C Angioni

E Fable

C Stephens

J F Artaud

C Bourdelle

P Maget

J Citrin

K Van De Plassche

F Felici

TUNGSTEN TRANSPORT IN TOKAMAKS: TOWARDS REAL-TIME KINETIC-THEORY-BASED PLASMA PERFORMANCE OPTIMISATION

Mechanisms by which tungsten dynamically accumulates in the core of tokamaks are identified in integrated modelling of an ASDEX-Upgrade discharge. Then the same modelling tools are applied to a WEST long pulse L-mode plasma. This modelling work on two disctinct tokamaks also stresses the validaty domain of reduced transport models, in this case QuaLiKiz. At AUG, a ramp-down of the Electron Cyclotron Resonance heating is analysed with a constant background Neutral Beam Injection heating until W accumulation is observed. It is found that the central ratio of the electron to the ion temperature, together with the central source of particles deposited by the beams play a key role. Indeed, while the central particle source yields more centrally peaked density profiles, leading to inward neoclassical convection of tungsten, large Te/Ti can compensate this unfavorable scenario by increasing turbulent particle diffusion. Such competition between Te/Ti and the central particle source is also investigated with fast transport models, in particular within the integrated modelling framework RAPTOR where the turbulent transport is computed from QuaLiKiz 10D neural network. This paves the way towards real-time-kinetic-theory-based plasma performance optimisation. Finally, modelled WEST plasmas with dominant electron heating showed the limits of the implemented collision operator in QuaLiKiz yielding an overstabilisation of Trapped Electron Mode, resulting in mispredictions of the kinetic profiles (electron density and temperature).

INTRODUCTION

Tungsten is foreseen as plasma facing material in next generation tokamaks (ITER/DEMO) due to better properties: high melting point, capable of sustaining high heat and particle fluxes from the core, low erosion rates and limits the fuel retention which is, in particular, relevant to prevent radioactive tritium accumulation in the wall components. In view of these overall good qualities and the need to gather experience on tokamak operation with W plasma facing components, in Europe AUG and JET have been already operated for several campaigns with a full W wall and a W divertor respectively, and WEST is recently operating with a W wall as well. Experiments in AUG and JET have shown that the presence of W significantly affects plasma operation, limits the routinely achievable parameter domain of the device and requires actions to avoid central accumulation, which leads to high levels of core radiation due to the high charge number and partial ionisation. Uncontrolled central W accumulation produces large radiation losses and can trigger the loss of the high confinement regime (H-mode) or even disruptions, which are both detrimental for ITER (marginal input power with respect to the L-H transition power threshold and severe damages caused by disruptions).

For these reasons it is necessary to predict such detrimental scenarios regarding W accumulation in the plasma core. For such predictions, integrated modelling frameworks are required, not only to predict W transport but also the evolution of the electron density and ion/electron temperature profiles to resolve the nonlinear interactions among all transport channels. These frameworks necessitate accurate models for turbulent and neoclassical transport, but also suficiently fast models to prepare plasma scenarios prior to operation or even control the W accumulation in real time. In this work, we analyse W accumulation in an ASDEX Upgrade discharge within the integrated modelling framework ASTRA to underline the dominant ingredients in developing such accumulations. Then faster transport models are benchmarked on such cases, in particular the turbulent transport from QuaLiKiz-10D neural network within the integrated modelling platform RAPTOR.

Then the same tools are used to model tungsten transport in a WEST long pulse L-mode. The absence of toroidal rotation sources, the large aspect ratio and dominant electron heating allows us to stress the predictive capabilities of the transport models used in view of extrapolation to future reactors, in particular the first prefusion power phase of ITER. In this case, mispredictions are found due to overstabilisation of Trapped Electron Mode by collisions. To characterise this behaviour, the flux driven simulations performed so far are also complemented with gradient driven simulations.

First in section 1.1, the experimental setup for the ASDEX Upgrade H-mode featuring a tungsten accumulation is described. In particular, the discharge analysed features a ramp-down of the ECRH power. In section 1.2 the assumptions used in the integrated modelling framework ASTRA are described. Then dominant ingredients found responsible for W accumulation are underlined, namely the central particle source from the neutral beams and the central ratio Te/Ti. Faster transport models are also qualitatively validated with respect to mechanisms described above. Finally in section 2, a WEST long pulse L-mode plasma is described where tungsten does not accumulate throughout the 33 seconds of the discharge. Flux driven and gradient driven simulations are performed to characterise the mispredictions obtained in the regime of dominant electron heating.

TUNGSTEN ACCUMULATION IN ASDEX UPGRADE NBI HEATED H-MODE PLASMA

Experimental setup

A particular ASDEX-Upgrade discharge featuring a ramp-down of the ECRH power is studied in the following, where previous analyses of tungsten accumulation were performed for steady phases [1,2]. It consists of an Hmode plasma with Ip = 1 MA, BT = 2.5 T, ne =9 x 10 19 m-3, 7.5 MW of injected NBI power. Here we focus on two time windows where PECRH = 200 kW and PECRH = 1.9 MW. The time traces of the NBI, radiated and ECRH power together with the central and mid-radius tungsten densities inferred from UV grazing incidence spectroscopy are shown in Fig. 1. It is observed that while the ECRH injected power is decreased, the central tungsten density (r/a = 0.2) is steadily increasing. On the other hand the mid-radius tungsten density is decreasing. When the ECRH power is completely switched off, a strong accumulation is observed first in the central region and then at mid-radius which causes a collapse of the electron temperature. Prior to this accumulation phase, it is also observed that the central tungsten density is increasing in between sawtooth crashes. Ion and electron temperature profiles for the two extreme phases of interest, i.e. for PECRH = 200 kW and PECRH = 1.9 MW are also gathered in Fig. 1. The electron density profiles are provided by the integrated data analysis IDA (interferometry+Thomson scattering). The toroidal Mach number and the ion temperature profiles are computed from Charge eXchange Recombination spectroscopy. The electron temperature are given by the Electron Cyclotron Emission measurements. It is shown that the the electron temperature is similar/lower than the ion temperature profile at low ECRH power. On the other hand, for the PECRH = 1.9 MW case, the ratio Te/Ti remains above unity in the radial domain r/a < 0.4. The role of the central electron to ion temperature ratio and the central particle peaking will be further discussed.

Integrated modelling

Central particle source and neoclassical convection

To investigate the impact of different mechanisms on the tungsten transport within this discharge, the integrated modelling framework ASTRA [3] has been used. The turbulent transport is modelled with the linear gyrokinetic code QuaLiKiz [4] and the neoclassical transport with the code NEO [5], both including the impact of toroidal rotation on the fluxes. Regarding the heat and particle sources, the TORBEAM code is used outside the integrated modelling framework to compute the deposited power on the electons for each steady state phases. The NBI deposited power is computed self consistently with the evolved plasma state inside the ASTRA framework, together with the particle source. The latter also includes the Ware pinch computed from NEO. The following channels are then evolved in time in the simulation: the electron temperature and density, the tungsten density, the ion temperature and the current. The toroidal rotation, though included in the simulation as measured by CXRS, is not evolving and is fixed for each steady state phases studied. The goal is to model the central accumulation or non accumulation of tungsten for a given injected ECRH power, and in particular for the two extreme phases of Fig 1 . As the neoclassical convection of W is unfavourable with respect to the electron density peaking, in contrast to temperature peaking which results in the so-called thermal screening, the evolution of the electron density in the simulation is tested for different particle sources. The first assumption follows the computation from the NBI module in ASTRA, giving a centrally peaked particle source. Then a reduction of a factor 2 of this particle source has been used and finally the central part r/a < 0.27 has been switched off. The profiles of these sources are shown in Fig. 2. The resulting predicted density profiles, at equilibrium between transport and sources, yield the central density peaking also shown in Fig. 2. It is found that the NBI central particle source is the main ingredient in setting the peaked density profiles and not the transport alone. Finally, inside r/a = 0.1, the Ware pinch is also an important ingredient as shown in the case where there is no turbulent transport nor particle sources (switched off). For the three particle source shapes described previously, the resulting central tungsten density is also predicted and shown in Fig. 2. It is found that tungsten accumulates in the simulation (up to a concentration of 10 -3 ) only when the nominal particle source is used. For the two other cases, the electron density peaking is not driving sufficiently inward neoclassical tungsten convection to trigger an accumulation. This clearly shows that the central electron density peaking and thus the particle source from the beam is the main ingredient in triggering central tungsten accumulation in this case. The role of the ECRH power regarding the onset of large electron density peaking is investigated in the following.

Core Te/Ti and density pump-out

The role of turbulence in developping peaked density profiles has been shown to be the main ingredient in front of the particle source. However the central accumulation problem is set up in a radial domain where turbulence is not the dominant transport process (central region inside r/a = 0.2) and where the source also becomes important. It is thus important to assess the role of turbulence at different central Te/Ti, i.e. at different injected central electron power. Integrated simulations with ASTRA and QuaLiKiz for the turbulent transport have shown that for the high ECRH power case, Electron Temperature Gradient driven instabilities were destabilised in the core. This results in large electron heat transport and thus central Te/Ti close to 1. This is in contrast to what is observed experimentally, where in the high ECRH case Te/Ti is larger than 1. Such discrepancies with experimental measurements of the ion and electron temperature profiles are shown in Fig. 3. When relaxing the quasilinear weight of ETGs in the turbulent transport, higher Te/Ti are recovered in the simulations, though the values are higher than in the experiment. This shows that a proper evaluation of the electron heat transport through the ETG instabilities have to be assessed which is a complex problem for quasilinear codes. For a proper treatment, nonlinear multiscale gyrokinetic simulations are required which cannot be used in an integrated modelling framework. Nevertheless the impact of Te/Ti and thus of the centrally deposited ECRH power can be underlined in the simulations. Indeed, the turbulent particle diffusion together with the predicted density peaking are shown in Fig. 3 with an without ETGs electron heat transport or in other word for large/low central Te/Ti. It is found that when the central Te/Ti is large, above 1, turbulent diffusion can compensate the central particle source which results in modest central density peaking. This low density peaking is favourable with respect to central neoclassical tungsten accumulation. On the other hand, when Te/Ti is close to 1 or below, the central turbulent diffusion is reduced yielding large centrally located density peaking which in turn results in large inward tungsten neoclassical convection.

Fast turbulent transport model: QuaLiKiz-10D neural network

To go towards real time control of tungsten transport in tokamaks, faster models for turbulent and neoclassical transport are required while retaining as much physics as possible, e.g. toroidal rotation. In this section, a neural network of QuaLiKiz [6] is used within the integrated framework RAPTOR [7]. In this framework, the transport equations for impurity densities are not solved but the interplay between density pump-out and central particle source observed in the previous sections and responsible for tungsten accumulation is investigated. To do so, transport equations for the electron density and temperature, the main ion temperature and the current diffusion are evolved in time. The sources are taken identical to the ASTRA simulations.

(right) Predicted electron density peakings at two radial position for various levels of ECRH power.

A scan in the injected ECRH power is then performed and the resulting temperature profiles are shown in Fig. 4. As expected, Te/Ti increases while increasing the ECRH power. The central density peaking at r/a=0.1 (also shown in Fig. 4) decreases which qualitatively reproduces the density pump-out with Te/Ti observed in ASTRA. At mid-radius, on the other hand, the density peaking increases which is consistent with changes in collisionality from increasing Te [8]. Again it is to be emphasized that the coupling of the central tungsten content (accumulation) and Te through radiation is currently missing in such simulations. Nevertheless, this qualitative agreement between ASTRA/QuaLiKiz and RAPTOR/QLKZ-10DNN is promising for fast tungsten accumulation predictions.

