Early blood profiles of virus infection in a monkey model for Lassa fever

Abstract

Acute arenavirus disease in primates, like Lassa hemorrhagic fever in humans, begins with flu-like symptoms and leads to death approximately 2 weeks after infection. Our goal was to identify molecular changes in blood that are related to disease progression. Rhesus macaques (Macaca mulatta) infected intravenously with a lethal dose of lymphocytic choriomeningitis virus (LCMV) provide a model for Lassa virus infection of humans. Blood samples taken before and during the course of infection were used to monitor gene expression changes that paralleled disease onset. Changes in blood showed major disruptions in eicosanoid, immune response, and hormone response pathways. Approximately 12% of host genes alter their expression after LCMV infection, and a subset of these genes can discriminate between virulent and non-virulent LCMV infection. Major transcription changes have been given preliminary confirmation by quantitative PCR and protein studies and will be valuable candidates for future validation as biomarkers for arenavirus disease.

FIG. 1.

Macaque PBMC genes that are differentially modulated in LCMV-infected cells and control uninfected cells. Virulent (LCMV-WE) and nonvirulent (LCMV-Arm) infections are compared in terms of the time courses of up- and downregulated genes. Values on the bar represent the ratios between up- and downregulated genes. There are more differentially expressed genes that are down- rather than upregulated. For these data we used rhesus macaques inoculated with a lethal dose (1 × 10³ PFU) of LCMV-WE (day 1 [n = 2], day 2 [n = 1], day 3 [n = 1], day 4 [n = 1], day 6 [n = 2], day 7 [n = 1]), three monkeys inoculated with 1 × 10³ PFU LCMV-Arm virus ([day 3 [n = 1], day 5 [n = 1], day 7 [n = 1]), and five uninfected control monkeys. “Early” means in the previremic stage of infection. The microarray data and the related experimental information from this study can be accessed at http://www.ncbi.nlm.nih.gov/geo/, platform number GSE5790.

FIG. 2.

Gene expression profiles distinguish between infection with hemorrhagic fever virus and infection with a benign virus. Blood was obtained from rhesus macaques on the days indicated, and the most prominent differentially expressed genes between the two viruses are represented here. Animal numbers are as described for Fig. 1, so for days 1 and 6 the geometric mean of data from two animals is presented. In addition to the 31 genes depicted here, there are approximately 450 genes that distinguish these two infections with a P value confidence level of <0.05 (see Tables SI to SV in the supplemental material). Differential expression, i.e., the level of a gene's expression after infection normalized to its expression level before infection, is shown. The green color represents downregulated gene expression, and the red color represents upregulated gene expression. White values are below the level of ±twofold deviation from baseline, and gray values are below white values in detection quality. Days postinfection (d1, etc.) are indicated.

FIG. 3.

Functional categories of transcripts (see Tables SI to SV in the supplemental material). Differentially expressed genes with confidence level P values of <0.05 were grouped into functional categories including cell adhesion (A), cell proliferation (B), inflammatory (C), IFN response (D), proteolysis (E), signal transduction (F), and transcription (G). On the heat map, the green color represents downregulated gene expression and the red color represents upregulated gene expression (the scale at the bottom of panel A indicates relative expression change). Days postinfection (d1, etc.) are indicated.

FIG. 3.

Functional categories of transcripts (see Tables SI to SV in the supplemental material). Differentially expressed genes with confidence level P values of <0.05 were grouped into functional categories including cell adhesion (A), cell proliferation (B), inflammatory (C), IFN response (D), proteolysis (E), signal transduction (F), and transcription (G). On the heat map, the green color represents downregulated gene expression and the red color represents upregulated gene expression (the scale at the bottom of panel A indicates relative expression change). Days postinfection (d1, etc.) are indicated.

FIG. 4.

Plasma protein level was affected by LCMV infection. Cytokine and chemokine concentrations in plasma samples of uninfected control and LCMV-infected monkeys were determined. Cytochemokines (A and B), IL-1β (C), IL-8 (D), and angiogenesis and growth factors (E) were measured using SearchLight protein arrays. Results are expressed as means ± standard deviations (error bars) from at least three monkeys and three replicates per time point. Based on the Student t test, we observed statistically significant differences (P < 0.05) between LCMV-infected plasma samples and control preinfection plasma. Vertical bars superimposed upon symbols signify average plasma protein levels of preinfection control samples (n = 21). Pre and post, preinfection and postinfection, respectively.

FIG. 4.

Plasma protein level was affected by LCMV infection. Cytokine and chemokine concentrations in plasma samples of uninfected control and LCMV-infected monkeys were determined. Cytochemokines (A and B), IL-1β (C), IL-8 (D), and angiogenesis and growth factors (E) were measured using SearchLight protein arrays. Results are expressed as means ± standard deviations (error bars) from at least three monkeys and three replicates per time point. Based on the Student t test, we observed statistically significant differences (P < 0.05) between LCMV-infected plasma samples and control preinfection plasma. Vertical bars superimposed upon symbols signify average plasma protein levels of preinfection control samples (n = 21). Pre and post, preinfection and postinfection, respectively.

FIG. 5.

Pathway analysis reveals interactions between the major gene products affected by virulent infection. EGFR and IL-1R are included in the diagram to show that our results corroborate those from Bowick et al. (10), in which guinea pigs infected with virulent Pichinde virus P18 have much lower levels of phosphorylated EGFR and IL-1R than guinea pigs infected with benign Pichinde virus P2. Arrows pointing from one gene product to another signify activation. Small gray arrows signify downregulation, with the open arrows derived from kinomic results from the work of Bowick et al. (10). EGR1 and EGR2 are transcription factors, NR4A is a retinoic acid receptor, avian sarcoma virus oncogene homolog JUN is a transcription factor, murine sarcoma virus oncogene homolog FOS is a transcription factor, PTGS2 encodes COX-2, and IL-1R is an IL receptor protein. A connection between PTGS2 and VEGF was published previously (2) but not described in the Ingenuity Pathway Analysis, so it is indicated by a stippled arrow.