Amblyomma americanum)   (2). Six3; normal range = 4,500–12,000 cells/mm3), and thrombocytopenia  without a more likely clinical explanation. Evidence of recent Heartland virus infection included 1) detection of viral RNA by reverse transcriptase–polymerase chain reaction on blood or tissue or 2) a ≥4-fold rise in virus-specific plaque reduction neutralization antibody titers between acute and convalescent serum specimens.A confirmed case of Heartland virus disease was defined as a clinically compatible illness in a person with laboratory evidence of recent Heartland virus infection. A clinically compatible illness was defined as fever (≥100.4°F [≥38.0°C]), leukopenia . Patients had symptom onset during May to September . All of the patients had fever, thrombocytopenia, and leukopenia when first evaluated. Of the five patients whose acute symptoms were systematically recorded, all reported fatigue and anorexia, and four reported headache, nausea, myalgia, or arthralgia. Four of the patients were hospitalized. One patient with multiple comorbidities died. All of the patients reported spending several hours per day outside , and five of the six patients reported tick bites in the 14 days preceding their illness onset.Ehrlichia and Anaplasma infection or have not responded to doxycycline therapy (No vaccine or medication is available to prevent or treat Heartland virus disease. Because the virus likely is transmitted through infected ticks or other arthropods, prevention depends on using insect repellents, wearing long sleeves and pants, avoiding bushy and wooded areas, and performing tick checks after spending time outdoors. Health-care providers should consider Heartland virus testing in patients who develop fever, leukopenia, and thrombocytopenia without a more likely explanation and who have tested negative for  therapy . Questio


Penicillium janthinillum strain GXCR by polyvinyl alcohol (PVA)-sodium alginate (SA) beads to remove heavy metals from an aqueous solution containing a low concentration (70 mg/L) of Cu, Pb, and Cd. The PVA-SA-conidia beads showed perfect characters of appropriate mechanical strength suitable for metal removal from the dynamic wastewater environment, an ideal settleability, easy separation from the solution, and a high metal biosorption and removal rate even after four cycles of successive sorption-desorption of the beads, overcoming disadvantages when fungal biomasses alone are used for heavy metal removal from wastewater. We also discuss the major biosorption-affecting factors, biosorption models, and biosorption mechanisms.The biomass of filamentous fungi is an important cost-effective biomass for heavy metal biosorption. However, use of free fungal cells can cause difficulties in the separation of biomass from the effluent. In this study, we immobilized the living conidia of the heavy metal-resistant  Heavy metals in aqueous solutions often originate from effluents discharged from many industries, and these heavy metals are usually characterized by their hazardous effects, persistency, and tendency to accumulate2Microbial biomass-based metal biosorption techniques, especially those employing filamentous fungi, are of low cost in comparison to sorption on commercial ion-exchange resins, activated carbon, and metal oxides456789101112131415161741313Penicillium janthinillum strain GXCR, which has very high resistance to multiple heavy metals and strong metal biosorption by the mycelial biomassPreviously, we reported a strain of filamentous fungus, 3BO3, and 3% CaCl2 through cross-linking for 20 min  lower than that by the PVA-SA-conidia beads at each time point of the corresponding treatments  increased by PVA-SA beads rather than PVA-SA-conidia beads  occurred between the treatments of the beads at 2.7 and 3.6 mm in diameter  increased  decreasing at 25 °C, in the meantime, its removal rate significantly (p < 0.05) increased (p < 0.05) decrease at 25 °C while the removal rate almost kept constant during increasing temperature  analysis. Consequently, based on changes in positions of infrared peaks of PVA-SA-conidia beads before and after biosorption of Cu , Pb Fig, and Cd sorption .The Langmuir sorption isotherm model is to characterize the sorption kineticsBy using the PVA-SA-conidia beads in the multi-component metal solution of Cu, Pb, and Cd, the biosorption was in order of Pb > Cu > Cd. However, with the PVA-SA beads, the adsorption was in order of Cu > Pb > Cd . The remAdditionally, the mycelia generated from PVA-SA-embedded GXCR conidia were still alive even after four cycles of successive sorption/desorption of the beads .Rhizopus sp.30The understanding of the mechanism in each biosorption system is a prerequisite for the engineering process application potential26272829263BO3, 3% CaCl2, and 1.9 × 104 conidia/mL for a cross-linking of 20 min to generate the beads to embed the conidia of heavy metal-resistant P. janthinillum strain GXCR. The further confirmed optimal conditions for high sorption and high removal efficiency by the PVA-SA-conidia beads included the solution of initial pH 3, pre-culture duration  of the beads before sorption, sorption of 100 min, and bead size of 2.7 mm in diameter.In this study, we determined the optimal conditions for preparation of the beads to be 2% PVA, 3% SA, 1% HUnder these conditions, the resulting beads were characterized by an ideal mechanical appropriate strength, a better settleability , a bette31326272829303132The metal-resistant strains seem, no doubt, to be more suitable for application in the toxic heavy metal solution systems especially when the living cells are usedUsually, metal biosorption by fungal biomass decreased as the temperature increased especially under high temperatureReportedly, higher fungal biomass concentrations in aqueous solutions lead to decrease in metal biosorption because of increase in the electrostatic interactions of the functional groups on the cell surfaces while accompanied by decreased cell surface area caused by attachment to each otherAspergillus niger biomass, but phosphate groups and the lipids fractions of the biomass do not participate in metal adsorptionSaccharomyces cerevisiae3, PO3, and 4 for Cd biosorption .The beads were pre-cultured for 1–4 d at 32 °C by shaking at 200 rpm in LPD medium, and then fully rinsed by sterilized water. Subsequently, the residual water present on surfaces of the beads was removed by using Whatman filter paper before biosorption. The fresh beads were put in the 250-mL flask containing 50 mL water solution with or without Cu, Pb, and Cd, and incubated at 200 rpm on a shaker. The metals in the solution were measured through the ZEEnit 700P atomic absorption spectrometry equipped with a flame graphite furnace . All sportion experiments were independently batch-repeated three times. The average values were used in this study.After metal sorption, the beads were collected by filtration, soaked for 12 h in the distilled water and then desorbed for 24 h in 0.5 M HCl for regeneration of the beads. The regenerated beads were stored at 4 °C for reuse.The beads were observed and photographed with an ordinary optical microscope. For observation of mycelial development inside the beads and analysis of the energy spectrum, the beads were cut with a scalpel, fixed for 24 h in 2.5% glutaraldehyde fixative, washed three times as 10 min for each time with 0.1 M phosphate buffer, subsequently dehydrated in gradient concentrations of ethanol, and then photographed to document fungal biomass morphology, while recording the energy spectra by using a HITACHI S-3400 scanning electron microscope (SEM) (Japan) equipped with an EDXS following the conventional procedures.TM atomic force microscopy (AFM)  under the three dimension -controlled parameters of 90 μm × 90 μm × 10 μm for II scanner, 35 μm × 35 μm × 3 μm for FastScan scanner, and the scanning speeds of >2 mm/s for Y-axle and 12 mm/s for Z-axle, respectively.For the surfaces of the beads, the beads after sorption were rinsed three times with physiological saline, and then observed and photographed by a Dimension FastScan−1 on a Nicolet 6700 Fourier transform infrared (FT-IR) spectrophotometer.The IS analysis followed the KBr method in the literaturet-test at p value of <0.05.Statistical analyses were performed with SPSS. Differences between two variables in the mean were evaluated by the Student’s How to cite this article: Cai, C.-X. et al. A novel approach of utilization of the fungal conidia biomass to remove heavy metals from the aqueous solution through immobilization. Sci. Rep.6, 36546; doi: 10.1038/srep36546 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


We show that a broad linear-elastic regime ends at a limiting strain of about 8%, whatever the stiffness of the agglomerate, which corresponds to the beginning of shear failure. The possibility to finely tune the stiffness, size and shape of this simple material makes it an ideal model system for investigations on, for example, fracturing of porous rocks, seismology, or root growth in cohesive porous media.By mixing glass beads with a curable polymer we create a well-defined cohesive granular medium, held together by solidified, and hence elastic, capillary bridges. This material has a geometry similar to a wet packing of beads, but with an additional control over the elasticity of the bonds holding the particles together. We show that its mechanical response can be varied over several orders of magnitude by adjusting the size and stiffness of the bridges, and the size of the particles. We also investigate its mechanism of failure under unconfined uniaxial compression in combination with  The mechanical and fracture properties of cohesive granular materials, where solid but deformable bonds ensure the rigidity of a granular packing, are relevant to a number of questions in powder aggregation679101213141612131821Here we describe a class of cohesive granular materials with tunable properties, and characterise the mechanical response of these materials to uniaxial compression. The materials are similar to wet granulates, like sand, but where the liquid in the capillary bridges between adjacent grains has been replaced by polydimethylsiloxane (PDMS), a curable elastomer, which is then cross-linked to produce “solid” capillary bridges see . Before EP ~ 1 kPa−1 MPa, is several orders of magnitude smaller than the Young’s modulus of the beads, Eb ~ 60 GPa. Thus, we ensure that the mechanical properties of the composite materials are dominated by the deformation of the bridges, and not by the indentation of the beads into each other, such as would be the case with powders18242767816For these materials, we focus our attention on conditions of soft bridges, and hard particles. In particular, in our system the Young’s modulus of the polymer bridges, E of such cohesive granular materials by over two orders of magnitude, by changing the modulus Ep and volume fraction W of the polymer phase, and the diameter D of the particles in the granular phase. A simple model for the micro-contacts, or bridges between particles, is also developed to explain how E should scale with these parameters. Finally, we report on the ultimate yielding of these materials, through shear failure under large strain, using in situ x-ray microtomography.We will show how one can control the