Acute infection with venezuelan equine encephalitis virus replicon particles catalyzes a systemic antiviral state and protects from lethal virus challenge

Abstract

The host innate immune response provides a critical first line of defense against invading pathogens, inducing an antiviral state to impede the spread of infection. While numerous studies have documented antiviral responses within actively infected tissues, few have described the earliest innate response induced systemically by infection. Here, utilizing Venezuelan equine encephalitis virus (VEE) replicon particles (VRP) to limit infection to the initially infected cells in vivo, a rapid activation of the antiviral response was demonstrated not only within the murine draining lymph node, where replication was confined, but also within distal tissues. In the liver and brain, expression of interferon-stimulated genes was detected by 1 to 3 h following VRP footpad inoculation, reaching peak expression of >100-fold over that in mock-infected animals. Moreover, mice receiving a VRP footpad inoculation 6, 12, or 24 h prior to an otherwise lethal VEE footpad challenge were completely protected from death, including a drastic reduction in challenge virus titers. VRP pretreatment also provided protection from intranasal VEE challenge and extended the average survival time following intracranial challenge. Signaling through the interferon receptor was necessary for antiviral gene induction and protection from VEE challenge. However, VRP pretreatment failed to protect mice from a heterologous, lethal challenge with vesicular stomatitis virus, yet conferred protection following challenge with influenza virus. Collectively, these results document a rapid modulation of the host innate response within hours of infection, capable of rapidly alerting the entire animal to pathogen invasion and leading to protection from viral disease.

FIG. 1.

Rapid, systemic activation of the host antiviral response following VRP footpad inoculation. Adult BALB/c mice (3 mice per group) were inoculated in the RRFP with 2 × 10⁶ IU of null VRP or were mock infected with diluent. At 1, 3, 6, 12, or 24 h following inoculation, the mice were euthanized and then perfused with PBS, and tissues were resected. Total cellular RNA was isolated, cDNA was synthesized, and the expression of a panel of IFN-stimulated genes (coding for IFN-β, IP-10, p56, and Isgf3γ) was assessed by TaqMan real-time PCR in the DLN (A), liver (B), and brain (C). The induction (fold) of each gene is represented as expression in VRP-infected animals relative to expression in mock-infected animals. Bars represent mean values ± the standard error of the mean.

FIG. 2.

High levels of biologically active IFN are present in the serum of VRP-infected mice. Adult BALB/c mice were inoculated in the RRFP with 2 × 10⁶ IU of null VRP. At 1, 3, 6, 12, or 24 h following inoculation, the mice were euthanized, followed by cardiac puncture and exsanguination to collect blood samples. Type I IFN present in the serum was measured by standard IFN biological assay on L929 cells, and the results are presented as IU per milliliter. Each bar represents an individual animal, with the limit of detection (LOD) for the assay indicated by a dotted line. The data in this and the previous figure correspond to the same group of mice.

FIG. 3.

VRP pretreatment prior to peripheral and intranasal challenge with virulent VEE protects mice from death and extends the average survival time of mice challenged intracranially. Adult BALB/c mice (5 to 6 mice per group) were pretreated by inoculation in the RRFP with 2 × 10⁶ IU null VRP for 6, 12, or 24 h prior to peripheral challenge with 10 PFU of VEE in the opposing LRFP (A), intranasal challenge with 10³ PFU of VEE (a dose necessary to achieve 100% mortality in mock-pretreated animals) (B), or intracranial challenge with 10³ PFU of VEE (C). Mock-pretreated mice received diluent in the RRFP 6 h prior to challenge. Animals were monitored daily for morbidity and mortality. The ASTs following intracranial challenge were as follows (mean ± standard error): mock-pretreated mice, 2.7 ± 0 days; VRP-pretreated mice (6 h), 5.0 ± 0 days (P < 0.001); VRP-pretreated mice (12 h), 5.0 ± 0.3 days (P < 0.001); and VRP-pretreated mice (24 h), 4.0 ± 0.4 days (P < 0.05). One-way analysis of variance was used for statistical analysis performed on AST values following intracranial challenge of VRP-pretreated animals compared to mock-pretreated animals.

FIG. 4.

