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. 2006 Jun;80(11):5338-48.
doi: 10.1128/JVI.00274-06.

Gamma interferon plays a crucial early antiviral role in protection against West Nile virus infection

Bimmi Shrestha  1 , Tian Wang, Melanie A Samuel, Kevin Whitby, Joe Craft, Erol Fikrig, Michael S Diamond
Affiliations

Gamma interferon plays a crucial early antiviral role in protection against West Nile virus infection

Bimmi Shrestha et al. J Virol. 2006 Jun.

Abstract

West Nile virus (WNV) causes a severe central nervous system (CNS) infection in humans, primarily in the elderly and immunocompromised. Prior studies have established an essential protective role of several innate immune response elements, including alpha/beta interferon (IFN-alpha/beta), immunoglobulin M, gammadelta T cells, and complement against WNV infection. In this study, we demonstrate that a lack of IFN-gamma production or signaling results in increased vulnerability to lethal WNV infection by a subcutaneous route in mice, with a rise in mortality from 30% (wild-type mice) to 90% (IFN-gamma(-/-) or IFN-gammaR(-/-) mice) and a decrease in the average survival time. This survival pattern in IFN-gamma(-/-) and IFN-gammaR(-/-) mice correlated with higher viremia and greater viral replication in lymphoid tissues. The increase in peripheral infection led to early CNS seeding since infectious WNV was detected several days earlier in the brains and spinal cords of IFN-gamma(-/-) or IFN-gammaR(-/-) mice. Bone marrow reconstitution experiments showed that gammadelta T cells require IFN-gamma to limit dissemination by WNV. Moreover, treatment of primary dendritic cells with IFN-gamma reduced WNV production by 130-fold. Collectively, our experiments suggest that the dominant protective role of IFN-gamma against WNV is antiviral in nature, occurs in peripheral lymphoid tissues, and prevents viral dissemination to the CNS.

