| Influenza A virus is still a major cause of morbidity and mortality worldwide. Today the antivirals mostly are designed according to different segment of influenza virus itself, and the resistanse strains apear quickly because of the arise of new influenza strains through antigenic ’drift’ or antigenic’shift’.So it is necessary to find more targets for developing antivirals. The host genes essential for the virus replication have attracted increasing interest, because a missing cellular function is more difficult for the virus to adapt to, and should affect replication independent of the type, strain and antigenic properties of the invading influenza virus. Genome-scale analyses of virus infections will help us to understand the complexities of virus-host interactions and may lead to the discovery of novel drug targets or antiviral therapies.In this study, A549 cells were mock infected or infected with influenza virus A/PR/8/34 at an moi of 1 TCID50 per cell. At 4 hours,12 hours,24 hours,48 hours post infection, the cells were collected, and total RNA was extracted by using TRIzol. Microarray experiments were performed on cells infected at 4 h,12 h,24 h and 48 h under the four pairwise conditions described above. Real-time PCR was performed to verify the microarray data. Then KEGG pathway and Gene Ontology analysis were performed to understand the gene function of these upregulated or downregulated genes in the microarray data. From the results we can see that virus replication resulted in the differential expression of 2,45,294, and 261 genes at the 4 h,12 h,24 h and 48 h time points, respectively. Interestingly, virus replication resulted host gene downregulation at 24-and 48-h post infection. Given that influenza virus generally stimulates IFN response and the upregulated genes mostly belong to the IFN-related genes which suggests that some may play essential or accessory roles in the host cell’s stress response, downregulated genes are attractive candidates for future functional studies as some of the downregulated genes play important roles in the lipid or saccharide synthesis.In order to screen the genes that can regulate the influenza virus replication from the downregulated genes, we developed a high-thought method to measure the influenza virus replication. In the present study, we approached In-Cell Western assay to measure influenza virus replication at different MOI infection and compared it with the TCID50 method. The results suggest that In-Cell Western can detect influenza virus replication at an moi of 0.01 TCID50/cell infection. At the same time, the replication measured by the In-Cell Western method is consistent with the result measured by TCID50. Therefore, In-Cell Western assay is a promising method for high through-put screen and we will apply it to screen the genes involved in influenza virus replication. To determine if the downregulated genes can regulate the replication of influenza,34 expression plasmids of downregulated genes were overexpressed and their effects on the influenza virus replication were measured by the In-Cell Western. The results showed that 14 out of 34 may regulate influenza virus replication. Then we further tested 14 genes mentioned above and confirmed 8 out of 14 genes can inhibit influenza virus replication. To further investigae the potential roles of APOH and SULF2 in influenza virus replication, we applied real-time PCR method to verify the inhibition effect and confirmed it.Taken together, we found 8 genes that can inhibit influenza virus replication and identified APOH and SULF2 as endogenous inhibitors of influenza viruses in vitro and their function in vivo need further research. |
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