Alpha/beta interferon protects against lethal West Nile virus infection by restricting cellular tropism and enhancing neuronal survival - PubMed (original) (raw)

Alpha/beta interferon protects against lethal West Nile virus infection by restricting cellular tropism and enhancing neuronal survival

Melanie A Samuel et al. J Virol. 2005 Nov.

Abstract

West Nile virus (WNV) is a mosquito-borne flavivirus that is neurotropic in humans, birds, and other animals. While adaptive immunity plays an important role in preventing WNV spread to the central nervous system (CNS), little is known about how alpha/beta interferon (IFN-alpha/beta) protects against peripheral and CNS infection. In this study, we examine the virulence and tropism of WNV in IFN-alpha/beta receptor-deficient (IFN- alpha/betaR-/-) mice and primary neuronal cultures. IFN-alpha/betaR-/- mice were acutely susceptible to WNV infection through subcutaneous inoculation, with 100% mortality and a mean time to death (MTD) of 4.6 +/- 0.7 and 3.8+/- 0.5 days after infection with 10(0) and 10(2) PFU, respectively. In contrast, congenic wild-type 129Sv/Ev mice infected with 10(2) PFU showed 62% mortality and a MTD of 11.9 +/- 1.9 days. IFN-alpha/betaR-/- mice developed high viral loads by day 3 after infection in nearly all tissues assayed, including many that were not infected in wild-type mice. IFN-alpha/betaR-/- mice also demonstrated altered cellular tropism, with increased infection in macrophages, B cells, and T cells in the spleen. Additionally, treatment of primary wild-type neurons in vitro with IFN-beta either before or after infection increased neuronal survival independent of its effect on WNV replication. Collectively, our data suggest that IFN-alpha/beta controls WNV infection by restricting tropism and viral burden and by preventing death of infected neurons.

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Figures

FIG. 1.

FIG. 1.

Induction of IFN-α/β mRNA in brains of mice infected with WNV. Mice were inoculated with 102 PFU of WNV by footpad injection and sacrificed on days 5, 8, and 10 p.i. Total RNA from frontal cortices (black bars) and cerebella (gray bars) was analyzed for the expression of 18S rRNA, IFN-α mRNA, and IFN-β mRNA by fluorogenic quantitative RT-PCR. Data are expressed as the relative increase (_n_-fold) over RNA isolated from uninfected controls. Average values are from three to four mice, and error bars indicate standard deviations. Asterisks indicate differences that are statistically significant (*, P < 0.05; **, P < 0.005).

FIG. 2.

FIG. 2.

Survival and virologic analysis of wild-type and IFN-αβR−/− 129Sv/Ev mice. (A) Eight- to 10-week-old mice were inoculated with 100 to 102 PFU of WNV by footpad injection and followed for 21 days. The survival curves were constructed using data from two to three independent experiments. The numbers of animals were as follows: 18 wild-type mice and 20 IFN-α/βR−/− mice infected with 102 PFU and 8 IFN-α/βR−/− mice infected with 101 and 100 PFU. Survival differences between wild-type and IFN-α/βR−/− mice were statistically significant at all viral doses (P < 0.001). (B to E) Viral burden after WNV infection. WNV RNA levels in the serum (B) and viral loads in the brain (C) and spinal cord (D) were determined from samples harvested on days 1, 2, and 3 p.i. with 102 PFU by quantitative RT-PCR or by plaque assay. (E) Viral burden in muscle, heart, lung, kidney, liver, and pancreas was determined on day 3 after infection for IFN-α/βR−/− mice and on days 3 and 8 for wild-type mice. Data are shown as the average PFU per gram of tissue and reflect four to eight mice per group. The dotted line represents the limit of sensitivity of the assay. The viral burden in IFN-α/βR−/− mice was statistically significant in tissue samples as indicated by asterisks (*, P < 0.05).

FIG. 3.

FIG. 3.

Immunohistochemistry of WNV-infected IFN-α/βR−/− and wild-type mice. (A) WNV antigen expression within CNS tissues of IFN-α/βR−/− and wild-type mice. Representative images from the cerebellum, brain stem, hippocampus, choroid plexus, and spinal cord are shown from day 3 p.i. for IFN-α/βR−/− and day 10 p.i. for wild- type mice after review of brains from three or four independent experiments for each group. Inset images show spinal cord staining at a higher magnification (×400). Staining in neurons (black arrows) and within cells of the choroid plexus (blue arrows) is indicated. (B) Detection of WNV antigen in livers of infected IFN-α/βR−/− and wild-type mice at day 3 and day 10 p.i., respectively. Infection of cells lining the sinusoids is indicated (red arrows). All sections were counterstained with hematoxylin; magnifications are ×200 to 400.

