extfieldsofstudy: Chemistry Medicine

text:
Sensing of EGTA Mediated Barrier Tissue Disruption with an Organic Transistor

Barrier tissue protects the body against external factors by restricting the passage of molecules. The gastrointestinal epithelium is an example of barrier tissue with the primary purpose of allowing the passage of ions and nutrients, while restricting the passage of pathogens and toxins. It is well known that the loss of barrier function can be instigated by a decrease in extracellular calcium levels, leading to changes in protein conformation and an increase in paracellular transport. In this study, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid (EGTA), a calcium chelator, was used to disrupt the gastrointestinal epithelial barrier. The effect of EGTA on barrier tissue was monitored by a novel label-free method based on an organic electrochemical transistor (OECT) integrated with living cells and validated against conventional methods for measuring barrier tissue integrity. We demonstrate that the OECT can detect breaches in barrier tissue upon exposure to EGTA with the same sensitivity as existing methods but with increased temporal resolution. Due to the potential of low cost processing techniques and the flexibility in design associated with organic electronics, the OECT has great potential for high-throughput, disposable sensing and diagnostics.

Introduction
Understanding and controlling the barrier function of epithelial tissue is of great importance for pharmaceutical research and drug development, as well as having applications in diagnostics and fundamental research. Manipulating barrier function is primarily applicable in targeted drug delivery, which involves the specific transport of a molecule across the lumen to the underlying tissue. Developing methods to achieve such transport is of particular relevance for drug delivery across gastrointestinal tissue. In some cases, an agent is used to temporarily increase permeability of the cell layer and allow passage of a drug [1]. Further, drug delivery assays require adequate in vitro cell models to assess drug transport. In vitro models for barrier tissue are also necessary to assess the effects of toxins or pathogens, for the purpose of both diagnostics and the study of diseases. Regardless of application, the goal of in vitro models is to mimic in vivo barrier tissue behavior.
Barrier tissue generally consists of tightly packed layers of epithelial cells. Individual cells are joined to one another by junctional proteins, which act as cell-cell seals [2]. In addition, the cells are anchored to underlying tissues. The anchoring provides an asymmetric architecture to the barrier, in which the apical side is exposed to the lumen, and the basal side is attached to the basal lamina [3,4]. This architecture provides selective transport across the barrier, which can be modulated to increase the passage of nutrients via transient opening of the apical junction [5,6]. The apical junction is composed of two distinct junctions; the tight junction (TJ), found closest to the apical side, and the adherens junction (AJ), found underneath the TJ [7]. These junctions comprise complexes of intracellular and transmembrane proteins. The major proteins involved in TJs are claudins [8], occludins [9] and ZO-1 [10,11], while AJs consist primarily of E-cadherin and catenin [12]. Beneath the apical junction are additional junctional complexes known as desmosomes, which contribute to cell integrity [13]. The integrity of junctional protein complexes, and hence the integrity of barrier tissue, is known to be affected by outside stimuli. In particular, the function of some proteins such as cadherins, are sensitive to the concentration of extracellular calcium. Cadherins, found in both the adherens junction and the desmosome contain multiple calcium binding domains [14]. When insufficient calcium is present, cadherins are not able to form homo or heterojunctions with adjacent cells [15]. As a consequence, the proteins are internalized, leading to an opening of the paracellular pathway. Other tight junction associated proteins that require the presence of calcium include G proteins, protein kinase C and calmodulin [16]. Hence, decreases in extracellular calcium concentration can lead to disassembly of TJs. In fact, a calcium switch assay is often used to study TJ reformation after removal and then replacement of calcium [16].
In this study, we use Caco-2 cells grown on permeable transwell filters. When cultured in this format, these cells are known to form polarized monolayers with an apical brush border, similar to that found in the human colon [17]. More specifically, differentiated monolayers of Caco-2 cells create a barrier similar to that observed in vivo. Confluent monolayers of Caco-2 cells are widely used by the pharmaceutical industry to evaluate the absorption of oral drugs: the fraction of drug absorption can be directly correlated with the apparent permeability of the drug molecule across the cell layer [18]. The dependence of barrier integrity of the Caco-2 cells on calcium concentration has been confirmed [19], with the internalization of TJ and AJ proteins occurring when calcium concentrations decrease to micromolar levels [20]. As discussed above, disruption of junctional protein complexes leads to opening of the intercellular junction. To mimic this effect in vitro, we exposed Caco-2 monolayers to EGTA (Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), a specific calcium chelator. EGTA has been shown previously to have dramatic effects on paracellular permeability and transepithelial resistance (TER) [1,21]. In particular, E-cadherin was found delocalized from the cell periphery after treatment with EGTA [22,23]. To monitor the barrier integrity of epithelial tissue on exposure to EGTA, we use a recently introduced method based on an organic electrochemical transistor (OECT) [24,25]. We validate the OECT results with traditional characterization techniques, including immunofluorescence staining of junctional adhesion proteins, permeability assays and measurement of transepithelial resistance (TER).

Experimental Section
Cell Culture. Caco-2 cells from ATCC were seeded at a density of 5 × 10 4 cells/insert (1.1 cm 2 ). Cells were routinely maintained at 37 °C in a humidified atmosphere of 5% CO 2 , in DMEM (Advanced DMEM Reduced Serum Medium 1×, Invitrogen) with 2 mM Glutamine (Glutamax™-1, Invitrogen), 10% FBS (Fetal Bovine Serum, Invitrogen) and Pen-strep (5,000 (U/mL) Penicillin-5000 (µg/mL) Streptomycin, Invitrogen). For all experiments, Caco-2 cell layers were used after 3 weeks in culture, corresponding to a TER of 400-500 Ω·cm 2 and a maximum apparent permeability of 1 × 10 −6 cm· s −1 , consistent with literature reports [26]. Cells were cultured on transwell filters with a 0.4 µm² pore size and area of 1.1 cm 2 . For OECT measurements only, 24 transwell filters were used. Cells were exposed various concentrations of EGTA, from a stock solution of 0.6 M EGTA in DI water, pH adjusted to 7.4 with 1 M Tris-HCl. EGTA was added to the basal side of the cell filter, without changing media. Controls confirmed that the act of addition/removal of basal solution alone did not disrupt the barrier tissue layer.
Immunofluorescence. After exposure to EGTA, Caco-2 cells grown on filters were fixed with 3-4% paraformaldehyde in PBS pH 7.4, for 15 min at room temperature. Permeabilization was performed using 0.25% Triton in PBS, for 10 min at room temperature and with a blocking step consisting of 1% BSA in PBST (0.05% Tween 20 in PBS), for 30 min at room temperature. Mouse monoclonal anti-E-cadherin and rabbit polyclonal anti-claudin-1 and anti occludin were used at 5 µg/mL (Invitrogen), in 1% BSA in PBST for 1 h at room temperature. Monolayers were then incubated for 1 h at room temperature with the secondary antibodies Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 568 goat anti-rabbit (Molecular Probes). Finally, the cells were incubated for 5 min at room temperature with Fluoroshield with DAPI (Sigma Aldrich), mounted and examined with a fluorescent microscope.
Permeability Assays. After exposure to EGTA, a permeability marker (Lucifer Yellow) was added to the apical side of the monolayer and fluorescence was measured after 1 h incubation at 37 °C in a humidified CO 2 incubator. The value of the apparent permeability (P app ) was calculated according to the following relationship: where LY bas and LY api are the concentration of Lucifer Yellow in the basal and apical sides of the hanging porous filter, respectively, V bas and V api are the volume in the basal and apical sides, respectively, t is the time of incubation, C o is the initial concentration of Lucifer Yellow (LY) on the apical side and A is the area of the filter. At least two samples were measured for each condition.
CellZscope Measurements. The CellZscope (Nanoanalytics) measures the impedance of barrier-forming cell cultures grown on permeable membranes and provides the transepithelial electrical resistance as output. Impedance of cell layers grown on filters as previously described, were measured in complete DMEM. During EGTA exposure, TER values were measured continuously.
OECT Fabrication. The conducting polymer formulation consisted of PEDOT:PSS (Heraeus, Clevios PH 1000), supplemented with ethylene glycol (Sigma Aldrich, 0.25 mL for 1 mL PEDOT:PSS solution), dodecylbenzenesulfonic acid (DBSA, 0.5 µL/mL), and 3-glycidoxypropyltrimethoxysilane (GOPS) (10 mg/mL), the latter serving as a heat activated cross-linker to ensure film stability in aqueous solutions. Devices were fabricated on glass slides with channel dimensions defined using a parylene peel-off technique described previously [27,28]. In this technique, a parylene film is deposited on glass and subsequently patterned using conventional photolithography techniques. PEDOT:PSS is deposited on the glass/parylene pattern. When the patterned parylene is removed from the glass substrate via mechanical peeling, PEDOT:PSS is left on the glass in the negative spaces. This technique allows the patterning of sensitive materials, in that it avoids exposure of the active layer (here, the conducting polymer) to harsh developers, solvents, or other etchants. Following PEDOT:PSS deposition, devices were baked for 1 h at 140 °C in atmospheric conditions. A PDMS well was added to confine the electrolyte and define the channel area. The OECTs in this study have a channel width of 2 mm and a channel length of approximately 6 mm, resulting in a channel area of approximately 12 mm 2 . PEDOT:PSS lines extending from the well served as the source and drain contacts.
OECT Measurements. For OECT measurements, Ag/AgCl served as the gate electrode. All measurements were made using a Keithley 2612 Source Meter and customized Labview software. Cell media (as described above) was used as the electrolyte. Measurements were performed at ambient temperature, but controls were conducted to ensure that temperature effects do not dominate changes in OECT response within the time required for measurements. Measurement parameters were chosen to avoid exposing the cell layers to a voltage drop above 0.5 V, as high voltages have been shown to damage bilayer membranes [29]. OECT data were collected using the following parameters: In this way, NR = 1 refers to no barrier properties, and NR = 0 refers to full barrier properties of the cell layer (although not full suppression of OECT modulation [24]). See Appendix for further description.

Results and Discussion
Barrier tissue cells grown on 24 well filters were integrated with an OECT as illustrated in Figure 1. A transwell filter hosting the cell layer is placed within an electrolyte well, between the Ag/AgCl gate electrode and the PEDOT:PSS channel.

