Alpha/beta interferon protects adult mice from fatal Sindbis virus infection and is an important determinant of cell and tissue tropism

Abstract

Infection of adult 129 Sv/Ev mice with consensus Sindbis virus strain TR339 is subclinical due to an inherent restriction in early virus replication and viremic dissemination. By comparing the pathogenesis of TR339 in 129 Sv/Ev mice and alpha/beta interferon receptor null (IFN-alpha/betaR(-/-)) mice, we have assessed the contribution of IFN-alpha/beta in restricting virus replication and spread and in determining cell and tissue tropism. In adult 129 Sv/Ev mice, subcutaneous inoculation with 100 PFU of TR339 led to extremely low-level virus replication and viremia, with clearance under way by 96 h postinoculation (p.i.). In striking contrast, adult IFN-alpha/betaR(-/-) mice inoculated subcutaneously with 100 PFU of TR339 succumbed to the infection within 84 h. By 24 h p.i. a high-titer serum viremia had seeded infectious virus systemically, coincident with the systemic induction of the proinflammatory cytokines interleukin-12 (IL-12) p40, IFN-gamma, tumor necrosis factor alpha, and IL-6. Replicating virus was located in macrophage-dendritic cell (DC)-like cells at 24 h p.i. in the draining lymph node and in the splenic marginal zone. By 72 h p.i. virus replication was widespread in macrophage-DC-like cells in the spleen, liver, lung, thymus, and kidney and in fibroblast-connective tissue and periosteum, with sporadic neuroinvasion. IFN-alpha/beta-mediated restriction of TR339 infection was mimicked in vitro in peritoneal exudate cells from 129 Sv/Ev versus IFN-alpha/betaR(-/-) mice. Thus, IFN-alpha/beta protects the normal adult host from viral infection by rapidly conferring an antiviral state on otherwise permissive cell types, both locally and systemically. Ablation of the IFN-alpha/beta system alters the apparent cell and tissue tropism of the virus and renders macrophage-DC-lineage cells permissive to infection.

FIG. 1

Effects of IFN-α/β on the replication and dissemination of Sindbis virus TR339 in vivo. 129 Sv/Ev IFN-α/βR^+/+ mice (○), IFN-α/βR^+/− heterozygous mice (▴), and IFN-α/βR^−/− mice (●) were inoculated s.c. with 100 PFU of TR339 and sacrificed at various times p.i., and virus titers from serum (A), brain (B), spleen (C), and liver (D) were determined. Values represent the geometric mean virus titer (log₁₀ PFU/ml or g) for two (IFN-α/βR^+/−) or three (IFN-α/βR^−/− and 129 Sv/Ev) mice as determined on BHK cells. Datum points are shown ± the standard deviation (SD), where n = 3. The lower limit of detection is indicated (broken line).

FIG. 2

Comparison of TR339 virus titers from multiple tissues at 72 h p.i. 129 Sv/Ev IFN-α/βR^+/+ mice (open bars), IFN-α/βR^+/− heterozygous mice (hatched bars), and IFN-α/βR^−/− mice (solid bars) were inoculated, and tissues were harvested as described in Fig. 1. The values represent the geometric mean virus titer (log₁₀ PFU/ml or g) for two (IFN-α/βR^+/−) or three (IFN-α/βR^−/− and 129 Sv/Ev) mice. Datum points are shown ± the SD, where n = 3. A 0.5 log₁₀ PFU/ml bar denotes a value below the limit of detection, i.e., <1.88 log₁₀ PFU/g for brain, <2.40 log₁₀ PFU/ml for serum, and <2.18 log₁₀ PFU/g for all other tissues.

FIG. 3

Replication of TR339 and glycoprotein mutant TRSB-R114 compared in IFN-α/βR^−/− mice. Comparison of TR339 (solid bars) and TRSB-R114 (cross-hatched bars) virus titers in multiple tissues from IFN-α/βR^−/− mice at 72 h p.i. The values represent the geometric mean virus titer (log₁₀ PFU/ml or g) ± the SD for three mice.