WEST LONG PULSE L-MODE WITH DOMINANT ELECTRON HEATING

To stress the validity of the transport models used to predict tungsten accumulation in AUG, the same tools are used for a WEST long pulse L-mode discharge where no accumulation is observed. This also allows us to cover a wider operational domain, that is considering larger aspect ratio, absence of injected torque and central particle sources from NBI, dominant electron heating from LHCD and ICRH heating systems. These plasmas are particularly relevant considering scenarios of the Pre-Fusion Power Operation phase 1 of ITER where cyclotron resonance heating will be used (20 to 30 MW of ECRH), without neutral beams.

Experimental setup

In this WEST plasma lasting over 30 seconds, the tungsten content is monitored through UV spectroscopy, SXR, bolometry and visible spectroscopy. The magnetic field was at its nominal value of 3.7 T, in upper single null configuration, the plasma current plateau at 400 kA and up to 2.8 MW of LHCD is delivered. It is observed (time traces in Fig. 5) that tungsten does not accumulate throughout this long pulse. More details on the tungsten transport modelling for this plasma can be found in [9]. An increased in the total radiated power is observed during the ICRH heated phase but is not considered in the following analysis. Electron temperature profiles (to be compared to the modelling in the next section) are extracted from Electron Cyclotron Emission (ECE) which makes the measurements reliable only in the region r/a<0.6 due to polluted signal from LHCD non-thermal electrons at larger radii. The electron density profiles were inverted from interferometry measurements using 8 lines of sight. Data consistency checks are performed with the METIS code [10] to compensate the lack of measurements such as the ion temperature profiles where the central Ti is made consistent with the measured DD neutron rate.

Predictive modelling of the electron temperature and density profiles

Flux driven modelling

Flux driven simulations are first considered using ASTRA with QuaLiKiz for the turbulent transport and NEO for the neoclassical transport. These simulations are carried out at 8 seconds during the Ip and LHCD plateaus, prior to the nitrogen injection phase. An off-axis LHCD power deposition has been assumed, at ρ = 0.2, with a Gaussian width of 0.3 and a current drive efficiency of 5 x 10 18 A/W/m 2 . It is found (Fig. 6) that the electron temperature profile is overestimated while the density profile is underestimated. This results from low turbulent electron heat flux with respect to the electron temperature gradient due to stabilised Trapped Electron Modes.

Gradient driven modelling: collisional stabilisation of Trapped Electron Modes

To further investigate discrepancies observed in the flux driven simulations, gradient driven simulations are performed with the gyrokinetic codes QuaLiKiz and GKW [11]. Parameters for such simulations are gathered in Tab. 1. A quasilinear approach is adopted for the two code comparisons. Table 1: Parameters taken at 8 seconds and used for gradient driven simulations with QuaLiKiz and GKW. The particle flux is then computed and the condition Γ e = 0 (no particle source and negligible Ware pinch) is found for the two codes and shown in the 2D space R/Ln and R/LTi in Fig. 7. While GKW recovers conditions close to the experimental R/Ln and R/LTi, QuaLiKiz finds much lower R/Ln fulfilling the condition Γ e = 0. This is also consistent with the flux driven simulations where the modelled density peaking (with QuaLiKiz) was much lower than the experimental one. A collisionless case with reduced Trapped Electron Mode drive 𝑅 𝐿𝑇𝑒 = 0 is also investigated. In this case, a good agreement is found between GKW and QuaLiKiz for the condition Γ e = 0. This underlines the role of the ∇𝑇𝑒 TEM and collisions in the correct predictions of turbulent transport when dominant electron heating is used. Further development of the collision operator in QuaLiKiz have been performed [12] and preliminar results look promising in this strong TEM regime.

CONCLUSIONS

Reduced kinetic-theory-based models for transport modelling in view of plasma scenario optimisation and tungsten accumulation avoidance, are proven to be powerful and accurate tools. Applied to an ASDEX Upgrade discharge with NBI heating it was shown with ASTRA and QuaLiKiz that the competition between turbulent particle diffusion (enhanced with Te/Ti) and the central particle source from the neutral beams were the key ingredients for neoclassical tungsten accumulation modelling. The qualitative behaviour of the central density peaking with Te/Ti was also reproduced with faster transport models, i.e. QuaLiKiz 10DNN paving the way towards real-time tungsten transport predictions.

In specific regimes relevant for ITER operations, such as the ones encountered in the WEST tokamak, i.e. low toroidal rotation, dominant electron heating, additional development of reduced transport models are still required with constant validation against first principle modelling. Indeed, it was shown that in these regimes, a proper treatment of collisional stabilisation of Trapped Electron Modes are necessary

Figure 1 :

 1 Figure 1: (left top panel) Time traces of the NBI, radiated and ECRH power for #32408. The two time windows scrutinized in the modelling section are highlighted. (left bottom panel) Time traces of the two radial locations of the W density reconstructed from the UV grazing incidence spectroscopy. (right) Ion and electron temperatures for the two time windows, at high and low ECRH injected power.

Figure 2 :

 2 Figure 2: (left top panel) Assumptions used for the central particle source in the integrated modelling of the W accumulation phase. (left bottom panel) Resulting electron density profiles. (right) Simulated central tungsten density, using the different assumptions for the particle source.

Figure 3 :

 3 Figure 3: (left) Modelled and experimental ion and electron temperature profiles, with and without ETG contributions in the computed electron heat flux. (right) Turbulent particle diffusion and resulting central density peaking.

Figure 4 :

 4 Figure 4: (left) Electron temperature profiles predicted with RAPTOR and QuaLiKiz 10D neural network. (right) Predicted electron density peakings at two radial position for various levels of ECRH power.

Figure 5 :

 5 Figure 5: Time traces of several quantities for the WEST long pulse #54178.

Figure 6 :

 6 Figure 6: Predicted electron temperature and density profiles against experimental fitted profiles.

Figure 7 :

 7 Figure 7: Comparison of the 𝛤 𝑒 = 0 condition between GKW (solid lines) and QuaLiKiz (dashed lines) for the nominal case (left) and a simpler case with 𝑅 𝐿𝑇𝑒 = 0 and the electron-ion collisionality 𝜈 = 0 (right).

ACKNOWLEDGEMENTS

This work has been carried out within the framework of the EUROfusion Consortium and has received funding from the Euratom research and training programme 2014-2018 and 2019-2020 under grant agreement No 633053. The views and opinions expressed herein do not necessarily reflect those of the European Commission.


Abdelhak Djemel

Olaf Stefanczyk

Cédric Desplanches

Kunal Kumar

Rachid Delimi

Farouk Benaceur

Shin-Ichi Ohkoshi

Guillaume Chastanet
email: guillaume.chastanet@icmcb.cnrs.fr

Switching on thermal and light-induced spin crossover by desolvation of [Fe(3-bpp) 2 ](XO 4 ) 2 •solvent (X = Cl, Re) compounds †

published or not. The documents may come

Switching on thermal and light-induced spin crossover by desolvation of [Fe(3-bpp)2](XO4)2×solvent (X = Cl, Re) compounds

Introduction

The research and development of molecular switchable materials is one of the major challenges in modern material chemistry due to their potential application in the construction of novel classes of data storage and processing devices and sophisticated sensors dedicated for specific applications. [START_REF]Spin Crossover in Transition Metal Compounds I-III[END_REF] In this respect, the molecular complexes exhibiting spin crossover (SCO) appear to be the closest to practical applications. 2 Typical SCO systems are based on pseudo-octahedral Fe(II) complexes ([Ar]3d [START_REF] Cook | Spin state behavior of iron(II)/dipyrazolylpyridine complexes. New insights from crystallographic and solution measurements[END_REF] ). They are able to switch between a highspin (HS) paramagnetic state with spin S = 2, and a low-spin (LS) diamagnetic state with spin S = 0, under the influence of external physical (temperature -T, pressure -P, magnetic field -H, light -hν) and chemical (chemical potential -μ, pH, absorption or desorption of host molecules) stimuli. [START_REF] Brooker | Spin crossover with thermal hysteresis: practicalities and lessons learnt[END_REF] These switching processes not only lead to the alteration of their magnetic properties but also alter many other physical properties (e.g., optical properties, conductivity, and heat capacity). [START_REF] Ridier | Unprecedented switching endurance affords for high-resolution surface temperature mapping using a spincrossover film[END_REF] It is worth pointing out that the spin crossover phenomenon is not exclusive for Fe(II) systems. In less common cases it can also be detected for other metal complexes with 3d 4 (i.e. Cr(II), Mn(III)), 3d [START_REF] Fitzpatrick | Electronic vs. structural ordering in a manganese(III) spin crossover complex[END_REF] (i.e. Mn(II), Fe(III)), 3d 6 (i.e. Co(III)), and 3d 7 (i.e. Co(II), Ni(III)) configurations. [START_REF] Fitzpatrick | Electronic vs. structural ordering in a manganese(III) spin crossover complex[END_REF] To promote spin crossover at room temperature, many families of compounds have been explored such as the 0D-coordination complexes based on the bpp tridentate ligand (bpp being either 2,6-di-(1H-pyrazol-3yl)pyridine (3-bpp) or 2,6-di-(1H-pyrazol-1-yl)pyridine (1-bpp)), [START_REF] Cook | Spin state behavior of iron(II)/dipyrazolylpyridine complexes. New insights from crystallographic and solution measurements[END_REF] the 1D-coordination polymer based on triazole ligands [START_REF] Roubeau | Triazole-Based One-Dimensional Spin-Crossover Coordination Polymers[END_REF] or the 2D and 3D-coordination networks of the Hoffman family. 3c The main strategy followed in the design of these families was to favour strong intermolecular interactions in order to promote a cooperative character of the spin crossover at the origin of memory effects. 8 Apart from coordination polymers that intrinsically afford cooperative interactions within the coordination network, molecules able to promote hydrogen bonding or π-π stacking for instance are highly investigated.