OECT Measurement of EGTA Mediated Barrier Tissue Disruption
In an OECT, the drain current (I D ) between the source and drain electrodes is modulated by the application of a gate voltage. The mechanism for current modulation relies on the electrochemical doping and dedoping of a degenerately doped conducting polymer film in contact with an electrolyte [30,31]. A positive gate voltage induces a flux of positive ions into the transistor channel, dedoping the polymer film and reducing conductivity. On release of this potential, the ions leave the film and the original doping level, and hence conductivity, is restored. The I D transient response to a square gate voltage pulse is directly correlated with the magnitude of ionic flux into the conducting polymer. In the present device architecture, the barrier properties of the cell layer modify this ionic flux. Thus, monitoring the I D response to square V G pulses, shown in Figure 2 as normalized response (NR), yields information about the barrier properties of the cells with respect to ions in the electrolyte. Figure 2(a) shows the OECT normalized response as a function of time in the presence and absence of a cell layer. As illustrated in Figure 2(a), operation of the OECT with no cell layer corresponds to NR = 1, which is associated with a high ionic flux through the electrolyte and into the polymer channel. When intact barrier tissue is present, the ionic flux is reduced, and NR = 0. Figure 2(b) shows the OECT response in the presence of barrier tissue on addition of varying concentrations of EGTA. On introduction of 1 mM EGTA to the basolateral side of the transwell filter, we observe negligible changes in the normalized response of the OECT, indicating little or no disruption of barrier properties. There is perhaps a slight, transient increase in TER, which returns to baseline levels after 15 min. It is possible that there is a very slight decrease in ion flux. This has been observed with other compounds at low concentrations and may be explained by a tightening of the ion permeable TJ pores due to subtoxic exposure of certain chemicals [32]. Increases in TER have also been observed with an increase in permeability upon overexpression of occludin, illustrating functional decoupling of these two parameters [33].

Figure 2. Normalized response of the OECT. (a) Control NR of OECT with cells (white) and without cells (black), with no exposure to ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid (EGTA). (b)
In situ monitoring of NR on addition of 1 mM (dark cyan), 10 mM (orange) and 100 mM (violet) EGTA. EGTA is added at time = 0, as indicated by the arrow.
On addition of 10 and 100 mM EGTA, we observe a concentration dependent reduction in NR, indicating increasing barrier disruption for higher concentrations of EGTA, with the presence of 100 mM EGTA destroying barrier integrity within 45 min. These experiments were carried out for 60 min only due to concerns that extended exposure of the cells to room temperature conditions would induce a change in barrier tissue properties [34]. It should be noted that in all experiments carried out in this study, EGTA was added directly to the cell medium. We anticipate that the use of EGTA in complete DMEM, as opposed to a calcium free buffer, will somewhat mitigate the effect of EGTA. However, as changes in both media and ionic composition are known to affect TER, we wanted to ensure that we saw effects due to calcium chelation alone. As a consequence, it is not surprising that we measure no effect on addition of 1 mM EGTA, as the calcium concentration in complete DMEM is approximately 1-2 mM and EGTA chelates calcium at a ratio of 1:1. In the case of 10 mM EGTA, the signal appears to become saturated but at an NR of slightly greater than 0.5. It is possible that this signal would continue to rise slowly, however we have previously observed a similar situation with low concentrations of hydrogen peroxide [24], where the signal reaches an intermediate level and then does not increase. This could represent a partial opening of the TJ or an inhomogeneous chelation of the calcium across the barrier tissue layer. An important feature of the OECT is the time in which breaches in barrier tissue integrity are detected. In the case of 100 mM EGTA, a 50% increase in NR is observed within 5 min, and the NR plateaus at a value slightly less than 1.0. We believe that the OECT can detect barrier tissue damage more rapidly because it is sensitive to more subtle breaches in barrier tissue integrity. The improved sensing speed has been observed on addition of a variety of toxic compounds (manuscripts in preparation). Of note, while the device is more sensitive to subtle barrier damage, the sensitivity is expected to suffer at the other extreme, suggesting that the present OECT is a poor sensor for the formation of barrier tissue and gross cell death/detachment. Figure 3 shows the immunofluorescence staining of adherens and tight junction proteins carried out after 2 h of exposure to EGTA using antibodies against E-cadherin, occludin and claudin-1. Cell nuclei were stained with DAPI. Control staining on Caco-2 cells without EGTA exposure shows that there is at least partial colocalization of E-cadherin with both occludin (Figure 3(a)) and claudin (Figure 3(b)), and that all three proteins are present at the cell peripheries. The colocalization of E-cadherin with occludin and claudin-1 is indicative of normal maturation and polarization of the cell layer as the presence of AJs are necessary to recruit TJs on the apical side to form apical junction complexes, resulting in an alignment of the AJs and TJs [35]. In the case of occludin the staining is slightly diffuse, the control included. This may partly be because immunofluorescence was carried out on filters rather than on coverslips, leading to slight warping of the substrate. On exposure to 1 mM EGTA, the proteins remain colocalized, with negligible difference compared to the control. On the addition of 10 mM EGTA, delocalization of proteins from the periphery is observed, indicating that the apical junctional complexes have been compromised. E-cadherin is considerably more diffuse in the sample exposed to 10 mM EGTA compared to the control. On exposure of 100 mM EGTA, E-cadherin, occludin and claudin-1 are present, but no longer localized at the cell borders. Previous studies have demonstrated that a 30 min treatment of 4 mM EGTA added to DMEM on the apical and basal side results in the displacement of occludin to the intracellular compartment [36], while a study which exposed Caco-2 cells to EGTA in media without calcium for 20 min [37] showed a normal localization of occludin with aberrant localization of claudin-1. Figure 3. Immunofluorescence of proteins in the apical junction upon exposure to EGTA. Monolayers were exposed to various concentrations of EGTA for 2 h and then stained with antibodies against apical junction proteins. In panel a: DAPI (blue), E-cadherin (green) and occludin (red); in panel b: DAPI (blue), E-cadherin (green) and claudin-1 (red). Cells were exposed to 0, 1, 10 and 100 mM EGTA from top to bottom. The scale bar is 10 µm.

Validation of EGTA Effect Using CellZscope Measurement of TER and Permeability Assays
Figure 4(a) shows the effect of EGTA on TER, as measured with the CellZScope. As seen with the OECT, exposure to 1 mM EGTA shows a slight change in TER with respect to the control. On exposure of 10 mM EGTA for two and half hours, the mean TER is reduced to approximately 20% of the initial value. On addition of 100 mM EGTA, the reduction in TER is faster, stabilizing at 10% of the initial value. To directly compare the rate of sensing with the OECT, a useful parameter to consider is the time taken to reach a 50% decrease in TER. As mentioned above, the OECT detects a 50% change (increase in NR) within approximately 5 min. In the case of the CellZscope, this change takes approximately 15 min. The time taken to reach a minimum TER value with 100 mM EGTA was 1.5-2 h, compared to 45 min with the OECT. It was previously demonstrated by the EVOM (epithelial Volt-Ohm meter) technique that in the presence of 1 mM EGTA on both sides of the monolayer, a rapid drop in TER value was observed in 10 min [38]. The discrepancy between these results and what we present here can be explained by the increased potency of EGTA when presented to both the apical and basal sides, further exaggerated by the use of a calcium free buffer [23]. Figure 4(b) shows results of permeability assays carried out on Caco-2 monolayers after 2 h of EGTA exposure. The addition of 1 mM EGTA leads to a permeability of (1.08 ± 0.81) × 10 −6 cm· s −1 , a slight increase compared to the control permeability of (8.81 ± 7.94) × 10 −7 cm· s −1 . The addition of 10 mM EGTA leads to an increase in permeability to (6.30 ± 1.95) × 10 −6 cm· s −1 , while treatment with 100 mM EGTA results in a permeability of (1.64 ± 0.14) × 10 −5 cm· s −1 , 18 times larger than the control value. For comparison, a filter alone typically has a permeability of 1 × 10 −4 cm· s −1 . Previous studies of Caco-2 permeability have demonstrated a mild increase on exposure of 2 mM EGTA in Krebs buffer, with a more marked increase at 20 mM EGTA (up to 4 hour exposure) [23].
Measurement of permeability, although a valuable parameter, must be considered carefully as the permeability measured is molecule-specific (depending on charge and size). Therefore, care must be taken when comparing these data with other parameters that describe barrier tissue integrity, such as TER. In general, a direct correlation between the solute permeability of a cell layer and the TER exist; tight cell layers exhibit high electrical resistance and low permeability, although there are certain conditions where these two parameters can be decoupled [39]. In fact, if the slight increase in permeability can be taken as statistically significant, it is in agreement with literature demonstrating that increases in TER can sometimes be accompanied by increases in permeability (discussed above), thus illustrating decoupling of these two parameters. This can be explained in part by the fact that TER is a parameter that describes an instantaneous snapshot of ion flux through claudin based pores in epithelial tissue layers [40], while permeability is a parameter that describes the transport of larger molecules, most likely through a different pathway, one theory being that larger molecules go through barrier tissue layers via dynamic opening and closing of tight junction strands. For this reason, permeability is a parameter that must be measured over longer time periods, e.g., 1 h. As indicated above, the permeability of Lucifer Yellow was measured after incubation of this compound for 1 hour (after 2 h exposure to EGTA). Therefore, for a fairer comparison, the latter time points should be considered for TER measurements. In the case of the results shown here, a clear increase in permeability is observed with 10 mM and 100 mM EGTA, in line with the trends observed with the OECT and the CellZscope. . % TER value and permeability assays of cells upon exposure to EGTA. Monolayers were exposed to various concentrations of EGTA for 2.5 h. Panel a shows normalized TER value + standard deviation upon exposure to 0 mM (black), 1 mM (dark cyan), 10 mM (orange) and 100 mM EGTA (violet) for 2.5 h. During the experiment, measurements were taken continuously. Panel b shows the apparent permeability after exposure to EGTA. Data represents mean value + standard deviation of experiments made in duplicate.

Conclusions
In this study, we integrate cells with OECTs and demonstrate that the presence of a cell layer modulates the OECT transient response. Upon a decrease of extracellular calcium concentration due to the presence of EGTA, the OECT rapidly senses the breach in the epithelial cell layer in a concentration dependent manner. We validated these results by comparison with traditional techniques such as immunofluorescence, TER measurement and a Lucifer Yellow permeability assay, and found that the data correlates with the results obtained using the OECT. The OECT was able to detect EGTA-induced breaches in epithelial layers with equal sensitivity to the three techniques tested. Of the traditional techniques, only TER measurements (CellZScope) allow dynamic monitoring of barrier tissue integrity. Importantly, the OECT was found to be able to detect EGTA-induced breaches in epithelial layers with increased temporal resolution compared to the CellZscope. The OECT appears to be uniquely sensitive to subtle breaches in barrier tissue integrity, and measures with an extremely fine temporal resolution. We previously showed that the OECT was capable of measuring disruption of barrier tissue caused by exposure to hydrogen peroxide with 30 s [24]. Currently, the device probes the barrier tissue for a duration of 2 s, followed by a 28 off time, to realize a 30 s temporal resolution. The off time of the device serves two purposes: to give the cells adequate time to recover from the voltage pulse, and to allow for the recovery of the PEDOT:PSS film, bringing the drain current back to a baseline value. Duty cycle, and pulse duration could be optimized to observe more dynamic behavior if desired, with the limiting parameter being recovery of baseline current. For sake of comparison, the CellZscope requires 40 s to collect an impedance scan, which puts a hard limit on its temporal resolution.
The CellZscope has an additional advantage in that it has been developed for use in an incubator, allowing continuous, real time monitoring of barrier tissue properties. Although the current prototype of the OECT allows dynamic measurement, it is limited to operation at room temperature. Work is ongoing to transition the OECT to an in situ measurement system to allow integration of traditional cell culture materials and electronics into an incubator to allow longer term measurements and further to streamline the operation for medium to high throughput testing. Future optimization of the OECT device for sensitivity is ongoing;the device is also being adapted for use with additional cell lines to create new tools in different diagnostic applications. In addition, we intend to capitalize on the advantages in low cost fabrication and ease of device design facilitated by the use of conducting polymers towards the goal of high throughput, disposable sensing and diagnostics.


