Using single cell spatial and transcriptional analysis to understand early host response to RNA virus infections

Abstract

Abstract The RIG-I like receptor pathway, which signals through the mitochondrial anti-viral signaling (MAVS) protein, initiates the type I interferon (IFN) response to RNA viruses such as vesicular stomatitis virus (VSV) and Ebola Virus (EBOV). To investigate the mechanism of MAVS-mediated control of VSV and EBOV infections in vivo, we utilized single cell RNA sequencing and multiplex immunohistochemistry (mIHC) in C57Bl/6J and MAVS−/− animals to understand the cellular response spatially and transcriptomically in the draining lymph nodes (dLN). Identification of virally infected cells through scRNA-Seq allowed contrast of infected cells and bystander cells. Viral quantification of EBOV and VSV samples showed an increased viral load in MAVS−/− conditions and distinct patterns of dissemination through the LN. The relationship between viral load and IFN signaling revealed a negative correlation in EBOV infected cells with no relationship in VSV infected cells highlighting the importance of viral inhibition of the IFN response in EBOV infections. To assess these patterns spatially, mIHC antibody panels have been implemented on control and experimental tissue. An in-house algorithm for cell segmentation called RAPID was applied to a controlled experiment and revealed extensive batch effects from the staining process. Current work to perform batch normalization is being carried out to better assess changes in protein marker expression. Combining information gained from the sequencing analysis with the observed spatial organization through mIHC, we aim to develop a model illustrating the role of the MAVS-dependent pathway in controlling early virus replication. Supported by Intramural research program of the NIH