Banana trees, citrus fruit trees, pome fruit trees, grapevines, mango trees, and stone fruit trees are major fruit trees cultured worldwide and correspond to nearly 90% of the global production of woody fruit trees. In light of the above, the present manuscript summarizes the viruses that infect the major fruit trees, including their taxonomy and morphology, and highlights selected viruses that significantly affect fruit production, including their genomic and biological features. The results showed that a total of 163 viruses, belonging to 45 genera classified into 23 families have been reported to infect the major woody fruit trees. It is clear that there is higher accumulation of viruses in grapevine (80/163) compared to the other fruit trees (each corresponding to less than 35/163), while only one virus species has been reported infecting mango. Most of the viruses (over 70%) infecting woody fruit trees are positive-sense single-stranded RNA (+ssRNA), and the remainder belong to the -ssRNA, ssRNA-RT, dsRNA, ssDNA and dsDNA-RT groups (each corresponding to less than 8%). Most of the viruses are icosahedral or isometric (79/163), and their diameter ranges from 16 to 80 nm with the majority being 25–30 nm. Cross-infection has occurred in a high frequency among pome and stone fruit trees, whereas no or little cross-infection has occurred among banana, citrus and grapevine. The viruses infecting woody fruit trees are mostly transmitted by vegetative propagation, grafting, and root grafting in orchards and are usually vectored by mealybug, soft scale, aphids, mites or thrips. These viruses cause adverse effects in their fruit tree hosts, inducing a wide range of symptoms and significant damage, such as reduced yield, quality, vigor and longevity. Banana trees (banana and plantain), citrus fruit trees , pome fruit fruits , grapevines, mango trees, and stone fruit trees  have a global production of 147.33, 108.01, 74.28, 129.76, 50.64 and 43.13 million metric tons, respectively, according to the statistical data published in 2017 . The total production of these fruit trees is 553.15 million metric tons and accounts for 86.55% worldwide, illustrating their major significance.Citrus, family Rutaceae), including oranges, lemons, grapefruits, tangerines, and limes, are native to Southeast Asia, New Caledonia, and Australia, and are now grown throughout the tropics and subtropics worldwide [Musa, family Musaceae) are native to tropical Indomalaya and Australia [Vitis, family Vitaceae) originated in the south Caspian belt, Turkey, and the Balkans [Mangifera indica, family Anarcardiaceae) is native to East Asia, specifically the Indo-Burma region [Citrus fruit trees  or single-stranded (ss), positive-sense (+) or negative-sense (–), linear or circular. The severity of the disease and the symptoms caused, and subsequently the agricultural impact on the fruit production, may also vary. The viruses occurring in major fruit trees, together with their taxonomy and molecular and biological traits, are reviewed here.Twenty-two viruses, belonging to fifteen genera classified into fifteen families, have been identified infecting citrus .Closterovirus, family Closteroviridae) is a flexuous filamentous virus (2000 × 11−12 nm in size), with a +ssRNA genome (approximately 20 kb in length), containing twelve open reading frames (ORFs) flanked by 5′- and 3′-untranslated regions (UTRs) [Aphis gossypii and Toxoptera citricida) [Citrus tristeza virus  . CTV is Ophiovirus; family Aspiviridae) is a -ssRNA virus consisting of three genomic segments  encapsidated in filamentous nucleocapsids (approximately 760 × 3–4 nm in size) [Citrus psorosis ophiovirus  have vastly different bipartitie genomes  and membrane-bound bacilliform virions (120–130 × 50–55 nm in size in case of CiLV-C) [Brevipalpus in a persistent manner [Brevipalpus, in a persistent manner [Citrus leprosis virus C, C2 and N  is a +ssRNA virus (5983 nt in length) with spherical particles (28 nm in diameter) [T. citricida, Myzus persicae, and A. gossypii [Citrus vein enation virus  . CVEV isiameter) . The disgossypii .Capillovirus, family Betaflexiviridae) is a flexuous filamentous virus (600–700 × 12 nm in size), with a +ssRNA genome (6496 nt in length excluding the poly (A) tail). CiTLV causes stunting or dwarfing, necrosis at the bud union, and virus-induced bud union incompatibility on scions grafted onto P. trifoliata, citrange, or citrumelo rootstocks [Citrus tatter leaf virus  has filamentous virions (approximately 960 × 12–15 nm in size) and a +ssRNA genome (8747 nt in length). CLBV was associated with bud union crease of sweet orange, grapefruit and Clementine plants grafted on P. trifoliata or trifoliate hybrid rootstocks [Citrus leaf blotch virus  is a filamentous virus (960 × 14 nm in size) with a +ssRNA genome (7531 nt in length). CYVCV occasionally causes yellow veins, ringspots and venial necrosis in the leaves, irregular yellow blotches in the rind, and smaller and flatter fruits on many varieties, especially on limes and lemons. CYVCV is considered an emerging threat for the citrus fruit industry [Citrus yellow vein clearing virus  is a nonenveloped bacilliform virus (150 × 30 nm in size) with a dsDNA genome (7559 bp in length) [Planococcus citri [Citrus yellow mosaic virus  . CYMV inus citri .Sadwavirus, family Secoviridae) has an isometric virion (26 nm in diameter) and two +ssRNAs  as its genome [Satsuma dwarf virus  has a tripartite +ssRNA genome . CVV causes a range of symptoms; usually mild on oranges and mandarins but potentially severe on citrons and lemons, associated with fruit malformation and yield reduction [Citrus variegation virus  and citrus sudden death-associated virus  may be associated with citrus sudden death disease, affecting four million orange trees in a very important citrus region in Brazil [Citrus endogenous pararetrovirus , apple stem grooving virus  and apple stem pitting virus  all belong to the family Betaflexiviridae, and have +ssRNA genomes  encapsidated in filamentous virions . These viruses have similar molecular and biological features, and while they remain generally latent in most commercial apple cultivars, they may cause disease when grafted on sensitive rootstocks [Apple chlorotic leaf spot virus  has a tripartite +ssRNA genome  encapsidated in icosahedral virions (26–35 nm in diameter). ApMV infects a large number of woody hosts and is frequently found in mixed infections with ACLSV, ASPV, ASGV, and other apple-infecting viruses. ApMV causes pale yellow to bright cream irregular spots or bands together with major veins on spring apple leaves, with the severity depending on cultivar susceptibility. The symptomatic leaves may be distributed randomly on the tree or limited to a single branch, and drop prematurely. ApMV is asymptomatic in pear [Apple mosaic virus  named temperate fruit decay-associated virus  was identified infecting apple, pear and grapevine trees in Brazil . TFDaV cThe remaining viruses infecting pome fruit trees are of minor significance or to date there is not enough research data available to access their significance.Thirty-five viruses, belonging to fifteen genera classified into nine families, have been identified infecting stone fruit trees .Potyvirus, family Potyviridae) has a +ssRNA genome (approximately 9786 nt in length) encapsidated in filamentous virions (750 × 15 nm in size), and causes ‘’sharka’’ disease, the most devastating disease of stone fruit trees worldwide which causes severe damages and has enormous economic and social impact. Tens of millions of euros and dollars have been spent for controlling this pathogen without success [A. spiraecola and M. persicae) in a nonpersistent manner. There is no evidence for either pollen or seed transmission [Plum pox virus  and are frequently found in mixed infections on stone fruit hosts. PNRSV is a nonenveloped isometric and quasi-isometric (bacilliform) virus , while PDV is a multicomponent virus with five types of particles differing in size  [ApMV (described in the section ‘Viruses infecting pome fruits’), prunus necrotic ringspot virus (PNRSV), and prune dwarf virus (PDV) all belong to the genus  length) . Both vi length) ,35. PDV Robigovirus, family Betaflexiviridae) has a +ssRNA genome (8372 nt in length) encapsidated in filamentous virions (1000–2000 nm in length). CGRMV is generally latent in most Prunus species, and causes green islands and rings cast against a yellow mottle background on leaves, together with misshapen and bitter fruits with pitted and necrotic fruit flesh [Cherry green ring mottle virus  is associated with severe fruit deformations, yield reduction, graft incompatibility, and bud necrosis in some stone fruit species. Some ACLSV isolates cause “pseudopox” disease in apricot and plum, characterized by depressions and protuberances that deform the fruit, which is easily confused with the PPV-caused “sharka” disease .Trichovirus, family Betaflexiviridae) has a +ssRNA genome (8018 nt in length) encapsidated in filamentous virions , and represents the most severe disease on cherry cultivars in some regions. CMLV causes chlorotic mottling and distortion of the leaves, and smaller, flavorless and delayed ripening fruits. CMLV can be transmitted by grafting and also efficiently by the bud/scale mite Eriophyes inaequalis [Cherry mottle leaf virus  has a bipartite +ssRNA genome  encapsidated in icosahedral virions (28 nm in diameter). ToRSV is considered one of the most devastating members of the genus with a worldwide distribution. The virus is found in woody and semi-woody hosts, and also in herbaceous ornamental and weedy species [Prunus spp [Tomato ringspot virus  have +ssRNA genomes  encapsidated in filamentous virions . Both viruses are associated with little cherry disease, which is a complex and serious viral disease with a large impact on fruit quality of infected cherry trees worldwide. LChV-1 and LChV-2 can dramatically reduce size, color, and taste of the fruits, and a damage vegetative growth of susceptive cherry varieties especially as mixed infections [Phenacoccus aceris and Pseudococcus maritimus) [Little cherry virus 1 and 2  .Recently, thirteen novel viruses belonging to eight families have been reported by next-generation sequencing from stone fruit trees ,40, and A total of 80 viruses (including five unnamed ones), belonging to twenty-nine genera classified into eighteen families, have been identified infecting grapevines. Some of them cause serious diseases including leafroll, rugose wood (RW), fanleaf, decline and other symptoms  Table S.Closteroviridae , with +ssRNA genomic segments  encapsidated in long flexuous virions (1350–2000 × 12 nm in size). GLRaVs are associated with grapevine leafroll disease (GLRD), which is characterized by reddening (red-berried