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Immunostimulatory Effects of Silica Nanoparticles in Human Monocytes

Amorphous silica particles, whose applications are increasing in many biomedical fields, are known to be less toxic than crystalline silica. In this study, the inflammatory effects of amorphous silica nanoparticles were investigated using 30-nm amorphous silica nanoparticles and human peripheral blood mononuclear cells (PBMCs) or purified monocytes. As a result, production of IL-1β and IL-8 were increased. In addition, the mitochondrial reactive oxygen species (ROS) was detected, which may lead to mitochondrial membrane disruption. Most importantly, inflammasome formation was observed. Therefore, these results provide immunological information about amorphous silica nanoparticles and suggest that amorphous silica nanoparticles can evoke innate immune reactions in human monocytes through production of IL-1β and IL-8.

As commodities containing silica nanoparticles have been increasing, chances of exposure to silica nanoparticles have also been increasing. To date, research into toxicity of silica particles has focused on 0.5− to 10-μm crystalline silica. However, nano-sized (smaller than 100 nm) silica particles, which are becoming more popular these days, apparently possess unique toxicological properties compared to 0.5− to 10-μm silica particles (10). Among crystalline and amorphous forms of silica (silicon dioxide, SiO2), the commonest forms of free crystalline silica include quartz, tridymite, and cristobalite. Natural quartz exists in sandstone (67% silica) and granite (25∼40% silica). Amorphous silica can be found in diatomaceous earth, opal, silica, and glass (11).
Inhalation of small silica crystals causes acute pulmonary inflammation. Chronic occupational exposure to silica crystals may induce pneumoconiosis silicosis, which results in progressive lung fibrosis. However, underlying mechanisms for pulmonary inflammation and fibrosis have not yet been fully identified (12). After inhaled silica crystals are deposited in small airways and phagocytized by resident macrophages, pulmonary alveolar macrophages secrete IL-1β or TNF (tumor necrosis factor)-α to induce inflammation and TGF (transforming growth factor)-β to induce fibrosis. During the process, apoptotic cell death occurs to remove unhealthy cells containing silica particles. Then, silica particles released from dying cells are phagocytized by neighboring macrophages, which evokes further inflammation (13).
Amorphous silica particles are known to be less toxic than crystalline silica. However, the applications of amorphous silica have been increasing, and nano-sized amorphous silica particles have become popular in many biomedical fields. Therefore, we investigated the inflammatory effects of amorphous silica nanoparticles using 30-nm amorphous silica nanoparticles and assessed their effects on human peripheral blood mononuclear cells (PBMCs) or purified monocytes. Cytotoxicity and production of IL-1β and IL-8 were evaluated. The mitochondrial ROS, mitochondrial membrane integrity, inflammasome formation, and endosomal localization of silica nanoparticles were also assessed.

MATERIALS AND METHODS
Cell culture and cell purification After internal review board approval and informed consent (No: 4-2012-0088), 100-ml blood was obtained from ten healthy donors. PBMCs were separated using ficoll/hypaque density gradient (density=1.070−1.074) by centrifugation at 1,600 rpm for 25 min. PBMCs were cultured in RPMI 1640 containing 10% FBS and streptomycin/penicillin (each 100 IU/mL) at 37 o C in a moisturized 5% CO2 incubator. Although endotoxin was not detected in silica nanoparticles used in this study, polymyxin B (InvivoGen, San Diego, CA, USA) at a concentration of 10 ng/ml was added as an endotoxin neutralizer. To purify monocytes, PBMCs were incubated for 2 h in RPMI 1640 containing 1% FBS at 37 o C in a moisturized 5% CO2 incubator, and adherent cells were obtained after discarding non-adherent cells. Staining for CD14, a monocyte marker, was performed to identify the monocyte population. Staining with MitoSOX and JC-1 Monocytes were treated with silica nanoparticles (125 and 250 μg/ml for 30-nm silica particles) for 40 min for MitoSOX staining (for 15 min). After treatment with silica nanoparticles, blood monocytes were stained with fluorescent dyes (MitoSOX) diluted in Hank's balanced salt solution for 15∼30 min at 37 o C in the dark. Then, 2.5μM MitoSOX (Invitrogen, San Diego, CA, USA) was applied to detect mitochondrial superoxide. After exposure to silica nanoparticles, staining with 2μM JC-1 (Invitrogen) was performed to determine mitochondrial depolarization. Flow cytometric analysis was performed using a FACScan (Becton Dickinson, Franklin Lakes, NJ, USA), and the results were analyzed using WinMDI software.

TEM analysis
Monocytes were treated with 125μ/ml of silica nanoparticles for 15 min and fixed with Karnovsky solution (2% glutaraldehyde, 2% paraformaldehyde, 0.5% CaCl2) for 6 h. After washing with PBS for 2 h, cells were treated with 1% OsO4 in 0.1M PBS for 2 h. Dehydration of each sample was performed gradually with 50%, 60%, 70%, 80%, 90%, 95% and 100% alcohol for 10 min, followed by a final addition of propylene oxide for 10 min. Samples were then treated with a mixture of EPON (EPON 812, MNA, DDSA, DMP30) and propylene oxide (1：1) for 18 h and heated in an embedding oven at 35 o C for 6 h, 45 o C for 12 h, and 60 o C for 24 h. The cell block was trimmed and sectioned at 0.25μm using an ultramicrotome (Leica Ultracut UCT, Leica Microsystems PBMCs were treated with 30-nm amorphous silica nanoparticles for 6 h and cytotoxicity was determined by CCK-8 assay. (b, c) PBMCs were treated with silica nanoparticles for 6 h and supernatant levels of IL-1β (b) and IL-8 (c) were assessed by ELISA. LPS (25 ng/ml) was pretreated for 3 h before nanoparticle exposure. Data represent means± S.D. of three independent experiments. One-way ANOVA analysis shows significance (p＜0.0001) (b, c) and student's t-test between certain pairs (b, c) was used for statistical analysis.
GmbH, Wetzlar, Germany). These ultra-thin sections were stained with 1% toluidine blue and put on a copper grid. The samples were then stained with both uranyl acetate (6%) and lead citrate, and analyzed using a TEM (JEM-1011, JEOL, Tokyo, Japan).

Statistical analysis
Independent Student's t test and one-way analysis of variance (ANOVA) were used to compare the differences between the control and study groups. A p-value of ＜0.05 was consid-ered significant.

RESULTS
Cytotoxicity and cytokine production by PBMCs after exposure to 30 nm amorphous silica nanoparticles The cytotoxicity of 30-nm silica nanoparticles in PBMCs has increased dose-dependently (Fig. 1a) starting at a concentration of 31.3μ/ml. Similar results were obtained after pretreatment with a low concentration (25 ng/ml) of LPS for 3 h (Fig.  Figure 2. Production of mitochondria ROS in blood monocytes. Monocytes were treated with silica nanoparticles or silver nanoparticles for 40 min. Cells were stained with 2.5μM MitoSOX (for mitochondrial superoxide) for 15 min and analyzed by flow cytometry. MI, mean intensity. Data shown here are representative results from three independent experiments. 1a). The LD 50 values of silica nanoparticles were 41.8μg/ml without LPS and 41.4μg/ml with LPS. Therefore, the pretreatment with LPS at a low concentration did not influence cytotoxicity following exposure to silica nanoparticles.
After exposure to silica nanoparticles, the levels of IL-1β and IL-8 produced by PBMCs increased dose-dependently. The IL-1β levels were 0.4, 1.3, and 1.7 ng/ml after treatment with silica nanoparticle at concentrations of 31.3, 125, and 250μg/ml, respectively. When LPS was pretreated, the IL-1β levels were higher (6.6, 11.6, and 7.9 ng/ml after treatment with silica nanoparticles at concentrations of 31.3, 125, 250μg/ml, respectively) than those treated with silica nanoparticles alone (Fig. 1b). The IL-8 levels were 4.9, 10.7, and 2.4 ng/ml after treatment with silica nanoparticles at concentrations of 31.3, 125, and 250μg/ml, respectively. When LPS was pretreated, the IL-8 levels were higher (13.1, 19.4, and 6.3 ng/ml after treatment with silica nanoparticles at concentrations of 31.3, 125, and 250μg/ml) than those treated with silica nanoparticles alone (Fig. 1c). After pretreatment with LPS, production of IL-8 started to decrease at a silica nanoparticle concentration of 250μg/ml. After pretreatment with LPS, production of IL-1β or IL-8 started to decrease at a silica nanoparticle concentration of 250μg/ml. These results suggest that PBMCs primed with LPS become vulnerable to the fol-lowing stimuli. Therefore, PBMCs respond more sensitively at lower concentrations of the following stimuli, but they become exhausted and do not respond to higher concentrations of the following stimuli.

Superoxide generation and deterioration of mitochondrial membranes in monocytes
After purification of monocytes from PBMCs, generation of superoxide was detected by MitoSOX and expressed as MI. As shown in Fig. 2, the MIs were 497 at a silica nanoparticle concentration of 125μg/ml and 555 at a silica nanoparticle concentration of 250μg/ml. When compared to the results of 5-nm silver nanoparticles, which were used as a positive control, silica nanoparticles induced significantly lower levels of superoxide. Silica nanoparticles operated at higher concentrations than silver nanoparticles, which was due to different characteristics of individual nanoparticles. The LD 50 of 5-nm silver nanoparticles is 0.48μg/ml (14), whereas that of 30 nm silica particles is 41.8μg/ml. In our study, 100-nm silver nanoparticles were used as a negative control because they do not trigger monocytes at 0.6∼0.9μg/ml (14).
Since MitoSOX detects superoxide at mitochondrial membrane, which may induce deterioration of mitochondrial membrane, we assessed mitochondrial membrane potential. JC-1 staining revealed the disturbance of mitochondrial membrane after exposure to 30-nm silica nanoparticles. The percentage of mitochondrial staining decreased by 51.5% at 1 h, by 50.4% at 1.5 h, and by 50.0 % at 2 h (Fig. 3). The control level was 60.5%, which was detected without exposure to silica nanoparticles.