Fe(II) complexes with the 3-bpp ligand are known to exhibit promising properties (e.g., near-room temperature transitions, [START_REF] Marcén | Critical temperature of the LIESST effect in a series of hydrated and anhydrous complex salts [Fe(bpp)2]X2[END_REF][START_REF] Jornet-Molla | A Reversible Hydrogen-Bond Isomerization Triggered by an Abrupt Spin Crossover near Room Temperature[END_REF][START_REF] Jornet-Mollá | Synthesis, Structure, and Photomagnetic Properties of a Hydrogen-Bonded Lattice of [Fe(bpp)2] 2+ Spin-Crossover Complexes and Nicotinate Anions[END_REF] thermal hysteresis [START_REF] Sung | A Study of the Spin-State Transition and Phase Transformation in[END_REF][START_REF] Marcén | Critical temperature of the LIESST effect in a series of hydrated and anhydrous complex salts [Fe(bpp)2]X2[END_REF][START_REF] Jornet-Molla | A Reversible Hydrogen-Bond Isomerization Triggered by an Abrupt Spin Crossover near Room Temperature[END_REF][START_REF] Djemel | Solvatomorphism-Induced 45 K Hysteresis Width in a Spin-Crossover Mononuclear Compound[END_REF][START_REF] Jornet-Molla | Interplay between spin crossover and proton migration along short strong hydrogen bonds[END_REF] ) and solvent dependent behaviours. 10,12d,e,13a Indeed, the cooperative and hysteretic transformation principally promoted by elastic interactions between complexes through weak interactions (van der Waals interactions, hydrogen bonds, π-π stacking), is strongly stimulated by the trisheterocyclic N,N,N-tridentate 3-bpp ligand with several aromatic rings, ready to form π-π, anion-πring and σsolvent⋯πring interactions, as well as up to four Nring-H⋯X hydrogen bonds per one [Fe(3-bpp)2]. This finds practical confirmation in the observation of the 14 K-wide thermal hysteresis loop for [Fe(3-bpp)2](triflate)2•H2O for instance. [START_REF] Sung | A Study of the Spin-State Transition and Phase Transformation in[END_REF] For such [Fe(3-bpp)2] 2+ complexes, there are two main ways of modulating the intermolecular interactions network. The first one is the impact of solvation on spin crossover characteristics. 10,12d,e,13a This stems from the fact that solvent molecules present in the crystal structures are deeply involved in the intermolecular interactions. Frequently, more cooperative transitions occur after desolvation; however, it comes with a significant decrease in the spin transition temperature (T1/2) -the temperature at which half of the complex is in a LS state. For instance, [Fe(3-bpp)2]I2•4H2O exhibits a gradual SCO at T1/2 > 320 K, the same material with two water molecules shows an abrupt SCO at T1/2 = 256 K, while the anhydrous one reveals an abrupt SCO at T1/2 = 204 K and narrow thermal hysteresis. [START_REF] Marcén | Critical temperature of the LIESST effect in a series of hydrated and anhydrous complex salts [Fe(bpp)2]X2[END_REF] In the most extreme case, the pristine diamagnetic compound [Fe(3bpp)2](triflate)2•3H2O, after the loss of two water molecules, exhibits an abrupt SCO with large thermal hysteresis (T ↓ 1/2 = 147 K and T ↑ 1/2 = 285 K). [START_REF] Sung | A Study of the Spin-State Transition and Phase Transformation in[END_REF] A second way to tune the intermolecular interaction network is to vary the anions, also involved in the construction of such an intermolecular network. Up to now, more than 20 types of organic and inorganic anions have been applied in the syntheses of [Fe(3-bpp)2] SCO systems. [START_REF] Sung | A Study of the Spin-State Transition and Phase Transformation in[END_REF][START_REF] Marcén | Critical temperature of the LIESST effect in a series of hydrated and anhydrous complex salts [Fe(bpp)2]X2[END_REF][START_REF] Jornet-Molla | A Reversible Hydrogen-Bond Isomerization Triggered by an Abrupt Spin Crossover near Room Temperature[END_REF][START_REF] Jornet-Mollá | Synthesis, Structure, and Photomagnetic Properties of a Hydrogen-Bonded Lattice of [Fe(bpp)2] 2+ Spin-Crossover Complexes and Nicotinate Anions[END_REF][START_REF] Djemel | Solvatomorphism-Induced 45 K Hysteresis Width in a Spin-Crossover Mononuclear Compound[END_REF][START_REF] Jornet-Molla | Interplay between spin crossover and proton migration along short strong hydrogen bonds[END_REF][START_REF] Baldé | The role of iron(II) dilution in the magnetic and photomagnetic properties of the series [Fex,Zn1-x(bpp)2[END_REF][START_REF] Sugiyarto | Spin transition centres linked by the nitroprusside ion. The cooperative transition in bis(2,6-bis(pyrazol-3-yl)pyridine)iron(II) nitroprusside[END_REF][START_REF] Buchen | Non-First-Order Kinetics of the High Spin-Low Spin Relaxation in [Fe(bpp)2](BF4)2 after LIESST and Thermal Spin Trapping[END_REF][START_REF] Halcrow | Relationship between the Molecular Structure and Switching Temperature in a Library of Spin-Crossover Molecular Materials[END_REF][START_REF] Jornet-Molla | Temperature dependence of desolvation effects in hydrogen-bonded spin crossover complexes[END_REF] This results in the following series of anions ordered according to increasing temperatures T1/2 for the most solvated phases of Fe(II) complexes: [Fe(CN)5(NO)] 2-(183 K) [START_REF] Sugiyarto | Spin transition centres linked by the nitroprusside ion. The cooperative transition in bis(2,6-bis(pyrazol-3-yl)pyridine)iron(II) nitroprusside[END_REF] 10b From this series it can be concluded that the halides and oxygen-rich anions are the most promising counterions for the observation of room temperature SCO. However, above room temperature switching might also be promoted by a concomitant desolvation. [START_REF] Jornet-Molla | Temperature dependence of desolvation effects in hydrogen-bonded spin crossover complexes[END_REF] This desolvation also strongly impacts the lifetime of the photo-induced state. Indeed, in special cases, Fe(II) complexes can reveal a light-induced excited spin-state trapping (LIESST) effectswitching between the LS ground state and the metastable HS state after irradiation with light at low temperature, at which the metastable HS state exists long enough to be detected in the experiment. [START_REF] Decurtins | Light-induced excited spin state trapping in a transition-metal complex: The hexa-1-propyltetrazole-iron(II) tetrafluoroborate spin-crossover system[END_REF] The lifetime of the photo-induced state can be estimated through the measurement of the relaxation temperature T(LIESST). [START_REF] Chastanet | A critical review of the T(LIESST) temperature in spin crossover materials -What it is and what it is not[END_REF] This relaxation temperature is linked to T1/2 according to the inverse energy gap law: if upon desolvation T1/2 decreases therefore, T(LIESST) should increase. 10a As part of our long term study of such [Fe(3-bpp)2] 2+ complexes, [START_REF] Djemel | Solvatomorphism-Induced 45 K Hysteresis Width in a Spin-Crossover Mononuclear Compound[END_REF][START_REF] Baldé | The role of iron(II) dilution in the magnetic and photomagnetic properties of the series [Fex,Zn1-x(bpp)2[END_REF] we focused on [Fe(3-bpp)2](XO4)2•S compounds with X = Cl and Re. The perchlorate-based compound showing gradual SCO above room temperature for the dehydrated phase has already been tested by Goodwin et al. in 1988, 12e but magnetic data need to be re-measured using more sophisticated equipment and missing structural, spectroscopic and photomagnetic data should be completed. Consequently, we decided to investigate this compound from the crystal structure, spectroscopic, magnetic and photomagnetic points of view, on the solvated and desolvated phases, and to compare its features to a newly synthesized perrhenate derivative. [START_REF] Shakirova | Spin Crossover in New Iron(II) Coordination Compounds with Tris(pyrazol-1-yl)Methane[END_REF] This allowed us to determine the impact of anion substitution and desolvation on SCO and LIESST properties of these two compounds. As a result, we obtained and comprehensively characterised [Fe(3-bpp) (2•sol) and their desolvated phases which will be described in detail later in this work.

< BPh4 -(ca. 200 K) 12c < NCS -(227 K) 10a,15d ≈ NCSe -(230 K) 10a,15 ≈ [Au(SCN)2] -(232 K) 12c < [Ag(CN)2] -(251 K) 12c < BF4 -(288 K) 10 ≈ [Au(CN)2] -(291 K) 12c < PF6 -(ca. 300 K) 10b,12e < ClO4 -(above 313 K) 12e < I -(above 320 K) 10b < Br -(340 K) 10 < NO3 -(ca. 350 K).
2](ClO4)2•2.5H2O•MeOH (1•sol) and [Fe(3-bpp)2](ReO4)2•3H2O

Results and discussion

Syntheses

The first preliminary attempts to obtain monocrystalline [Fe(3-bpp)2](ClO4)2•solvent compounds were carried out by Sugiyarto and co-workers, using water as the solvent. 12e Contrary to the previous method, in the present study, a methanolic solution of Fe(II) chloride and 3-bpp ligand was treated with an aqueous solution of NaClO4, resulting in the formation of dark red prismatic crystals [Fe(3-bpp)2](ClO4)2•2.5H2O•MeOH (1•sol). The analogous synthesis, replacing NaClO4 by KReO4, yields dark red prismatic crystals of the [Fe(3-bpp)2](ReO4)2•3H2O complex (2•sol). At this point, it is worth emphasizing the lack of chlorides in the final products, despite their presence in the mother liquors. This directly confirms a stronger affinity of the O-donor anions than the halogen-donor ones and the efficiency of the anion exchange, most likely thanks to the formation of stronger hydrogen bond interactions between 3-bpp ligands and anions (hydrogen bond acceptors).

Thermal stability, colour changes and spectroscopy

In order to determine the composition and thermal stability of both compounds, thermogravimetric analyses were performed in dry nitrogen flow on pristine samples 1•sol and 2•sol in the temperature range of 20-400 °C with stabilisation at 25 °C for 1 hour (ESI, Fig. S1 †). After isothermal desolvation at 25 °C, 1•sol loses half methanol and two water molecules (6.9% of the initial mass), and 2•sol releases 1.25 water molecules (2.2% of the primary mass). A further increase in temperature resulted in the release of additional 1.5 water molecules from 1•sol (3.6% of the original mass) at just above 54.5 °C and the loss of the residual two water molecules from 2•sol (3.8% of initial mass) in two steps at 50.5 °C and 66 °C. Above 75 °C, 1•sol and 2•sol reach an anhydrous state [Fe(3-bpp)2](XO4)2 (X = Cl, 1 and Re, 2). Next, both samples are stable up to 220 °C and 310 °C for 1 and 2, respectively. Above these temperatures perchlorates and 3-bpp start to decompose. The total weight losses during desolvation are estimated to be around 10.5% and 6.0%, which correspond to the release of approximately 0.5 MeOH and 3.5 H2O for 1•sol and 3.25 H2O for 2•sol. These values are slightly different from those estimated on the basis of elemental analysis (1 MeOH and 1.75 H2O for 1•sol and 2.5 H2O for 2•sol) and X-ray crystallography (1 MeOH and 3 H2O for 1•sol and 3 H2O for 2•sol); however they are still within the limits of detection of each technique. Moreover, the amounts of solvent molecules detected by thermogravimetric and elemental analyses may be disturbed by the inevitable evolution of the sample composition during the sample preparation stage. Thus, in this manuscript, the amount of solvent molecules with the chemical formula of [Fe(3-bpp)

2](ClO4)2•2.5H2O•MeOH (1•sol) and [Fe(3-bpp)2](ReO4)2•3H2O (2•sol) is determined based on the most reliable X-

ray crystallography technique (vide infra).

Additionally, it should be noted that the desolvation is accompanied by a colour change from dark red for the solvated forms to orange-yellow when heated to 120 °C for the desolvated forms. The yellow powder remains unchanged after 2 hours at room temperature for 1 but turns back to red for 2 (ESI, Fig. S2 and S3 †). In order to understand the causes of colour changes, infrared (IR) and ultraviolet-visible-near infrared (UV-Vis-NIR) spectroscopy studies were performed at room temperature for the different phases (ESI, Fig. S4 and S5 †).

