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. 2016 Jul 15:220:33-8.
doi: 10.1016/j.virusres.2016.04.004. Epub 2016 Apr 5.

Immune gene expression in swine macrophages expressing the Torque Teno Sus Virus1 (TTSuV1) ORF-1 and 2 proteins

Pankaj Singh  1 , Sheela Ramamoorthy  2
Affiliations

Immune gene expression in swine macrophages expressing the Torque Teno Sus Virus1 (TTSuV1) ORF-1 and 2 proteins

Pankaj Singh et al. Virus Res. .

Abstract

Torque Teno viruses (TTVs) are small DNA viruses which are ubiquitous in nature. Recent reports indicate that swine torque teno viruses (TTSuVs) can act as primary pathogens or play a role in exacerbating co-infections. However, very little is known about the TTSuV host-viral interaction or how they so successfully establish chronic infections in the host. To determine whether the major viral proteins can modulate host immunity, recombinant TTSuV1 ORF1 and 2 proteins were expressed in a swine macrophage cell line (3D4/31). The differential expression of a panel of innate, adaptive, regulatory and inflammatory immune genes was studied by quantitative PCR; using cDNA samples collected at 6, 12, 24 and 48h post-transfection. The ORF1 protein induced an early anti-viral response. However, at 6h post-transfection it also upregulated IL-10, PD-1 and SOCS-1, the suppressors of T cell mediated immunity. An ensuing diminishment of the early protective response was noted. The TTSuV1 ORF2 protein suppressed IFN-β and IL-13 responses but did not significantly influence anti-viral immunity otherwise. These findings indicate that the TTSuV1 ORF1 protein plays a significant but dual role in viral immunity.

Keywords: Cytokine; ELISA; Gene expression; Porcine; TTSuV1; Torque teno viruses; qPCR.

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Figures

Fig 1
Fig 1
Immunofluorescent images of the expression of TTSuV1 ORF1 and 2. The swine macrophage cell line 3D4/31 was transfected with TTSuV1 ORF1 (left image) or ORF2 (right image). Protein expression was detected with either a rabbit anti-ORF1 antibody (ORF1) or mouse anti-V5 tag monoclonal antibody (ORF2). Apple green fluorescence in the nucleus for ORF1 and in both the nucleus and cytoplasm for ORF2 is indicative of expression of the relevant protein. Representative images at 24 hrs post-transfection are presented. Fluorescence was not detected in untransfected cells (image not shown).
Fig 2
Fig 2. Differential expression of Type I Interferons
The mean relative fold change values for IFN-α and IFN-β at 6, 12, 24 and 48 hrs post-transfection of 3D4/31 swine macrophage cells expressing the TTSuV1 ORF1 or ORF2 proteins is depicted. A mean of 4 values is shown. Values ≥ 2 fold changes are considered significant (solid bar). Insignificant changes are not depicted. * p ≤ 0.05.
Fig 3
Fig 3. Expression of interferon-induced innate genes
The differential expression of Mx1, Mx2, OAS-1, PKR and RNaseL at 6, 12, 24 and 48 hrs post-transfection is shown as the mean of four replicates. The swine macrophage cell-line, 3D4/31, was transfected with constructs expressing the TTSuV1 ORF1 or ORF. Values ≥ 2 two-fold changes when compared to control cells transfected with the empty vector are considered significant (solid line). No significant regulation was detected in cells transfected with the TTSuV1 ORF2 expression plasmid (data not shown). * p ≤ 0.05.
Fig 4
Fig 4
Regulation of pro-inflammatory cytokines by the TTSuV1 ORF1: The mean fold change values of four replicates at 6, 12, 24 and 48hrs after transfection of the porcine 3D4/31 macrophage cell line, calculated by the ΔΔ Ct method is shown. Fold change values ≥ 2 in comparison to the control cells transfected with the empty vector are considered significant (solid line). Insignificant data points are not depicted. No significant changes were noted in TTSuV1 ORF2 transfected cells. * p ≤ 0.05.
Fig 5
Fig 5
Differential expression of immune regulatory genes: Fold change values for SOCS-1 and PD-1 (calculated by the ΔΔCt method) as the mean of four replicates of 3D4/31 swine macrophage cells, transfected with the TTSuV1 ORF1 protein at 6, 12, 24 and 48hrs are depicted. Fold change values ≥ 2 are considered significant (solid line). Insignificant values are not depicted. No significant changes were noted for cells transfected with the TTSuV1 ORF2 expression plasmid. * p ≤ 0.05.
Fig 6
Fig 6
Differential expression of pattern recognition receptors: The mean fold change values of the cytosolic DNA sensors TLR9 and DAI-ZBP1 calculated by the ΔΔCt method for are shown. Swine 3D/4 31 swine macrophage cells were transfected with a TTSuV1 ORF1 or ORF2 expression construct and samples collected at 6, 12, 24 and 48hrs. Fold change values ≥ 2 are considered significant (solid bar). No significant changes in expression were noted for ORF2. * p ≤ 0.05.
Fig 7
Fig 7
Detection of IL-10 and TNF-α by ELISA: The mean value of two replicates of cell culture supernatants from 3D4/31 cells transfected with the TTSuV1 ORF1 is shown. The average of the negative control values from cells transfected with the vector alone was subtracted from data for the treatment groups. IL-10 or TNF-α were not detected in the supernatants of TTSuV1 ORF2 transfected cells (data not shown). * - p ≤ 0.05 (Student T test).
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