Guillaume Gouget

Patricia Beaunier

David Portehault

Clément Sanchez

New route toward nanosized crystalline metal borides with tuneable stoichiometry and variable morphologies †

published or not. The documents may come

New route toward nanosized crystalline metal borides with tuneable stoichiometry and variable morphologies

Introduction

The family of metal-boron alloys spans a wide range of compounds, including various stoichiometries. This diversity results from the high density of covalent bonds in metal borides structures. By increasing the boron content (Fig. 1), the structure evolves from linear B-B chains, to two-and three-dimensional boron frameworks including interconnected boron clusters 1 in which B-B lengths are shorter than in onedimensional chains. This structural observation is commonly related to the mechanical properties of metal borides, such as super-hardness in WB 4 2 or high temperature refractory behavior in metallic ceramics as HfB 2 . 3 Covalence also results in high melting temperatures of borides (2600 °C for YB 6 ) and pure boron allotropes (2075 °C for β-B). [START_REF] Lide | CRC Handbook of Chemistry and Physics[END_REF] Noteworthy, the metal-boron interaction also brings specific behaviors. For instance, in OsB, metal atoms bring high electron concentration, so that electrostatic repulsions prevent compression, thus yielding ultra-incompressibility. 2 More generally, boron-poor Fig. 1 Crystal structures of NiB (space group Amam), YB 4 (P4/mbm) and YB 6 (Pm-3m).

(elemental ratio M:B ≥ 1:1) borides of transition metals are drawing attention in a range of fields from magnetism [START_REF] Rades | [END_REF] to catalysis. [6][7][8][9] In this latter case, B-poor phases such as NiB and CoB are potential low price catalysts for dehydrogenation and Fisher-Tropsch reactions. 10 In these phases, boron may prevent coking and deactivation of the active sites by sequestering carbon atoms. 11,12 On the other side, boron-rich borides are actively studied and used, such as hexaborides for field emission 13 and thermoelectric energy conversion. 14 Richer borides comprising icosahedral B 12 boron clusters, for instance YB 66 , are also strongly investigated for thermoelectricy. 15 Thus, original properties of metal borides are deeply related to both elements and the way they interact. This is particularly exemplified by MgB 2 superconductivity 16 : its remarkably high critical temperature of 39 K for classical conductors was not expected, given the apparent simplicity of the composition 17 compared to usual cuprate superconductors. The superconductivity of MgB 2 is related to both elements being light, as suggested in the BCS theory, 18 together with anisotropic boron organisation in metallic honeycomb layers 19 stabilized by electron transfer from magnesium to boron. All in all, metal borides exhibit a large diversity of technologically relevant mechanical and transport behaviours (Fig. 2) that originate from the complex interplay between the covalent boron framework and the metal atoms. Interestingly, all the above mentioned properties are related to bulk phases. The example of other materials families, including gold [20][21][22] and chalcogenides, 23 shows how properties, e.g. in catalysis and optics, can be impacted when nanoparticles are scrutinized. However, such studies are still extremely scant for crystalline metal borides. [START_REF] Rades | [END_REF]24 Such scarcity originates from the harduous synthesis of crystalline metal borides, which has been discussed recently. 24 Only few phases are known at the nanoscale, especially rare earth and alkaline earth hexaborides, produced by chemical vapor deposition (CVD) 25,26 , solid state reactions, often under autogeneous pressure. [27][28][29] Some transition metal borides (VB 2 30 , FeB 5 , Co 2 B 31 ) have been reported at the nanoscale from solid state chemistry, while MgB 2 has been obtained via a range of methods involving the reactivity of gaseous magnesium. [32][33][34] In most cases the synthetic processes cannot be generalized to a wide range of metal borides, does not enable size control (except CVD), and particle sizes are rarely below 30 nm, and often above one micrometer. Besides, the control of the final stoichiometry for nanoscaled crystalline metal borides is currently uncharted. Fig. 2 Attractive properties of metal borides, known for bulk phases. The periodic table of elements is restricted to boron and metals. Recently, a route was proposed toward a wide range of metal boride nanocrystals, 35 based on the reaction between metal salts and an alkali borohydride below 900 °C, at atmospheric pressure in an inorganic molten salt mixture, composed of LiCl/KCl at the eutectic composition (T f = 353 °C). This onepot process enables production of nanocrystalline metal borides at temperatures between 400 °C and 900 °C, with a readily available, thermally stable and no oxygen-containing solvent, easily eliminated after reaction by using water. In a typical synthesis schematized Fig. 3, the solid precursors and salts are heated together over the melting temperature of the reactant medium in argon atmosphere. After cooling down to room temperature, a black block is obtained. It contains salts that are dissolved afterwards with a polar solvent such as deionized water. Washing and drying the remaining black suspension lead to a black powder consisting of metal boride nanoparticles. The particle size could be controlled and decreased below 20 nm. However, each metal studied was associated to a precise M:B ratio, so that stoichiometry control for a given metal was not provided. Besides, all reported nanoparticles were round shaped. In this article we present the first evidence of a general synthesis of nanocrystalline metal borides with controlled stoichiometry using syntheses in inorganic molten salts, by focusing on the B-poor Ni-B and Brich Y-B systems. Depending on the ratio between metal and boron precursors, the temperature and reaction time, NiB, Ni 4 B 3 , Ni 2 B, Ni 3 B, YB 6 and YB 4 can be selectively fabricated (Fig. 3). Furthermore, we highlight the occurrence of a peculiar anisotropic uniaxial defective crystal structure within spherical particles that originate from crystallization in confined space. Finally, we report for the first time the ability to reach anisotropic nanoparticles, as a first step toward morphological control of crystalline metal boride nanostructures.

Results

Briefly, all syntheses were performed under inert argon atmosphere. Prior to heating, metal chlorides and sodium borohydride were mixed under argon with the powder mixture of LiCl and KCl at the eutectic composition (45/55 wt.). After reaction and cooling, the frozen salt matrix was washed with deionized water, the remaining black powders were dried under vacuum and stored in air.

Nickel borides

Starting from NiCl 2 as metal precursor, the reaction medium was heated at 750 °C during 1.5 h. Different NiCl 2 :NaBH 4 (Ni:B) molar ratios were investigated. The corresponding X-Ray diffraction (XRD) patterns (Fig. 4) show that for Ni:B = 1:2, NiB is the major product, with broad reflections suggesting nanosized crystallites. The apparent crystallite size calculated according to the Scherrer formula is 9±2 nm. Minor peaks of Ni 4 B 3 are also observed. For Ni:B = 1:1. Transmission electron microscopy (TEM) of the NiB (NiCl 2 :NaBH 4 = 1:2) sample (Fig. S1) reveals spherical crystalline nanoparticles of 2 to 40 nm, in agreement with the crystallite size, dispersed within an amorphous matrix of 1 to 2 nm thickness. The smaller the particles, the more they tend to perfect spheres. Particles bigger than 20 nm are slightly faceted. High resolution TEM (HRTEM) (Fig. 5) highlights lattice fringes in a spherical NiB nanoparticle. Many defects are observed and demonstrate the polycrystalline nature of each particle. Noteworthy single crystalline domains appear as parallel stripes within the single isotropic particles. When comparing two domains aside, the orientation of B-B bonds changes. For instance (Fig. 5c ande) the two domains with [0-12] and [-1-12] zone axes differ from each other by rotating the structure 17.2 ° along the [021]* direction. Note that such a defect does not yield coherent domains, as the zig-zag chains are discontinued at the interface (Fig. 5d). Each single domain can be indexed according to the NiB structure for all particles with diameter below 20 nm (Fig. S2). A minority of bigger faceted particles are indexed as Ni 4 B 3 (Fig. S3). Brunauer-Emmett-Teller analysis of the N 2 sorption isotherms provides a specific surface area value of 57 m 2 •g -1 for the NiB sample (NiCl 2 :NaBH 4 = 1:2), almost three times higher than values reported for analog amorphous 6 or crystalline 36 NiB nanoparticles (20 m 2 •g -1 ). Fig. S4 displays TEM and HRTEM pictures of the samples obtained for higher proportions of sodium borohydride. In agreement with XRD results, 50-200 nm crystals of Ni 4 B 3 and Ni 2 B embedded in an amorphous layer (1 to 2 nm thick) are observed for initial NiCl 2 :NaBH 4 ratios 1:1.2 and 1:1. The sample with a NiCl 2 :NaBH 4 ratio of 1:0.65 is made of particles bigger than 100 nm (Fig. S5).

SEM-EDX analyses show that all samples are composed of boron, nickel, and oxygen. For NiCl 2 :NaBH 4 = 1:2, quantitative analysis provides an elemental composition B 58 Ni 31 O 11 . We showed previously that the amorphous matrix embedding the particles is partially oxidized boron. 35 Consequently, by considering that the crystalline phase is in the vast majority NiB, then the material composition is equivalent to (NiB) 31 (B 27 O 11 ), reflecting the biphasic crystalline-NiB/amorphous-B (B am ) character of the sample. 54 % of the B atoms are involved in the crystalline phase, in accordance with the initial excess of boron introduced. The O/B content within the amorphous phase is O/B am = 0.4, which suggests that the amorphous layer is composed of partially oxidized amorphous boron, in agreement with previous reports. Unfortunately, the boron content could not be quantitatively evaluated by SEM-EDX in the samples from initial NiCl 2 :NaBH 4 ratios 1:1.2, 1:1 and 1:0.65 as the B content was too low to unambiguously deconvolute the EDX peaks of B and C Kα edges (Fig. S6).