Young’s modulus The mechanical properties of cohesive granular materials depend on their microscopic structure and composition. Briefly, if the polymer bridges between particles are stiffer, or larger, we found the Young’s modulus of the bulk material to be higher, as might be expected. There is also some dependence of the material stiffness on the size of the particles used.D, polymer fraction W , the Young’s modulus of the polymer Ep, and packing fraction ϕb. Details of the material preparation are given in the Methods section, at the end of this paper. Similar materials could also be made with other polymers (such as polyurethane or polybutadiene), extending the range of bulk elastic properties even further than those presented here. Additionally, the wetting properties of the beads and bridges could be adjusted by judicious choice of ingredients  to cross-linker, prior to curing. By varying the mass ratio of base to cross-linker from 66:1 to 10:1, we measured Young’s moduli of bulk PDMS samples from μm diameter glass beads. For these experiments the polymer content was fixed at W = 2.3%. This choice ensures that well-defined capillary bridges connect neighbouring beads, without merging into clusters; as will be described in the next section, we are in the pendular regimeϕb was measured in these samples to be, on average, 0.574, with a standard deviation (representing the reproducibility between samples) of ±0.01. In general, ϕb did not vary strongly with EP. However, as Material samples were made by mixing different preparations of PDMS with 210 E of the cohesive granular material was about an order of magnitude higher than that of the PDMS in its bridges, but very much lower than that of the glass beads. In other words the elasticity of the composite material increases with, and is controlled by, that of the PDMS, despite the fact that the polymer represents only a small minority phase. By varying EP from 1 kPa to 1.5 MPa, we could tune E from 90 kPa to 6 MPa, respectively. Finally we note that at the lowest concentration of cross-linker the PDMS remains somewhat liquid-like even after curing; it still made a rigid cohesive sample, but the properties of this material may be controlled more by capillary forces, rather than the elasticity of the bridges.For these conditions, as summarised in i.e. as long as the fluid remains in individual capillary rings, like those shown in E, with polymer content W.Next, we studied how the size and shape of the polymer bridges affects the elasticity of the cohesive granular material, while keeping all other properties of the polymer and beads constant. For comparison, it is well-known that the mechanical properties of wet granular materials are linked to their internal liquid morphology2434W = 0.3% to 8.2%, for fixed bead size D = 210 μm and polymer modulus EP = 250 kPa. The range of W studied allows us to probe the pendular regime, starting from the first observable capillary bridges at low W, but also extends to the funicular regime, as we will shortly demonstrate. As shown in W between 0.3 and 2.7%, E depends strongly on W, and varies by more than an order of magnitude over this range. Above W = 2.7%, E continues to increase with added polymer, but much more slowly than below the threshold value.For this set of experiments we prepared a range of samples with different polymer contents, from W = 0.7% the polymer was homogeneously distributed throughout the sample, forming well-defined bridges between adjacent beads. The samples were then ground up in a mortar, with a pestle, and a single layer of detached beads was spread on a glass slide, for each sample. Images of these beads were taken by digital microscopy, the radii r of the polymer bridges remaining adhered to the particles were measured manually using ImageJA, increases linearly with W, up to several percent of polymer content. Trimers were first observed at W* = 2.7%, and this critical value is shown on W* in the dependence of the packing fraction of the beads, ϕb, with the amount of polymer , we sought to minimise boundary effects, and keep ϕb constant. As demonstrated in ϕb = 0.584 ± 0.01, regardless of particle diameter. These two sets of experiments, summarised in E on ϕb, but instead observed an unexpected variation in E, of nearly one order of magnitude, across the range of bead sizes tested.In a first series of experiments we kept the geometry of the samples fixed to a cylinder of 4.65 mm × 4.9 mm (base diameter × height), while increasing the bead size from d and D expected for capillary bridges39This dependence of material elasticity on bead size is somewhat surprising, as linear elasticity is inherently scale-free. In other words, if all lengths in the system are scaled up by a constant factor, we would expect the elastic response to remain unchanged. That this is not, in fact, true here suggests that there is an additional length-scale in the system, such as a minimum bead separation. One length-scale that can be excluded from consideration here is the capillary length. For the samples discussed above we also measured the average bridge and bead radii. As shown in have observed a strong dependance of E on the bead size, which implies that there is some additional length-scale that is relevant to the physics here. Although the origins of this effect remain unclear, we have ruled out a possible influence of drainage.To summarise, we have shown that perturbations of the average volume fraction of the beads appear to have little observable influence on the mechanical properties of the material. However, we So far we have limited our study to the linear regime of the stress-strain curves, from which the Young’s modulus of a sample can be extracted. Now, we briefly investigate the behaviour of the cohesive granular material for larger compressive strains, focusing on the end of the linear regime. Although deviation from a linear elastic response is not necessarily equivalent to yielding of the sample, we shall presently show how it corresponds to the onset of shear failure in the compressed sample. We therefore take it to be a good approximation of the yielding point.εL by the first point of the stress-strain curve that deviates significantly from a linear fit, as demonstrated in εL = 8% . This suggests that the plasticity of the material is controlled by a limiting strain, rather than a limiting stress.For each of the compression experiments discussed above we determined the limit strain in situ uniaxial compression experiment on one of our samples using micro-computed x-ray tomography (microCT); details of this test are given in the Methods section. The use of precision monodisperse beads here allowed us to reconstruct the positions of nearly all the beads in the sample after a series of compressive steps. Four x-ray scans were made at different strains, as indicated in i after each compression step n. The relative displacements of the beads, i.e. we subtract the sample-wide average displacement).To explore further the internal deformations of the sample, in this limit of large strains, we performed an εL. Now we can clearly see the intermediate stages of shear failure, demonstrated by the shear band, or slip plane, that has developed across the sample. This type of yielding behaviour is expected for homogeneous and isotropic solids failing under compression, when the deviatoric stress (or strain) reaches the yield stress (or strain). Several amorphous materials, such as metallic glasses44The relative displacements within the sample are visualised in We have shown how to make stiff, porous granular materials, and characterised their mechanical properties. As demonstrated above, the elastic response of this class of cohesive material can be tuned by changing the stiffness and size of its polymer bonds, as well as the diameter of its beads. A full micro-mechanical model, which would link macroscopic responses such as elasticity, fracture and plasticity to microscopic modes of deformations, is beyond the scope of this paper. Nevertheless, we will see here how simple models of elastic bridges, combined with finite element simulations (performed using Comsol Multiphysics), can be used to derive simple scaling relations that can explain most of the observed findings.Ep/Eb < 10−4. Since the modulus of the composite material also scales as E ~ Ep, this suggests that we can neglect the deformation of the beads when considering elastic responses. Indeed, the stress transmitted by actual contact of the beads should obey the Hertz theory of contactsIn these materials there is a large difference between the Young’s moduli of the glass beads and of the polymer bonds: E ~ Eb. Again, this is not what we see: here E/Eb < 10−3.A linear elastic response can also be observed when granular materials are cemented with a hard cement, when a and height h with bonded surfaces . Williams and GamonpilasEa, and the true Young’s modulus of the material composing it, Ep:Therefore, we will instead consider a model where the elastic response of the aggregate is only due to elastic deformations of the polymer bridges. Interestingly, the effective Young’s modulus of such a model can still be an order of magnitude higher than that of the bridge material, a result similar to the data shown in ν is the Poisson ratio of the material and S = a/h the aspect ratio of the cylinder. We show the results of Ea can be significantly higher than Ep, especially for large aspect ratios and for materials with a Poisson ratio close to 0.5, such as is the case for elastomers. For perfectly incompressible materials (ν = 0.5), Ea/Ep = 1 + S2/2, and for large aspect ratios, S ≫ 1, it further shows a scaling of the apparent stiffness with the cross-sectional area of the cylinder. Although a cylinder is only a rough approximation of a solid capillary bridge, this example shows how bonding between the beads and the bridges, along with incompressibility of the polymer bridges and their large aspect ratios, can lead to a Young’s modulus of the aggregate that is higher than that of the polymer. Also, the model predicts a linear relation between Ea and Ep, and a scaling of Ea with the cross-sectional areas of the polymer bridges, in fair agreement with the measurements reported in where R, initially separated by a surface-to-surface distance l0 and connected by an elastic bridge consisting of a cylinder of radius r, height H and Poisson ratio ν, which has been truncated by the spherical caps of the intruding beads . We thus define an effective Young’s modulus as E* = σ*/ε*, for small strains. The results of these simulations are shown in l0 and bridge sizes r, and taking nearly incompressible bridges with ν = 0.49. As in the case of the bonded cylinder, we see that here E* can be significantly higher than Ep, in particular for beads close to contact (e.g. for l0/R = 0.02). Specifically, like the data shown in To go towards a more realistic description of the mechanics of the polymer bridges, we consider now a system made up of two beads of radius eads see . As in ti.e. untruncated) cylinder of radius r and height H  but that it fails to describe the cases of large bridges and beads close to contact. Over a larger range of parameter space the simulations instead match the relation E*/Ep ∝ (r/R)2, which is in good agreement with our measurements of E ∝ r2 