Development and evaluation of a SYBR Green real-time RT-PCR assay for evaluation of cytokine gene expression in horse.

Cytokine secretion is one of the main mechanisms by which the immune system is regulated in response to pathogens. Therefore, the measurement of cytokine expression is fundamental to characterizing the immune response to infections. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) is widely used to measure cytokine mRNA levels, but assay conditions should be properly evaluated before analyzing important equine infections through relative quantification of gene expression. The aim of this study was to develop and evaluate a set of RT-qPCR assays for a panel of the most common cytokines in horses involved in innate and adaptive immune responses. Eight cytokines (interleukin (IL)-1ß, IL-2, IL-4, IL-10, IL-12, TNFα, IFNß and IFNγ) and a housekeeping gene (ß-actin) were detected and amplified with the same annealing temperature in a SYBR Green RT-qPCR assay of samples of mitogen-stimulated peripheral blood mononuclear cells from a healthy horse and whole blood from a horse infected with African horse sickness virus. The method gave good efficiency for all genes tested, allowing quantification of relative expression levels. These SYBR Green RT-qPCR assays may be useful for examining cytokine gene expression in horses in response to exposure to economically important pathogens.

An active DNA vaccine against infectious pancreatic necrosis virus (IPNV) with a different mode of action than fish rhabdovirus DNA vaccines.

Although there are some commercial vaccines available against infectious pancreatic necrosis virus (IPNV), the disease still continues to be a major problem for aquaculture development worldwide. In the current work, we constructed a DNA vaccine against IPNV (pIPNV-PP) by cloning the long open reading frame of the polyprotein encoded by the viral RNA segment A. In vitro, the vaccine is properly translated giving the functional IPNV polyprotein since preVP2, VP2 and VP3 proteins were detected because of the VP4-protease cleavage. EPC cells transfected with the vaccine plasmid expressed the viral proteins and induced the expression of type I interferon (IFN)-induced Mx genes. Furthermore, IPNV synthesized proteins seemed to assemble in virus-like particles as evidenced by electron microscopy. In vivo, rainbow trout specimens were intramuscularly injected with the vaccine and expression of immune-relevant genes, the presence of neutralizing antibodies and effect on viral load was determined. The pIPNV-PP vaccine was expressed at the injection site and up-regulated MHC Ialpha, MHC IIalpha, type-I interferon (IFN), Mx, CD4 and CD8alpha gene expression in the muscle, head kidney or spleen, although to a much lower extent than the up-regulations observed in response to an effective DNA vaccine against viral hemorrhagic septicaemia virus (VHSV). However, the IPNV vaccine was also very effective in terms of acquired immunity since it elicited neutralizing antibodies (in 6 out of 8 trout fingerlings) and decreased 665-fold the viral load after IPNV infection. The effectiveness of this new IPNV DNA vaccine and its possible mechanism of action are discussed and compared to other viral vaccines.

Salmonid fish viruses and cell interactions at early steps of the infective cycle.

A flow cytometric virus-binding assay that directly visualizes the binding and entry of infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and virus haemorrhagic septicaemia virus (VHSV) to several cell lines was established. The highest efficiency of binding was shown by the BF-2 cell line and this was used to study, at the attachment level, the interactions of these cells with salmonid fish viruses in coinfections, and to further determine if the earliest stage of the viral growth cycle could explain the previously described loss of infectivity of IHNV when IPNV is present. Our results demonstrated that IPNV binds to around 88% of cells either in single or dual infections, whereas IHNV attachment always decreased in the presence of any of the other viruses. VHSV binding was not affected by IPNV, but coinfection with IHNV reduced the percentage of virus-binding cells, which suggests competition for viral receptors or co-receptors. Internalization of the adsorbed IHNV was not decreased by coinfection with IPNV, so the hypothetical competence could be restricted to the binding step. Treatment of the cells with antiviral agents, such as amantadine or chloroquine, did not affect the binding of IPNV and VHSV, but reduced IHNV binding by more than 30%. Tributylamine affected viral binding of the three viruses to different degrees and inhibited IPNV or IHNV entry in a large percentage of cells treated for 30 min. Tributylamine also inhibited IHNV cytopathic effects in a dose-dependent manner, decreasing the virus yield by 4 log of the 50% endpoint titre, at 10 mm concentration. IPNV was also inhibited, but at a lower level. The results of this study support the hypothesis that IHNV, in contrast to VHSV or IPNV, is less efficient at completing its growth cycle in cells with a simultaneous infection with IPNV. It can be affected at several stages of viral infection and is more sensitive to the action of antiviral compounds.

Comparative expression analysis of the renin-angiotensin system components between white and brown perivascular adipose tissue.

Recent studies have demonstrated that the rat adipose tissue expresses some of the components necessary for the production of angiotensin II (Ang II) and the receptors mediating its actions. The aim of this work is to characterize the expression of the renin-angiotensin system (RAS) components in perivascular adipose tissue and to assess differences in the expression pattern depending on the vascular bed and type of adipose tissue. We analyzed Ang I and Ang II levels as well as mRNA levels of RAS components by a quantitative RT-PCR method in periaortic (PAT) and mesenteric adipose tissue (MAT) of 3-month-old male Wistar-Kyoto rats. PAT was identified as brown adipose tissue expressing uncoupling protein-1 (UCP-1). It had smaller adipocytes than those from MAT, which was identified as white adipose tissue. All RAS components, except renin, were detected in both PAT and MAT. Levels of expression of angiotensinogen, Ang-converting enzyme (ACE), and ACE2 were similar between PAT and MAT. Renin receptor expression was five times higher, whereas expression of chymase, AT(1a), and AT(2) receptors were significantly lower in PAT compared with MAT respectively. In addition, three isoforms of the AT(1a) receptor were found in perivascular adipose tissue. The AT(1b) receptor was found at very a low expression level. Ang II levels were higher in MAT with no differences between tissues in Ang I. The results show that the RAS is differentially expressed in white and brown perivascular adipose tissues implicating a different role for the system depending on the vascular bed and the type of adipose tissue.