Probiotics expressing double-stranded RNA targeting VP28 efficiently protect shrimps from WSSV infection

Abstract

Envelope protein VP28 of white spot syndrome virus (WSSV) serves as an excellent target of viral control via RNA interference (RNAi) in shrimps. In this study, double-stranded RNA targeting VP28 (dsVP28) was successfully produced by a hairpin expression vector (pWH1520-VP28) in Lactobacillus plantarum and Lactococcus lactis. Although leaky expression of xylose-inducible promoter in pWH1520 was detected, it was an application advantage as addition of xylose is not necessary. The probiotics produced dsVP28 at ranges of 0.78-1.82 ng per 10(12) CFU. Shrimps were submerged in two doses of probiotics (approximately 4.5 x10(8) [1X] CFU/ 2 L seawater or 4.5 x 10(10) [100X] CFU/2 L seawater) for 5 days prior to oral challenge with WSSV-infected tissues (approximately log copies of 10.2-10.7/tank). Shrimps receiving ds-L. lactis showed dose-dependent survival with the lowest mortality rate (33%) and the lowest WSSV copies (2.88 +/- 0.18 logs per 100 ng total DNA from shrimp tissue) after receiving 100X ds-L. lactis. Both strains of L. plantarum could decrease shrimp mortalities and ds-L. plantarum could reduce viral loads significantly. To better understand the role of dsVP28 expressing L. lactis against WSSV, qRT-PCR assays were performed to assess expression levels of shrimp immune genes. Shrimps receiving 100X of ds-L. lactis significantly increased expression of (i) systemic RNA interference defective protein-1 (SID-1) and Dicer-2 (Dcr-2) genes in RNA interference and (ii) peroxinectin (PX) activation, prophenoloxidase-1 (proPO I), and anti-lipopolysaccharide factor-1 (ALF1) innate immune genes. Our overall findings suggest that not only ds-L. lactis probiotics can reduce shrimp mortality due to WSSV, the strain can also instigate RNAi and activate shrimp innate immune systems.