Effects of reticuloendotheliosis virus infection on cytokine production in SPF chickens

Abstract

Infection with reticuloendotheliosis virus (REV), a gammaretrovirus in the Retroviridae family, can result in immunosuppression and subsequent increased susceptibility to secondary infections. The effects of REV infection on expression of mRNA for cytokine genes in chickens have not been completely elucidated. In this study, using multiplex branched DNA (bDNA) technology, we identified molecular mediators that participated in the regulation of the immune response during REV infection in chickens. Cytokine and chemokine mRNA expression levels were evaluated in the peripheral blood mononuclear cells (PBMCs). Expression levels of interleukin (IL)-4, IL-10, IL-13 and tumor necrosis factor (TNF)-α were significantly up-regulated while interferon (IFN)-α, IFN-β, IFN-γ, IL-1β, IL-2, IL-3, IL-15, IL-17F, IL-18 and colony-stimulating factor (CSF)-1 were markedly decreased in PBMCs at all stages of infection. Compared with controls, REV infected chickens showed greater expression levels of IL-8 in PBMCs 21 and 28 days post infection. In addition, REV regulates host immunity as a suppressor of T cell proliferative responses. The results in this study will help us to understand the host immune response to virus pathogens.

Figure 1. REV genome load in infected PBMCs.

Chickens were infected with the HLJ07I strain of REV and sampled at 7, 14, 21 and 28 days post-infection. Gag copy numbers in 10⁶ PBMCs were quantitated using real-time RT-PCR. At least three samples were analyzed in duplicate at each sampling time point. The error bars represent standard error of the mean.

Figure 2. The relative cytokine and chemokine mRNA levels in PBMCs of chickens infected with REV-A strain HLJ07I or uninfected controls for 7, 14, 21 and 28 days.

Values were normalized to the endogenous GAPDH control and were presented as the log₂ mean fold-change in mRNA expression (relative to the uninfected control). Data are the means of three independent experiments. * indicates P < 0.05 and ** indicates P < 0.01 when the REV-infected group was compared with the control group.

Figure 3. The proliferation of PBMCs post infection of REV.

The PBMCs (1 ×10⁷ cells/ml) were isolated from heparinized peripheral blood of REV-A infected or uninfected control chickens. The mean fluorescence intensity (MFI) was statistically analyzed. * indicates P < 0.05 when the REV-infected group was compared with the control group.

Figure 4. The subpopulation ratios of CD4+/CD8+ in the PBMCs of chickens infected with REV detected by flow cytometry.

The PBMCs were isolated from the heparinized peripheral blood and stained with mouse monoclonal antibodies against chicken CD3, CD4, and CD8. CD4+/CD8+ ratios were calculated from the number of cells labeled with the fluorescent monoclonal antibodies of anti-CD4 or anti-CD8 analyzed using a flow cytometer. All data were expressed as mean ± standard error. * indicates P < 0.05 when the ratio of the REV-infected group was compared with that of the control group.