*2.1. Clinical Evaluation of Sows Infected with Pinar del Rio (PdR) vs. Margarita CSFV Strains*

In the first experiment, aiming to determine the capacity of CSFV strains of different virulence levels to induce trans-placental infection, two groups of pregnant sows were inoculated with CSFV at 74 days of gestation. Group A (Sows 1 and 2) was infected with the highly virulent CSFV Margarita strain, while Group B (Sows 3 and 4) were inoculated with the low virulence PdR strain. Clinical signs were recorded daily by a trained veterinarian in a blinded manner.

After inoculation, both CSFV Margarita-infected sows (Group A) showed anorexia and apathy between 6 and 11 days post-infection (dpi). Subsequently, Sow 2 started to eat normally, whereas the clinical condition of Sow 1 deteriorated progressively, showing constipation/diarrhoea, some peaks of fever, evident weight loss, and, eventually, weakness of the hindquarters. This animal was euthanised at 17 dpi (91 days of gestation) for animal welfare reasons, while the remaining sows were euthanised at 22 dpi (96 days of gestation). Both Margarita infected sows showed similar lesions at necropsy, consisting of petechiae in the kidneys, stomach, and intestine, and, in the case of Sow 1, also in the urinary bladder. Conversely, Sows 3 and 4, inoculated with the PdR strain, remained healthy throughout the study, and no lesions related to CSFV infection were found at necropsy.

#### *2.2. CSFV RNA Level Detected in Sows after Infection with Margarita or PdR Strains*

CSFV RNA was evaluated by reverse transcription-quantitative PCR (RT-qPCR) [18] in serum samples collected weekly and tissue samples collected at necropsy. The RNA load was characterised as high, moderate or low in accordance with the cycle threshold (Ct) value, as described in the materials and methods section. The CSFV RNA load detected in sera oscillated from moderate to low load (Ct value from 28 to 35) regardless of the virulence degree of the strain used to infect the sows. The RNA was detected at 8 dpi in all the animals in Group A and B (infected with Margarita or PdR strains, respectively). However, at 14 dpi, and until the end of the experiment, samples from the two animals in Group B and from Sow 2 (Group A) were negative (Figure 1A). Notably, only Sow 1 infected with

the CSFV Margarita strain was positive at 14 dpi and at the time of euthanasia (17 dpi), although with low RNA load (Ct value 34).

**Figure 1.** Classical swine fever virus (CSFV) RNA detection by RT-qPCR in sow samples. (**A**) RNA levels detected in sera at different times post-infection. (**B**) RNA levels detected in tissues from sows infected with either the CSFV Margarita (black symbols) or PdR strain (grey symbols). Cycle threshold (Ct) values over 42 (dotted line) were considered as negative. Asterisk indicates the animal that was euthanised at 17 dpi.

In the tissue samples, the CSFV RNA load detected in the tonsils samples was similar in both experimental groups, with Ct value around 27 (moderate RNA load). The viral RNA load in Peyer's patch samples was also similar for both groups, with the exception of Sow 4 (PdR infected), which was negative (Figure 1B).

*2.3. The High Virulence CSFV Strain Margarita Elicited Faster and Higher Humoral Response than PdR Strain in the Infected Sows*

Specific anti-E2 and neutralising antibodies were evaluated weekly in sera by ELISA and neutralisation peroxidase linked assay (NPLA) [19], respectively. Anti-E2 antibodies were detected in both of the CSFV Margarita-infected sows (Group A) at 14 dpi and at the time of euthanasia. In Group B, infected with the PdR strain, only one animal showed anti-E2 antibodies at 22 dpi (Figure 2A).

**Figure 2.** Immune response of sows after CSFV infection. (**A**) CSFV specific anti-E2 antibody response against the E2 glycoprotein detected by ELISA (in blocking %), values above 40% (dotted line) being considered as positive. (**B**) Interferon alpha (IFN-α) response in serum determined by ELISA test from sows infected with either the CSFV Margarita (black symbols) or PdR strain (grey symbols). The IFN-α concentration in sera is expressed as units/mL. Asterisk indicates the animal that was euthanised at 17 dpi.

Similarly, both of the Margarita-infected sows showed neutralising antibody titers by NPLA assay starting at 14 dpi, which increased at 17 and 22 dpi for Sows 1 and 2, respectively. In the case of PdR

infected sows, neutralising antibody response was only detected in Sow 3 at 22dpi, while sow 4 did not show neutralising antibodies throughout the whole trial (Table 1).


**Table 1.** Neutralising antibody response in sows after CSFV infection.

\* sample taken at 17 dpi.

#### *2.4. IFN-*α *and IFN-*γ *Response in Sows Infected with High or Low Virulence CSFV Strains*

Interferon alpha (IFN-α) and interferon gamma (IFN-γ) were evaluated by ELISA test in sera from sows at different time-points after infection. IFN-α was detected in the sera of all the sows from Groups A and B at 4 and 8 dpi. Notably, the highest levels were registered in sows from Group A (Sows 1 and 2) at 4 dpi (Figure 2B). For IFN-γ, no detectable levels in sera were found in either experimental group after infection.