cultivars) or yellowing (white-berried cultivars) of the leaves, while the veins remain green and the leaf margins roll downward in late summer and autumn, resulting in decreased vigour and yield by 15–20% on average [Ampelovirus, are transmitted through vegetative propagation, grafting, and insect vectors including mealybugs and soft scale species, while no vector has been identified for GLRaV-7 (genus Velarivirus), GLRaV-2 (genus Closterovirus), and GLRaV-4 strain Car [Grapevine leafroll-associated viruses (GLRaVs) 1, 2, 3, 4, 7 and 13 are filamentous viruses belonging to the family  average . GLRaV-1 average . Some GL average . GLRaVs rain Car .Vitivirus), and grapevine rupestris stem pitting-associated virus  belong to the family Betaflexiviridae. They have +ssRNA genomes  encapsidated in filamentous virions (600–1000 × 10–15 nm in size). GVA, GVB, and GVD are associated with the RW complex symptoms, characterized by alterations of the woody cylinder such as pits and grooves on the scion or/and rootstock, together with an overall decrease in vigour and yield, and possibly graft incompatibility [Grapevine virus A, -B, and -D  has a bipartite +ssRNA genome  encapsidated in icosahedral virions (30 nm in diameter). GFLV is the main etiological agent of fanleaf degeneration disease, characterized by a distortion with toothed margins, closer primary veins, and widely open petiolar sinuses [Xiphinema index) that feeds on roots [Grapevine fanleaf virus , is not associated with pathological symptoms on mango fruits or leaves . This noyviruses .Eighteen viruses, belonging to four genera classified into six families, have been identified infecting banana worldwide . These aBabuvirus, family Nanoviridae) is a circular ssDNA virus (18–20 nm in diameter), consisting of six different components referred to as DNA-R, -S, -C, -M, -N, and -U3 (each 1060 ± 50 nt in length). BBTV is the most devastating virus; it causes banana bunchy top disease and leads to severe reductions in the productivity. For instance, a yield loss of 50–100% in severely infected plants of highly susceptible cultivars was reported from affected regions [P. nigronervosa and P. caladii) in a persistent and circulative manner [Banana bunchy top virus , including banana streak GF virus, banana streak IM virus, banana streak MY virus, banana streak OL virus, banana streak UA virus, banana streak UI virus, banana streak UL virus, banana streak UM virus, banana streak VN virus) have bacilliform-shaped virions (130–300 × 30 nm in size), and noncovalently closed circular dsDNA genomes (approximately 7.2–7.8 kb in length). BSVs are responsible for banana streak disease, which is widely distributed worldwide. BSVs infect the natural and synthetic hybrids of Musa plants and cause initially yellow and finally necrotic dots or streaks on the leaves, and pseudo-stem splitting. Some isolates may cause stunting, leaf necrosis and a premature death [ in a semipersistent manner [Banana streak viruses  has a +ssRNA genome  tail) encapsidated in flexuous and filamentous particles (660–760 × 12 nm in size). BBrMV is among the most economically important viruses, resulting in yield losses ranging from 30% to 70% for infected banana plants according to previous records [P. nigronervosa, Rhopalosiphum maidis, A. gossypii, and A. craccivora) in a nonpersistent manner [Banana bract mosaic virus , sugarcane mosaic virus (SCMV) strain SCMV-Ab , banana mild mosaic virus and banana virus X , and cucumber mosaic virus  are of minor significance [Abaca bunchy top virus , and root grafting in orchards. Some of them are also transmitted by pollen grains and seeds and are vectored by mealybugs, soft scales, aphids, mites or thrips.The review summarizes the currently available information on the biological, genomic and morphological traits of viruses infecting woody fruit trees, which can be utilized to help the prevention and control of numerous ecologically and economically important diseases. Detailed knowledge of the viral diseases potentially affecting specific woody fruit trees will lead to faster identification of the causative agents. Additionally, known genomic sequences will facilitate the design of oligonucleotide primers targeting specific viruses or groups of viruses enhancing detection.


In this study, the effect of the ionic cross-linking mode on the ability to control physical properties and in vitro release behavior of the dexamethasone (DEX) drug from chitosan (CS) and chitosan/hydroxyapatite (CS/HA) beads was investigated. CS solutions without and with HA and DEX were dripped into two coagulation solutions, prepared with a non-toxic ionic crosslinker  and distilled water, one at pH = 9.0 and other at pH = 6.0. Optical microscopy (OM) and scanning electron microscopy (SEM) results showed changes on the surface topology of the beads, with a reduction of roughness for beads prepared at pH = 6.0 and an increase for the one prepared at pH = 9.0. The diameter and sphericity of the beads prepared at pH = 6.0 proved more uniform and had a larger pore size with a good interconnectivity framework. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) suggested a higher crosslinking degree for beads prepared at pH = 6.0, corroborated by X-ray diffraction profiles (XRD) analysis that indicated a decrease in the crystalline structure for such beads. In in vitro drug release data, all beads presented a sustained release during the studied period (24 h). The drug release rate was affected by the pH of the coagulation solution used in the preparation of the beads. The in vitro kinetics of the release process was of the Peppas–Sahlin model, controlled by both diffusion and relaxation of polymer chains or swelling . Our results suggest that DEX-loaded CS/HA beads, crosslinked in TPP coagulation solution at pH = 9.0, led to a decrease in the DEX release rate and prolonged the release period. Thus, this composition might have prospective as a functional material for bone and cartilage tissue engineering. Polymeric material suitable for biomedical applications must be biocompatible and biodegradable . Due to Chitosan can be modified in different ways to control the rate of release and efficiency of the bioactive agent in the delivery systems, for example, from cross-linked beads . As it hThe practice of the beads-based delivery system allows control of the drug release profile and the specific target site by carefully adapting the formulation of various drug combinations and polymeric materials. This type of delivery system provides longer life, controlled release rate, and in addition, directs the specific drug. There are different methods for forming beads, for example, interaction with counterions, such as anions , cross-linking, solvent evaporation, ionic gelling, spray drying, emulsion polymerization, and precipitation, etc. .To improve the properties and increase the application of chitosan potential, it is common to combine materials with different characteristics in the manufacture of spheres, such as the combination of calcium phosphates (such as hydroxyapatite) with polymers . One of Hydroxyapatite (HA) is one of the most commonly used ceramics for tissue engineering because of its close resemblance to the chemical composition of the inorganic part of natural bone, which is formed by calcium phosphate (about 60% by weight), collagen (20–30 wt%) and water 10–20 wt%) . Th. Th−1  also exhibited a new band at 1210 cm−1 a, consis−1 corresponded to the P–O bond in the HA bulk spectrum  and crosslinked CS beads at coagulation solution pH = 9.0  and pH = 6.0  were compared  to charaThe diffraction pattern of CS, as shown in After the incorporation of HA into CS and CS/DEX beads , the two main peaks at 2θ value of 25.6° and 31.7° which are (002) and (211) reflections of HA, respectively ,60,61,62XRD spectra of the CS/DEX-TPP9, CS/DEX-TPP6, CS/HA/DEX-TPP9, and CS/HA/DEX-TPP6 beads , show peThe in vitro release behavior of dexamethasone (DEX) from the CS and CS/HA beads prepared at coagulation solution pH = 9.0 (TPP9) and pH = 6.0 (TPP6) was investigated through UV-visible spectroscopy in PBS up to 24 h at pH 7.4 and 37 °C . Accordi10(PO4)6(OH)2, might interact with the amino and hydroxyl ions of chitosan by the formation of hydrogen bonds [en bonds ,69. Thisen bonds  reported2), the lower value of Akaike information criterion (AIC), and the largest value of model selection criterion (MSC) [To analyze the release mechanism of the dexamethasone (DEX) from chitosan (CS) and the chitosan/hydroxyapatite (CS/HA) beads, prepared at two coagulation solutions (pH = 9.0 and pH = 6.0), over release behavior, the experimental drug release data were fitted to three kinetic models: zero-order, Higuchi, and Peppas–Sahlin ,72,73,74on (MSC) .2, AIC, and MSC considering the CS and CS/HA beads crosslinked at pH = 9.0 (CS/DEX-TPP9 and CS/HA/DEX-TPP9) and pH = 6.0 (CS/DEX-TPP6 and CS/HA/DEX-TPP6). The results show that the DEX release from CS and CS/HA beads, prepared at both pH, showed good fitting with Peppas–Sahlin model, as is exhibited in 2, lower values of AIC, and higher values of MSC were recorded , 80–93% deacetylation degree, and viscosity average molecular weight of 1.7 × 105 Da, kindly donated by Northeastern Biomaterials Evaluation and Development Laboratory—CERTBIO . This chitosan is accredited by the National Institute of Metrology, Quality, and Technology (INMETRO) at the International Organization of Standardization (ISO)/International Electrotechnical Commission (IEC) 17025:2005 and is used for medical applications. Synthetic hydroxyapatite (HA) with particle size <200 nm, sodium tripolyphosphate , and dexamethasone D1756 (DEX) were purchased from Sigma–Aldrich® . Phosphate buffered saline solution (PBS) at pH = 7.4 and acetic acid  were purchased from Vetec® . Hydrochloric acid (HCl) (98%) was obtained from Nuclear  and ethanol  was obtained from Neon® .Chitosan (CS), derived from the exoskeleton of w/v) chitosan solution was prepared by dissolving chitosan in a 1% (v/v) aqueous solution of glacial acetic acid. The polymer solution was maintained under constant mechanical stirring  at 75 rpm, at room temperature (25 ± 1 °C) until it became a homogeneous solution. Thereafter, HA powder with a concentration of 20% (w/v) was added to the chitosan solution, which was maintained under mechanical stirring at 75 rpm until it became a homogeneous mixture. The CS/HA suspension was dripped, using a syringe pump  and 22G × ½ gauge needles at 25 mL/h, into two coagulation baths (TPP/distilled water solutions). One at pH = 9.0 (coded as TTP9) and other at pH = 6.0 (coded as TPP6) with the same TPP concentration in both baths concentrations (0.1 M). The beads were left in the precipitating solutions for 24 h, at 75 rpm, and 25 ± 1 °C to crosslinking of the beads. Then, the beads were washed with distilled water and PBS solution to a neutral pH (7.4). Afterward, frozen in a freezer , at approximately −18 °C for 24 h and lyophilized in a lyophilizer  at −56 °C for 48 h. Subsequently, they were dried at room temperature. The schematic representation of the fabrication of the CS/HA beads through two coagulation baths (TTP9 and TPP6) is shown in Chitosan/hydroxyapatite beads were manufactured by ionotropic gelation followed by lyophilization. First, a 2% (The CS/HA/DEX beads were prepared by the same method described in Dv), surface diameter (Ds), volume (V), surface area (As), and sphericity (S) of the samples were calculated by equations:A is the area of the particle, and P is the perimeter.The dimensions of the beads were determined using an optical microscope . About 10 beads were placed on a dry glass slide, positioned at the base of the microscope. The images were made by applying the 5040Z lens and a 100× magnification. The measurements of the beads were carried out using a 5040LOW lens with magnification 20× using the 2D_MEASURE software . The volumetric diameter  Phenom-World, Pro-X800-07334 . The beads were placed in double carbon tape. The central part of the beads was sectioned with a surgical blade to examine the cross-sectional morphology. SEM images of each bead were taken by applying an electron beam accelerating voltage of 15 kV, with a depth of focus of 1 mm and a resolution of 30 nm. The beads surface deposition of HA was investigated using SEM with energy dispersive spectrum (EDS) .