Bovine Viral Diarrhea Virus Strain- and Cell Type-Specific Inhibition of Type I Interferon Pathways

The interaction of noncytopathogenic bovine viral diarrhea virus type 1 (BVDV-1ncp) with antigen-presenting cell (APC) subsets is of great interest due to the recent increase in severe acute BVDV outbreaks attributed to this genotype (1, 12, 13). We report the effect of a BVDV-1ncp strain (Ho916ncp [13]) causing severe acute infection on in vitro type I interferon (alpha/beta interferon [IFN-α/β]) production compared to that of a mild-acute strain (Ky1203ncp) (15). Ho916ncp but not Ky1203ncp induced IFN-α/β in only lineage-negative (LIN⁻) cells, a recently described enriched IFN-α/β-producing cell population in cattle (A. Gibson, S. Miah, P. Griebel, J. Brownlie, and D. Werling, submitted for publication). In agreement with published data, monocytes and monocyte-derived dendritic cells (moDC) did not produce IFN-α/β in response to either strain (4, 11, 17).

Experimental infection of calves with BVDVncp results in the detection of IFN-α/β and the IFN-α/β-inducible gene Mx (5, 20) as well as serum IFN-α/β activity in pregnant dams and the fetus (22). The identification of IFN-α/β-producing cells within the lymph nodes of BVDVncp-infected calves (4) and of enriched circulating IFN-α/β-producing cells (Gibson et al., submitted) points to an APC subset as a cellular source of IFN-α/β during in vivo BVDVncp infections. In the present study, monocytes, moDC, and LIN⁻ cells, generated from the same animal (11, 24-26, 28; Gibson et al., submitted), were exposed to either strain at a multiplicity of infection (MOI) of 0.1 and IFN-α/β was assessed after 48 h, using a previously described Mx-CAT (chloramphenicol acetyltransferase) reporter assay (4-6, 9). LIN⁻ cells, but not monocytes or moDC, produced significant amounts of IFN-α/β in response to Ho916ncp but not to Ky1203ncp or mock-infected control (Fig. 1). To assess whether these observed differences in IFN-α/β production were due to differences in viral replication, the presence of BVDV was determined by immunoperoxidase staining using a bovine hyperimmune serum, similar to what was described previously (11). Astonishingly, no clear differences in abilities to infect the different cell types were seen for the two BVDVncp strains (Fig. 2). As differences in viral replication did not explain differences in IFN-α/β production, we next assessed the potential effects of both BVDVncp strains on interferon response factors (IRFs). BVDVncp is known to inhibit IFN-α/β production through viral proteins N^pro (2, 3, 10, 14) and E^rns (16, 18, 19), which block IFN-α/β production by targeting IRF-3 for degradation by polyubiquitination (7, 14) or by degrading viral RNA (19). Translocation of IRF-3 occurs during infection but not binding to DNA (2). In contrast, IRF-7 is neither activated nor translocated to the nucleus (2).

FIG. 1.

Type I interferon response to BVDVncp. Monocytes (MON), moDC, and LIN⁻ cells (LIN) were isolated from the same animal and stimulated with 50 μg ml⁻¹ of poly(I:C), BVDVncp strains Ho916ncp and Ky1203ncp at an MOI of 0.1, and corresponding volumes of mock-infected supernatant for 48 h. Supernatants were harvested for the detection of IFN-α/β by an Mx-CAT reporter assay where n = 4. *, P value of <0.05 at a 95% confidence interval.

FIG. 2.

Antigen-presenting cell susceptibility to BVDVncp. Monocytes (A), moDC (B), and LIN⁻ cells (C) were infected with BVDVncp strains Ho916ncp and Ky1203ncp for 48 h (MOI, 0.1). Cells were fixed and stained for the presence of BVDV antigen using a bovine hyperimmune serum, and the antigen was detected either with anti-bovine alkaline phosphatase-conjugated secondary antibody (monocytes and LIN⁻ cells) or with anti-bovine horseradish peroxidase (HRP)-conjugated secondary antibody (moDC). Dark blue/gray cytoplasmic staining or red/brown cytoplasmic staining indicates the presence of BVDV. Light microscopy images were captured with a MiniVID lens camera (LW Scientific Inc.) and further analyzed with Scope Photo image software version 2.0.4. The left panels display mock-infected cells, the center panels display Ho916ncp-infected cells, and the right panels display Ky1203ncp-infected cells. Displayed data are representative of 4 individual animals for monocytes and LIN⁻ cells and 2 individual animals for moDC.

We assessed the impact of both strains on IFN-α/β signaling components IRF-3 and IRF-7 by Western blotting, which showed that IRF-3 and IRF-7 were increased in all three cell types infected with Ho916ncp compared to mock-infected controls. In contrast, Ky1203ncp induced a reduction of IRF-7 expression in monocytes and moDC and a reduction in IRF-3 expression in all three cell types. IRF-7 remained unchanged in LIN⁻ cells, potentially indicating cell type-specific degradation in monocytes and moDC for Ky1203ncp (Fig. 3). Ho916ncp-induced IFN-α/β production by LIN⁻ cells appears to be independent of IRF-3 or IRF-7 modulation; however, LIN⁻ cells, unlike monocytes or moDC, express TLR7 (27; Gibson et al., submitted), which has been recently implicated in the recognition of other pestiviruses, such as West Nile virus and dengue virus (8, 21, 23). Ho916ncp, as a severe acute BVDV-1ncp strain could be more readily accessible to the endosomal compartment within LIN⁻ cells, thus producing cell type-restricted IFN-α/β through TLR7. Our data show for the first time differences in BVDV-1ncp strains with regard to a cell-specific IFN-α/β response and offer some suggestions for this modulation.

FIG. 3.

Relative expression of IRF-3 and IRF-7 in response to BVDVncp. Monocytes (MON), moDC, and LIN⁻ (LIN) cells were isolated from the same animal and stimulated with Ho916ncp and Ky1203ncp alongside mock-infected controls for 48 h. Protein lysates were prepared with protease inhibitors and analyzed by Western blotting for the expression of IRF-3 and IRF-7, detected by enhanced chemiluminescence and further analyzed using Quantity One densitometry software version 4.6.6 (Bio-Rad Laboratories, United Kingdom). Relative expression of IRF-3 and IRF-7 is displayed normalized to β-actin and mock-infected control for each viral supernatant, where n = 3.