Fig 1: AcMNPV elicits IFN-α-mediated antiviral activity in porcine PBMCs in vitro. PBMCs from three piglets were either mock infected or infected with AcMNPV for 18 h and supernatants were subsequently collected. (A) The antiviral activity against VSV serotype Indiana of the supernatants was assayed in MDBK cells. (B) Dilutions of supernatants from PBMCs infected with AcMNPV or standard porcine IFN-α (100 U/mL) as control were pretreated with neutralizing anti-IFN-α antibodies or NMS at room temperature for 1 h and assayed for antiviral activity in a bioassay in MDBK cells against VSV infection. The samples were fixed and the assay was revealed with crystal violet; the dye was resuspended and read at 595 nm. (C) IFN-α levels were quantified by ELISA. Each symbol represents PBMCs from a distinct pig. (D) Antiviral activity against FMDV O1 Campos in the samples was assayed in LFBK cells. Standard porcine IFN-α was used for the quantification of antiviral activity units in A and D (1 U corresponds to 2.16 pg IFN-α; IC50 ~ 7.22 U/mL). The results are from an experiment that is representative of at least two independent experiments. One-sample t-test was performed to identify differences in antiviral activity between supernatants from AcMNPV- and mock-treated cells. Student’s t-test was performed to compare IFN-α between AcMNPV- and mock-treated cells or antiviral activity between samples pretreated with anti-IFN-α K9 mAb and those pre-treated with NMS. * p < 0.05; ** p < 0.01; *** p < 0.001.
Fig 2: Type I and type II IFNs are detected in sera from AcMNPV-inoculated pigs. Five piglets were intravenously injected with 1 × 109 PFU of AcMNPV and serum samples were collected both before the administration and at 3, 6, and 9 hpi. IFN-α and IFN-γ levels in sera were quantified by ELISA.
Fig 3: Antiviral activity against VSV and FMDV is detected in sera from AcMNPV-inoculated pigs. Five piglets were intravenously injected with 1 × 109 PFU of AcMNPV and serum samples were collected both before the administration and at 3, 6, and 9 h post-inoculation. The antiviral activity against VSV serotype Indiana in sera was assayed in MDBK cells. The antiviral activity against FMDV O1 Campos in sera was assessed in LFBK cells. In both cases, standard porcine IFN-α was used for the quantification of antiviral activity units (1 U corresponds to 2.16 pg IFN-α; IC50 ~ 7.22 U/mL).
Fig 4: The pseudotyping of AcMNPV with G-VSV, but not the enrichment with immunostimulatory CpG ODNs, improves the induction of IFN-α. (A) Schematic representation of the recombinant baculoviruses Ac-GVSV and Ac-pCpG. The main components are pointed out. (B) Western blots of sucrose-cushion purified Ac-GVSV particles and AcMNPV- and Ac-GVSV-infected Sf9 cells were revealed with anti-GVSV mAb to evidence the presence of the glycoprotein and the density reading of each band was represented in the graph below. (C) Sf9 cells were infected with AcMNPV or Ac-GVSV at 27 °C and pH 6.2 for 7 days and observed by optical microscopy (200×). (D–G) PBMCs from three piglets were either mock-infected or infected with AcMNPV (3.37 × 108 ± 1.2 × 107 PFU/mL), Ac-pCpG (2.69 × 108 ± 5.1 × 107 PFU/mL), or Ac-GVSV (2.44 × 108 ± 2.55 × 107 PFU/mL) at the indicated moi for 16 h to subsequently collect the supernatants. (D,F) IFN-α levels in supernatants were quantified by ELISA. (E,G) The antiviral activity against VSV serotype Indiana of the supernatants was assayed in MDBK cells. Standard porcine IFN-α was used for the quantification of antiviral activity units (1 U corresponds to 2.16 pg IFN-α; IC50 ~ 7.22 U/mL). The results are from one experiment that is representative of at least three independent experiments. Paired sample t-test was performed in all the experiments to identify differences between viruses. * p < 0.05.
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