−1 at 4 cm−1 resolution, and each spectrum represents 64 co-added scans referenced against an empty ATR cell spectrum.The beads , finely grinded using a mortar and pestle, and their pure components  were analyzed by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) using a Perkin Elmer 400 FTIR Spectrometer  equipped with attenuated total reflectance (ATR) accessory employing the Zn-Se crystal. Spectra were recorded over the range of 4000 to 600 cm−1. For this characterization, the beads were cryogenically ground and then placed in the sample holder of the diffractometer.X-ray diffraction profiles (XRD) were recorded on a Shimadzu diffractometer model XRD-7000  with Ni-filtered Cu-Kα radiation, a voltage of 40 kV and a current of 30 mA, to identify the composition and crystallinity of the beads , prepared into two coagulation baths (TPP6 and TPP9). The scanning range was from 2θ = 2° to 60° at a scan speed of 0.033°sThe amount of dexamethasone (DEX) released from CS and CS/HA beads was measured using an ultraviolet-visible (UV-VIS) spectrophotometer . First, the bead samples  were incubated in a volumetric flask in a 50 mL PBS solution (pH = 7.4), as a release medium, and kept under constant stirring of 150 rpm in an IKA shaker model KS4000i  at 37 °C ± 0.5 °C to simulate body temperature. Subsequently, an aliquot of the release medium (4 mL) was withdrawn at fixed time intervals , and replaced with 4 mL of a fresh release medium (at 37 °C ± 0.5 °C) each time. The collected samples were then analyzed for DEX content by measuring the absorbance at 242 nm (λmax of DEX in phosphate buffer solution pH 7.4 measured using a UV-VIS spectrophotometer). All the release experiments were carried out in triplicate, and the average values were used for further data treatment and plotting. The drug concentration was calculated according to a standard curve, and Equation (6) determined the cumulative release. The corresponding drug-release profiles were represented through plots of the cumulative percentage of drug release  versus time.0 is the sampling volume (4.0 mL), Ci is the concentration (mg/L) of the released drug collected at time t, and m is the mass of the drug incorporated in the polymer (1.25 mg).2); (ii) lower value of Akaike information criterion (AIC), and (iii) largest value of model selection criterion (MSC) [In vitro release data were analyzed by zero-order (Equation (7)), Higuchi (Equation (8)), and Peppas–Sahlin (Equation (9)) kinetic models ,78,79,80on (MSC) ,82.tQ is the amount of drug released at time t; Q0 the amount of drug in the solution at t = 0;  and k0 the zero-order release constant.Hk representing the Higuchi rate constant.Q∞ is the maximum amount of the drug released measured at the equilibrium, k1 and k2 are the release constants, and m is the release exponent.Beads of chitosan (CS) and chitosan/hydroxyapatite (CS/HA) loaded with steroid anti-inflammatory agent, dexamethasone (DEX), crosslinked into two sodium tripolyphosphate (TPP) coagulation solutions, one at pH = 9.0 and other at pH = 6.0, with the same concentration of TPP in both solutions (0.1 M), were fabricated by freezing and lyophilization method. The morphology of the beads was influenced by the coagulation solution pH. The beads crosslinked at neutral pH had a rougher surface, whereas the ones crosslinked at acid pH had a smooth surface. The diameter and sphericity of the beads prepared at acid pH proved uniform. Likewise, at this pH, SEM data suggested a larger pore size with a good interconnectivity framework. The crosslinking degree was higher for the beads prepared at acid, as suggested by FTIR-ATR data, corroborating with XRD analysis that indicated a decrease in the crystalline structure for the beads prepared at pH = 6.0. According to in vitro drug release data, all beads presented a sustained release during the studied period (24 h). However, the ones prepared with HA at basic pH (pH = 9.0) led to a decrease in the DEX release rate and prolonging the release period. The in vitro kinetic of DEX from CS and CS/HA beads, prepared at both pH, was best described using the Peppas–Sahlin model. This kinetic model points to a release mechanism that relies on the effects of Fickian diffusion and relaxation of polymer chains or swelling . Thus, neither absolute Fickian diffusion (as a result of pure drug diffusion) nor Zero-order (because of polymer chain relaxation) was the predominant mechanism in this case. This can be assumed as a combination of both contributions of polymer relaxation and drug diffusion factors that determine the drug release mechanism. All of these results demonstrate that DEX-loaded CS/HA beads, crosslinked at TPP coagulation solution at basic pH, may be a promising drug carrier for bone tissue engineering.


To fully unravel the ixodid ticks’ role as vectors of viral pathogens, their susceptibility to new control measures, and their ability to develop acaricide resistance, acclimatization of ticks under laboratory conditions is greatly needed. However, the unique and complicated feeding behavior of these ticks compared to that of other hematophagous arthropods requires efficient and effective techniques to infect them with tick-borne viruses (TBVs). In addition, relatively expensive maintenance of animals for blood feeding and associated concerns about animal welfare critically limit our understanding of TBVs. This mini review aims to summarize the current knowledge about the artificial infection of hard ticks with viral pathogens, which is currently used to elucidate virus transmission and vector competence and to discover immune modulators related to tick–virus interactions. This review will also present the advantages and limitations of the current techniques for tick infection. Fortunately, new artificial techniques arise, and the limitations of current protocols are greatly reduced as researchers continuously improve, streamline, and standardize the laboratory procedures to lower cost and produce better adoptability. In summary, convenient and low-cost techniques to study the interactions between ticks and TBVs provide a great opportunity to identify new targets for the future control of TBVs. Asfarviridae, Nairoviridae, Peribunyaviridae, Phenuiviridae, Flaviviridae, Orthomyxoviridae, Rhabdoviridae, and Reoviridae  and the Crimean–Congo hemorrhagic fever virus (CCHFV), which are known to cause severe clinical symptoms in humans  by allowing them to feed on THOV-infected Syrian hamsters  and bank voles (Clethrionomys glareolus) (Erinaceus europaeus), striped field mouse (A. agrarius) European pine vole (Pitymys subterraneus), and common pheasant (Phaseanus colchicus)  , 1997b, areolus) , Europealchicus)  were useI. ricinus was also evaluated in mountain hares (Lepus timidus), New Zealand white rabbits (Oryctolagus cuniculus) and red deer (Cervus elaphus) . Co-feeding transmission was also observed for Bhanja virus and Palma virus in D. marginatus, D. reticulatus, and I. ricinus ticks infested on mice (Amblyomma americanum infested on rabbits (H. longicornis ticks infested on BALB/c mice (Co-feeding transmission of the Louping ill virus on elaphus) . Non-viriculatus , 1997 anpeltatum  and Amblriegatum  using gu on mice  and Hear rabbits . Lastly,B/c mice .Another alternative to tick infestation is through membrane feeding. Membranes from animal and non-animal origin  are usually utilized, with variable success, to feed ticks. This method could also be used for studies on the dynamics of pathogen transmission, since it can reduce the variation within a given treatment group because the blood meal from the same donor reduces the variation that may arise from individual tick–host relationships .I. ricinus, I. hexagonus, D. reticulatus, and R. bursa with the Bluetongue virus  has the advantage of estimating the viral dose received by the ticks Figure . Howeverat virus , 2017a, to virus , I. riciith TBEV , and theith TBEV . D. marg viruses . Alterna viruses , though th CCHFV , I. riciith TBEV , and H. at virus  and THOVat virus .I. scapularis infected with Langat virus (A. americanum infected with the Heartland virus (Infection of ticks through immersion provides a low cost and relatively simple artificial method, since it can synchronously infect a large number of ticks with a defined virus stock. The ticks are believed to be infected when they successfully swallowed the immersion medium containing the virus; with the ingested virus ultimately reaching the midgut . The virat virus , the denat virus , and TBEat virus  and for nd virus .To fully understand the interaction of ticks with TBVs, efficient techniques for the artificial infection and maintenance of tick colonies under laboratory conditions are crucial. As emphasized in this mini review, it is the unique but complicated feeding behavior of ixodid ticks that makes studies related to virus transmission, vector competence, and other aspects of tick–virus interaction a challenging endeavor. However, with the availability of these alternative feeding methods and techniques to infect ticks with different viruses of public health importance, the potential for studies on TBVs to catch up with the advances in mosquito-borne viral disease research is no longer a far-fetched scenario. In addition, the limitations of current techniques do not outweigh importance of studying TBVs. Understanding the interactions between ticks and the TBVs they transmit offers a great opportunity to identify new targets for the future control of TBVs.