Co-culturing blood monocytic cells with immortalized mesenchymal cells.

<p>CD163 expression. Each point represents an individual pig (three independent experiments). FI: fluorescent intensity.</p

Characterization of immortalized mesenchymal cells by flow cytometry.

<p>Immunophenotyping was performed for detection of specific cell markers known to be expressed by mesenchymal cells (CD44, CD105, CD90 and CD29) and non-mesenchymal cell markers (CD16 and CD11b) (A). Three independent experiment (B). The percentages of CD105⁺, CD90⁺, CD44⁺ and CD29⁺ immortalized mesenchymal cells were 97±3%, 94±4%, 96±3% and 42±9%, respectively, in cells from nasal mucosa; 84±11%, 96±3%, 260 93.8±% and 29±3%, respectively, in cells from lungs; 86±6%, 85±3%, 81±1% and 41±2%, respectively, in cells from spleen; 92±0.5%, 95±0.6%, 91±2% and 34±2%, respectively, in cells from lymph nodes; 70±11%, 82±5%, 70±4% and 34±2%, respectively, in cells from red bone marrow.</p

Productive replication of PRV and HSV-1 in porcine TG neurons.

<p>Confocal images of TG neuronal cultures in the inner chamber at 24hpi with PRV (A,B) and 48hpi with HSV-1 (D,E) stained for neurofilament (red) and late viral antigens (green) (bar  = 50 µm). Percentage of neurons with axons growing out to the outer chamber that show viral antigens at 24hpi with PRV (C) and 48hpi with wt HSV-1 (F, left bar) and beta-galactosidase activity at 24hpi with SΔUS5-LacZ HSV-1 (F, right bar). Data represent the mean ± s.e.m. of three independent experiments.</p

<i>In vitro</i> differentiation of immortalized mesenchymal cells.

<p>Immortalized mesenchymal cells differentiation into chondrocytes (see dark-blue color-chondrocytes), osteoclast (see red color-calcium deposit), and adipocytes (see lipid droplets in the cells) confirmed by staining with alcian blue, alizarin red and oil red, respectively. Scale bar ꞊꞊ 10 μm.</p

RT-PCR specifications.

<p>Primer sequences and annealing temperatures (°C) used in RT-PCR and predicted length (bp) of amplified fragments.</p

IFNalpha induces a reactivatable, latent PRV and HSV-1 infection in porcine TG neurons.

<p>Percentage infected neurons that are late viral antigen positive at 1, 5 and 8dpi with PRV (A) and at 2, 5 and 12dpi with HSV-1 (B) in the presence or absence of 500 U/ml IFNalpha. For the neurons fixed at 8dpi with PRV and 12dpi with HSV-1, medium containing IFNalpha was washed out at 5dpi and replaced with new culture medium or new culture medium supplemented with forskolin (200 µM). Data represent the mean ± s.e.m. of three independent experiments.</p

Co-culturing blood monocytic cells with immortalized mesenchymal cells triggers siglec-1 expression.

<p>Each point represents an individual pig (three independent experiments). FI: fluorescent intensity.</p

Proliferation analysis of the immortalized mesenchymal cells.

<p>Growth pattern of the five immortalized mesenchymal cells. The data are represented as mean +/- SD of three independent experiments.</p

Cell cycle analysis of immortalized mesenchymal cells.

<p>Most cells of the five mesenchymal cells were at the G0/G1 phase of cell cycle (A). The data are represented as mean of three independent experiments (B).</p

PRV and HSV-1 express LATs during <i>in vitro</i> latency.

<p>(A,B) RT-PCR analysis of actin and viral immediate early (IE180 and ICP0), late (gB and gD) and LAT transcript RNA isolated from neuronal cultures that were either mock infected, productively infected with PRV (A, 1dpi) or HSV-1 (B, 2dpi), or latently infected with PRV (A, 5dpi with IFNalpha) or HSV-1 (B, 9dpi, 4 days post IFNalpha withdrawal). For each condition three different samples were analyzed and representative gels are shown. For HSV-1, two samples of 9dpi, 4 days post IFNalpha withdrawal are shown, one without and one with detectable ICP0 transcript expression. Specific bands are marked with a black arrowhead. (C) Percentage of infected neurons positive for LAT promoter-driven beta-galactosidase at 2 and 5dpi with HSV-1 LbetaA in the presence or absence of 500 U/ml IFNalpha. Data represent the mean ± s.e.m. of three independent experiments. (D) Light microscopic images of uniform (i,ii) and focal (iii) LAT promoter-driven beta-galactosidase distribution during the acute stage (2dpi without IFNalpha, i, ii) or the latent stage (5dpi with IFNalpha, iii) of infection with HSV-1 LbetaA. Arrows point to infected non-neuronal cells (i), dashed line marks contour of neuronal cell body in (iii) (bar  = 20 µm).</p