Eastern and Venezuelan equine encephalitis viruses differ in their ability to infect dendritic cells and macrophages: impact of altered cell tropism on pathogenesis

Abstract

Eastern and Venezuelan equine encephalitis viruses (EEEV and VEEV, respectively) cause severe morbidity and mortality in equines and humans. Like other mosquito-borne viruses, VEEV infects dendritic cells (DCs) and macrophages in lymphoid tissues, fueling a serum viremia and facilitating neuroinvasion. In contrast, EEEV replicates poorly in lymphoid tissues, preferentially infecting osteoblasts. Here, we demonstrate that infectivity of EEEV for myeloid lineage cells including DCs and macrophages was dramatically reduced compared to that of VEEV, whereas both viruses replicated efficiently in mesenchymal lineage cells such as osteoblasts and fibroblasts. We determined that EEEV infection of myeloid lineage cells was restricted after attachment, entry, and uncoating of the genome. Using replicon particles and translation reporter RNAs, we found that translation of incoming EEEV genomes was almost completely inhibited in myeloid, but not mesenchymal, lineage cells. Alpha/beta interferon (IFN-alpha/beta) responses did not mediate the restriction, as infectivity was not restored in the absence of double-stranded RNA-dependent protein kinase, RNase L, or IFN-alpha/beta receptor-mediated signaling. We confirmed these observations in vivo, demonstrating that EEEV is compromised in its ability to replicate within lymphoid tissues, whereas VEEV does so efficiently. The altered tropism of EEEV correlated with an almost complete avoidance of serum IFN-alpha/beta induction in vivo, which may allow EEEV to evade the host's innate immune responses and thereby enhance neurovirulence. Taken together, our data indicate that inhibition of genome translation restricts EEEV infectivity for myeloid but not mesenchymal lineage cells in vitro and in vivo. In this regard, the tropisms of EEEV and VEEV differ dramatically, likely contributing to observed differences in disease etiology.

FIG. 1.

Morbidity and mortality differ following infection of outbred mice with VEEV or EEEV. Adult, outbred CD-1 mice were infected with cDNA clone-derived ZPC738 VEEV or FL93-939 EEEV and monitored for morbidity and mortality as follows. (A and B) Percent survival following subcutaneous (A) or intracerebral (B) inoculation of CD-1 mice with 10³ PFU of VEEV (white circles, dashed line) or EEEV (black circles, solid line). (C and D) Clinical score over the disease course of mice infected subcutaneously with 10³ PFU of VEEV (C) or EEEV (D). Clinical scores to assess signs of disease in the mice were as follows: 0, healthy (white); 1, ruffled fur and behavioral changes (hatched); 2, paresis or ataxia (crosshatched); 3, moribund (gray); 5, dead (black). (E and F) Percent weight change over the disease course of mice that were infected subcutaneously (E) or intracerebrally (F) with 10³ PFU of VEEV (white circles) or EEEV (black circles). Data are representative of at least two independent experiments.

FIG. 2.

Replication and dissemination profiles of VEEV and EEEV differ in subcutaneously inoculated mice. CD-1 mice, infected with 10³ PFU of ZPC738 VEEV (white circles) or FL93-939 EEEV (black circles) subcutaneously in each rear footpad, were sacrificed at various times p.i. Titers of virus from DLN (A), serum (B), spleen (C), ankle joint (D), bone aspirate (E), and brain (F) were determined. Values represent the geometric mean virus titers (log₁₀ PFU/ml or g) for three mice, determined on BHK cells, and are shown ± standard deviations. Dotted line denotes inoculum dose (A) or limit of detection (B to F).

FIG. 3.

EEEV replication within DLN cells is reduced compared to that of VEEV. CD-1 mice were inoculated subcutaneously in each rear footpad with EEEV-based (EREP) or VEEV-based (VREP) replicon particles expressing a reporter of infection, GFP or fLUC. (A) Diagrammatic representation of replicon genome and bipartite helper system. (B and C) Mice infected with fLUC-expressing VREP (B) or EREP (C) were inoculated intraperitoneally with luciferin substrate as described in Materials and Methods and subjected to in vivo imaging at 8 h p.i. The intensity of the luciferase signal is depicted by a pseudocolor map. All animals were imaged for 2 s to prevent signal saturation. (D to F) DLNs were dissected from these mice as well as SBREP-inoculated controls, and fLUC activity was quantified by in vitro luciferase assay (F). Datum points are presented as RLU per DLN ± standard deviation where n = 8, and confidence limits are indicated. DLNs from mice similarly inoculated with GFP-expressing VREP (D) or EREP (E) were harvested 8 h p.i., flattened with a coverslip, and photographed using a Nikon inverted fluorescence microscope to reveal GFP-positive cells.

FIG. 4.

EEEV productively infects mesenchymal, but not myeloid, lineage cells. Primary CD-1 bone marrow-derived DCs (A), RAW 264.7 monocytes/macrophages (B), primary CD-1-derived osteoblasts (C), and BHK-21 fibroblasts (D) were infected with VEEV (white circles) or EEEV (black circles) at equal MOIs (0.1). Titers of progeny virus released into the supernatant were determined by plaque assay. Values represent the geometric mean virus titers (log₁₀ PFU/ml). Datum points are shown ± standard deviations where n = 3.

FIG. 5.