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Screening solid-phase combinatorial libraries of bioactive compounds against fluorescently labeled target biomolecules is an established technology in ligand and drug discovery. Rarely, however, do screening methods include comprehensive strategies—beyond mere library blocking and competitive screening—to ensure binding selectivity of selected leads. This work presents a method for multiplexed solid-phase peptide library screening using a ClonePix 2 Colony Picker that integrates (i) orthogonal fluorescent labeling for positive selection against a target protein and negative selection against competitor species with (ii) semi-quantitative tracking of target vs. competitor binding for every library bead. The ClonePix 2 technology enables global at-a-glance evaluation and customization of the parameters for bead selection to ensure high affinity and selectivity of the isolated leads. A case study is presented by screening a peptide library against green-labeled human immunoglobulin G (IgG) and red-labeled host cell proteins (HCPs) using ClonePix 2 to select HCP-binding ligands for flow-through chromatography applications. Using this approach, 79 peptide ligand candidates  were identified as potential HCP-selective ligands, enabling a potential rate of >3,000 library beads screened per hour. The screening of solid-phase combinatorial peptide libraries has found wide application in the development of biospecific agents, including ligands for the purification of biomolecules ,4,5,6,7,The polydispersity between library beads and the diversity of proteins present major challenges for fluorescence-based library screening in the form of variable (i) polymer bead autofluorescence, (ii) fluorescence intensity of the target biomolecule, and (iii) fluorescence intensity of the ensemble of contaminant proteins. These challenges pose the need for a novel technology that can survey the range of bead aspect ratios and fluorescent signals for both target and contaminants across the whole pool of library beads prior to screening, and utilize this information to tailor the selection criteria of average bead fluorescence intensity and ratio of target vs. contaminant fluorescence intensity. This approach eliminates outliers, minimizes the loss of false negatives, and greatly promotes the selection of beads carrying candidate peptide ligands with desired binding strength and specificity. Ensemble imaging of combinatorial libraries followed by informed selection of beads with fluorescent intensity adjusted to the remaining population has been successfully demonstrated by Heusermann et al.  This sysIn this work, we demonstrate the use of ClonePix 2 for library screening by selecting peptide ligands for protein purification by flow-through mode chromatography. To this end, we designed a multiplexed dual-fluorescence screening approach to identify an ensemble of peptides that cooperatively target a set of protein contaminants, while showing no affinity for the molecule to be enriched. In this study, we adopted a model screening feed containing >300 host cell proteins (HCPs) from a null  Chinese hamster ovary (CHO) cell culture harvest  as proteThe identification of an ensemble of ligands for negative chromatography represents a paradigmatic case study of multiplexed library screening. The mixture of Chinese hamster ovary (CHO) host cell proteins (HCPs) and human IgG in a 1:4 weight ratio used as model feedstock in this study has been characterized in previous work . This feA hexametric combinatorial OBOP (one-bead-one-peptide) library X1-X2-X3-X4-X5-X6-G-S-G was first synthesized on HMBA-ChemMatrix resin via Fmoc/tBu synthesis following the “split-couple-and-recombine” method proposed by Lam et al. ,20. SpecWhile initially developed by Molecular Devices to conduct high-throughput screening of mammalian cells, the ClonePix 2 colony picker is amenable to OBOC screening. The ClonePix 2 system, in fact, can fully automate bead imaging and selection, while requiring minimal modifications to process ChemMatrix beads, whose diameter (~100–300 µm) resembles that of cell colonies. To ensure that the polymers present in the CloneMatrix do not interfere with the subsequent peptide sequencing by LC/MS/MS by reduction of the peptide MS signal, beads carrying a known sequence (HWRGWVGSG) were sequenced pre- and post-casting in CloneMatrix, with no impact on sequencing. In addition to the library beads, the sequence GSGSGSGSG, known to have minimal interaction with the model proteins utilized in this work, was also synthesized on HMBA-ChemMatrix and incubated with the fluorescent proteins as a negative control.To select ligands that selectively bind the whole spectrum of HCP proteins, we established gates for a maximum allowable AF488 intensity and a minimum AF546 intensity detected as measures of IgG and HCP capture, respectively. These pick gates were based on the distribution of interior mean fluorescent intensity a calculaFor detection of HCP-bound beads, the geometric mean for interior mean intensity from imaging with the ClonePix 2 Rhodamine filter (detection of AF546-HCPs) was measured at 620.1 b with anTo determine whether all beads selected were effectively deposited, the 96-well plates were visually inspected to confirm the presence or absence of a single bead in each designated well. Plate transfer efficiency, defined as the ratio of the number of wells confirmed to be occupied by a bead divided by the number of beads transferred, was measured by imaging the resulting 96-well plates using the Molecular Devices CSI-FL fluorescent imager; the percentage of occupied wells that had more than one bead deposited was also monitored. The plate transfer efficiency was calculated at 71% ± 5.9%, with 14% ± 16% of occupied wells carrying more than one bead. Bead transfer efficiency can be improved by optimizing matrix curing to obtain a more rigid support during picking, or adjusting the parameters for bead aspiration and depositing.The comparison between the beads selected for specific binding of HCPs and the full library is shown in The throughput of the “cast-image-pick” strategy presented here is heavily dependent on the gate criteria used for selecting positive beads. In this workflow, the main bottleneck is represented by the total number of beads to pick as opposed to the total number of beads in the library screened. While the ClonePix 2 instrument is capable of picking beads on the order of 200 positive beads picked per hour , the gatHMBA-ChemMatrix resin, 1,2-ethanedithiol (EDT), and triisopropylsilane (TIPS) were sourced from MilliporeSigma . N′, N′-dimethylformamide (DMF), methanol, N-methyl-2-pyrrolidone (NMP), and dichloromethane (DCM) were sourced from Fisher Chemical . Fmoc/tBu-amino acids, O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), diisopropylethylamine (DIPEA), piperidine, and trifluoroacetic acid (TFA) were sourced from Chem-Impex International . CloneMatrix semi-solid media was generously donated by Molecular Devices . Lyophilized human polyclonal antibody was obtained from Athens Research . 0.2 µm bottle top filters, N-methylpyrrolidone (NMP), and Grenier Bio-One 6-well plates were obtained from VWR International . Sodium phosphate (monobasic), and Tween 20 3 kDa MWCO Amicon filters were obtained from MilliporeSigma . Sodium chloride, sodium phosphate (dibasic), sodium hydroxide, and hydrochloric acid were obtained from Fisher Chemical . CHO-S null cell culture harvest was provided by the Biomanufacturing Training and Education Center (BTEC) at NC State University. Macrosep Advance 3 kDa MWCO centrifugal filters were obtained from Pall Corporation .The solid-phase peptide libraries, and HWRGWVGSG and GSGSGSGSG control peptides were synthesized on HMBA-ChemMatrix (loading = 0.6 mmol/g) by Fmoc/tBu strategy on a Syro II automated peptide synthesizer (Biotage) as described in detail in prior work ,20. Brieg for 30 min then filtered with a 0.2 µm PES membrane using VWR Full Assembly Bottle-Top vacuum filters, followed by concentration to 2.3 g/L and diafiltration into 50 mM sodium phosphate, 20 mM sodium chloride, pH 8.3 using Macrosep Advance 3 kDa Centrifugal Filters. Human polyclonal IgG was dissolved in 50 mM sodium phosphate, 20 mM sodium chloride, pH 8.3 at 5 g/L. Alexa Fluor 546 and Alexa Fluor 488 were dissolved at 1 mg/100 µL extra dry DMF and immediately combined with 1 mL of the diafiltered harvest for AF546 (HCP-AF546) and 1 mL IgG for the AF488 (IgG-AF488) and incubated on a rotator at room temperature and light protected for 1 hour. Each sample was then diafiltered into PBS, pH 7.4 using Amicon Ultra 3 kDa MWCO filters to remove unreacted dye.The CHO-S cell culture harvest  was clarFollowing equilibration in PBS, pH 7.4, the deprotected library (~100 µL) and control resins (~20 µL) were individually mixed with the labeled proteins in 0.2% Tween 20 in PBS, added to obtain a final concentration of 1.3 mg/mL IgG-AF488 and 0.58 mg/mL HCP-AF546, and incubated overnight at 2–8 °C. Resin beads were then washed with 0.1% Tween 20 in pH 7.4 (PBS-T) and suspended in a semi-solid CloneMatrix solution. The matrix was prepared from two parts Molecular Devices CloneMatrix and three parts 83.3 mM sodium phosphate, 250 mM NaCl, 0.17% Tween 20. Aliquots of 5 to 10 µL of settled library beads were gently incorporated into the matrix solution, and evenly aliquoted on a 6-well plate. The plates were then incubated at 37 °C for 2–18 h to cure the matrix. A ClonePix 2 colony picker  was used for fluorescent imaging and selection of library and control beads. Specifically, the plates were imaged using the ClonePix FITC  and Rhod  laser lines to monitor AF488 and AF546, respectively. Due to slight autofluorescence of the ChemMatrix beads under the FITC filter, bead location  was assigned based on fluorescence intensity from the FITC filter. Beads were picked for further processing based on the following characteristics: (i) FITC interior mean intensity <2500, (ii) Rhodamine interior mean intensity >100, (iii) 0.05–0.25 mm radius. Picking was performed in suspension mode, with 20 µL aspiration volume to pick up the bead, and a 60 µL expel volume (the excess volume above the aspirated liquid was water). Picked beads were further gated at >620 Rhodamine interior mean intensity for subsequent sequencing.Menegatti, Stefano; Lavoie, R. Ashton; di Fazio, Alice; Carbonell, Ruben G. Peptide Ligands for Capture of Host Cell Proteins. U.S. Provisional Patent Application No. 62/784,104, 21 December 2018.


