then constructed corresponding regular expression patterns . FinallyTo evaluate the performance of ViMRT, we compared ViMRT with three other classic tools . Moreover, we used the additional dataset including the abstracts of influenza virus (IV) and SARS-CoV-2 to further evaluate the recognition effect in other viruses. We calculated three different parameters: Precision, Recall and F1-score. The three parameters were defined as follows:where TP, FP and FN represent the numbers of true positive , false positive  and false negative , respectively.https://www.ncbi.nlm.nih.gov/gene/) due to the particularity of different viral genes and then developed Python scripts to identify the virus genes from the sentences including virus mutation.We first built the virus gene corpus from PubMed and the NCBI gene database (https://github.com/stanfordnlp/stanza) for many human languages to identify the diseases from the sentences including virus mutation and further developed Python scripts to extract related-disease information based on disease corpus collected from CTD (http://ctdbase.org).We used the Stanza natural language processing (NLP) library  from the ViMIC database. The development dataset and test dataset were allocated in a ratio of 1:2. In total, SM accounted for 62.60% of total annotations. SSM was 36.45% and the fraction of NLM was only 0.95% . The fraTo evaluate the performance of ViMRT, we compared recognition results with three other conventional tools, tmVar, nala and MutationFinder in the test dataset. Notably, We further compared the recognition results of four methods in three mutation signatures . For SM,We developed the ViMRT web search engine by identifying mutations in 9629 abstracts and 3160 full texts and covered the annotations of viral genes and related diseases. It can be accessed through an easy-to-use graphical web interface, as shown in First, for each result, it returns the searched entity with a red background as well as other entities with different colors including diseases, genes and other mutations , which chttp://bmtongji.cn:1225/mutation/Docs.The usage of ViMRT can realize three recognitions: virus mutation recognition, virus gene recognition and disease recognition. Firstly, virus mutation recognition includes downloading literature, optimizing the recognition results of tmVar by rule patterns and recognizing virus mutation by regular expression patterns. Secondly, the virus gene is identified by virus gene vocabulary and a gene match program. Thirdly, disease recognition is based on Stanza 1.4.0 and a normalizing script written by Python (v3.8.5). Full usage of ViMRT is minutely displayed on the ‘Docs’ page: This study is the first to gather abundant virus mutation information and develop ViMRT to identify virus mutation by precise rules and regular expressions. It has achieved a higher performance for virus mutation recognition than other conventional tools. At the same time, we are the first to develop a search engine web about a variety of viruses for researchers to quickly search mutation-specific information that includes virus gene and related diseases as reported in the biomedical literatures.ViMRT displays some notable advantages in identifying virus mutation recognition. Firstly, ViMRT curated and created virus mutation information from five viruses closely related to human disease from the literature to comprehensively cover the majority of written forms. Next, based on that information, we designed rule patterns to optimize recognition results of tmVar, and further summarized three mutation signatures termed SM, SSM, NLM which characterized the written form preference of virus mutation in different literatures. Then we developed regular expressions to recognize mutations for each mutation signatures . As we hIn addition, we observed that ViMRT showed a slightly decreased but still good performance  to recognize the mutation of SARS-CoV-2 and IV compared to five viruses , while tSeveral future directions can extend this work. Firstly, future study is warranted to expand the rules and regular expressions for more viruses with the emergence of special written forms. With the rapid growth of literature data, we will continue to expand our corpus of viruses, add more virus types and regularly update the literature of our search engine. Secondly, due to the particularity of writing styles of virus mutations, the current version of ViMRT mainly used the rule-based NLP method. In a preliminary study, we have explored whether it is possible to perform a binary classification task using machine learning models for discriminating sentences containing the mutation information to reduce the false positive. Both F1-scores of machine learning methods based on TF-IDF word embedding and TextCNN model could reach 0.8. In the future, with continuous updates and expansion of the virus data set constructed in this study, the state-of-the-art pre-trained language models, such as BERT (This work was supported by the National Natural Science Foundation of China [81972914 to X.Y.Z. and 81573023 to X.Y.Z.]; the Fundamental Research Funds for the Central Universities [22120200014 to X.Y.Z.]; and Shanghai ‘Rising Stars of Medical Talent’ Youth Development Program [2019-72 to Y.W.].Conflict of Interest: none declared.


Cefotaxime by novel nanocomposites beads composed of iota carrageenan (IC), sulfonated poly vinyl alcohol (SPVA) and nano sulfated zirconia (SZrO2) was evaluated in this study. SZrO2 was synthesized from solvent-free and easy calcination technique then embedded with 1–2.5 wt.% into the polymeric matrix. A batch adsorption experiment was carried out to investigate the effects of dosage, pH, beginning concentration, and time on Cefotaxime antibiotic adsorption. The ideal conditions to achieve complete removal are 88.97 mg L−1 initial cefotaxime concentration at time 3.58 h with 11.68 mg of beads composite with 2.5 wt.% of SZrO2. The pseudo second order kinetics model better illustrated the adsorption of cefotaxime on nanocomposite beads, and the maximum adsorption capacity are 659 mg g−1 for the composite with 2.5 wt.% of SZrO2. The mechanism of adsorption process depend mainly on the interactions between the different functional groups of SPVA, IC and SZrO2. The nanocomposites beads also exhibit excellent reproducibility after ten adsorption cycles. This type of nanocomposites beads can be easily separated from water without leaving any residue, verifying this novel nanocomposite beads has strong potential in water treatment for the antibiotic contaminant removal.Adsorption efficiency of  These water-related issues are better understood and handled as a result of enhanced detection and awareness of the toxicological, environmental, and biological effects of an ever-growing list of substances dubbed Emerging Pollutants (EPs). EPs are a group of man-made chemicals, such as pharmaceuticals, pesticides, flame retardants, detergents, and cosmetics, that are essential to contemporary living and whose manufacture and use have risen dramatically in the previous decades3. The uncontrolled and continuous release of these new compounds into the environment may have an impact on aquatic biota, human health5 and also the performance and costs of drinking water treatment technologies7.Water treatment processes confront significant challenges in terms of optimizing technology to avoid human health concerns and to ensure environmental sustainability, as population grows, water sources become scarcer and water quality deteriorates due to land use and climate changeCefotaxime is important type of cephalosporin antibiotics for the treatment of gram negative and positive bacterial infections with a lengthy half-life owing to its aromatic-ring structure. It is usually detected in wastewater and drinking water and is very difficult to totally remove8. Nowadays, there are different techniques have been applied to eliminate antibiotics from aquatic system, such as oxidation9, adsorption and degradation11. Between these techniques, adsorption is cheap, simple, promising method for antibiotics, organic and inorganic contaminants removal from water15. The polymeric beads as adsorbent have been more favorable than the nanoparticles, film or fiber forms because of its easy fabrication, excellent dispersion, controllable size dimension and quick recovery process.Antibiotics are classified as a type of persistent pollutant that lead to fast spread of different types of resistant bacteria. 