#### *2.5. Evaluation of the Foetuses from CSFV Infected Sows at Necropsy*

Foetuses from the CSFV Margarita-infected sows showed internal haemorrhages in tonsil, intestine, kidneys, lymph nodes and spleen. Furthermore, four of the foetuses from Sow 1 and three foetuses from Sow 2 had generalised haemorrhagic lesions in the skin (data not shown). Additionally, one mummified foetus was found in both of them. Conversely, foetuses from Sows 3 and 4, inoculated with the PdR low virulence strain, showed no lesions at necropsy.

## *2.6. Vertical Transmission and CSFV Replication in the Foetuses*

Following hysterectomy, serum and tissue samples were collected from all the foetuses, about two weeks before the expected delivery day, in order to determine CSFV transmission from sows to their foetuses. All the foetuses from Group A were RT-qPCR positive with high CSFV RNA load (Ct values between 15.67 and 23) in the majority of sera, tonsil, spleen and thymus samples (see Table 2). In the different organs, the mean Ct value ranged from 17.28 to 20.33 for foetuses from Sows 1 and 2, respectively. By contrast, only 3 out of 13 (23%) foetuses from each of the PdR-infected sows were positive in sera by RT-qPCR, ranging from high to low RNA load (Table 2). However, after analysis by RT-qPCR of tonsil, spleen and thymus samples, the number CSFV positive foetuses increased to 11 out of 13 (Sow 3) and 9 out of 13 (Sow 4), respectively, with high to low CSFV RNA load in the positive tissues.

#### *2.7. Immune Response in the Foetuses from CSFV-Infected Sows*

Absence of CSFV specific humoral response was found in sera from all the foetuses in the study. However, IFN-γ levels were detected only in serum sample of five out of the 13 foetuses from the Sow 1 (infected with Margarita strain), in values ranging between 23.2 and 130.9 pg/ml. In addition, detectable levels of IFN-α were also registered in 11 samples in foetuses from both Margarita-infected sows (Table 3). Interestingly, foetuses from sows infected with the low virulence strain (PdR) showed higher levels of IFN-α (between 100 to 200 units/ml). Notably, the positive values were found in the foetuses that were CSFV RNA positive for the four samples analysed or in those that showed the higher CSFV RNA load in the tissue samples (Tables 2 and 3). Finally, detectable levels of soluble CD163 (sCD163) were found in foetal sera samples from both experimental groups, being about 10 times higher the concentration in samples from Group A (Figure 3).


**Table 2.** Detection of CSFV RNA in foetuses from sows infected with CSFV Margarita or PdR strains.

Undet: undetectable, negative sample.

**Table 3.** IFN-α levels in foetal serum.


**Figure 3.** sCD163 levels in foetal sera. Foetuses from sows infected with either the Margarita (black bars) or PdR (white bars) CSFV strains are represented. Results are expressed as the equivalent number of copies of CD163 transfected cells.

#### *2.8. Phenotypical Profile in Foetal PBMCs after CSFV Infection*

Samples from whole blood were obtained from three foetuses in each infected group, and peripheral blood mononuclear cells (PBMCs) were isolated. Flow cytometry analysis was performed to study the phenotypical profile in these cells. The PBMCs analysed corresponded with foetuses that showed CSFV RNA levels in serum samples. Additionally, PBMCs of three foetuses from uninfected sows, from the same farm of origin, were also analysed to use as reference, uninfected controls. The CD4<sup>+</sup> T-cell subset ranged from 4% to 16% of PBMC from the Margarita infected foetuses (Group A), showing a reduction in two out of three samples analysed with values below 5% (Figure 4). This cell population ranged from 14% to 17% in the three foetal PBMC tested from Group B, while a wider range was detected in the PBMC from naïve samples (from 11% to 38%). By contrast, the CD8<sup>+</sup> T-cells were increased in the CSFV infected foetuses, with percentages between 29% and 56% in Group A, and 20% to 27% in Group B (infected with PdR strain), whereas it was always below 15% for the naïve samples (Figure 4).

#### *2.9. Infection with the CSFV Moderately Virulent Strain: Clinical Signs and CSFV Replication in Sows*

In the second experiment, in order to evaluate the capacity of a CSFV moderately virulent strain to induce trans-placental infection and congenital viral persistence, two pregnant sows (Sows 5 and 6) were inoculated with the Catalonia 01 (Cat01) strain. As in Experiment 1, the infection was carried out at 74 days of gestation, and a trained veterinarian recorded clinical signs daily.

The Cat01 infected sows did not show any clinical signs after inoculation. However, at 34 dpi (108 days of gestation), Sow 6 went into early labour and gave birth to eight stillbirths and two live piglets. All the stillbirths showed haemorrhagic lesions, whereas the live piglets were very weak and had to be euthanised on the same day for ethical reasons. The sow was also euthanised at this time. Both Cat01 infected sows were CSFV RNA positive in sera, and rectal and nasal swabs at 7dpi. The CSFV RNA load was low in all the samples, with Ct values ranging from 31 to 37. Afterwards, both sows cleared

the virus, only Sow 6 was positive in rectal swab at 28 dpi, although at low RNA concentration (Ct 35.59) (data not shown).

**Figure 4.** Comparative expression of the CD4+ and CD8+ T-cell subsets in PBMCs from uninfected foetuses (white dots), foetuses infected with the Margarita (black dots), or the PdR (grey dots) CSFV strain.