The IR spectra in KBr of 1•sol and 2•sol are similar (ESI, Fig. S4 †) with characteristic absorption peaks of water molecules (intense broad peaks around 3500 cm -1 and weak sharp peaks at 1640 cm -1 ), the 3-bpp ligand (broad peaks in the 3400-2500 cm -1 range and numerous peaks in the 1600-500 cm -1 "fingerprint" region) and the anions (perchlorate in 1•sol -two intense peaks with maxima around 1100 and 620 cm -1 , and perrhenate in 2•sol a single intense peak at ca. 900 cm -1 ). Interestingly, the IR spectra of both samples after 24 hours of storage in air under ambient conditions (T = 25 °C, relative humidity 40 ± 5%) indicate a decrease of [ν(O-H)] and [γ(O-H)] absorption bands for 1•sol, which is a consequence of sample desolvation during storage, while 2•sol is stable in air. Moreover, both samples after 2 hours of heating at 120 °C are almost completely desolvated which is inferred from the lack of characteristic water absorption peaks after such a thermal treatment, in agreement with TGA analysis.

Complementary studies of solid-state UV-Vis-NIR spectra of 1•sol and 2•sol for pristine, ambient condition stored and desolvated forms gave better insight into the spin state of the Fe(II) centre for each phase (ESI, Fig. S5 †). High-spin octahedral Fe(II) complexes are known to have one low-intense broad spin-allowed d-d transition ( 5 T2g → 5 Eg) in the Vis/NIR region, while low-spin octahedral Fe(II) complexes are characterised by two intense bands from 1 A1g → 1 T2g and 1 A1g → 1 T1g transitions in the UV/Vis region. [START_REF]Spin Crossover in Transition Metal Compounds I-III[END_REF] The absorption spectra for both fresh 1•sol and 2•sol are very similar with a low intensity broad metal-to-ligand charge transfer (MLCT) band in the NIR range (I) with a maximum of about 800 nm, several strong visible light absorption bands (II) with maxima at around 410, 460, 535 and 620 nm, and two intense π-π* ligand bands in the UV region (III) with maxima at 250 and 310 nm. Spectra of 1•sol and 2•sol are in agreement with the intense colour of crystals. Interestingly, after 1 day of storage under ambient conditions, 1•sol shows a decrease in absorption bands around 535 nm (consisting of MLCT and 3-bpp ligand-localized transitions) and a slight increase in the broad absorption band in the NIR region, indicating a change of the local environment of Fe(II) centres, probably correlated with the slow conversion of LS to HS spin state Fe(II) centres. Concerning 2•sol, no changes were observed after 24 h hours of storage under ambient conditions, which proves the durability of the material. Heating the samples at 120 °C resulted in a significant reduction in the intensity of all bands in the II range and the appearance of new bands with a maximum above 1000 nm ( 5 T2g → 5 Eg d-d transition), witnessing the decrease of the LS state in favour of the HS state of Fe(II) at room temperature.

In order to better understand the solid-state optical spectra of 1•sol and 2•sol and their evolution during the desolvation process, additional quantum chemical calculations (the single point DFT and TD-DFT calculations) were performed for [Fe(3-bpp)2] 2+ units of 1•sol and 2•sol and the HS state reference system of [Fe(3bpp)2][Au(CN)2]2•2H2O 13a (see the ESI † for more details). First of all, these studies confirmed a low spin state of 1•sol and 2•sol, which can be easily identified from the spin unrestricted energy level diagrams and related molecular orbitals (ESI, Fig. S8 and S9 †). Moreover, the ground state to the lowest 100 electronic excited states were calculated with oscillator strength magnetic moment and constituting molecular orbitals, allowing to calculate absorption spectra for all systems (ESI, Fig. S7 †). The calculated spectra of 1•sol and 2•sol are very similar to each other with several absorption bands consisting of LMCT and d-d transition ( 1 A1g → 1 T2g and 1 A1g → 1 T1g) in combination with MLCT and 3-bpp ligand-localized transitions. The spectrum of the HS state reference system is different with mainly MLCT and 3-bpp ligand-localized transitions, and d-d transition ( 5 T2g → 5 Eg) in the lower energy region. Moreover, the calculated spectra are shifted by ca. 200 nm in relation to the experimental spectra due to the applied single-point DFT calculation method, which despite omitting the presence of anions and solvent molecules and the periodicity of the structure in the solid phase, still well reproduce the experimental spectra.

As a summary, all of these studies are consistent and provide a unified image of the processes occurring in 1•sol and 2•sol during desolvation. Both fresh compounds contain low-spin Fe(II) complexes, hence the colours observed are dark red at 20 °C. When stored at room temperature, 1•sol spontaneously loses most of the solvent molecules and turns to orange, while 2•sol remains stable in this condition. A complete desolvation can be achieved by heating the samples at 120 °C and their colours evolve to yellow and orange for 1 and 2, respectively. This effect is associated with spin crossover as evidenced by UV-Vis spectrometry. Both compounds clearly show different thermal behaviours which we have investigated further by magnetometry (vide infra).

X-ray crystal structure analysis

The crystal structures of 1•sol and 2•sol were determined by single-crystal X-ray diffraction measurements. For 1•sol, being more sensitive to desolvation under ambient conditions, data collection was performed at 90 and 250 K and 250 K for 2•sol. Both compounds crystallize in the triclinic space group P , and their crystallographic parameters are listed in ESI, Table S1. † The asymmetric units of both complexes are almost identical (Fig. 1) and consist of the Fe(II) ion coordinated by two 3-bpp organic ligands, two ClO4 -and 2. 13) and 1.712( 6) Å for 1•sol and 2•sol, respectively, and the ClO4 -anions, even at 90 K, are much more disordered than ReO4 -ones at 250 K. (d). Legend to all figures: C -grey, Cl -green, Fe -yellow, N -blue, O -red, and Re -violet. For clarity, hydrogen atoms have been omitted in all figures. 2) and 2.747(2) Å, respectively. Moreover, the mean hydrogen bond lengths between the solvent molecules are 2.763(2) and 2.861(2) Å for 1•sol and 2•sol, respectively. Interestingly, the hydrogen bonds involving perrhenate are shorter than perchlorate ones.

Crystal

Unfortunately, under desolvation, crystals lose their crystallinity and the crystal structure of the desolvated phases was not reachable. In order to better understand the desolvation-induced transformation of 1•sol and 2•sol, powder X-ray diffraction studies were performed at T = 25 °C in a relative humidity of 40 ± 5% (ESI, Fig. S11 †). The perfect match between the measured diffractograms and those calculated from the X-ray structures confirms the homogeneity and phase purity of the pristine samples 1•sol and 2•sol used in all experiments. Moreover, diffractograms measured at 10-minute intervals for fresh samples indicate that 1•sol loses solvent molecules practically after the first scan, reaching the new partially desolvated stable phase within 2 hours, while 2•sol does not change. These results are in line with TGA and spectroscopic measurements. Finally, powder X-ray diffraction measurements for thermally desolvated samples at 120 °C for 2 hours (1 and 2) confirmed that the materials were stable under the experimental conditions (do not return to 1•sol and 2•sol phases), and showed lower crystallinity manifested by broadening of the diffraction peaks.

Fig. 2 The π-π stacking of 1•sol (a) and 2•sol (b) (d). The numbers correspond to the distances between the centroids of the aromatic rings and the hydrogen and oxygen atoms directly located above them.

located in the ab-plane. The dashed lines represent the paralleldisplaced (red lines) and the T-shaped π-π stackings (green lines). The anion-π interactions (blue dashed lines) and the σ⋯π interactions (green dashed lines) around [Fe(3-bpp)2] 2+ entities of 1•sol (c) and 2•sol

Magnetic properties

These experiments were performed using a vibrating sample magnetometer (VSM) under a nitrogen flow with a 2 K min -1 sweep rate for polycrystalline samples of 1•sol and 2•sol. The magnetic behaviour of 1•sol in the temperature range (120-370 K) is shown in Fig. 3c. Initially, the product χMT at a low temperature of 120 K (0.1 cm 3 K mol -1 ) is characteristic of the low-spin state of the Fe(II). With increasing temperature, the magnetic response increases gradually from around 260 K up to 363 K to reach a χMT value of 3.5 cm 3 K mol -1 , which corresponds to all Fe(II) centres thermally converted into a high-spin state. After cooling, the magnetic signal gradually decreases until it reaches 0.1 cm 3 K mol -1 at 120 K, which means that Fe(II) is in a low-spin state, but it does not follow the initial curve recorded upon warming. The following thermal cycles superimposed on this second curve, demonstrating the irreversibility of the first warming. The first transition at 340 K can therefore be attributed to the desolvation observed by thermogravimetric analysis (ESI, Fig. S1 †). Compound 1 exhibits, after desolvation, a gradual spin transition with T1/2 ≈ 210 K which confirms and complements the earlier observations given by Goodwin et al. for the desolvated sample of [Fe(3-bpp)2](ClO4)2. 12e These results demonstrate, on one hand, that the phenomenon of spin conversion is associated with the complex desolvation process, and on the other hand, that the desolvated compound [Fe(3-bpp)2](ClO4)2 (1) exhibits a gradual and complete spin crossover. It should be noticed that the derivative curve does not show a symmetric shape. This might be an indication that several sites are switching, as suspected from UV-Vis studies. It is however difficult to conclude without the crystal structure of this desolvated phase. In order to check the stability of this desolvated form, we restudied the magnetic properties of the same sample resulting from the VSM treatment and kept in a container for 5 days. The results shown in Fig. 3c evidence that the two measurements are almost identical which confirms the stability of the desolvated form, when it is stored in the air. Measurements of the χMT product as a function of temperature were also performed for compound 2•sol (Fig. 3d). At room temperature, the χMT product (0.05 cm 3 K mol -1 ) is characteristic of Fe(II) in a low-spin state. An increase in temperature above 350 K causes an abrupt change in the magnetic response followed by a more gradual change upon additional warming. The χMT product reaches 3.2 cm 3 K mol -1 at 410 K, which corresponds to Fe(II) centres converted into a high-spin state. As the temperature is lowered, χMT gradually decreases down to 150 K to reach 0.1 cm 3 K mol -1 , without overlapping the first warming curve, showing the irreversibility of this first warming. Subsequent cycles show a reproducible gradual conversion. As for 1•sol, the first heating (red curve) causes a desolvation that changes the magnetic properties of 2•sol. Compound 2 exhibits a gradual a reversible SCO with a transition temperature T1/2 = 304 K. Furthermore, 2 also exhibits good air stability confirmed by magnetic measurement carried out on the material kept in the open thin-walled quartz tube for 5 days.

In conclusion, the analysis of the magnetic properties revealed similar desolvation-assisted SCO effects for both compounds, occurring at 340 and 350 K for 1•sol and 2•sol, respectively. After complete desolvation, both systems exhibit a reversible gradual spin conversion with T1/2 ≈ 210 K and 304 K for 1 and 2, respectively. Remarkably, replacing the perchlorate with perrhenate increased the transition temperature by 85 K, moving it slightly above the room temperature region. This implies that the same approach in other SCO systems, based on different organic ligands, could be equally promising. Moreover, the comparison of the T1/2 values for the desolvated 1, 2 and previously reported [Fe(3-bpp)2]X2 systems led to the completion of a series of anions, ordered according to increasing temperatures T1/2, for the desolvated phases of iron(II) complexes: BF4

-(175 K) 10a,17 ≈ PF6 -(177 K) 10 < I -(204 K) 10b < 1-ClO4 -(210 K) ≈ NCSe -(231 K) 10a,15 < [Ag(CN)2] -(251 K) 12c ≈ Br -(252 K) 10 < [Au(CN)2] -(291 K) 12c < 2-ReO4 -(304 K)
, and a discovery of the highest value of T1/2 for 2 among the known desolvated [Fe(3-bpp)2] systems.