16. Iota carrageenan is the most often utilized, which comprise two sulfated groups per disaccharide unit. Because of its chemical functionality and natural abundance, it is widely used for water treatment, namely in the removal of pharmaceutical contaminants and pesticides17.Carrageenan is a sulfated polysaccharide made up of anhydrogalactose and galactose units found in red seaweed extracts18.Poly vinyl alcohol (PVA) is an eco-friendly, biodegradable and cost-effective polymer with outstanding chemical stability and hydrophilicity properties; therefore, it is widely used to eliminate various pollutant from water. This type of polymer contains abundant hydroxyl groups and can be easily coagulated and cross-linked, however, by mixing PVA with IC lead to create porous network rich with hydroxyl and sulfate groups, which can be effective for the adsorption capacity enhancement23. Among metal oxide nanoparticles, ZrO2 has received a lot of interest in recent years due to its unique properties such as good chemical and mechanical stability with high hydrophilic properties, in addition to their availability, low cost and simple synthesis method21. However, sulfated zirconia as the adsorbent consider as a cation exchanger that promotes more elimination of the cationic counterpart such as wide sector of antibiotics which contain amine groups.The presence of metal oxide nanostructures in the surface and structure of polymeric matrix leads to the production of nanocomposite polymeric-based matrix, which has been proposed as one of the most potent tools for preventing membrane fouling in various water treatment processesCefotaxime antibiotic contaminant from water. PVA was chosen as the key polymer in the beads due to its good interaction with IC polymer to form compatible composite rich with functional groups, in addition to supporting SZrO2 nanoparticles. To our knowledge, this is the first time to SZrO2 nanoparticles have been incorporated into PVA/IC blending polymers matrix in different concentrations to create nanocomposite beads named as SPVA/IC/SZrO2 and use it as adsorbent for antibiotics in water treatment.This work aimed to synthesis eco-friendly and effective green adsorbent nanocomposite beads prepared by using water as a solvent to process biodegradable and low-cost polymers to remove Cefotaxime sodium salt and 4-sulphophthalic acid (SPA) (99.9 wt.% in H2O) were purchased from  and Glutaraldehyde (GA) (50 wt.% in H2O) was purchased from Alfa Aesar.Iota carrageenan (type V), PVA (99% hydrolysis and medium MW), 2 was prepared using a simple calcination method in the absence of any solvent between zirconium oxychloride octahydrate ZrOCl2.8H2O and ammonium sulfate (NH4)2SO4 with 1:5 molar ratio were mixed and ground in a mortar then placement for 12 h at room temperature, after that calcined for 5 h at 600 °C. Finally, the powder was ground in ball mill at 1500 rpm for 30 min to obtain nano size.Nano SZrO2O at 90 °C for 2 h then blending with percentage PVA: IC (95:5) wt.%. After that, crosslinking the polymers blend by certain amount of GA as covalent crosslinker and SPA as ionic crosslinker and sulfonating agent for PVA, to convert to sulfonated polyvinyl alcohol (SPVA)22. Then the prepared nanocomposite was incorporated with different concentrations of SZrO2 (1wt.% and 2.5wt.%) in polymeric blend. The two solution samples were named SPVA/IC/SZrO2-1, SPVA/IC/SZrO2-2.5. The composite solution was put in syringe, dropped in saturated solution of boric acid to form beads, and left for overnight then washing.First, PVA and IC were dissolved separately in 100 mL deionized H2 beads, esterification reactions between carboxylic groups of SPA and hydroxyl groups of two polymers were used to ionically crosslink IC and PVA. Furthermore, acetal interactions between GA's aldehyde groups and the hydroxyl groups of the two polymers covalently crosslinked the two polymers. While SZrO2 was bonded to polymeric matrix by hydrogen bond interactions between its oxygenated groups and the -OH groups of two polymers.As illustrated in Fig. 2 powder and the nanocomposite beads were monitored by Fourier transform infrared spectrophotometer (Schimadzu FTIR-8400 S-Japan), while the structures were evaluated by X-ray diffractometer (Schimadzu7000-Japan). Thermal changes of SPVA/IC/SZrO2 beads were traced by using thermo-gravimetric analyzer  the range of the temperature was 25–800 °C, under nitrogen atmosphere and the heating rate at 10 °C min−1. Morphological structure of the SPVA/IC/SZrO2-2.5 beads was shown by scanning electron microscope (SEM) combined with energy-dispersive analysis X-ray (EDX) (Joel Jsm 6360LA-Japan). Visualization of the nano SZrO2 was done by using transmission electron microscopy .The characteristic groups of SZrOWw and Wd were the masses of the swollen and dry PVA/IC/SZrO2 nanocomposite bead, respectively.To investigate the beads stability, pieces with diameter of 1 mm were immersed in 100 mL of deionized water at 35℃ for 24 h. The swelling coefficient S was calculated as follows:2 nanocomposite bead, 5 mg of beads were added up to 10 mL of solutions containing the pH values ranging from 3 to 9. The suspensions were then shaken gently for 24 h, then final pH values of every solution were recorded and the pHPZC values was calculated.To determine the point of zero charge (pHpzc) of PVA/IC/SZrOCefotaxime antibiotic were diluted to different degrees by adding deionized water. However, 50 mL of Cefotaxime antibiotic solutions with different concentrations  at 25 ˚C were added with a specific amount of prepared nanocomposite beads, with the help of a thermostated shaker running at 150 rpm. After reaching equilibrium, the adsorbent beads were separated and the remaining antibiotic concentration in the aqueous phase was measured using a UV spectrophotometer at 235 nm.Adsorption tests were carried out by using a batch equilibration technique. One gram per liter stock solutions of −1) were the amounts of antibiotic adsorbed per unit weight of adsorbent at time t and equilibrium; C0, Ct and Ce mg L−1 were the antibiotic concentrations at initial time, time t and the equilibrium time, respectively; V (L) was the volume of antibiotic solution; and m (g) was the amount of adsorbent. The removal efficiency R was determined as follows:The amounts of adsorbed antibiotic were calculated according to the following formulas:Cefotaxime antibiotic removal conditions, we tried to establish a relationship between