s are inappropriate for use in cases of severe infection and may be BMS-540215 BMS-540215 VEGFR inhibitor VEGFR inhibitor limited due to the risk of rapid emergence of drug resistant viruses. Thus there is an obvious need to complement existing therapies with new antiinfluenza drugs. To search for new antivirals, we hypothesized that common viral effects on cell metabolism should occur after infection with different avian and human influenza viruses and that this pattern should lead to the identification of drugs effective on all influenza A viruses potentially. We first sought to identify a common gene expression signature following the infection with different human and avian influenza A viruses.
While several microarray analyses have already compared the pandemic 1918 H1N1 Cryptotanshinone virus or some H5N1 strain to other less pathogenic strains, our study is the first to demonstrate that a global influenza induced gene expression signature can be defined.
This proof of concept study was conducted on a home made nylon Cryptotanshinone array using a human pulmonary epithelial cell line infected by five influenza A virus subtypes. Using this signature, we determined if molecules disturbing this pattern of infection would have a broad influenza antiviral effect. By consulting the Connectivity Map, a database of drug associated gene expression profiles, we identified molecules that induced gene expression changes after cell treatment that were mainly opposite to those induced by infection.
These molecules were tested in vitro for their effect on the five different viruses.
To confirm our methodology, we took the opportunity of using the new emerging pandemic H1N1 virus as a model to test the effect of these molecules on a new unknown virus. Materials and Methods 1 Cell lines and viruses Cells of the human lung epithelial cell line A549 were grown as monolayers in Dulbecco,s modified Eagle,s medium supplemented with 10% fetal bovine serum, 2 mM L glutamine, 100 U of penicillin/mL, and 100 mg of streptomycin sulfate/mL at 37uC. Influenza viruses A/New Caledonia/20/99, A/Moscow/ 10/99, A/Lyon/969/09, A/Turkey/ 582/2006, A/Finch/England/2051/94, and A/ Chicken/Italy/2076/99 were produced in MDCK cells in EMEM supplemented with 2 mM L glutamine, 100U of penicillin/ mL, 100 mg of streptomycin sulfate/mL and 1 mg of trypsin/ mL.
Viruses were titrated to determine tissue culture infection dose 50% in MDCK cells as described in our previous study.
For the microarray analysis, A549 cells were infected for 24 h at 37uC with influenza viruses at a multiplicity of infection of 1 in DMEM supplemented with 2 mM L glutamine, 100 U of penicillin/ mL, 100 mg of streptomycin sulfate/mL and 0.5 mg of trypsin/ mL. This moi was chosen to ensure that 100%of the cells were infected 24 h postinfection. The microarray experiments were performed in five independent replicates. For kinetics on A549 cells, confluent cells were infected with influenza viruses at a moi of 0.1 or 2 for one hour under a minimal volume of infection medium at 37uC. The cells were then overlaid with fresh infection medium and incubated at 37uC. Samples of supernatants were collected at defined time points and stored at 280uC until end point titration assays in MDCK cells. 2 RNA preparation and hybridization to the gene chip Total RNA was extracted from cell pellets using an RNe