Photomagnetic properties

The effect of light irradiation at low temperature was studied on the solvated (1•sol and 2•sol) and desolvated and 2) forms at 10 K in the cavity of the SQUID magnetometer coupled by optical fibre to a laser diode emitting at 650 nm light (P = 5 mW cm -2 ). Fig. 3a shows the study of the photomagnetic properties carried out on the pristine phase of 1•sol. At 10 K, the sample is initially in the low-spin state. Under the effect of irradiation, the magnetic response increases strongly (ESI, Fig. S12a †), which is interpreted by the population of a metastable high-spin state of Fe(II) centres. In the photostationary state, the χMT product reaches 3.5 cm 3 K mol -1 . During the measurement of the relaxation temperature T(LIESST), the magnetic response of compound 1•sol remains almost unchanged up to 40 K then gradually drops to reach its initial value before photoexcitation. The curve shows a shoulder leading to the estimation of two values of T(LIESST) of 67 and 82 K. This shoulder might come from a partial desolvation occurring during the SQUID ventilation steps necessary to remove atmospheric oxygen and overcome its magnetic response around 50 K (20 minutes purge at room temperature). An attempt was made to study the photomagnetic properties on the desolvated phase (sample from magnetic analysis in VSM). The LIESST effect was observed with T(LIESST) = 66 K (the inset of Fig. 3a). The major difference between the two experiments concerns the shape of the T(LIESST) curves, which is more gradual for the solvated derivative than for the desolvated one. The T(LIESST) of 66 K, close the value observed for the shoulder in 1•sol supports that this shoulder comes from a partially desolvated sample.

In the case of 2•sol, no LIESST effect was observed on the solvated compound. An attempt to desolvate the material was performed at 380 K, then another T(LIESST) experiment was performed (Fig. 3b and ESI, Fig. S12b †). After irradiation, almost all molecules are converted to the metastable high-spin state. This irradiation is then stopped and the T(LIESST) is recorded. The T(LIESST) value of 50 K is obtained (the inset of Fig. 3b). The lower T(LIESST) value compared to compound 1 may be due to the fact that the transition temperature T1/2 is higher for 2 (304 K) than for 1 (210 K). In accordance with Hauser's inverse energy law and the T(LIESST) database vs. T1/2 by Létard, a higher T1/2 favours low T(LIESST), for the same family of compounds. [START_REF] Létard | A Guideline to the Design of Molecular-Based Materials with Long-Lived Photomagnetic Lifetimes[END_REF] This is also supported by slightly lower T(LIESST) values for 1 and 2 than reported ones of 75 K for [Fe(3-bpp)2](NCSe)2 (T1/2 = 231 K), 10a,15 77 K for [Fe(3-bpp)2]Br2 (T1/2 = 252 K) 10a and 82 K for [Fe(3-bpp)2](PF6)2 (T1/2 = 177 K). 10a (2•sol) were synthesised as a result of self-assembly of Fe(II) with 3-bpp in the presence of perchlorate and perrhenate anions for 1•sol and 2•sol, respectively. Fresh compound 1•sol is susceptible to partial desolvation in air at room temperature, while 2•sol is air stable. Moreover, both samples after thermal treatment at 120 °C were completely desolvated and could be stored under ambient conditions for a long time without loss of quality. All desolvation processes are accompanied by a colour change from dark red to yellow, and the evolution of IR and UV-Vis-NIR spectra suggests the occurrence of a desolvation-assisted process. X-ray diffraction studies have shown that the structures of both pristine materials are similar and consist of layers of complex cations [Fe(3bpp)2] 2+ separated by solvent molecules and charge-compensating XO4 -anions (X = Cl, 1•sol and Re, 2•sol). All of these building blocks are linked by complex networks of π-π, anion-π and σ⋯π interactions, and hydrogen bonds. Magnetic measurements indicate that 1•sol and 2•sol show desolvation-assisted SCO from the low-spin (LS, S = 0) to high-spin (HS, S = 2) state at 340 and 350 K, respectively, followed by a reversible gradual spin conversion with T1/2 ≈ 210 and 304 K for 1 and 2, respectively. Interestingly, the 304 K transition temperature for 2 is the highest value among the established desolvated [Fe(3-bpp)2] systems. Finally, photomagnetic studies confirmed the LIESST phenomenon with T(LIESST) = 82 K and 66 K for 1•sol and 1 phases, respectively. This strongly differs from the perrhenate derivative for which the LIESST effect is exclusively observed for the desolvated sample 2 with T(LIESST) ≈ 50 K. This is the first observation of the photomagnetic effect in a perrhenate-based spin crossover system, an effect that is switched on by desolvation.

Conclusions

Novel compounds [Fe(3-bpp)2](ClO4)2•2.5H2O•MeOH (1•sol) and [Fe(3-bpp)2](ReO4)2•3H2O

Experimental section

Materials

Reagents were purchased from commercial sources (Sigma-Aldrich and Alfa Aesar) and used without further purification. The 2,6-di-(1H-pyrazol-3-yl)pyridine ligand (3-bpp) was prepared according to a literature procedure. [START_REF] Lin | Novel two step synthesis of pyrazoles and isoxazoles from aryl methyl ketones[END_REF] Caution! We did not encounter any problems during our studies; nevertheless, the perchlorate salt compounds are potentially explosive and should be handled with care.

Synthesis of [Fe

(3-bpp)2](ClO4)2•2.5H2O•MeOH (1•sol) and [Fe(3-bpp)2](ReO4)2•3H2O (2•sol)
The complexes 1•sol and 2•sol were obtained by following the same procedure in a nitrogen atmosphere: solid FeCl2•4H2O (20 mg, 1 mmol) was added to a 6 ml methanol solution of 3-bpp ligand (42.2 mg, 2 mmol), resulting in the formation of a dark red clear solution. After a few minutes of stirring, a methanol solution of [Fe(3bpp)2]Cl2 was treated with 6 ml of an aqueous solution of NaClO4 (24.5 mg, 2 mmol) or KReO4 (57.9 mg, 2 mmol) for 1•sol and 2•sol, respectively. The resulting dark red solution was allowed to evaporate slowly at room temperature, forming dark red crystals. After two weeks, both products were collected. Pristine crystals of 1 have the tendency to lose methanol spontaneously in air, which is associated with the colour change from dark red to orange, thus, it is recommended to not let the solution dry completely and investigate. The average yield is around 75% (50 mg) for 1•sol and 90% (90 mg) for 2•sol. Elemental analysis calculated (%) for C23H25.5Cl2FeN10O10.75 (1•sol-0.25 H2O, 740.8 g mol -1 ): C, 37.29; H, 3.47; N, 18.91; found: C, 37.38; H, 3.74; N, 18.91. Elemental analysis calculated (%) for C22H23FeN10O10.5Re2 (2•sol-0.5 H2O, 1023.8 g mol -1 ): C, 25.81; H, 2.26; N, 13.68; found: C, 25.83; H, 2.47; N, 13.64.

Single crystal X-ray structure determination

Suitable crystals of 1•sol and 2•sol were attached to a MiTeGen microloop using immersion oil under ambient conditions, and were measured on a Rigaku R-AXIS RAPID diffractometer at 90 K for 1•sol and on a Bruker APEX II diffractometer at 250 K for 2•sol, using graphite monochromated MoKα (λ = 0.71075 Å) radiation. Data collection and reduction were performed using a Rigaku RAPID AUTO and a BRUKER SUIT (APEX II) programs for 1•sol and 2•sol, respectively. Structures were solved by direct methods using the SHELXL-T package incorporated in the WinGX-2020.1 crystallographic collective package, then refined against F 2 using all data by full-matrix least squares techniques with SHELXL-2014/7 included in the OLEX-2 1.2 software package. 26a,b Non-hydrogen atoms were refined anisotropically. All hydrogen atoms were positioned with idealized geometry and refined using a riding pattern. The crystal structure of 1•sol was refined as a two-component twin considering the twin law (1 0 0 0-1 0 0 0-1) with a twin scale factor (BASF) of 0.0090 (6). Moreover, in the crystal structure of 1•sol, both perchlorate anions (Cl1 and Cl2) are disordered at two positions with occupancies of 0.55/0.45 and 0.50/0.50 for Cl1 and Cl2, respectively. A similar behaviour is also observed for solvent molecules of 1•sol: the water molecules are disordered with occupancies 0.25 for O1A, 0.75 for O1B, and 0.5 for O2, and the ethanol molecule is disordered with occupancy 0.5. All figures were prepared with Mercury 3.9 CCDC visualization software. 26c Crystal data, data collection, and refinement parameters for 1•sol and 2•sol are listed in ESI, Table S2. † CCDC 2073012 and 2073013 † contain the supplementary crystallographic data for 1 and 2, respectively.

Characterization techniques

Elemental analyses (C, H, N) were carried out in a Thermo Fisher FlashEA-1112 microanalyzer with a Mettler Toledo MX5 microbalance. Thermogravimetric analysis (TGA) for the complexes was carried out in a Rigaku Thermo Plus TG8120 apparatus, under an atmosphere of dry N2 (flow 100 mm min -1 ) at a heating rate of 2 K min -1 over a range from 20 to 400 °C with stabilisation at 25 °C for 1 hour. The infrared (IR) transmission measurements were performed on powdered microcrystals mixed with KBr using a JASCO FT/IR-4100 spectrometer in the range of 4000-400 cm -1 . Solid state UV-Vis-NIR diffuse-reflectance spectra for ca. 10 wt% of samples dispersed in BaSO4 were collected with a JASCO V-670 UV-Vis spectrophotometer equipped with an ISN-723 integrating sphere accessory, converted with the Kubelka-Munk function. Powder X-ray diffraction (PXRD) data were recorded on a RIGAKU MiniFleX600 diffractometer equipped with monochromated Cu-Kα radiation (λ = 1.5406 Å), the powder samples were packed into a zero-diffraction plate holder and diffraction patterns were collected over a 2θ range of 3-90° at a scan rate of 10° min -1 .

Magnetic measurements

Magnetic measurements were carried out on polycrystalline samples of 1•sol and 2•sol, using a vibrating sample magnetometer (VSM) from MicroSense EZ7, with an EV1-LNA temperature controller. Magnetic susceptibility measurements were performed in the temperature range of 120-370 K for 1•sol and 120-410 K for 2•sol, respectively, in an applied magnetic field of 10 kOe and a temperature ramp of 2 K min -1 . The flow of nitrogen gas was used for temperature control. Samples were placed in thin-walled quartz tubes and immobilised with quartz wool. The magnetic data were corrected from the sample holder and the diamagnetic contributions (using Pascal's constants). [START_REF] Kahn | Molecular Magnetism[END_REF]

Photomagnetic studies

Photomagnetic measurements were carried out on polycrystals of 1•sol (0.157 mg) and 2•sol (0.315 mg) blocked between two transparent polypropylene films. Masses of samples were determined using a microbalance from Mettler Toledo, Mettler ME30 (d = 0.1 mg). Photomagnetic measurements were performed with laser diodes coupled to an optical fibre through the cavity of a MPMS-5 Quantum Design SQUID magnetometer. The standardized procedure for registering the T(LIESST) properties was as previously published. [START_REF] Chastanet | A critical review of the T(LIESST) temperature in spin crossover materials -What it is and what it is not[END_REF][START_REF] Létard | A Guideline to the Design of Molecular-Based Materials with Long-Lived Photomagnetic Lifetimes[END_REF] After cooling slowly to 10 K (in the LS state), samples were irradiated at a given wavelength, and the laser power was adjusted to 5 mW cm -2 at the sample surface to minimize and prevent the effect of heating. The magnetization change was followed until the photo-saturation point was reached, then the laser was turned off to stop irradiation. After that, the magnetization was measured in the dark every 1 K in the range of 10-100 K with a temperature ramp of 0.3 K min -1 to determine the T(LIESST) value given by the extreme of the δχMT/δT versus T curve. 15b,c In the absence of irradiation, magnetism was also measured over the temperature range of 10 to 150 K to follow the thermal spin transition and obtain a low temperature baseline.