in the pendular regime. E ∝ r2 in our experimental results could then indicate that the elastic behaviour of the aggregate is mainly governed by these extended bridges.These simulations allow us to test the range of the analytic model that treats an elastic bridge as a simple bonded cylinder. To do so, we replace the bridge between the beads by a full  to the macroscopic elastic properties of the sample could be investigated using lattice-solid models, which simulate large ensembles of discrete particles interacting via springs, and which are widely used to model the mechanical properties of rocks5253We have seen that simple contact-mechanics models can enlighten the underlying physics of our system and provide good quantitative comparisons to our experimental findings. They even suggest the importance of small gaps between beads, when bridges are made, to the macroscopic response of the material. An additional characteristic length scales, such as this gap width, is necessary to explain the observed dependence of ead size . HoweverεL ~ 8%, compared to typical values of 1–2% of yield strain observed for aggregates cemented with hard cemente.g. PDMS can be reversibly stretched by more than 40% of its lengthW, Ep and D is remarkable. This behaviour indicates that yielding of the aggregate could be a purely geometric effect linked to local rearrangement of the beads, in a similar manner to the yielding of amorphous systems such as colloidal suspensionsIn several places we have noted that our materials can behave differently to the more well-known cemented aggregates. One useful feature of our cohesive aggregates worth highlighting is the large strains that they can accommodate. The value of the limiting strain is In summary, we have investigated the mechanical properties of a cohesive granular material obtained by mixing glass beads with a curable elastomer, and shown how its elasticity can be finely tuned by controlling the stiffness of the polymer phase and its volume fraction within the sample. We have seen how scaling relations for the Young’s modulus of the aggregate can be obtained by using simple models, which in turn show good agreement with our experimental findings. We have also found that the stiffness of the aggregate depends on the bead size, while remaining largely insensitive to variations in the volume fraction of the beads. Finally, we have shown that this material exhibits a linear elastic response, even for large strains, and a strain at yield that is independent of its strength and microscopic details. The high tunability of the properties of this model cohesive granular material makes it a good candidate for investigations on problems involving cohesive porous media, such as the hydraulic fracture of porous rocks, seismology, bio-fouling, or root growth in porous matrices.All beads were made of soda-lime glass with a Young’s modulus between 60 GPa and 70 GPa (manufacturer’s info). Prior to use, beads were first cleaned for two hours in a strong surfactant solution , rinsed, further cleaned with a solution of 1M NaOH, and rinsed again several times with water, until reaching neutral pH. Deionized (Millipore) water was used for all cleaning steps. Finally, the beads were dried in an oven at 90 °C overnight.3 using a pycnometer, and the results are summarised in μm beads were used for most tests, while the monodisperse 200.9 μm beads were reserved for the tomography experiments. The remaining beads were used to test the effects of particle size on material properties. The particle size distributions of the beads were measured by optical microscopy using a minimum of 100 beads per bead type. The mean diameters agreed with manufacturer specifications to better than 5%, and are shown in The densities of each type of bead were measured with a precision of ±5 kg/m3, and did not vary within instrument precision over the range of mixing ratios used. Samples were made by mixing a set amount of PDMS uniformly into glass beads  in a mortar with a pestle, taking care that the PDMS was imbibed directly into the bead pack, rather than adhering to the pestle or mortar wall. A subset of the mixture was then cast into a desired mould, where it was gently compressed into shape. To ensure sample homogeneity, the moulded samples were vibrated at a fixed acceleration of 2 g for two minutes at each of the following frequencies: 20 Hz, 100 Hz, 200 Hz, and 500 Hz. This provided an acceleration that is close to, but below, the fluidisation onset of wet granulatesϕb, in any sample was obtained by measuring the mass (ms) and volume (Vs) of that sample, the density of the beads ρb, and the masses of PDMS (mp) and beads (mb) mixed in the mortar prior to curing, and using the relation ϕb = ρsmb/ρb(mp + mb), where ρs = ms/Vs.PDMS was prepared by thoroughly mixing Sylgard 184 (Dow Corning) base and curing agent in the desired weight ratio (from 10:1 to 66:1) and then degassing the resulting mixture under vacuum. The density of the degassed PDMS was measured to be 1010 kg/mEP, was determined from cylindrical samples of dimensions (base diameter × height) of 15.00 mm × 8.00 mm, while the Young’s modulus of the cured aggregate samples, E, was measured on cylinders of dimensions 4.65 mm × 4.90 mm, unless stated otherwise. The cylinder heights could vary by ~10% between samples, but were measured individually for each test, and the measured values were used in calculating the sample strain. A droplet of sunflower oil was deposited on the surface of the pistons before each experiment, to prevent sticking of the sample during compression. The results do not depend on the sample size or aspect ratio, as one can see on Ep for cylinders of diameter 15.00 mm × height 7.70 mm, at W = 2.3%, D = 210 μm and ϕb = 0.56–0.57.Unconfined uniaxial compression tests were performed using two different custom-made testing machines. The results from the two instruments showed no noticeable differences, for tests on samples prepared under identical conditions. The Young’s modulus of PDMS, via a stepper motor of submicron resolution, while the restoring force was monitored using either an analytical balance (Denver Instruments) or a compact high-precision load cell , with an effective force resolution of 5 mN. Compression consisted of discrete steps of 7.6 μm. After each step the load cell was continuously monitored, until the restoring force reported by it had reached an equilibrium value. Only then was the next compression step made. This led to an average compression rate μm/min. Tests made with significantly longer E given here is the result of at least three independent measurements, with standard deviations shown as error bars.During a mechanical test compression was controlled in situ x-ray microtomography was prepared with monodisperse beads of diameter 200.9 ± 1.9 μm (see W = 3.7 % of PDMS prepared with a 10:1 weight ratio of base to curing agent (EP = 1.5 MPa). The Young’s modulus of the sample was measured as E = 7.9 MPa and the bead volume fraction was ϕb = 0.59 ± 0.01. A series of four x-ray computed microtomography scans (GE Nanotom) was performed at different fixed strains /(δπR2).Comsol Multiphysics 4.0 was used to build a 3-D finite-element model of an elastic bridge. The bridge was generated by subtracting two spheres of radius us r see . A finitE* leads to:We also performed simulations of full cylinders to compare with analytical results obtained by Williams and Gamonpilas see . AdaptinHow to cite this article: Hemmerle, A. et al. A cohesive granular material with tunable elasticity. Sci. Rep.6, 35650; doi: 10.1038/srep35650 (2016).


The prevalence of atrial fibrillation is increasing rapidly; however, to date, population-based data are lacking on the attributable cost of illness of atrial fibrillation from a societal perspective, including both direct and indirect costs.The study was an incidence-based cost-of-illness study based on national registries covering the entire population of Denmark. We identified all patients with a first-time hospital diagnosis of atrial fibrillation between 2001 and 2012. For every atrial fibrillation patient, we identified three age- and sex-matched controls from the general population. Both the total and the attributable costs of atrial fibrillation were estimated based on individual level information on hospital care (in- and out-patient contacts), primary sector care, use of prescription drugs and productivity loss.Average 3-year societal costs per patient attributable to atrial fibrillation were estimated to be €20,403–26,544 during the study period. The costs were highest during the first year after diagnosis of atrial fibrillation. Admission costs constituted the largest cost component, whereas primary sector costs and medicine costs only constituted minor components. The attributable costs were more than two-fold higher among patients experiencing a stroke. The total 3-year cost attributable to atrial fibrillation in Denmark was estimated to be €219–295 million.The societal costs attributable to atrial fibrillation are significant. Reducing the need for hospitalizations, in particular from stroke, is a key factor in controlling the costs. Atrial fibrillation (AF) is the most common cardiac arrhythmia and a major modifiable risk factor for ischaemic stroke. The prevalence of diagnosed AF in the general population was 2.9% in a recent Swedish study, which strongly indicates that the prevalence has been underestimated in previous studies , 2. The AF represents a challenge not only for the individual patient and family, but also constitutes a major economic challenge for healthcare systems and societies as a whole because of the high prevalence and the constrained public budgets. Consequently, there is an increasing need to estimate the costs of AF and determine the distribution of different cost components, as it is becoming increasingly critical to take costs into account in clinical decision making –8. A numWe, therefore, designed a nationwide cost-of-illness study using Danish registries covering the entire population with the aim of estimating the societal costs of AF, including both a total and attributable cost approach.This study was designed as a historical, registry-based cost-of-illness analysis. Healthcare provision in Denmark is publicly funded with equal access and includes no co-payment by the patient in the hospital sector and only a small amount of co-payment for some services in the secondary healthcare sector. Hence, except for some minor co-payment for out-of-hospital pharmacological therapy, all services related to AF  are free of charge from the patient perspective. In Denmark, there is a long tradition of systematic recording of health service provisions, for example, and it is possible to access and combine a number of high-quality, exhaustive registries at the individual-patient level, with the use of the patient’s encrypted social security number. Danish legislation permits researchers and others to access the databases. Ethics committee approval and written informed consent are not required for registry-based research, according to Danish law.International Classification of Diseases, 10th edition [ICD-10] code I48) [New (incident) AF patients diagnosed in the period 2001–2012 (inclusive of both years) were identified in the National Patient Registry  . For