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Figures

FIG. 1.
FIG. 1.
Survival analysis of wild-type, IFN-γ−/−, and IFN-γR−/− mice after WNV infection. (A) Wild-type, IFN-γ−/−, and IFN-γR−/− mice were infected subcutaneously with 102 PFU of WNV (New York) and monitored for 28 days for morbidity and mortality. The survival curves were constructed using data from three to five independent experiments. The survival differences between wild-type, IFN-γ−/−, and IFN-γR−/− mice were statistically significant (P < 0.0001). The numbers of animals were n = 50 for wild-type, n = 37 for IFN-γ−/−, and n = 29 for IFN-γR−/− mice. (B) Wild-type and IFN-γ−/− mice were infected i.c. with 101 PFU of WNV (Madagascar) and monitored for 28 days for morbidity and mortality. The survival curves were constructed with data from two independent experiments. The survival differences between wild-type and IFN-γ−/− mice were not statistically significant (P = 0.7). The numbers of animals were n = 20 for wild-type mice and n = 20 for IFN-γ−/− mice.
FIG. 2.
FIG. 2.
WNV burden in peripheral and CNS tissues of wild-type and IFN-γ−/− mice. (A) Levels of viral RNA in serum detected by fluorogenic real-time RT-PCR. The kinetics and level of viral RNA were determined from the sera of wild-type and IFN-γ−/− mice after WNV (New York) infection at the indicated time point by fluorogenic real-time RT-PCR. The data are expressed as number of copies of WNV RNA per milliliter of serum (n = 10 mice per time point). (B) Levels of viral RNA in inguinal lymph nodes. Viral RNA was extracted from the draining inguinal lymph nodes of wild-type and IFN-γ−/− mice at the indicated time points after WNV infection and quantitated by fluorogenic real-time RT-PCR. The data are expressed as the number of copies of WNV RNA per microgram of rRNA after normalization for tissue content (n = 6 mice per time point). (C) Levels of viral RNA in the spleen on day 1 postinfection. Viral RNA was extracted from wild-type or IFN-γ−/− spleens at day 1 postinfection and quantitated by fluorogenic real-time RT-PCR. The data are expressed as the number of copies of WNV RNA per microgram of rRNA after normalization for tissue content (n = 6 mice per group). (D to F) Levels of infectious virus in tissues. Infectious WNV levels were measured in the spleen (D), brain (E), and spinal cord (F) by using a viral plaque assay in BHK21 cells after tissues were harvested and homogenized at the indicated time points after WNV infection. The data are shown as the average PFU per gram of tissue (n = 10 mice per time point). The dotted line represents the limit of sensitivity of the assay (✽, P ≤ 0.05; ✽✽, P ≤ 0.005 [compared to wild-type mice]).
FIG. 3.
FIG. 3.
Level of infectious WNV burden in the peripheral and CNS tissues of wild-type, IFN-γ−/−, and IFN-γR−/− mice. The level of infectious virus was measured from the spleens (A), brains (B), and spinal cords (C) of wild-type, IFN-γ−/−, and IFN-γR−/− mice by using a viral plaque assay in BHK21 cells at the indicated time points after WNV (New York) infection. The data are shown as the average PFU per gram of tissue (n = 5 to 10 mice per time point). The dotted line represents the limit of sensitivity of the assay (✽, P ≤ 0.05; ✽✽, P ≤ 0.005 [compared to wild-type mice]).
FIG. 4.
FIG. 4.
WNV-specific antibody responses in wild-type and IFN-γ−/− mice. Wild-type and IFN-γ−/− mice were infected with WNV (New York), and sera were collected at the indicated time points. The development of specific IgM (A) or IgG (B) antibodies to WNV was determined by ELISA using purified WNV E protein. The data are the averages of at least eight mice per time point. (C) Neutralizing antibody titers. Neutralizing titers were determined by using a PRNT assay. All serum samples were performed in duplicate, and the data are expressed as reciprocal PRNT50s (i.e., the antibody titers that reduced the number of plaques by 50%). The data are the average of eight mice per time point.
FIG. 5.
FIG. 5.
Cytokine profiles in the sera of wild-type and IFN-γ−/− mice. Wild-type and IFN-γ−/− mice were infected with WNV (New York) via the footpad, and sera were collected at the indicated time points after infection. (A) Type I IFN activity was measured by bioassay using encephalomyocarditis virus and L929 cells. The data are an average of three to five mice per time point. (B to D) Other inflammatory cytokines and chemokines. The levels of MCP-1 (B), IL-6 (C) and TNF-α (D) in serum were determined by using a flow cytometric bead array. The data are expressed as an average of six mice per time point from two independent experiments. The dotted line represents the limit of sensitivity of the assay (✽, P ≤ 0.05 compared to wild-type mice).
FIG. 6.
FIG. 6.
WNV burden in peripheral and CNS tissues of bone marrow chimeras. Bone marrow chimeras were prepared as described in Materials and Methods using donors and recipients (see Table 1). Reconstituted mice were infected subcutaneously with 102 PFU of WNV (New York). Viral loads were measured at the indicated days in the blood (A), spleens (B), and brains (C) of mice by using quantitative RT-PCR. The y axis depicts the ratio of the amplified WNV-E cDNA to β-actin cDNA of each sample. At each time point, five mice per group were analyzed (✽, P ≤ 0.05 indicates a significant difference in viral RNA levels between reconstituted groups).
FIG. 7.
FIG. 7.
Inhibition of WNV production from the bone marrow-derived DCs treated with IFN. DCs were generated from the bone marrow of wild-type mice. DCs were pretreated with 100 IU of IFN-α, IFN-β, or IFN-γ 24 h before infection. Cells were infected at an MOI of 0.2, and the production of infectious WNV (New York) was determined 1 day later by plaque assay in Vero cells. The data are expressed as an average of two independent experiments performed in triplicate (✽✽, P ≤ 0.005 compared to untreated cells).
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