  5 water molecules and disordered methanol in the case of 1•sol or two ReO4 -counterions and three water molecules in the case of 2•sol. The [FeN6] units have a pseudo-octahedral geometry in which each Fe(II) ion is coordinated by six nitrogen atoms belonging to two pyridine (py) and four pyrazole (pyr) groups. The two 3-bpp ligands are in the meridional positions of the distorted octahedron. This distortion is generally observed due to the low mean values of Npy-Fe-Npyr angles within 3-bpp ligands of 79.2(2)° and 79.5(2)° for 1•sol and 2•sol, respectively (ESI, Table S2 †). Furthermore, this deformation is also illustrated by a slight difference in the average Fe-Npy and Fe-Npyr bond lengths: 1.926(5) and 1.954(5) Å for 1•sol, and 1.924(5) and 1.962(4) Å for 2•sol. Mean Fe-N distances (1.944(5) and 1.949(5) Å for 1•sol and 2•sol, respectively) are consistent with the low-spin state of Fe(II). 1a Calculations of the distortion parameters using Octadist give Σ = 94.31°, Θ = 307.65° and ξ = 0.076 Å for 1•sol and Σ = 91.93°, Θ = 30.91° and ξ = 0.100 Å for 2•sol, 23 indicating a slightly more distorted octahedron in 1•sol than in 2•sol. This may indicate that the Fe(II) centre in 1•sol is more prone to experience the LS to HS SCO than in 2•sol. This observation is in line with the conclusions drawn from the UV-Vis-NIR spectroscopy of fresh samples. At this point it should be noted that the structural parameters related to the complexes [Fe(3-bpp)2] of both materials are very similar to previously described low-spin state phases of [Fe(3-bpp)2]X2•nH2O (X = Cl, Br and I), 10,18i [Fe(3bpp)2](BF4)2•nH2O, 10 [Fe(3-bpp)2](CF3SO3)2•nH2O, 10 and [Fe(3-bpp)2](NCX)2•nH2O (X = S and Se). 10a,15 However, the parameters describing anion environments vary considerably. The average Cl-O and Re-O distances are 1.409(

Fig. 1

 1 Fig. 1 Crystal structures of 1•sol and 2•sol. Asymmetric unitsof 1•sol (a) and 2•sol(b) with selected atom labelling. Thermal ellipsoids with 50% probability are shown. Packings along the crystallographic a-axis for 1•sol(c) and 2•sol

packings of 1 •

 1 sol and 2•sol along the crystallographic a-axis are shown in Fig.1. It is noteworthy to mention that both packings are comparable. The layers of Fe(II) complexes lie in the ab-plane and are well separated by alternating layers of anions and solvent molecules. Interestingly, the alternating anionic layer of 1•sol contains only one of two types of perchlorates (Cl1 or Cl2) -Fig.1c, while 2•sol consists of both types of perrhenates simultaneously (Re1 and Re2) -Fig.1d. The layered arrangement of elements in 1•sol and 2•sol is the result of the presence of extensive π-π interactions between [Fe(3-bpp)2] 2+ entities (Fig.2a and b), anionπ interactions involving perchlorates/perrhenates and 3-bpp ligands and σ⋯π interactions between solvent molecules and 3-bpp ligands (Fig.2c and d), and hydrogen bond networks including 3-bpp ligands, solvent molecules and anions (ESI, Fig.S10 †). Each [Fe(3-bpp)2] 2+ unit of 1•sol and 2•sol interacts via parallel-displaced and T-shaped π-π interactions with four adjacent Fe(II) complexes to form two-dimensional layers in the abplane (Fig.2a and b). The average distances between the adjacent pyrazole mean planes (the parallel-displaced π-π interaction distances) are 3.377(2) and 3.575(2) Å for 1•sol and 2•sol, respectively. Meanwhile, the average lengths between the pyrazole ring centroids and the hydrogen atoms directly above them (the T-shaped π-π interaction distances) are 2.951(2) and 2.800(2) Å for 1•sol and 2•sol, respectively. These relatively small distances indicate the existence of strong π-π stackings, contributing significantly to the stabilization of the overall structures of 1•sol and 2•sol. Additionally, the medium-strength anion-π interactions between the lone pairs in the oxygen atoms of XO4 -anions and pyridine rings of the 3-bpp ligands with the average distance between the pyridine rings centroids and the oxygen atoms directly located above them of 3.029(2) and 3.169(2) Å for 1•sol and 2•sol (Fig.2c and d), respectively, and the intermediate-strength σ⋯π interactions between the hydrogen atoms of solvent molecules and the pyridine rings of 3-bpp ligands with average distances of3.337(2) and 3.370(2) Å for 1•sol and 2•sol, respectively, also actively participate in the stabilization of 1•sol and 2•sol. Furthermore, one-dimensional hydrogen bond networks along the a-direction (for anionic layer with Cl1) or the b-direction (for anionic layer with Cl2) for 1•sol, and the local hydrogen bond networks for 2•sol can be recognized (ESI, Fig. S6 †), in which each [Fe(3-bpp)2] 2+ entity of 1•sol interacts through Npyr-H⋯OClO4 and Npyr-H⋯Owater connections at a distance of 2.952(2) and 2.697(2) Å, respectively, while each [Fe(3-bpp)2] 2+ unit of 2•sol interacts via Npyr-H⋯OReO4 and Npyr-H⋯Owater bonds at a distance of 2.730(

Fig. 3

 3 Fig. 3 Results of magnetic studies and photomagnetic studies at Hdc = 10 kOe of pristine (1•sol and 2•sol) and desolvated phases (1 and 2). (a) and (b) Temperature dependences of χMT before (black squares), during (red circles) and after photoirradiation with 650 nm light (blue down triangles) of 1•sol, and after desolvation (green up triangles), during photoirradiation with 650 nm light (light blue left triangles), after photoirradiation (purple right triangles) and after thermal relaxation at 120 K of 1 and 2. (c) and (d) The temperature dependence of χMT during desolvation (red open circles), two consecutive cooling (T↓) and heating (T↑) cycles (triangles) and after five days of storing the sample in the air (yellow Insets to (c) and (d) contain the first derivatives of the χMT vs. T plots after photoirradiation with 650 nm light for pristine 1•sol (blue down triangles) and desolvated 1 and 2 (purple right triangles).

Acknowledgements

The present research was supported by several sources: the CNRS, the University of Bordeaux, the "Agence Nationale de la Recherche" (ANR project BISTA-MAT: ANR-12-BS07-0030-01 ANR project FEMTOMAT: ANR-13-BS04-0002), the "Region Nouvelle Aquitaine", a JSPS Grant-in-Aid for Specially Promoted Research (Grant Number JP15H05697), a Grant-in-Aid for Scientific Research on Innovative Area Soft Crystals (Area No. 2903, JP17H06367), a Grant-in-Aid for Scientific Research(A) (Grant Number JP20H00369), IM-LED LIA (CNRS) and the Algerian General Directorate of Scientific Research and Technological Development (DGRSDT). We acknowledge the Cryogenic Research Center, The University of Tokyo, the Center for Nano Lithography & Analysis, The University of Tokyo supported by MEXT, and the MEXT Quantum Leap Flagship Program (MEXT Q-LEAP) (JPMXS0118068681). K. K. is grateful to a Grant in-Aid for JSPS fellows (Grant Number JP20J20245). O. S. is thankful for JSPS KAKENHI Grant Number JP21K14582.

Conflicts of interest

There are no conflicts to declare.


Pierre Bessière
email: pierre.bessiere@envt.fr

Maxi Fusade-Boyer
email: maxime.fusade-boyer@envt.fr

Mathilda Walch
email: mathilda.walch@envt.fr

Laetitia Lèb
email: laetitia.lebre@envt.fr

Jessie Brun
email: jessie.brun@envt.fr

Guillaume Croville
email: guillaume.croville@envt.fr

Séverine Boullier
email: severine.boullier@envt.fr

Marie-Christine Cadiergues
email: marie-christine.cadiergues@envt.fr

Jean-Luc Guérin
email: jean-luc.guerin@envt.fr

Household Cases Suggest That Cats Belonging to Owners with COVID-19 Have a Limited Role in Virus Transmission

Keywords: COVID-19, cats, reverse-zoonosis

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for COVID-19 and spread rapidly following its emergence in Wuhan in 2019. Although cats are, among other domestic animals, susceptible to SARS-CoV-2 infection, little is known about their epidemiological role in the dynamics of a household infection. In this study, we monitored five cats for viral shedding daily. Each cat was confined with its COVID-19 positive owners in separate households. Low loads of viral nucleic acid were found in two cats, but only one developed anti-SARS-CoV-2 antibodies, which suggests that cats have a limited role in COVID-19 epidemiology.

Introduction

Since its emergence in late 2019, SARS-CoV-2, the etiological agent of COVID-19, has infected millions of people throughout the world. The virus spread from an animal reservoir, probably of bats, and might have involved an intermediate host. This highlights the importance of animals and the One Health perspective in the control of a zoonotic and emerging disease [START_REF] Zhou | A pneumonia outbreak associated with a new coronavirus of probable bat origin[END_REF]. In the past few months, spillover events have resulted in several countries reporting a viral transmission from humans to animals. These events mostly involved cats [START_REF] Musso | New SARS-CoV-2 Infection Detected in an Italian Pet Cat by RT-QPCR from Deep Pharyngeal Swab[END_REF][START_REF] Sailleau | First Detection and Genome Sequencing of SARS-CoV-2 in an Infected Cat in France[END_REF][START_REF] Segalés | Detection of SARS-CoV-2 in a Cat Owned by a COVID-19-affected Patient in Spain[END_REF][START_REF] Pagani | Human-to-Cat SARS-CoV-2 Transmission: Case Report and Full-Genome Sequencing from an Infected Pet and Its Owner in Northern Italy[END_REF][START_REF] Patterson | Evidence of Exposure to SARS-CoV-2 in Cats and Dogs from Households in Italy[END_REF], but also dogs [START_REF] Patterson | Evidence of Exposure to SARS-CoV-2 in Cats and Dogs from Households in Italy[END_REF][START_REF] Sit | Infection of Dogs With SARS-CoV-2[END_REF] and minks [START_REF] Oreshkova | SARS-CoV-2 Infection in Farmed Minks, The Netherlands[END_REF]. Since other domestic animals, such as ferrets [START_REF] Schlottau | SARS-CoV-2 in Fruit Bats, Ferrets, Pigs, and Chickens: An Experimental Transmission Study[END_REF] and hamsters [START_REF] Sia | Pathogenesis and Transmission of SARS-CoV-2 in Golden Hamsters[END_REF], are also susceptible to SARS-CoV-2 infection, reverse zoonosis events involving these animals will likely occur. In most case reports involving cats, animals are sampled only once and few studies perform daily monitoring of viral shedding. Human-to-cat transmission might be underestimated and there is a strong need to better understand the role of cats in COVID-19 epidemiology [START_REF] Leroy | The Risk of SARS-CoV-2 Transmission to Pets and Other Wild and Domestic Animals Strongly Mandates a One-Health Strategy to Control the COVID-19 Pandemic[END_REF]. In this study, we monitored viral shedding in five different cats living in five different households with their quarantined and COVID-19-positive owners.