eacAll included patients had at least 1 year of history and follow-up included in the registries (i.e. a patient diagnosed in November 2012 was ‘followed’ in the registries until November 2013).Primary sector costsOutpatient costsHospital admission costsMedicine costsHome care costsProductivity loss (productivity costs)A societal perspective was applied including the following six cost types:Primary sector costs were obtained from the National Health Service Registry, which holds data on all contacts in the primary care sector, including contacts with general practitioners and private practice specialists . The feeData on hospital costs were obtained from the National Patient Registry. We used the Danish outpatient charges (DAGS charges) as unit cost estimate for each outpatient contact (including emergency room visits) and the Diagnosis Related Groups (DRG) charges for admissions.Data on medicine costs were obtained from the Registry of Medicinal Product Statistics, which holds information on all acquisitions of prescription medicine at Danish pharmacies, including prices . The marHome care costs included both costs at own home and from nursing homes. The Danish municipalities provide these home care services, and the data originate from the municipalities’ own registry, which was accessed via Statistics Denmark. The data were available from 2008 onwards. Data on care in nursing homes were, however, only available until 2012. The registry includes information about the services (measured in minutes) each individual has been granted every month with respect to both nursing and practical help. In order to approximate this into a cost estimate, gross wages for nurses and home-helpers were applied from the Salary Data Office of Municipalities and Regions. In order to estimate an hourly salary, the annual salary was divided by 1628 h, which is the number of effective working hours per year given a 37-h working week. To translate the wage into a cost, a 75% overhead charge was added covering administration costs and transportation.Productivity losses were estimated using weekly employment data from the DREAM database . The DREAM database includes all individuals who have received labour market-related transfer payments since 1991 . With DRThe productivity value for each individual was estimated by multiplying the estimated yearly employment fraction with a gender-specific gross average wage, adjusted for the number of effective working hours per week performed by each gender. With the productivity value of each individual, the productivity loss in year t was estimated as the productivity value in year t minus the productivity value in the baseline year. This was defined as the total productivity loss (productivity costs). The attributable cost in terms of productivity loss was the productivity loss for the AF patient minus the productivity loss for the controls. All costs were inflated to 2013 price levels. Primary sector costs, outpatient costs and hospital admission costs were inflated using the price and salary index from the Danish Regions. Similarly, medicine costs were inflated using the medicine price index from Danish Regions. For the estimates of home care costs and productivity losses, constant 2013 prices were used for salaries and earnings, respectively. The salaries for nurses and home-helpers were identified using yearly gross wages for municipally employed nurses and home-helpers, whereas the productivity values were estimated using per-hour earnings from the private sector. Future costs in year 2 and in year 3 were discounted by 4% per year.We first determined the yearly incidence and prevalence of AF. The AF prevalence estimates were based on the AF patients diagnosed between 2001 and 2012. The number of prevalent AF patients in a given year (by the end of the year) was, thus, the number of new cases plus the number of AF patients from previous years minus the number of deaths. The denominator was the total Danish population aged 18 years or older in the given year. The mortality among AF patients relative to the controls was measured as the percentage of both AF patients and controls who died in the 1st, 2nd and 3rd year after AF diagnosis. Information about deaths was retrieved from the National Causes of Death Registry. Information on the general population, including age- and sex-distribution, was obtained from Statistics Denmark.We then estimated the costs of AF, including the costs in the 1st, 2nd and 3rd year after the AF diagnosis, using both a total cost and attributable cost approach.i in year t was defined as:The total costs for every individual The attributable costs are the total costs of AF minus the total costs among the controls in accordance with previous publications , 18. HenSince AF patients and controls were not necessarily part of the analysis during the entire 3 years, due to either death or the conclusion of the study, their costs were weighted by the fraction of the time they were part of the analysis.Finally, we performed a subgroup analysis among AF patients subsequently diagnosed with ischaemic stroke . This was done in order to assess the cost impact of ischaemic stroke in AF patients. That is, in the National Patient Registry for the 1st, 2nd or 3rd year following the AF diagnosis, we identified all AF patients, with a subsequent hospital contact for ischaemic or non-specified stroke. In order to ensure that only new cases of stroke were included, a 5-year wash out period was applied.The total cost of AF and subsequent stroke was, for every patient, a measure of his or her costs in year t (after the AF diagnosis) minus his or her costs in the baseline year (i.e. the year up to the AF diagnosis). In this subanalysis, patients were also categorized according to the incidence year of stroke .The attributable cost was estimated as the total cost of an AF patient with subsequent stroke minus the total cost among AF patients without stroke (i.e. AF patients without stroke are the controls). Again, attributable costs were estimated as a difference-in-difference.A total of 193,265 patients were registered with a first-time hospital diagnosis of AF during the study period from 2001 to 2012. The yearly incidence varied from 15,066 to 18,094, which corresponded to incidence rates of 3.6–4.1 per 1000 person-years  was 107,526, corresponding to a prevalence proportion of 24.6 per 1000 persons  comprised €194–263 million. For patients diagnosed in 2010, the attributable costs were €289,684,981.AF is characterized by a high incidence and prevalence, particularly among the elderly, as demonstrated in our nationwide study. The societal costs for handling AF and its consequences for the individual patient are substantial, particularly the high costs of hospital admissions in relation to AF, whereas other cost components including cost for primary care and medicine only play a minor role. Prevention of stroke is a key component as illustrated by the huge difference in costs for AF patients with and without a subsequent stroke. Due to the high incidence and prevalence, the costs of AF at a national level were very high.Our findings confirm and extend previous cost-of-illness studies , 9–11. OThe total healthcare costs in Denmark are approximately €14 billion per year. According to our estimates, the total healthcare costs attributable to AF amounted to €194–263 million during our study period, corresponding to 1.3–1.7% of the total healthcare costs. This estimate is in accordance, but most likely more accurate, than a previous UK study, which, based on data from 1995, estimated that the direct costs of AF constituted 0.9–2.4% of the UK healthcare budget in 2000 . The disLooking at the distribution of cost components, it is somewhat difficult to make direct comparisons between the existing cost-of-illness studies due to differences in methodology; however, it is a strikingly consistent finding that hospital admission for AF and AF-related complications is the dominant cost component –8. StrokThe strengths of our study included the nationwide, population-based, controlled study design with inclusion of three matched general population controls for each AF patient, as well as the use of prospectively collected registry data. There is a long tradition for registry-based research in Denmark and the included registries are, in general, considered to be highly accurate as they are all used for administrative purposes. The predictive values of the AF and stroke diagnoses recorded in the National Patient Registry have been examined previously and reported to be more than 90% , 22.The limitations include the use of standard charges and fees as unit costs for admissions, outpatient contacts and GP visits. These may not reflect the true resource use accurately; however, they were considered to be the best available proxies for the true resource use. Furthermore, even though our study applies a broad perspective, including the cost of home care and cost of lost productivity, all costs (e.g. costs for short-term sick leave) were not included. The applied registry (the DREAM database) only included information on long-term sick leave/absence. Other costs for patients and relatives  and possible costs of informal care provided by spouse or another close relative were also not included. Our estimates for the costs of AF are, therefore, likely to be conservative.Our study documents the substantial societal costs attributable to AF using nationwide Danish registries covering the entire population. The costs represents a huge challenge for societies in the coming decades due to increasing incidence of AF. Healthcare costs, particularly hospital admissions, are the main cost component and these costs are strongly influenced by the occurrence of stroke, the most important medical complication of AF. Effective prevention of stroke in patients with AF is therefore paramount both from a clinical and economical perspective.