factors and responses, according to a response surface methodology (RSM) model. The selected matrix for the response surface methodology followed the Box-Behnken design24, with 17 trials. To evaluate the adsorption process performance, three factors were used: A ; B ; and C , at three levels illustrated in Table To optimize the Cefotaxime antibiotic varied from 25 to 150 mg L−1. The pH was fixed at 5 and dose of adsorbent was 2 mg mL−1, while the adsorption time was varied from 0 to 24 h. In order to elucidate the adsorption kinetics, the pseudo-first-order, pseudo second-order and intraparticle diffusion models were applied, as illustrated in Table 2 composite bead using Langmuir and Freundlich isotherms.The rate and mechanism of the adsorption process could be elucidated based on kinetic studies with different initial concentrations of 2 composite bead, 10 mg of beads was mixed with 50 mL of 20 mg L−1 antibiotic. The mixture was shaken for 15 min. after adsorption, the beads was separated. Then 0.01 M NaOH was added and the solution was shaken again for 15 min. Finally, the beads was repeatedly washed by 70% (v/v) ethanol aqueous solution, until antibiotic was no longer detected in the solution. The cyclic adsorption–desorption processes were conducted up to 10th cycle successfully to study the reusability of the nanocomposite.To investigate the reusability of PVA/IC/SZrO2 and PVA/IC/SZrO2 composite beads. SZrO2 spectrum shows a board peak at 3250 cm−1 and sharp peak at 1630 cm−1, that may be referred to the adsorbed moisture and hydrogen bonds formed, however, the peak around 500 cm−1 return to Zr–O bond, while, peaks at 1217, 1128 and 1016 cm−1 are characteristic for S–O. On the other hand, the FTIR spectra for PVA/IC/ SZrO2 nanocomposite beads, the bands around 3250 cm−1 are attributable to the O–H bonds from water molecules that become more adsorbed as the concentration of sulfated zirconia increases due to its hydrophilic properties, and the bands at 1600 cm−1 are indicative of –OH groups of PVA and IC. The sulphate groups of iota carrageenan have a distinctive peak at 830 cm−1. The C–H bonds in the polymers structure can be attributed to bands at 2840 and 2300 cm−125, while the weak bands at 1700 and 1750 cm−1 correspond to C=O bonds and C–H bending, respectively in the aromatic structure of sulfophithalic acid (SPA), which demonstrate that the crosslinking process has been completed. Sulfate groups of SZrO2 are responsible for the bands at 950 and 1100 cm−1.Figure 2 concentration, while the sulfated zirconia powder curve shows characteristic peaks intensity of SZrO2 at a 2θ angle 54, 28, 3827.In Fig. 4ZrO2 composite beads and porous future for inside surface Fig. 2, proving the synthesize of sulfated zirconia.Figure 2shows about 8% loss in weight at ~ 150 ºC and that returns to the moisture evaporation21. The second loss stage of composite beads occurred in the range of 150—270 ℃ and is related to the degradation of functional groups, while the third loss stage is characterized by a remarkable decomposition from 270 to 360 ℃ and is related to the decomposition of polymeric chains, which began at 230 ℃ for the undoped beads and at 270 ℃ with a lower weight percentage for the doped beads. This behaviour demonstrates that the addition of SZrO2 improves the composite's temperature stability by boosting covalent, ionic, and hydrogen bonding in the nanocomposite.As shown in Fig. 2-1 and SPVA/IC/SZrO2-2.5 in water with respect to time was studied at room temperature. After a set amount of time had passed, the degree of swelling was assessed, and the immersion experiment lasted 24 h. SPVA/IC beads showed maximum degree of among other beads with SZrO2 and that can be explained by adding SZrO2 to the polymeric mixture of SPVA and IC may result in an increase in the amount of hydrogen bonds formed between polymer chains and SZrO2, resulting in increased chemical stability in polymer chains.As shown in Fig. PZC) is used for that purpose in addition, it can be used to comprehending the electrostatic interactions of the beads with Cefotaxime molecules. The point of zero charge of SPVA/IC nanocomposite beads was found around pH 6.5 while for SPVA/IC/SZrO2-1 and SPVA/IC/SZrO2-2.5 nanocomposite beads was found around pHs 6.1 and 5.3 respectively as shown in Fig. 2 affects the PZC of SPVA/IC polymeric matrix and due to the greater percent of oxygenated groups content in SZrO2 lowers the value of PZC to 6.1 and 5.3 for SPVA/IC/SZrO2-1 and SPVA/IC/SZrO2-2.5 nanocomposite beads respectively. Two processes are involved in the absorption of Cefotaxime from aqueous medium by nanocomposite beads. The first is the diffusion of Cefotaxime into nanocomposite beads with water molecule penetration, and the second is associated with electrostatic attractive forces. The pH has a significant and observable impact on the absorption process. The influence of the pH of the solution on Cefotaxime adsorption process was studied in the range of 3 to 9 to find the optimal pH value, as shown in Fig. Cefotaxime followed by hydrolysis of the β-lactam ring and the acetoxy ester. In addition, increasing pH elevate the deprotonation of Cefotaxime, accordingly that will increase the repulsion between Cefotaxime molecules and negatively charged beads above its PZC values. On the other hand, at very low pH values less than 4, the protonation rate of Cefotaxime amine groups will increased, which increases its solubility and decreases its adsorption rate, besides the surfaces of SPVA/IC/SZrO2 would be surrounded by many hydronium ions that compete with Cefotaxime for active sites. While, the high adsorption capacity at solution pH ranged between 4 and 6 can be referred to maximum stability of Cefotaxime at pH ranged between 4 and 6 (pKa for Cefotaxime is 3.1)28 and that increase the possibility to form hydrogen bond and electrostatic attraction between nanocomposite beads and Cefotaxime molecules as shown in Fig. Cefotaxime.It is crucial to investigate the beads' main surface charge at a certain pH. The calculation of the point of zero charge  beads dosage in mg on dependent response, which is removal efficiency (%). To explain Cefotaxime elimination using SPVA/IC/SZrO2-2.5 beads, the following regression equation for the link between response and variables  was utilized:The RSM is a model based on statistics and it is often used to clarify the interaction between reaction parameters for optimization since it is faster, practical and all of the components' effects and potential interactions may be evaluated. The adsorption of Cefotaxime removal efficiency with SPVA/IC/SZrO2-2.5 nanocomposite beads as shown in Fig. Cefotaxime concentrations increases the removal efficiency decreases, while factors time and beads dose have significant effect on removal efficiency whereas they increase, the removal efficiency increased and then decreased.Moreover, according to the perturbation plot for 2 (0.9566). The p value of the quadratic model was less than 0.05, therefore, this quadratic model was found to be significant in addition this model is confirmed due to the F-value is found to be 17.16.ANOVA analysis of variance is well-known for determining the statistical significance of the quadratic response surface model. As shown in Table Cefotaxime concentration is 88.97 mg L−1 at time: 3.58 h and adsorbent dosage: 11.68 mg to achieve full removal. The efficiency of Cefotaxime elimination was discovered to be impacted by all variable