Materials and Methods

Owners and Cats

All of the owners developed COVID-19-like symptoms (fever, cough, headache, fatigue, and a loss of taste or smell) and subsequently tested positive for SARS-CoV-2 infection by means of RT-qPCR performed on nasopharyngeal swabs in a medical facility in accordance with the French governmental guidelines [START_REF]Prise En Charge En Ambulatoire Du Covid-19-Recommandations Aux Professionnels De Santé En Charge Des prélèvements De dépistage Par RT-PCR[END_REF]. They all belonged to clusters involving friends and relatives who were never in contact with the cats. Four owners were veterinary students, while a cat from case 5 was owned by a veterinarian's relative.

Viruses 2021, 13, 673 2 of 8 All of the cats lived strictly indoors. For one week during their quarantine, the owners performed daily oropharyngeal and rectal swabs on their cats and the cats' food bowls (environmental swabs) following veterinary instructions (EC approval SSA_2020_0010). These instructions were given by phone and through a document, available in Supplementary File S1. Swabs were collected daily and stored dry at 4 • C in the meantime. The delay between the onset of the owners' symptoms and the first cat swabbing ranged from four to eight days. Blood samples were taken from all of the cats at least six weeks after their owners' recovery to assess the cats' serological status. For more details about the owners, see Supplementary Table S1.

RT-qPCR from Feline and Environmental Swabs

Swabs were briefly vortexed in 500 µL PBS and viral RNA was extracted from 140 µL in accordance with the manufacturer's instructions (QIamp viral RNA; Qiagen, Toronto, ON, Canada). RNA extractions were performed within the 48 h following sampling (swabs were kept at 4 • C in the meantime). RT-qPCR was performed on 96-well plates with a final volume of 20 µL in a Light Cycler system (Roche, Penzberg, Germany). The mixes were prepared in accordance with the manufacturer's instructions (QuantiNova Probe; Qiagen, Toronto, ON, Canada) with 2 µL of RNA and primers that targeted the E-gene. To obtain human-derived viral RNA and perform phylogenetic analysis, the owner from case 3 self-performed a nasal swab which was treated as a feline one. To check whether the material in the oropharyngeal and rectal swabs originated from felines, RT-qPCR targeting the feline 40S ribosomal protein S7 (RPS7) gene was also performed. SARS-CoV-2-positive swabs were further tested for human ribosomal protein L30 (RPL30) mRNA expression, to rule out human contamination. RPS7 and RPL30 RT-qPCR were performed on 96-well plates in a final volume of 10 µL in a Light Cycler System (Roche, Penzberg, Germany). Mixes were prepared according to the manufacturer's instructions (QuantiFast; Qiagen, Toronto, ON, Canada). The primers' sequences are listed in Supplementary Table S2.

Virus Isolation

Vero E6 cells were plated in a 12-well plate and cultured in Dulbecco's modified Eagle medium (DMEM) and complemented with 10% of heat-inactivated fetal bovine serum and 1% of penicillin-streptomycin at 37 • C and with 5% of CO 2 . Cells at 90% confluence were incubated with 100 µL of each RT-qPCR positive viral sample in DMEM complemented the 2% of heat-inactivated fetal bovine serum with 1% of penicillin-streptomycin, 2 µg/mL of ciprofloxacin, 10 µg/mL of BM-cyclin 1, and 1 µg/mL of amphotericin B (Merck, Darmstadt, Germany) for 72 h. Infectivity was assessed by screening cells for cytopathic effects and, at the end of the 72-h waiting period, the presence of SARS-CoV-2 nucleic acids was confirmed by RT-qPCR. Experiments were carried out in a biosafety level 3 facility at the National Veterinary School of Toulouse.

ELISA

The serological status of the cats was assessed using a commercial SARS-CoV-2 N double antigen enzyme-linked immunosorbent assay (ELISA) kit in accordance with the manufacturer's instructions (ID Screen; ID-Vet, Montpellier, France).

Serum Neutralization Assay

Vero-E6 cells were plated in 96-plates and cultured DMEM complemented with 10% of heat-inactivated fetal bovine serum and 1% of penicillin-streptomycin at 37 • C and with 5% of CO 2 . The samples and controls were heat-inactivated at 56 • C for 30 min, serially diluted in DMEM starting at 1:10, mixed with 100 TCID 50 of SARS-CoV-2 (previously amplified and titrated on Vero-E6 cells), incubated for 2 h at 37 • C, and transferred to a culture plate. Following a 1-h incubation at 37 • C, the virus-serum dilutions were removed, and the cells were washed with PBS. Cells were incubated in a growth medium (DMEM complemented with 2% of heat-inactivated fetal bovine serum and 1% of penicillin-streptomycin) for 72 h at 37 • C and with 5% of CO 2 and were screened for cytopathic effects. Serum from a SARS-CoV-2 immunized mouse (kindly provided by JC. Guéry) was used as a positive control. PBS was used as a negative control. Experiments were carried out in a biosafety level 3 facility at the National Veterinary School of Toulouse.

Whole-Genome Sequencing

A SARS-CoV-2 whole-genome amplification was performed according to the ARTIC amplicon sequencing protocol for MinION for nCoV-2019 [START_REF] Quick | Sequencing Protocol (single Sample) V1 (protocols.io.Bdbfi2jn)[END_REF]. Briefly, reverse transcription of viral RNA was performed using an SSIV Reverse Transcriptase (Thermo Fisher Scientific, Inc., Mississauga, ON, Canada) and random hexamers followed by a PCR with the Q5 Hot Start DNA Polymerase (New England Biolabs, Ipswich, MA, USA) and the hCoV-2019/nCoV-2019 Version 3 Amplicon Set. Samples were submitted to this protocol and the amplification was visually checked by running 5 µL aliquots on 1% agarose gel. DNA libraries were prepared using an SQK-LSK109 Ligation sequencing kit supplied by ONT (Oxford Nanopore Technologies, Oxford, UK) associated with an EXP-NBD104 (ONT) native barcoding kit for the multiplexing of 3 samples. In total, 30 fmol DNA libraries were loaded on a FLO-FLG001 flongle and were run on a MinION Mk1C device (ONT) for 10 h. Fast base-calling was performed in real-time with Guppy (v3.5) embedded in the MK1C software (v19.12.12) with the 'Trim Barcode' option on (ONT). Fastq files were mapped on the MW420003.1 reference genome using bwa-sw (v0.7.15) and consensus genomes were produced with bcftools mpileup (v1.6). Consensus sequences were then checked using the Geneious (v.2019.03) software (Biomatters, Ltd., Auckland, New Zealand). The owner-and feline-derived viral sequences were submitted to GenBank (accession numbers MW505982.1 and MW513511.1 respectively).

Phylogenetic Analysis

Whole-genome sequences of the 50 strains most closely related to hCoV-19/cat/France/ LHOOQ/2020 and hCoV-19/human/FRA/66JQ-O/2020 (the strains isolated from case 3 and its owner, respectively) were downloaded from NCBI using a BLASTn search. All cat-isolated SARS-CoV-2 sequences also were loaded, as well as one strain representative of each clade according to GISAID classification. All of the sequences were aligned using the MAFFT online server and tested for recombination events using the Recombination Detection Program 4 (RDP4) [START_REF] Martin | RDP4: Detection and Analysis of Recombination Patterns in Virus Genomes[END_REF]. When several of the collected sequences in the same host and place were similar, only one was kept for the following steps. Phylogenetic analysis was performed using a generalized time-reversible (GTR) model with discrete gamma distribution and four categories implemented in MegaX (MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms; Kumar, Stecher, Li, Knyaz, and Tamura 2018) performing a 1000-bootstrap resampling analysis-a process visualized with Figtree (v.1.4.2).

Results

Case Description

Five neutered European cats (two females and three males) belonging to owners with COVID-19 were enrolled in this study. The animals ranged in age from 1 to 10 years old. None of the cats developed any symptoms. Age, breed, gender, and samples are recapitulated in Table 1.

Two Cats Out of Five Shed SARS-CoV-2

Cases 3 and 5 tested positive for SARS-CoV-2 RNA. For case 3, low quantities of viral nucleic acids were detected in a rectal swab (Ct 37.0) and an oropharyngeal swab (Ct 35.6) three and four days, respectively, after the owner's diagnosis. Viral nucleic acids were also detected in the cat's feeding bowl on days 5 and 6 after the owner's diagnosis (Ct 33.6 and 34.2 respectively). For case 5, viral nucleic acid was detected once on an oropharyngeal swab (Ct 36.7) and once on an environmental swab (Ct 37.5) (Table 1). All cat and environmental swabs were tested for feline 40S ribosomal protein S7 (RPS7) mRNA expression to check whether swabbing was performed appropriately: feline RPS7 mRNA expression was detected in all feline swabs and most environmental swabs (including SARS-CoV-2-positive ones). SARS-CoV-2-positive samples were further tested for human ribosomal protein L30 (RPL30) mRNA expression, which was not detected, suggesting that the presence of viral RNA was not due to human contamination. Virus isolation from positive swabs on Vero E6 cells were unsuccessful. All other swabs were negative at the other time points.

One Cat Out of Five Developed Anti-SARS-CoV-2 Antibodies

Antibodies were found in case 3 only and the other samples were negative. Serum from case 3 yielded positive ELISA titers (OD value: 1.088; % positivity: 121%), which was confirmed by the detection of neutralizing antibodies (neutralization titer > 1:1280). These results suggest that SARS-CoV-2 was effectively transmitted in only this case, despite the proximity of the other owners with their cats.

Phylogenetic Analysis

A partial viral genome (accession number MW513511.1) was obtained from the positive oropharyngeal swab of case 3, while a whole genome (accession number MW505982.1) was obtained from the owner's nasopharyngeal swab. Phylogenetic analysis performed on the whole genome showed that both viruses in our study had clustered with viruses from Switzerland and belonged to the clade GH according to GISAID nomenclature. This clade is characterized by the nucleotide substitutions C241T, C3037T, A23403G, G25563T, the amino acid substitution Q57H in the NS3 protein, and the substitution D614G in the spike protein, which is known to enhance infectivity and viral fitness [START_REF] Hou | SARS-CoV-2 D614G Variant Exhibits Efficient Replication Ex Vivo and Transmission In Vivo[END_REF]. Phylogenetic analysis also showed that both sequences from case 3 were very closely related, sharing more than 99.99% genetic identity (Figure 1). Both sequences differed only by the synonymous G26031A mutation and an amino acid substitution F843L in the ORF1ab protein that was not described in the literature. Nevertheless, since only a partial genome was obtained from the cat sample, two regions (from amino acid (aa) 262 to aa 318, and from aa 448 to aa 520) were not successfully sequenced and other differences may have been missed.

by the synonymous G26031A mutation and an amino acid substitution F843L in the ORF1ab protein that was not described in the literature. Nevertheless, since only a partial genome was obtained from the cat sample, two regions (from amino acid (aa) 262 to aa 318, and from aa 448 to aa 520) were not successfully sequenced and other differences may have been missed.