Vespa velutina nigrithorax, a major predator of honeybees, is spreading in Europe in part due to a lack of efficient control methods. In this study, as a first step to identify biological control agents, we characterized viral RNA sequences present in asymptomatic or symptomatic hornets. Among 19 detected viruses, the honey bee virus Deformed wing virus-B was predominant in all the samples, particularly in muscles from the symptomatic hornet, suggesting a putative cause of the deformed wing symptom. Interestingly, two new viruses closely related to Acyrthosiphon pisumvirus and Himetobi Pvirus and viruses typically associated with honey bees, Acute bee paralysis virus and Black queen cell virus, were detected in the brain and muscles, and may correspond to the circulation and possible replication forms of these viruses in the hornet. Aphid lethal paralysis virus, Bee Macula-like virus, and Moku virus, which are known to infect honey bees, were also identified in the gut virus metagenome of hornets. Therefore, our study underlined the urgent need to study the host range of these newly discovered viruses in hornets to determine whether they represent a new threat for honey bees or a hope for the biocontrol of V. velutina.The Asian yellow-legged hornet  Vespa velutina nigrithorax, native to Southeast Asia, ranges from Nepal to South Eastern China. However, in 2004, it was first observed near Agen, France, [The original distribution area of the Asian yellow-legged hornet  France, , and one France, . Its acc France, .Vespidae under temperate climates, the colonies last only one season. Every year, the surviving yellow-legged hornet-mated queens disperse in order to initiate new nests after hibernation. Each colony, initiated by this single individual, develops until the following autumn, first producing several thousand workers, and then hundreds of males and new founders, which mate in autumn [Vespidae in Europe resulted in a rapid colonization of France and neighbouring countries , with substantial population levels and impacts in most of the colonized regions [Since a single individual can found a colony, this outbreak had a high invasive potential, even though, like other social n autumn . Despiten autumn ,5,7, no  regions ,9,10. Mo regions ,15,16,17V. velutina predates on a wide diversity of insects such as hoverflies and houseflies, but with a preference for aggregative species, including social Hymenoptera species such as honey bees and common wasps [V. velutina pressure strongly impacts the colonies [V. velutina establishment and dissemination [V. velutina populations [During nest development, on wasps . Althougcolonies . To datemination ,21. Howeulations ,24,25,26ulations ,28,29, tulations , thus liV. velutina [Deformed wing virus [Israeli acute paralysis virus (IAPV) [Apis mellifera) have also been identified in wild Hymenoptera species [Vespula spp. [Vespa genus, Vespinae. Because V. velutina is an important predator of A. mellifera, we can expect pathogen transfers from target prey to predator. Among the seven viruses commonly found in honey bee colonies, Deformed wing virus (DWV), Black queen cell virus (BQCV) and Sacbrood virus (SBV) have the highest prevalence, as they are detected in up to 90% of western honey bee colonies [Varroa destructor virus 1 (VDV-1) recently renamed DWV-B, and DWV-C [Acute bee paralysis virus (ABPV), Kashmir bee virus (KBV), Israeli acute bee paralysis (IAPV)) have a worldwide distribution. While ABPV is more prevalent in Europe and South America, KBV prevails in North America and South America and IAPV in the Middle East and Australia [Chronic bee paralysis virus (CBPV) is widespread, although not highly prevalent in western honey bees [Vespa velutina, which may also be affected by some specific pathogens.As observed with other wasp introduction events , the lacvelutina ,9,21,23.ng virus , Moku ving virus  and Isras (IAPV)  have bee species  includinula spp. ,37,38, wcolonies ,40,41. Dcolonies ,43,44,45colonies , Varroa nd DWV-C . Acute pustralia . Chronicney bees , but hasney bees . Such wiV. velutina in order to identify specific viruses or strains that could be used as potential biocontrol agents of V. velutina populations. A number of viral microbial control agents have been successfully used against invasive species [In this study, we investigated viral agents of  species  or patho species . Therefoad libitum to foster elimination of previous feeding before dissections and then frozen at 20 °C. One hornet sampled by a beekeeper (in southeast France) exhibited deformed wings  in front of honey bee colonies in two apiaries from the south-east of France during autumn 2014 . Live hogs 159+,  and was gs 159+,  and imme®-ICE . Head, thorax and abdomen of non-symptomatic individuals from the same apiary (140-3 and 144-1) were cut (avoiding thawing) and separately incubated in 1.7 mL RNAlater®-ICE at −80 °C for 18 h, then for 20 h at −20 °C. We then dissected the gut, the abdomen cuticle and fat bodies, and the six legs, which where pooled with the wings and thorax cuticle, the thorax muscles, the mandibles and the brain. Leftover samples of thorax and head were also kept for later analysis. The detailed list of the hornet and honey bee samples can be found in Dissections of two asymptomatic hornets  and the hornet 159+ exhibiting deformed wings were performed on ice and resulted in brain, muscle, gut and mandible samples. The remaining external parts (cuticle and legs) were pooled together. For the deformed wings hornet, only muscle, intestine and external parts could be sampled . For the® Reagent , depending on sample size) with a 0.8 cm-diameter. bead in a 2 ml tube and a TissueLyser (Qiagen) . The tube was then centrifuged for 2 min at 12,000× g at 4 °C, and the supernatant was collected into a new tube to be processed for RNA extraction. RNA was extracted using an RNeasy mini kit  and DNase according to the manufacturer’s instructions. The final suspension volume was 100 µL for whole hornet and 50 µL for the dissected parts. RNA was quantified with a Nanodrop2000®  and RNA quality was assessed with RNA Chips  using an Agilent 2100 Bioanalyzer.All the hornet samples  were ground in 500 to 800 µL TRIzolNext generation RNA-sequencing was performed by MGX-Montpellier GenomiX  on four samples showing the best RNA yield and quality: muscle from the symptomatic specimen (159M), and brain (140C), muscle (140M) and gut (144I) from non-symptomatic specimens. To enrich the samples for polyA and mRNA, ribosomal RNA was first depleted twice with a Ribo-zero kit . The library was prepared with a TruSeq Stranded mRNA Sample Preparation kit (Illumina). Samples were paired-end sequenced (2 × 125 nt) with a Illumina HiSeq 2500 sequencer. E. coli, yeast and phiX) and stored in ng6.toulouse.inra.fr [Short reads were quality checked  were then detected using BLASTx program [A two-step mapping method was used as in previous virus discovery studies in mosquitoes  and wild program  on the nVirus genome reconstruction was performed in three steps. First, for each potential virus found, the nucleotide genomic sequence of the virus species detected by BLAST as the first hit was retrieved from GenBank and used as a viral reference genome for further reconstruction. Paired-end reads from each library were mapped using Bowtie2 V2.3.3  against Pi statistics was estimated using a Popoolation software with the disable correction parameter set to 1 [A more stringent mapping was also performed to calculate the mean coverage and expression level FPKM of the reconstructed genome and to estimate the quantity of polymorphism. For this mapping, paired-reads ends were trimmed (five bases at 5′ ends and 9 bases at 3′ ends) using the fastx-trimmer program from the standalone FASTX-Toolkit 0.0.13 when the median quality showed the lowest values (Q < 38) and when the quality variance was high. The number of bases trimmed was chosen according to Illumina Quality plots generated with fastx_quality_stats and Fastq_quality_boxplot_graph.sh programs implemented in the FASTX-Toolkit . Trimmedset to 1 . To analA Spearman correlation test between polymorphism and expression level was performed using the PAST v3 program , using 0Sequences were aligned with the Muscle program . Site seReverse transcription of either 1 µg RNA from the whole hornet samples or 144 ng to 1 µg RNA from the dissected parts was performed in 20 reaction volume using random priming and according to the manufacturer’s protocol .® G2 Flexi DNA polymerase (Promega) in 25 µl total volume. PCR reactions were performed using an Eppendorf thermocycler. The following program was used: one cycle at 94 °C for 5 min, 35 cycles at 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 45 s, and a final elongation cycle at 72 °C for 10 min. The reactions were then held at 10 °C. Five microliters of PCR products were run in 4% agarose gel, or one microliter was analysed with DNA Chips using an Agilent DNA 1000 reagent (Agilent technologies). The presence of the virus was determined according to the expected sizes of the fragments . Multiplex PCR was performed as previously described ,76 to deFor SANGER sequencing, PCR was carried out with specific virus primers in the same conditions, except for DWV sequencing, which was performed from the (conserved) helicase coding region for partial sequencing as described in . PCR proRNAseq data were used for identifying viruses from hornet tissues sampled from one symptomatic and two asymptomatic hornets  was clearly predominant both in muscle sample 159M (40.3% of the classified contigs) and gut sample 144I (93.2% of the classified contigs) .Firstly, complete DWV-B genomes could be reconstructed from the RNA libraries of individual organs  . In the Phylogenetic analysis from the DWV-B assembled contigs showed that the brain (140C), muscle , and gut (144I) samples clustered together with the DWV-B reference sequence (NC006494-DWV-B) and not with DWV recombinant variants  . In the Secondly, multiplex PCR was used to study the presence of DWV in both hornets and honey bees sampled at the same location. DWV was detected from all the hornets and bee samples except for the dissected parts of sample 140-1, where only gut and muscle were positive .In order to determine whether the DWV strains in hornets and bees sampled together were the same, 11 helicase coding regions were sequenced. This confirmed that all the sequences from this study belonged to DWV species (Genbank Submission 2248150), but no strongly supported clusters could be identified in the phylogenetic tree because helicase is too conserved for fine phylogenetic resolution.Aphid lethal paralysis virus , Moku virus  and a Bee macula-like virus  were found in the hornet gut (144I). Complete genome could not be assembled for these viruses, but 4.2, 6.3 and 3.1 Kb contigs of the corresponding genomes could be assembled  was found in the brain (sample 140C), but not in the muscle of the same hornet individual. A partial 3.5-kb genome could be assembled  and an 8.5-Kb complete genome of Black queen cell virus  were found in the gut of hornet (144I) (A 9.5-Kb complete genome of t (144I) . The taxt (144I) .Vespa velutina associated acypi-like virus were detected in both the brain (140C) and muscle (140M) from the same hornet and in the gut of the 144I individual (Vespa velutina associated acypi-like virus clustered in a single clade (aLRT node statistics = 