Differential Innate Immunity-related Gene MRNA Expression In HD-11 Cells After Infected With H9N2 AIV

The low pathogenic avian influenza virus H9N2 subtype is a candidate of threat to poultry industry as well as human health. Influenza virus is responsible for immune suppression of birds, and increases susceptibility to secondary infection which may lead to serious clinical illness. HD-11 cells, a chicken macrophages cell line, are important avian innate immune cells.In this study, infection of H9N2 to HD-11 cells, resulted in activation of cellular antiviral immune response. HD-11 cells were infected with H9N2 at multiplicity of infection of 1, and were specifically detected by direct immunofluorescence assay. Cells were collected at 0, 6, 12, 24, 36, 48, and 72 hours post infection(h p i), and total RNA was extracted. One μg RNA was used to synthesize cDNA by reverse transcriptase PCR. The effects of H9N2 on HD-11 cells was detected by comparing the relative expression of pathogen recognition receptors, inflammatory cytokines, Chemokines, and iNOS mRNAs by real-time fluorescence quantitative RT-PCR.At 12 hpi, HD-11 cells exhibited cytopathic changes, including rounded shape, shrinkage, jagged membrane, agglomeration, and vacuolation. Direct immunofluorescence staining showed that a bright green color was observed around the nucleus of the infected cells at 36 hpi.. These results demonstrated that HD-11 cells are susceptible to H9N2 infection.Moreover, infected HD-11 cells showed a significantly higher expression of some genes related to cellular immune response, including chTLR7, chMDA5, IFN-α, IFN-β, IL-1β, IL-2, IL-6, XCL1, CXCLi1, and iNOS, suggesting their crucial roles in antiviral and inflammatory response against H9N2 infection. In detail, chTLR7 was observed to be significantly upregulated at 12 hpi, and reached at maximum level at 48 hpi, and up to 3.8-fold change. The maximum expression of chMDA5 mRNA was noticed at 72 hpi. Interestingly, expression level of chMDA5 was stronger than chTLR7, suggesting the importance of chMDA5 in mediating the antiviral immune response against H9N2 infection. In the downstream antiviral signaling pathway, IFN-α and-β showed maximum expression levels at 72 hpi, up to 48.8-fold change and up to 11.3-fold change, respectively.In addition, H9N2 infected cells showed significantly elevated levels of inflammatory cytokines that includes IL-1β, IL-2, and IL-6. Among the chemokines, XCL1, XCLi1, and CXCLi2 mRNA were significantly upregulated, and their levels reached maximum at 36, 48, and 72 hpi, respectively. Furthermore, iNOS mRNA was significantly upregulated at 12 hpi, and reached maximum at 36 hours, up to 4.3-fold change, then expressed stably at 48 and 72 hours.Taken together, these data demonstrated that H9N2 replicates effectively in HD-11 cells and this replication is associated with the production of cytopathic effects and induction of some genes involved in antiviral signaling pathways.