Discussion

Cats belonging to COVID-19-positive owners were monitored for viral shedding and clinical signs. None of the cats developed symptoms. SARS-CoV-2 nucleic acids were found by RT-qPCR in two cats, for two consecutive days in cat 3, and only once in cat 5, and both had high Cts values. They were also found on the dinner bowls of both cats, suggesting that the virus was shed in the house, although it could have emanated from the owners as well as from the cats. We detected RPS7 mRNA using RT-qPCR in all of the oropharyngeal and rectal swabs, demonstrating that the owners managed to swab their animals correctly. Furthermore, human RPL30 mRNA was not detected in any SARS-CoV-2-positive feline or environmental swabs, thus decreasing the probability that viral RNA presence was due to human contamination. Case 3 had the only cat that tested positive for anti-SARS-CoV-2 antibodies using both ELISA and serum virus neutralization assays. The other cats tested negative by means of RT-qPCR for SARS-CoV-2 and, according to serological tests made a few weeks later, the cats also tested negative for antibodies. This suggests that the cats were never infected, rather than that the shedding window was missed when swabbing the animals. Since viral shedding was weak and transient, cats may not be efficient at shedding SARS-CoV-2.

In experimental studies, cats inoculated intranasally or intratracheally with SARS-CoV-2 shed viruses up to 10 days post-infection [START_REF] Gaudreault | SARS-CoV-2 Infection, Disease and Transmission in Domestic Cats[END_REF][START_REF] Shi | Susceptibility of Ferrets, Cats, Dogs, and Other Do-Mesticated Animals to SARS-coronavirus 2[END_REF] and were able to infect non-inoculated cats through direct or indirect contact [START_REF] Halfmann | Transmission of SARS-CoV-2 in Do-Mestic Cats[END_REF]. However, the inoculum dose and the

Discussion

Cats belonging to COVID-19-positive owners were monitored for viral shedding and clinical signs. None of the cats developed symptoms. SARS-CoV-2 nucleic acids were found by RT-qPCR in two cats, for two consecutive days in cat 3, and only once in cat 5, and both had high Cts values. They were also found on the dinner bowls of both cats, suggesting that the virus was shed in the house, although it could have emanated from the owners as well as from the cats. We detected RPS7 mRNA using RT-qPCR in all of the oropharyngeal and rectal swabs, demonstrating that the owners managed to swab their animals correctly. Furthermore, human RPL30 mRNA was not detected in any SARS-CoV-2-positive feline or environmental swabs, thus decreasing the probability that viral RNA presence was due to human contamination. Case 3 had the only cat that tested positive for anti-SARS-CoV-2 antibodies using both ELISA and serum virus neutralization assays. The other cats tested negative by means of RT-qPCR for SARS-CoV-2 and, according to serological tests made a few weeks later, the cats also tested negative for antibodies. This suggests that the cats were never infected, rather than that the shedding window was missed when swabbing the animals. Since viral shedding was weak and transient, cats may not be efficient at shedding SARS-CoV-2.

In experimental studies, cats inoculated intranasally or intratracheally with SARS-CoV-2 shed viruses up to 10 days post-infection [START_REF] Gaudreault | SARS-CoV-2 Infection, Disease and Transmission in Domestic Cats[END_REF][START_REF] Shi | Susceptibility of Ferrets, Cats, Dogs, and Other Do-Mesticated Animals to SARS-coronavirus 2[END_REF] and were able to infect non-inoculated cats through direct or indirect contact [START_REF] Halfmann | Transmission of SARS-CoV-2 in Do-Mestic Cats[END_REF]. However, the inoculum dose and the infection route may not reflect the natural circumstances in which a cat could be naturally infected. In a more recent study, three cats living together in one household with COVID-19-positive owners were monitored for viral shedding and the SARS-CoV-2 RNA was sequentially detected in all three cats-whether this was due to a cat-to-cat transmission remains unknown. Interestingly, viral RNA was detected up to 11 days after the onset of the owners' symptoms [START_REF] Neira | A Household Case Evidences Shorter Shed-Ding of SARS-CoV-2 in Naturally Infected Cats Compared to Their Human Owners[END_REF].

In our study, the cats lived in small to moderate-sized households and the owners did not take particular hygiene measures while interacting with their pets. Despite these elements, in only two cats was viral RNA detected. Cat 5 did not develop anti-SARS-CoV-2 antibodies and a viral genome was found only once in an oropharyngeal swab and once in an environmental swab. It remains unknown whether the swabs were contaminated with viral RNA from the owner. In addition, human cases with transient SARS-CoV-2 shedding and an absence of antibody responses have been reported [START_REF] Alonso | Recurrence of SARS-CoV-2 Infection with a More Severe Case After Mild COVID-19, Reversion of RT-QPCR for Positive and Late Antibody Response: Case Report[END_REF][START_REF] Lin | A Case of Transient Existence of SARS-CoV-2 RNA in the Respiratory Tract with the Absence of Anti-SARS-CoV-2 Antibody Response[END_REF] and the same phenomenon could occur in cats. Notably, Temman and colleagues failed to detect anti-SARS-CoV-2 antibodies in nine cats belonging to COVID-19 positive students [START_REF] Temmam | Absence of SARS-CoV-2 Infection in Cats and Dogs in Close Contact with a Cluster of COVID-19 Patients in a Veterinary Campus[END_REF].

The infected cats did not display any clinical signs, which is consistent with previous experimental studies [START_REF] Gaudreault | SARS-CoV-2 Infection, Disease and Transmission in Domestic Cats[END_REF][START_REF] Shi | Susceptibility of Ferrets, Cats, Dogs, and Other Do-Mesticated Animals to SARS-coronavirus 2[END_REF][START_REF] Halfmann | Transmission of SARS-CoV-2 in Do-Mestic Cats[END_REF]. However, subclinical manifestations cannot be excluded and it is worth noting there have been some reports of symptomatic cats in households with COVID-19 patients [START_REF] Musso | New SARS-CoV-2 Infection Detected in an Italian Pet Cat by RT-QPCR from Deep Pharyngeal Swab[END_REF][START_REF] Sailleau | First Detection and Genome Sequencing of SARS-CoV-2 in an Infected Cat in France[END_REF][START_REF] Segalés | Detection of SARS-CoV-2 in a Cat Owned by a COVID-19-affected Patient in Spain[END_REF][START_REF] Neira | A Household Case Evidences Shorter Shed-Ding of SARS-CoV-2 in Naturally Infected Cats Compared to Their Human Owners[END_REF]. Age and comorbidities are likely involved in these differences and further studies are required to better understand SARS-CoV-2 pathogenesis in cats.

In addition to the epidemiological context, phylogenetic analysis supports the humanto-cat transmission hypothesis since the strains detected in the owners and the cats shared a high genetic identity. The feline viral genome differed only by one amino-acid and one nucleotide substitution. A sequence comparison with the other available feline-derived SARS-CoV-2 genomes did not reveal any shared cat-specific mutation. None of the available viral sequences from cats harbored this substitution, which suggests a random event rather than an adaptation to cats despite the low number of available sequences.

Our study has a number of limitations. The sample size was limited due to the constraining nature of the study's protocol, which renders it difficult to generalize our findings to a larger cat population. Due to the strict lockdown measures at the time of this study, the owners performed the swabbing unsupervised. They received detailed instructions, but we cannot exclude the possibility that the samples were not taken thoroughly enough, despite the RPS7 mRNA presence, or that they were contaminated by the owners' hands or fomites, despite the human RPL30 mRNA absence. In addition, there is a lack of evidence regarding the sensitivity of oropharyngeal swabbing compared to nasal or nasopharyngeal swabbing in cats.

In conclusion, the SARS-CoV-2 genome was detected by RT-qPCR in two cats out of five, which suggests that human-to-cat transmission is not an infrequent event. Given the variability of clinical manifestations, COVID-19 in cats is likely under-diagnosed. However, viral shedding was weak and transient, which hints that, even when infected, cats probably play a limited role in COVID-19 epidemiology.

Supplementary Materials:

The following are available online at https://www.mdpi.com/article/ 10.3390/v13040673/s1, Table S1: Households descriptions, Table S2: primers and probe for RT-qPCR [START_REF] Corman | Detection of 2019 Novel Corona-Virus (2019-NCoV) by Real-Time RT-PCR[END_REF][START_REF] Penning | A Validation of 10 Feline Reference Genes for Gene Expression Measurements in Snap-Frozen Tissues[END_REF][START_REF] De Jonge | Evidence Based Selection of Housekeeping Genes[END_REF], file S1: swabbing instructions given to the owners. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to the interests of retaining patient confidentiality.

Figure 1 .

 1 Figure 1. Whole-genome maximum likelihood phylogenetic tree of SARS-CoV-2. The viruses in Table 1, sequence from case 3, and the owner-derived sequence are both labelled in red. Other feline-derived viral sequences are labeled in purple. Bootstrap supports >60% are indicated next to the nodes; the scale bar indicates the numbers of nucleotide substitutions per site. The strain hCoV-19/Wuhan/WIV04/2019 was used as the outgroup.

Figure 1 .

 1 Figure 1. Whole-genome maximum likelihood phylogenetic tree of SARS-CoV-2. The viruses in Table 1, sequence from case 3, and the owner-derived sequence are both labelled in red. Other feline-derived viral sequences are labeled in purple. Bootstrap supports >60% are indicated next to the nodes; the scale bar indicates the numbers of nucleotide substitutions per site. The strain hCoV-19/Wuhan/WIV04/2019 was used as the outgroup.

Author Contributions:

  Conceptualization, P.B., M.-C.C., J.-L.G.; experiments, P.B., M.F.-B., M.W., L.L., G.C., J.B.; writing-original draft preparation, P.B., M.F.-B.; writing-review and editing, P.B., S.B., M.-C.C., J.-L.G.; supervision, M.-C.C., J.-L.G.; funding acquisition, J.-L.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the research unit IHAP (Université de Toulouse, INRAe, ENVT) and by the department of dermatology of ENVT.Institutional Review Board Statement:The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee Sciences et Santé Animale n • 115 (protocol code COVIFEL approved on 1 September 2020, registered under SSA_2020_010).

Table 1 .

 1 Summary of the cases, including breed, age, gender, the time between the onset of owner's symptoms and cat's first swab, number, and RT-qPCR Ct values of positive swabs (Day 1 corresponding to the first day the cat was enrolled in the study) and serological status determined by ELISA and serum neutralization assays.

	Case Breed	Age (Years)	Gender	Days Between Owners' Symptoms Onset and Cat Swabbing	OP	Positive Swabs R	Env	ELISA/ SN (Days 1 )
	1	DSH	1	Female	4	None	None	None	Neg/Neg (54)
	2	DSH	10	Male	6	None	None	None	Neg/Neg (62)
	3	DSH	3	Female	5	Day 2: 35.6	Day 1: 35.7	Day 1: 33.6 Day 2: 34.2	Pos/Pos (47)
	4	DSH	1	Male	8	None	None	None	Neg/Neg (51)
	5	DSH	6	Male	7	Day 3: 36.7	None	Day 6: 37.5 Neg/Neg (174)

DSH: domestic shorthair cat; OP: oropharyngeal; R: rectal; Env: environment; Neg: negative; Pos: positive; SN: serum neutralization assay.

1

: time between the onset of owners' symptoms and the ELISA test.

Acknowledgments:

We warmly thank the cat owners for their involvement in the study. We are very thankful to J.-C. Guéry for providing serum from a SARS-CoV-2 immunized mouse.

Conflicts of Interest:

The authors declare no conflict of interest.