Bovine Viral Diarrhea Virus Type 2 Impairs Macrophage Responsiveness to Toll-Like Receptor Ligation with the Exception of Toll-Like Receptor 7

<div><p><i>Bovine viral diarrhea virus</i> (BVDV) is a member of the <i>Flaviviridae</i> family. BVDV isolates are classified into two biotypes based on the development of cytopathic (cp) or non-cytopathic (ncp) effects in epithelial cell culture. BVDV isolates are further separated into species, BVDV1 and 2, based on genetic differences. Symptoms of BVDV infection range from subclinical to severe, depending on strain virulence, and may involve multiple organ systems and induction of a generalized immunosuppression. During BVDV-induced immune suppression, macrophages, critical to innate immunity, may have altered pathogen recognition receptor (PRR) signaling, including signaling through toll-like receptors (TLRs). Comparison of BVDV 2 strains with different biotypes and virulence levels is valuable to determining if there are differences in host macrophage cellular responses between viral phenotypes. The current study demonstrates that cytopathic (cp), noncytopathic (ncp), high (hv) or low virulence (lv) BVDV2 infection of bovine monocyte-derived macrophages (MDMΦ) result in differential expression of pro-inflammatory cytokines compared to uninfected MDMΦ. A hallmark of cp BVDV2 infection is IL-6 production. In response to TLR2 or 4 ligation, as might be observed during secondary bacterial infection, cytokine secretion was markedly decreased in BVDV2-infected MDMΦ, compared to non-infected MDMΦ. Macrophages were hyporesponsive to viral TLR3 or TLR8 ligation. However, TLR7 stimulation of BVDV2-infected MDMΦ induced cytokine secretion, unlike results observed for other TLRs. Together, these data suggest that BVDV2 infection modulated mRNA responses and induced a suppression of proinflammatory cytokine protein responses to TLR ligation in MDMΦ with the exception of TLR7 ligation. It is likely that there are distinct differences in TLR pathways modulated following BVDV2 infection, which have implications for macrophage responses to secondary infections.</p></div

Expression of proinflammatory cytokine gene transcription in MDMΦs inoculated with high and low virulence BVDV2 strains.

<p>MDMΦs were differentiated in 96 well plates for 7 days and inoculated with BVDV2 strains in duplicate at an MOI of 1 with RNA harvested at 2, 6, 18, and 24 h after inoculation. Cytokine mRNA was analyzed by qPCR using ribosomal protein s9 (RPS9) as an endogenous control with fold change expressed relative to uninfected control MDMΦs harvested at the corresponding time points. <i>il1β</i> (A), <i>tnfα</i> (B), <i>il6</i> (C), <i>il8</i> (D), <i>il12p40</i> (E), <i>il10</i> (F) were measured using primer sets specific for bovine genes using SYRB Green chemistry. Bars represent the mean value ± SEM from four different experiments from 9 total donor cattle. *** P < 0.001.</p

Proinflammatory cytokine secretion of BVDV2 inoculated or LPS stimulated MDMΦs 24 h after treatment.

<p>MDMΦs were differentiated in 96 well plates for 7 days and inoculated with BVDV2 strains in duplicate at an MOI of 1 or 2 μg/mL LPS with cell supernatants harvested at 24 h after treatment. Cytokine protein was analyzed by Searchlight Array using analytes specific for bovine IL-1β (A), IL-6 (B) and TNFα (C) with 50 μL of cell supernatant analyzed in duplicate. Cytokines were quantified by generation of standard curves against recombinant bovine cytokines provided by the manufacturer of the Searchlight platform. Bars represent the mean value ± SEM from four different experiments from 9 total donor cattle. ** P < 0.001.</p

Primer sequence for bovine targets and control genes.

<p>Primer sequence for bovine targets and control genes.</p

TNFα protein secretion from BVDV2 infected monocyte derived macrophages after stimulation with bacterial TLR agonists.

<p>MDMΦs were differentiated in 96 well plates for 7 days and inoculated with BVDV2 strains with an MOI of 1 for 48 h prior to stimulation with Pam3Cys [5 μg/mL], <i>M</i>. <i>haemolytica</i> LPS [10 μg/mL], or <i>E</i>. <i>coli</i> (055:B5) LPS [1 μg/mL]. Cell supernatants were analyzed for TNFα protein concentration 24 h after TLR stimulation and measured by Searchlight Array platform. Data from infection with cytopathic or noncytopathic strains are in the top panel (A) and from high or low virulence strains indicated in the lower panel (B). Bars represent the mean value ± SEM from four different experiments from 9 total donor cattle. ** P < 0.001 compared to uninfected, TLR stimulated cells.</p

TNFα protein secretion from BVDV2 infected MDMΦ after stimulation with viral TLR agonists.

<p>MDMΦs were differentiated in 96 well plates for 7 days and inoculated with BVDV2 strains with an MOI of 1 for 48 h prior to stimulation with Poly I:C [50 μg/mL], Imiquimod [10 μg/mL], ssRNA40 LyoVec [10 μg/mL]. Cell supernatants were analyzed for TNFα protein concentration 24 h after TLR stimulation and measured by Searchlight Array platform. Incubation with cytopathic and noncytopathic strains are in the top panel (A) or high and low virulence strains indicated in the lower panel (B). Bars represent the mean value ± SEM from four different experiments from 9 total donor cattle. ** P < 0.001 compared to uninfected, TLR stimulated cells.</p