Yttrium borides

By using YCl 3 as metal precursor, yttrium borides can be targeted through synthesis in molten salts. In order to reach different boron-rich compounds, extensive screening of experimental parameters was performed, by focusing on the yttrium-to-boron precursor ratio YCl 3 :NaBH 4 , as in the Ni-B system, but also on the temperature and the dwell time. Below, the different samples are identified as "YCl 3 :NaBH 4 -Tt", where T and t are respectively the reaction temperature (°C) and duration (h). By screening these conditions, speciation diagrams can be mapped (Fig. 6), where relative amounts of crystalline phases are qualitatively evaluated from the relative intensities of the corresponding phases in the XRD patterns. Overall and as expected, YB 6 is favored at high proportions of NaBH 4 , but also at high T and t. The XRD diagrams of three typical powders are displayed Fig. 7. The sample 1:12-1000-4 exhibits the cubic structure of YB 6 as the only crystalline phase. For 1:8-900-4, the reflections are attributed to YB 4 and YB 6 . For 1:5-800-1, two crystalline phases are observed: YB 4 is the only metal boride phase, while the yttrium borate Y(BO 3 ) is also observed. More generally, at low temperature and short time corresponding to a low energy input, oxidized phases (yttrium borate or oxide Y 2 O 3 ) are obtained. As the reaction media do not contain any source of oxygen, then these byproducts originate from poorly stable yttrium species that react with water during the washing step. Efforts to selectively dissolve this oxidized phase, using acidic or reductive conditions, were inefficient. Apparent crystallite sizes ε hkl were calculated for the different crystalline phases (Table 1). Smaller YB 6 particles are synthetized at 1000 °C (ε hkl = 49 nm for YCl 3 :NaBH 4 = 1:12) than 900 °C (ε hkl = 59 nm for YCl 3 :NaBH 4 = 1:8). In other common systems, an increase in the temperature usually yields extensive growth and bigger particles, thus suggesting in the specific case of this boride synthesis in molten salts that the initial reagent ratio (e.g. YCl 3 :NaBH 4 ) is the key parameter for size control. For YB 4 (1:8-900-4 sample), anisotropic crystallites are detected, with ε 00l = 81 nm and ε hk0 = 26 nm, thus indicating faster growth along the [001] axis. TEM observation of the 1:12-1000-4 sample (Fig. 8a-c) highlights cubic particles with diameters ranging from 30 to 200 nm. The selected area electron diffraction (SAED) pattern (Fig. 8a inset) on an ensemble of particles shows that the particles are YB 6 cubes, in agreement with XRD. HRTEM (Fig. 8d) of one nanocube highlights 0.41 nm (100) lattice fringes showing that the nanocubes expose (100) faces, which is consistent with the cubic structure of metal hexaborides (Fig. 1) and with previous reports on bigger hexaboride particles. 27 A thin amorphous layer is distinguished at the surface of the crystals, with thickness below 1 nm. Note that although the cubes are well faceted, they exhibit rough surfaces and round angles (Fig. 8b andc). Slight amounts of amorphous matter are also observed next to the cubes (Fig. 8b andc). TEM on the 1:8-900-4 sample (Fig. 9a andc) displays two populations of crystalline particles, consisting in approximately 100 nm long and 30 nm large nanorods on one side, and 80 nm nanocubes on the other side. All crystals are embedded in a nanotextured amorphous solid, already encountered for Ni-B systems, that is made of partially oxidized boron. Again, the nanocubes are YB 6 (Fig. 9e) particles. On the contrary, HRTEM (Fig. 9b) performed on the tip of one nanorod shows lattice distances of 0.50 nm and 0.40 nm, which correspond to (110) and (001) planes of YB 4 , respectively. Consequently, these YB 4 nanorods exhibit preferential growth along the [001] axis, in accordance with XRD results. To our knowledge, we report here the first occurrence of a metal tetraboride at the nanoscale with well-defined morphology.

Discussion

Composition selectivity in the crystalline phase

For a given metal-boron binary system, the results presented herein indicate the feasibility to control the stoichiometry of crystalline nanoparticles by using a set of synthesis parameters, such as the ratio of metal and boron precursors, the temperature and the heating duration. This composition control is first shown for the Bpoor nickel borides, with NiB nanospheres of 2 to 20 nm diameter (NiCl 2 :NaBH 4 = 1:2). This is, to our knowledge, the smallest crystalline nickel boride nanoparticles synthesized so far. 36 Ni 4 B 3 , Ni 2 B and Ni 3 B can also be selectively fabricated by only varying the precursor NiCl 2 :NaBH 4 ratio from 1:1.2, 1:1 and 1:0.65 ratios, respectively. In these latter cases, particles are bigger than for NiB but are still below 100 nm. To our knowledge, we show here the first occurences of Ni 4 B 3 and Ni 2 B nanocrystals with such a small size, while crystalline Ni 3 B particles were previously reported. 36 On the side of B-rich phases, we explored the yttrium-boron system. YB 6 nanocubes of ca. 80 nm are reachable, while YB 4 nanorods are synthesized for the first time. In this latter case, oxidized phases, especially yttrium borate YBO 3 , are observed as side product and suggest that the powder prior to washing exhibits reactive yttrium species, presumably poorly crystalline solid phases that oxidize spontaneously in contact with air and water. Although more efforts are required to isolate single crystalline phases in the case of yttrium, the Ni-B and Y-B systems clearly demonstrate that increasing the relative amount of borohydride precursor favors the synthesis of higher borides, as expected intuitively. In addition, a temperature increase drives the reaction towards B-rich phases, in agreement with the high energy barrier to crystallization exhibited by such compounds that usually require synthesis at high temperature, above 1000 °C in solid state reactions. 37 Noteworthy, an amorphous phase made of partially oxidized boron 35 is always observed, whether as a 1 to 2 nm-thick shell (Fig. 5 and8d) or as particles separated (Fig. 8 and9) from the crystalline phases. As oxidation can only occur during the washing step, then amorphous boron is obtained at the end of the synthesis, prior to purification. The occurrence of this amorphous component is consistent with the fact that an excess of boron precursor is always required to reach a given metal-to-boron ratio in the crystalline phase (e.g. NiCl 2 :NaBH 4 = 1:2 for NiB, YCl 3 :NaBH 4 = 1:12 for YB 6 ). Hence, part of the boron atoms initially introduced is segregated into the amorphous shell in the final product. Consequently, we hypothesize that this amorphous phase is reminiscent of the growth mechanism, which will be discussed elsewhere. Nevertheless, this amorphous shell has been shown 35 to be sufficiently porous to provide access of ionic species to the surface of the nanocrystals, so that it enables future investigation of catalytic properties.

Crystal habit: faceting and occurrence of anisotropy in metal boride nanocrystals

Particle size control. As exemplified previously on NbB 2 nanocrystals, 35 an increase of the metal-to-boron ratio in the precursor ratio yields a decrease in the size of crystalline YB 6 nanoparticles. Two explanations can be proposed to account for this size reduction. First, as the concentration of metal precursor is constant, then an increase in the relative amount of borohydride results in an overall increase in the reactant concentrations, thus favoring nucleation over growth and yielding smaller particles. A second explanation may originate from the amorphous boron shell formed in the course of the reaction. Indeed, an increase of the borohydride content should yield an increase in the volume fraction of the amorphous boron phase, which may act as a template restricting nanocrystal growth.