Inflammasome formation in monocytes
To determine the inflammasome formation by silica nanoparticles, monocytes were stained with anti-NLRP3 and anti-caspase-1 antibodies after exposure to nanoparticles. As a result, oligomerization of NLRP3 and caspase-1 was detected 15 min after exposure to silica nanoparticles at a concentration of 125μg/ml (Fig. 4). Oligomerization of NLRP3 and caspase-1 was not found in control cells which were not exposed to silica nanoparticles.

Endocytosis of silica nanoparticles in monocytes
Next, the presence of silica nanoparticles and intracellular changes in monocytes were assessed via TEM. Within 30 min after treatment with silica nanoparticles at a concentration of 125μg/ml, double membrane structures suggestive of autophagosomes were found as indicated with white arrows (Fig.   5a). Silica nanoparticles also were detected in endosomes (Fig. 5b).

DISCUSSION
The aim of this study was to provide information on inflammatory effects of amorphous silica nanoparticles. As reported previously, crystalline silica nanoparticles induce NLRP3 inflammasome to secrete IL-1β, which is mediated by particle uptake, rupture of lysosomal membrane, and release of cathepsin B (15,16). The subsequent secretion of IL-1β is known to be responsible for silicosis (17). In contrast, the results from amorphous silica nanoparticles vary depending on particle size and cell types (18)(19)(20). Therefore, in our study using human PBMCs or purified monocytes, the effects of amorphous silica nanoparticles were assessed.
Maturation of IL-1β is tightly regulated by NLRP3, 1 of the 4 inflammasome types (21). Thus, even if pro-IL-1β protein is present inside a cell, it cannot be released until NLRP3 inflammasomes are formed. Upon proper assembly of NLRP3 inflammasomes, caspase-1 is activated and cleaves the proform of IL-1β to generate mature IL-1β. Mature IL-1β is then released from cells to perform their respective immunological  functions. Therefore, factors triggering inflammasome formation are also critical for producing functional IL-1β.
We assessed the responses of human PBMCs after treatment with silica nanoparticles. As expected, 30-nm amorphous silica nanoparticles induced IL-1β production in human PBMCs. We further speculated that silica nanoparticles may increase IL-1β production when pretreated with a low concentration of LPS. These findings suggest that the immunological relevance of silica nanoparticles should be considered with regard to their synergistic effects on minimally activated immune responses.
To simulate an in vivo environment we purified monocytes from PBMCs and assessed inflammasome formation. Previous immunological studies with nanoparticles mainly focused on macrophage cell lines, but not primary monocytes. It has been reported that macrophages and monocytes have different pathways to produce inflammasomes. While macrophages require 2 signals to release mature IL-1b, the first signal to produce pro-IL-1β protein and the second signal to activate NLRP3, monocytes constitutively express active casapse-1 and require only 1 signal for IL-1β release (22).
To activate caspase-1, inflammasome formation is a prerequisite. During inflammasome formation, oligomerization of NLRP3 occurs simultaneously with oligomerization of caspase-1. Confocal findings of monocytes treated with 30-nm silica nanoparticles suggest that oligomerization of NLRP3 or caspase-1 occurs rapidly as early as 15 min after exposure to nanoparticles. We investigated mechanisms underlying inflammasome formation following exposure to silica nanoparticles. ROS has been suggested to be a causative factor in the induction of the inflammasome complex. There have been many reports on the sources of ROS, such as peroxisomes, nitric oxide synthases, oxidoreductin 1, and cytosolic enzymes including cyclooxygenase, in the endoplasmic retic-ulum (23,24). However, the majority of ROS are produced as a by-product of the mitochondrial electron transport chain in cells undergoing aerobic metabolism (25). Although TiO2 (26), silica (27) and silver nanoparticles (14) produce ROS, there have been few reports that have identified the types of ROS produced. In our study, ROS was evaluated by assessing the production of mitochondria superoxide. Recently, superoxide in the mitochondrial membrane was reported to trigger inflammasome formation (14,28) in specific structures called mitochondria-associated ER membranes (29). Our results showed that 30-nm silica nanoparticles more increased production of mitochondrial superoxide than 100-nm silver nanopaticles and that collapse of the mitochondrial membrane was observed in blood monocytes 1 h, 1.5 h, and 2 h after treatment with 30-nm silica nanoparticles.
Collectively, inflammasome formation in monocytes was observed after exposure to amorphous silica nanoparticles. However, rupture of the endolysosomal membrane which was shown in crystalline silica was not detected in our study. These findings suggest that induction of superoxide and subsequent deterioration of the mitochondrial membrane may be one of the major factors in the induction of inflammasome formation in amorphous silica nanoparticles.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

text:
Epidemiology, molecular virology and diagnostics of Schmallenberg virus, an emerging orthobunyavirus in Europe

After the unexpected emergence of Bluetongue virus serotype 8 (BTV-8) in northern Europe in 2006, another arbovirus, Schmallenberg virus (SBV), emerged in Europe in 2011 causing a new economically important disease in ruminants. The virus, belonging to the Orthobunyavirus genus in the Bunyaviridae family, was first detected in Germany, in The Netherlands and in Belgium in 2011 and soon after in the United Kingdom, France, Italy, Luxembourg, Spain, Denmark and Switzerland. This review describes the current knowledge on the emergence, epidemiology, clinical signs, molecular virology and diagnosis of SBV infection.

Introduction
At the end of the summer and in the autumn 2011, hyperthermia and drop in milk production were reported in adult dairy cows in north-west Germany and The Netherlands [1]. In some cases, transient diarrhoea was also recorded in the Netherlands [2]. Some of the symptoms observed were similar to the disease caused by Bluetongue virus (BTV) and a re-emergence of this virus that led to a major epizooty in 2006-2008 in Europe was feared. Surprisingly, no known bovine pathogen was identified in samples from symptomatic cattle [3][4][5]. In November 2011, the Friedrich-Loëffler Institute (FLI) in Germany detected viral RNA belonging to a new virus in a pool of blood samples from clinically affected dairy cows using a metagenomic approach [3]. This new virus was called Schmallenberg virus (SBV) after the place of origin of the collected samples. Analysis of viral genomic sequences revealed similarities with Akabane, Aino and Shamonda viruses, all belonging to the Orthobunyavirus genus from the Bunyaviridae family. Douglas, Sathuperi and Shamonda viruses were later identified as closer relatives of SBV [6]. A specific realtime quantitative reverse transcription PCR (RT-qPCR) was then developed by FLI to detect the SBV genome and the protocol shared with many European partners. The inoculation of 9-month old calves with blood of cattle that were RT-qPCR positive for SBV or with the virus isolated in Culicoides variipennis larvae cells (KC cells) caused fever and mucous diarrhoea, providing experimental evidence that SBV might be responsible for the clinical signs observed [3].
This paper reviews current knowledge on the emergence, molecular virology, clinical signs, diagnosis and seroprevalence of SBV and is based on data published up to the end of January 2013 in peer-reviewed journals, internet-based reporting systems such as the Program for Monitoring Emerging Diseases (proMED-mail), communications from research institutes and official reports from governmental and European institutions such as the European Food and Safety Authority (EFSA).

Timeline of SBV infection in Europe
SBV was first detected in Germany and The Netherlands in 2011 [3]. In December 2011, The Netherlands reported a teratogenic effect of SBV in sheep with the birth of malformed lambs with crooked neck, hyrocephalus and stiff joints [2]. The presence of SBV was then reported in Belgium at the end of December 2011 and in the United Kingdom on the 22 nd of January 2012. France reported its first case of SBV on the 25 th of January 2012 after the virus genome was detected by RT-qPCR in brain samples from malformed lambs born on farms located in the territorial divisions of "Moselle" and "Meurthe et Moselle" in northeastern France [7]. The presence of SBV was then reported in Luxembourg on the 16 th of February [8]. On the 17 th of February, SBV was confirmed in a malformed goat in north-east Italy [8] and on the 12 th of March, in Spain (Andalusia), in a newborn lamb [9].
By the end of April 2012, SBV had been detected in 3628 herds in Europe [10]. SBV-infected holdings recorded up to this date corresponded to infections occurring in 2011. In May 2012, acute SBV infections were detected in cattle in south west France in the Pyrénées-Atlantiques territorial division [11], indicating that SBV was able to re-circulate after the winter period. Similar conclusions were also made after the detection of the virus in the United Kingdom in newborn lambs born in May and June 2012 [12,13] and in Germany in cattle, sheep and goat holdings sampled in 2012 [14].
Early 2012, the development of assays to detect anti-SBV antibodies, as discussed later in this review, provided a useful tool to show evidence of SBV infection since viraemia is transient [3,15].
On the 5 th of June, Denmark reported the presence of antibodies against SBV in two cattle from southern Jutland [16] and on the 20 th of July, Switzerland confirmed its first cases of acute SBV infection in cows from two farms in the canton of Berne [17].
By August 2012, more than 5500 cases of SBV infection in ruminants had been recorded across northern Europe [18].
In mid-September, anti-SBV antibodies were detected in Austria in cattle and sheep [19]. At the beginning of October 2012, the presence of antibodies to SBV was reported in western Poland in goats that were sampled at the end of July 2012 [20,21] and in Sweden in cows [22]. In mid-October, anti-SBV antibodies were detected in northern Scotland in a tup and in two cows from Finland that were sampled at the end of September 2012 [23]. Further studies suggested that the virus had spread to South Finland during the summer and early autumn of 2012 [24]. At the end of October 2012, the presence of SBV was detected in Ireland in a bovine foetus [25,26] and a few days later, in Northern Ireland in a malformed calf [27,28].
By the end of October 2012, SBV infection was confirmed by RT-qPCR and/or serology in approximately 6000 holdings in Europe [29].
In November 2012, antibodies against the virus were detected in milk from cattle herds in Norway [30] and an outbreak of SBV was reported in Italy (Sardinia) in a sheep flock with cases of abortion and foetal malformations [31]. At the end of December 2012, SBV was detected for the first time in the Czech Republic following the birth of malformed lambs [32]. In mid-January 2013, the first cases of SBV were confirmed in Estonia in sheep foetuses [33] and at the end of January the presence of SBV was confirmed in sheep in Slovenia [34].
All these reports show that SBV has spread rapidly in vast parts of Europe.

