Induction of antiviral immunity by double-stranded RNA in a marine invertebrate

Abstract

Vertebrates mount a strong innate immune response against viruses, largely by activating the interferon system. Double-stranded RNA (dsRNA), a common intermediate formed during the life cycle of many viruses, is a potent trigger of this response. In contrast, no general inducible antiviral defense mechanism has been reported in any invertebrate. Here we show that dsRNA induces antiviral protection in the marine crustacean Litopenaeus vannamei. When treated with dsRNA, shrimp showed increased resistance to infection by two unrelated viruses, white spot syndrome virus and Taura syndrome virus. Induction of this antiviral state is independent of the sequence of the dsRNA used and therefore distinct from the sequence-specific dsRNA-mediated genetic interference phenomenon. This demonstrates for the first time that an invertebrate immune system, like its vertebrate counterparts, can recognize dsRNA as a virus-associated molecular pattern, resulting in the activation of an innate antiviral response.

FIG. 1.

dsRNA induces an antiviral state in shrimp. Shrimp (1 to 2 g) were injected intramuscularly with either saline (positive [○] and negative [░⃞] controls) or 8 μg of dsRNA as indicated. At 72 h after dsRNA injection, animals (n = 38 to 42) were infected by intramuscular injection with TSV (a) or WSSV (b and c) as described in Materials and Methods. In panel a, the effect of a 1,000-fold increase in the dose of TSV (from 10⁻⁸ to 10⁻⁵ [wt/vol] dilutions) in both positive controls and dsRNA-treated shrimp is also shown in the dotted mortality curves. The dsRNA preparations were a 309-bp portion of duck Igυ (; accession no. AJ312200), a 1,316-bp genomic noncoding region of clone BAC6 from the catfish IgH locus (•; accession no. CC936713), a 1,079-bp portion of pig IgG cDNA (✽; accession no. U03778), and a 1,184-bp fragment of the BAC cloning vector pBeloBAC11 (26) (♦). The chi-square statistic was used to assess the significance of the observed antiviral protection by comparing the final cumulative mortality in dsRNA-treated groups with that of their respective positive controls: (a) duck Igυ dsRNA (χ² = 6.05 [0.01 < P < 0.025]) and duck Igυ dsRNA TSVx1000 (χ² = 0.75 [not significant]) and (b) duck Igυ dsRNA (χ² = 5.29 [0.01 < P < 0.025]), pig Ig dsRNA (χ² = 12.93 [P < 0.001]), pBeloBAC11 dsRNA (χ² = 8.67 [0.001 < P < 0.005]), and fish noncoding dsRNA (χ² = 15.39 [P < 0.001]). Panel c shows eosin-hematoxylin staining and anti-WSSV immunostaining of hemopoietic tissue sections from a control infected shrimp in comparison to a dsRNA-treated/WSSV-infected shrimp. The sections shown were obtained 72 h after viral infection. Arrows indicate the presence of intranuclear inclusions characteristic of WSSV infection and WSSV-positive immunoreactivity. Scale bars, 20 μm.

FIG. 1.

dsRNA induces an antiviral state in shrimp. Shrimp (1 to 2 g) were injected intramuscularly with either saline (positive [○] and negative [░⃞] controls) or 8 μg of dsRNA as indicated. At 72 h after dsRNA injection, animals (n = 38 to 42) were infected by intramuscular injection with TSV (a) or WSSV (b and c) as described in Materials and Methods. In panel a, the effect of a 1,000-fold increase in the dose of TSV (from 10⁻⁸ to 10⁻⁵ [wt/vol] dilutions) in both positive controls and dsRNA-treated shrimp is also shown in the dotted mortality curves. The dsRNA preparations were a 309-bp portion of duck Igυ (; accession no. AJ312200), a 1,316-bp genomic noncoding region of clone BAC6 from the catfish IgH locus (•; accession no. CC936713), a 1,079-bp portion of pig IgG cDNA (✽; accession no. U03778), and a 1,184-bp fragment of the BAC cloning vector pBeloBAC11 (26) (♦). The chi-square statistic was used to assess the significance of the observed antiviral protection by comparing the final cumulative mortality in dsRNA-treated groups with that of their respective positive controls: (a) duck Igυ dsRNA (χ² = 6.05 [0.01 < P < 0.025]) and duck Igυ dsRNA TSVx1000 (χ² = 0.75 [not significant]) and (b) duck Igυ dsRNA (χ² = 5.29 [0.01 < P < 0.025]), pig Ig dsRNA (χ² = 12.93 [P < 0.001]), pBeloBAC11 dsRNA (χ² = 8.67 [0.001 < P < 0.005]), and fish noncoding dsRNA (χ² = 15.39 [P < 0.001]). Panel c shows eosin-hematoxylin staining and anti-WSSV immunostaining of hemopoietic tissue sections from a control infected shrimp in comparison to a dsRNA-treated/WSSV-infected shrimp. The sections shown were obtained 72 h after viral infection. Arrows indicate the presence of intranuclear inclusions characteristic of WSSV infection and WSSV-positive immunoreactivity. Scale bars, 20 μm.

FIG. 2.

Induction of the antiviral state is due to RNA. Shrimp (2 to 3 g) were injected intramuscularly with either saline (positive [○] and negative [░⃞] controls) or dsRNA. At 72 h after this initial injection, animals were infected with WSSV either alone or mixed with dsRNA. (a) Animals (n = 26 to 31) were injected each time with 50 μl of a solution containing ca. 10 to 15 μg of dsRNA for duck υ and challenged with WSSV-positive extract. The 72-0 group (•) received dsRNA both 72 and 0 h (coinjection) before viral infection, the 72 group (gray triangles) received dsRNA only 72 h prior to infection, and the 0 group (▵) received dsRNA only mixed with the WSSV inoculum. (b) Shrimp (n = 20 to 28) were kept in a recirculation system (see Materials and Methods), injected with dsRNA 72 h before viral challenge, and then reinjected with dsRNA mixed with WSSV-positive extract. Positive (○) and negative (░⃞) controls were as described for panel a. dsRNA for the duck υ was used in every case and applied at 10 to 15 μg per injection. dsRNA was purified with phenol and chloroform (gray triangles), RNeasy columns (Qiagen) (•), or purified with RNeasy columns and treated with a cocktail of RNases (RNase A, RNase T₁, and RNase V1 from Ambion) (□). The Fisher exact test was used to assess the significance of the observed antiviral protection by comparing the final cumulative mortality in dsRNA-treated groups with that of their respective positive controls: (a) 72-0 treatment (P = 0.0003) and 72 treatment (P = 0.0001) and (b) dsRNA column (P = 0.0017) and dsRNA solvent (P = 0.0003).

FIG. 3.

Poly(C-G), but not poly(I-C) or poly(C), induces the antiviral state. Shrimp (1 to 2 g, n = 38 to 42) were injected with 8 μg (a) or 7 μg (b) of synthetic dsRNA analogues as indicated and infected 72 h later with WSSV. All RNA analogues were purchased from Sigma-Aldrich, reconstituted in 400 mM NaCl-10 mM Tris-Cl (pH 7.4), and annealed and quantified as described in Materials and Methods. dsRNA for the duck υ (7 μg) was included in the experiment on panel b for comparison. The chi-square statistic was used to assess the significance of the observed antiviral protection relative to positive controls: (a) poly CG (χ² = 16.92 [P < 0.001]) and (b) duck Igυ dsRNA (χ² = 32.17 [P < 0.001]) and poly(C-G) (χ² = 35.05 [P < 0.001]).