Neutral operator with variable parameter and third-order neutral differential equation

Additional file 1. The TRS sequences of structural genes in the HP-PRRSV/SD16 genome (GenBank: JX087437)

The impact that the lack of ORF5 has on the PCV2 replication.

<p>(A) PK-15 cells transfected with either recombinant PCV2 or mutant PCV2 DNA were detected by IFA. (a) Cells transfected with rPCV2 DNA were detected using anti-Cap pAbs, (b) Cells transfected with PCV2Δ DNA were detected using anti-Cap pAbs, (c) Cells transfected with PCV2Δ DNA were detected using anti-ORF5 pAbs. Bar = 100 μm for all the Figures. (B) PCV2-infected PAMs were assayed for viral DNA copy number by real-time PCR. (C) The cell cultures were harvested and PCV2 titers were detected as TCID₅₀. Results are presented as mean ± SD (n = 3).</p

Proteasome inhibitors block the degradation of GFP-ORF5 protein.

<p>PAMs transfected with pEGFP-C1 or pEGFP-ORF5 were treated with 10 μM MG132, or with 30 μM BAPTA-AM, 50 μM E64, or 20 mM NH₄Cl starting at 24 h after transfection. The cells were collected 12 h later and GFP-ORF5 proteins were detected by immunoblotting using anti-GFP antibody. EGFP was used as a control.</p

The GFP-tagged PCV2 ORF5 induces ER stress in PAMs.

<p>(A) Total RNA was extracted from cells expressing either GFP alone, GFP-ORF5 or untransfected cells. Real-time PCR analysis of GRP78 mRNA levels was normalized to the corresponding CT value for porcine β-actin mRNA. The results are mean ± SD and representative of three independent experiments. (B) The level of GRP78 expression was determined by western blot with the mouse monoclonal antibodies against GRP78. β-actin was used as an internal loading control. (C) NF-κB p65 activation was determined using the TransAM assay. The data represent the mean and standard deviation from three different experiments. (D) Cellular extracts were subjected to western blot analysis with antibodies specific for endogenous IκBα, NF-κB p65 and total p65, and anti-β-actin was used as a control for sample loading.</p

Cell proliferation assays of the GFP-ORF5 protein expressing cells.

<p>The CCK8 assay was used to measure proliferation of 3 × 10³ cells from PAMs transfected with pEGFP-ORF5 and control cells over time. Each data set represents the mean ± SD of three replicates.</p

Identification of Putative ORF5 Protein of Porcine Circovirus Type 2 and Functional Analysis of GFP-Fused ORF5 Protein

<div><p>Porcine circovirus type 2 (PCV2) is the essential infectious agent responsible for causing porcine circovirus-associated diseases in pigs. To date, eleven RNAs and five viral proteins of PCV2 have been detected. Here, we identified a novel viral gene within the PCV2 genome, termed ORF5, that exists at both the transcriptional and translational level during productive infection of PCV2 in porcine alveolar macrophages 3D4/2 (PAMs). Northern blot analysis was used to demonstrate that the ORF5 gene measures 180 bp in length and overlaps completely with ORF1 when read in the same direction. Site-directed mutagenesis was used to show that the ORF5 protein is not essential for PCV2 replication. To investigate the biological functions of the novel protein, we constructed a recombinant eukaryotic expression plasmid capable of expressing PCV2 ORF5. The results show that the GFP-tagged PCV2 ORF5 protein localizes to the endoplasmic reticulum (ER), is degraded via the proteasome, inhibits PAM growth and prolongs the S-phase of the cell cycle. Further studies show that the GFP-tagged PCV2 ORF5 protein induces ER stress and activates NF-κB, which was further confirmed by a significant upregulation in IL-6, IL-8 and COX-2 expression. In addition, five cellular proteins (GPNMB, CYP1A1, YWHAB, ZNF511 and SRSF3) were found to interact with ORF5 via yeast two-hybrid assay. These findings provide novel information on the identification and functional analysis of the PCV2 ORF5 protein and are likely to be of benefit in elucidating the molecular mechanisms of PCV2 pathogenicity. However, additional experiments are needed to validate the expression and function of the ORF5 protein during PCV2 infection in vitro before any definitive conclusion can be drawn.</p></div

The GFP-tagged PCV2 ORF5 up-regulates IL-6, IL-8 and COX-2 expression in PAMs.

<p>PAMs were transfected with pEGFP-C1 or pEGFP-ORF5 plasmids and harvested at 48 h post-transfection. (A to C) The effect of PCV2 ORF5 expression on porcine IL-6, IL-8 and COX-2 transcription in cultured PAMs. Total RNA was extracted from cells expressing either GFP alone, GFP-ORF5 fusion, or untransfected cells. Realtime RT-PCR analysis of IL-6, IL-8 and COX-2 mRNA levels were normalized to the corresponding CT value for porcine β-actin mRNA. The results are mean ± SD and representative of three independent experiments. (D to F) The concentrations of IL-6, IL-8 and COX-2 in GFP-ORF5 expressing PAMs or control cells culture supernatants were measured by ELISA. Data are mean ± SD and representative of three independent experiments. *<i>p</i> < 0.05 and **<i>P<</i>0.01 versus the control group.</p

Sequences of primer pairs used for qRT-PCR.

<p>Sequences of primer pairs used for qRT-PCR.</p

Analysis of the stages of cell division of expressing GFP-ORF5 protein by flow cytometry and effect of GFP-ORF5 expression on the porcine CycA protein expression.

<p>(A) Histograms from flow cytometry data for propidium iodide (PI) staining. (B) Quantitative analysis of the percentage of cells in each phase of the cell cycle from flow cytometry data. (C) Total RNA was extracted from cells expressing either GFP alone, GFP-ORF5 or untransfected cells. Real-time PCR analysis of CycA mRNA levels were normalized to the corresponding CT value for porcine β-actin mRNA. (D) The level of CycA expression was determined by western blot with the mouse monoclonal antibodies against CycA in all cell lines. β-actin was used as an internal loading control. The results are mean ± SD from three independent experiments. *<i>p</i> < 0.05 and **<i>P<</i>0.01 versus the control group.</p

mRNA expression curves.

<p>PAMs were infected with wild-type PCV2 (wPCV2), recombinant PCV2 (rPCV2), or a start codon mutant PCV2 (PCV2Δ) at an MOI of 1.0. Cellular total mRNA was harvested at six time points from 12 to 96 h post infection, treated with gDNA eraser and tested by real-time quantitative RT-PCR. (A) Expression of ORF5 mRNAs. (B) Expression of ORF1 mRNAs. (C) Expression of ORF2 mRNAs. (D) Expression of ORF3 mRNAs. (E) Expression of ORF4 mRNAs. Results are presented as group mean cDNA numbers ± SD (n = 3).</p