parameters.According to RSM analysis results as shown in Fig. Cefotaxime onto SPVA/IC/SZrO2-2.5 nanocomposite beads: pseudo-first-order, pseudo-second-order, and intraparticle diffusion models. For the three kinetic models at varying concentrations, the calculated sorption capacities, reaction rate constants, and R2 values of the linear correlation coefficients were shown in Table 2 values are generally greater than those obtained from pseudo-first-order kinetics, implying that the sorption process followed pseudo-second-order kinetics and that chemisorption was the rate-controlling phase. This appears to include valence forces as a result of chemical reactions or electron exchange between oxygenated groups on Cefotaxime molecules and hydroxyl groups on nanocomposite beads and that were responsible for the hydrogen bonds. Despite the fact that the pseudo-second-order-kinetics model had the best fit order, the findings produced from this model were insufficient to evaluate Cefotaxime molecules' diffusion mechanism onto SPVA/IC/SZrO2-2.5 nanocomposite beads. As shown in Fig. Cefotaxime molecules transport through the solution to the bead surface and form film diffusion at the boundary layer, or charged molecules diffuse from a bulk solution to the beads' exterior surface after that the Cefotaxime charged molecules transferred into the pores and intraparticular active sites of beads. Finally, the Cefotaxime molecules were chemically bound via small pores in the beads, followed by the final equilibrium adsorption.Three kinetic models were used to study the adsorption of Cefotaxime molecules and the bending sites of the beads, and the R2 values of the Langmuir model were extremely near to one. Furthermore, at low temperatures, RL values were higher than at high temperatures, and its values were found to be between zero and one, indicating that the adsorption process is chemical rather than physical22. However, the results showed that the adsorption process onto beads was best fitted by Freundlich isotherm model, with values of "n" ranging from 2 to 10, which indicate good adsorption and implying higher efficiency for Cefotaxime adsorption by nanocomposite beads as the values get increase.Langmuir and Freundlich models as shown in Fig. 2-2.5 nanocomposite beads had potential in wastewater application.After 10 cycles, the adsorbent's ability to regenerate was tested, and it was observed that the removal efficiency was still high, and that the fluctuations in the removal % after each desorption cycle were quite minor, as shown in Fig. 2-2.5 nanocompoaite beads for drug removal was compared with other adsorbent materials as illustrated in Table The efficiency of prepared SPVA/IC/SZrO4ZrO2 as doping agent into the polymeric PVA/IC blend improves the nanocomposite beads adsorption capacity towards Cefotaxime antibiotic. It was discovered that when the ultimate concentration of 100 mg L−1Cefotaxime solution was compared at varied contact times and pH 6 conditions, the outcomes were better in the case of SPVA/IC/SZrO2-2.5 nanocomposite beads. The increasing of SO4ZrO2 content in the PVA/IC matrix increases porosity and active functional groups such as oxygen containing groups, so that the adsorption efficiency improves. At various experimental conditions, the time-dependent adsorption capacity was assessed. According to the experimental optimization, it was observed that as Cefotaxime concentrations increases the removal efficiency decreases, while factors time and beads dose have significant effect on removal efficiency whereas they increase, the removal efficiency increased and then decreased. The adsorption kinetic of Cefotaxime was better fitted to pseudo-second-order model, and the adsorption isotherm was better fitted to the Langmuir and Freundlich models. The ability of SPVA/IC/SZrO2-2.5 nanocomposite beads to regenerate ten times further demonstrated its suitability for long-term use. The current study reveals that SPVA/IC/SZrO2-2.5 nanocomposite beads have a lot of potential in water treatment for removing antibiotic pollutants because of their fast separation, ease of operation, high capture capacity, and good cycle performance.Novel and low expensive nanocomposite beads was produced using a simple blending, eco-environmentally approach and available polymers as PVA and IC. It was appeared that the incorporation of different weight ratio of SOSupplementary Information.


Serine Incorporator (SERINC) family members are multi-pass transmembrane proteins, whose putative role is the incorporation of serine into membranes during phosphatidylserine and sphingolipid biosynthesis [Saccharomyces cerevisiae [ynthesis . SERINC revisiae . Mammalirevisiae . Of the SERINC5 SNPs with borderline personality disorder [SERINC5 encodes multiple splice isoforms, only the longest SERINC5 transcript variant encodes a stable protein comprised of 10 transmembrane domains (TMs) [SERINC5 was initially identified in cultured rat oligodendrocytes  and mousdisorder . While hns (TMs) . Recent ns (TMs) . AlthougDrosophila melanogaster SERINC, which shares only 36% amino acid sequence identity with human SERINC5, potently restricted HIV-1 infectivity [In 2015, 2 independent studies highlighted the antiviral function of SERINC proteins for the first time ,10. It wectivity . Similarectivity ,12. AddiSERINC5 expression levels are not induced by HIV-1, MLV, or SARS-CoV-2 infection [We recently found that SERINC5 restricts Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infectivity in the absence of ORF7a, a SARS-CoV-2 accessory protein . Recent nfection ,11,13.SERINC5 localizes to the plasma membrane lipid rafts (assembly sites for HIV-1 and IAV)  and the Recent reports have shown that SERINC5 can also modulate the type I IFN response . Differe160xxxLL165) mediates binding with AP2 [36XXL39) and 2 additional residues P31 and R63 of glycoGag are critical for glycoGag-mediated down-regulation of SERINC5 [Viruses have evolved mechanisms to counteract the inhibitory effect of SERINC5. To date, HIV-1/SIV Nef, MLV glycoGag, EIAV S2, and SARS-CoV-2 ORF7a proteins have been shown to antagonize the antiviral effect SERINC5 ,10,13,27with AP2 ,28. Furtwith AP2 . However SERINC5 . In the Viral envelope (Env) glycoproteins impart differential sensitivity to SERINC5 restriction. HIV-1/MLV pseudoviruses containing envelope glycoproteins from other viruses such as vesicular stomatitis virus (VSV), Ebola, avian leukosis virus (ALV), ecotropic MLV, human T-cell lymphoma virus-1 (HTLV-1), or parainfluenza virus 5 (PIV5) are resistant to SERINC5 restriction –11,18,30In summary, SERINC5, while initially identified as an antiretroviral restriction factor, growing evidence suggests that it has a broad antiviral activity targeting viruses from a wide number of virus families. Interestingly, it is not under positive selection and is not inducible by IFN. Although multiple studies have been conducted investigating the antiviral effect of SERINC5, its cellular functions remain largely unknown. Recent findings showing that SERINC5 modulates viral infection as an innate immunity factor suggest that the role of SERINC5 is more complex than previously thought. Future studies integrating the different aspects of SERINC5 during virus infection are needed. Finally, a better understanding on the repertoire of viruses affected by SERINC5 and the factors viruses encode to counteract it will provide a more accurate understanding on the antiviral role of SERINC5.


