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.

Prolactin peptide (pPRL) induces anti-prolactin antibodies, ROS and cortisol but suppresses specific immune responses in rainbow trout.

Prolactin has several immune functions in fish however, the effects on innate and specific components of rainbow trout immunity are currently unknown. Therefore in this study, prolactin peptide (pPRL) injection in rainbow trout generated anti-PRL antibodies that were confirmed through Western blot assays of fish brain tissue extract. At the same time, this group of fish was immunized with a viral antigen (VP2) and the specific antibody titer generated by the rainbow trout was subsequently determined, as well as the sero-neutralizing capacity of the antibodies. Interestingly, this group of fish (pPRL-VP2) generated approximately 150% less antibodies compared with fish immunized only with the viral antigen (VP2), and pPRL-VP2 fish increased their cortisol level by 4 times compared to the control. Additionally, through qPCR assay, we determined that the pPRL-VP2 fish group decreased pro-inflammatory transcript expression, and the serum of these (pPRL-VP2) fish stimulated ROS production in untreated fish leukocytes, a phenomenon that was blocked by the pharmacological cortisol receptor inhibitor (RU486). Collectively, this is the first report that indicates that pPRL could modulate both components of immunity in rainbow trout.

Sumoylation of nucleoprotein (NP) mediated by activation of NADPH oxidase complex is a consequence of oxidative cellular stress during infection by Infectious salmon anemia (ISA) virus necessary to viral progeny.

The study evaluated the effects of nucleoprotein viral and the infectious virus in SHK-1 cells. The results show a strong respiratory burst activation and the induction of p47phox, SOD, GLURED, and apoptotic genes. Additionally, the cells alter the profile of SUMOylated proteins by the effect of transfection and infection experiments. In silico analyses show a set of structural motifs in NP susceptible of post-translational modification by the SUMO protein. Interestingly, the inhibition of the NADPH oxidase complex blocked the production of reactive oxygen species and the high level of cellular ROS due to the nucleoprotein and the ISAv. At the same time, the blocking of the p38MAPK signaling pathway and the use of Aristotelia chilensis, decreased viral progeny production. These results suggest that the NP triggers a strong production of ROS and modifying the post-translational profile mediated by SUMO-2/3, a phenomenon that favors the production of new virions.

Molecular characterization of the bovine IER3 gene: Down-regulation of IL-8 by blocking NF-κB activity mediated by IER3 overexpression in MDBK cells infected with bovine viral diarrhea virus-1.

The immediate early response 3 (IER3) is a key regulatory factor in the immune response, particularly as related to homeostasis immunomodulation via the nuclear factor kappa B (NF-κB) signaling pathway. The IER3 gene has been identified in mammals and, more recently, in other higher vertebrates. Nevertheless, relatively little is known about this regulator in bovines. Therefore, this study explored, characterized, and compared the genetic context of bovine IER3 to homologous genes in the human, mouse, and canine chromosomes. In silico analysis identified several regions of interest preserved in phylogenetically distant species. Similar analyses were also conducted for interleukin-8, a cytokine in which several putative cis elements were identified for the inducible transcription factor NF-κB. Subsequent challenge assays against the bovine viral diarrhea virus-1 revealed NF-κB signaling pathway activation just 15min post-infection, a process blocked by the BAY-117085 inhibitor. Similarly, infection strongly increased IER3 expression. Interestingly, IER3 down-regulated interleukin-8 expression, as confirmed by IER3 gene inhibition using small interfering RNA, RT-qPCR, and luciferase assays. In conclusion, this is the first report to present data indicating that bovine IER3 is a strong regulator of immune-marker expression, specifically modulating bovine interleukin-8 activation through the NF-κB/IER3 pathway in response to the bovine viral diarrhea virus.

Infectious pancreatic necrosis virus infection of fish cell lines: Preliminary analysis of gene expressions related to extracellular matrix remodeling and immunity.

