Early production of type I interferon during West Nile virus infection: role for lymphoid tissues in IRF3-independent interferon production

Abstract

Infection of cells with flaviviruses in vitro is reduced by pretreatment with small amounts of type I interferon (IFN-alpha/beta). Similarly, pretreatment of animals with IFN and experiments using mice defective in IFN signaling have indicated a role for IFN in controlling flavivirus disease in vivo. These data, along with findings that flavivirus-infected cells block IFN signaling, suggest that flavivirus infection can trigger an IFN response. To investigate IFN gene induction by the very first cells infected during in vivo infection with the flavivirus West Nile virus (WNV), we infected mice with high-titer preparations of WNV virus-like particles (VLPs), which initiate viral genome replication in cells but fail to spread. These studies demonstrated a brisk production of IFN in vivo, with peak levels of over 1,000 units/ml detected in sera between 8 and 24 h after inoculation by either the intraperitoneal or footpad route. The IFN response was dependent on genome replication, and WNV genomes and WNV antigen-positive cells were readily detected in the popliteal lymph nodes (pLN) of VLP-inoculated mice. High levels of IFN mRNA transcripts and functional IFN were also produced in VLP-inoculated IFN regulatory factor 3 null (IRF3(-/-)) mice, indicating that IFN production was independent of the IRF3 pathways to IFN gene transcription, consistent with the IFN type produced (predominantly alpha).

FIG. 1.

Detection of IFN in sera and peritoneal fluids from mice inoculated with WNV VLPs. Pairs of BL6 mice were injected i.p. with the indicated doses (in IU) of WNV VLPs. Serum and peritoneal lavage samples were collected at the indicated times postinoculation and tested for IFN activity by a bioassay. (A) IFN levels detected in sera 24 and 48 h after inoculation. (B) IFN levels detected in the peritoneal fluids from the same animals. (C) IFN levels detected in serum samples from a second experiment targeting earlier times of collection, with demonstration that UV inactivation (4 W at 254 nm for 1 min at 10 cm, yielding an inactivation rate of >99.9985%) prevented IFN production. (D) IFN levels detected in serum samples collected at 24 h post-i.p. inoculation with the indicated amounts of VLPs, determined by an IFN-α ELISA (open bars) or an IFN activity bioassay (closed bars). Graphs in panels A, B, and C are truncated at the level of detection for the individual assays, and the filled and open bars in these panels represent data obtained for two mice. <, the values displayed were at the limits of detection of these assays (D).

FIG. 2.

Comparison of levels of IFN produced by BL6 and IRF3^−/− mice following inoculation with WNV VLPs by different routes. Pairs of mice were injected by the i.p. or f.p. route with the indicated doses of WNV VLPs. After 24 h, sera were collected and IFN levels were determined by bioassay (filled and open bars represent the values obtained for the two mice in each group).

FIG. 3.

Comparison of levels of serum IFN and levels of pLN mRNAs for WNV, IFN-α, IFN-β, and IRF7 following WNV VLP (or mock) inoculation into BL6 and IRF3^−/− mice. (A) IFN levels, determined by bioassay, in sera recovered 8 h after inoculation by the indicated routes. (B) IFN levels, determined by bioassay, in sera recovered 24 h after inoculation by the indicated routes. (C) WNV mRNA levels determined from RNAs isolated from pLN obtained from the left rear foot of animals 24 h after inoculation by the indicated routes. (D) IFN-α mRNA levels determined from RNAs isolated from pLN obtained from the left rear foot of animals 24 h after inoculation by the indicated routes. (E) IFN-β mRNA levels determined from RNAs isolated from pLN obtained from the left rear foot of animals 24 h after inoculation by the indicated routes. (F) IRF7 mRNA levels determined from RNAs isolated from pLN obtained from the left rear foot of animals 24 h after inoculation by the indicated routes. Bars represent three individual mice of each genotype, arranged in the same order in each panel.

FIG. 4.

IHC staining of pLN from VLP-inoculated mice. Mice were inoculated with VLPs or medium by f.p. injection, and the pLN were dissected and processed for IHC 24 h later, as described in Materials and Methods.