As of April 2022, the Omicron BA.1 variant of concern of SARS-CoV-2 was spreading quickly around the world and outcompeting other circulating strains. We examined its stability on various surfaces and found that this Omicron variant is more stable than its ancestral strain on smooth and porous surfaces. The Omicron SARS-CoV-2 variant of concern (VOC) is highly transmissible in humans. As of April 2022, it has outcompeted other known variants and dominated in different regions (7 50% tissue culture infectious dose [TCID50]/mL) on different surfaces in triplicate. We incubated the treated surfaces at room temperature (21°C–22°C) for different time points as indicated and then immersed them in viral transport medium for 30 min to recover the residual infectious virus. We titrated the recovered virus by TCID50 assays using Vero E6 cells, as described  and Omicron VOC (PANGO lineage BA.1) in this study –expressing Vero E6 cells to titrate infectious virus particles recovered from treated stainless steel and printing paper  Table. Ohttps://www.biorxiv.org/content/10.1101/2022.01.18.476607v1). More evidence is needed to account for the increased transmissibility of Omicron variant. The virus’s stability on surfaces may be one factor and should be taken into consideration when recommending control measures against infection. A recent study revealed that an infectious dose as low as 10 TCID50 units could infect >50% of human study participants (We noted that the cell line used for virus titration can affect our findings. It has been reported that Omicron variant is less dependent upon TMPRSS2 for cell entry (One limitation of our study is that the experiments were conducted in a well-controlled laboratory environment. Variations in environmental conditions would affect the rate of viral inactivation. Therefore, the time required for virus inactivation that we demonstrated may not reflect all real-life scenarios. In addition, the components of the viral droplet medium applied in this study were different from those of the respiratory droplets, which could also affect the stability of the virus. Nonetheless, our findings demonstrate that the Omicron variant is more stable than the ancestral SARS-CoV-2 on different surfaces, a finding that may be relevant for determining recommendations for public health measures to limit virus transmission.Additional information about the difference in stability of Omicron variant and ancestral SARS-CoV-2.


The study of IAV infections in poultry has been mostly limited to strains from the North American and Eurasian lineages, whereas limited information exists on similar studies with strains from the South American lineage (SAm). To better evaluate the risk of introduction of a prototypical SAm IAV strain into poultry, chickens were infected with a wild-type SAm origin strain (WT557/H6N2). The resulting virus progeny was serially passaged in chickens 20 times, and the immunopathological effects of the last passage virus, 20Ch557/H6N2, in chickens were compared to those of the parental strain. A comparison of complete viral genome sequences indicated that the 20Ch557/H6N2 strain contained 13 amino acid differences compared to the wild-type strain. Five of these mutations are in functionally relevant regions of the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). However, despite higher and more prolonged virus shedding in chickens inoculated with the 20Ch557/H6N2 strain compared to those that received the WT557/H6N2 strain, transmission to naïve chickens was not observed for either group. Analyses by flow cytometry of mononuclear cells and lymphocyte subpopulations from the lamina propria and intraepithelial lymphocytic cells (IELs) from the ileum revealed a significant increase in the percentages of CD3 Anseriformes and Charadriiformes. The spread and diversity of IAVs across the globe depends on the migratory patterns of wild birds that include both regional and intercontinental movement  and 11 NA (N1-N11) IAV subtypes have been identified in nature in multiple combinations. Except for the H17N10 and H18N11 IAVs identified in fruit bat species of Guatemala and Peru, , 2013 removement . From thmovement . Based omovement . The polIn Argentina, active influenza virus surveillance in wild birds was initiated in 2006  as an inThe A/rosy-billed pochard/Argentina/CIP051-557/2007 (H6N2) virus (WT557/H6N2) was isolated from waterfowl as described previously . Stock oad libitum throughout the experimental period. Animal care and experimental procedures were performed under ABSL3+ conditions with investigators wearing appropriate protective equipment and in accordance with the approved protocols N° 55/2013 of the National Institute of Agricultural Technology Ethics Committee .Specific-pathogen-free (SPF) White Leghorn embryonated eggs  were purchased, incubated, and hatched in an automatic incubator  under appropriate conditions, and randomly separated into groups of 10 chickens. Groups were housed separately in sterilized isolators for chickens under negative pressure conditions (Allentown CH8ISOL) with food and water 6 EID50 in 1 ml or 1 ml PBS, respectively. At 1 dpi, four naive chickens were placed together with the infected birds to monitor transmission. Tracheal and cloacal swabs were collected at 1, 3, 5, and 7 dpi in 1 ml freezing medium (50% glycerol in PBS containing 1% antibiotics) and stored at −80°C until use. Virus titers in the swabs were determined by equivalent EID50/ml as previously described , and serum samples were collected at 21 dpi.3-weeks old SPF White Leghorn chickens were used throughout the studies. Groups of chickens were inoculated intraocularly, intranasally, intratracheally, and orally with WT557/H6N2 or 20Ch557/H6N2 virus containing 5 × 10escribed . The limTo investigate the replication of WT557/H6N2 and 20Ch557/H6N2 viruses in the tissues of chickens (lung and intestine), six birds per group were euthanized using manual cervical dislocation at 3 dpi. This was performed only after chickens were anesthetized with isoflurane. Body and bursa of Fabricius were weighed and recorded in all euthanized animals (six birds/group at 3 dpi).Single cell suspensions of 10 centimeters of ileum were prepared as described previously . Briefly6 cells per well were seeded on 96 well-plates (V-shape) and washed 2 times with the same buffer. Staining was performed by resuspending the cellular pellet of each well with 100 μl of staining buffer including different combinations of antibodies, or as single-color staining for compensation. Cells were incubated at 4°C for 20 min and washed 2 times with staining buffer by centrifugation at 1,200 rpm for 3 min.Cells were diluted in staining buffer  and 1 × 10Monoclonal antibodies (mAbs)  were purchased from Southern Biotech. , CD25-Alexa 647 was purchased from AbD Serotec  and CD56-Alexa 647 was purchased from LSBio . Lymphocytes from lamina propria and epithelial cell compartments from the ileum were stained with three mAb mix: CD3-SPRD, CD4-FITC, CD8α-PE, and CD25-Alexa 647; CD3-SPRD, CD8α-FITC, TCRγδ-PE, and CD25-Alexa 647; and CD3-SPRD, BU1-FITC, KUL01-PE, and CD56-Alexa 647. All antibodies were titrated to determine the optimal staining concentration of each one.Positive cells were analyzed with a FACS Calibur flow cytometer  and CellQuest software. Analyses were done on 20,000 events and discrete viable lymphoid