*4.2. Experimental Design*

Six pregnant sows (Landrace) of 68 days of gestation, from a commercial farm, were housed in the biosafety level 3 (BSL3) animal facility at CReSA (Barcelona, Spain). The animals were purchased from pestivirus-free farms, and they were also checked for antibodies against CSFV before arriving at the CReSA facilities. Animals were numbered from one to six and distributed in three groups (from A to C), each group in a separate box with standard facilities for pregnant sows. In accordance with the previously established methodology to evaluate the capacity of CSFV for trans-placental transmission, two sows were included in each experimental group [24,46]. After five days of acclimatisation period (74 days of gestation), Sows 1 and 2 (Group A) were inoculated with the CSFV Margarita strain, Sows 3 and 4 (Group B) with the PdR strain and Sows 5 and 6 (Group C) with the Cat01 strain. The viral dose for all the inocula was 10<sup>5</sup> TCID <sup>50</sup> per animal, and the inoculation was carried out by intramuscular injection in the neck [22,24,47]. After infection, a trained veterinarian recorded clinical signs daily in a blinded manner. Two experiments were carried out, Experiment 1 included Groups A and B, while Experiment 2 included the Group C sows.

In Experiment 1 of the trial, serum and nasal and rectal swab samples were collected on the day of infection and at 4, 8, 14, and 22 dpi, which corresponded with days 74, 78, 82, 88 and 96 of gestation, respectively. At this time, the sows were euthanised, following the accepted procedures accordingly with the European Directive 2010/63/EU. Whole blood in EDTA was obtained in the day of infection and before euthanasia for ex vivo collection of PBMCs. After necropsy, tissue samples from tonsil and Peyer's patch were collected [24]. In parallel, the foetuses from all gilts were obtained, following procedures previously described to avoid foetal distress [24,48]. All foetuses were subjected to an exhaustive necropsy in which the presence of macroscopic lesions in different organs was evaluated [49]. Sera and whole blood samples and tissues (tonsil, spleen, and thymus) were collected from 13 foetuses per each sow.

In Experiment 2, sera samples were collected on the day of infection and at 7, 14, 21, and 28 dpi (days 81, 87, 95, and 102 of pregnancy, respectively). At farrowing, rectal swabs were collected from all piglets. Sows were kept with their litters for 21 days, and, after removal of the sow, piglets were fed an age-appropriate diet (StartRite, Cargill, Spain) until the end of the trial. The handling of the piglets was performed following previously described protocols [11].

Serum and nasal and rectal swabs were collected from piglets at 7, 15, 23, 27, and 32 dpb. At this time, whole blood samples were collected and piglets were euthanised following procedures according to the European Directive, using a pentobarbital overdose of 60–100 mg/kg of weight, administered via the jugular vein. In addition, sows and piglets were euthanised before the end of the trial if they presented clinical signs compatible with severe CSF or exhibited prostration behaviour, in accordance

with previous studies [22]. The experiment was approved by the Ethics Committee for Animal Experiments of the Autonomous University of Barcelona (UAB), according to existing Spanish and European regulations.

#### *4.3. Detection of CSFV RNA*

The NucleoSpin RNA isolation kit (Macherey-Nagel, Düren, Germany) was used in order to extract RNA from sera and nasal and rectal swab samples, as well as from organ samples, following the protocol provided by the manufacturer. In all cases, a final volume of 50 μL of RNA was extracted from an initial sample volume of 150 μL. The detection of viral RNA was carried out by a previously described RT-qPCR assay [18], validated in our laboratory for the detection of CSFV RNA in sera, nasal, and rectal swabs and tissue samples [11,22]. Samples were considered positive when the Ct values were equal to or less than 42. In addition, using the Ct value, samples were determined to have either high (Ct value below 23), moderate (between 23 and 28), or low (Ct value above 28) CSFV RNA load, as previously described [23,50]. Samples in which fluorescence was undetectable (Undet) were considered negative.

#### *4.4. Determination of E2-Specific and Neutralising Antibodies*

CSFV E2-specific antibodies were evaluated in sera from sows, foetuses, and piglets, using a commercial ELISA kit (IDEXX Laboratories, Liebfeld, Switzerland). Positive results were considered when the blocking percentage was ≥40%, following the manufacturer's recommendations. Additionally, neutralising antibodies against the respective infecting strain were determined using an NPLA assay [19]; thus, animals from Groups A, B, and C were evaluated for neutralising antibodies against the Margarita, PdR, and Cat01 strain, respectively. The neutralising antibody titres were expressed as the reciprocal dilution of serum that neutralised 100 TCID of 50% of the culture replicates.

