Suppression of adenosine deaminase acting on RNA (ADAR) expression stimulates immunity and enhances white spot syndrome virus (WSSV) resistance in Penaeus vannamei

Abstract

Adenosine Deaminase Acting on RNA (ADAR) is an enzyme that converts adenosine (A) to inosine (I) in RNA nucleotides, which can inhibit Dicer function during RNA interference, promoting viral replication and reducing the immune response. This research identified and characterized ADAR in the whiteleg shrimp, Penaeus vannamei, and investigated its role in regulating innate immunity during infection with the white spot syndrome virus (WSSV). The complete cDNA of P. vannamei ADAR (PvADAR) consisted of 2567 nucleotides encoding 690 amino acids with a molecular weight of 77.5 kDa. PvADAR was highly expressed in hemocytes. Downregulation of PvADAR by injection of PvADAR-specific double-stranded RNA (dsPvADAR) at concentrations of 1 μg/g body weight resulted in upregulation of proPO, lysozyme, caspase-3, and argonaute-2 gene expression. Moreover, the total hemocyte count increased significantly from 6 to 72 h post dsPvADAR injection. The WSSV challenge was performed by immersion (10⁵ and 10⁶ viral copies/ml of WSSV). Samples were collected at 6-h intervals for up to 72 h post-infection to evaluate immune-related gene expression of proPO, lysozyme, caspase-3, and argonaute-2. During WSSV infection, PvADAR expression decreased, while proPO, caspase-3, and argonaute-2 increased at early stages, with lysozyme showing late-stage upregulation. In addition, the WSSV challenge was conducted in PvADAR-downregulated shrimp; dsPvADAR (1 μg/g body weight) was injected 24 h prior to the challenge. The results showed significant upregulation of immune-related genes, except for argonaute-2. Notably, shrimp injected with dsPvADAR showed significantly reduced viral loads and improved survival probability, particularly in the shrimp challenged with 10⁶ viral copies/ml of WSSV. These results indicated that suppressing PvADAR increased expression of immune-related genes under both normal conditions and during WSSV infection, suggesting that PvADAR negatively affects the shrimp immune response. Moreover, suppressing PvADAR levels in shrimp could lower viral replication and promote survival probability. This approach may lead to a new strategy for promoting shrimp immunity, thereby increasing shrimp resistance to pathogens and benefiting aquaculture.