0.99) and showed similarities to the Acyrthosiphon pisum virus and the Rosy apple aphid virus, both proposed as Acypiviridae (node aLRT statictic = 0.95)  and RNA-dependent RNA polymerase (PF00680), a genomic organization similar to that of APV [Vespa velutina associated acypi-like virus is most certainly a new hornet virus species from the Picornavirales order and belongs to the newly proposed Acypiviridae family.Three complete 9.7–9.9 Kb genomes of dividual a–c. The  = 0.95) a. The Vet of APV   and gut (144I) (Himetobi P virus (HPV)-like sequences did not allow the reconstruction of the whole virus genome, but only four annotated contigs from both muscle (140M) and gut (144I) samples, covering 2.4 kb. RNA-dependent RNA polymerase domains (PF00680) were identified in contig 1 and 3 from samples 140M and 144I, respectively, and phylogeny clearly showed that both Vespa velutina associated triato-like viruses belonged to genus Triatovirus, and are closely related to the Bat dicistrovirus species (node aLRT = 1)  (node aLRT = 0.97) and shares only 50% identity with HPV.t (144I) d–e. CoveLRT = 1) a. Along Vespa velutina associated permutotetra-like virus 1 and 2 are two full-length genomes of 4.7–4.8 Kb (Vespa velutina associated permutotetra-like virus 1 and 2 was similar to that of the Drosophila A virus (unassigned) and the other permutotetra-like viruses [Vespa velutina associated permutotetra-like virus 1 and 2 are two distinct species and are the sister group of the Drosophila A virus   b. Vespa  domains f. Maximu species , a membe species  . These results show that viral sequences found in brain and muscles have different genetic diversity signatures compared to the ones found in intestine, and thus that they did not result from simple cross-contaminations.Viral nucleotide diversity was estimated using Pi polymorphism in the three virus species found in two or more samples, i.e., ke virus . FurtherVespa velutina associated acypi-like virus. DWV-B and Vespa velutina associated triato-like virus were not included in this analysis since conditions on coverage and/or minimum variant frequency thresholds were not met. To further investigate this point, specific patterns of single nucleotide polymorphisms (SNPs) were analyzed in Eight hornet samples were positive for SBV, as determined by RT-PCR . A similAcute bee paralysis virus isolate Hungary 1 (GenBank AF486072.2) isolated from bees (Genbank Submission 2248150). However, they were different from other ABPV sequences obtained from bees in the same experiment (29 informative positions out of 431). From the multiplex PCR method, eleven hornets out of twelve tested positive for ABPV, . HoweverSeven hornet samples were positive for BQCV, as determined by RT-PCR, and this virus was detected not only in the gut, as suggested from RNA-sequencing data in the sample 144I, but also in muscles (hornet 140-1) . For both BQCV and ABPV, similar patterns of BQCV or ABPV presence or absence were found in bee samples at the same locations than hornets . Vespa velutina associated acypi-like virus, DWV-B, and Vespa velutina associated triato-like virus sequences in the brain and muscle samples were not a result of simple cross-contamination that could have occurred during sample processing or library preparation. Instead, this study indicates that viruses typically associated with honey bees were actively infecting several hornet tissues. To date, oral inoculation has been the only known virus transmission route in hornets. The presence of viruses in the brain and muscle tissues combined with intra-species levels of polymorphism (SNP variation) indicate virus multiplication in hornets. The putative DWV circulation in the insect was further emphasized by the identification of DWV in other organs like mandibles or thorax cuticle and legs. Furthermore, DWV-B was shown to be predominant in all the RNA-sequencing samples. DWV-B read counts in the muscle sample were much higher in the hornet exhibiting symptoms of deformed wings (159M) than in the sample that did not show any symptoms (140M). The high DWV-B expression level may be related to the symptoms, when a low level of expression of the virus may correspond to a latent infection, as defined by de Miranda and Genersch [V. velutina would clarify whether V. velutina is an usual host for DWV or if it only became infected from A. mellifera honeybees after its introduction in Europe.Polymorphism and SNP analyses of viral signatures between samples indicated that the detection of the Genersch , especiaGenersch . HoweverGenersch . To our Genersch . FinallyApis species , but also social wasps, with prevalence ranging from 2.2% to 67% prevalence [A. mellifera, A. ceranae [Vespidae may be another host of such viruses. As a follow up, experimental infections will be required to assess the pathogenicity of these honey bee viruses (BQCV and ABPV) in hornets. ABPV and, to a lesser extent, BQCV were both identified in muscles and/or cuticle, legs, and mandibles, as well as in the guts. Similarly, food contamination alone seems unlikely for the presence of these viruses in hornets. BQCV is known to infect many evalence . Convers ceranae , and bum ceranae . Our datVespa velutina associated acypi-like virus sequences, identified in both brain and muscle from 140C and 140M and whose whole genome could be reconstructed, may be an infective virus. In aphids, APV is known to reduce growth, to increase developmental time and possibly related to an increased mortality rate [Vespidae and to bees, such as A. mellifera. If we assume that circulation of viruses in insect body should be related to the infection cycle, then ity rate . FurtherVespa velutina associated triato-like virus genome could not be reconstructed, the identification of sequences of this virus both in muscles and guts suggests that it may infect V. velutina. However, larger samplings are required to confirm its prevalence. Furthermore, the phylogenetic tree showed that these sequences are more similar to the Bat dicistrovirus, which has, to date, only been described twice in the feces of the bat species Pipistrellus pipistrellus [Additionally, even if the whole strellus ,91. Of nMotts Mill virus, Drosophila A virus (DAV-like), the Aphid lethal paralysis virus (ALPV), the Bee Macula-like virus (BeeMLV), and Moku virus. The presence of Moku virus has already been described in an Asian yellow-legged hornet (Vespa velutina nigrithorax) collected in Belgium [In contrast, the presence of viruses exclusively in the hornet gut may result from food contamination (infected preys or contaminated pollen) ,53. The  Belgium , but theVespa velutina. DWV-B was predominant and may cause symptoms. Because DWV variants exhibited various pathogenicity levels, it is of great importance to assess their virulence for this new host species and to study their evolution over time, as recombination was already shown among variants [V. velutina have to be investigated to better assess the impact they may have on European honeybee and hornet populations. Moreover, new viruses were described, and the Vespa velutina associated acypi-like and Vespa velutina associated triato-like viruses were, for the first time identified in Vespidae species. In sum, there is an urgent need to study their host range and evaluate whether they represent a new threat to honeybees or a potential solution for the biocontrol of V. velutina in Europe, where they are dramatically spreading.Our results from the French samples showed the presence of a large diversity of viruses in the invasive hornet variants . Other v


Bunyavirales, Picornavirales, Articulavirales, and Tymovirales, and to the realm Ribovira. Hence, through this study, we expand the knowledge of the virome composition of different mosquito species in Sweden. In addition, by providing viral reference genomes from wider geographic regions and different mosquito species, future in silico recognition and assembly of viral genomes in metagenomic datasets will be facilitated. Metagenomic studies of mosquitoes have revealed that their virome is far more diverse and includes many more viruses than just the pathogenic arboviruses vectored by mosquitoes. In this study, the virome of 953 female mosquitoes collected in the summer of 2017, representing six mosquito species from two geographic locations in Mid-Eastern Sweden, were characterized. In addition, the near-complete genome of nine RNA viruses were characterized and phylogenetically analysed. These viruses showed association to the viral orders  Peribunyaviridae, Flaviviridae, Reoviridae, and Togaviridae and are thought to be ancestral to arboviruses. With this in mind, ISVs could be a potential source of new arboviruses if they acquire dual-host tropism [Mosquitoes are known to host and transmit numerous arthropod-borne viruses (arboviruses) that cause disease both in humans and animals, such as West Nile virus (WNV) , Zika vi tropism ,17,18. TCulex (Cx.) pipiens and Cx. Torrentium, are already present in the country [The majority of the metagenomic surveys of mosquitoes have been conducted in countries where arboviruses of human and animal health concern circulate, such as China, Australia, Mozambique, and the US ,6,19,20. country ,25.Bunyavirales, Picornavirales, Articulavirales, and Tymovirales, and the realm Ribovira. In this study, we aimed to provide additional knowledge of viruses circulating in Northern Europe, as well as investigate the virome composition of different mosquito species in this region by using a viral metagenomic approach. In total, the virome composition of six different mosquito species collected in two locations in Mid-Eastern Sweden were investigated. In addition, we have further genetically characterised nine near-complete viral genomes associated with the orders ® was placed close to a bird breeding ground from Monday to Friday every second week, from July to the end of August. The Mosquito Magnet® was emptied every 24 h. The mosquitoes were euthanized and stored at −80 °C after collection. Female mosquitoes were determined to species based on morphological character, using a taxonomic key [Culex (Cx) torrentium and Cx. pipiens were identified to species using a molecular method previously described [Coquillettidia (Cq) richardii, Aedes (Ae) communis, Ae. annulipes, Ae. cantans and all Cx. pipiens and Cx. torrentium) were used in further analyses.Mosquitoes were collected in the summer of 2017 at two different geographic locations in Mid-Eastern Sweden, close to the small town Flen (coordinate: 59°04′08.6″ N 16°31′14.1″ E) and in the nature reserve Hammarskog (coordinate: 59°46′31.9″ N 17°35′01.3″ E) close to the city of Uppsala. In the location close to Flen, mosquitoes were collected once every second week from May to the end of August, using dry-ice-baited Centers for Disease Control and Prevention (CDC) miniature light traps and CDC gravid traps . In Hammarskog, a Mosquito Magnetomic key , stereomescribed . Eight t® evolution  at two cycles of 5500 RPM for 30 s with a 40 s pause between each cycle. In addition, to keep the temperature at 10 °C a Cryolys  cooled with liquid nitrogen was coupled to the Precellys® evolution. The homogenates were centrifuged at 8000× g for 5 min at 4 °C and the supernatant was collected for future analysis. Total RNA was extracted with TRIzol™ according to the manufacture’s protocol. The aqueous phase obtained after the addition of chloroform and