#### *4.5. IFN-*α *ELISA Test in Serum Samples*

IFN-α concentration was determined in sera from foetuses and sows from Groups A and B, as well as piglets from Group C at 7 and 15 dpb, using a previously described in-house ELISA test [11,51]. Briefly, plates were coated overnight with an anti-IFN-α monoclonal antibody (K9 clone, PBL Biomedical Laboratories, Piscataway, New Jersey, USA). After washing, 50 μl of serum samples and serial dilutions of IFN-α recombinant protein (PBL Biomedical Laboratories) were plated by duplicate and incubated for 1 hour at 37 ºC. Afterwards, plates were washed, and a biotinylated anti-IFN-α antibody was added (F17 clone, PBL Biomedical Laboratories). Following an incubation of 1 hour at 37 ◦C, the plates were washed, and streptavidin-HRP was added. Finally, after a 30 minute incubation, 3,3- ,5,5- -tetramethylbenzidine (TMB) was used for revealing the technique, using H2SO4 1N as a stop solution. Plates were read at 450 nm, and cytokine concentrations (units/ml) were determined using a regression line built with the optical densities of the cytokine standards used in the test.

#### *4.6. ELISA Detection of IFN-*γ *and sCD163*

IFN-γ and sCD163 were analysed in sera from foetuses and sows from Groups A and B. Commercial ELISA test was used for detection of IFN-γ (IFN-γ ELISA Kit, Porcine, Life Technologies), following the manufacturer's instructions and the results were expressed as picograms per millilitre (pg/ml). Finally, a formerly described ELISA using lysates from CD163 transfected CHO cells as standard was used to quantify sCD163 [39,52]. Results were expressed as the equivalent numbers of CD163-transfected CHO cells (ENC).

#### *4.7. PBMCs Collection and Flow Cytometry Assay*

PBMCs were obtained from whole blood collected at the time of necropsy from three animals of each group in Experiments 1 and 2 of the trial, previously characterised by RT-qPCR. Cells were separated by density-gradient centrifugation with Histopaque 1077 (Sigma-Aldrich St. Louis, MO, USA), followed by osmotic shock in order to eliminate the remaining red blood cells. The number and viability of the PBMCs were determined by staining with Trypan Blue [21]. Additionally, thymocytes were obtained from three uninfected foetuses, and whole blood samples were also collected from three uninfected foetuses and piglets at the same time of gestation/days after birth as the foetuses from Experiment 1 or the piglets from Experiment 2, respectively.

The phenotypic profile of PBMCs from foetuses and piglets was evaluated by flow cytometry. Single staining was performed using the mAbs to porcine CD4 (74-12-4, IgG2b) Alexa Fluor 647 conjugate (BD Biosciences), and CD8-α (76-2-11, IgG2a) FITC-labelled (BD Biosciences, Franklin Lakes, NJ, USA).

The staining protocols were performed as previously described [11,12]. After staining, cells were filtered and passed in the cytometer (FACSAria IIu, BD Biosciences), with 10,000 cell events being recorded for each sample. The cells were analysed by FACSDiva software, version 6.1.2 and the results were expressed as the percentage of positive cells obtained for each staining, using irrelevant isotype-matched mAbs as staining controls.

**Author Contributions:** Conceptualization, L.G.; Investigation, J.A.B., S.M.-G., and L.G.; Methodology, L.G., S.M.-G., J.A.B., M.P.-S., I.M., R.R., L.C., and M.D.; Formal Analysis, J.A.B., S.M.-G., and L.G.; Resources, L.G. and R.R.; Writing—Original Draft Preparation, J.A.B., S.M.-G., and L.G.; Writing—Review and Editing, J.A.B., S.M.-G, M.D., and L.G.; Supervision, L.G.; Project Administration, L.G.; Funding Acquisition, L.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by grant RTI2018-100887-B-I00, Ministerio de Ciencia, e Innovación from the Spanish government. J.A.B has a pre-doctoral fellowship FPI 2016, Ministerio de Ciencia e Innovación from the Spanish government. The authors thank Ivan Cordón and the CReSA animal facility staff for their help during the in vivo part of the experiment. IRTA is supported by CERCA Progamme/Generalitat de Catalunya.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


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