Molecular virology
Analysis of viral sequences has led to the classification of SBV in the Bunyaviridae family and the Orthobunyavirus genus.

Genome and structure
The bunyavirus genome consists of 3 segments of negativesense single-stranded RNA: the L (large), M (medium) and S (small) segments [35] ( Figure 1A). Phleboviruses and tospoviruses differ from other bunyaviruses since their S segment adopts an ambisense coding strategy. The L segment encodes the RNA-dependent RNA polymerase (RdRp) L (or L protein), the M segment encodes a precursor polyprotein that is co-translationally cleaved into the envelope glycoproteins Gn and Gc and the non-structural protein NSm and the S segment encodes the nucleoprotein N and the non-structural protein NSs in an overlapping open reading frame. The three segments of the SBV genome have been fully sequenced [3,36] but its structure and the different encoded proteins are not yet wellcharacterised and can only be predicated from the data available on related viruses ( Figure 1B).
Virions from bunyaviruses are enveloped, spherical and have a diameter of approximately 80 to 120 nm.
They acquire their membrane when budding at the Golgi apparatus lumen [37][38][39]. Electron microscopy performed at the FLI confirmed that, similarly to other bunyaviruses, the SBV virus particle has a diameter of approximately 100 nm and is membrane-enveloped [40]. Virus particles of bunyaviruses are constituted of 4 structural proteins: the two surface glycoproteins Gn and Gc and the viral polymerase complex composed of the polymerase L protein and the nucleoprotein N. This complex is responsible for the transcription and replication of the viruses that occur exclusively in the cytoplasm. Inside the virus particle, the viral genome is present as a ribonucleoprotein (RNP) associated with many copies of the nucleoprotein N and a few copies of the polymerase L.

Life cycle
Since SBV was discovered recently, very little is known on its life cycle and knowledge accumulated on other bunyaviruses can only be discussed.
The replication cycle of bunyaviruses is exclusively cytoplasmic. It begins with the recognition of the cellular receptor, unknown for the majority of bunyaviruses, by the Gn/Gc heterodimers present at the surface of the virion membrane [41,42]. Virions enter the cell via endocytosis. Change of pH in vesicles induces conformational modifications of the viral glycoproteins and the exposure of the Gc fusion peptide [43]. The viral envelope fuses with the membranes of the endosomes and the RNP are released inside the cytoplasm. The primary transcription can start and produce viral mRNA via a mechanism of catch-snatching [44]. The L protein cleaves a sequence of 10 to 18 nucleotides from the 5' end of capped mature cellular mRNA to use it as a primer for the initiation of viral transcription. Viral mRNA are synthesised and translation by host cell ribosomes leads to the production of viral proteins. The L and N proteins are needed for replication of the viral genome and the glycoproteins Gn and Gc form heterodimer complexes in the endoplasmic reticulum. Both glycoproteins are then transported to the Golgi apparatus via a Golgi-retention signal, located on Gn for most bunyaviruses, where their glycosylation is completed [45].
Assembly of bunyaviruses is thought to occur mostly in tubular virus factories containing both cellular and viral components situated around the Golgi complex [37,46,47]. From free nucleotides, the viral L polymerase produces complementary copies of the whole viral genome called antigenomes that are also present as RNP and are needed for the production of high quantities of viral genomes. Newly-formed genome RNP then accumulate in the Golgi complex where they directly interact with the C-terminal domains of the glycoproteins Gn and Gc [48][49][50][51]. Maturation of viral particles occurs via budding through the modified membrane of the Golgi apparatus. For orthobunyaviruses, these steps depend on the NSm protein that is associated with the Golgi apparatus via three transmembrane domains [52,53]. Mature virus particles are then transported in vesicles to the plasma membrane where they are released in the extracellular compartment by exocytosis. Further morphological changes then occur resulting in the release of fully infectious extracellular virus particles.

Pathogenicity
The pathogenicity of orthobunyaviruses is dependent on multiple viral factors encoded by the three genomic segments. For example, the neuroinvasive ability of La Crosse virus, another orthobunyavirus from the California serogroup, is determined by the polymerase and/or the glycoproteins whereas the host immune response is inhibited by NSs that antagonises the expression of type I interferon (IFN-I) and the transcription mediated by RNA polymerase II [54][55][56][57][58][59]. NSs from Bunyamwera (Bunyamwera serogroup), the prototype virus for the Orthobunyavirus genus and the Bunyaviridae family, also contributes to viral pathogenesis and seems to be a major virulence factor. This non-structural protein inhibits protein synthesis and the host cell antiviral response by interfering with RNA polymerase II-dependent transcription, IFN-I production and apoptosis mediated by IRF-3 [60][61][62][63][64][65].
Although there is no conservation of sequence, NSs from other bunyaviruses are also involved in pathogenesis and inhibition of the host cell antiviral response. For example, NSs from Rift Valley fever virus suppresses host transcription by interfering with the subunits of the transcription factor II H (TFIIH) complex, degrades dsRNA-activated protein kinase (PKR) and represses the activation of the promoter IFN-β via its association with a subunit of the Sin3A repressor complex [66][67][68][69][70].
However, little is known about the viral factors involved in the pathogenicity of viruses involved in veterinary medicine, such as Akabane virus, Shamonda virus and SBV. Recently, it was shown that IFN-I receptor (IFNAR) knock-out mice [71] are susceptible to SBV infection and can develop fatal disease as previously reported for La Crosse virus [72] and that intracerebral injection of SBV is lethal for NIH-Swiss mice [36]. Moreover, a study has suggested that infectious serum from cattle is more suitable for standardised SBV infection model than culture-grown virus [73]. These models could be useful in the future to study SBV pathogenesis and contribute to the design of vaccines. Reverse genetic systems have also been developed for SBV and provide a powerful tool to characterise the virus [36,74]. Recombinant viruses lacking NSs have already been generated to study the role of the viral protein as a virulence factor. It was subsequently shown that NSs is not essential for the virus to replicate in vitro [36,74] but a virus lacking the viral protein is attenuated in newborn mice [36]. NSs was found to block protein synthesis [74] and to interfere with the production of IFN [36,74] suggesting that, similarly to other bunyaviruses, NSs of SBV is able to modulate the host innate immune response.

Phylogeny
Initial phylogenetic analysis of the SBV genomic segments indicated that SBV displays 69% identity with Akabane virus for the L segment, 71% identity with Aino virus for the M segment and 97% identity with Shamonda virus for the S segment [3]. After analysis of additional sequence data, it was reported that the M segment of the Sathuperi and Douglas orthobunyaviruses display higher identity with SBV whereas the S and L segments are closer to the Shamonda virus [75]. All these viruses belong to the Simbu serogroup, although no crossprotection has been reported between them. All together, these studies suggested that the 3 genomic segments of SBV could be the result of a reassortment between the segment M of the Sathuperi virus and segments S and L of the Shamonda virus. Nevertheless, another group has recently determined the almost full-genome sequences of 9 viruses from the Simbu serogroups belonging to five species (i.e. species Shamonda virus (Shamonda virus, Peaton virus and Sango virus), species Sathuperi virus (Douglas virus and Sathuperi virus), species Shuni virus (Aino virus and Shuni virus), species Akabane virus (Sabo virus) and species Simbu virus (Simbu virus)) [6]. Phylogenetic analysis of all of these sequences has shown that SBV belongs to the Sathuperi virus species. This conclusion is also supported by a serological investigation showing that Douglas and Sathuperi viruses, but not Shamonda virus, are neutralised by anti-SBV serum [6]. In this study, it was also suggested that SBV is an ancestor of Shamonda virus, the latter being a reassortant containing the S and L genomic segments from SBV and the M segment from an unclassified virus.

Clinical signs
Sheep and goats seem to be very mildly affected by SBV infection. Symptoms are more apparent in adult cows, and include loss of appetite, hyperthermia and diarrhoea, which can lead to a 50% reduction in milk production [76][77][78]. Symptoms usually disappear within a few days. The viraemia induced by SBV is short-lived, lasting for 2 to 6 days in cattle [3,78].
In December 2011, The Netherlands reported a teratogenic effect of SBV in sheep with manifestations comparable to those observed for Akabane and Aino viruses [79][80][81][82]. Infected females are able to transmit the virus to foetuses (ovine, caprine and bovine), which developed atypical malformations leading most frequently to intra-uterine death or death immediately after birth. Common congenital malformations and clinical signs in aborted and stillborn animals include a neuro-musculo-skeletal disorder called arthrogryposis, severe torticollis, ankylosis, kyphosis, lordosis scoliosis, brachygnathia inferior and neurological disorders such as amaurosis, ataxia and/or behavioral abnormalities ("dummy syndrome" as observed during the epizooty caused by BTV serotype 8 (BTV-8) [83][84][85] (Figure 2).
In case of twin gestation, one twin can suffer from arthrogryposis and the other from neurological disorders. One twin can also be born malformed and the other one viable or only affected by a delayed growth.
Newborns suffer from severe neurological disorders that generally lead to death of the animal several hours to several days after birth. It was reported that a SBVpositive one-week old calf born at term showed severe central nervous system lesions, severe dysfunctions of the cerebral cortex, basal ganglia and mesencephalon, severe porencephaly or hydranencephaly but no arthrogryposis [86]. Interestingly, the SBV genome was still detectable in the CNS, suggesting that it is able to persist in the infected foetus after birth.
Histological studies have revealed lymphohistiocytic inflammation in the central nervous system and glial nodules in the mesencephalon and hippocampus in ovine species. Astrogliosis and microgliosis were detected in both calves and lambs and some cases of myofibrillar hypoplasia of skeletal muscles were reported for both species [84]. Histological examination of the brain and spinal cord of a ten-day old SBV RT-qPCR-positive calf has also reported the presence of meningoencephalitis and poliomyelitis [89]. Furthermore, immunohistochemistry and in situ hybridisation methods performed on brain sections have suggested that neurons are the major target for SBV replication in naturally infected newborn lambs and calves [36,88].
In the case of Akabane virus, infection of the foetus occurs between the 28 th and 36 th day of gestation in sheep, the 30 th and 50 th day in goats and the 76 th and 174 th day in bovines [81]. The severity of foetal injury depends on the time of infection during gestation and maximal damage occurs when neuronal tissues are differentiating [78,81,90]. Detection of the SBV genome in brain, blood or spleen samples in malformed aborted or stillborn ruminants, which represent the majority of the diagnosed cases from December 2011 to March 2012 for lambs and March to June 2012 for calves, indicates that infection of the mother occurred during gestation, in autumn 2011 [29]. A study has estimated the risk of SBV infection for foetuses born from cows primo-infected after formation of the placenta to 28% [91]. No visible clinical signs are present at birth if in utero infection occurs while the immune system of the foetus is able to control the infection [91].
SBV can infect bison as reported in Germany [92]. The virus is also able to infect wild cervids and llamas but no clinical signs or macroscopic abnormalities were recorded for these species [93,94]. The virus might infect other wild species and domestic animals such as horses or dogs, as reported for viruses belonging to the Orthobunyavirus genus. However, SBV infection has not yet been reported in these species. Most of the viruses from the Simbu serogroup are not considered to be zoonotic, with the exception of Oropouche virus, which can infect humans and provoke severe flu-like symptoms. To date, no evidence of SBV infection in humans has been reported and no SBV-neutralising antibodies have been detected in sera from persons (farmers and veterinarians) exposed to the virus [95,96].

