PMC

Immunohistochemical staining of infected immune and epithelial cells. Paraformaldehyde-fixed cryosections of Peyer's patches and small intestine harvested on day 7 after infection with 5804PeH (A and C) or 5804PeH_EpR-blind (B and D) were stained with markers for T cells (CD3; Santa Cruz) (A and B) or cytokeratin (C2562; Sigma) (C and D), followed by an Alexa Fluor 568-labeled secondary antiserum. Nuclei were counterstained with DAPI.

Immunohistochemical detection of CDV in ferret lymphatic and epithelial tissues. (A to D) Lymph node sections from animals infected with either 5804PeH (A and C) or 5804PeH_EpR-Blind (B and D) and sacrificed at either day 7 (A and B) or day 12 (C and D) postinoculation. (E to H) Lung sections from animals infected with either 5804PeH (E and G) or 5804PeH_EpR-blind (F and H) and sacrificed at either day 7 (E and F) or day 14 (G and H) postinfection. (I to L) Intestinal sections from animals infected with either 5804PeH (I and K) or 5804PeH_EpR-blind (J and L) and sacrificed at either day 7 (I and J) or day 14 (K and L) postinfection. Black arrows in panels J and L indicate infected cells that are of lymphoid morphology. (M to P) Bladder sections from animals infected with either 5804PeH (M and O) or 5804PeH_EpR-blind (N and P) and sacrificed at either day 7 (M and N) or day 14 (O and P) postinfection. All pictures were taken at a magnification of ×1,000, under oil immersion.

Characterization of recombinant viruses. (A and B) Growth kinetics in VerodogSLAMtag and MDCK cells and FtAEpCs. Cells were infected at an MOI of 0.01 (A) or 0.1 (B), and samples were collected daily for 4 days for VerodogSLAMtag cells or every second day for 8 days for epithelial cell lines. The cell-associated virus titer was determined by a limiting dilution method and expressed as the TCID₅₀. The average values for 4 experiments are shown, and error bars indicate standard deviations. Each dotted line represents the detection limit of the assay. (C to E) Syncytium formation in VerodogSLAMtag cells infected at an MOI of 0.01 (C) and in FtAEpCs (D) and MDCK cells (E) infected at an MOI of 0.1. Photographs were taken at the indicated time points, using fluorescence excitation and phase-contrast. Magnification, ×100. Overlays of both types of photograph are shown.

Transport and function of mutant H proteins. (A) Amino acid alignment of the MeV IC-B H protein and the corresponding region of the CDV 5804P H protein. Residues identified by Leonard et al. () (L482, P497, and Y543) as important for interaction of MeV H with nectin-4 are highlighted with asterisks above the residues. The corresponding residues (V478, P493, and Y539) were mutated in 5804P H. (B and C) Cell surface biotinylation of CDV and MeV H proteins. Cells transfected with expression plasmids for CDV (B) or MeV (C) parental and mutant H proteins were labeled with NHS-LC-LC-biotin, immunoprecipitated with an antiserum recognizing either the MeV H or CDV H protein cytoplasmic tail, and detected with a streptavidin-HRP conjugate. Western blots of cell lysates from the same experiment were analyzed for total H protein expression. The relative surface expression of each protein was calculated by normalizing the biotinylation or Western blot signal to the signal obtained for the respective parental H protein. The relative surface expression represents the ratio between the normalized biotinylation signal and the normalized Western blot signal for each replicate. Average relative surface expression levels were calculated from four independent experiments and are shown in bar graphs below the blots. (D) Cell-cell fusion observed for wild-type and mutant CDV or MeV H proteins. Confluent monolayers of VerodogSLAMtag cells were transfected with the respective H protein plasmid and the corresponding CDV or MeV F protein expression plasmid. Pictures were taken at 24 h posttransfection. Magnification, ×100.

Pathogenesis of recombinant CDVs in ferrets. Groups of five and eight animals were infected intranasally with 10⁵ TCID₅₀ of the parental strain 5804PeH and the recombinant virus 5804PeH_EpR-blind, respectively. Previously published data for three 5804PeH-infected animals () were also included, resulting in a group of eight animals for each virus. Only data for the five animals followed until day 12 after infection and until death are shown in panels A and B, respectively. (A) Clinical scores observed for wild-type virus- and 5804PeH_EpR-blind-infected animals. Scores are shown for the five animals followed until day 12 after infection or until death. Each square represents one animal. White squares indicate no changes, and black squares indicate severe rash, fever above 40°C, and weight loss above 10%. (B) Survival of animals infected with the different viruses. The death of an animal is indicated by a step down on the curve. (C) The course of cell-associated viremia is shown as the log₁₀ of the virus titer per 10⁶ PBMC. (D) The percentage of infected PBMC was quantified by flow cytometry. (E) Total leukocyte counts from infected animals, shown as 10³ leukocytes per mm³. (F) In vitro proliferation activity of lymphocytes from infected animals. Days postinfection are indicated on the x axes of the graphs, and error bars represent standard deviations. (G) Quantification of virus shedding in epithelial tissues on day 12 after infection. Titers are expressed as TCID₅₀/ml. Each symbol represents one animal, and geometric means are indicated by horizontal black lines.