Virus binding to the cell surface triggers an array of host responses, including activation of specific signaling pathways that facilitate steps in virus entry. Using mouse polyomavirus (MuPyV), we identified host signaling pathways activated upon virus binding to mouse embryonic fibroblasts (MEFs). Pathways activated by MuPyV included the phosphatidylinositol 3-kinase (PI3K), FAK/SRC, and mitogen-activated protein kinase (MAPK) pathways. Gangliosides and α4-integrin are required receptors for MuPyV infection. MuPyV binding to both gangliosides and the α4-integrin receptors was required for activation of the PI3K pathway; however, either receptor interaction alone was sufficient for activation of the MAPK pathway. Using small-molecule inhibitors, we confirmed that the PI3K and FAK/SRC pathways were required for MuPyV infection, while the MAPK pathway was dispensable. Mechanistically, the PI3K pathway was required for MuPyV endocytosis, while the FAK/SRC pathway enabled trafficking of MuPyV along microtubules. Thus, MuPyV interactions with specific cell surface receptors facilitate activation of signaling pathways required for virus entry and trafficking. Understanding how different viruses manipulate cell signaling pathways through interactions with host receptors could lead to the identification of new therapeutic targets for viral infection. Virus binding to cell surface receptors initiates outside-in signaling that leads to virus endocytosis and subsequent virus trafficking. How different viruses manipulate cell signaling through interactions with host receptors remains unclear, and elucidation of the specific receptors and signaling pathways required for virus infection may lead to new therapeutic targets. In this study, we determined that gangliosides and α4-integrin mediate mouse polyomavirus (MuPyV) activation of host signaling pathways. Of these pathways, the PI3K and FAK/SRC pathways were required for MuPyV infection. Both the PI3K and FAK/SRC pathways have been implicated in human diseases, such as heart disease and cancer, and inhibitors directed against these pathways are currently being investigated as therapies. It is possible that these pathways play a role in human PyV infections and could be targeted to inhibit PyV infection in immunosuppressed patients. Virus binding to cell surface receptors often activates signaling cascades that promote virus entry . Many enMyc, Fos, Jun) upon binding to cells (–Polyomaviruses (PyV) are nonenveloped, double-stranded DNA viruses that rapidly induce primary host response genes  binds to cell surface gangliosides and the α4-integrin receptor through specific sites on the VP1 capsid protein –12, and –13–Both gangliosides and integrins modulate GFR activation –25. Gang–We describe a diverse signaling network activated immediately following MuPyV binding to the cell surface. We present evidence that interactions between VP1 and glycan receptors, gangliosides and integrins, stimulate specific signaling events required for MuPyV infection. Furthermore, we identify a subset of these signaling pathways that are critical for MuPyV entry and downstream trafficking of virus into infectious pathways.Inhibition of tyrosine kinases during virus binding to the cell surface blocks JCPyV and SV40 infection, suggesting that PyV early signaling events are essential for polyomavirus infection , 9. We cIn order to identify specific signaling pathways activated during MuPyV binding and entry, we profiled the phosphorylation of 43 different tyrosine, threonine, and serine kinases via a phosphokinase array method (R&D Systems) after pseudovirus addition to MEFs. Pseudoviruses (PsVs) are virus-like particles that are assembled from the major (VP1) and minor (VP2/3) capsid proteins but lack an encapsidated viral genome . Using t−/− MEFs) that are resistant to MuPyV infection . The MuPyV α4-integrin binding site is an LDV motif within VP1 that is distinct from the sialic acid binding site of VP1 (−/−). Electron micrographs of purified wild-type and mutant pseudoviruses showed intact 50 nM capsids  or ganglioside (SA−/−) mutant pseudoviruses was greatly reduced. Furthermore, the LNV SA−/− mutant pseudovirus elicited little to no AKT phosphorylation , indicating that MuPyV activation of EGFR does not require ganglioside interactions , the FAK−/− MEFs unexpectedly displayed a complete loss of GD1a (see y axis), rendering them uninfectable by MuPyV. FAK−/− MEFs have not been previously reported to lack cell surface gangliosides. However, even after ganglioside supplementation, confirmed by flow cytometry with a GD1a antibody , a non-cell-permeable reducing agent that removes biotin from virus on the cell surface while internalized virus retains the biotin tag. Whole-cell lysates (WCL) were collected and incubated with streptavidin-coated beads. The virus bound to the streptavidin-coated beads (pulldown) was eluted in 50 mM TCEP to isolate the internalized fraction. Whole-cell lysates  and the streptavidin pulldown product  were then immunoblotted with anti-VP1. As expected, we observed that the fraction of internalized virus increased from 30 min to 3 h in the DMSO control . However−/− MEFs versus that with wild-type MEFs. Wild-type cells displayed increasing virus internalization (pulldown) from 1 h to 5 h post-virus addition with only one degradation band at less than 42 kDa  , 42. The43–We identified a diverse signaling network activated by MuPyV cell surface binding, including the MAPK, PI3K, and FAK/SRC pathways. Activation of the PI3K and FAK/SRC pathways was required for early steps of MuPyV infection, while the MAPK pathway was not essential. PI3K activation was dependent upon VP1 interactions with both cell surface gangliosides and the α4-integrin receptor, while VP1 interactions with either gangliosides or α4-integrin were sufficient to activate the MAPK pathway. Finally, we defined the contribution of each signaling pathway to early steps of infection. We found that PI3K activation was required for virus internalization, whereas the FAK/SRC pathway contributed to virus trafficking along microtubules. These results indicate that VP1 cell surface binding activates specific signaling pathways essential for early steps of MuPyV infection.MuPyV activation of the MAPK, PI3K, and FAK/SRC pathways is likely initiated by GFRs on the cell surface, but how the virus may activate GFR signaling is unclear, given that the capsid does not contain specific GFR binding sites. A likely possibility is that MuPyV multivalent binding to gangliosides and α4-integrin facilitates activation by indirectly clustering GFRs located in cholesterol-rich microdomains of the plasma membrane. For example, previous results showing increased transcriptional responses to complete viral capsids versus capsomere subunits suggested that clustering is important for signaling . In suppThe MAPK pathway, measured by ppERK, was activated rapidly upon virus addition to cells  through Myc, Fos, and Jun) upon cell surface binding  and JCPyV, can lead to major complications in immunosuppressed patients . Thus, u−/−) MEFs, obtained from Thomas Benjamin at Harvard Medical School  and maintained in complete growth medium. The α4-integrin KD MEFs were generated in our laboratory. Lentiviruses containing shRNAs directed against α4-integrin (RefSeq accession number NM_010576) were prepared at the Functional Genomics Facility at the University of Colorado.MEFs and ganglioside KO  at 10,000 × g. The virus was then salt extracted (washed in 850 mM NaCl), resuspended in phosphate-buffered saline (PBS), and washed an additional 2 times through the 100-kDa filter to remove contaminants. Pseudoviruses were generated following a standard protocol (http://home.ccr.cancer.gov/lco/production.asp).Wild-type virus was NG59RA. Prior to addition to cells, the virus was sonicated at 70 W for 1 min and incubated at 45°C for 20 min. The solution was centrifuged at 10,000 × protocol  publiclyLyophilized gangliosides were obtained from Matreya LLC (GD1a 1062 and GM1 1061) and MyBiosource (GT1a MBS663096). Gangliosides were resuspended in serum-free DMEM and supplemented into cells for 6 h at the indicated concentrations.3VO4) and a protease inhibitor cocktail . Lysates were separated by 8 to 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Membranes were incubated with primary antibody for 16 h at 4°C  or at 37°C for 1 h (for Santa Cruz Biotechnology antibodies anti-ERK sc-93 and anti-tubulin sc-8035). Immunoblots underwent chemiluminescent development and images were obtained on the Image Quant LAS400 imager. ImageJ was used to quantify the integrated density of bands.Cells were collected in RIPA buffer containing phosphatase inhibitors  , GD1a  followed by incubation with primary antibodies. Cells were processed on a CyAn ADP analyzer.Text S1 Supplemental materials and methods. Download Text S1, DOCX file, 0.02 MBFigure S1  Bar graph for the dose response of neutralizing I58 anti-VP1 antibody. Virus was mixed with antibody prior to addition to cells. The I58 antibody was 100% neutralizing to a dilution of 12,500. (Right) The time course of infection at the time of neutralizing antibody addition. (B) Diagram of the drug treatment experimental protocol. (C) Genistein, a tyrosine kinase inhibitor, blocked MuPyV infection in a dose-responsive manner when treatment was added during virus binding and entry (0 to 2 h). Infection was quantified at 24 h p.i. with immunofluorescence and based on the percentage of T-ag-positive nuclei, and results were normalized to those of the DMSO control. Green, T-ag; blue, 4′,6-diamidino-2-phenylindole (DAPI). (D) The prefuse force-directed network of MuPyV-activated kinases identified in the kinase array. The network was generated based on the experimental evidence of interactions; the size of the nodes corresponds to the fold change in activation found in the kinase array, and the thickness of the edges corresponds to the overall score of the interaction. Members of the MAPK pathway are shown in blue, members of the PI3K pathway are in pink, and members of the FAK/SRC pathway and SRC family kinases are shown in green. Significantly enrichment for pathway protein-protein interactions (PPI) is shown. (E) Immunoblot of pEGFR activation 30 min and 2 h post-virus addition. Download Figure S1, TIF file, 1.9 MBFigure S2 t test showed signficance at P = 0.009 and P = 0.016. (C) Immunofluorescence images for GD1a (green) and DAPI (blue) stains. (D) Flow cytometry staining for cell surface VP1 30 min post-virus addition in control and α4-integrin KD1 MEFs. Geometric means of uninfected cells and at 30 min p.i. were plotted . (E) Immunoblot of MuPyV in control and α4-integrin KD1 MEFs. Download (A)Immunoblot for α4-integrin of KD1, KD2, and control MEF lysates. The bar graph shows integrated densities of bands normalized to the control; error bars are standard errors. (B) Representative immunofluorescence images of infection. Nuclei were labeled with by 4′,6-diamidino-2-phenylindole  and T-ag (red). The bar graph shows quantification of infection at 24 h p.i. A Figure S2, TIF file, 1 MBFigure S3 n = 3). (B) Immunoblot of ppERK and p-cJun 15 min and 24 h p.i. in the presence or absence of MEK1/2 inhibitor U0126 (20 µM). ERK was phosphorylated at 15 min p.i. The MEK1/2 inhibitor U0126 blocked ERK phosphorylation when added with the virus. However, it had no effect on virus infection, as shown by T-ag staining of lysates 24 h p.i. c-Jun was phosphorylated in both DMSO and iMEK1/2 samples at 15 min and 24 h p.i. (C) MEFs were treated with virus either in the presence or absence of inhibitors for 30 min at 4°C. Cells were fixed and stained for cell surface-bound virus (anti-VP1) and the receptor GD1a (anti-GD1a). (D) Inhibition of the EGFR, caspases, Rho-GTPases, or actin polymerization (latrunculin) during virus binding and entry (0 to 2 h) or post-virus entry (0 to 4 h). Infection was quantified at 24 h p.i. as the percentage of T-ag-positive nuclei, and treatments were compared to results with the DMSO control. Error bars are standard errors (n = 3). Download (A) Dose-response curves of MAPK inhibitor treatments . Inhibitors were present either during virus binding  or post-virus binding . Results were normalized to those for the DMSO controls, and error bars are standard errors  and FAK−/− MEFs (green) 30 min post-virus addition , displayed as a contour plot with GD1a levels on the y axis (anti-GD1a) and virus binding on the x axis (anti-VP1). The geometric means of the negative controls are plotted as the gray dashed line. (B) Flow cytometry data showing GD1a staining of FAK−/− MEFs and GD1a-supplemented FAK−/− MEFs 5 h post-ganglioside addition. (C) Infection of FAK+/+ MEFs, FAK−/− MEFs, and FAK−/− MEFs supplemented with gangliosides GD1a or GT1b. Nuclei were stained with 4′,6-diamidino-2-phenylindole  and T-ag (Red). The bar graph shows infection of T-ag-positive nuclei at 24 h p.i. (n = 3). The right panel shows representative slides from the infections. Also shown are infections of wild-type, ganglioside−/− MEFs, and ganglioside−/− MEFs supplemented with GD1a or GT1b prior to infection. These infections were carried out alongside FAK−/− infections as a positive control. (C) Immunoblot of the time course of MuPyV in FAK+/+ and FAK−/− MEFs. Download A) Flow cytometry data for FAKFigure S4, TIF file, 1.3 MBFigure S5 ) Confirmation of biotin-SS-MuPyV linkage, determined by SDS-PAGE and Coomassie staining after pulldown with streptavidin-coated beads. (B) Internalization assay in wild-type, ganglioside−/−, and α4-integrin knockdown MEFs. (C) Infectivity of ATTO-565 MuPyV at a molar ratio of 0, 10, 20, or 40. The gel shows a Typhoon scanner image with a 560 laser. Download (AFigure S5, TIF file, 0.6 MB


