Figure 1.

Overexpression of IFITMs inhibits classical swine fever virus (CSFV) replication in porcine alveolar macrophages (PAMs). (A) Cell viability of cell lines stably overexpressing IFITMs. (B) Confirmation of CMV-IFITM1, CMV-IFITM2, and CMV-IFITM3 lentivirus transfection by detection of enhanced green fluorescent protein (EGFP) reporter. (a) Mock-transfected PAMs. (b) PAMs transfected with lentiviruses expressing CMV. (c) PAMs transfected with CMV-IFITM1 lentivirus, CMV-IFITM2 lentivirus (d), and CMV-IFITM3 lentivirus (e). Scale bars, 100 μm. (C) Western blot analysis of PAM cell lines stably expressing Flag-tagged IFITM1, IFITM2, or IFITM3 or CMV alone using an anti-Flag antibody. (D) Western blot analysis of IFITMs in PAM cell lines stably expressing Flag-tagged IFITM1, IFITM2, or IFITM3 or CMV alone using an IFITM1-specific antibody and an IFITM2/3 antibody against IFITM2 and IFITM3, respectively. β-actin served as an internal control. (E) CSFV genomic RNA in CMV-IFITM1, CMV-IFITM2, and CMV-IFITM3 cell lines. The CMV, CMV-IFITM1, CMV-IFITM2, and CMV-IFITM3 cell lines were infected with CSFV at a multiplicity of infection (MOI) of 1. CSFV genomic RNA levels were determined by real-time quantitative PCR (RT-qPCR) at 12 and 24 h post-infection (hpi). Data were normalized to β-actin expression. (F) Infectious progeny viral titers in culture medium from CMV-IFITM1, CMV-IFITM2, and CMV-IFITM3 cells. The viral titers of CSFV in supernatants were quantified by an immunofluorescence assay (IFA) and expressed as tissue culture infective dose (TCID₅₀)/mL. Data (A,E,F) represent the mean ± SD of three independent experiments and were measured in technical duplicate. Comparisons between groups were performed by Student’s t test. * p < 0.05, ** p < 0.01 and *** p < 0.001.