scow/10/99 and avian A/Turkey/582/2006, A/Finch/England/2051/94, and A/Chicken/ Italy/2076/99 influenza viral strains. These CAY10505 PI3K inhibitor viruses are herein referred to as H1N1, H3N2, H5N1, CAY10505 PI3K inhibitor H5N2 and H7N1. A549 cells express both sialic acid a2,6 and a2,3 galactose receptors and were shown to be infected by human, avian and swine influenza viruses. Infections were performed at 37uC, a temperature at which both human and avian influenza viruses efficiently infect cell cultures and at a moi of 0.1. In these conditions, there was evidence of productive viral replication of all viruses but with some kinetic and yield differences between viruses, as determined by infectious titers of supernatants of influenza virus infected A549 cells.
The H5N1 virus titers CYC116 peaked higher and earlier compared to other viruses titers.
Avian H7N1 and H5N2 viruses replicated with correct efficiencies, similar to the human H3N2 virus. In contrast, the human H1N1 virus strain replicated slower and grew to lower titers than other viruses. To determine the host gene response CYC116 to infection, total cellular RNA was extracted at 24 hpi and submitted to reverse transcription in the presence of 33P. Each condition was performed in 5 independent replicates. All labeled cDNAs provided a good radioactive intensity and were hybridized onto home made nylon microarrays containing 8782 IMAGE cDNA clones.
All hybridizations were of good quality according to signals within acceptable range, number of features present, and signals from control spots. Supervised analysis of normalized gene expression data was conducted using the SAM algorithm.
This algorithm was used to identify genes whose expression levels were significantly altered by influenza infection. We set the delta threshold in the SAM analysis to allow an acceptable false discovery rate of 10%. We found that the expression levels for a total of 300 genes differed significantly between mock and infected samples. Using the DAVID Bioinformatics Resources database, we annotated this signature using the gene ontology terms.
This revealed an enrichment of genes related to various cellular processes such as protein complex biogenesis, membrane and microtubule organization, DNA metabolic and catabolic processes, cell proliferation regulation, cell cycle and cell death.
A subset of six genes with absolute fold changes in log2 above 2 was selected to validate the microarray analysis by quantitative RT PCR analysis: DNMT1, NTE and CAPN1 that were found downregulated in infected cells and G1P2, OAS1 and ICAM1 that were upregulated. The 6 genes were chosen at random among the most 20 dysregulated genes upon infection. This quantification was performed on new samples equivalent to those used for the microarray analysis. Figure 3 shows the confirmation by RT qPCR of the microarray data. For each gene and each strain, microarray FCs are presented as a black boxplot and RT qPCR results are depicted as a gray histogram. Results from RT qPCR were in good agreement with the cDNA microarray analyses for five out of six genes tested. Indeed, except for CAPN1, significant difference between infected and non infected cells was also observed in quantitative RT PCR analysis, similar to DNA microarray analysis. This result was acceptable considering that samples anal