Background: The objective of the present study was to evaluate the acid tolerance response and pH adaptation whenEnterococcusfaecalis interacted with extract of lime (Citrus aurantiifolia).Methods: We usedE. faecalis ATCC 29212 and lime extract from Aceh, Indonesia. The microbe was analyzed for its pH adaptation, acid tolerance response, and adhesion assay using a light microscope with a magnification of x1000. Further, statistical tests were performed to analyze both correlation and significance of the acid tolerance and pH adaptation as well as the interaction activity.Results:E. faecalis was able to adapt to a very acidic environment (pH 2.9), which was characterized by an increase in its pH (reaching 4.2) at all concentrations of the lime extract (p < 0.05).E. faecalis was also able to provide acid tolerance response to lime extract based on spectrophotometric data (595 nm) (p < 0.05). Also, the interaction activity ofE. faecalis in different concentrations of lime extract was relatively stable within 6 up to 12 hours (p < 0.05), but it became unstable within 24–72 hours (p > 0.05) based on the mass profiles of its interaction activity.Conclusions:E. faecalis can adapt to acidic environments (pH 2.9–4.2); it is also able to tolerate acid generated byCitrus aurantiifolia extract, revealing a stable interaction in the first 6–12 hours. Enterococcus faecalis is a significant agent in the pathogenesis of root canal infections, especially in post-endodontic treatment, with a prevalence of 24–77% in these infections1. This bacterium is very difficult to eliminate because the pathogen can survive in poor nutrient conditions. It can adapt to acidic conditions, including living in the dentin tubule of a closed root canal with a smear layer. It can also express the dominant biofilm protein to maintain its attachment to host cells2.E. faecalis has been shown tolerate to acidic environments as well as to adapt to pH changes, which are the essential virulence factors in maintaining antibacterial balance3. Fisher reported thatE. faecalis could survive in environments with high NaCl concentrations at extreme temperatures of 5–65°C with a pH of 4.5–10.04. Stuartet al.1 reported thatE. faecalis are less sensitive, with a pH of 5.0 at 25°C after it has been incubated for 10 h. The author also found that it has an excellent growth capability at pH 8.5 and low adhesion at pH 7.1 in a medium coated with bovine serum albumin (BSA).E. faecalis is resistant to medication materials such as calcium hydroxide5 and chlorhexidine (CHX)6. The long-term use of both medication materials can lead to parachloroaniline (PAC), causing blockage of the dentinal tubules and eventually becoming toxic7. Fosfomycin may also interfere with acid tolerance systems and pH changes ofE. faecalis in tooth root canals by inhibiting phosphoenolpyruvate synthetase8.Citrus aurantiifolia). Lime peel extract contains phenols, flavonoids, hydrogen peroxide, tannins, alkaloids, and saponins that have antibacterial, antioxidant, antifungal, analgesic, and anti-inflammatory properties9. Nwankwo10 reported that lime extract helped to preventKlebsiella pneumonia,Salmonella, andEscherichia coli. Here, the acid in lime extract influenced the bacterial development and cell metabolism. The present study evaluates the acid tolerance response and pH adaptation ofE. faecalis when the bacterium grows as biofilm in the presence of lime extract with different concentration.Indonesia, especially in Aceh has a tropical climate with a variety of plants that can be utilized in medical treatment, including lime  were used in this study. The extractions were conducted at the Laboratory of Microbiology at the Faculty of Veterinary, University of Syiah Kuala, Darussalam, Banda Aceh, Indonesia. The material and bacterium were preparedin vitro to analyze the pH adaptation, acid tolerance response, and interaction activity ofE. faecalis in different concentration of lime extracts.The lime extract andLime peel was separated from the flesh then dried using dehydrator until the water content reduced to 10%. Dried lime peel was grinded into powders. The powder was put into a glass container and macerated with ethanol 70% for two days and then strained using a gauze. Filtrate was evaporated using a rotary evaporator at 80°C to obtain the pure lime extracts.E. faecalis bacteria was subsequently re-cultured in 5 ml of Mueller-Hinton Broth (MHB) medium  in anaerobic conditions at a temperature of 37°C for 48 hours. Afterward, theE. faecalis grown on the liquid medium was synchronized further with McFarland 0.5 (1 × 108 CFU/ml) . The accurate density of McFarland standard was checked using a spectrophotometer with an absorbance reading of 0.08 to 0.1 at 625 nm11.One colony ofE. faecalis in MHB (1:10) were added to each of the beakers. The initial pH of each mixture prior to incubation (0 hours) was 2.89, 2.75, 2.91, 2.92, 2.95, 2.98 and 3.10, respectively. Next, bacterium containing beaker was put into incubator (37°C) for 6 hours, 12 hours, 24 hours, 48 hours, and 72 hours in an anaerobic atmosphere using Anaerogen TM GasPack , at each of these times, the beakers’ pH was measured using a pH meter . Various changes in pH from 0 hours to the specified time can be used as an indicator of whetherE. faecalis has a tolerance response to the acidic environment and can adapt to changing pH12.A total of 50 ml of lime extracts in several different concentrations  was put into different beaker glasses. Then, 5 ml ofE. faecalis to lime extract utilizing the principle of spectrophotometry13. The analysis was performed based on the incubation time that had been determined following the measurement of pH shaken at 500 rpm. Here, 50 ul of MHB was put into a microplate in triplicate . The microplate was incubated at room temperature for 15 minutes and vacuumed. The materials tested andE. faecalis derived from the incubation processes at 6 h, 12 h, 24 h, 48 h, and 72 hours were added to each well. The microplate was put into an incubator 37 °C for 15 minutes. Then bio-tolerant activity was measured by Elisa Reader  at a wavelength of 595 nm.The cultures of the pH measurements were used to measure the acid tolerance response of14. This incubation time-based interaction activity on the microplate 96 wells series was done following the Gamble’s working principle15. It was modified using violet crystalline and safranin staining to confirm the possible occurrence of bacterial contaminants. First, microplate in triplicate wells were coated with 50 μl of MHB  for 15 min, and aspirated. Second, 50 uL ofE. faecalis in MHB were added and incubated for 15 min at room temperature. Third, 100 uL of different concentrations of LE were added and incubated for 6 h, 12 h, 24 h, 48 h, and 72 hours (as adapted from research conducted by Bachtiar)16. All residues of the test materials  in the microplate were aspirated and the plate was settled for 10 min at room temperature. Then, 50 μL of 2% violet crystalline were added to each well for 5 minutes; the wells were washed with phosphate buffer saline (PBS) two times . A total of 100 μL of Lugol solution was added for 1 minute and washed with PBS. Cell metabolites and dye were removed by the addition of 100 μL of 96% alcohol for 20 seconds. Safranin solution, 50 μL, were added for 2 minutes and washed with PBS18. The microplate was measured using an Elisa reader using with optical density of 595 nm13. For each period, the turbidity of the medium was visually compared with a 0.5 McFarland standardAdhesion assay was conducted based on the principles of Gram-stainingE. faecalis acid tolerance and adhesion to lime extract were calculated to determine average values and standard deviations for each concentration. Two-ways analysis of variance (ANOVA) was performed with significance set at p < 0.05. The analysis was performed using SPSS ver. 20.0 software.E. faecalis to adhere and form biofilm, compared to the control (fosfomycin). All results  (ontrol) , althougontrol)  and robuontrol) .E. Faecalis in lime extract based on replicationspH adaptation ofClick here for additional data file.Copyright: © 2018 Mubarak Z and Soraya C2018Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).E. Faecalis in lime extract based on replicationsOptical density (OD) of acid tolerance respond ofClick here for additional data file.Copyright: © 2018 Mubarak Z and Soraya C2018Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).E. Faecalis in lime extract based on replicationsThe OD value of the interaction activity ofClick here for additional data file.Copyright: © 2018 Mubarak Z and Soraya C2018Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).E. faecalis to ethanolic extract of lime peel, the initial pH of concentrated extract (100% v/v) withoutE. faecalis was 2.89. This is in agreement with those reported by Sitangganget al.19 that the water extract of lime has highly acidic pH ranges (1.7–3.1). As illustrated inE. faecalis suspension prior to incubation (0 hour).In searching for pH adaptation ofE. faecalis might be related to the presence of buffering compounds in the extract that is able to maintain pH. According to Bolhariet al. the juice of lime (C. aurantiifolia) contains 88% water, 6–8% citric acid, 2% potassium citrate and calcium, 0.4–0.6% and other substances20. The water extract of the fruit contains a number of bioactive compounds such as alkaloids, phenols, flavonoids, steroids, terpenoids, reducing sugar, saponins and cardiac glycosides21. Moreover, the peel ofC. aurantiifolia contained 7 percent essential oil consisting of 46 compounds, most of which are terpenes22. The acidity is generated by citric acid and amino acids, while the essential oils contribute to maintaining its acidic pH23. Citric acid is reported to play a crucial role as a natural material to maintain pH balance and possesses antibacterial activity24.This relatively stable pH of lime extract after serial dilution in water and after the addition ofE. faecalis compared to lime extract alone (as a negative control) (p < 0.05). The increased pH occurred in all different concentration of lime extracts, from 6.25% (v/v) to 100% (v/v)  at a temperature of 37°C. The ability ofE. faecalis to adapt to situations with a low pH and temperature has been reported by Morandiet al.25 in the experiment