Study On The Role Of Host SRC In Avian Infectious Laryngotracheitis Virus Infection

Avian infectious laryngotracheitis(AILT) is a major respiratory disease of chickens induced by ILT herpesvirus. ILTV is an Iltovirus within the Herpesviridae family. Infection with ILTV can induce a mild to severe upper respiratory tract disease in chickens, depending on the virulence of the virus, and causes heavy economic losses in the poultry industry worldwide. At present, the control of AILT rely mainly on the body’s immune response caused by vaccination, however, immunization can’t eliminate latent infection virus in host cell and inhibit virus replication in cell, it is difficult to stem the epidemic. Given the side effects of vaccination against infectious laryngotracheitis(ILT), novel strategies for ILT control and therapy are urgently needed.The manipulation of the cell-intrinsic barrier mediated by host factors regulating the interaction between ILTV and host cells can provide a basis of another promising option. In the present study, we used LMH cells infected with ILTV LJS09 as an in vitro experimental model to investigate the molecular mechanisms of host cell responses to ILTV infection. To better illustrate the related molecular events of viral infection of the host cell, we determined the levels of virus by qPCR assays and TCID50 firstly to understand the biological characteristics of ILTV-LSJ09 infection in LMH cells, and then we used genome-wide transcriptome studies in combination with bioinformatic analysis and predicted SRC maybe an important modulator of ILTV infection. SRC is the first oncogene identified, it is an important factor for a wide range of biological processes, such as cell proliferation, adhesion, organization of the cell skeleton, cell division and cell death. Western blotting show that phosphorylation of SRC in LMH cells was greatly increased by 2 dpi, which indicated that ILTV infection can activate SRC in host cells. Growth curve for LMH cells after ILTV infection show that ILTV infection significantly reduced the number of cells by 3 dpi. Combined with the SRC influence on cell proliferation, cell cycle analysis was performed by flow cytometry and result showed that With the increase of ILTV infection time, host cells in G1 phase significantly reduced, apoptotic cells were increasing, however cells to S/M and G2 phase the proportion is not reduced. In order to further understand the specific role of SRC in ILTV infection, we tested the biological functions of SRC in the process of LMH response to ILTV infection by small molecule inhibitors and little interference fragment specific targeting chicken SRC. ILTV specific real-time quantitative PCR shows that levels of virus in LMH significantly reduced after SRC was inhibited. Flow cytometry result shows that SRC inhibition can promote ILTV-induced cell death. Therefore, SRC is essential for the maintenance of virus replication but is not required for ILTV-induced cell death. We can conclude that SRC is a key determinant of virulence and replication of ILTV in LMH cells, it suggests that SRC prolongs the survival of LMH cells in response to ILTV infection by improving threshold of ILTV-induced cell death. SRC influence on ILTV infection also confirmed in embryos by survival analysis of embryos and virus replication in allantoic fluids and CAMs of embryos treated with specific small molecule inhibitors of SRC. To determine the mechanism by which SRC affects the biological outcome of ILTV infection in LMH cells, we re-analyzed the transcriptome data and build the protein-protein interaction network of the significant genes identified, according to the analysis we suppose that focal adhesion kinase is required for SRC to modulate ILTV infection in LMH cells. We confirmed the inference in cells and embryos by functional studies of small molecule inhibitors and RNAi, further study identify a positive feedback loop between SRC and tyrosine kinase focal adhesion kinase(FAK). Function study shows that this process is required for SRC to modulate ILTV infection.Our study have advanced our understanding of host-ILTV interactions on a molecular level, expanded the present perception of the molecular mechanisms of ILTV interactions with host, provided certain theoretical support for establishing a more safe and long-lasting AILT prevention method.