Postnatal Persistent Infection with Classical Swine Fever Virus and Its Immunological Implications

Muñoz-González, Sara; Ruggli, Nicolas; Rosell, Rosa; Pérez, Lester Josué; Frías-Leuporeau, Maria Teresa; Fraile, Lorenzo; Montoya, Maria; Cordoba, Lorena; Domingo, Mariano; Ehrensperger, Felix; Summerfield, Artur; Ganges, Llilianne

Bone marrow haematopoietic cells (BMHCs) were obtained from the femurs of selected pigs (pigs 1 and 3: infected with the PR strain; pigs 19 and 23: infected with the Cat01 strain; and two non-infected pigs) at 6 weeks of age, following the protocol previously described [43, 44] . To phenotype these cells, flow cytometry was performed using the corresponding hybridoma supernatants in the indirect labelling for SLA-I (74-11-10, IgG2b), SLA-II (1F12, IgG2b), CD163 (2A10/11, IgG1), CD172a (BA1C11, IgG1), granulocyte precursors (6D10, IgG2a), and c-kit or CD117 (2B8/BM IgG1); all of the hybridoma supernatants were kindly donated by Dr. J. Dominguez (INIA, Madrid, Spain). For the detection of CSFV-infected cells, a polyclonal FITC-labelled anti-CSFV conjugate (PrioCON FITC conjugate PAb-CSF, Prionics, Switzerland) was used. For the cellular markers, the secondary antibody was R-phycoerythrin goat anti-mouse IgG (Jackson ImmunoResearch, Suffolk, UK). Briefly, 2.5 × 10 5 cells/50 μl/well were labelled for 1 h at 4°C. The anti-CSFV conjugate was diluted 1:100 in cold PBS with 2% FBS. After 1 h of incubation at 4°C, the cells were washed with cold PBS with 2% FBS by centrifugation at 450 × g, at 4°C for 5 min. Then, the secondary antibody conjugated with R-phycoerythrin diluted 1:200 was added for SLA-I, SLA-II, CD163, CD172a, 6D10 and CD117 markers. The cells were incubated for a further 45 min at 4°C and then were washed as before and resuspended in PBS with 2% FBS. SLA-I, SLA-II, CD163, CD172a, 6D10, CD117, and CSFV-positive cells and unstained cells were counted using FACSaria I (Becton Dickinson), and the data were analysed by FACSDiva software, version 6.1.2. Irrelevant isotype-matched mAbs, unlabelled or labelled with the different fluorochromes, were used as negative controls. The gate strategy was applied in 90% of living cells using the forward and side scatter (FS/SS) characteristics. For two colour immunolabelling, the same procedure described above for incubation and washing was followed. To 2.5 × 10 5 cells/50 μl/well, 50 μl of SLA-II marker was added, followed by the secondary antibody conjugated with R-phycoerythrin diluted 1:200. Mab CD172a was biotinylated using standard protocols (CD172a_b). After the third wash, the cells were incubated with CD172a_b for 1 h at 4°C. Finally streptavidin-allophycocyanin (APC) was added at a 1:100 dilution. For 6D10 + /CSFV + labelling, the polyclonal FITC-labelled anti-CSFV conjugate and 6D10 hybridoma supernatants revealed with R-phycoerythrin conjugate were used. CD172a + /SLA-II + or 6D10 + /CSFV + was acquired using FACSAria I (Becton Dickinson, San Jose, California, USA), and the positive percentages were analysed by FACS-Diva software, version 6.1.2.