The restricted tropism of EEEV for myeloid lineage cells is not mediated by the virus-receptor interaction. (A and B) Cells of the mesenchymal (A) (represented by BHK-21 fibroblasts) or myeloid (B) (represented by RAW 264.7 monocytes/macrophages) lineage were infected at equal multiplicities (MOI = 1) with GFP-expressing parental or chimeric replicon particles and photographed using a fluorescence microscope to reveal GFP-positive cells. Replicons are as follows: VEEV replicon genome with VEEV structural proteins (VREP/Vgp) or EEEV structural proteins (VREP/Egp), SB replicon genome with SB structural proteins (SBREP/SBgp), and EEEV replicon genome with EEEV structural proteins (EREP/Egp) or SB structural proteins (EREP/SBgp). Inset panels for EREP/Egp and EREP/SBgp depict infection at 100-fold-higher MOIs than those of other infections. (C and D) Parallel experiment in which fibroblasts (C) or CD-1-derived cDCs (D) were infected with fLUC-expressing parental and chimeric replicons: VREP/Vgp (white bars), SBREP/SBgp (gray bars), EREP/Egp (black bars), VREP/Egp (horizontally hatched bars), and EREP/SBgp (diagonally hatched bars). Cells were harvested for luciferase activity assay at various times p.i. Values represent the geometric mean fLUC activities (log₁₀ RLU/μg). Datum points are shown ± standard deviations where n = 3. For panel D, a single asterisk indicates that VREP/Egp is not significantly reduced compared with VREP/Vgp (P > 0.5) and a double asterisk indicates that EREP/SBgp is not significantly increased versus EREP/Egp (P > 0.5); EREP/Egp values are significantly reduced compared with those for SBREP/SBgp and VREP/Vgp at all time points (P < 0.01).

FIG. 6.

Translation of an EEEV-based reporter RNA is dramatically reduced in myeloid lineage cells. (A) Diagrammatic representation of in vitro-transcribed reporter RNA expressing fLUC. (B and C) Cells of the mesenchymal (B) (represented by BHK-21 fibroblasts) or myeloid (C) (represented by RAW 264.7 monocytes/macrophages) lineage were electroporated with equal concentrations of RNA reporters based upon the genomes of VEEV (white bars), SB (gray bars), or EEEV (black bars). Cells were harvested for luciferase activity assay at various times p.i. Values represent the geometric mean fLUC activities (log₁₀ RLU/μg). Datum points are shown ± standard deviations where n = 3. In panel C, a single asterisk indicates that translation of SB RNA reporter is significantly reduced compared with that of VEEV reporter (P < 0.01) and a double asterisk indicates that translation of EEEV reporter is significantly reduced compared with that of VEEV and SB reporters (P < 0.01).

FIG. 7.

EEEV genome translation is blocked in myeloid lineage cells following introduction of the genome by infection. (A) Diagrammatic representation of the EREP-nsP3-fLUC replicon genome. (B) EEEV replicon particles containing this genome were packaged in the standard EEEV structural protein coat (Egp) and used to infect BHK-21 fibroblasts (black circles), primary osteoblasts (white circles), or RAW 264.7 macrophages (black squares) at an MOI of 1. Cells were harvested for luciferase activity assay at various times p.i. Values represent the geometric mean fLUC activities (log₁₀ RLU/μg). Datum points are shown ± standard deviations where n = 3.

FIG. 8.

The absence of IFN-α/β pathway components neither restores EEEV infectivity for DLN cells in vivo nor alleviates the restriction on EEEV productive infection and genome translation in myeloid lineage cells in vitro. (A and B) Mice lacking PKR, RNase L, and Mx (TD); mice lacking IFN-α/β receptor-mediated signaling (IFNAR1^−/−); or normal congenic controls (129/Pas) were inoculated subcutaneously in each rear footpad with equal doses of EREP (black bars in panels A and B), VREP (white bars in panel A), or SBREP (hatched bars in panel B) replicon particles expressing fLUC. Data comparing EREP to VREP (A) and EREP to SBREP (B) are presented in separate panels as these were independent experiments. For panels A and B, DLNs were harvested at 8 h p.i. and fLUC activity was quantified by in vitro luciferase assay. Datum points are presented as RLU per DLN ± standard deviation where n = 8. (C) Primary cDCs generated from 129/Pas (black bars) or IFNAR1^−/− (white bars) mice were infected with EEEV, SB, or VEEV at equal MOIs (0.1). Titers of progeny virus released into the supernatant were determined by plaque assay. Values represent the geometric mean virus titers (log₁₀ PFU/ml) at 48 h p.i. ± standard deviation where n = 3 and confidence limits are indicated. (D) Primary cDCs generated from 129/Pas (black bars), TD (hatched bars), or IFNAR1^−/− (white bars) mice were infected with fLUC-expressing EREP or SBREP conventional replicon particles (MOI, 1). Cells were harvested for luciferase activity assay at 24 h p.i. Values represent the geometric mean fLUC activities (log₁₀ RLU/μg) ± standard deviations where n = 3, and confidence limits are indicated. (E) Primary cDCs derived from 129/Pas (black circles), TD (white circles), or IFNAR1^−/− (black squares) mice were infected with EREP-nsP3-fLUC replicon particles (MOI, 1). Cells were harvested for luciferase activity assay at various times p.i. For panels C to E, values represent the geometric mean fLUC activities (log₁₀ RLU/μg) ± standard deviations where n = 3. A single asterisk in panel E indicates that there was no significant elevation of fLUC activity in the absence of PKR/RNase L or the IFN-α/β receptor versus 129/Pas control cells (P > 0.5).

FIG. 9.

EEEV infection does not result in systemic induction of IFN-α/β in vivo. CD-1 mice were inoculated subcutaneously with 10³ PFU of EEEV (black bars) or VEEV (white bars), and serum was collected at intervals p.i. Serum levels of IFN-α/β were measured by bioassay. Values represent the geometric mean cytokine levels (log₁₀ IU/ml of IFN-α/β) for three mice and are shown ± standard deviations where n = 3. The limit of detection for each data set is indicated by the dotted line.