Lambda interferon (IFN-lambda) in serum is decreased in hantavirus-infected patients, and in vitro-established infection is insensitive to treatment with all IFNs and inhibits IFN-gamma-induced nitric oxide production

Abstract

Hantaviruses, causing hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), are known to be sensitive to nitric oxide (NO) and to pretreatment with type I and II interferons (alpha interferon [IFN-alpha]/IFN-beta and IFN-gamma, respectively). Elevated serum levels of NO and IFN-gamma have been observed in HFRS patients, but little is known regarding the systemic levels of other IFNs and the possible effects of hantaviruses on innate antiviral immune responses. In Puumala virus-infected HFRS patients (n = 18), we report that the levels of IFN-alpha and IFN-beta are similar, whereas the level of IFN-lambda (type III IFN) is significantly decreased, during acute (day of hospitalization) compared to the convalescent phase. The possible antiviral effects of IFN-lambda on the prototypic hantavirus Hantaan virus (HTNV) replication was then investigated. Pretreatment of A549 cells with IFN-lambda alone inhibited HTNV replication, and IFN-lambda combined with IFN-gamma induced additive antiviral effects. We then studied the effect of postinfection treatment with IFNs. Interestingly, an already-established HTNV infection was insensitive to subsequent IFN-alpha, -beta, -gamma, and -lambda stimulation, and HTNV-infected cells produced less NO compared to noninfected cells when stimulated with IFN-gamma and IL-1beta. Furthermore, less phosphorylated STAT1 after IFN treatment was observed in the nuclei of infected cells than in those of noninfected cells. The results suggest that hantavirus can interfere with the activation of antiviral innate immune responses in patients and inhibit the antiviral effects of all IFNs. We believe that future studies addressing the mechanisms by which hantaviruses interfere with the activation and shaping of immune responses may bring more knowledge regarding HFRS and HCPS pathogenesis.

FIG. 1.

The level of IFN-λ in serum is decreased during the acute phase of HFRS. The data shown are means plus the standard deviations (SD) of the percentages of IFN-α, IFN-β, and IFN-λ at acute phase compared to the convalescent phase for the individual patients.

FIG. 2.

IFN-λ inhibits HTNV replication in A549 cells. Titers of HTNV in supernatants sampled 30 h postinfection of A549 cells incubated in medium with or without various concentrations of IFN-α, -β, -γ, λ1, or -λ2. Cells were treated with IFNs for 24 h before infection with HTNV. The data represent the means plus the SD of three individual experiments.

FIG. 3.

IFN-λ combined with IFN-γ has additive antiviral effect on HTNV replication. Cells were treated with IFN-α, -β, or -γ alone and with different combinations of IFN-α, -β, or -γ, -λ1, and -λ2 (10 ng of each IFN/ml) for 24 h and were then infected with HTNV. Supernatants were collected 30 h later and titrated. The data represent the medians of three individual experiment.

FIG. 4.

An established HTNV-infection is insensitive to IFN treatment. The titers of HTNV in supernatants sampled 10, 24, and 48 h after treatment with IFN-α, -β, -γ, -λ1, or -λ2 were determined. A549 cells were infected with HTNV for 24 h before treatment with 10 ng of the different IFNs/ml. The data represent means plus the SD of three individual experiments.

FIG. 5.

Established HTNV infection inhibits IFN-γ- and IL-1β-induced NO production. The concentration of nitrite was measured in supernatants from cells stimulated with IFN-γ and IL-1β. Vero E6 cells were infected with HTNV for 30 h before stimulation or left uninfected as a control. The data represent means plus the SD of three individual experiments.

FIG. 6.

Established HTNV infection inhibits IFN-γ-induced STAT1 phosphorylation. A549 cells were infected with HTNV and stimulated 30 h later with 100 ng of IFN-γ/ml for 30 min, fixed, and stained for DNA (A), HTNV (B), phosphorylated STAT1 (C), and merged (D). Infected cells (positive for HTNV antigen) show less nuclear phosphorylated STAT1 than do noninfected cells.