Transcriptomic Characterization of the Novel Avian-Origin Influenza A (H7N9) Virus: Specific Host Response and Responses Intermediate between Avian (H5N1 and H7N7) and Human (H3N2) Viruses and Implications for Treatment Options

Josset, Laurence; Zeng, Hui; Kelly, Sara M.; Tumpey, Terrence M.; Katze, Michael G.

In vitro evaluation of minocycline antiviral activity. To validate our drug prediction, we evaluated the ability of minocycline, a tetracycline, to inhibit H7N9 viral replication in vitro. This molecule was chosen because it was predicted to reverse H7N9 signatures by both computational methods but had not been tested previously against influenza virus. Using a colorimetric viability assay, the 50% cytotoxicity concentration of minocycline on Calu-3 cells was evaluated to be around 1,000 M, and we observed no significant cytotoxicity up to 266 M (see Fig. S6 in the supplemental material). To evaluate potential antiviral effect of minocycline, polarized Calu-3 cells were pretreated with increasing concentrations of minocycline for 3 h and infected with An-hui01 at an MOI of 0.01 (Fig. 5) . At 24 hpi, there was a significant decrease of 1.3 log 10 (17%) of Anhui01 viral titers in cells treated with 200 M minocycline (P Ͻ 0.01). Lower concentrations of minocycline did not inhibit Anhui01 viral replication. Ribavirin, an antiviral drug with in vitro activity against both DNA and RNA viruses, was used as a positive control and induced a more robust decrease of 5.3 log 10 (71%) of the log 10 viral titer (Fig. 5) . Overall, these data show that minocycline exerts a modest but significant within the IPA database. A negative z score indicates that the regulator is known to downregulate the same genes that were significantly upregulated after infection and/or to upregulate genes that were downregulated after infection. Dashed lines depict the limit of significance (|z score| Ͼ 2). For each virus, z scores were calculated using log 2 FC expression of DE genes at each time point, and molecules with significant negative z scores for at least 2 time points were selected for this representation (29 drugs). Potential antivirals were defined as drugs with a z score Ͻ Ϫ2 for at least 2 time points and no positive z score (molecules highlighted in color). Twenty-six regulators were predicted to have an effect opposite to that of infection for at least one IAV. Molecules were ranked from most-to least-negative mean z score across all conditions. (B) The Connectivity Map (Cmap) was used to confirm potential anti-IAV effects of regulators predicted in IPA. Gene expression profiles for 10 molecules (out of the 26) were found in the Cmap database and compared with IAV-infected profiles. Cmap scores go from Ϫ1 to 1, with positive scores for drugs inducing changes similar to the viral signature and negative scores for opposite changes. Relationships between viral signatures and drugs were depicted in a network with a circular layout. Edges between virus and drugs are colored based on the Cmap scores comparing drug and viral profiles at each time point. Drugs are colored based on the mean of the Cmap scores for all time points. Drugs colored in orange induced gene expression changes that are the reverse of those for IAVs after cell treatment. Note that the Cmap query requires a list of up-and downregulated genes and was therefore not performed for VN1203 at 7 and 12 hpi, for which there were too few downregulated genes. antiviral effect against H7N9, validating our in silico drug screening.