The pathogenic infectious pancreatic necrosis virus (IPNV) causes high economic losses in fish farming. This virus can modulate several cellular processes during infection, but little is known about the infection mechanism. To investigate gene activation in response to IPNV, CHSE/F and SHK-1 cell line were infected with a cytopathic Sp field isolate of IPNV, and the expression profiles of proinflammatory, antiviral cytokine, and extracellular matrix markers were analyzed. IPNV induced the production of perlecan, fibulin-1, matrix metalloproteinase-2, 14-3-3ß, interleukin-1ß, Mx1, and interferon regulatory factors-1, -3, and -9. Interestingly, IPNV-mediated activity was 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 IPNV regulates gene expressions in fish through the activation of several key transcription factors. Collectively, these data indicate that IPNV is a viral regulator of expression for extracellular-matrix and immune markers, even during early infection. Finally, this is the first report in fish to find IPNV modulating the activation of interleukin-1ß production primarily through the NF-κB pathway.

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.

Molecular characterization of infectious pancreatic necrosis virus strains isolated from the three types of salmonids farmed in Chile.

BACKGROUND: The infectious pancreatic necrosis virus (IPNV) causes significant economic losses in Chilean salmon farming. For effective sanitary management, the IPNV strains present in Chile need to be fully studied, characterized, and constantly updated at the molecular level. METHODS: In this study, 36 Chilean IPNV isolates collected over 6 years (2006-2011) from Salmo salar, Oncorhynchus mykiss, and Oncorhynchus kisutch were genotypically characterized. Salmonid samples were obtained from freshwater, estuary, and seawater sources from central, southern, and the extreme-south of Chile (35° to 53°S). RESULTS: Sequence analysis of the VP2 gene classified 10 IPNV isolates as genogroup 1 and 26 as genogroup 5. Analyses indicated a preferential, but not obligate, relationship between genogroup 5 isolates and S. salar infection. Fifteen genogroup 5 and nine genogroup 1 isolates presented VP2 gene residues associated with high virulence (i.e. Thr, Ala, and Thr at positions 217, 221, and 247, respectively). Four genogroup 5 isolates presented an oddly long VP5 deduced amino acid sequence (29.6 kDa). Analysis of the VP2 amino acid motifs associated with clinical and subclinical infections identified the clinical fingerprint in only genogroup 5 isolates; in contrast, the genogroup 1 isolates presented sequences predominantly associated with the subclinical fingerprint. Predictive analysis of VP5 showed an absence of transmembrane domains and plasma membrane tropism signals. WebLogo analysis of the VP5 BH domains revealed high identities with the marine birnavirus Y-6 and Japanese IPNV strain E1-S. Sequence analysis for putative 25 kDa proteins, coded by the ORF between VP2 and VP4, exhibited three putative nuclear localization sequences and signals of mitochondrial tropism in two isolates. CONCLUSIONS: This study provides important advances in updating the characterizations of IPNV strains present in Chile. The results from this study will help in identifying epidemiological links and generating specific biotechnological tools for controlling IPNV outbreaks in Chilean salmon farming.

Transcriptomic analysis of responses to cytopathic bovine viral diarrhea virus-1 (BVDV-1) infection in MDBK cells.

The bovine viral diarrhea virus (BVDV) is responsible for significant economic losses in the dairy and cattle industry; however, little is known about the protective and pathological responses of hosts to infection. The present study determined the principal molecular markers implicated in viral infection through meta-transcriptomic analysis using MDBK cells infected for two hours with a field isolate of BVDV-1. While several immune regulator genes were induced, genes involved in cell signaling, metabolic processes, development, and integrity were down-regulated, suggesting an isolation of infected cells from cell-to-cell interactions and responses to external signals. Analysis through RT-qPCR confirmed the expression of more than one hundred markers. Interestingly, there was a significant up-regulation of two negative NF-κB regulators, IER3 and TNFAIP3, indicating a possible blocking of this signaling pathway mediated by BVDV-1 infection. Additionally, several genes involved in the metabolism of reactive oxygen species were down-regulated, suggesting increased oxidative stress. Notably, a number of genes involved in cellular growth and development were also regulated during infection, including MTHFD1L, TGIF1, and Brachyury. Moreover, there was an increased expression of the genes ß-catenin, caprin-2, GSK3ß, and MMP-7, all of which are crucial to the Wnt signaling pathway that is implicated in the embryonic development of a variety of organisms. This meta-transcriptomic analysis provides the first data towards understanding the infection mechanisms of cytopathic BVDV-1 and the putative molecular relationship between viral and host components.

