Fig 1.

Ovine Nectin-4 can function as a receptor for PPRV. (A) Schematic representation of the lentivirus constructs encoding ovine SLAM or Nectin-4 ORFs. The bicistronic constructs encoded the encephalomyocarditis virus internal ribosome entry site (IRES) and a puromycin resistance gene. (B) Oligo(dT)-primed RT-PCR analysis of total cell RNA extracted from transduced or wild-type (wt) PO cell lines confirming SLAM or Nectin-4 mRNA expression. (C) PPRV infection in PO cell lines. Wild-type or lentivirus-transduced cells were infected at high MOI (4) with PPRV (Ivory Coast 1989 strain). Cytopathic effect was observed by phase-contrast microscopy at 48 h p.i. All images were equally manipulated for brightness and contrast using MS Powerpoint. (D) High-MOI single-step growth curve of PPRV in PO cell lines. Wild-type or transduced PO cells were infected at an MOI of 4. Virus was harvested at 0, 6, 12, 24, 48, and 72 h p.i. by freeze-thawing. PPRV replication rates were compared using an unpaired two-tailed t test comparison of the mean rates of viral exponential growth (12 to 48 h p.i.). Individual slopes were calculated by fitting a first order polynomial straight line to each data set; *, P < 0.05. (E) PPRV infection in PO and CHO cells expressing ovine Nectin-4. Wild-type or transduced PO or CHO cell lines were infected at high MOI (4) with PPRV (Ivory Coast 1989 strain). Virus was harvested at 48 h p.i. by freeze-thawing. (F) MeV and CDV replicate efficiently in PO cell lines expressing ovine SLAM or Nectin-4. Wild-type or transduced PO cell lines were infected at low MOI (0.1) with MeV (Dublin strain) or CDV (5804P strain). Virus was harvested at 72 h p.i. by freeze-thawing. (E and F) An unpaired two-tailed t test was used to compare the viral titers; *, P < 0.05; **, P < 0.005. (D, E, and F) Virus titration was performed on Vero cells expressing canine SLAM with a detection limit of 10 TCID₅₀. All infections were performed in triplicate; the error bars denote standard deviations from the means.