cell populations were gated according to the forward/side scatter characteristics. Percentages of different lymphoid cell subpopulations in the ileum were determined by multiparametric analysis.Sera were tested by competitive enzyme-linked immunosorbent assay (ELISA) for specific antibodies against Influenza A Virus according to the manufacturer’s recommendations  using 10-fold dilutions of each serum sample.Serum samples were collected to determine the antigenic relatedness between WT557/H6N2 and 20Ch557/H6N2 viruses at the end of the experiment. Sera were treated with receptor destroying enzyme  to remove sialic acid receptors. The anti-viral antibody titers developed were evaluated using the HI assay system outlined by the WHO Manual on Animal Influenza Diagnosis and Surveillance . HomologTotal RNA was extracted from the stock virus in an allantoic fluid using the QIAamp Viral RNA Mini Kit  according to the manufacturer’s instructions. Reverse transcription and PCR amplification were performed using the universal primers described by The consensus amino acid and nucleotide sequences for all eight gene segments of WT557/H6N2 and 20Ch557/H6N2 viruses were generated using BioEdit 7.CY067691 to CY067698 and ON306370 to ON306377.The genome sequences obtained in this study for WT557/H6N2 and 20Ch557/H6N2 stock viruses are available from GenBank under accession numbers p < 0.05 were considered statistically significant.Data were tested for normal distribution (Shapiro–Wilk test) and homogeneity of variance (Leven’s test) prior to analysis using Statistical Package for the Social Sciences for Windows . An ANOVA test was used, and a Tukey’s test was applied when differences were detected. Variables that did not fit the ANOVA’s assumptions were analyzed by Kruskall–Wallis and Dunn’s test. Chi-Square tests were used to compare the number of animals that were positive for IAV between different experimental groups.p < 0.05), confirming better fitness of this virus in comparison to parental WT557/H6N2. Virus titers in trachea from 20Ch557/H6N2 inoculated chickens were significantly higher than WT557/H6N2 inoculated chickens at 1 and 3 dpi . Also, virus titers in cloaca from 20Ch557/H6N2 inoculated chickens were significantly higher than WT557/H6N2 inoculated chickens at 1 dpi (p = 0.0372). Although overt signs of disease were detected in neither group of the inoculated chickens, the results indicate that the chicken-adapted virus replicates better than WT557/H6N2 in trachea and cloaca, increasing viral shedding and for a longer time post-infection; thus, our laboratory adaptation strategy has increased viral fitness of the duck origin SAm H6N2 virus in chickens.We followed a strategy that was previously described for adaptation of a domestic duck origin H9N2 virus to Japanese quail and chickens using lung homogenates from the previous passage . In thisn = 6/group) was sacrificed at 3 dpi and bursa of Fabricius, lung, and intestinal tissues were collected. Mean bursa-to-body weight ratios were reduced in both IAV-inoculated groups in comparison to the negative control group , consistent with IAV infection in chickens. However, no significant differences in bursa-to-body weight ratios were observed between challenge viruses. All lung samples (n = 6) from the 20Ch557/H6N2 inoculated group was RNA virus positive, in contrast to a single lung sample (n = 1 out 6) from WT557/H6N2 inoculated group (p < 0.05) (p < 0.05). No significant differences were observed in either the number of positive samples or in levels of virus titers in intestinal samples for either IAV inoculated group. This is also consistent with the PCR results, in which significantly higher replication was observed in tracheal swabs, and only a slightly higher replication was observed in cloacal swabs. These results are consistent with the strategy of virus adaptation that selects for strains with improved respiratory tropism. In this case, such improvement does not appear to alter the natural tropism for the intestinal tract typical of wild aquatic bird origin IAVs.To better determine the extent of virus replication of WT557/H6N2 and 20Ch557/H6N2 strains, a subset of chickens from each inoculated group ( < 0.05) . In addip < 0.05). Furthermore, an increase in the percentages of CD3+TCRγδ+ lymphocytes in the ileum of chickens inoculated with 20Ch557/H6N2 virus was observed (p < 0.05), being in part due to an increase in CD3+TCRγδ+ CD8a+ subpopulation. Besides, the macrophage population positive for staining of KUL01 marker showed a trend to increase in proportion in lamina propria from ileum from 20Ch557/H6N2 chickens  cell compartments from the ileum were analyzed. Remarkably, in both compartments, there was an increase in total mononuclear cells in response to the infection, being significantly higher only in the case of 20Ch557/H6N2 infected animals , we compared deduced amino acid sequences from all open reading frames of WT557/H6N2 and 20Ch557/H6N2 viruses.Amino acid mutations were observed in the internal components of the chicken-adapted virus, either in structural or in non-structural proteins . The onlWe also found amino acid mutations in the superficial genes from the 20Ch557/H6N2 virus . The HA To study direct contact transmission of WT557/H6N2 and 20Ch557/H6N2 viruses, four naïve chickens were placed together with infected birds at 1 dpi. Trachea and cloacal swabs were collected at different days post-contact (dpc) and sera collected at 20 dpc were tested by competitive enzyme-linked immunosorbent assay (ELISA) for specific antibodies against influenza A virus. Transmission studies indicated that the SAm wild type and chicken-adapted H6 viruses were not transmissible through direct contact. All swabs from direct contact animals were negative for the presence of viral RNA and blood samples did not show the presence of antibodies against the influenza virus (data not shown). When we analyzed serum samples from infected chickens, we observed 100% of infection with 20Ch557/H6N2 virus  and 75% of infection with WT557/H6N2 virus  . These rThe antigenic properties of the SAm H6 viruses were investigated by using chicken antisera generated from the replication studies mentioned above. In general, antisera against the WT557/H6N2 and 20Ch557/H6N2 viruses showed low HI titers against the homologous virus . It is nin vivo chicken studies with H6 IAV strain of South American lineage isolated from wild duck and its adapted version in chickens, thereby getting valuable and unknown knowledge on the pathogenesis and transmission of influenza A viruses isolated from wild birds in the region. Our results showed that, even though there was no evidence of clinical signs of disease compatible with IAV infection, both viruses infected chickens (observed mainly by seroconversion) but were not transmitted to other birds through direct contact. This, together with the significant bursa-to-body weight ratios reduction observed in both infected groups, demonstrated that WT557/H6N2 and 20Ch557/H6N2 viruses behave like LPAIV in chickens as observed with viruses from other lineages  (in vivo. The mutation observed in the NS1 (A86T) protein of the 20Ch557/H6N2 virus has been already described in other influenza viruses, however, no clear association to species-specificity has been attributed to this variation (Sequencing data from WT557/H6N2 and 20Ch557/H6N2 