The MRNA And Proteins Expression Levels Analysis Of TC-1 Cells Immune Response To H9N2 Avian Influenza Virus

H9N2 subtype avian influenza virus was low pathogenic virus, and just caused a mild symptoms post infection. However, it has have been widely circulated in the world, and brought huge losses to the aquaculture industry, which were still need to pay more attention to prevent and control of the disease in poultry. Moerover, H9N2 subtype AIVs can also crossed the space barrier and infected mammals and human, which made a great threat to public health. Since 1999, the cases of H9N2 subtype avian influenza virus infection in humans have been reported in mainland C hina and Hong Kong. Thence more and more human cases were reported later. H9N2 subtype avian influenza virus can only cause some mild respiratory symptoms and low productirity, but co-infection with other pathogens will increase the symptoms. Furthermore, it is easily to produce mutation and recombination, and produce a new, more harmful variant of the strain, such as H7N9 that six internal gens were derived from H9N2 subtype AIVs. So that H9N2 AIVs were a potential factor that endangers public health and safety.At the present stage, the researchers were paying more attention to the pathogenicity and pathological changes of host. However, little is known about the immune response process and its mechanism of host infection. Therefore, preliminary study on the host immune response was made in this study. Firstly, in order to pick out the virus which were more sensitive to the TC-1 cells. In this study, 5 strains of H9N2 subtype avian influenza virus, preserved in laboratory, were detected by virus titer assay and indirect immunofluorescence assay. By comparison, the virus titer of CK/SD/ch was the highest, followed by CK/SD/w3, CK/SD/01, CK/SD/836-F1, CK/SD/w4. The virulence of avian influenza virus is different, but it can be effectively replicated in TC-1 cells, which lays the foundation for further study on the spread of the virus.In order to reveal the host immune response post infection, the expressions of PRRs were detected. q RT-PCR and ELISA methods were used to detect the three kinds of pattern recognition receptors TLR-3, RIG-I and MDA-5 in m RNA level and protein level, respectively. Results showed that MDA-5 have a substantial up-regulatetion in m RN A and protein levels, and MDA-5 plays a key role in the innate immunity, there were 11.5 and 7.5 fold in the m RN A and protein levels, respectively. RIG-I and TLR-3 were moderately up-regulated, the maximum up regulations were just 4.8 and 5.6 fold in m RNA and protein levels. According to the comparison of three strains influenza virus, CK/SD/w3 induced obvious increase of PRRs in m RNA level, and there were 0.2-1.4 times higher than other AIVs in each hp i. However, there were no significantly different in protein level at each point. The expressions of TLR-3 induced by three AIVs were just 1.16-fold in 18 hpi, the expression of MDA-5 was just 6.4-fold at the 24 hpi, and there was no significantly different between the three strain AIVs.In order to clarify the inflammatory reaction of the host against AIV, the concentration of cytokines was used to indicate the degree of inflammation. q RT- PCR and ELISA methods were used to detect the response of 4 cytokines(IL-1?, IL-6, TNF-? and IFN-?). The results showed that IL-1?, IL-6 and IFN-? were relatively up-regulated, the maximum up-regulation were 19.0, 21.9 and 24.1 folds, respectivley, However, TNF-? was in a moderately upregularion, the maximum up-regulation was just 6.3-fold. By the contrast of three strains of H9N2 AIVs, the results indicated that IL-1?, IL-6 and IFN-? were in high levels(18.0-25.0 fold-changes). At the protein levels, the up regulation of various cytokines was similar, there were no significant up-regulation at 6, 12 and 18 hpi, some cytokines were even downregulated. It is worth noting that among the three H9N2 AIVs test, the difference between the cytokines was not significant at the each time point in protein levels, and the up-regulation was moderately, only 2-4 fold changes. These cytokines are proinflammatory cytokines, participates in the immune response against influenza virus. Under physiological conditions, the content of these cytokines is very low, but in the pathological conditions, large amounts of cytokines were rapaidly released which promote the progression of the disease.Chemokines were also an important immune factor in the host body, they play an important role in directional trend to imflammatory response site. In this study, 5 chemokines(IL-8, Ccl-2, Ccl-3, IP-10 and RAN TES) were detected. The results showed that: in the m RNA levels, these chemokines have a significant increase, the maxmiun increase and the most obvious was Ccl-3 which maximum up-regulation about 19.1-fold. By the comparison of three H9N2 AIVs test, the Chemokines were significantly up-regulated in CK/SD/w3 and CK/SD/w4 tests group, but it's higher in CK/SD/w3 test group. However, CK/SD/ch only on the increase of Ccl-3 was more obvious(about 12.5- fold). At the protein levels, the upregulation of RANTES and IP-10 were most obvious, and Ccl-2 and Ccl-3 are relatively small. There were no significantly different between the three strains H9N2 AIVs in up-regulation of chemokines. These chemokines were secreted by monocytes and macrophages, which could recruit free monocytes, neutrophils and lymphocytes through the vascular endothelium to reach the site of inflammation. The more obvious the upregulation of chemokines, the more inflammatory cells and infla mmatory factors were recruited, and the more serious the inflammatory response was.