VRP pretreatment dramatically reduces the viral load in the serum and brain of animals challenged with VEE. Adult BALB/c mice (3 mice per group) were pretreated by inoculation in the RRFP with 2 × 10⁶ null VRP for 6, 12, or 24 h prior to challenge with 10 PFU of virulent VEE in the opposing LRFP. Mock-pretreated mice received diluent 6 h prior to challenge. At 24 h postchallenge, serum was collected by tail vein bleed. At 88 h postchallenge, mice were euthanized and then perfused with PBS, and the brain (including the olfactory bulbs) was resected. Challenge virus titers were determined by standard plaque assay on BHK cells. Bars represent mean values ± the standard error of the mean. The assay limit of detection (LOD) is indicated by the dotted line.

FIG. 5.

VRP pretreatment dose is a critical parameter in mediating protection against VEE challenge. Adult BALB/c mice (5 mice per group) were pretreated with decreasing doses of null VRP (2 × 10⁶ IU, 10⁵ IU, or 10⁴ IU) inoculated in the RRFP for 6 h prior to challenge with 10 PFU of virulent VEE (LRFP). Mock-pretreated mice received diluent 6 h prior to challenge. (A) Animals were monitored daily for morbidity and mortality. (B) At 24 h postchallenge, serum was collected by tail vein bleed. At 88 h postchallenge, mice were euthanized and then perfused with PBS, and the brain (including the olfactory bulbs) was resected. At each dose of VRP pretreatment, subsequent challenge virus titers were determined by standard plaque assay on BHK cells. Bars represent mean values ± the standard error of the mean. The assay limit of detection (LOD) is indicated by the dotted line.

FIG. 6.

VRP-induced antiviral gene induction in the brain is diminished and VRP-mediated protection from challenge is abolished in IFN-α/βR knockout mice. Adult wild-type 129 Sv/Ev or IFN-α/βR knockout (IFN-α/βR^−/−) mice were inoculated in the RRFP with 2 × 10⁶ IU of null VRP or were mock infected with diluent. (A) At 6 h following inoculation, the mice were euthanized and then perfused with PBS, and brain tissue was resected. Total cellular RNA was isolated, and the expression of a panel of IFN-stimulated genes (coding for IFN-β, IP-10, p56, and Isgf3γ) was assessed by TaqMan real-time PCR. Black bars represent gene expression in wild-type 129 mice, and striped bars represent gene expression in IFN-α/βR^−/− mice. The induction (fold) of each gene is represented by expression in VRP-infected animals relative to expression in mock-infected animals. Bars represent mean values (3 animals per group) ± the standard error of the mean. (B) Adult wild-type (WT) 129 Sv/Ev (5 mice per group) or IFN-α/βR^−/− mice (8 mice per group) were pretreated by inoculation in the RRFP with 2 × 10⁶ IU null VRP for 6, 12, or 24 h prior to challenge with 10 PFU of virulent VEE in the opposing footpad. Mock-pretreated mice received diluent 6 h prior to challenge. Animals were monitored for morbidity and mortality every 6 h for the first 48 h and every 24 h thereafter.

FIG. 7.

VRP pretreatment differentially protects mice from challenge with heterologous viruses. (A) VRP pretreatment does not protect mice against heterologous challenge with VSV. Adult BALB/c mice (7 or 8 mice per group) were pretreated by inoculation in the RRFP with 2 × 10⁶ IU of null VRP for 24 h prior to intranasal challenge with 2 × 10⁶ PFU of VSV. Mock-pretreated mice received diluent 24 h prior to challenge. Animals were monitored daily for morbidity and mortality. (B) Adult BALB/c mice (3 mice per group) were pretreated by inoculation in the RRFP with 2 × 10⁶ IU of null VRP for 6 or 24 h prior to intranasal challenge with 2 × 10⁶ PFU of VSV. Mock-pretreated mice received diluent 24 h prior to challenge. At 88 h postchallenge, mice were euthanized and then perfused with PBS, and the brain (including the olfactory bulbs) was resected. Challenge virus titers were determined by standard plaque assay on BHK cells. Bars represent mean values ± the standard error of the mean. (C) VRP pretreatment is capable of protecting mice from death following lethal influenza virus challenge. Adult BALB/c mice (6 mice per group) were pretreated by inoculation in the RRFP with 2 × 10⁶ IU null VRP for 6, 12, or 24 h prior to intranasal challenge with 12 PFU of influenza virus. Mock-pretreated mice received diluent 6 h prior to challenge. Animals were monitored daily for morbidity and mortality.