Anisotropic shapes: the case of yttrium boride YB 4 . Isotropic YB 6 nanocubes could be obtained from the synthesis in molten salts at ambient pressure. These particles are similar to other hexaboride particles as previously reported from solid-state methods under autogeneous pressure. 27 They exhibit {100} facets and a morphology related to the cubic symmetry of the crystal structure of hexaborides. More interestingly, YB 4 nanocrystals could be observed for the first time. Moreover they exhibit a nanorod shape with an anisotropy factor of ca. 3. Noteworthy, the only processes reported so far to produce anisotropic one-dimensional (1D) nanostructures of metal borides employed reactive gaseous species, for instance through chemical or physical vapor deposition. 24 They were mostly focused on metal hexaborides. Herein, we report the first example of 1D crystalline metal boride nanoparticles from a liquid synthesis. Contrary to reactions based on gaseous species and that often yield 1D nano-objects through the mediation of a substrate, the YB 4 are likely to grow through a specific crystal habit related to the crystal structure of YB 4 . Indeed, its tetragonal structure differentiates the (001) plane on one side and the (hk0) planes on the other side. More in-depth observation of the reported crystal structure (Fig. 1 and10) shows that chains of B 6 octahedra clusters run along the c axis (orthogonal to the (001) plane), while the (a,b) plane can be considered as sheets of B 6 clusters connected by B 2 dumbbells. The Y atoms are located between the B 6 sheets, on top of the holes left within the sheets between B 6 clusters and B 2 dumbbells (Fig. 10). The nanorods show {001} tip facets and {110} lateral faces. Interestingly, the B-terminated {110} planes show the highest densities of B 6 clusters and B atoms (0.05 B 6 clusters•nm -2 and 0.35 B atoms•nm -2 ) in YB 4 , while {001} and {100} planes show the second (0.04 B 6 clusters•nm -2 and 0.32 B atoms•nm -2 ) and third (0.03 B 6 clusters•nm -2 and 0.28 B atoms•nm -2 ) highest B 6 and B densities, respectively. Consequently, one can draw a parallel with the crystal habit of more common compounds such as oxides, for which growth directions in classical crystallization are often driven by surface energies (Wulff Law), [START_REF] Cölfen | Mesocrystals and Non-classical Crystallization[END_REF] in turn related (in the absence of surface directing ligand) to the atom density in each plane. Indeed, the most stable faces are usually those showing the highest atom densities. The correlation of the B 6 and B density with the relative amount of the different facets in YB 4 nanorods ({110} in majority, {001} (tips) in minority, while no {100} face is observed) indicates that the surface energy is driven by the density of B 6 clusters or B atoms, as building-blocks of the YB 4 structure. B-terminated facets are indeed expected from the excess of boron source introduced. Although the consideration of relative surface energies requires confirmation by first principle calculations, it is consistent with the high strength of the B-B bonds in such compounds and with the high stability of B 6 clusters in borides, [START_REF] Albert | [END_REF] which suggest that the boron covalent framework should drive the relative surface energies, so that the role of the metal atoms would be secondary. Noteworthy, the largest facets in bulk tetraboride grown by a metal flux method are also {110}, 40 confirming their low surface energy. Anisotropic structures within isotropic nanoparticles: the case of nickel boride NiB. In contrast to YB 4 and YB 6 , the spherical morphology of the NiB nanoparticles is not related to the orthorhombic crystal structure (Amam). This conclusion is supported by the large density of crystalline defects (Fig. 5) that precludes any structure-driven faceting. The well-shaped spherical particles are unusual in the case of an anisotropic crystal structure, such as NiB. Hence, this morphology suggests solidification from molten metal or alloy droplets. 41 The melting point of bulk NiB is tabulated at 1035 °C, [START_REF] Lide | CRC Handbook of Chemistry and Physics[END_REF] close to the value for bulk gold. As known for gold 42 and covalent solids, 43 strong melting point depression can occur in this size range. Although physical data are missing in the case of NiB to evaluate the expected melting point of 10 nm NiB nanoparticles, one can point out that the synthesis temperature of 750 °C lies in the range where melting can be expected for 10 nm nanoparticles of metalliccovalent cohesion. 42,43 Following the assumption of NiB crystallization from molten droplets and considering the very high melting point of boron allotropes, [START_REF] Lide | CRC Handbook of Chemistry and Physics[END_REF] then the droplets should be confined within the solid amorphous boron matrix. Solidification and crystallization within the confined droplets could explain the high density of crystal defects observed within the NiB nanoparticles after cooling. On the contrary, YB 4 and YB 6 exhibit melting points higher than 2300 °C, which precludes any process involving a molten Y-B alloy. Hence, the growth mechanism follows a more conventional route that yields to faceting according to relative facet energies and the Wulff Law.

Experimental Syntheses

The precursors NaBH 4 (min 98 %, Alfa Aesar), anhydrous YCl 3 (99.99 %, Alfa Aesar) and anhydrous NiCl 2 (99.99 %, Alfa Aesar) were stored in an argon filled glovebox (M-Braun) and used as received. A LiCl/KCl mixture at the eutectic composition (wt. 45/55) was prepared from the commercially available salts (Aldrich). The mixture was evacuated under vacuum at 200 °C for 1 day, and then stored in a glovebox. Powder mixtures were manipulated in an inert atmosphere. They were finely ground in a ballmill (Retsch MM400) for 2 min at 20 Hz, and then transferred into a glassy carbon crucible. The crucible was maintained in a quartz tube, which was connected to an argon ramp. During heating in a vertical oven (Carbolite), the medium was stirred with a glassy carbon rod rotating at 100 rpm. After the apparatus cooled below 100 °C, the frozen salt medium was washed with deionized water, the suspension was centrifuged and the powder was separated from the supernatant. The obtained powder was then washed, centrifuged and recovered from the supernatant three times. The powder was evacuated at 40 °C overnight. Nickel boride syntheses. Following the general procedure, 2.5 g of eutectic mixture were mixed to 2.0 mmol of nickel (II) chloride and 4.0, 2.4, 2.0 and 1.3 mmol of sodium borohydride for experiments 1:2, 1:1.2, 1:1 and 1:0.65 respectively. The mixture was heated from ambient temperature to 750 °C (20 °C.min -1 ), then maintained at 750 °C for 1.5 h before cooling. Yttrium boride syntheses. Each synthesis is named after the molar ratio of inserted precursors, the temperature and the dwell time. The parameters of the yttrium borides syntheses are detailed in Table S1.

Techniques

Powder X-ray diffraction (XRD). XRD measurements were performed on a D8 Bruker apparatus operating at the Cu-Kα1 radiation. Patterns were indexed according to ICDD files: NiB (01-074-1207), Ni 4 B 3 (01-073-2551), Ni 2 B (04-001-0967), Ni 3 B (04-014-0853), YB 6 (04-003-0596), YB 4 (00-007-0057), YBO 3 (01-089-3501), Y 2 O 3 (01-071-0049). Apparent crystallite sizes ε were calculated using the Scherrer formula and pseudo-Voigt functions to fit peaks.

Transmission electron microscopy (TEM).

A 120 kV Tecnai Spirit microscope was used for TEM and Selected Area Electron Diffraction (SAED) pictures. High Resolution-TEM (HRTEM) was also operated on a 200 kV JEOL JEM 2011 apparatus, at the Institut des Matériaux de Paris Centre. Samples were prepared by evaporating a drop of suspension in ethanol on a carbon-coated copper grid. Energy Dispersive X-Ray Spectrometry (EDX). Elemental EDX analyses were obtained on a Scanning Electron Microscope (SEM) S-3400-N (Hitachi), equipped with an EDX detector (Oxford) calibrated prior measurements to allow for retrieving quantitative data. 100 x 50 μm 2 areas were probed. Acceleration tension of the source was 10 kV. A titanium grid was used as quantitative reference. Samples were fixed to a conductive carbon scotch. Nitrogen sorption. Samples were degassed at 150 °C overnight before measuring on an ASAP 2000 apparatus (Micromeritics).

Conclusions

To conclude, this study shows for the first time that the stoichiometry of metal boride nanocrystals can be tuned. To achieve this goal, syntheses are performed in inorganic molten salts that are stable in temperature, with negligible vapor pressure, thus ensuring a convenient, low cost and scalable method. Single crystalline phases can be isolated in most Ni-B systems and in YB 6 , although NiB is obtained with slight amounts of Ni 4 B 3 as side-product, while YB 4 could not be isolated properly. Besides, the crystalline metal borides are always obtained along with amorphous boron, which in some cases (e.g. NiB) play an important role on the particle shape and size. This points out different crystallization mechanisms, which might depend on the melting point of the nanoscaled alloys. When the synthesis temperature is lower than the melting point (e.g. the Y-B system), then classical particle growth may occur, then leading to faceting and anisotropic shapes expected from the Wulff Law, without any surface-directing ligand. On the opposite, if the synthesis is performed at a temperature exceeding the melting point, then amorphous boron may act as a template confining alloy droplets, which would in turn crystallize upon cooling in the space defined by the droplets, thus yielding metal boride spheres. In the case of an anisotropic crystal structure (e.g. NiB), the competition between the structure-driven tendency to anisotropic growth and the spherical shape imposed by the template results in strains, released in the form of uniaxial crystal defects. To confirm these mechanistic conclusions, in situ characterizations are under way, by using spectroscopic, scattering and electron microscopy techniques.

New route toward nanosized crystalline metal borides with tuneable stoichiometry and variable morphologies

Supplementary information

Table S1 Reaction conditions for the synthesis of yttrium borides. Table S1 Reaction conditions for the synthesis of yttrium borides. YCl 3 :NaBH 4 is the yttrium-to-boron source molar ratio, T and t are the temperature and dwell time, respectively. The corresponding masses are reported for each synthesis.

Fig 3 .

 3 Fig 3. Synthesis of metal borides in inorganic molten salts. LiCl/KCl (45/55 wt.) melts at T f = 353 °C. After cooling down the reactant medium and washing out the salts, various metal boride nanoparticles are obtained in term of compositions, stoichiometries and shapes. a Results from Portehault et al. 35.

  2; 1:1 and 0.65, Ni 4 B 3 , Ni 2 B and Ni 3 B are isolated as single crystalline phases, respectively. Minor parasite peaks are observed in the two last patterns. They could not be indexed along with a known phase. In contrast to NiB, Ni 4 B 3 , Ni 2 B and Ni 3 B patterns show narrow peaks that suggest larger particles. The calculated crystallite sizes for Ni 4 B 3 , Ni 2 B and Ni 3 B are 29±6, 33±3 and 40±2 nm, respectively.

Fig. 4

 4 Fig. 4 Powder XRD patterns for NiCl 2 :NaBH 4 molar ratios 1:2, 1:1.2, 1:1 and 1:0.65.

Fig. 5

 5 Fig. 5 HRTEM picture of a NiB nanoparticle from the initial ratio NiCl 2 :NaBH 4 = 1:2 and orientations of B-B bonds for two consecutive single crystal domains. a) Black arrows highlight the direction of uniaxial defects within the crystalline NiB particle. b) For each single domain stripe, the zone axes and lattice planes are indexed according to the NiB structure. c-e) Example of a misfit at the interface between two domains, viewed orthogonally to the (021) interface: c) ahead the interface, d) at the interface and e) behind of the interface. Nickel atoms are omitted for clarity.

