| Avian reovirus (ARV) is an important pathogen of poultry, mainly infecting chickens and turkeys. It induces chicken arthritis, chicken little syndrome, respiratory syndrome, immune suppression, and causes huge economic losses to the global poultry industry. However, current research is mainly on ARV pathogen isolation and identification, genome sequencing, development of detection methods. To efficially prevent and control infection of ARV and promote healthy development of the global poultry industry, the research of pathogenesis on avian reovirus is urgently needed. Because cytokines have a wide biology activities, they are important mediators of immune response and inflammation. Currently, it has been demonstrated that cytokines play important roles in pathogenesis of rheumatoid arthritis, tuberculosis, AIV infections, infectious bursal virus infection, coccidia infections and other diseases. But the role of cytokines in pathogenesis of ARV infection mediated widespread inflammation in body and immune suppression remain unclear.To investigate the influence of ARV infection on expression of cytokines (IL-1β, IL-6, IL-17, IL-18, IFN-γ, and TNF-α), we established real-time PCR assay on IL-1β, IL-6, IL-17, IL-18, IFN-γ, and TNF-α using SYBR Green I. By using infected chicken embryo fibroblasts and7-day-old SPF chicks infected with ARV standard virulent strain S1133, and we detected the changes in mRNA level of IL-1β, IL-6, IL-17, IL-18, IFN-γ, TN F-α genes in different cells and tissues and organs (joints, heart, spleen, thymus, bursa and WBC) in SPF chickens.Based on sequences deposited in GenBank of chicken interleukin-1(3(IL-1β), interleukin-17(IL-17), interleukin-18(IL-18), interferon-γ (IFN-γ), tumor necrosis factor-a (TNF-a) and3-glyceraldehyde phosphate dehydrogenase (GAPDH) genes, we designed and synthesized corresponding specific primers and generated recombinant plasmids as the standard by chicken embryo fibroblast cDNAs. We established SYBR Green I dye fluorescence quantitative PCR standard curve and analyzed the melting curves. The results showed that the dissolution profile of each gene showed a single melting peak, the standard amplification efficiency was between95.6%-102.0%, and R2of linear correlation was greater than0.99, the detection limit of each gene was10copies and the coefficient of variation within groups was less than2.0%. The results showed that the established methods had a good specificity, sensitivity and repeatablity.We detected dynamic changes of mRNA levels of ARV structural proteins gC, as well as IL-1β, IL-6, IL-17, IL-18, IFN-γ and TNF-α in ARV S1133infected-CEF-cells by real-time PCR. The results showed that,10h post-infection of CEF with ARV-S1133, the relative expression levels of viral σC mRNA began to rise rapidly, and reached the peak at48h (13162.73times, P<0.01); the infection also induced changes of mRNA expression of IL-1β, IL-6, IL-17, IL-18, IFN-γ and TNF-α in CEF cells. At36h,48h and60h post-infection with ARV-S1133, mRNA expression levels of IL-1β, IL-6, IL-17, IFN-γ and TNF-α genes substantially increased and reached a peak at48h, the changes in expression were88.78,40.43,97.19,111.58and4.4times(P<0.05), respectively throughout the course of infection the mRNA levels of IL-18was low, and its expression levels decreased at the late stage of infection; there was a linear correlation between cytokines transcript levels and six virus titers after CEF cells were infected with five different titers of ARV for24h. There was a positive correlation between mRNA levels of IL-1β, IL-6, IL-17, IFN-γ genes and virus titers, and a negative correlation between mRNA levels of IL-18, TNF-α genes and virus titers. The results showed that L-1β, IL-6,IL-17, IL-18, IFN-γ and TNF-α might be involved in ARV replication and pathogenesis.In this study, we detected the dynamic changes of mRNA expression of IL-1β, IL-6, IL-17, IL-18, IFN-γ and TNF-α genes in different tissues and organs (joints, heart, spleen, thymus, bursa and WBC) of7-day-old SPF chickens infected with ARV S1133through footpad-injection by real-time PCR. The results showed that ARV infection might lead to changes of transcription leves of these cytokines, but these changes varied among different tissues. These results suggested that after ARV infection, cytokines might participate in inflammiation and damage processes among various tissues and organs. During the late stages of infections, in the thymus and bursa, the expression levels of IL-17were upregulated; during the early stages of infections in the thymus, spleen and bursa, the expression levels of IL-18, IFN-y and TNF-a were upregulated, whereas their expression levels were downregulated at the late stage of infection. Theses results indicated that IL-17, IL-18, IFN-y, TNF-a may be involoved in the immunosppression of ARV.In this study we examined the amount of peripheral blood lymphocytes CD4+, CD8+T cells by flow cytometry in the process of SPF chickens infected with ARV. Our results showed that7d and14d post-infections CD4+and CD8+T cell ratio was higher than the control group. Seven days and fourteendays post-infection, CD8+T cells were significantly increased(P<0.05). it suggested that post infection7-14d is an important period to clear ARV in the body of infected chicken; Id,21d,28d,35d post-infection, CD4+and CD8+T cell ratio in experiment group was lower than control group. One day post-infection, CD4+and CD8+ratio was significantly decreased (P<0.05). It show that ARV infection resulted in infected chickens’immune function disorder in those time points, and suggested that the number of CD4+and CD8T T cells decreased maybe an important manifestation of the immune suppression of ARV Iinfection.This research obtained the data of IL-1β, IL-6, IL-17, IL-18, IFN-γ, TNF-α mRNA dynamic expression in avian reovirus infection of cells in vitro and SPF chicken tissues and the change information of peripheral blood T cell subsets of SPF chicken post-infected with ARV. Our study showed that different cytokines in ARV-infected cells and SPF chickens’tissues/organs may play an important role in inflammatiary and process of tissue damage, the change information of CD4+, CD8+T cells may reflect ARV infected chickens’immune level, and will help understand the mechamisms of pathogenesis of avian reovirus. |
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