FIG. 4

Cytokine levels in serum from TR339-infected and mock-infected mice. 129 Sv/Ev IFN-α/βR^+/+ mice (○), IFN-α/βR^+/− heterozygous mice (▴), and IFN-α/βR^−/− mice (●) were inoculated s.c. with 100 PFU of TR339 (solid line) or with PBS (dashed line), and the serum was harvested at various times p.i. The serum levels of proinflammatory cytokines were determined by ELISA, as follows: (A) IL-12 p40 (confidence limit of detection, 223.2 pg/ml), (B) IFN-γ (confidence limit of detection, 48.83 pg/ml), (C) TNF-α (confidence limit of detection, 97.66 pg/ml), and (D) IL-6 (confidence limit of detection, 195.31 pg/ml). The values represent the mean cytokine level for two (IFN-α/βR^+/−) or three (IFN-α/βR^−/− and 129 Sv/Ev) mice. Datum points are shown ± the SD, where n = 3. The cytokine levels in PBS-inoculated controls were below the limit of detection except for Fig. 4A, where IL-12 p40 was detectable in 129 Sv/Ev mice at 72 and 96 h p.i.

FIG. 5

Histopathological and ISH analyses of tissue sections from TR339-infected IFN-α/βR^−/− mice. Magnification, ×200. ISH analyses were counterstained with hematoxylin. (A) ISH showing extensive virus replication in large cells of the DLN at 24 h p.i. The B-cell follicle stained more intensely with hematoxylin. (B) ISH showing virus replication in the spleen at 24 h p.i. Again, the more intensely stained region is the follicle with central arteriole. (C) ISH analysis of the spleen at 72 h p.i. showing a more widespread virus infection. (D) H&E stain of the same spleen section at 72 h p.i. revealing a severe loss of splenic architecture and reduced cellularity; the more intensely staining triangular region in the center is the B-cell follicle. (E) ISH showing virus replication in large discrete cells of the liver at 72 h p.i., some of which have an elongated appearance, suggesting that they may be sinusoid lining cells. (F) ISH analysis of a sagittal section of the right forelimb joint at 72 h p.i. Extensive virus replication is evident in the periosteum of the bone. (G) ISH showing virus replication at 72 h p.i. in the nasal turbinates, both in the lamina propria and extending into the darker-staining neuroepithelial layer. Virus signal is also seen in the periosteum of the bone. (H) ISH showing an isolated viral lesion in the olfactory bulb of one mouse at 72 h p.i.

FIG. 6

IHC analyses of tissue sections from TR339-infected IFN-α/βR^−/− mice. Magnification, ×400. (A) Distribution of Sindbis virus antigen in the DLN at 24 h p.i. (detected by using CY2-conjugated secondary antibody and visualized by using an FITC filter). (B) Spleen at 24 h p.i. costained for Sindbis virus antigen (CY2) and B-cell marker, B220 (Texas red). Micrographs were taken with FITC and Texas red filters superimposed to demonstrate localization of Sindbis virus antigen in the splenic marginal zone surrounding B-cell follicle. Panels C and D represent TR339-infected spleen, at 24 h p.i., costained for Sindbis virus antigen (CY2) and macrophage marker, Mac-1 (Texas red), respectively. Cells on which virus antigen and Mac-1 colocalize are indicated with paired arrows. Panels E and F show anti-Sindbis (CY2) and Mac-1 (Texas red) costaining, respectively, in the spleen at 72 h p.i. Extensive colocalization of signal is evident, as indicated by paired arrows, and the tissue appears to be severely damaged in keeping with ISH and H&E data. Panels G and H show colocalization of Sindbis virus antigen (CY2) and the Küpffer cell marker, F4/80 (Texas red), in virus-infected liver at 72 h p.i.

FIG. 7

Effects of IFN-α/β on TR339 replication in primary cell cultures. Primary peritoneal macrophage cultures were infected with TR339 virus (MOI = 10), and titers of progeny virus released into the supernatant were determined. Virus growth curves were determined in cells harvested from IFN-α/βR^−/− mice (●, n = 3) and 129 Sv/Ev IFN-α/βR^+/+ mice (○, n = 3). Parallel growth curves were determined in the presence of 8,000 neutralizing U of anti-IFN-α/β antiserum in both IFN-α/βR^−/− mice (▴, n = 2) and 129 Sv/Ev IFN-α/βR^+/+ mice (▵, n = 2). Values represent the geometric mean virus titer (log₁₀ PFU/ml). Data points are shown ± the SD, where n = 3.