Studies On Toll-Like Receptor Transcriptic Level In Vitro And In SPF Chickens Infected With Avian Reovirus

Avian reovirus (ARV) is an important pathogen of poultry, causing various of disease syndromes, including viral arthritis, immunesuppression and respiratory syndrome and results in huge economic losses to the poultry industry. However, currently studies on ARV infection focus on ARV pathogen isolation and identification, genome sequencing and development of detection methods. To effectively prevent and control ARV infection, it is necessary and urgent to furthermore explore ARV pathogenesis. As toll-like receptors are a class of pattern recognition receptors with various biological functions in the innate immune system, they can recognize pattern associated molecular patterns, induce acquired immune response, and play important roles in innate immune system. It has been well demonstrated that chicken Toll-like receptors play important roles in a number of disease development, for example, rheumatoid arthritis, tuberculosis, AIV infections, infectious bursal virus infection and coccidia infections. However, it is still unknown that the effects of ARV infections on toll-like receptor transcriptic expression and the regulation of Toll-like receptors on ARV pathogenesis as well as immune suppression.To examine the transcriptic expression profiles of toll-like receptors after ARV infection, initially the quantitative real-time PCR assays for ChTLR3> ChTLR5、ChTLR7、ChTLR15 and ChTLR21 were developed based on SYBR Green ||.And then, it was aimed to dectect the mRNA level of ChTLR3、 ChTLR5、ChTLR7、ChTLR15 and ChTLR21 genes in the chicken embryo fibroblasts and different organs (peripheral blood lymphocytes,liver,heart, thymus,spleen, bursa and joints) of 7-day-old SPF chicks infected with ARV S1133 during the experiment course.According to the sequences in GenBank of ChTLR3、ChTLR5、ChTLR7、 ChTLR15 and ChTLR21 as well as phosphate dehydrogenase (GAPDH) genes, specific primers were designed and synthesized, recombinant plasmids carrying targent genes were generated as a positive standard using spleen cDNA of 10-day-old chick. The quantitative real-time PCR was established using SYBR Green Ⅱ dye and assesssed by its standard curve and melting curves. The results showed that the dissolution profile of each gene exhibited a single melting peak, the standard amplification efficiency was approached to 100.0%, and R2 of linear correlation was greater than 0.99, the detection limit of the assay for each gene meet the requirement of real-time PCR. The results suggest that the established quantitative real-time PCR can be useful for quantification of chicken Toll-like receptors.Next, the developed quantitative real-time PCR was used to analyze the mRNA transcriptic changes of ARV structural protein σC gene and chicken Toll-like receptors in ARV S1133-infected CEF cells. The results showed that the relative expression levels of ARV σC gene were elevated at 10h post infection (hpi), and reached a peak at 48hpi (P< 0.01). During this period, the mRNA transcriptic levels of ChTLR3, ChTLR5, ChTLR7, ChTLR15 and ChTLR21 genes were changed significantly in the infected CEF cells with different patterns.The mRNA transcriptic level of ChTLR3 reached the peak at 24 hpi 6.5 times higher than the control. The mRNA expression level of ChTLR5 gene reached the peak at 10hpi, which was 20.67 times higher than the control.The mRNA expression level of ChTLR7 gene reached the peak at 6hpi,which was 9.22 times higher than the control.The mRNA expression level of ChTLR15 gene reached a peak at 12 hpi,which was 14.23 times higher than the control.The mRNA expression level of ChTLR21 gene reached the peak at 10hpi,which was 13.74 times higher than the control. At the same time, the analysis of different ARV dose infection experiment revealed that the mRNA transcriptic level of ChTLR3、ChTLR5、ChTLR7、ChTLR15 and ChTLR21 genes were positively correlated with the virus doses.These data above suggest that ARV infection could effectively increase the mRNA expression changes of ChTLR3、ChTLR5、ChTLR7. ChTLR15 and ChTLR21. Furthermore, ChTLR、ChTLR5、ChTLR7、ChTLR15 and ChTLR21 may play important roles in ARV replication and pathogenesis.The mRNA expression levels of ChTLR3、ChTLR5、ChTLR7、ChTLR15 and ChTLR21 genes were tested in different tissues and organs (peripheral blood lymphocytes,liver,heart, thymus,spleen, bursa and joints) of 7-day-old SPF chickens infected with ARV S1133 through footpad-injection by the quantitative real-time PCR. The results showed that the ARV infection readibly induced the mRNA transcriptic changes of ChTLRs, with different patterns in different organs and tissues. These results suggest that after ARV infection, ChTLRs might be associated with inflammiation and damage in various tissues and organs. In joints, the mRNA expression levels of ChTLR3、ChTLR5、 ChTLR7、ChTLR15 and ChTLR21 changed significantly, especially rised on 3d post infection (dpi) significantly, and then significantly decreased on 14dpi (P<0.05 and P< 0.01, respectively).These data suggest that the expression change of ChTLRs may be associated with tissue damage after ARV infection. In hearts, the mRNA expression levels of ChTLR3、ChTLR5、ChTLR7、 ChTLR15 and ChTLR21 genes rised significantly on 3dpi (P< 0.05 or P< 0.01), indicating that the expression change of ChTLRs may be associated with the heart injury. In liver, the mRNA expression level of ChTLR3 gene was increased significantly on 1dpi (P<0.05), but ChTLR7、ChTLR 15 and ChTLR21 genes induced significantly on 3dpi (P<0.05 or P<0.01), suggesting that their expression may be associated with liver damage. In immune organs, including spleen, thymus and bursa, the mRNA expression levels of ChTLR3、ChTLR5、 ChTLR7、ChTLR 15 and ChTLR21 genes changed significantly, especially the ChTLR3 changed significantly on 1dpi (P<0.05 or P<0.01).The mRNA expression of ChTLR7 gene rise significantly on 5dpi in spleen (P<0.05),whereas it rise significantly on 1dpi in thymus and bursa (P<0.05), suggesting that ChTLR7 may be involved in immunosuppression caused by ARV. In peripheral blood, the mRNA expression level of ChTLR3 gene significantly reduced on 1dpi, and ChTLR21 gene decreased significantly on7dpi (P<0.05). The mRNA expression level of ChTLR7 gene increased significantly on 7dpi (P<0.01),and other receptors also similarly change. These data above suggest that the change of their expression may be associated with tissue damage induced by ARV infection and immune regulation.In this study, we analyzed the mRNA dynamic expression of ChTLR3-. ChTLR5、ChTLR7、ChTLR15 and ChTLR21 in CEF cells and organs and peripheral blood cell of SPF chicken infected with, and explored the effects of different toll-like receptors in ARV infection,which might contribute to further reveal ARV pathogenesis.