Transmission
The majority of bunyaviruses are transmitted by arthropod vectors and, in particular, mosquitoes, phlebotoms, culicoides, ticks and thrips, with the exception of hantaviruses which are transmitted by rodents. Studies have shown that viruses within the Simbu serogroup are mostly transmitted by culicoides, but also by mosquitoes from the Aedes and Culex genus and by several species of ticks [97,98]. Recently, a study has reported the presence of the SBV genome in a pool of culicoides (C. obsoletus complex, C. chiopterus and C. dewulfi) trapped from July to October 2011 in Belgium [99]. Culicoides from the C. obsoletus group trapped in Denmark during the same period also contained SBV RNA [100]. Furthermore, SBV RNA was detected in C. obsoletus complex and C. chiopterus collected in August-September 2011 in the Netherlands where the prevalence of SBV among Culicoides at this period was estimated to be around 0.25% [101]. The virus has also been found in biting midges in Norway, Poland and Sweden [102][103][104]. These studies suggest that species of culicoides identified as vectors for BTV also act as vectors for the transmission of SBV [105][106][107][108]. To date, no studies have been carried out to assess the ability of other arthropods, such as mosquitoes and ticks, to act as vectors for the transmission of SBV.
Cases of acute SBV infection were recorded following the start of the 2012 vector season in France, the United Kingdom, Switzerland, Germany and Italy [11][12][13][14]17,29,31]. It is not yet known how SBV is able to persist despite the winter season. It could be the result of the vector population surviving the cold season or the virus persisting in the cattle population or in other reservoirs. It has been reported that some culicoides species are present inside farm buildings during the winter and are able to complete their life cycle in animal enclosures [109,110]. It is then possible that SBV is able to persist from year to year in the vector population despite winter temperatures.

Origin
The history and geographical origins of SBV raise numerous questions. As for the introduction of BTV-8 in The Netherlands and Belgium in 2006, the causes of SBV emergence in northern Europe remain unknown. The closest relatives to SBV are Sathuperi, Douglas, Shamonda, Akabane, Aino, Peaton or Sango viruses but, to date, they have not been identified in Europe [111]. These viruses are present in other parts of the world and seem to be able to emerge in regions far from their areas of enzootic distribution. Akabane and Aino viruses are distributed in the Far East and Australia, and Akabane virus has been found recently in Israel and also in Turkey. Similarly, Shamonda virus was detected in Nigeria in the 1960s and has been isolated since only in Japan in 2005 [112]. Although SBV was discovered only recently, there is no doubt that its origins are more ancient and that it might have co-evolved with other closely related viruses. Viruses belonging to the Simbu serogroup have not been well studied and epidemiological data are poor. Nevertheless, phylogenetic analyses based on samples taken in different regions of the world at different periods of time suggest that these viruses evolve slowly. For example, the nucleotide sequences of Japanese strains of the Shamonda virus isolated in 2005 differ by only 3% from those of strains isolated in Nigeria 30 years before [112].
As discussed above, Shamonda virus is thought to be a reassortant containing the S and L genomic segments from SBV and the M segment from an unclassified virus [6]. Such reassortment phenomena have been described previously between viruses of the Orthobunyavirus genus [113]. In the Simbu serogroup, it is thought that Peaton virus is derived from an ancestor generated by a reassortment between Akabane and Aino viruses [114] and that a virus circulating in Japan is the product of reassortment between a Japanese strain of the Aino virus and an Australian strain of the Peaton virus [113].
SBV emergence is a reminder that bunyaviruses and particularly orthobunyaviruses are a potential threat for European livestock. Akabane and Aino viruses are already present in the Mediterranean region and might be introduced into Europe. Consequently, the surveillance of livestock and vector populations is critical to monitor the emergence of such viruses in Europe. The identification of the Batai virus, an Orthobunyavirus belonging to the Bunyamwera serogroup, in populations of Anopheles maculipennis trapped in the south-western part of Germany in 2009 illustrates the importance of this type of surveillance [115].
It is surprising that SBV has emerged in the same region of Europe as BTV-8 in 2006 and later BTV-6 and −11, and may suggest that the viruses followed the same route of introduction. SBV might have been present before in a region of the world where no and/or rare clinical signs were manifested or reported in the native population. Considering the increase in international trade of animals and products of animal or vegetal origin, it is of no doubt that Europe will face more and more frequently this type of emergence.

Diagnosis
Diagnosis of SBV infection relies on the detection of the viral genome by RT-qPCR, as mentioned above. It is a duplex assay that was initially developed by the FLI [3,116]. The technique is based on the simultaneous amplification of a SBV gene and an endogenous gene, β-actin or GAPDH, which is used as an internal positive control (IPC) to ascertain RNA integrity and the absence of PCR inhibitors. Primers amplifying a part of the L gene segment were first used as a template for the detection of the SBV genome. A protocol targeting the S segment was then optimised and showed higher sensitivity [15]. This protocol was implemented in most laboratories in Europe. Brain samples from aborted or stillborn lambs, kids and calves have mainly been used for diagnosis of SBV. Studies have shown that samples from the cerebrum, external placental fluid and the umbilical and spinal cord are suitable for the detection of SBV [116] and that the highest concentration of SBV RNA is found in the brainstem [117,118].
Viral isolation requires the inoculation of Vero (African green monkey kidney epithelial), BHK-21 (baby hamster kidney fibroblast) or KC (Culicoides variipennis larvae) cells with brain, serum or blood samples.
Once SBV strains were available, virus neutralisation tests (VNT) and a plaque reduction neutralisation test were developed to detect antibodies present in the serum of infected animals [96,119,120]. These methods are time-consuming (4 to 6 days) and cannot be automated. A diagnostic test that allows serological testing of a large number of samples, not possible by VNT, was needed to diagnose SBV infection and estimate SBV seroprevalence in infected areas. An indirect ELISA test based on a recombinant SBV nucleoprotein antigen produced by ID-VET (Montpellier, F-34070) was designed. This test was validated by the Animal Health laboratory at the French agency for food, environmental and occupational health and safety (ANSES) in Maisons-Alfort (Alfort ANSES laboratory) in April 2012 and currently provides a rapid and less expensive tool for serological diagnosis [121]. A Dutch group has developed an ELISA based on SBV that has been cultured on partially purified and completely inactivated Vero cells [122]. This assay was shown to be a sensitive test to detect antibodies in foetal or procolostral sera and diagnose SBV in newborn calves and lambs. ELISA tests have also been developed to detect anti-SBV antibodies in milk [123,124].
Interestingly, in a study looking at anti-SBV antibodies in foetal blood samples from calves and lambs, it was shown that 74% of the samples tested positive for SBV infection by ELISA whereas only 27% were SBV-positive by RT-qPCR [122]. Using a VNT assay [121], the Alfort ANSES laboratory also found that from 71 aborted or stillborn animals with malformations evocative of SBV, for which the laboratories had received brain samples and serum samples from their "mothers", 100% of the mother sera were VNT positive whereas only 61.9% (44/71) of the offspring brain samples were SBV-positive by RT-qPCR (unpublished data). These different results suggest that the diagnosis by RT-qPCR might have underestimated the real number of SBV cases. It is possible that the foetus is able to produce neutralising antibodies that will clear the virus. Neutralising antibodies against Akabane virus can be found in foetal and precolostral sera from calves [79,125]. This would explain why, in some cases, aborted or stillborn animals with clinical signs of SBV infection are found RT-qPCR-negative for the virus and the difficulties to isolate the virus. Other explanations would be that the genome of the virus is destroyed after the death of the foetus or during sampling and processing of the samples or that SBV infection is not the cause of the malformations observed.

Surveillance
Sharing knowledge and tools has allowed affected and neighbouring regions to efficiently and rapidly monitor the progression of SBV infections in Europe as shown in Figure 3A.
For example, in France, a national passive SBV surveillance system coordinated by the Ministry of Agriculture was launched on the 4 th of January 2012 in consultation with the French epidemiological surveillance platform for animal health. Veterinarians were asked to send samples (brain, spleen) from stillborns or malformed ruminant offspring showing clinical signs such as arthrogryposis, torticolis, scoliosis, brachygnathia and/or hydranencephaly. RT-qPCR was performed to detect SBV RNA and virus was isolated using the protocols kindly provided by Dr Martin Beer and Dr Bernd Hoffmann from the FLI, as described above [3,116]. From December 2011 to the beginning of March 2012, the Alfort ANSES laboratory was the laboratory designated by the Ministry of Agriculture to diagnose SBV infection in France. In mid-March 2012, a network of 66 local laboratories was established for SBV testing. This laboratory network is similar to the one established for  Figure 3B). SBV-infected farms are localised mainly in the north-east and the central-west of France [127]. Following the new cases of SBV recorded in different regions of France since the beginning of September 2012, surveillance measures were re-established from the 1 st of November 2012 to monitor congenital forms of SBV. They will take into account all cases detected from the 1 st of September 2012 [128]. To date, France remains the country with the highest number of SBV-infected farms recorded.

