The early involvement of CNS-draining lymphatics and host response to West Nile virus infection Open Access

O'Neal, Troy (Fall 2019)

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West Nile virus (WNV) is a mosquito-borne flavivirus of global importance, which can result in neuroinvasive infection leading to encephalitis, prolonged neurological dysfunction or death. The early induction of the host response and efficient CD8+ T cell responses are both required for protection. We found that viral RNA and activated conventional dendritic cells (cDCs) accumulated in the meninges and CNS-draining lymph nodes (LNs) prior to neuroinvasion. Furthermore, WNV-specific CD8+ T cells accumulated in the CNS-draining LNs and the spleen with similar kinetics. WNV-specific CD8+ T cells from the spleen and brain exhibited dramatically different activation and inhibitory marker expression and cytokine production, whereas WNV-specific CD8+ T cells from the meninges and CNS-draining LNs displayed unique intermediate phenotypes. Notably, meningeal and brain WNV-specific CD8+ T cells produced both tumor necrosis factor (TNF)-α and interferon (IFN)-γ and had the highest fraction of cells expressing PD-1. CNS-localized WNV-specific CD8+ T cells were more efficient at controlling viral infection of cortical neurons than peripheral WNV-specific CD8+ T cells. These findings characterize the early involvement of CNS-draining lymphatics during WNV pathogenesis and indicate that anatomic localization influences CD8+ T cell programming during WNV infection. The type I IFN (IFN-I) response promotes induction of innate antiviral defenses, restricting early viral replication. We developed WNV- inclusive single-cell RNA sequencing (scRNA-seq), an approach to examine transcriptional heterogeneity in IFN-stimulated gene (ISG) induction and viral RNA abundance across single cells. We observed that only a small fraction of cells within the bulk population produced high levels of IFN-β transcript. Genes associated with the IFN- I response exhibited both high unimodal and bimodal variation. The majority of ISGs negatively correlated with viral RNA abundance, often displaying a sharp decline in expression for cells with high levels of viral RNA. WNV-inclusive scRNA-seq represents a robust approach for parallel single-cell transcriptomics and WNV RNA detection, which can be implemented in other systems to identify novel therapeutic targets or extend the resolution of in vivo studies to single infected cells.

Table of Contents

Introduction (1-16)

Flaviviruses (1)

WNV ecology and pathogenesis (2-5)

The host response to WNV infection (5-7)

Integrating single-cell approaches (7-9)

Dendritic cells and WNV infection (9-10)

CD8+ T cell-mediated immunity during WNV infection (10-13) Meningeal immunity and neuroinflammation (13-16)

1 West Nile virus-inclusive single-cell RNA sequencing reveals heterogeneity in the type I interferon response within single cells (17-51)

1.1 Introduction (20-23)

1.2 Results (24-30)

1.3 Discussion (31-33)

1.4 Materials and methods (34-39)

1.5 Figure 1: Population-level analysis of WNV infection in L929 cells (40, 44) 1.6 Figure 2: WNV-inclusive single-cell RNA sequencing (40-41, 45)

1.7 Figure 3: Cellular heterogeneity in IFN-stimulated gene induction following WNV infection (42, 46)

1.8 Figure 4: Unimodal and bimodal variation in antiviral effector gene expression at single-cell following WNV infection (42, 47)

1.9 Figure 5: ISGs negatively correlate with WNV RNA abundance (42-43, 48)

1.10 Figure 6: Sharp downward trends for ISGs negatively correlated with viral RNA (43, 49)

2 The early involvement of CNS-draining lymphatics and CD8+ T cell control during West Nile virus infection (52-93)

2.1 Introduction (54-57)

2.2 Results (58-68)

2.3 Discussion (69-73)

2.4 Materials and methods (74-78)

2.5 Figure 1: Early viral dissemination and ISG expression in peripheral lymphoid tissues (79, 84)

2.6 Figure 2: Early differential activation of DC subsets in peripheral lymphoid tissues (79, 85)

2.7 Figure 3: Early viral RNA detection and myeloid cell accumulation in CNS-draining lymph nodes and meninges (80, 86)

2.8 Figure 4: Early cDC activation in CNS-draining lymph nodes and meninges (80, 87)

2.9 Figure 5: Accumulation of WNV-specific CD8+ T cells in the meninges, brain, and peripheral and CNS-draining lymphoid tissues (80-81, 88)

2.10 Figure 6: CD8+ T cells in the CNS have altered surface marker expression compared to those in the periphery (81, 89)

2.11 Figure 7: W4B Cells from the CNS have polyfunctional cytokine secretion (82, 90)

2.12 Figure 8: W4B cells isolated from the brain are transcriptionally distinct from splenic T cells (82-83, 91)

2.13 Figure 9: Brain W4B cells more effectively control WNV replication than Splenic W4B cells (83, 92)

Discussion (94-102)

Probing WNV infection at single-cell resolution (94-96)

Exploring the role of meningeal immunity during WNV infection (96-100)

Figure 1: Microenvironmental cues within the CNS re-program WNV-specific CD8+ T cells resulting in transcriptional, phenotypic, and functional differences (101-102)

Bibliography (103-145)

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