Fig. 6

 6 Fig. 6 Speciation diagrams of yttrium borides and other yttrium phases obtained by synthesis in salt melts in respect to the molar ratio of precursors (YCl 3 :NaBH 4 ), the temperature (T) and the time (t) of heat treatment. Left to right: YCl 3 :NaBH 4 is fixed at 1:5, T and t vary; YCl 3 :NaBH 4 = 1:8, T and t vary; and t = 4 h, YCl 3 :NaBH 4 and T vary. For each experiment, the phase proportion is qualitatively represented in a histogram pie.

Fig. 7

 7 Fig. 7 XRD patterns of yttrium boride samples a) 1:12-1000-4 (YB 6 ), b) 1:8-900-4 (YB 4 and YB 6 ) and c) 1:5-800-1 (YB 4 and YBO 3 ).

Fig. 8

 8 Fig. 8 TEM analysis of YB 6 particles (1:12-1000-4 sample). a-c) Low magnification images of a region exhibiting nanocubes, the SAED pattern in the inset is indexed along the YB 6 structure. d) HRTEM picture of a nanocube and the corresponding Fast Fourier Transform in inset.

Fig. 9

 9 Fig. 9 Left: TEM analysis of the 1:8-900-4 sample. a) Low magnification picture of a region exhibiting nanorods dispersed in an amorphous oxidized boron matrix, b) HRTEM picture of a nanorod with fringes corresponding to YB 4 , c) and d) TEM pictures of a region exhibiting a nanocube partially embedded in an amorphous oxidized boron matrix and e) HRTEM picture of a nanocube indexed along the YB 6 structure. Right: typical morphologies of yttrium borides observed in the 1:8-900-4 sample.

Fig. 10

 10 Fig. 10 Facets {110}, {001} and {100} of yttrium tetraboride and density of boron octahedra and boron atoms for the three facets. The facet proportion in YB 4 nanorods is evaluated according to dimensions observed Fig. 9. It increases with the boron surface density. Especially no {100} face is observed by TEM.

Fig. S1

 S1 Fig. S1 Low magnification TEM picture of the sample obtained with initial ratio NiCl 2 :NaBH 4 = 1:2.

Fig. S2

 S2 Fig. S2 HRTEM pictures of NiB nanoparticles obtained with initial ratio NiCl 2 :NaBH 4 = 1:2.

Fig. S3

 S3 Fig. S3 HRTEM picture of Ni 4 B 3 30 nm particle, minor product in the sample obtained with initial ratio NiCl 2 :NaBH 4 = 1:2.

Fig. S4

 S4 Fig. S4 TEM pictures of Ni 4 B 3 and Ni 2 B particles, obtained for initial ratios NiCl 2 :NaBH 4 = 1:1.2 and 1:1, respectively.

Fig. S5

 S5 Fig. S5 TEM picture of Ni 3 B particles obtained for an initial ratio NiCl 2 :NaBH 4 = 1:0.65.

Fig. S6

 S6 Fig. S6 EDX spectra of samples NiB, Ni4B 3 , Ni 2 B and Ni 3 B.

Fig. S2

 S2 Fig. S2 HRTEM pictures of NiB nanoparticles obtained with initial ratio NiCl 2 :NaBH 4 = 1:2. Spherical particles 2 to 6 nm in diameter are indexed along the NiB structure. Some crystal defects are observed in the particles. c) The crystalline spheres are surrounded by a ca. 1.5 nm thick amorphous shell.

Fig. S3

 S3 Fig. S3 HRTEM picture of a 30 nm particles, minor product in the sample obtained with initial ratio NiCl 2 :NaBH 4 = 1:2. The inset shows the corresponding Fourier transform indexed on the Ni 4 B 3 structure.

Fig. S4

 S4 Fig. S4 TEM pictures of (a) Ni 4 B 3 and (b) Ni 2 B particles, obtained for initial ratios NiCl 2 :NaBH 4 = 1:1.2 and 1:1, respectively. The particles are faceted and exhibit diameters between 50 nm and 200 nm. They form dense aggregates and show evidences of sintering with clear grain boundaries. They are embedded in an amorphous shell that crystallizes under the electron beam, possibly related to nickel hydroxide. HRTEM pictures and corresponding Fourier transforms (insets) highlight the crystal structures of c) Ni 4 B 3 for NiCl 2 :NaBH 4 = 1:1.2 and of b) Ni 2 B for NiCl 2 :NaBH 4 = 1:1.

Fig. S5

 S5 Fig. S5 TEM picture of Ni 3 B particles obtained for an initial ratio NiCl 2 :NaBH 4 = 1:0.65.

Fig. S6

 S6 Fig. S6 EDX spectra of samples NiB, Ni4B 3 , Ni 2 B and Ni 3 B, obtained with initial ratios NiCl 2 :NaBH 4 = 1:2, 1:1.2, 1:1 and 1:0.65, respectively.

Table 1

 1 Apparent crystallite sizes of yttrium borides calculated from the XRD diagrams.

	Sample	YB 6	YB 4
		ε hkl (nm)	ε 00l (nm)	ε hk0 (nm)
	1:12-1000-4	49	-	-
	1:8-900-4	59	81	26
	1:5-800-1	-	51	18

Fig. S1 TEM picture of the sample obtained with initial ratio NiCl 2 :NaBH 4 = 1:2. The sample is composed of crystalline particles dispersed in an amorphous boron phase. The particles can be divided in two groups, depending on size and morphology: the major one is composed of particles around 5 nm in diameter, with a spherical morphology, that are NiB particles according to HRTEM (Fig. S2 and Fig. 3). Other particles in minority exhibit diameters above 20 nm (black arrows) and are slightly faceted. They are indexed on the Ni 4 B 3 structure. (Fig. S3).


C Kaplon

S Fremy

P Aho

M.-C Pothier

A Legrand-Guillien

C Minoui-Tran

A Payan

L Vabret

Mendes-Martins

Alexis De Rougemont

Jérôme Kaplon

Céline Fremy

Serge Aho

Pierre Pothier

Marie-Christine Legrand- Guillien

Adissa Minoui-Tran

Christopher Payan

Astrid Vabret

Lucile Mendes-Martins

Mondher Chouchane

Raphaëlle Maudinas

Frédéric Huet

François Dubos

Didier Hober

Mouna Lazrek

Christelle Bouquignaud

Anne Decoster

Sophie Alain

Jeanne Languepin

Yves Gillet

Bruno Lina

Yahia Mekki

Florence Morfin-Sherpa

Aurélie Guigon

Jérôme Guinard

Vincent Foulongne

Michel Rodiere

Véronique Avettand-Fenoel

Stéphane Bonacorsi

Antoine Garbarg- Chenon

Dominique Gendrel

Pierre Lebon

Mathie Lorrot

Patricia Mariani

Jean-François Meritet

Aurélie Schnuringer

Gérard Agius

Agnès Beby-Defaux

Denis Oriot

Ronald Colimon

Gisèle Lagathu

Olivier Mory

Sylvie Pillet

Bruno Pozzetto

Jean-Louis Stephan

Dr Alexis De Rougemont
email: alexis.de-rougemont@u-bourgogne.fr.

Clinical severity and molecular characteristics of circulating and emerging rotaviruses in young children attending hospital emergency departments in France

Clinical severity and molecular characteristics of circulating and emerging rotaviruses in young children attending hospital emergency departments in France

INTRODUCTION

Group A rotavirus (RVA) is the leading cause of acute gastroenteritis (AGE) in young children worldwide, and is estimated to cause around 453,000 deaths every year in children under 5 years old, mostly in developing countries (1). Although fast and appropriate care has considerably reduced mortality in industrialised countries, rotaviruses are still responsible for considerable morbidity in infants and generate significant health costs.

RVAs belong to the Reoviridae family and possess a genome composed of 11 dsRNA genomic segments encoding six structural (VPs) and five/six non-structural (NSPs) proteins.

On the basis of the outer capsid proteins VP7 and spikes VP4, RVA can be classified into G and P genotypes, respectively. Nucleotide differences in these two genes currently allow the classification of RVA in 27 G-and 37 P-genotypes, among which 12 G and 15 P are associated with infections in humans, thus showing a considerable diversity of strains (2, 3) .

Both viral outer layer proteins elicit the production of neutralizing antibodies in the host, but no precise G or P type clearly correlates with the severity of the disease. The five RVA genotype combinations G1P [8], G2P [START_REF] Iturriza-Gomara | Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network[END_REF], G3P [8], G4P [8] and G9P [8] are responsible for approximately 90% of RVA infections in children (4). Of note, uncommon G types such as G5, G8, G10 and G12 have emerged in various areas of the world, notably in tropical regions [START_REF] Santos | Global distribution of rotavirus serotypes/genotypes and its implication for the development and implementation of an effective rotavirus vaccine[END_REF][START_REF] Todd | Rotavirus strain types circulating in Africa: Review of studies published during 1997-2006[END_REF].