Seroprevalence
Serologic investigations are needed to determine seroprevalence in affected and neighbouring regions and monitor the spread of SBV infection. They are also useful to improve modelling predictions and assess the overall impact of SBV. Serological studies were conducted in several European countries following the design of new tools to detect anti-SBV antibodies.
A study investigating the prevalence of antibodies against SBV among dairy cattle in The Netherlands during winter 2011-2012 has reported an estimated seroprevalence of 72.5% [129].
Antibodies against SBV have been detected in 91% of cows sampled within 250 km of the location where SBV emerged in Belgium in February-April 2012 [91]. Additional studies performed in all of Belgium have reported a between-herd and within-herd seroprevalence of 99.76% and 86.3%, respectively in cattle (11 635 cattle from 422 herds sampled between the 2 nd of January and the 7 th of March 2012) [130]. A between-herd seroprevalence of 98.03% and a within-herd seroprevalence of 84.31% were also found in sheep (1082 sheep from 83 flocks sampled between the 4 th of November 2011 and the 4 th of April 2012) and a within-herd seroprevalence of 40.68% was found in goats (142 goats from 8 flocks sampled during the same period) [131]. These reports suggest that most host animals have been in contact with SBV in Belgium.
Preliminary seroprevalence studies were also undertaken by the Alfort ANSES laboratory in farms from different French territorial divisions using ELISA and VNT [132]. A high seroprevalence of SBV in cattle and sheep herds (32 to 100%) was found in northern and eastern regions of France as shown on Figure 3B. SBV seroprevalence seems lower in farms situated in southern parts of France (7.5 to 14.9%). These data are in accordance with the introduction of SBV in France from the north-east part of the country where the first cases were diagnosed. This suggests that, like BTV in 2007, SBV spread from the north-east of France to the rest of the territory. However, more serological studies need to be undertaken to confirm these findings.
The high seroprevalence of SBV found in The Netherlands, Belgium and in several French farms, indicates a widespread exposure to SBV during the biting insect season in 2011. Retrospective and prospective serological studies on ovine, bovine and caprine livestock will help to elucidate the period of SBV introduction in Europe. Similarly, it would be interesting to study the seroprevalence of SBV in wild species to evaluate their involvement as a reservoir of the virus. A study has reported the presence of anti-SBV antibodies in roe and red deer from southern Belgium sampled from October to December 2011 [93]. An average seroprevalence of 43.1% was found in the cervids sampled suggesting that SBV had spread rapidly among wild deer and that wild species could be involved as reservoirs of the virus.

Impact
SBV has a low or limited impact on animal health. Reports submitted by different European countries affected by SBV have estimated morbidity and mortality rates of less than 3% [133]. The highest proportion of SBV-confirmed herds in comparison with the total number of herds per region is 6.6% for sheep and 4% for cattle [29]. Nevertheless, it remains difficult to provide an accurate (See figure on previous page.) Figure 3 Location of SBV infection in Europe and France. (A) Spread of SBV infection for all ruminants in Europe from September 2011 to October 2012 (source: European Food and Safety Authority [29]). Affected countries reported the location and the time of the first report of confirmed SBV infection. (B) Location of SBV infected farms in France as of August 31, 2012 (source: French Ministry of Agriculture [127]). A total of 3197 farms have been reported to be SBV-infected. The seroprevalence of SBV in several farms from different French territorial divisions (northern regions (02 and 76), eastern region (54 and 57) and central region (18 and 87)) where SBV had been previously detected or neighbouring farms is also shown [132]. The presence of anti-SBV antibodies in cattle or sheep were detected by ELISA and VNT. The first, second and third farms where SBV was detected in France are also indicated. estimation of the number of herds affected by the virus and to determine the economic impact of the disease on the livestock industry since cases were most likely underreported or underdetected [132,134,135].
Few data are available on the impact of SBV within herds. A preliminary survey investigating the impact of SBV infection in sheep flocks in France has been conducted recently. This study showed that in SBV RT-qPCR-positive flocks, an average of 85% lambs were healthy while 13% were born dead or died rapidly after birth and 2% were born with malformations but survived for more than 12 h (data from 363 flocks and 64 548 lambs) [136]. However, the clinical impact was very variable between flocks. This might be due to differences in the proportion of ewes at a susceptible stage of pregnancy at the time of exposure to SBV.

Prevention
Considering SBV has a limited impact on animal health, trade restrictions have not been advised and are regarded as unjustified by the European Union (EU) and the World Organisation for Animal Health (OIE) [137]. SBV is transmitted by a vector that is widespread within Europe and movement bans would be ineffective. However, many countries outside the EU have imposed restrictions on the import of live animals and products from the EU such as semen and embryos. Recently, SBV RNA has been detected in the semen of naturally infected bulls and SBV infection was reported in calves inoculated experimentally with SBV RT-qPCR-positive semen [138][139][140]. These findings show that the semen of bulls naturally infected with SBV can be infectious and suggest that SBV and Akabane virus differ in terms of semen contamination since a group has reported previously that the semen from bulls infected experimentally with Akabane virus is not infectious [141]. The export of semen from countries where SBV is present might represent a risk of contamination. It is then essential to develop a sensitive test for the detection of SBV RNA in semen and to perform further studies to determine the risk of transmission of SBV via this route and the impact of the virus on fertility.
Vaccination is a preventive measure that could reduce the impact of SBV [142]. The development of vaccines is in progress and a vaccine should be available commercially in the future [30,143]. Nevertheless, the cost of vaccination for the livestock industry might not be justified since SBV seems to be a low impact disease. Vaccines have been developed against Akabane disease but management of outbreaks relies mainly on the sentinel monitoring of vectors and cattle [142,144,145].

Conclusions
The emergence of SBV at the end of 2011 in Europe is a reminder that the introduction of new diseases remains a threat for European countries. The rapid response to the emergence of SBV established by affected European countries has shown that an efficient network of laboratories is in place to face the emergence of new animal viruses. SBV was able to re-circulate after the winter season and is still circulating in Europe. A certain level of protection exists within the ruminant populations but it is likely that a significant percentage of animals remains susceptible to SBV in areas where no or few cases of SBV have been reported. This suggests that new congenital cases of SBV infection will occur during the winter 2012-2013. Maintaining an efficient surveillance would be essential to further describe the progression of the epidemic and its impact on the breeding industry. More studies are needed to determine the regions in which SBV is present, to understand its geographical and genetic origin and to identify its putative reservoirs. A better understanding of the pathogenesis associated with SBV infection and the ability of SBV antibodies to protect animals against the disease will also be useful to control the disease.


Moroccan Leishmania infantum: Genetic Diversity and Population Structure as Revealed by Multi-Locus Microsatellite Typing

Leishmania infantum causes Visceral and cutaneous leishmaniasis in northern Morocco. It predominantly affects children under 5 years with incidence of 150 cases/year. Genetic variability and population structure have been investigated for 33 strains isolated from infected dogs and humans in Morocco. A multilocus microsatellite typing (MLMT) approach was used in which a MLMtype based on size variation in 14 independent microsatellite markers was compiled for each strain. MLMT profiles of 10 Tunisian, 10 Algerian and 21 European strains which belonged to zymodeme MON-1 and non-MON-1 according to multilocus enzyme electrophoresis (MLEE) were included for comparison. A Bayesian model-based approach and phylogenetic analysis inferred two L.infantum sub-populations; Sub-population A consists of 13 Moroccan strains grouped with all European strains of MON-1 type; and sub-population B consists of 15 Moroccan strains grouped with the Tunisian and Algerian MON-1 strains. Theses sub-populations were significantly different from each other and from the Tunisian, Algerian and European non MON-1 strains which constructed one separate population. The presence of these two sub-populations co-existing in Moroccan endemics suggests multiple introduction of L. infantum from/to Morocco; (1) Introduction from/to the neighboring North African countries, (2) Introduction from/to the Europe. These scenarios are supported by the presence of sub-population B and sub-population A respectively. Gene flow was noticed between sub-populations A and B. Five strains showed mixed A/B genotypes indicating possible recombination between the two populations. MLMT has proven to be a powerful tool for eco-epidemiological and population genetic investigations of Leishmania.

Introduction
Leishmaniasis constitutes a group of diseases caused by obligatory, intracellular, protozoan parasites of the genus Leishmania, that cause a spectrum of diseases, ranging from self-limiting, self-curing cutaneous leishmaniasis (CL) to visceral leishmaniasis (VL) with fatal spontaneous evolution [1]. In areas bordering the Mediterranean Sea, Leishmania infantum is the etiologic agent of VL, but can also cause CL. VL is endemic in these countries, affecting the most vulnerable young children and adults, normally associated with HIV/AIDS [2]. The recent global VL and CL incidence were estimated by Alvar et al in 2012. Based on these estimates, approximately 0.2 to 0.4 million VL cases and 0.7 to 1.2 million CL cases occur each year. More than 90% of global VL cases occur in just six countries: India, Bangladesh, Sudan, South Sudan, Brazil and Ethiopia [2].Most strains of L. infantum isolated from Mediterranean foci belong to the predominant zymodeme MON-1 [3], despite their very wide geographical distribution.  . Epidemiological patterns of leishmaniasis in Mediterranean area are changing dramatically, due to several factors, such as widespread migration from rural to urban and peri-urban areas, socio-political situation [4,5], climatic changes increasing exposure to the sandfly and also, in urban areas, increasing HIV infection.
VL is widespread in northern Morocco (Chefchaoun, Taounate, Taza, Fes, My yacoub, Meknes, Sefrou, Al Houceima and sidi kacem) ( Figure 1). Few epidemiological data are available concerning its epidemiology and clinical features. Also a new focus of CL due to L. Infantum was recently described [6]. Before 1995, human VL was not an obligatory reportable disease. The incidence of VL was over 150 cases/years in 2006-2008 [2] of which children under 4 years old are most affected. Expansion of arid zone and increase of global temperature increases the incidence of leishmaniasis in this region [7]. Dog is the main reservoir of L.infantum in Morocco as in all the Mediterranean basin [8,9].
The sand fly vectors of L. infantum in this region are: P. perniciosus [10], P.longicuspis [11] and P. ariasi [9]. Zymodeme MON-1 is predominant in Morocco, however, zymodeme MON-24 has been occasionally isolated from a dog [12]. This zymodeme was considered to cause CL sporadically. Different methods have been used for the identification and classification of Leishmania parasites as reviewed [5]. These methods, including multilocus enzyme electrophoresis (MLEE) which is still considered as the gold standard for species and strain typing of Leishmania, are limited in the intrinsic level of polymorphism they can detect and are, only in exceptional cases, able to differentiate strains in the zymodeme MON-1 [5]. Epidemiological studies on VL caused by L. infantum require the use of highly discriminative techniques that are able to differentiate MON-1 strains. Multilocus Microsatellite Typing (MLMT) has proven to be a powerful tool for population genetic and epidemiological studies of Leishmania spp. For review see Schoenian et al. [5]. Ochsenreither et al. [13], Kuhls et al. [14,15] developed panel of 14 microsatellite markers which are able to discriminate L. Infantum strains and have been useful for investigating genetic polymorphism and population structure of L.infantum in various endemic areas [16][17][18].
In the present study, we used the above mentioned 14 microsatellites markers to investigate the genetic polymorphism and population structure of Moroccan L. infantum strains isolated from human and canine cases (CanL). We have compared the Moroccan MLMT profiles to those of Tunisian, Algerian and European strains belonging to previously characterized MON-1 and non-MON-1 isoenzyme groups. This comparison was done to understand VL transmissions scenarios in North Africa and South Europe.