using pH adjusted to pH of 5.0 at 25°C within 10 hours.After incubation there was a significant increase in the acidic pH of lime extract containingv/v) see indicateE. faecalis growth was observed in different lime extract concentrations with long exposure, increased acid tolerance response shown by the microbe as the concentration of the lime extract increased  within 48–72 hours12.Molecularly, the acid tolerance response of of 7.2 E. faecaE. faecalis showed progressively decreased adhesion ability in the presence of different concentrations of lime extract within 6 – 24 hours  have various therapeutic properties such as antibacterial, antioxidant, antifungal, analgesic, and anti-inflammatory9.Varoni31.The mechanism utilized by bacteria to survive heat and low-pH of the environment operate in many different ways. The most successful means of surviving low-pH stress is the complete avoidance of extremely acidic environments. However, none more critical than the sensing of mild acidification to prevent the potentially lethal consequences of the inappropriate production of potentially antigenic proteins. Bacteria that are forewarned by mild acidification can prepare through the induction of a wide range of protective measures. It can alter the composition of the cell membrane, extrude protons, protect macromolecules, alter metabolic pathways, and generate alkalineE. faecalis exposed to different concentrations of lime extract compared to that exposed to fosfomycin indicated better antibacterial activity of lime extract on the microbe than that of fosfomycin. Among active ingredients contained in the lime extract are flavonoids (polyethoxylated flavones and flavanones), coumarin, and terpenoids, all of which act as antibacterials32. Extract prepared from peel, fruit and leaves of lime show promising antibacterial activity against some microbes belong to both gram positive bacteria  and gram negative bacteria (Klebsiella pneumonia andProteus vulgaris)33.The lower adhesion activity observed inEnterococcus faecalis can adapt to environments with a pH of 2.9–4.3 generated by lime extracts. In additionE. faecalis also expressed a tolerance response to the acidic environment. The interaction activity ofE. faecalis in different concentrations of lime extract become stable within 6–12 hours at a temperature of 37°C. Therefore, the lime extract can be used to inhibit theE. faecalis growth.E. Faecalis in lime extract based on replications10.5256/f1000research.13990.d19664334.Dataset 1: pH adaptation ofE. Faecalis in lime extract based on replications10.5256/f1000research.13990.d19664435.Dataset 2: Optical density (OD) of acid tolerance respond ofE. Faecalis in lime extract based on replications10.5256/f1000research.13990.d19664636.Dataset 3: The OD value of the interaction activity of I have read the revised manuscript, and the authors have made modifications to the manuscript, and have no further comments. Thus, I recommend this work for publication in F1000Research. Thank you for considering me as a reviewer. Kindly Regards Boy M BachtiarI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In general, the paper would still benefit from a review by a native English speaker to correct the language. Additional recommended actions to consider are the following:Abstract in Methods (2nd line): instead of “Both materials were analyzed for their adaptation, …” please use “The microbe was analyzed for its adaptation, …”  Methods E. faecalis bacteria: please rephrase the last sentence “The Accurate of the density ...” to make it more understandable. in Culture of in Adaptation to pH assay: first sentence: how do the concentration values, given here in %, compare with units of Figures 1 to 3 where concentration is given in micrograms per milliliter?Results, Discussion  Figure 1: why is the initial pH value (at 0 hours) about the same at all lime extract concentrations? Are there any known inhibitor (buffering) substances in the lime extract  that could explain relatively constant initial pH at a range of concentrations? Figure 2: why is the indicated acid tolerance apparently higher towards increasing lime extract concentration also at 0 hours of exposure? Figure 3: why is the indicated adhesion level apparently higher towards increasing lime extract concentration also at 0 hours of exposure? Where are the error bars mentioned in the text?E. faecialis. However, could the results also involve a concentration-dependent interaction between e.g. citric acid and other antibacterial compounds of the extract? The paper is referring in many places to “interaction activity” between lime extract andI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We have made the revision based on suggestion and comment from reviewers Introduction The last paragraph: ………..acid tolerance response and pH adaptation of E. faecalis  when it interacts with lime extract.    Reviewer’s comment:  please change the bold word with: when the bacterium grows as biofilm in the presence of lime extract with difference concentration.Method -           “Both materials were made in vitro to analyze”. Reviewer’s comment: please change with: The material and bacterium were prepared - In lime extract, typo……………………………………into a glass - Culture of E. faecalis bacteria; please discard; “bacteria” - E. faecalis ATCC 29212 taken from glycerol ……………………………………………………………………………………………..and incubator .  Reviewer’s suggestion: discard this part, and please replace with: One colony of E. faecalis bacteria was subsequently re-cultured in 5 ml of Mueller-Hinton Broth (MHB) medium  in anaerobic conditions at a temperature of 37°C for 48 hours. Afterward, the E. faecalis grown on the liquid medium was synchronized further with McFarland 0.5 (1 x 108 CFU/ml) . The Accurate of the density of McFarland standart (typo: it should be standard) can be checked using a spectrophotuometer with an absorbance reading of 0.08 to 0.1 at 625 nm11. Adaptation to pH assay A total of 50 ml of lime extracts in several different concentrations  was placed into different beaker glasses. Then, 5 ml containing  1 X 108 CFU/mL of E. faecalis in MHB (1:10) were added to each of the beakers. The initial pH Reviewer’s suggestion: please indicate the initial pH that was measured at the zero h.,  before incubation. Reviewer’s suggestion:  Next, bacterium-containing beaker was put into incubator (37°C)  for 6 hours, 12, hours, 24 hours, 48 hours, and 72 hours in an anaerobic atmosphere using Anaerogen TM GasPack , at each of these times, the beakers’ pH was measured using a pH meter . Various changes in pH from 0 hours to the specified time can be used as an indicator of whether E. faecalis has a tolerance response to the acidic environment and can adapt to changing pH. Acid tolerance assay -           “Here, 96 wells of the triple microplate series were coated with 50 μl of MHB”. Reviewer suggestion: Please change to: Here, 50 ul of MHB was put into microplate in triplicate. -           “the test materials  derived……. Reviewer suggestion: please change to: material tested  and E. faecalis……………………………………………”Each well” change to the microplate was put into incubator for 15 min. -           “Then bio-tolerant activity was measured using the Elisa Reader  at a wavelength of 595 nm”. I suggest to discard this part, as it does not has a clear meaning, ….activity was measure by ELISA reader…….. Adhesion assay -           “The standard protein concentration of the E. faecalis and the active component concentration of the lime extract were measured via the Bradford method” It is not clear, why the authors need to analyze (by measuring) the E. faecalis’s proteins (I assume the whole cell proteins). Thus, what does the authors would like to say with "the standard protein concentration of E. faecalis and the concentration of active component of the lime extract”?. My suggestion is please discard this part. -           “Spectrophotometry detected the interaction of E. faecalis with lime extract at a wavelength of 595 nm” It is rather confusing. Spectrophotometry was used to detect the interaction between the bacterium and the lime extract. Did  you use this method to determine the bacterium growth rate? I suggest to discard this spectrometric method. Otherwise, please focus the methods  used only to address your hypothesis; "that biofilm formation or bacterial adherence ability contributes to E. faecalis survival in biofilm-related environment in the presence of lime extract." -           The principle of incubation time-based interaction activity on the microplate 96 wells series used in this research based on Gamble’s working principle. I think you only tested the bacterial adherence capability, not interaction activity between E. faecalis and……….? -           It was modified using violet crystalline and safranin staining. First, 96 wells of the triple microplate series were coated with……….. Suggestion: Please change this part: “First, microplate in triplicate wells. ...................Second, 50 uL of E. faecalis in ( ADD IN WHAT MEDIUM USED) ...........Third, 100 uL of different concentration of LE was added and incubated for.......... -           “A total of 100 μL of Lugol solution was added for 1 minute and then washed with PBS. The rest of the cell metabolism that was not bacterial cells was dissolved in 96% alcohol for 20 seconds until the dye completely removed. 50 μL of safranin solution was added for 2 minutes and then washed again with PBS18. The interaction activity between the lime extract and the E. faecalis bacteria in the microplate wells was assessed via an Elisa reader using a spectrophotometer  at a wavelength of 595 nm”As you only tested mono-species biofilm  ve bacterium), please add an explanation, why you used both crystal violet and safranin solution?, and you did it similar to the procedure for Gram staining method. Please discard the word: “The interaction activity between the lime extract and the E. faecalis Bacteria”. It would better to say: the bacterial adherence was assessed using crystal violet and measured using ELISA reader with optical density of 595 nm.Observation of adhesion mass. This method is not necessary for this study, as you have used crystal violet (CV) assay to measure biofilm mass. CV provides a good measure of biofilm mass, although it cannot measure biofilm viability.  This means, CV stains both  bacteria cells and the polymer substance (carbohydrate) as part of the extra cellular matrix.   Suggestion:Statistical analysis ………..standard deviations for each concentration. Reviewer comment: why there are no SD displayed in each graph shown in the figuresResults-The experiment was conducted in three replicates Reviewer suggestion:Please changed:  the experiment was performed in triplicate wellsThis study showed that the presence of lime extracts decreased pH, but reduction of low pH did not have a significant effect on the ability of E. faecalis to adhere and form biofilm, compared to the control (fosfomycin). All result  are shown in Fig .1-3).Interestingly, the tolerance effect was not influenced by exposure time and the concentration of LE set in this study , and the correlation between time exposure and LE concentration was positive . Otherwise, please exclude this part.