Local data on the current burden of rotavirus disease, including both virological and clinical aspects, are important for decision-making and optimization regarding immunization strategies (7). Hence, knowledge of the molecular epidemiology and antigenic diversity of cocirculating rotaviruses is necessary to ensure the suitability and the efficacy of vaccines. Indeed, RVA diversity is constantly generated by positive selection of single amino acid mutations in defined epitopes, and particularly in highly divergent regions of the outer capsid protein VP7 (8). Programs to monitor rotavirus antigenic drifts that might be caused by Acute gastroenteritis was defined by at least three soft or liquid stools or three bouts of vomiting in 24h. Children presenting with chronic diarrhoea, immune deficiency, inflammatory disease of the digestive tract, or nosocomial infections were excluded.

Clinical data, including personal identification and clinical symptoms, were collected from children presenting to paediatrics departments. Informed consent was obtained for all subjects. Disease severity was calculated using the Vesikari scale, a 0-20 point numerical score which assesses the clinical severity of rotavirus infections (where higher scores indicate greater severity), as previously described (11). An episode of gastroenteritis with a score 11 is considered a severe episode.

The stool samples were routinely screened for RVA using mainly immunochromatographic (ICG) tests or enzyme immuno-assays (EIA). All rotavirus-positive samples were stored at -20°C until genotyping by the National Reference Centre for Enteric Viruses, University Hospital of Dijon, France. The rotavirus strains were genotyped using RT-PCR according to the EuroRotaNet methods (www.eurorota.net/docs.php) and using the Qiagen OneStep RT-PCR kit (Qiagen, Hilden, Germany). The VP7, VP4, VP6 and NSP4 PCR products from strains of interest were sequenced with the same primers as for amplifications. All the sequencing reactions were performed using the ABI ® PRISM ® Terminator Cycle Sequencing Kit on a 3130XL DNA Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The NV, Sint-Martens-Latem, Belgium) and a selection of sequences from rotavirus reference strains available from the GenBank database. Phylogenetic analysis was performed using MEGA6 software (12). After sequence alignment using the MUSCLE programme (13), phylogenetic tree was inferred using the Maximum Likelihood method based on the Tamura 3-parameter model, which was the best-fit DNA substitution model for the nucleotide dataset submitted. Bootstrap values were calculated for 1000 replicates. The nucleotide sequences of representative G12 strains from this study have been deposited in the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/) under the following accession numbers: KU291317 to KU291349.

Statistical analyses were performed using STATA ® v11.0 software from StataCorp (College Station, TX, USA). Comparisons of clinical data and severity scores between G1 and G12 RVA, and between all RVA genotypes were performed using the Fisher-exact test for categorical data and the Kruskal-Wallis test for quantitative data. Fractional polynomials were used to model the relationship between children's ages and severity scores using a linear regression model including a non-zero intercept. P values of 0.05 were considered significant.

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8

RESULTS

From July 2009 to June 2014, 4800 stools specimens were collected with a mean rate of 73.8 samples per centre and per season (i.e. a 12-month period that begins in July and ends in June of the year after). The mean and median ages of the young patients were 13.7 and 10.5 months, respectively (range, 0.2 to 59.9 months) and the male/female ratio was 1.34. Most rotavirus infections occurred in children under 2 years old (83.9%). From the 4800 rotaviruspositive stool samples, 92 (1.9%) could not be genotyped by RT-PCR, and 4708 were successfully genotyped. Of these, 4578 (97.2%) faecal specimens contained only one RVA strain, 64 (1.4%) were mixed infections of G and P types, and 66 (1.4%) were partially genotyped.

RVA infections occurred in France throughout the year with a peak between January and April (Figure 1A). However, in most of the provincial cities, RVA infections were peaking 2 months later (i.e between February and March) than in the largest cities in our network (i.e. between January and February in Paris and Lyon) (Figure 1B).

Molecular distribution of circulating rotavirus strains

G1 strains were the most predominant during the course of the study and in most cities with a mean prevalence of 63.5% [53. 5-73.8]. The four other major G genotypes (G2, G3, G4 and G9) accounted for 34.0% [17.9-44.7] of infections with wide individual amplitude according to the season (Tables S1 andS2). Of note, G12 strains accounted for 1.7% [0.0-3.3]. Mixed infections involved associations of G1 strains with one of the other major G genotypes in 75.5% [57.1-100.0]. In addition, the distribution of P genotypes showed a clear predominance of P [8] strains with a mean detection rate of 91.2% [76.6-97.9] followed by P [START_REF] Iturriza-Gomara | Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network[END_REF] strains with 8.0% [1.4-22.1]. The study of the distribution of G genotype prevalence per month showed that G2 and G9 RVA infections were more frequently detected during the first part of the

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ACCEPTED MANUSCRIPT 9 epidemic season (i.e. from September to January) whereas G3, G4, G12 and to a lesser extent G1 RVA infections were more frequent during the second part and at the peak of the epidemic (from January to June) (Figures S1A andS1B). Moreover, no predominant RVA genotype was detected according to the age group.

Distribution of the genotype combinations showed a clear predominance of G1P [8] S2).

Combinations of major human RVA genotypes (i.e. P [START_REF] Iturriza-Gomara | Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network[END_REF] with either G1, G3, G4 and G9; and P [8] with G2) were regularly detected during the study with a mean prevalence of 1.7% [0.6-3.2] but were decreasing over time (Table 1).

Emergence of G12P[8] rotavirus strains

During the study period, 78 G12P [8] strains were detected with a mean prevalence of 1.6% [0.0-4.1], of which 68 were detected during the 2011-12 and 2012-13 seasons with 4.1% (n=35) and 3.0% (n=33) of prevalence, respectively. At the regional level, G12P [8] strains emerged in 2011-2012 at various rates in all regions except Dijon and Charleville-Mézières (Figure S3). They were still detected during the subsequent season in only five cities (Brest,

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Lille, Paris, Poitiers and Saint-Etienne). VP7 phylogenetic analysis showed that all these strains belonged to lineage III and segregated into two relatively distinct main clusters that differed from those of the older G12P [8] strains circulating in France between 2005 and 2009, albeit, only three strains detected in 2014 (R8391, R8422 and R8964) were more closely related to these older G12P [8] strains (Figure S4). All G12P [8] strains harboured VP6 genes of human origin (genotype I1). However, among these, two strains harboured NSP4 genes of animal origin (genotype E2), whilst all of the others were of human origin (genotype E1).

Human and animal reassortant rotaviruses

Forty (0.8%) atypical RVA strains, of which 27 (67.5%) were P [START_REF] Todd | Rotavirus strain types circulating in Africa: Review of studies published during 1997-2006[END_REF] strains, were detected during the surveillance and further characterized according to their VP6 and NSP4 coding genes (Table S3). Among these, 20 (51.3%) strains bore genotypes commonly found in cattle: 13 (33.3%) G6, six (15.4%) G8 and one (2.6%) G10 strains. Among the G8 strains, three were combined with a major human P type: two with P [8] and one with P [START_REF] Iturriza-Gomara | Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network[END_REF] in three different locations and seasons. The single G10 strain was combined with P [8] and harboured VP6 and NSP4 genes of human origin. Other strains were unusual combinations of common G types with common human or potential zoonotic P types such as P [START_REF] Matthijnssens | Multiple reassortment and interspecies transmission events contribute to the diversity of feline, canine and feline/canine-like human group A rotavirus strains[END_REF], P [START_REF] Todd | Rotavirus strain types circulating in Africa: Review of studies published during 1997-2006[END_REF], P [START_REF] Melliez | Morbidity and cost of rotavirus infections in France][END_REF] and P [START_REF] Koopmans | Seasonality and diversity of Group A rotaviruses in Europe[END_REF].

Clinical characteristics and severity of rotavirus infections

Clinical records could be collected from 624 children who were thereafter hospitalized in paediatrics departments. Comparisons of clinical records in relation to genotypes are reported in Table 2. No difference was found between RVA genotypes for the clinical presentation.

None of the patients had been vaccinated. No deaths were reported during the study period.

Vesikari severity scores could be calculated for 282 children from the same cohort. In all, 148 (51.7%) infants, most of whom were more than 6 months old (91.9%), hospitalized with where G1P [8] score was 10.24 ±3.9 and G12P [8] score was 10.13 ±2.8. No difference in Vesikari severity score was found between G1P [8], G2P [START_REF] Iturriza-Gomara | Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network[END_REF], G3P [8], G4P [8], G9P [8] and G12P [8] (P=0.604), between G1P [8] and G12P [8] (P=0.813) or between P[4] (9.93 ±2.9) and P [8] (10.38 ±3.4) RVA infections (P=0.386).

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12

DISCUSSION

During this 5-year study, we have showed that G12P [8] RVA strains have slightly emerged in France during two consecutive seasons, and confirmed that RVA infection severity is not related to genotype.

Although rotavirus infections in Europe have been reported to occur with a geographical gradient of increasing incidence from the south-west towards the north (14), at the country level, they seem to spread more according to centrifugal progression from areas where the population density is the highest towards the lowest. In France, the winter rotavirus epidemics occurred globally 2 months earlier in large cities than in smaller provincial cities, as previously reported (15). Indeed, high population densities in the large cities facilitate contact between individuals and may thus play a substantial role in shifting the epidemic peak.

