Characterization of the host response to pichinde virus infection in the Syrian golden hamster by species-specific kinome analysis

Abstract

The Syrian golden hamster has been increasingly used to study viral hemorrhagic fever (VHF) pathogenesis and countermeasure efficacy. As VHFs are a global health concern, well-characterized animal models are essential for both the development of therapeutics and vaccines as well as for increasing our understanding of the molecular events that underlie viral pathogenesis. However, the paucity of reagents or platforms that are available for studying hamsters at a molecular level limits the ability to extract biological information from this important animal model. As such, there is a need to develop platforms/technologies for characterizing host responses of hamsters at a molecular level. To this end, we developed hamster-specific kinome peptide arrays to characterize the molecular host response of the Syrian golden hamster. After validating the functionality of the arrays using immune agonists of defined signaling mechanisms (lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α), we characterized the host response in a hamster model of VHF based on Pichinde virus (PICV(1)) infection by performing temporal kinome analysis of lung tissue. Our analysis revealed key roles for vascular endothelial growth factor (VEGF), interleukin (IL) responses, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, and Toll-like receptor (TLR) signaling in the response to PICV infection. These findings were validated through phosphorylation-specific Western blot analysis. Overall, we have demonstrated that hamster-specific kinome arrays are a robust tool for characterizing the species-specific molecular host response in a VHF model. Further, our results provide key insights into the hamster host response to PICV infection and will inform future studies with high-consequence VHF pathogens.

Fig. 1.

Characterization of cellular responses to LPS or TNF-α treatment validates the functionality of the hamster-specific kinome arrays. IPA generated functional network for the BHK-21 cellular response to known immune agonists LPS and murine TNF-α. A Top functional network for LPS-treated cells. Biological responses associated with this network include: cell death and survival, cell-mediated immune response, cellular development. B Top functional network for murine TNF-α treated cells. Biological responses associated with this network include: infectious disease, cellular assembly and organization, cell signaling.

Fig. 2.

Study design for PICV infection of Syrian golden hamsters and lung viral titers. A At D0, three uninfected hamsters were euthanized to serve as baseline controls. The remaining hamsters were challenged with 300 PFU of PICV and euthanized in groups of three at D1, D3, D5, and D7. B Lung tissue was processed and titrated for PICV as described in Materials and Methods. Samples were titrated three times on three different days. Results are plotted as the average ± one standard deviation.

Fig. 3.

Hierarchal clustering and principal component analysis (PCA) of temporal kinome data from PICV-infected hamster lung tissue. A Hierarchal clustering analysis of kinome profile of individual PICV-infected hamster lungs. B Hierarchal clustering analysis of PICV-infected hamster groups based on day of euthanasia. The letter and number combination designate the day of euthanasia. The distance metric used was (1 − Pearson correlation), while McQuitty linkage was used as the linkage method. Rows correspond to probes (phosphorylation targets), and columns correspond to samples. Colors indicate the averaged (over three intra-array replicates) phosphorylation intensity of each target, with red indicating greater amounts of phosphorylation and green indicating lesser amounts of phosphorylation. The intensity of the color corresponds to the measured level of phosphorylation. C Three-dimensional principal component analysis of PICV-infected hamster data sets across the time course of the study.

Fig. 4.

Representative core nodes of functional networks from temporal kinome data of PICV-infected hamster lung tissue. Kinome data sets were uploaded to IPA for functional network analysis. These nodes are derived from the full functional networks identified for each data comparison. A D1 versus D0; B D3 versus D0; C D5 versus D0; D D7 versus D0.

Fig. 5.

Temporal Western blot analysis of claudin-1, VE-cadherin, ICAM-1, and VCAM-1 expression. Lung samples were processed and Western blot analysis was performed as described in Materials and Methods. Spot intensities were quantified and normalized to the loading control. Data were averaged for each group (D0, D1, D3, D5, and D7) and the plotted pixel intensities represent the average intensity of the expressed marker for each animal from the respective group (n = 3 for each group). Standard deviations were calculated using the Student's t test.