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

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

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

RT-PCR specifications.

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

Occurrence of apoptosis in the epithelium and lamina propria in human nasal respiratory explants during in vitro cultivation.

<p>Occurrence of apoptosis in the epithelium and lamina propria in human nasal respiratory explants during in vitro cultivation.</p

Evolution of HSV1 mucosal spread.

<p>(A) Kinetic evolution of HSV1 plaque formation. Explants were inoculated with 1 ml virus suspension containing 10⁷ TCID₅₀/ml HSV1 VR-733 and sampled at 0, 12, 16, 20, 24 and 36 h post inoculation (pi). Serial 20 µm cryosections were made and plaque latitude (white bars) and plaque depth underneath the basement membrane (BM), distance covered by HSV1 in the lamina propria, (black bars) were measured using ImageJ. Data are represented as means of 10 plaques of triplicate independent experiments+SD (error bars). *, Significant differences at the 0.05 level. (B) Representative confocal photomicrographs of the evolution of HSV1 VR-733 spread in human nasal respiratory explants at 0, 12, 16, 20, 24 and 36 h pi. Collagen IV is visualised by red fluorescence. Green fluorescence visualises HSV1 antigens. Bar, 100 µm. Abbreviations: Ep, epithelium; LP, lamina propria. The BM is marked with a dashed line.</p

Rate (%) of TUNEL-positive cells ± SD in both the epithelium and lamina propria of a region of HSV1-negative and of a region of HSV1-positive cells (virus plaque) at 0, 12, 24 and 36 h post inoculation (pi).

<p>ND = not determined; no HSV1-positive cells were found.</p><p>* Significant differences compared with the control (HSV1-negative tissue) at the 0.05 level.</p

HSV1 invasion score and epithelial damage score.

<p>Explants were inoculated with 1 ml 10⁷ TCID₅₀/ml HSV1 VR-733 and sampled at different time points post inoculation (pi). Serial 20 µm cryosections were made and evaluated. HSV1 mucosal invasion in the depth was graded on a 6-point scale, as follows: 0 = epithelium not infected, 1 = columnar cell(s) infected, 2 = suprabasal cell(s) infected, 3 = basal cell(s) infected, 4 = basement membrane and HSV1 colocalisation, HSV1 does not cross the basement membrane, 5 = HSV1 penetrates the basement membrane into the lamina propria. Epithelial damage was graded on a 4-point scale, as follows: 0 = no damage, 1 = superficial damage, 2 = epithelial damage involving basal cells, basal epithelial cells partly detached, 3 = epithelium severely damaged, loose. Both scales were combined on the same axis. The scores represent the mean of the scores of 10 different regions of HSV1-negative (100 cells) and HSV1-positive (virus plaque) cells per person at 0, 12, 16, 20, 24 and 36 h post inoculation (pi); experiments were performed in triplicate. Error bars indicate SD. *, Significant differences compared with the control (HSV1-negative epithelium) at the 0.05 level.</p

Light photomicrographs of the human model.

<p>Representative light photomicrographs of human nasal respiratory mucosa explants at 0 (A, B, C) and 96 h (D, E, F) of in vitro cultivation are illustrated. Light microscopical morphometric analysis was performed to evaluate the maintenance of structural integrity of the explants during in vitro cultivation. The three-dimensional organization of the explants was assessed by evaluating the morphometry of the epithelium, lamina reticularis and lamina propria. Eight-micron-sections were stained with haematoxylin-eosin (A and D) for evaluation of the epithelial thickness (indicated by black arrows). A reticulin staining (B and E) was performed to measure the thickness of the lamina reticularis (indicated by white arrows). A Van Gieson staining (C and F) was used to count the relative amounts of collagen and nuclei within a region of interest (roi indicated by a rectangle) of the lamina propria. By setting a threshold, different colors were assigned to collagen and nuclei, respectively, and the percentages of collagen and nuclei were determined within this roi. Bar, 50 µm.</p

Morphometric evaluation of the human model.

<p>Maintenance of the structural integrity of the explants during in vitro cultivation was evaluated by assessing the three-dimensional organization of the explants. Epithelial thickness (A), thickness of the lamina reticularis (B) and percentages of nuclei and collagen within a region of interest of the lamina propria (C) were evaluated in explants at different time points of in vitro cultivation. Data are represented as means+SD (error bars) of triplicate independent experiments.</p