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White spot syndrome virus (WSSV) is a potent shrimp viral pathogen responsible for significant economic losses to shrimp aquaculture all over the world. Several studies have demonstrated efficient RNAi-based approaches for suppressing viral genes and reducing shrimp mortality. However, the application has been difficult in real practical use. The present work aimed to develop an efficient approach for the delivery of VP28 specific-siRNA using Bacillus subtilis, a novel live oral vaccine vehicle, to protect shrimp against WSSV infection. A plasmid that can function in both bacteria and eukaryotic cells was developed by combining pBE-sDNA and the pCMV promoter from the pcDNA4 plasmid to obtain pBE:cDNA4. Subsequently, to evaluate the efficacy of pBE:cDNA4 in vivo, dsGFP-expressing pBE:cDNA4 (pBE:cDNA4-dsGFP) and dsVP28-expressing pBE:cDNA4 (pBE:cDNA4-dsVP28) were used to knockdown target transcripts in WSSV-infected shrimp. A high survival rate was shown for WSSV-infected shrimp injected with pBE:cDNA4-dsVP28 at 7-14 days post-infection (dpi). The pBE:cDNA4-dsVP28 plasmid was transformed into B. subtilis, and B. subtilis was used as a delivery vehicle during oral feeding. WSSV-infected shrimp fed with B. subtilis carrying pBE:cDNA4-dsVP28 showed 91.67% survival compared to the control, which showed only 28.57% survival. The results here demonstrate the efficient delivery of specific siRNAs by using bactofection with B. subtilis.

Gamma-interferon-inducible lysosomal thiol reductase (GILT) is involved in the adaptive immune response via its effects on major histocompatibility complex (MHC)-restricted antigen presentation. In addition to antigen presentation, GILT exerts its antiviral activity by reducing disulfide bonds in proteins involved in viral infection and assembly, thereby inhibiting viral envelope-mediated infection and viral progeny production. In black tiger shrimp, Penaeus monodon GILT (PmGILT) was cloned and characterized, and found to be involved in the shrimp innate immune response and to exert neutralizing activity against white spot syndrome virus (WSSV) infection. However, the anti-WSSV mechanism of PmGILT in the shrimp innate immune response has not been defined. To explore the anti-WSSV activity of PmGILT, a yeast 2-hybrid (Y2H) assay was performed to identify WSSV proteins targeted by PmGILT. The assay revealed 4 potential PmGILT-interacting WSSV proteins: WSSV002, WSSV164, WSSV189, and WSSV471. Three of these 4 WSSV proteins (WSSV002, WSSV164 and WSSV189) were successfully produced and confirmed to interact with PmGILT in in vitro pull-down assays. WSSV189 and WSSV471 were previously identified as structural proteins, whereas WSSV164 is an immediate -early protein which has anti-melanization activity, and WSSV002 is an unknown. Because of the thiol reductase activity of PmGILT, WSSV164 and WSSV189, both of which are cysteine-containing WSSV proteins, were chosen for disulfide bond reduction assays. PmGILT reduced intrachain disulfide bonds in both WSSV proteins, suggesting that PmGILT exerts its anti-WSSV activity via its thiol reductase activity to disrupt the WSSV protein complex and restore the melanization activity of PmproPO1 and PmproPO2.

In a previous study, the gene EgP450 that encodes the proteins of 505 amino acids was isolated from oil palm. The recombinant protein EgP450 is bound to phenylurea-like herbicides which detoxify the substance. Aflatoxin B1 (AFB 1), a mycotoxin produced by Aspergillus sp., is another toxic compound that is known to cause acute toxic effects and act as a hepatocarcinogenic agent. This study aimed to examine the role of EgP450 enzyme in mycotoxin bioactivation in human mesenchymal stem cells (hMSCs). Docking analysis showed that EgP450 is bound to the group of carcinogens, which includes AFB 1 , n-(2-fluorenyl) acetamide, n-n-butyl-n-butan-4-ol-nitrosamine, n-nitrosodiethylamine, n-nitrosodiethylamine and n-nitrosodimethylamine. An in vivo aflatoxin toxicity test on hMSCs and AFB 1 induces the expression of Bmi-1 which is one of the markers for the development of cancer. The presence of EgP450 at 0.15 µg/mL could reduced the Bmi-1 expression in AFB 1 induced cells. Moreover, this protein also showed some antioxidant activity. These results exhibited the enormous potential of EgP450 in the detoxification processes.

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