FIG. 4.

FIG. 4.

IFN-α/βR−/− mice show increased viral replication and altered tropism in the spleen. (A) Viral burden in the spleen of IFN-α/βR−/− and wild-type mice was determined from samples harvested on days 1, 2, and 3 p.i. by plaque assay. Data are shown as the average PFU per gram of tissue and represent average values from five mice per group. Error bars indicate standard deviations, and the dotted line represents the limit of sensitivity of the assay. (B) Immunohistochemical analysis of WNV antigen within spleen sections from WNV-infected IFN-α/βR−/− and wild-type mice at day 3 p.i. At low magnification, marked destruction of splenic architecture is observed in infected IFN-α/βR−/− mice with disrupted follicle structures (black arrows). Data are representative of staining performed on tissues from three mice per group. (C to F) Analysis of splenocytes derived from 8- to 9-week-old wild-type and IFN-α/βR−/− mice at day 3 p.i. Cells were costained for WNV antigen and cellular markers to determine the total percentage of splenocytes infected (C) and the percentage of the individual cell population infected (D). Data were averaged from four independent experiments performed on a total of five to nine age- and sex-matched mice per group. Error bars indicate standard deviations, and asterisks indicate differences that are statistically significant (*, P < 0.05). (E) Representative flow cytometry histograms of B220+ and WNV antigen-positive costaining in wild-type and IFN-α/βR−/− mice are shown. The numbers indicate the percentages of positive cells in each quadrant. (F) Total RNA from CD11b+ and CD19+ cells purified from infected spleens was analyzed for the levels of WNV-positive and -negative strands using strand-specific real-time RT-PCR. Data were normalized to 18S rRNA levels and are expressed as the relative severalfold increases in WNV RNA detected in IFN-α/βR−/− mice compared to that in infected wild-type mice. Average values are from six mice per group.

FIG. 5.

FIG. 5.

Survival data for wild-type and IFN-α/βR−/− mice, following intracranial inoculation. (A) Eight- to 10-week-old mice were inoculated with 101 PFU of WNV by intracranial injection. The survival curves were constructed using data from two independent experiments with 20 wild-type mice and 11 IFN-α/βR−/− mice. The difference in the mean time to death between wild-type and IFN-α/βR−/− mice was statistically significant (P < 0.001). (B and C) Viral burden in CNS tissues after intracranial WNV infection. Viral load in the brain (B) and spinal cord (C) was determined by plaque assay from samples harvested on days 1, 2, and 3 p.i. for IFN-α/βR−/− and wild-type mice as well as day 6 p.i. for wild-type mice. Data are shown as the average PFU per gram of tissue and reflect four to eight mice per group. The dotted line represents the limit of sensitivity of the assay; asterisks indicate values that are statistically significant (*, P < 0.05) compared to data for wild-type mice.

FIG. 6.

FIG. 6.

IFN-α/β inhibits WNV infection in neurons. Primary cultures of SCG neurons were prepared from wild-type and IFN-α/βR−/− mice. (A) Neurons were pretreated 24 h prior to infection with 100 IU/ml of the indicated mouse IFN. Cells were infected at an MOI of 10 and evaluated for production of infectious WNV at 24 h. (B) Neurons were infected at an MOI of 10 for 24 h, treated with 100 IU/ml of the indicated mouse IFN for 12 h, washed, and then evaluated for WNV production at 48 h. Data represent an average of three independent experiments, and asterisks indicate values that are statistically significant (*, P < 0.05) compared to untreated cells.

FIG. 7.

FIG. 7.

IFN-β prolongs neuronal survival following WNV infection. SCG neurons from wild-type mice were infected with WNV at an MOI of 10. Neurons were either untreated (black bars) or treated 24 h prior to (gray bars) or after (white bars) infection with 100 IU IFN-β. Cells were subsequently maintained in the presence of IFN-β throughout the time course. (A) Neuronal survival was quantitated on days 4, 8, and 11 p.i. Approximately 1,000 to 2,000 cells were counted per condition in each replicate. Data are expressed relative to survival of uninfected neurons and represent the average of four independent experiments. Error bars indicate standard deviations. (B) Infectious virus production at 24 intervals following IFN-β treatment prior to or after infection was assayed on days 1 to 2, 2 to 3, 3 to 4, 7 to 8, and 10 to 11 p.i. Viral production from days 0 to 1 was assayed for IFN-β pretreatment only, as posttreatment occurred at day 1 p.i. (C) WNV antigen expression (red) was determined on day 4 p.i. by immunofluorescence microscopy. DAPI was used as a nuclear counterstain (blue). Asterisks indicate differences that are statistically significant (*, P < 0.05).

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