During the study, the circulation of the G1P [8] strain in France was overall predominant and similar to that in the rest of the world despite the unexpected transient emergence of new major genotypes. This predominance may be due to the emergence of new antigenic G1 variants appearing or disappearing alternately under the influence of natural immune-pressure mechanisms (16).

Following their emergence in France, G9P [8] strains maintained a relatively high incidence level (15), before strongly decreasing during the current study period. Interestingly, this decline occurred much later in France than in other European countries, where, after emergence, the prevalence of G9P [8] strains progressively decreased season after season (17).

However, during the last season in France, high G9P [8] prevalence was observed again. To date, there is no evidence of any particular environmental or immune factors that could explain the long circulation of G9 strains or their renewed circulation in French infants.

During the study, G2P [START_REF] Iturriza-Gomara | Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network[END_REF] prevalence was markedly high during the 2009-2010 season, whilst it was negligible in the rest of the study period. These fluctuations reflect here the normal While the detection rate for G4P [8] was relatively negligible during the study, the circulation of G3P [8] strains increased and then stabilized at around 14%. This increased frequency was supposedly at the expense of the decrease in the frequencies of G1P [8] and G9P [8] (19, 20), but could just be related to the re-emergence of G3 antigenic mutants.

G12 strains, first detected in France in 2004, were the most frequent among the uncommon strains detected in Europe and Australia (17, 21). Recently, G12P [8] have emerged during the 2011-12 season in 11 of the 13 monitored regions but were only persistent in 5 regions during the subsequent season. This particularly mild emergence may be probably explained by both a herd immunity against P [8] types and an acquired cross-reactive immunity of the population. Indeed, it differs from the intense emergence reported in the North of Spain during the 2010-2011 season (22), although it could be related the more so as the two countries share the same borders and the Spanish G12 emergence preceded only by 1 season the French one.

Phylogenetically, these G12 strains belong to the lineage III but diverge from the older strains previously detected, suggesting that an evolution of VP7 antigens or an introduction of new strains in the country have occurred.

Infections with viruses of possible zoonotic origin was regularly observed during the 5 seasons, notably G6 and G8 strains associated with P [START_REF] Todd | Rotavirus strain types circulating in Africa: Review of studies published during 1997-2006[END_REF] or P [START_REF] Koopmans | Seasonality and diversity of Group A rotaviruses in Europe[END_REF], which harboured NSP4 and VP6 genes from porcine and bovine hosts, and might have resulted from natural reassortment during animal-human transmissions or mixed human rotavirus infections from environmental reservoirs (23, 24). These observations demonstrate the interspecies transmission of RVAs, notably from animals living in close contact with humans, resulting in the introduction of new rotavirus genotypes in humans. Interestingly, two G8P [8] and one G8P [START_REF] Iturriza-Gomara | Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network[END_REF] strains were

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ACCEPTED MANUSCRIPT 14 detected in three different places showing that some G8 strains might initiate their adaption to humans, as previously observed for G4, G9 and G12 strains. While the G8P [START_REF] Iturriza-Gomara | Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network[END_REF] strain was associated with animal VP6 and NSP4 and was seemingly related to African strains (25), both G8P [8] strains harboured human VP6 and NSP4 genes, thus showing active recombination with human P [8] strains, as previously reported (26). These human G8P [8] strains require further investigations to evaluate their potential emergence; their fitness is currently insufficient to allow easy transmission to humans.

Analysis of the clinical records showed no difference in clinical manifestations or severity in relation to genotype. These results are consistent with our previous findings (15, 27). More than half of the rotavirus infections in these children were considered severe, which is consistent with previous studies (28, 29). RVA infections in children under 6 months old were, however, less severe than those in any other age group, probably thanks to the protection provided by maternal antibodies. Commonly, differences in severity may be explained by variations in virulence between strains and by the immune status of the population towards a serotype which is partially dependent upon the infecting strain. However, globally, most studies on rotavirus infection severity have been either inconclusive or contradictory (27, 30-32). Nevertheless, both VP7 and VP4 antigens induce antibodies, and most of the genotypes compared had the same P [8] type, thus abrogating the differences between G types. As the severity of the disease does not depend on age but on the infecting virus and previous exposure (33), severe infections may be found mostly during primary infection or exposure to new antigens, whilst less severe infections occur after second exposure, during which heterotypic protection may reduce the symptoms and shorten the infection. These data therefore suggest that VP4 and VP7 antigens are not the only factors responsible for the severity of rotavirus infections. Other strain-specific viral factors and

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15 immune status, whether naive or after previous infections, influence the intensity and the severity of the clinical symptoms. The data also strongly suggest that all RVA genotypes may cause acute gastroenteritis, as their G and P types cannot be dissociated, and thus reinforce the need for vaccines that ensure effective protection, whether directly or by cross-reaction, against all rotavirus infections. Today, rotavirus vaccination is still too low in France to have any impact on annual epidemics other than locally. The relative stability of RVA genotypes currently co-circulating and the large predominance of P [8] type strains may ensure vaccine effectiveness, and, given the good outcomes of vaccination in countries with sufficient coverage (34), extensive vaccination should be encouraged in France. The surveillance of rotavirus infections during ongoing and future vaccination programs will ensure the detection of emergent new reassortants and will help the health authorities to optimize appropriate vaccination strategies against rotavirus diseases, all the more so as all genotypes can cause severe infections in infants.

6 METHODS

 6 and to monitor potential reassortments between human or human and animal strains have to be carefully developed.In France, rotavirus infections occur mostly during the winter and spring seasons. Although deaths remain exceptional, RVA are responsible for about 300,000 acute diarrhoea episodes, half of which are severe, 140,000 consultations and 18,000 hospitalisations each year (9). In spite of the introduction of RVA vaccines in 2006, coverage remains particularly low in France and was estimated at around 8.9% in French infants under 12 months old in 2011 (10). Since the establishment of the French rotavirus surveillance network by the National Reference Centre for Enteric Viruses (Dijon, France), it has become possible to assess the nationwide circulation of RVA strains, especially in infants. This prospective study was designed to monitor and characterize rotavirus infections, including both viral and clinical data, in children under 5 years old suffering from community-acquired acute gastroenteritis and attending paediatric emergency units during the rotavirus vaccine era in France. Special attention was paid to the detection of uncommon strains and emerging reassortants. After its approval by the local ethical committee (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale de Bourgogne, Dijon, France, on the 24 th of November 2005), the surveillance study was conducted during five consecutive seasons from July 2009 to June 2014, and involved 4800 children under 5 years old suffering from rotavirus-induced acute gastroenteritis and attending the paediatric emergency units of 16 French Hospitals in 13 regions, including Paris.

  edited and genotyped using BioNumerics software (Applied Maths

  classified as severe according to the Vesikari scale (i.e. a score 11 points). Severity was significantly lower in children aged 0-6 months with a mean score of 8.9 ±2.7 (P<0.0001) than in the three other age groups. A linear regression model using fractional polynomials was used to study this relationship, i.e. between the children's age in months and the severity score, and no linear relation was found as shown in FigureS5. The mean Vesikari severity score of the 282 rotavirus infections was calculated at 10.30 ±3.3

  strains driven by the reemergence of VP7 antigenic mutants, or potentially due to reassortment among co-circulating strains or an introduction of new lineages of G2 strains or antigen combinations (18).

Figure 1 . 24 Figure S5 .

 124S5 FIGURE LEGENDS

a

  These strains are also included in mixed infections; b Details on mixed infection strains are reported in

Table 1 )

 1 . The five major genotype combinations accounted for 93.3% [92.3-96.2] of strains, showing a relatively stable detection rate from season to season. Of note, G2P[4] strain circulation rapidly decreased from 17.5% during the 2009-2010 season to reach 1.1% during the last season, whilst G9P[8] strains, which remained stable at 8.9% [6.3-11.5] during the first four seasons, increased to 22.0% during the 2013-2014 season. The circulation of G3P[8] strains increased significantly during the last three seasons to reach a mean detection rate of 15.5% [13.2-19.3] (Figure

	strains

TABLE 1 .

 1 Distribution and detection rates of G and P genotype combinations of group A rotavirus detected in France from 2009 to 2014 No. of strains detected during the 5 consecutive seasons (%)

	Total strains	(%)

Table S1 .

 S1

													1
	ACCEPTED MANUSCRIPT	T	I P	R	C	M A N U S	D	E	T	P	E	C	A C

TABLE 2 .

