Article Figures & Data
Figures
- FIG. 1.
End-point titration. BHK-21 and SK6 cells were infected with A12-IC (WT) or A12-LLV2 at an MOI of 10 at 37°C. After 1 h, unabsorbed virus was removed by washing with 150 mM NaCl-20 mM MES (pH 6.0), and MEM was added. Supernatants were collected at 24 hpi, and the virus titers were determined on BHK-21 cells.
- FIG. 2.
IFN secretion after FMDV infection. SK6 (A) or PK (B) cells were infected with A12-IC (WT) or A12-LLV2 at an MOI of 1. Supernatants were collected at different times postinfection, and pIFN-α was measured by antigen capture ELISA. The results are representative of three independent experiments. Error bars refer to the standard deviation for each mean data point (n = 3).
- FIG. 3.
RNAi of porcine PKR. SK6 cells were transfected twice with 200 nM of a pool of siRNAs specific for porcine PKR (siPKR). Transfected cells were infected with A12-IC (WT) or A12-LLV2 at MOIs from 0.0001 to 1, and after 24 h the virus titer was measured on BHK-21 cells (B). A control experiment with siGLO (Risc-free) was performed simultaneously (A). The results are representative of two independent experiments.
- FIG. 4.
IFN-α/β response in porcine cells. IFN-α1/β mRNA expression was measured by real-time RT-PCR in SK6 cells (A and C) or PK cells (B and D) infected at an MOI of 1 with A12-IC (WT) or A12-LLV2. Porcine GAPDH was used as an internal control. Results are expressed as the increase (n-fold) of gene expression for virus-infected cells relative to mock-infected cells at the indicated time points. Error bars indicate the standard deviation for each mean data point (n = 3). Similar results were obtained in at least two independent experiments.
- FIG. 5.
Induction of IFN-β mRNA expression in the presence of cycloheximide. (A) SK6 cells were treated with 10 μg/ml of poly(rI · C) in the absence or presence of 100 μg/ml cycloheximide. (B) SK6 cells were infected with A12-IC (WT) or A12-LLV2 (MOI of 1; 9 h) in the absence or presence of 50 μg/ml cycloheximide. The levels of IFN-β mRNA were measured by real-time RT-PCR as indicated in Fig. 4. Error bars indicate the standard deviation for each mean data point (n = 3). chx, cycloheximide.
- FIG. 6.
Expression of ISG products in response to FMDV infection. SK6 or PK cells were infected with A12-IC (WT) and A12-LLV2 at an MOI of 1. Expression of PKR, OAS, and Mx1 mRNAs was measured by real-time RT-PCR using GAPDH or 18S rRNA as an internal control. Results are expressed as the increase (n-fold) for each specific gene in virus-infected cells relative to mock-infected cells. Error bars indicate the standard deviation for each mean data point (n = 3). Similar results were obtained in at least two independent experiments.
Tables
- TABLE 1.
Oligonucleotide primer and probe sequences for amplification of porcine genes used in real-time RT-PCR
Gene type Primer and probe sets Sequence GenBanka GAPDH Porcine GAPDH-327Fb 5′-CGTCCCTGAGACACGATGGT-3′ AF017079 Porcine GAPDH-380Rc 5′-CCCGATGCGGCCAAAT-3′ Porcine GAPDH-348Td 5′-AAGGTCGGAGTGAACG-3′ Mx1 Porcine Mx1-803Fb 5′-GAGGTGGACCCCGAAGGA-3′ M65087 Porcine Mx1-859Rc 5′-CACCAGATCCGGCTTCGT-3′ Porcine Mx1-824Td 5′-AGGACCATCGGGATC-3′ OAS Porcine OAS-889Fb 5′-CTGTCGTTGGACGATGTATGCT-3′ AJ225090 Porcine OAS-954Rc 5′-CAGCCGGGTCCAGAATCA-3′ Porcine OAS-919Td 5′-TCAAGAAACCCAGGCCT-3′ PKR Porcine PKR-968Fb 5′-GGAAGAAAACAAACACAGCTTGAA-3′ AB104654 Porcine PKR-1048Rc 5′-CCAAATCCACCTGAGCCAATT-3′ Porcine PKR-994Td 5′-CCAGGTT TGTCGAAGAT-3′ pIFNα set#1 Porcine IFNα-236Fb 5′-TGGTGCATGAGATGCTCCA-3′ M28623 Porcine IFNα-290Rc 5′-GCCGAGCCCTCTGTGCT-3′ Porcine IFNα-256Td 5′-CAGACCTTCCAGCTCT-3′ pIFNα set#2 Porcine IFNα-449Fb 5′-TCACCCTCTATCTGCAAGAGAAGA-3′ M28623 Porcine IFNα-514Rc 5′-TGACTTCTGCCCTGACGATCT-3′ Porcine IFNα-478Td 5′-AGCCCTGTGCCTG-3′ pIFNβ Porcine IFNβ-11Fb 5′-AGTGCATCCTCCAAATCGCT-3′ M86762 Porcine IFNβ-69Rc 5′-GCTCATGGAAAGAGCTGTGGT-3′ Porcine IFNβ-32Td 5′-TCCTGATGTGTTTCTC-3′ - TABLE 2.
Antiviral activity in supernatants of porcine cellsa
Inducing virusb Total antiviral activity (U) Antiviral activity (U) in cells incubated with: Normal serum Neutralizing anti-IFN-α Neutralizing anti-IFN-β Neutralizing anti-IFN-α/β A12-LLV2 16 16 <2 16 <2 A12-IC (WT) <2 <2 NA NA NA
