Quantification of interleukine-8 and interferon-γ expression in BVDV infected animals.

In this study we investigate mRNA expression of interleukin-8 (IL-8), a potent neutrophil chemoattractant, and interferon-gamma (IFN-γ), one of the major responsible of antiviral effect, in Bovine Viral Diarrohea Virus (BVDV) infection. Total RNA was isolated from brain, cerebellum and in lymphatic organs from immune, digestive and respiratory apparatus from 12 BVDV infected and 3 healthy animals. The samples were DNase treated, retrotranscripted and quantified by SYBR Green real time PCR performed on Rotor-Gene 3000. The primers for each target gene, GADPH as housekeeping and BVDV were designed using the Oligo software on sequences available from Genbank database. A recombinant plasmid was constructed for each target gene and serial dilutions were used to construct a standard curve. Melting curve analysis was performed for each assay. The number of copies of target genes and of BVDV genome were normalized by the ratio with the number of copies of housekeeping gene. All the values obtained were statistically analysed by One-way ANOVA. In healthy animals IL-8 was not or weakly expressed; this is not unusual because it is a proinflammatory cytokine that is expressed during inflammation. IFN-γ, instead, showed a basal level of expression in the healthy animals, in fact, it is a cytokine normally expressed by Th1 lymphocytes. Eight BVDV infected animals showed a strong downregulation of IL-8 expression. In some organs of 4 animals the expression of IL-8 were less inhibited; in these organs we observed purulent lesions: neuthophils’ attraction is, in fact, mediated by IL-8. In 8 infected animals also IFN-γ levels were lower than the levels in healthy animals. In some organs of the other 4 animals the IFN-γ expression levels were similar to the values in healthy animals; in these organs we also detected a low BVDV amount. We, in fact, observed a correlation between the cytokines’ expression and virus level: a higher downregulation of both cytokines was evidenced in organs with greater BVDV levels. This study permitted to analyse and confirms the interference of BVDV on immunology systems in natural infected animals. Particularly downregulation of IFN-γ showed the effect of BVDV on Th1 response and downregulation of IL-8 showed its inhibition of inflammatory response.