“the results showed that the interaction activities of E. faecalis in Lime extract will decrease from 6, 12, 24, and 72 hours. Reviewer‘s comment: I am wondering what the authors mean with the interaction activity. I suggest to discard this part.The mass profile also indicated interaction activity between lime extract and E. faecalis. Antibiotics are capable of forming a covalent bond to activate the cysteine residue of a bacterial cell, triggering UDP-N-acetylglucosamine to form hydrogen bonds. It inhibits the synthesis of peptidoglycan as an antibacterial defense. Reviewer’s comment: Please discard this part, as 1. I cannot see the data (mass profile). 2. Antibiotic are capable........this is not the result of this study. When necessary please put the sentence in the discussion section.    Discussion Reviewer’s comment: please focus the results you got. Especially, please do not repeat the result, and exclude: “The interaction activity between lime extract and E. faecalis can be assumed to be the antibacterial activity, because there was a decrease in the interaction activity between E. faecalis and the biological components of lime extract within 6–24 hours and again within 48–72 hours. The interaction activity is related to the activity of the active ingredients contained in the lime extract, such as flavonoids (polyethoxylated flavones and flavanones), coumarin, and terpenoids, all of which act as antibacterials”. I suggest to rewrite this part ……….The survival of E. faecalis in the presence of LE………..Conclusion E. faecalis can adapt to environments with a pH of 2.9–4.3 generated by lime extracts. In addition E. faecalis also expressed a tolerance response to the acidic environment. The interaction activity between E. faecalis and lime extract become stable within 6–12 hours at a temperature of 37°C. Therefore, the lime extract can be used to inhibit the E. faecalis growth. Reviewer’s comment: Your conclusion should succinctly describe the overall result of the experiment. Please delete: 1. The interaction activity between E. faecalis and lime extract become stable within 6–12 hours at a temperature of 37°C. 2/.  Therefore, the lime extract can be used to inhibit the E. faecalis growth. Please refer your conclusion to the title that reflects the result.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Revision has been made according to the comment of the reviewer


The affected trees show trunk necrosis, cracking and canker before collapse in summer. In this study, we discovered and characterized a new luteovirus from apple trees in RAD-affected orchards using high-throughput sequencing (HTS) technology and subsequent Sanger sequencing.Illumina NextSeq sequencing was applied to total RNAs prepared from three diseased apple trees. Sequence reads were de novo assembled, and contigs were annotated by BLASTx. RT-PCR and 5′/3’ RACE sequencing were used to obtain the complete genome of a new virus. RT-PCR was used to detect the virus.Luteovirus in the family Luteoviridae. The virus is graft transmissible and detected by RT-PCR in apple trees in a couple of orchards.Three common apple viruses and a new luteovirus were identified from the diseased trees by HTS and RT-PCR. Sequence analyses of the complete genome of the new virus show that it is a new species of the genus A new luteovirus and/or three known viruses were found to be associated with RAD. Molecular characterization of the new luteovirus provides important information for further investigation of its distribution and etiological role.The online version of this article (10.1186/s12985-018-0998-3) contains supplementary material, which is available to authorized users. Malus domestica L.) is the most widely cultivated fruit crop worldwide , herbicide damage, winter and drought injuries in RAD has been largely ruled out through rigorous observations and/or testing. This prompted us to use high-throughput sequencing (HTS) to further investigate possible causal agent(s) of RAD.HTS combined with bioinformatic analyses is becoming a routine technology in the field of plant virology for the discovery of many new and emerging viruses, detection of known viruses and investigation of viral genetic diversity and evolution . In thisIn June 2016, samples of branches were collected from six RAD symptomatic apple trees , CPAS1  and CPAS2 (scion samples of PA7–9), were processed at SeqMatic . Plant ribosomal RNAs (rRNA) were removed from total RNAs using Illumina Ribo-Zero rRNA Removal Kit for cDNA library construction. The samples were sequenced on the Illumina NextSeq sequencing platform with 15-sample bar-coded multiplexing.https://www.qiagenbioinformatics.com/). The raw reads were filtered to remove the failed reads, and qualified reads were assembled de novo into contigs with a cut-off of 150-nt. Contigs were annotated by BLASTx comparisons to Viruses_NR and Viroids databases downloaded from NCBI GenBank databases.Analyses of total sequence reads were performed using the CLC Genomics Workbench 9.5.2 platform  were predicted using the Open Reading Frame Finder at ng MEGA7 . RecombiTo verify the luteovirus sequences are associated with graft-transmissible agents, dormant buds of each of four selected trees from the PSU block  in West Virginia Table . The FRELuteovirus in the family Luteoviridae from CPAR (2 contigs), CPAS1 (6 contigs) and CPAS2 (2 contigs), respectively. Approximately 335,446 (86×), 48,908 (12×) and 778,086 (199×) reads were mapped to contig CPAS1–8, the longest luteovirus contig, supporting the presence of the virus in the three samples. Multiple contigs with identities of 83–99% to ACLSV, ASGV and ASPV were also identified from all three samples, respectively, but the data are not presented here. No viroid was detected from any of the HTS samples.Total reads of 27,727,559 (CPAR), 28,817,295 (CPAS1) and 30,614,167 (CPAS2) were obtained after removing the failed reads. Assembly de novo of the reads generated a total of 59,415 (CPAR), 60,992 (CPAS1) and 65,115 (CPAS2) contigs larger than 150 nt. Blastx searches against the Viruses_NR database revealed contigs with amino acid (aa) sequence identities of 29–76% to peach associated luteovirus (PaLV), cherry associated luteovirus (ChaLV) and rose spring dwarf associated virus (RSDaV) of the genus The luteovirus was detected in all six HTS samples 6/6, 100%), whereas ACLSV 2/6, 33%), ASGV  and ASPV  were only found in some of these trees  and contains all cis-acting elements of the luteoviruses [1364GGAUUUUUAGAGGGGCU1380 and 1392CCGGCUUUGAAUCCCCUUU1410 known to be responsible for the − 1 ribosomal frameshift are located at the junction of ORF1 and ORF2. A tract of ten tandem CCXXXX (X is any base) repeats that is required for the ORF3 stop codon readthrough starts at nt 3695. The 3′ terminal region contains all conserved elements [5503GUACGUCCUGGUAGAACAGG5522 . These two insertions, 5506CG5507 and 5516G, are unique to ALV-1.Complete genome of the new virus, with the proposed name apple luteovirus 1 (ALV-1), is 6001 nucleotides (nt) in size (GenBank no. MF120198), encoding ten open reading frames (ORFs). The genome starts with a conserved elementEnamovirus [The arrangement and structure of six of these ORFs  resembles that of other luteoviruses Fig.  8, 10–1–110–14. amovirus  but its Luteoviridae confirmed that ALV-1 is most closely related to PaLV  [Luteovirus.Comparisons of genomic and individual protein sequences among species in the family aLV Fig. . The genny gene) , ALV-1 sLuteoviridae by RDP4 did not detect any recombination breakpoints.Phylogenetic analyses conducted using the genomic sequences of ALV-1 and other luteoviruses placed the virus with ChALV, PaLV, RSDaV and nectarine stem pitting-associated virus (NSPaV) in a cluster distinct from that of BDYVs and two legume luteoviruses Fig. . TopologLuteovirus in the family Luteoviridae, particularly with four known luteoviruses  that infect woody plants [5503GUACGUCCUGGUAGAACAGG5522 in ALV-1 due to two insertions , making it a unique motif among the luteoviruses. The ALV-1 genome also contains two additional ORFs (1a and 5a) that are not present in any known luteoviruses. Pairwise comparisons showed that ALV-1 has the highest genomic sequence identity (52.8%) with PaLV [Luteovirus. Phylogenetic analysis also placed ALV-1 with the woody plant-infecting luteoviruses, suggesting that these viruses share a common ancestor.A novel luteovirus provisionally named apple luteovirus 1 and three common viruses  were identified by HTS of total RNA extracted from RAD-affected apple trees. BLAST search in pathogen databases identified several contigs showing similarities with members of the genus y plants –14. The y plants –11. The Recombination plays an important role in evolution of plant RNA viruses. Both bean leafroll virus and SMV are recombinants occurred before the species separation . It is aGraft inoculation of apple seedlings demonstrated that ALV-1 is graft transmissible, and the virus could be spread by vegetative propagation of scions or rootstock or both. The source of this luteovirus is unknown, and subsequent study of rootstock and mother trees of different apple cultivars is necessary to determine the original infection.Citrus sinensis L. OSb.) propagated on sour citrus rootstock (C. aurantium L.) worldwide many years ago [C. limonia L. OSb.) in Brazil since 1999 [Marafivirus in the family Tymoviridae. RAD is only observed on apple trees grafted on certain dwarf rootstock cultivars such as M9. Both T-QD and CSD also occurred only on sweet orange grafted on certain rootstock [Symptoms of RAD are very similar to apple union necrosis and decline (AUND) described in New York in early 1980s  and citrnce 1999 . T-QD isootstock , 21. T-Qootstock . Trunk sALV-1 was detected in both PSU-FREC and AFRS orchards, but RAD was only observed in the PSU-FREC orchard. Although most cultivars in the two orchards are different and have different infection rates, ALV-1 is detected in the common cv. Fuji trees. However, RAD was not observed in the Fuji tree block in the AFRS orchard where EMLA 7 rootstock was used. Therefore, M9 may be susceptible to ALV-1. Occurrence of AUND, T-QD and CSD had been associated with trees grafted on virus-susceptible rootstocks, respectively –21. ReplMetapolophium dirhodum Walker) and yellow rose aphid (Rhodobium porosum Sanderson). The aphid transmission of ChALV, NSPaV and PaLV has not been studied yet. Although aphids were observed in some RAD-affected orchards, their role in spreading ALV-1 in orchards needs to be defined.Nothing is known about the vector transmission of ALV-1. Salem et al.  reportedKoch’s postulates have not yet been fulfilled so we cannot yet conclude that ALV-1 is a causal agent of RAD. The ALV-1 infection in the PSU-FREC orchard affected with RAD is very common, with 93% of the trees tested positive for ALV-1 and 57–64% tested positive for the three apple latent viruses Additional file 2:Virus detection by RT-PCR in RAD-affected apple trees used for high-throughput sequencing. The four viruses are apple luteovirus 1 (ALV-1), apple chlorotic leaf spot virus (ACLSV), apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV), respectively. Lanes M) 1 kb plus DNA ladder, and W) water. Arrow indicate the DNA fragment with labeled size. (PPTX 226 kb)Additional file 3:Verification of graft transmission of apple luteovirus 1 to apple seedlings by RT-PCR using primers AluDetF6/R6 (Additional file