 2 Clinical characteristics according to rotavirus genotypes in 624 hospitalized diarrheal children between 2009 and 2014

	P a P b		--	0.512 d 0.675 d	0.520 e 0.495 e	0.607 e 0.962 e	0.433 d 0.078 d	0.575 e 0.498 e	0.114 d 0.516 d	0.306 e 0.464 e	0.349 e 0.361 e	0.719 d 0.308 d	0.139 e 0.131 e	0.545 d 0.223 d	0.345 e 0.158 e	0.337 e 0.265 e	0.553 e 0.402 e	0.740 e 0.628 e	0.372 d 0.550 d	using Kruskal-Wallis
	G1P[8] G2P[4] G3P[8] G4P[8] G9P[8] G12P[8] Total	n=407 n=59 n=74 n=5 n=52 n=27 n=624	Prevalence 64.2% 9.3% 11.7% 0.8% 8.2% 4.3% 100%	Mean age, months 14.2 ±11.0 15.6 ±13.4 15.6 ±11.9 17.5 ±15.6 11.9 ±8.8 15.5 ±11.2 14.3 ±11.0	Sex ratio 1.6 1.3 1.3 0.7 2.5 1.8 1.6	Diarrhea 93.9% 94.9% 97.3% 100.0% 96.2% 96.0% 94.7%	Mean number of diarrhea 6.0 ±3.9 bouts per day 4.5 ±3.0 8.3 ±5.9 6.4 ±5.9 7.5 ±3.3 5.9 ±5.8 6.4 ±4.3	Vomiting 80.0% 84.7% 89.0% 100.0% 78.8% 80.0% 81.4%	Mean number of vomiting 4.6 ±3.4 bouts per day 5.3 ±5.5 6.1 ±4.0 2.5 ±0.7 5.8 ±5.2 7.7 ±6.3 5.1 ±4.2	Blood in stool 1.2% 0.0% 0.0% 0.0% 0.0% 4.0% 1.0%	Fever >38.5°C 72.3% 73.9% 61.0% 75.0% 64.3% 77.8% 70.5%	Temperature, °C 38.5 ±1.0 38.6 ±0.9 38.3 ±1.0 38.4 ±0.1 38.3 ±0.8 38.5 ±0.9 38.5 ±0.9	Dehydration 54.7% 53.4% 75.7% 40.0% 65.4% 56.0% 57.7%	Dehydration degree, % 7.2 ±3.5 6.0 ±2.5 7.0 ±3.4 3.5 ±2.1 6.6 ±3.6 6.4 ±2.8 6.9 ±3.4	Hypovolemia 37.9% 25.9% 64.8% 20.0% 46.0% 54.2% 41.0%	Intravenous rehydration 61.2% 65.5% 74.3% 40.0% 60.8% 68.0% 63.2%	Oral rehydration 13.6% 19.0% 12.2% 20.0% 21.6% 12.0% 14.4%	Intensive care 1.2% 1.7% 2.7% 0.0% 1.9% 0.0% 1.4%	Hospitalization duration, 2.8 ±1.7 days 2.8 ±1.6 2.7 ±1.4 1.8 ±1.1 3.2 ±1.7 2.8 ±1.6 2.5 ±1.5	Comparison within all genotype groups; d a Comparison between G1P[8] and G12P[8] groups; b Comparison between all genotype groups; c	test; e using Fisher's exact-test; Statistically significant if P<0.05.

ACKNOWLEDGMENTS

We would like to thank Philip Bastable for editorial assistance. A part of the results of this study was presented on the 19 th of May 2015 at the 6 th European Rotavirus Biology Meeting in Dijon, France.

Data from 2001 to 2009 were extracted from our previous studies for comparison (15, 35) . Detailed numbers of G12 strains for each season are indicated in the bottom table. * first detection of G12P [8]; ** emergence of G12P [8] strains in France.


Khaled Medini
email: khaled.medini@emse.fr

Sophie Peillon
email: peillon@emse.fr

Xavier Boucher
email: boucher@emse.fr

Hervé Vaillant
email: vaillant@emse.fr

Performance Measurement for the Design of Product-Service Systems

Keywords: Product-Service Systems, decision making, performance, sludge treatment, recycling

Resources depletion, emerging competition, and increasing individual customer requirements are among the most common trends shaping nowadays manufacturing industry. Product-Service Systems (PSS) are put forth as a potential means for meeting these challenges due to their intrinsic characteristics such as dematerialization, multi-actor perspective, inimitable service know-how, and customer closeness. The scope of this paper falls under a wider topic relating to the development of PSS solutions for the steel sludge treatment sector. More specifically, the paper reports on a combined performance measurement and PSS design approach to support the decision making process on the PSS implementation. The approach takes into account the need for a collaborative effort among the PSS value network actors to successfully implement PSS offers. A case study from the sludge treatment sector is used to illustrate the synergies between performance measurement and decision making, in the PSS context.

Introduction

The last few decades witnessed a paradigm shift through the development and spread of servitization and Product-Service System (PSS) concepts. Goedkoop et al. [START_REF] Goedkoop | Product Service systems -Ecological and Economic Basics[END_REF] define PSS as 'a system of products, services, networks of 'players' and supporting infrastructure that continuously strives to be competitive, satisfy customer needs and have a lower environmental impact than traditional business models'. Since then, most contributors have broadly adopted this definition. As already stressed by many authors such as Goedkoop et al. [START_REF] Goedkoop | Product Service systems -Ecological and Economic Basics[END_REF] and Meier et al. [START_REF] Meier | Industrial Product-Service Systems-IPS2[END_REF], PSSs are generally delivered by a network of partners. A PSS is the result of a value co-production process within such a partnership. Therefore, PSS business models call for a continuous collaboration process that involves both the buying and the selling organizations, and other external and complementary partners. The stakeholders, whether they are producers, retailers, customers or end-of-life managers, require connected economic interests and shared vision of desirable outcomes for a system resource optimization [START_REF] Xing | A service innovation model for synergistic community transformation: Integrated application of systems theory and product-service systems[END_REF]. The scope of this paper falls under a wider topic relating to the development of PSS solutions for the steel sludge treatment sector. More specifically, the paper reports on a combined performance measurement and PSS design approach to support the decision making process on the PSS implementation. The approach takes into account the need for a collaborative effort among the PSS value network actors to successfully implement PSS offers. A case study from the sludge treatment sector is used to illustrate the synergies between performance measurement and decision making, in the PSS context. The novelty of this paper lies in the use of performance measurement and analysis at the PSS design stage. The remainder of the paper is organized as follows: section 2 sheds more light on performance measurement from a PSS perspective. Section 3 describes the proposed approach combining PSS design and performance measurement. Section 4 illustrates the approach with a case study. Conclusions and research perspectives are detailed in section 5.

Performance Measurement from a PSS Perspective

Performance measurement is a well-established concept in the operations management literature. The ultimate goal of the current research is to extend performance measurement to the PSS context; in particular by supporting the decision making process during PSS design.

Performance measurement definition and issues relating to PSS. Performance measurement is an activity aimed at reaching predefined goals that are derived from the company's strategic objectives by using performance indicators (PIs) [START_REF] Lohman | Designing a performance measurement system: A case study[END_REF]. A PI is a variable that expresses quantitatively the effectiveness or efficiency or both, of a part of or a whole process or system against a given norm or target [START_REF] Fortuin | Performance indicators -Why where and how[END_REF]. Performance measurement represents the concrete formulation of firm's strategic choices and has been closely related to supply chain and extended enterprise [START_REF] Folan | A review of performance measurement: Towards performance management[END_REF]. Lohman et al. [START_REF] Lohman | Designing a performance measurement system: A case study[END_REF] highlighted the need to measure the supply chain performance as a whole and to be able to drill down to different measures and different levels of details. Folan and Browne [START_REF] Folan | A review of performance measurement: Towards performance management[END_REF] assume that performance measurement has moved towards examination of the organization as a whole and impacting to a greater extent upon strategy. They assume that performance measurement would have an impact outside organization (i.e. external environment).

Among the difficulties shaping the performance measurement in the PSS context, and in extended enterprises at large, is the decentralized nature of the value network leading to uncontrolled growth in indicators. Basically, this stems from the connection of several companies to deliver the PSS [START_REF] Morlock | Performance Measurement for Robust and Agile Scheduling and Control of Industrial Product-Service Systems[END_REF]. This potentially leads to inconsistencies among the indicators used by the value network actors. The impact on the decision making is thus obvious as the performance is evaluated from single points of view. A multi-actor based indicators system reflecting all points of view of the value network actors is thus required and would be of much support to the PSS network management, which is a crucial task for the PSS [START_REF] Morlock | Performance Measurement for Robust and Agile Scheduling and Control of Industrial Product-Service Systems[END_REF]. Such indicators systems can act as a common platform from which all members of the value network can draw knowledge [START_REF] Folan | A review of performance measurement: Towards performance management[END_REF][START_REF] Morlock | Concept for a Performance Measurement Method for the Organization of the IPS² Delivery[END_REF].

Performance Measurement as a Support for the Decision Making Process. As the performance measurement has evolved from measuring and monitoring to supporting decision making, proper tools and methods should be created as substitute of historical data [START_REF] Lohman | Designing a performance measurement system: A case study[END_REF][START_REF] Folan | A review of performance measurement: Towards performance management[END_REF][START_REF] Morlock | Concept for a Performance Measurement Method for the Organization of the IPS² Delivery[END_REF]. Consistently with the above evolution, simulation is among the most used approaches to support the decision making. It allows replicating the actual behaviour of the real system at different granularity levels, ranging from value network actors down to production and service delivery activities. As such, the simulation, be it a discrete-event, an agent-based or a system dynamic based one, is a corner stone of the performance measurement, and thus of the decision making process [START_REF] Mourtzis | Design and operation of manufacturing networks for mass customisation[END_REF][START_REF] Medini | Product Service Systems value chain configuration -a simulation based approach[END_REF], and the performance indicators are its backbone. Several research works used the simulation to support the decision making process in the context of PSS [START_REF] Medini | Product Service Systems value chain configuration -a simulation based approach[END_REF][START_REF] Marques | PSS Production Systems: a simulation approach for change management[END_REF][START_REF] Medini | Transition to Product Service Systems: methodology based on scenarios identification, modelling and evaluation[END_REF] . Among the most common questions addressed in this context is the shift from a mere product oriented offer to a PSS oriented offer. The simulation here supports the performance measurement by evaluating different envisioned shifting scenarios using quantitative measures. Yet, in order for the decision makers to measure and improve the performance of their production systems, firms or even supply chains, there is a need to capture the most impacting performance drivers and align the decision making process with them. Consequently, several methods and tools have been used to deal with this appealing concern of exploiting performance measurement by decision makers. An example of these are multi-criteria decision methods which allow to combine a set of performance measures into a fewer ones, or even a single holistic index [START_REF] Medini | Tailoring performance evaluation to specific industrial contexts -application to sustainable mass customization enterprises[END_REF]. Another relevant means for understanding the most impacting performance drivers are decision trees which are the result of different classification algorithms [START_REF] Theil | Principles of Econometrics[END_REF]. These trees allow for a quick and clear graphical representation of the impact of different decision levels. The nodes of the trees represent the performance drivers, classified in order of their relevance; the most relevant drivers are linked to the root of the tree, while drivers with the lowest importance are at the bottom. The branches are labelled with the separating input variables (i.e. drivers).