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.

ISAV infection promotes apoptosis of SHK-1 cells through a ROS/p38 MAPK/Bad signaling pathway.

The impact that the infectious salmon anemia virus (ISAV) has on the immune response of Salmo salar, from the perspective of activating/inactivating cellular processes, is currently unknown. Therefore, the present study evaluated this interaction and found that SHK-1 cells infected with ISAV resulted in respiratory burst activation and the induction of a strong pro-apoptotic imbalance through an increased expression of the Bad protein and decreased transcripts of Bcl-xl. Interestingly, the pharmacological inhibition of the p38 MAPK protein through SB203580 blocked the production of reactive oxygen species, the activity of caspase 3, and the formation of apoptotic nuclei in SHK-1 cells. Additionally, when the NADPH oxidase complex, a producer of superoxide anions, was blocked through apocynin, decreased apoptotic activity was observed in infected cells without significant modifications to viral amplification. These results, together with bioinformatics analysis performed for the Bad gene of fugu, suggest that the ISA virus triggers a strong production of oxygen radicals capable of activating transduction signaling pathways and mediating the expression and activation of pro-apoptotic proteins through the p38 MAPK pathway, all of which results in the apoptosis of ISAV infected cells.

ISA virus regulates the generation of reactive oxygen species and p47phox expression in a p38 MAPK-dependent manner in Salmo salar.

Several viruses, including Orthomyxovirus, utilize cellular reactive oxygen species (ROS) for viral genomic replication and survival within host cells. However, the role of ROS in early events of viral entry and signal induction has not been elucidated. Here, we show that ISA virus (ISAV) induces ROS production very early during infection of CHSE-214 and SHK-1Ycells, and that production is sustained over the observed 24h post-infection. The mitogen-activated protein kinase (MAPK) family is responsible for important signaling pathways. In this study, we report that ISAV activates ERK and p38 in Salmo salar. In salmonid macrophages, while ERK was required for SOD, GLURED, p47phox expression, p38 regulated the ROS production by the NADPH oxidase complex activation. These results, together with the presence of several consensus target motifs for p38 MAPK in the promoter of the S. salar p47phox gene, suggest that p38 MAPK regulates p47phox gene expression in fish through the activation of this key transcription factor.

Herpes simplex virus type 1 induces simultaneous activation of Toll-like receptors 2 and 4 and expression of the endogenous ligand serum amyloid A in astrocytes.

Herpes simplex virus type 1 (HSV-1) is the most common pathogenic cause of sporadic acute encephalitis and it produces latent persistent infection lifelong in infected individuals. Brain inflammation is associated with activation of glial cells, which can detect pathogen-associated molecular patterns (PAMPs) through a variety of pattern-recognition receptors (PRR), including Toll-like receptors (TLRs). In this study, we evaluated the expression and activation of TLR2, TLR3, and TLR4 in HSV-1-infected astrocyte and neuronal primary cultures. Our results showed a clear induction in TLR2 and TLR4 expression in astrocytes as early as 1 h after HSV-1 infection, whereas no significant change was observed in neurons. In addition, infected astrocytes showed increased levels of interferon regulatory factors IRF3 and IRF7, interferon ß (INFß), interleukin 6 (IL6), and serum amyloid A (SAA3) transcripts, as well as phospho-IRF3 protein. These effects seemed to be dependent on viral replication since previous treatment of the cells with acyclovir resulted in low levels of TLRs expression and activation even after 4 h post-infection. These results suggest that reactivation of HSV-1 at the central nervous system (CNS) would likely induce and activate TLR2 and TLR4 receptors directly through interaction of astrocytes with the pathogen and also indirectly by endogenous ligands produced locally, such as serum amyloid protein, potentiating the neuroinflammatory response.