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.

The global transcriptomic response to H7N9 is specific and is intermediate between the cell responses to avian and human IAVs. To assess the whole transcriptomic cellular response to IAV infection, we used several metrics (Fig. 2) . First, multidimensional scaling (MDS) was used to visualize Euclidian distances between each transcriptomic profile in two dimensions ( Fig. 2A) . In this plot, each sample is represented by a dot; samples with similar transcriptomic profiles (i.e., a small Euclidian distance) are close together, while increasing distance is relative to increasing transcriptomic dissimilarity. We observed that samples clustered by time point and viral treatment, indicating a good agreement between biological replicates. At 3 and 7 hpi, IAV-infected samples were very similar to mock-infected ones (mocks), suggesting few transcriptomic changes early after infection, while the distance from mocks increased at 12 and 24 hpi. At 24 hpi, each viral condition was in a distinct location, indicating a specific response to each IAV. However, while avian H5N1 and H7N7 samples were relatively close together, H7N9 samples were located relatively far from each of the other IAVs, indicating a specific host response to this virus. To determine whether the host response to H7N9 infection was closer to the avian or human IAV response, we measured the transcriptomic distance between Anhui01-infected and other IAV-infected samples at each time point (Fig. 2B) . At 12 hpi, H7N9 was significantly closer to both H7N7 and H5N1 than to H3N2. Surprisingly, at 24 hpi, the shortest distance was between H7N9 and H3N2, while the largest distance was between H7N9 and H7N7, indicating that responses to H7N7 and H7N9 were the FIG 1 H7N9 replicates to a level similar to that of other influenza A viruses (IAVs) in polarized human bronchial epithelial Calu-3 cells. Polarized Calu-3 cells were infected apically with Anhui01 (H7N9), NL219 (H7N7), Pan99 (H3N2), or VN1203 (H5N1) at an MOI of 1 for an hour. After washing, medium was added and supernatants were collected at 3, 7, 12, and 24 h for determination of viral titers by standard plaque assay. Cells were harvested at the same time postinfection for transcriptomic profiling. Values represent means of titers in PFU/ml from quadruplicate wells Ï® SD. most distinct. Of note, the response to H7N9 infection was as different from that of mock samples as that to avian IAV at 24 hpi, suggesting very specific response to H7N9 infection. Finally, to determine transcriptomic distances between all IAV-and mockinfected samples at each time point, we used hierarchical clustering (Fig. 2C ). This analysis confirmed that H3N2 induced the largest host response early after infection and that at 24 hpi, responses to H7N7 and H5N1 were the most similar, while the response to H7N9 was closer to that to H3N2.