A Combined Design and Performance Measurement Approach for the PSS

This section reports on a combined design and performance measurement approach for PSS. Unlike the traditional and most common performance measurement systems, the current approach uses performance measurement a-priori, as a supporting tool for the decision making process regarding the relevance of PSS solutions and delivery systems. The foundations of the current approach are detailed further in [START_REF] Lohman | Designing a performance measurement system: A case study[END_REF][START_REF] Folan | A review of performance measurement: Towards performance management[END_REF][START_REF] Medini | Product Service Systems value chain configuration -a simulation based approach[END_REF][START_REF] Medini | Transition to Product Service Systems: methodology based on scenarios identification, modelling and evaluation[END_REF]. More specifically, the methodological guidance combines the steps of building and using performance measurement systems [START_REF] Lohman | Designing a performance measurement system: A case study[END_REF] with the methodological support for defining PSS scenarios [START_REF] Medini | Transition to Product Service Systems: methodology based on scenarios identification, modelling and evaluation[END_REF]. The performance measurement is enabled by a simulation approach inspired by Medini et al. [START_REF] Medini | Product Service Systems value chain configuration -a simulation based approach[END_REF]. The combined approach is structured in four steps which are detailed in the following. Context analysis combines the firm's mission, objectives and functional areas relating to these objectives, in keeping with [START_REF] Lohman | Designing a performance measurement system: A case study[END_REF]. More broadly, context analysis consists in understanding the company's industrial context and competition factors. This relies on semi-structured interviews with the PSS key actors. This step provides insights into the PSS development opportunities and the main strategic capabilities of the involved actors with regards to the PSS. Usage analysis and scenario prioritization are needed in order to define different PSS variants based on the possible different uses of the PSS, and to identify the valuecreation potential for the actors involved (provider, customer, and other stakeholders). This step relies primarily on semi-structured interviews, brainstorming, and questionnaires to capture expectations of both customer and other actors of the PSS value network. Afterwards, several scenarios are defined consistently with the expected uses of the PSS. Each of the scenarios is defined by a combination of actors and roles within the value network. Finally these scenarios are filtered in order to narrow the scope of the subsequent quantitative evaluation. The filtering criteria stems from the context analysis and the stakeholders' experience, and are defined during face-to-face meetings. Select or develop performance measures consistently with the firm's objectives behind the PSS implementation. More specifically, this step aims to define the performance indicators for each actor involved, then identify physical and financial flows that should be modelled in order to enable indicators calculation by use of simulation. Then, questionnaires are built upon these models and are used for data collection. Indicators should comply with the multi-actor perspective, meaning that the final set of performance measures should accommodate the points of view of all the actors of the PSS value network.

Evaluate scenario performance: this step is critical in supporting the decision making process as it provides an evaluation of several alternative scenarios, thus helping to put the focus on the most interesting ones according to the performance measures. To this end, a three-stage process is deployed: ̶ Building an experimentation plan is concerned with the a priori identification of potential performance drivers according to PSS actors' know-how. ̶ Evaluating scenario performances uses simulation to compute the performance measures, based on replication of the real operations within the PSS production and delivery network. ̶ Identification of performance drivers is performed out of the simulation results.

The tools used at this stage are decision trees, which form an efficient and comprehensive tool to identify the impact of different decision levels on the performance. Thresholds can be defined based on this analysis, in order to specify the circumstances under which the PSS implementation would be potentially successful.

Illustrative Case Study

Usually, machining sludge generated by manufacturers is collected and treated by specialized companies. The envisioned PSS solution is built around a briquette-making equipment which allows the compacting and briquetting, and makes the sludge reusable. The compacting and briquetting result in two reusable products, i) briquettes, which can be sold to and used by smelters, and ii) cutting fluid extracted from the sludge, which can be used by the manufacturers themselves. The actors involved in the envisioned PSS value network are: i) an equipment provider (i.e. briquette-making), ii) manufacturers producing sludge and representing potential customers of the envisioned PSS solution, and iii) smelters using electric arc furnaces for melting steel scrap and other metals who are potential customers of the produced briquettes.

Context analysis relies on semi-structured interviews with manufacturers, (who generate different types of sludge), with equipment provider, and with smelters. The interviews resulted in the identification of several alternative organizational scenarios that were filtered to come up finally with 6 relevant ones. The current paper is limited to the two following ones:

-U1a: the briquette-making equipment is sold to a manufacturer who is in charge of the compacting, briquetting and maintenance operations, retrieves cutting fluids and sells briquettes to smelters. -S1a: the briquette-making equipment remains the provider's property, and the manufacturer pays for its use in his premises according to a "rental" contract. The equipment maintenance can be included as a service in the contract and is then performed by the briquette-making equipment owner; otherwise, it is considered as an internal activity of the manufacturer. The selection of proper performance indicators was straightforward since the main concerns of the involved actors (i.e. equipment provider, manufacturer, and smelter) relate basically to costs and benefits. In the following, the analysis will be focused on the profit indicator, which is a result of the above ones. A deterministic continuous simulation model is used to evaluate the performance of the scenarios and is implemented under Excel in Visual Basic language. The back bone of the simulation model is a set of mathematical equations depicting the interdependencies between the physical and financial flows [START_REF] Medini | Product Service Systems value chain configuration -a simulation based approach[END_REF]. The simulation inputs are data about the value network activities (e.g. cost, input and output flows), contracts (e.g. duration, installation costs, and ascribed services), services (e.g. costs), market (e.g. scrap cost, market demand, sludge characteristics), roles (e.g. actors responsibilities in terms of services and activities). The aim of the evaluation is to identify the main economic performance drivers for the value network actors, so as to provide a support for the decision making process of these actors regarding PSS implementation alternatives. To this end, an experimentation plan was adopted and is represented in Figure 1, showing the combination of the input variables for each of the two scenarios. The simulation horizon covers a 10 years period.

The simulation results are processed using the R software 1 and the Analysis of Variance (ANOVA) method [START_REF] Speed | Introduction to Fisher (1926) The Arrangement of Field Experiments[END_REF]. The output is structured in three regression trees representing the performance drivers of the profit indicators. Figure 2a indicates that organizational scenarios are the main drivers of the equipment provider profit. Generally, the PSS scenario (S1a) allows for more profit which ranges from 80݇€ when services are not included in the offer, to 130݇€ when they are included. The sales scenario (U1a) allows for a lower profit which culminates at about 60݇€, when the services are also offered in addition to the sale contract. Manufacturers' and smelters' profits are mainly impacted by the market volume, as shown in Figures 2b and2c; the bigger are the market volumes, the higher is the generated profit. The other drivers of profit for these two actors are the waste treatment cost (wCost) and the scrap cost (sCost), respectively. The manufacturers profit reaches its highest values when waste treatment costs are high.

Indeed, the PSS helps avoiding the costs that are traditionally incurred by the manufacturers when they want to get rid of their produced sludge. Further on, for the smelters, the scrap cost is an important driver, after the market. This is explained by the fact that smelters profit comes from the savings generated out of purchasing the briquettes at a lower price than traditional steel scrap, thus the higher the scrap costs are, the higher is the smelters profit. The above analysis provides the different value network actors with some useful inputs regarding their decision making process, through the most relevant drivers for profit. The focus can thus be put on these specific drivers to define trade-offs taking into account all the actors points of view and thus easing the implementation of a winwin PSS solution.

Conclusion

The current paper reports on a combined performance measurement and design approach to support the decision making process and thus mitigate the uncertainty at the PSS design stage. The basic prerequisite of this approach is a strong collaboration among the PSS stakeholders from the early design phases of PSS solutions.The case study shows the ability of the approach to determine the key performance drivers for the PSS actors and to identify the most relevant PSS value network configurations prior to the PSS implementation. The current work, however, has limitations and thus, opens further research perspectives. First of all, the performance system implemented (based on the industrial consortium requirements) can be enriched further with other indicators in order to better reflect the service perspective. Additionally, the simulation model opens many opportunities regarding the quantification of uncertainty and its impact on performance measurement. Basically, this would strengthen the decision-aid provided to PSS actors.

Fig. 1 :

 1 Fig. 1: Experimentation plan

Fig. 2 :

 2 Regression trees of the a. equipment provider profit, b. manufacturers profit, c. smelters profit

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