1: Source of Leishmania and DNA extraction
Thirty-three strains of Moroccan L. infantum were analysed. Thirty strains were isolated from human VL cases aged between 1-7 years old, and three strains from canine cases. The strains came from all endemic areas in Northern Morocco; 11 from Taounate, eight from Fes, seven from My Yacoub, one from Al Houceima, one from Taza, and for five strains the exact origin was unknown. Table 1 list the strains, their origin and geographical distribution, the clinical form of the disease, and the source of strains used in this study and the reference strains. Isoenzyme profiles were available for only 2 Moroccan strains which belonged to the MON-1 zymodeme.
For 27 strains, DNA was extracted from promastigotes grown in Novye MacNealeNicolle biphasic culture medium (NNN) [19]. Furthermore, DNA was isolated from amastigotes in bone marrow aspirates spotted on six glass slides. DNA extraction from cultures was done by using phenol-chloroform extraction method and from slides, with some modifications, as described previously [20,21]. Figure 1 shows geographical distribution of the studies strains and VL endemic areas in Morocco.
The microsatellite profiles obtained previously for ten strains from Tunisia, ten from Algeria and 20 from different European countries were used as controls for comparison [15][16][17] and to investigate gene flow amongst North African and South European strains. These strains represent different clinical forms (CL and VL) and belonged to zymodeme MON-1 and different non-MON-1 zymodemes, see Table 1.

2: L. infantum identification and microsatellite genotyping
The internal transcribed spacer 1 (ITS-1) was amplified for all samples. The PCR product was digested with the restriction endonuclease HaeIII as described previously [22]. The resulting RFLP profile was identical to those of the WHO reference strain of L. infantum MHOM/TN/1980/IPT1 (data not shown).

3: Microsatellite data analysis
Two models were used to analyse the microsatellite data: (1) Population structure analysis was investigated using the program STRUCTURE 2.2, which implements a Bayesian model-based clustering method, using genotype data consisting of unlinked markers [23]. The admixture model was used. Markov chain Monte Carlo (MCMC) searches consisted of a burn-in length of 10,000 iterations followed by a run of 100,000 replications for each setting of K (the number of populations) from 1 to 10 with 10 replicate runs of each. The most appropriate number of populations was determined based upon ad hoc statistic ⎢K that evaluates the second order rate of change of the likelihood function with respect to the number of populations (K) [24]. (2) Microsatellite-based genetic distances analysis was based on the proportion of shared alleles distances. Phylogenetic trees were constructed using Neighbour-joining (NJ) method by the help of the softwares MSA 3.0, POPULATIONS 1.2.28 and MEGA version 3.1, as described previously [16].
Descriptive statistics for the observed genetic populations were calculated with the help of GDA software [24]. This includes allelic diversity (number of allelic variants per marker and mean number of alleles (MNA) per population), proportion of polymorphic loci (P), expected (He) and observed (Ho) heterozygosity and inbreeding coefficient (F IS ). The degree of genetic differentiation and gene flow among populations were assessed by calculating F ST values with corresponding pvalues, FST values higher than 0.25 indicate strong genetic differentiation [25].

4: Ethics statement
According to ethical approval of this study, all samples were anonymized. Written informed consent was obtained from each study participant or the parents/guardians on behalf of the children under 15 years old. Study design and procedures were revised and approved by the Institutional Review Board (IRB), Institut Pasteur du Maroc, Casablanca, Morocco, and the Ethical Committee of Charitè University Medicine, Berlin, Germany.

Results
Every analysed Moroccan strain had an individual microsatellite profile (33 genotypes). Thirteen markers were polymorphic, only marker Li 71-33 was monomorphic. Marker Lm4TA was the most polymorphic one presenting seven alleles, whereas markers Li41-56, Li46-67, Li 71-5/2 and LIST7039 were least polymorphic presenting only two alleles for each. For the 33 Moroccan strains, the mean number of allelic variants per locus (A) was 3.21. The observed heterozygosity (Ho) was between 0 and 0.15 and the expected heterozygosity (He), representing the probability that an individual will be heterozygous over the loci tested, ranged from 0 to 0.81 and was in most cases much higher than Ho. Inbreeding coefficient per locus (F IS ) was positive for 11 markers and ranges from 0.93 to .037, for the other three markers (F IS ) values was zero. This indicates a large number of homozygotes in the investigated strains (data not shown). Two main populations were detected by Structure for the data set analysed; Population 1 consisted of all strains from Morocco and the Tunisian, Algerian and European strains of MON-1 type and those of MON-77, 89 and 108, closely related to MON-1 as described previously [15,18]. Population 2 consisted of all non-MON-1 strains from Tunisia, Algeria and Europe which were added as controls; see Figure 2A and Table 1. These two populations were genetically different as shown by their F ST value (0.35) and P-value 0.0001.
When re-analysed separately by Structure, Population 1 was subdivided into two sub-populations ( Figure 2B). Subpopulation 1A consisted of 13 Moroccan strains; three from Fes, three from My Yacoub, one from Taounate, one from Taza, five of unknown origin in the endemic area and all European strains of MON-1 type that have been added for comparison. Sub-population 1B comprised 15 Moroccan strains; eight from Taounate, five from Fes, two from My Yacoub, and all Tunisian and Algerian MON-1 control strains ( Figure 2B and Table 1). These two sub-populations were as well significantly different as shown by their F ST value (0.34) and P-value 0.0001. Five Moroccan strains have shown mixed genotypes between sub-populations 1A and 1B; two strains from My Yacoub, two from Taounate and one from Al Houceima (see Table 1, Figure 2 and Figure 3). Theses strains shared allele's characteristic for each sub-population and showed significant shared membership in both sub-populations ( Table 2).
Measures for genetic diversity have been calculated for the two Populations 1 and 2 and for Sub-populations 1A and 1B, the five mixed genotypes were excluded from this analysis ( Table 3). Population 2 (non-MON-1) was found to be more diverse than Population 1, while Sub-population 1A was more diverse than Sub-population 1B (Table 3). Inbreeding coefficient (F IS ) was highest for Population 1 (MON-1 strains), and equally for both sub-populations of this population (Table  3).
Three main clusters were detected in the NJ distance tree based on the proportion of shared alleles (D AS ) measure ( Figure 3). The first cluster comprised all non-MON-1 strains from Tunisia, Algeria and Europe and was identical with Strains isolated from canine cases grouped together with strains from humans in Sub-population 1A. No correlation was found between a particular MLMT profile and host background.

Discussion
In this study, a panel of 14 microsatellite markers was applied to investigate the genetic polymorphism and population structure of Moroccan L. infantum strains and to compare them with strains from North African and European countries.
Both phylogenetic analysis based on genetic distances as well as Structure analysis using a Bayesian clustering approach grouped the Moroccan strains together with MON-1 strains or strains closely related to MON-1 from Tunisia, Algeria and Europe in Population 1. None of the Moroccan strains were assigned to the non MON-1 population (Population 2). Isoenzyme analysis had been performed for only two of the Moroccan strains studied which were typed as MON-1. MLMT analysis suggests that the remaining Moroccan strains analysed here also represent MON-1 and/or MON-1 closely related zymodyme types.
Surprisingly, two sub-populations were detected in Morocco. Sub-population 1A consisted of 13 Moroccan strains and that were related to the European MON-1 strains added for comparison. On the other hand, Sub-population 1B consisted of 15 Moroccan strains that were more similar to the Tunisian and Algerian MON-1 strains included in this study. The two sub-populations were genetically different from each other as confirmed by their F ST value and the corresponding P-value.
Phylogenetic analysis based on proportion of shared alleles was compatible with structure analysis based on Bayesian clustering model. The detected clusters were identical with identified populations and sub-populations hence confirming the presence of two sub-populations of L. infantum in Morocco and the presence of gene flow and mixed genotypes between them.
The presence of two L. infantum sub-populations co-existing in Moroccan endemic foci suggests multiple introduction of L. infantum from/to Morocco. Two scenarios are possible: (1) introduction from/to the neighboring North African countries, this is supported by the presence of Sub-population 1B (2), introduction from/to the European Mediterranean countries, this is supported by the presence of Sub-population 1A. Since Morocco is the African country closest to the European continent, through the Strait of Gibraltar, and due to the frequent migration of humans and their animals from and to Europe, back and forth introduction of the disease cannot be excluded. Due to their high variability and extensive size homoplasy, microsatellite markers are not suitable for estimating evolutionary history and are not able to confirm the origin and direction of introduction of Moroccan L. infantum from/to Europe. However, genomic sequencing data from North African and European strains is needed to verify these scenarios.
The presence of mixed genotypes between the Moroccan sub-populations indicates the presence of gene flow between them. These strains might represent either mixed infections or 1A/1B hybrid strains. Since all mixed Moroccan genotypes  were isolated from amastigotes in bone marrow aspirate spotted on slides, cloning of these strains to test whether they are true hybrids was not possible. Hybrids seem to occur quite frequently. Previous studies in Algeria [16] and Tunisia [17] revealed hybrid genotypes for 12 of the 82 Algerian and Tunisian strains (14.6% ) with one Tunisian strain being cloned and proven as real hybrid. Since multiple heterozygous loci were identified among hybrids and gene flow was detected between different populations, recombination between strains with different alleles seems to be the most parsimonious explanation [26]. The occurrence of gene flow and genetic recombination is increasingly being suggested for different Leishmania species [27] although the mechanism of genetic exchange remains to be established. As L. infantum infects humans and dogs and continues its life cycle in sandfly midgut, it is vital to investigate the compartments where genetic recombination of Leishmania could take place. It was recently reported that L. major is capable of having asexual cycle consistent with a meiotic process in sandfly vector [28].
Three sandfly species transmit L. infantum in Morocco; P. perniciosus [10], P.longicuspis [11] and P. ariasi [9]. One can speculate whether the sub-populations of Moroccan L. infantum might be related to the existence of different sand fly vector species involved in transmitting the respective parasites, as previously described for L .tropica, in North Israel [29]. To prove this hypothesis, it is needed to isolate strains of L. infantum from the respective sandfly species and investigate them with MLMT. Moreover, a new focus of cutaneous   [16] and Tunisia [17]. All Moroccan VL and CanL cases analyzed in this study have shown typical symptoms of visceral and canine leishmaniasis without significant differences in disease outcome between cases belonging to the two sub-populations, however, L. infantum strains that have caused CL in Morocco have not yet been analyzed by MLMT . Human and canine strains presented identical MLMT profiles. This highlights the key role of dogs as reservoirs of L.infantum in Morocco. To our knowledge, this is the first study that investigates the population structure and genetic diversity of L. infantum in Morocco by applying MLMT. We were able to detect two different sub-populations of L. infantum in Morocco and correlated them with strains isolated from North African countries and Europe. Putative hybrid strains indicated the presence of gene flow among the two sub-populations. However, it is needed to investigate more strains representing different hosts, clinical forms and zymodyme types to better understand the overall population structure and molecular epidemiology of L. infantum in Morocco and the other North African countries.