FIG 7.

MHV3-induced breakdown of BMEC barrier is related to downregulation of barrier protective IFN-β production by infected BMECs. (A and B) bEnd.3 cells were infected with MHV3, MHV-A59, and 51.6-MHV3 at an MOI of 0.1, and mRNA expression (A) and protein expression (B) of IFN-β was evaluated at 24 h p.i. by qRT-PCR and ELISAs, respectively. All samples were run in duplicate. All experiments were conducted in triplicate. (C to H) Evaluation of barrier integrity of bEnd.3 monolayers infected for 48 h with MHV3, MHV-A59, and 51.6-MHV3 at an MOI of 0.1 in the presence of recombinant IFN-β (100 pg/ml) or anti-IFN-β monoclonal antibodies (1 μg/ml). (C) Recordings of TEER in infected bEnd.3 cultures. (D and E) Evaluation of the paracellular permeability of infected bEnd.3 monolayers to NaF (D) or Evans blue (E) dyes. (F to H) mRNA expression levels of occludin (F), ZO-1 (G), and VE-cadherin (H) in bEnd.3 cells evaluated by qRT-PCR. Values represent fold change in gene expression relative to uninfected cells (control [CTRL]) after normalization with HPRT expression. Results are representative of two independent experiments. Values that are significantly different are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.