BVDV-1 induces interferon-beta gene expression through a pathway involving IRF1, IRF7, and NF-κB activation.

The bovine viral diarrhea virus (BVDV-1) is a pathogen with the capacity to modulate the interferon type I system. To further investigate the effects of BVDV-1 on the production of the immune response, the Madin-Darby bovine kidney cell line was infected with the cytopathic CH001 field isolate of BVDV-1, and the IFNbeta expression profiles were analyzed. The results showed that cpBVDV-1 was able to induce the production of IFNbeta in a way similar to polyinosinic-polycytidylic acid, but with less intensity. Interestingly, all cpBVDV-1 activities were blocked by pharmacological inhibitors of the IRF-1, IRF-7, and NF-κB signaling pathway, and the level of IFNbeta decreased at the level of transcript and protein. These results, together with in silico analyses showing the presence of several regulatory consensus target motifs, suggest that cpBVDV-1 regulates IFNbeta expression in bovines through the activation of several key transcription factors. Collectively, the results suggest that during cpBVDV-1 infection, cross talk is evident between various signaling pathways involved in transcriptional activation of IFNbeta in cattle.

Bovine A20 gene overexpression during bovine viral diarrhea virus-1 infection blocks NF-κB pathway in MDBK cells.

Viruses have developed cellular strategies to ensure progeny survival. One of the most interesting is immune camouflage, where the virus triggers a controlled-intensity immune response that prevents total destruction of the infected cell, thus "winning time" for the virus. This study explored the regulatory contexts of the bovine A20 gene during bovine viral diarrhea virus (BVDV)-1 infection, using IL-8 as an immune-response sentinel molecule. Assessments were conducted through RT-qPCR, Western blotting, gene silencing/overexpression, luciferase assays, and the use of pharmacological inhibitors, among other approaches. The results demonstrated that a) BVDV-1 increased A20 levels in Madin-Darby bovine kidney cells, b) increased A20 led to decreased IL-8 expression, and c) the virus affected the NF-κB signaling pathway. Collectively, these data identify bovine A20 as a strong regulator of immune marker expression. In conclusion, this is the first report on BVDV-1 modulating bovine IL-8 activation through the NF-κB/A20 pathway.

Characterization of bovine A20 gene: Expression mediated by NF-κB pathway in MDBK cells infected with bovine viral diarrhea virus-1.

Cytokine production for immunological process is tightly regulated at the transcriptional and posttranscriptional levels. The NF-κB signaling pathway maintains immune homeostasis in the cell through the participation of molecules such as A20 (TNFAIP3), which is a key regulatory factor in the immune response, hematopoietic differentiation, and immunomodulation. Although A20 has been identified in mammals, and despite recent efforts to identify A20 members in other higher vertebrates, relatively little is known about the composition of this regulator in other classes of vertebrates, particularly for bovines. In this study, the genetic context of bovine A20 was explored and compared against homologous genes in the human, mouse, chicken, dog, and zebrafish chromosomes. Through in silico analysis, several regions of interest were found conserved between even phylogenetically distant species. Additionally, a protein-deduced sequence of bovine A20 evidenced many conserved domains in humans and mice. Furthermore, all potential amino acid residues implicated in the active site of A20 were conserved. Finally, bovine A20 mRNA expression as mediated by the bovine viral diarrhea virus and poly (I:C) was evaluated. These analyses evidenced a strong fold increase in A20 expression following virus exposure, a phenomenon blocked by a pharmacological NF-κB inhibitor (BAY 117085). Interestingly, A20 mRNA had a half-life of only 32min, likely due to adenylate- and uridylate-rich elements in the 3'-untranslated region. Collectively, these data identify bovine A20 as a regulator of immune marker expression. Finally, this is the first report to find the bovine viral diarrhea virus modulating bovine A20 activation through the NF-κB pathway.

Cytopathic BVDV-1 strain induces immune marker production in bovine cells through the NF-κB signaling pathway.

The bovine viral diarrhea virus (BVDV-1) is a pathogen responsible for high economic losses in the cattle industry worldwide. This virus has the capacity to modulate the immune system of several higher vertebrates, but there is little information available on the cell infection mechanism. To further investigate the effects of BVDV-1 on the activation of the immune response, the Madin-Darby bovine kidney cell line was infected with the cytopathic CH001 field isolate of BVDV-1, and the proinflammatory and antiviral cytokine expression profiles were analyzed. The results showed that BVDV-1 was able to induce the production of BCL3, IL-1ß, IL-8, IL-15, IL-18, Mx-1, IRF-1, and IRF-7 in a way similar to polyinosinic-polycytidylic acid. Interestingly, all BVDV-1 activities were blocked by pharmacological inhibitors of the NF-κB signaling pathway. These results, together with in silico analyses showing the presence of several regulatory consensus target motifs, suggest that BVDV-1 regulates gene expression in bovines through the activation of several key transcription factors. Collectively, these data identified BVDV-1 as a viral regulator of immune marker expression, even from early infection. Additionally, this is the first report to find BVDV-1 modulating the activation of cytokine production and transcriptions factors mainly through the NF-κB pathway in vertebrates.

The effects of reference genes in qRT-PCR assays for determining the immune response of bovine cells (MDBK) infected with the Bovine Viral Diarrhea Virus 1 (BVDV-1).

The bovine viral diarrhea virus (BVDV) causes significant economic losses to the dairy industry worldwide, and understanding its infection mechanisms would be extremely useful in designing new and efficient treatments. Due to the limited number of specific antibodies against bovine proteins, differential gene expression analyses are vital for researching host immune responses to viral infection. qRT-PCR provides a sensitive platform to conduct such gene expression analyses, but suitable housekeeping genes are needed for accurate transcript normalization. The present study assessed nine reference genes in bovine kidney cells under conditions of BVDV-1 infection, incubation with pathogen-associated molecular patterns, and co-incubation with BAY117085, a pharmacological inhibitor of the NF-κB signaling pathway. Analyses of Ct values using the BestKeeper and Normfinder programs ranked CD81, RPL4, and GAPDH as the most reliable reference genes. This determination of a stable set of reference genes in this culture system will facilitate analyses of expression levels for genes of interest.