viruses was important to correlate the mutations observed (after 20 passages in chickens) to the higher viral replication observed in the respiratory tract of chickens infected with 20Ch557/H6N2 virus , which eiformes) . Howeverariation . Furtherariation  or NS1 pariation  were fouariation . For thi+TCRγδ+ lymphocytes observed in the epithelial cell compartment from our study could be related to the higher viral replication of 20Ch557/H6N2 virus and also to the acquisition of an additional glycosylation site in HA protein after chicken adaptation.With respect to the superficial genes, the HA protein of the 20Ch557/H6N2 virus differed from the WT557/H6N2 virus in the HA2 region at positions 127 (V127I), 153 (Y153H), 182 (K182N), and 346 (L346I). Interestingly, the three first amino acid substitutions in HA are part of the Receptor Binding Site (RBS). Functional studies are necessary to determine if these mutations increase the infective capacity of the adapted strain to chicken epithelial cells through better interaction with the sialic acid receptor. Considering that previous results showed that the interaction of recombinant HA proteins with sialic acid receptors may play a critical role in γδ T-cell activation , togetheGalliformes family, whereas S in position 400, as found in our chicken-adapted strain, is mainly found in viral variants identified in isolates from chickens . This mutation in NA protein is not associated with drug resistance . Intereschickens . This miNote worthily, we could not discard the possibility that certain parameters of H6 IAVs, such as infectivity, pathogenicity, and/or transmission in chickens, might be different if we had used another H6 virus isolated in Argentina for the adaptation strategy. However, the H6 viruses that we have been detecting in this country do not acquire significant amino acid mutations along their genome to date (>98% sequence identity in the H6 segment), which might be indicative that these viruses are well adapted to duck species found in Argentina. More important, none of the H6 isolates showed the mutations we found through our adaptation strategy in chickens. In addition, it is difficult to ensure if more passages or recombination between IAVs would enable virus transmission and/or increase pathogenicity. Secondary infections, immunosuppression, or adverse environmental conditions commonly found under field conditions might enable the acquisition of other mutations, or even fasten the occurrence of the mutations here reported. Therefore, biosecurity and management practices in commercial and backyard flocks should be considered in Argentina.in vivo studies with IAV of SAm lineage showed that WT557/H6N2 and 20Ch557/H6N2 viruses replicated in SPF chickens but do not transmit by direct contact. The genetic modifications acquired by the 20Ch557/H6N2 virus provided greater fitness in terms of infection, replication, and excretion in SPF chickens, triggering a greater immune response in the intestinal epithelium of this host at least at 3 dpi but showing lower immunogenicity. Taken together, these results provide valuable information regarding the immunopathogenesis and transmission of avian influenza viruses from SAm lineage, which may be useful for future prevention strategies to improve the health status of poultry in the region. Finally, considering that IAVs belonging to the H5, H7, and H9 subtypes have been found circulating in wild birds from South America and that HPAIV outbreaks in poultry were reported in the region  can be found in the article/The animal study was reviewed and approved by National Institute of Agricultural Technology Ethics Committee .AR, MR, and DP: study design. AR and VO: virus sequencing. AR, VO, and IS: animal studies with sample and data collection. AR, GP, and MR: interpretation of protein mutations. AR, IS, and MR: data analysis and interpretation of flow cytometry. AR: writing of the manuscript. MR and DP: revision of the manuscript. All authors approved the manuscript before its submission.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.


Heating-resistance test showed that FMDV infectivity was maintained when formulated with formulation. Additionally, the half-life of FMDV inactivation was prolonged remarkably. Sequencing analysis demonstrated that viral genome could not be altered in serial passages. Vaccine stability was monitored for up to 1 year at 4 °C, with a higher level of 146S content remained. This study suggested that the formulation could protect FMDV against massive structural breakdown and extend the shelf life of vaccine. Our findings could provide strategy to develop more solutions for the stabilization of viral vaccine.Foot-and-mouth disease (FMD) is a highly contagious viral disease that affects cloven-hoofed animals. Vaccination is the most effective measure to control FMD. However, FMDV particles are prone to dissociation, leading to insufficient potency of vaccine. Based on this characteristic, a combination of twenty percentage trehalose, 500 mM NaCl and 3 mM CuSOThe online version contains supplementary material available at 10.1186/s12985-022-01928-6. Foot-and-mouth disease (FMD) is a highly contagious disease that affects cloven-hoofed animals, including pigs, cattle, sheep and many other non-domesticated species . FollowiVaccination is a crucial strategy to prevent FMDV infection. Inactivated whole virus vaccine is superior to other vaccines due to the advantage of immunogenicity . HoweverThe development of thermally stable formulations could alleviate the bottlenecks of virus stabilization. For example, Polioviruses exhibited enhanced stability when mixed with 87% deuterium oxide . Cryo-pr4·5H2O, aiming to stabilize FMDV and prolong vaccine shelf life.Compared with a single stabilizer used alone, a mixture of stabilizers could significantly enhance stabilization of virus. The combinational use of lactalbumin hydrolysate and sucrose has better stabilized effect on attenuated peste des petits ruminants than trehalose alone . Similar2 at 37 °C. A PrimeScriptTM RT reagent kit containing gDNA Eraser and SYBR Premix Ex TaqTM II (Tli RNaseH Plus) was purchased from TaKaRa . MTS assay was available from Abcam . The 50% tissue culture infectious dose (TCID50) was measured with the Reed and Muench method. Trehalose, CuSO4·5H2O were purchased from sigma . All other chemicals were analytical grade reagents purchased from Beijing Chemical works  and all solutions were prepared using Milli-Q grade water .Baby Hamster Syrian Kidney (BHK-21cells) were cultured in Dulbeccoʼs modified Eagleʼs medium (DMEM) supplemented with 10% FBS. FMDV strains, O/MYA98/BY/2010 and Asia1/JSL/ZK/06 investigated extensively as vaccine strains, were provided by Zhongnongweite biotechnology Co., Ltd. Each strain was propagated in BHK-21 cells at a multiplicity of infection of 0.001 and incubated in 5% COConcentrations of Trehalose: 5%, 10%, 20% and 30%Concentrations of NaCl: 100 mM, 200 mM, 500 mM and 1 M4·5H2O: 0.5 mM, 1 mM, 1.5 mM and 3 mMConcentrations of CuSO4·5H2O on virus were evaluated by anti-aging test with high performance size exclusion chromatography (HPSEC) method [2SO4. The peak area at 259 nm was linearly proportional to 146S concentration with established standard curve. Therefore, 146S content could be calculated according to the peak area. In order to quickly assess the efficiency of