Pathophysiologic and Transcriptomic Analyses of Viscerotropic Yellow Fever in a Rhesus Macaque Model

<div><p>Infection with yellow fever virus (YFV), an explosively replicating flavivirus, results in viral hemorrhagic disease characterized by cardiovascular shock and multi-organ failure. Unvaccinated populations experience 20 to 50% fatality. Few studies have examined the pathophysiological changes that occur in humans during YFV infection due to the sporadic nature and remote locations of outbreaks. Rhesus macaques are highly susceptible to YFV infection, providing a robust animal model to investigate host-pathogen interactions. In this study, we characterized disease progression as well as alterations in immune system homeostasis, cytokine production and gene expression in rhesus macaques infected with the virulent YFV strain DakH1279 (YFV-DakH1279). Following infection, YFV-DakH1279 replicated to high titers resulting in viscerotropic disease with ∼72% mortality. Data presented in this manuscript demonstrate for the first time that lethal YFV infection results in profound lymphopenia that precedes the hallmark changes in liver enzymes and that although tissue damage was noted in liver, kidneys, and lymphoid tissues, viral antigen was only detected in the liver. These observations suggest that additional tissue damage could be due to indirect effects of viral replication. Indeed, circulating levels of several cytokines peaked shortly before euthanasia. Our study also includes the first description of YFV-DakH1279-induced changes in gene expression within peripheral blood mononuclear cells 3 days post-infection prior to any clinical signs. These data show that infection with wild type YFV-DakH1279 or live-attenuated vaccine strain YFV-17D, resulted in 765 and 46 differentially expressed genes (DEGs), respectively. DEGs detected after YFV-17D infection were mostly associated with innate immunity, whereas YFV-DakH1279 infection resulted in dysregulation of genes associated with the development of immune response, ion metabolism, and apoptosis. Therefore, WT-YFV infection is associated with significant changes in gene expression that are detectable before the onset of clinical symptoms and may influence disease progression and outcome of infection.</p></div

Circulating levels of inflammatory cytokines in YFV-DakH1279-infected rhesus macaques.

<p>Serum levels of IL-6 (A), IL-15 (B), MCP-1 (C), and IFNγ (D) increase sharply in animals requiring euthanasia following infection with YFV and significantly correlate with viral loads. Filled circles denote animals that required euthanasia and open circles denote animals that survived YFV infection.</p

YFV-DakH1279 infection results in severe lymphopenia.

<p>(A) Fold change in white blood cells, (B) lymphocytes and (C) neutrophils were measured at the indicated time points and calculated as the ratio of cell counts/mL for each day relative to the 0 dpi count. (D) A negative correlation between viral load and lymphocyte count (p<0.0001, R² = 0.46) (D), but no correlation was noted between numbers of circulating neutrophils and viral loads (E). Filled circles denote animals that required euthanasia and open circles denote animals that survived YFV infection.</p

Functional characterization of PBMCs transcriptomic response to YFV-17D infection at 3 days post-infection.

<p>The 46 DEGs after YFV-17D infection were grouped into functional categories based on enriched gene ontology (GO) terms (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003295#pntd.0003295.s003" target="_blank">Table S1</a>) and their expression value in log₂FC is depicted in a green to red gradient color scheme. Three animals were challenged with YFV-17D (orange), while another three animals were infected with YFV-DakH1279 (cyan). Color on the left of each heatmap indicates whether the gene was found differentially expressed after YFV-17D infection (orange), or both YFV-17D and YFV-DakH1279 (grey). Genes were functionally categorized into: (A) immune response category (these genes were either involved in cytokine signaling pathways (Cy), or specifically up-regulated in innate immune cells (In) or adaptive immune cells (Ad)); (B) epigenetics; 9C) Ubiquitination-ISGylation; (D) cytoskeleton-adhesion; or (E) did not map to a specific functional category.</p

YFV-DakH1279 infection results in severe liver damage in rhesus macaques.

<p>(A–B) Images of liver in a representative uninfected (A) and YFV-DakH1279 infected animal that required humane euthanasia (B). The infected liver is discolored with signs of hemorrhagic foci. (C–D, 400×) H&E staining of liver sections from an uninfected (C) and YFV-DakH1279-infected (D) animal. Extensive hepatocytes necrosis (1) along with eosinophilic degeneration of liver cells (Councilman bodies, 2), and fatty changes (3) are noted by the black arrows in panel D. (E–H) Histological analysis of YFV antigen in an animal that survived (E 100× & G 400×) or required humane euthanasia following YFV-DakH1279 infection (F 100× &H 400×).</p

Summary of histological analysis of liver, lymphoid tissue, and kidney sections.

<p>Liver + = mid-zonal necrosis severity; H = hemorrhage; L = lymphatic infiltration; V = vacuolization; R = regeneration; Spleen & Lymph Node + = apoptosis severity; M = mitosis; Kidney + = granular and proteinaceous cast severity.</p><p>Summary of histological analysis of liver, lymphoid tissue, and kidney sections.</p

Analysis of cell cycle markers suggests an age associated delay in CD4+ proliferation.

<p>We used multi-parameter flow cytometry to evaluate the proliferation kinetics of CD4+ cells in each of our cohorts. The dark lines illustrate the two old animals whereas the lighter lines illustrate the two adult animals. The cell cycling is normalized to the baseline cycling recorded during this challenge for each animal, setting this to 0%. This illustration reveals that post WNV infection CD4+ cell cycling decreased followed by a marked increase on days 7, 10 and 14 which then wanes. Of particular interest is the age associated shift in kinetics both older animals (dashed lines) had a slower increase in CD4+ cycling than the adult animals (solid lines) and a prolonged phase prior to returning to a steady state post infection.</p

Immunodeficient animals – CD8 depleted animals survive WNV infection.

<p>CD8+ Tcells from previously thymectomized and CD8 depleted animals were enumerated from total PBMC using multi-parameter flow cytometry prior to WNV infection. Twelve months prior to infection animals were thymectomized (N = 4), thymectomized and CD8 depleted (N = 4), only CD8 depleted (N = 3) or controls (N = 4) (dark filled circles) Light circles illustrate thymectomized animals, dark circles illustrate control (sham operated) animals and light filled circles represent CD8 depletion.</p