the subsequent centrifugation step was collected and diluted 1:1 with freshly prepared 70% ethanol and purified with GeneJet spin columns. The purified RNA was further pooled with RNA from the same mosquito species, location, and time point, resulting in a total of 12 pools , following the manufacture’s instruction. The amplified RNA was submitted to SciLifeLab for library preparation and sequencing using the Ion 530™ Chip kit . The 12 pools were dived on four Ion 530™ chips and sequencing was carried out with an Ion S5 XL sequencing system running the protocol for 400 bp-long reads.Prior to homogenisation, all mosquitoes were washed once in 70% ethanol and then twice in milliQ water to remove potential surface microorganisms. The mosquitoes in each pool were then transferred to Precellys tubes (soft tissue homogenization CK14) and 700 μL PBS supplemented with Amphoterecin (250 μg/mL) and Penicillin-Streptomycin (100 U penicillin/mL + 100 μg streptomycin/mL) was added. Homogenisation was performed mechanically with a Precellys12 pools . These phttps://github.com/bbuchfink/diamond/blob/master/diamond_manual.pdf). For the de-novo assembled contigs, an additional Diamond run was performed to output a DAA (Direct Access Archive) format that could be imported into MEGAN6 (version 6.15.2) for further investigation. Potential viral sequences were analysed with NCBI’s ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/), functional annotation of the ORFs was performed using NCBI’s BLASTp, EMBL-EBI’s protein sequence analysis and classification tool InterProScan [The Ion S5 XL sequence data were analysed in a metagenomics pipeline starting with quality control using FastQC (v1.2.1). Reads of low quality were trimmed with sickle (version 1.210) using a cut-off PHRED score of 20. The mean reads length after trimming was 359–380 nt. The good quality reads were de-novo assembled using MEGAHIT (v1.1.2). Both the good quality reads and the de-novo assembled contigs were taxonomically assigned in separate runs using Diamond version 0.9.10 with the blastx option using the default settings  and the NCBI database from October 2018. In order to visualize the results with the R-package Pavian (v0.8.4), the output format taxonomy (102) was used in Diamond. This option performs a LCA (Last Common Ancestor) algorithm to assign the taxonomy of each read and output a tabulated file . PCR reactions were carried out with KAPA2G Robust Hotstart ReadyMix . PCR products were analysed with electrophoresis and bands of correct size were cut out and extracted with a GenJet gel extraction kit . Purified PCR products were sent to Macrogen for Sanger sequencing and the returning sequences were analysed in DNASTAR, SeqMan Pro (Version 2.1.0.97).We further investigated the Hammarskog picorna-like virus (HPLV) with PCR and Sanger sequencing. Viral hits were inspected and retrieved in Megan, overlapping contigs that mapped to the same virus were manually assembled into longer sequences. The extended sequences were subsequently used to design primers pairs to fill gaps and cover as much as possible of the genome using the primer3 software. The original RNA extractions of interest were reverse transcribed to cDNA using SuperScriptThe raw sequence reads generated in this study are available at the NCBI sequence read archive (SRA) database under Bioproject accession (PRJNA574583). All virus genome sequences retrieved in this study have been deposited in GenBank under the accession numbers: MN513369–MN513382.Cq. richardii (433), Ae. cantans (407), Ae. communis (106) and Ae. annulipes (72). The less abundant mosquito species out of those collected were Ae. vexans (45), Ae. intrudens (44), Ae. punctor (29), Cx. pipiens (22) and Cx. torrentium (4)  . Iflaviridae, Nodaviridae, Orthomyxoviridae, Partitviridae, Peribunyaviridae, Phasmaviridae, Reoviridae, Rhabdoviridae, Solemoviridae, and Tombusviridae ), Ae. communis (0.112% TR) and Ae. annulipes mosquitoes (0.721% TR)  (0.016–2.844% TR), Xincheng anphevirus (0.004–0.142% TR), Anopheles darlingi virus (0.001–0.381% TR), and Wuhan mosquito orthophasmavirus 2 (0.003–0.215% TR)  compared to the Cx. pipiens (0.003% TR). Many of the viral hits were restricted to one mosquito species, such as similarity hits to the Hubei virga-like virus 21, which was only observed in the Cx. pipiens mosquitoes and in those, represented more than half of the viral reads in that pool (4.916% TR) , Hubei diptera virus 13 (0.022–0.074% TR), Hubei sobemo-like virus 8 and 9  and the Hubei tetragnatha maxillosa virus 8 (0.015–0.48% TR)  and lower abundance in those collected in Flen (0.002% TR)  and the Whidbey virus (0.007–0.102% TR) and two negative-sense RNA viruses, Kinkell virus (0.087–0.109% TR) and the unclassified virus Shuangao insect virus 12 (0.024–0.166% TR). Comparing the virus composition and abundance between the species and genera in this dataset, one can observe some differences. The Aedes genus seems to harbour a more diverse negative-sense RNA virome compared to the Culex and Coquillettidia genera. The Culex mosquitoes had few but very abundant viruses, one that represented more than half of the total viral reads in the Cx. pipiens pool. Many viral hits could only be observed in one of the mosquito species, indicating a host restriction. No significant differences could be observed comparing the locations or time point of collection in the Cq. richardii pools and Ae. cantans pool  . In term215% TR) . However916% TR) . Moreove.48% TR) . Viral r002% TR) . The Ae.ans pool . Iflaviridae, Nodaviridae, Tombusviridae, Solemoviridae, and Picornavirales and represented between 0.03–47% of the viral reads in the mosquito pools. Four of the positive-sense RNA viruses detected were further characterized and phylogenetically analysed and fell within the viral orders Picornavirales and Tymovirales.The positive-sense RNA viruses observed in our mosquitoes were within the viral familes/orders Cq. richardii mosquitoes collected in Uppsala showed many contigs that mapped to the Yongsan picorna-like virus 3 (YPLV3) (NC_040584.1). Using these contigs, combined with PCR and Sanger sequencing, the near-complete genome of a picorna-like virus, that we named “Hammarskog picorna-like virus” (HPLV)  A, was reof Korea  and has of Korea . HoweverAe. cantans collected late in the season in Flen and one genome was found in the Cq. richardii mosquitoes collected early in the season in Uppsala. The tombus-like viral contig retrieved from Ae. cantans mosquitoes was 4520 nt long and the virus was named Flen tombus-like virus (FTLV), while the tombus-like viral contig retrieved from the Cq. richardii mosquitoes was 4166 nt long and this virus was named Hammarskog tombus-like virus (HTLV)  (NC_040725) were identified. One of the genomes was found in the s (HTLV) B. Sequens (HTLV) . The InterProScan analysis of each ORF’s aa sequences revealed no predicted function for the first ORF, the second ORF showed a detailed signature match of a RdRp, hepatitis C virus (IPR002166) and the third ORF resulted in a detailed signature match of the Nodavirus capsid (IPR024292). Phylogenetic analysis of the two predicted RdRp sequences of the second ORF showed that our sequences cluster to the YTLV1 (YP_009553261) and the Wenzhou tombus-like virus 11 (YP_009342051). Based on this, we classified our near-complete viruses as tombus-like B.Ae. cantans mosquitoes, collected early and late in the summer, had many viral hits corresponding to the unclassified RNA virus Wuhan fly virus 4 (WFV4)  . From onavirales . The phyllection C.Orthomyxoviridae, Peribunyaviridae, Phasmaviridae, Mononegavirales, Articulavirales, and Bunyavirales and represented between 0.1–39% of the viral reads in the mosquitoes pools. Two of the negative-sense RNA viruses detected were further characterized and phylogenetically analysed and fell within the viral orders Bunyavirales and Articulavirales.The negative-sense RNA viruses observed in our mosquitoes belonged to the viral families/orders Cx. torrentium mosquitoes , respectively [Quaranjaviruses Wuhan mosquito virus 4 and 6 (KX883866 and MF176380). Based on the characterization of each segment and the phylogenetic analysis, our partial genome was classified as orthomyxo-like and named the Culex orthomyxo-like virus after the mosquito genera it was found in  was detected in the squitoes A. WMV4 iectively ,34. The found in A. Ae. cantans mosquitoes collected early in the summer in Flen, we identified near-full-length genome segments with high similarity to the Yongsan bunyavirus 1 (YBV1). The YBV1 is an unclassified bunyavirus with a linear single-stranded, negative-sense genome divided into three segments [In the segments . The segCx. torrentium mosquitoes , Mononegavirales mRNA-capping domain V (IPR026890), and mononegavirus L protein 2-O-ribose methyltransferase (IPR025786). Phylogenetic analysis of the 2074 aa sequence containing the RdRp gene showed the closest association to the CMLV2 (ASA47413), which is an unclassified virus. Two more distant associations were to the unclassified Anphevirus, Anopheles marajoara virus (QBK47216) and the drosophilid anphevirus, Drosophila unispina virus 1 (YP_009666282). As our near-complete genome showed such close genetic identity to other strains of CMLV2 (96% nt identity) [A large proportion of all viral reads were labelled as unclassified RNA viruses A. By invsquitoes A. BLASTndentity) , we consCq. richardii mosquitoes had viral reads mapping to the unclassified RNA virus Hubei noda-like virus 12 (HNLV 12). However, most of the reads were found in the pool of Cq. richardii mosquitoes collected late in the season in Uppsala  (KX883775.1). The HVLV 21 has a linear RNA genome of 10,072 nt, with four ORFs coding for one larger hypothetical polyprotein and three smaller hypothetical proteins [In the  21) KX8875.1. TheCulex mosquitoes and viral hits to the Yongsan bunyavirus 1 in the Aedes mosquitoes. Further, many viral hits could only be observed in one of the mosquito species, indicating a host restriction. One example is that only the Coquillettidia mosquitoes had viral reads that mapped to the YPLV3 ssRNA viruses in the orders Bunyavirales and Articulavirales and three viruses could not be classified to an order and have instead been classified in the realm Riboviria, which is the basal rank for RNA viruses [The near-complete genomes of nine RNA viruses were characterized and phylogenetically analysed in greater detail. Four of these viruses were associated to (+)ssRNA viruses in the orders  viruses . Phyloge viruses , that in viruses ,39. Furt viruses . The majTombusviridae virus YTLV1 (NC_040725), with 67.63% and 75.17% nt identity. The family Tombusviridae is currently associated with plant viruses [Two of the nine characterized viruses in this study showed association to a viral family related to plant viruses. The FTLV and HTLV were closely associated to the TLV1 NC_0725, withThis study expands the knowledge of viruses circulating in different mosquito species in Northern Europe. It provides information on the diversity of the viromes of the respective mosquito species investigated, as well as shows the presence of a number of viruses not known to circulate in this region and that are highly divergent to previously characterized viruses. Whether any of these viruses have any direct or indirect potential veterinary or public health-relevance remains to be further investigated.