stabilizers, formulated virus was incubated at 37 °C for 5 h to accelerate dissociation. Briefly, virus was supplemented with trehalose, NaCl and CuSO4·5H2O at various concentrations respectively, as detailed in Table 4·5H2O with optimal concentration screened.The thermal stabilization effect of trehalose, NaCl and CuSO) method . Briefly4 of BHK-21 cells in 100 μl of complete medium were seeded into each well of a 96-well plate. After incubated at 37 °C for 24 h, the cells were incubated with 100 μl of formulation at various concentrations  for another 72 h. The control cells were treated with DMEM. Then, the supernatants were discarded and 100 μl of DMEM along with 20 μl MTS solution were added to each well for additional 3 h. The cell viability was determined by the percentage of absorbance of the treated cells at 490 nm to that of the control cells.MTS assay was performed to measure the cytotoxicity of formulation. In brief, 5 × 105 cells (BHK-21) in 100 μl complete medium were seeded into each well of a 96-well plate. On the following day, the supernatants were discarded, and the cells were washed three times with MEM. Then, the cells were infected with treated virus at serially dilution (eight wells for each concentration) for 1 h. After removing inoculum and adding MEM, the plates were incubated for an additional 24–48 h at 37 °C. Until the cytopathic effect (CPE) was observed, TCID50 value was calculated with Reed and Muench method [Infectivity assay were carried out to further confirm the thermal stabilization effect of formulation on virus. After exposed to formulation for 16 h, virus were incubated at room temperature for 3, 5, 10 and 15 h respectively and then used for cells infection. The control virus were incubated for the same periods, but without exposure to the formulation. Briefly, 4 × 10h method . The sup50 value. According to the log of TCID50/ml value over time, the linear curve of inactivation on virus infectivity was established, and the time when the virus lost 50% infectivity was obtained and compared.The inactivation rate of virus both in formulated and non-formulated virus were determined after incubation at 25 °C, 37 °C for 36 h and 10 h respectively. Virus were sampled at indicated time points to measure virus titers, referred to TCIDTo validate if the formulated virus has stable antigen expression throughout multiple passage, the virus genomes of different passage were determined. The conserved VP1 gene was amplified with PCR. Briefly, the formulated virus was continuously passaged for 12 times, then the genomic RNA in every 3 generation were isolated for reverse transcription. The VP1 gene was amplified by PCR in a final volume of 50 μl, containing 1 μl of each primer, 5 μl of PCR buffer, 4 μl of dNTP, 0.25 μl of DNA polymerase, 1 μl of DNA, and 37.75 μl of RNase-free water. The thermocycler conditions were an initial denaturation at 95 °C for 3 min, denaturation with 35 cycles at 95 °C for 50 s.Having shown that the formulation could increase the thermostability of infectious FMDV, we speculated that the thermostability of inactivated vaccine would also be enhanced. To facilitate this, both non-formulated and formulated virus were inactivated and then emulsified with adjuvant ISA206 to produce vaccine. The 146S content in vaccine was monitored via HPSEC method during storage at 4 °C for up to 1 year. Samples were collected at indicated time points and then mixed with 1-pentanol in a 10 ml centrifuge tube at a ratio of 9:1. The mixture was shaken fully to break the emulsion. After being placed at 4 °C for 1 h, the upper oil phase was removed and the aqueous phase at the bottom was absorbed slowly for 146S determination.P < 0.05.The means and standard deviations (n = 3) of all values were analyzed with descriptive statistics. The statistical analysis was performed with a t-test, and differences between samples were considered statistically significant at 4·5H2O were selected as protectant agents and the optimized concentrations were determined using anti-aging test by detecting residual 146S concentration. The test was performed at 37 °C to quickly screen reasonable concentration. As indicated in Table 4·5H2O.This study was aimed to develop a formulation for FMD virus which has poor thermal stability. Trehalose, NaCl and CuSOCytotoxicity assay were carried out to determine whether the formulation had toxic effects on BHK-21 cells and then attenuated virus infection. As is shown in Fig. 50 of formulated virus stock at different temperatures. As shown in Fig. 50 values of both formulated and non-formulated virus stock declined in a time-dependent manner during storage at room temperature, but the log10TCID50/ml values of formulated virus stock  were higher than the non-formulated one  after 3, 5, 10 and 15 h storage. The stable effect of formulation was then verified using real-time PCR analysis. Consistent with the TCID50 value, the cell infected with formulated virus had higher relative mRNA level of 4.2%, 2.8%, 0.011% and 0.008%, compared to non-formulated virus . Microscopy showed that after treatment at 37 °C for 10 h, the cells inocubated with formulated virus had a higher proportion of infected cells compared to non-formulated virus. Encouragingly, the formulation also show preserving activity for FMDV Asia1/JSL/ZK/06 strain, with log10(TCID50 /ml) values of 6.6, 5.7, 3.7 and 2.6 (formulated virus) as opposed to 4.2, 3.6, 1.7 and 0 (non-formulated virus). Similarly, the relative mRNA level is higher in formulated virus  than that of non-formulated virus    formulation, with cell viability of 95.3%, 94.8%, 97.2% and 94.2%. In addition, TCIDAlthough the formulation was identified to protect virus against dissociation, it was uncertain about the situation on vaccine. In this study, we monitored the changes of 146S content in vaccine via real-time analyses and the result showed that half of 146S was remained after stored at 4 °C for up to 1 year. Overall, in the present study, we validated that the adoption of formulation for FMDV preservation is valuable for enhancing virus stabilization and meanwhile prolonging vaccine shelf life.Purification is a crucial production process determining vaccine quality. Owing to the advantage of scalability and high selectivity, ion exchange chromatography is an alternative strategy for FMDV antigen purification. However, Shanqing Liang et al. reported that the interaction between inactivated FMDV and ion media could cause capsid denaturation and then induced dissociation, hardly obtaining high recovery and stable native structural antigen. In thisIn summary, a formulation for FMDVstability was screened and determined with HPSEC method. The formulation could maintain viral integrity effectively even with elevated temperature and has no influence on genetic stability during viral passages. Monitoring of vaccine stability on long-term storage was further confirmed its stable effect on FMD vaccine. This research is a step forward in vaccine preservation protocols and could be a potential application in vaccine production regimens for FMD prevention.Additional file 1. Genetic stability of formulated FMDV. (a) Amplification of VP1 genes from different viral passages. Lane M: 2000bpDNA Maker; Lane1: F3; Lane2: F6; Lane3: F9; Lane4: F12. (b) Comparison of virus titer between non-formulated and formulated viruses during 12 passage cycles.