Staphylococcus aureus (MRSA) in Dutch long-term care facilities (LTCF).Highly resistant microorganisms (HRMOs) are of high concern worldwide and are becoming increasingly less susceptible for antibiotics. To study the cost effectiveness of infection prevention measures in long-term care, it is essential to first fully understand the impact of HRMOs. The objective of this study is to identify the costs associated with outbreaks caused by Methicillin-resistant After an outbreak of MRSA, Dutch LTCF can submit a reimbursement form to the Dutch Healthcare Authority  to get a part of the total costs reimbursed. In this study, we requested NZa forms for financial impact analysis. Details regarding the costs of the outbreak have been extracted from these forms and additionally specific LTCF have been visited in person to validate the data.34 complete reimbursement forms from the period between 2011 and 2016 were received from the NZa and have been included. The median cost per patient per day was estimated at €83.80, varying between €16.89 and €1,820.09. We validated five reimbursement forms by visiting the facility and recalculating the costs. We found a non-significant positive difference of €26.07 compared with the original data (p = 0.068).This study is to our knowledge the first to give a national overview of total costs associated with an MRSA outbreak in LTCF in the Netherlands. Overall, costs per patient per day seem lower than in a hospital setting, although total costs are much higher due to the long term of care. Due to the use of antibiotics, treatment options for infections caused by microorganisms are decreasing. Microorganisms that once were susceptible, have now become resistant to most of the commonly used antibiotics . Highly Difficult to treat infections occurring in LTCF that are caused by HRMOs pose a big problem, not just for LTCF, but for hospitals as well. LTCF, even those that are not connected to a hospital by direct patient transfers, play an important role in the transmission of HRMOs in a specific area and can thus be key in regional and hospital infection control strategies ,10.Staphylococcus aureus (MRSA) and other factors related to the structure of the healthcare system, the rates of MRSA and other HRMOs in the Netherlands are relatively low, compared to other countries in Europe and beyond ). Through this procedure, a lot of data is available on these outbreaks in the standardized forms used for this purpose. This study aims to analyze these data on the costs of MRSA outbreaks with the explicit objective to identify the costs associated with multiple outbreaks caused by MRSA in Dutch LTCF.On behalf of the government, the NZa facilitates reimbursements for MRSA outbreaks in LTCF. For this purpose, a form is filed by the facility to the NZA comprising an inventory of initiated actions and costs. Data from all reimbursements between January 2011 and December 2016 were requested and kindly provided by the NZa. Only the forms that were a 100% complete have been included. The data from these reimbursement forms comprise the national data used in this study. There are fixed categories for costs and, since every outbreak is unique, sometimes not all costs can be accommodated in the forms. Also, facilities interpret the form in their own way. As it is therefore not exactly known how these reimbursement forms compare to reality, a subset of five facilities from the national data was included in an on-site audit to validate the reimbursement forms.This research has been carried out according to the Dutch guideline for conducting economic evaluations in healthcare where applicable ,22. All The healthcare authority compensates a facility for the costs made by an outbreak with MRSA. These costs can include several posts: medical microbiology, antimicrobial resources, personnel- and material, personal protective equipment, cleaning and costs regarding the closure of a facility or certain departments of a facility. The reference price for closure varies per year and is set by the NZa. All other costs are filled out on a reimbursement form by the facility itself and are based on the facilities actual expenses. The reimbursement forms are checked by an external committee, which advises the healthcare authority on acceptability before reimbursements are made. 1% of the accepted costs, to a maximum of €25,000, must be paid by the facility itself.All facilities included in the national dataset were contacted and asked if they would participate with an on-site visit to establish the real costs of an outbreak regardless of the data submitted in the reimbursement forms.All identifiable extra costs made by the LTCF caused by the outbreak were included and analyzed from the LTCF perspective. This contrasts with the reimbursement forms submitted at the NZa, where facilities can only fill in certain costs that fit into the pre-specified categories. Therefore, the on-site audits potentially include costs that are not mentioned in the reimbursement form. The start of an outbreak was defined as the day at which several carriers with MRSA were recognized. The end of the outbreak was the day at which no further measures were in place to control the outbreak. Every included outbreak has been checked by the NZa and has been confirmed as an outbreak. Costs incurred by the LTCF due to an outbreak have been included till one year after the end date.Costs were divided into separate categories, namely: microbiological diagnostics, personnel, materials, cleaning and disinfection, missed income due to closed beds and other costs. Patient isolation was not included as a specific category. Costs for isolation, such as specific materials, fit into the previously mentioned categories.All relevant documents and financial information from the facilities were gathered, to analyze what extra resources were used to control and eliminate the outbreak. Different sources for data were used to make sure no costs were overlooked. The additional amount of cultures and other microbiological diagnostics were acquired in corporation with the local laboratory. Interviews with managers, nurses and other relevant personnel took place to calculate personnel costs. Also, opportunity costs for personnel were included. Additional costs for materials were based on interviews and documentation, including personal protective equipment for personnel and prescribing of extra medication and antimicrobial agents. The extra costs made for cleaning were based on documentation and invoices. Missed income was calculated per empty bed per day in comparison with normal occupation. The total number of empty bed days was multiplied with the reference price (€168) set by the Dutch guideline for conducting economic evaluations in healthcare and is corrected for inflation ,22. If tFor the financial analysis, the total costs for all categories of the different outbreaks were calculated. If the data was not normally distributed, the median and range were used.The national data and the data from the on-site audit were compared with a Wilcoxon Signed Rank Sum Test. A sensitivity analysis was performed to see what the total costs would be if the reference price for missed income would be 25% lower or higher. Calculations were performed with Microsoft Excel  and SPSS . A significance level of p <0.05 was applied to test statistical significance.In total, 46 reimbursement forms from the period between 2011 and 2016 were received from the NZa. Of these, 34 forms were complete and were analyzed. Since the data was not normally distributed, medians have been calculated. The total costs per patient per day from the national data have been compared with the data from the on-site audit. The four marked outbreaks that are included in the on-site audit and in the national data were not normally distributed and have therefore been compared with a Wilcoxon Signed Ranked Sum test. There was no significant difference between the total costs per patient per day from the four selected outbreaks in the national data (median €81.89) and the data from the on-site audit (median €109.87), however, there was a trend towards significance .A sensitivity analysis with a 25% margin of the reference price was performed . The senLiterature on the costs of outbreaks with MRSA in long-term care is scarce. This research provides an overview of such costs in the Netherlands. The goal is to give decision-makers in LTCF evidence for better choice in the implementation of infection prevention and control measures. Total costs were divided into the separate categories of diagnostics, personnel, cleaning and disinfection, material and closed beds.The costs found lie between €16.89 and €1,820.09 with a median of €83.80 per patient per day. Significant cost drivers are personnel, diagnostics and closed beds. Compared to outbreaks in the hospital setting, the costs per patient per day seem lower in LTCF. An on-site audit showed that the reimbursement forms might provide an underestimation of the costs and total costs might be even higher than presented here.Further research is needed to investigate whether the costs of implementation of infection prevention measures outweigh the potential costs associated with an outbreak. With the current system, LTCF get the costs of an outbreak reimbursed, except for 1% of the acceptable costs with a maximum of €25,000. It is questionable if this refunding is enough to create a financial incentive for LTCF to improve their infection prevention measures. A prevention-driven incentive could be to reward facilities as long as no outbreak occurs, or to reimburse resources spend on infection prevention measures.Controlling outbreaks requires a coordinated set of actions and stakeholders working together on diagnostics, infection prevention and antibiotic therapy. Nowadays, antimicrobial stewardship is hailed as one of the most important solutions of resistance. We argue that diagnostic stewardship and infection prevention measures are equally important, especially in LTCF where antibiotic therapy is more rare ,25. OnlyWe included 34 outbreaks in the Dutch national data in this study, that were subsequently analyzed. The completeness of the costs of these outbreaks has been validated by analyzing five outbreaks in an on-site audit. There was no significant difference found between the total costs based on the national data and the on-site audit, although there is a trend towards significance that shows that the reimbursed costs are lower than the actual costs (p = 0.068). However, this trend is probably caused by the outbreak included in the on-site audit that was not yet finished and was not included in the national data.There is not much known about the costs of outbreaks in healthcare facilities, especially in long-term care. Dik et al. analyzed seven outbreaks in the hospital setting. One of these outbreaks was an outbreak with MRSA. They found a total cost per patient per day of €657.08 (2015 price level). This is higher than the median of the costs we found. However, the duration of the outbreak was only 16 days. At the beginning of an outbreak, a lot of costs are made. After a certain period, only the fixed costs will remain. This causes the total costs per patient per day to drop, but the total costs of the outbreak to keep rising. Therefore, it is not possible to compare outbreaks that occurred in a hospital with outbreaks that occurred in LTCF using total costs per patient per day. The total costs estimated by Dik et al amounted to €31,539.84. Compared with this result, the total costs in the LTCF analyzed here are much higher at €111,568.71 .Staphylococcus aureus (MSSA) in a long-term care facility. The costs for MRSA were 1.95 times greater than the costs for MSSA at a median of $2,607  versus MSSA at a median of $1,332 . Nursing care with more than 2 times higher costs in the MRSA group and infection management with more than 6 times higher costs in the MRSA group were the largest and second largest components of the total costs of the infection, respectively [In 2003, Capitano et al. published an article comparing the costs per patient between MRSA and Methicillin-susceptible ectively . If we cectively ,27,28. OThe duration of outbreaks with MRSA differs between hospitals and nursing homes. Dik et al. describes an outbreak with MRSA in a hospital with a duration of 16 days and van der Bij et al. found an average duration of 40 days ,29. An oStrength of this study is the fact that multiple outbreaks with MRSA nationwide have been evaluated. All LTCF used the same forms to file the costs of the outbreak at the NZa and therefore these forms give a good overview of the costs associated with an outbreak with MRSA nationwide. As far as we know this is the first study to calculate a median and range for the costs of MRSA in LTCF. Limitations are the small sample size of the on-site audit, which could have influence on the comparison with the national data. With a sample size of five in the on-site audit group, it was only possible to compare these five outbreaks with the reimbursement forms from the same outbreaks in the national data. The comparison showed that there is a trend towards a significant difference (p = 0.068). Overall, it looks like the outbreaks included in the on-site audit last longer, include more positive patients and are more expensive compared to the outbreaks included in the national data. The levels of Charlsson comorbidity index from the LTCF have not been compared. It is possible that there is a difference, however the impact on the costs of an outbreak will be small since this study looks at carriers and not at infections. There are several risk factors for the spread of microorganisms, like the use of antibiotics, but these are not included in the Charlsson comorbidity index. Furthermore, there is a potential bias regarding the on-site audit. Facilities were asked to join the audit and were not randomly selected. It is plausible that these facilities had their records in order and knowingly aimed for a higher outcome, or vice versa.In principle, all facilities follow the national infection prevention protocols for management of MRSA in LTCF –33. Most