Immunostimulation of shrimp through oral administration of silkworm pupae expressing VP15 against WSSV.

White spot syndrome virus (WSSV) is one of the most concerning pathogens in penaeid shrimp and can cause severe loss in shrimp aquaculture worldwide. Among the WSSV structural proteins, VP15, a DNA-binding protein located in the WSSV nucleocapsid, is an antiviral protein candidate to protect kuruma shrimp (Marsupenaeus japonicus) from WSSV infection. We identified that the truncated VP15, VP15(26-57), is responsible for the protective effect against the WSSV. This study attempts to develop an immunizing agent against WSSV using silkworm pupa as a delivery vector through oral administration. The VP15, VP15(26-57), and SR11 peptide derived from VP15(26-57) were expressed in silkworm pupae. Oral administration of feed mixed with the powdered pupae that expressed VP15-derived constructs enhanced the survivability of kuruma shrimp with an overall relative percent survival (RPS) higher than 70%. There is no death for the group receiving pupa/VP15(26-57), and the RPS is 100%. In addition, we also investigated the relative mRNA expression levels of immune-related genes by qPCR at different time points. Our results indicate that the oral administration of pupa/VP15-derived products could provide a high protective effect against WSSV and be a practical approach for controlling WSSV in aquaculture.

In vivo enzymatic digestion of HRV 3C protease cleavage sites-containing proteins produced in a silkworm-baculovirus expression system.

Baculovirus expression vector system (BEVS) has been recognized as a potent protein expression system in engineering valuable enzymes and vaccines. Various fusion tags facilitate protein purification, leaving the potential risk to influence the target protein's biological activity negatively. It is of great interest to consider removing the additional tags using site-specific proteases, such as human rhinoviruses (HRV) 3C protease. The current study validated the cleavage activity of 3C protease in Escherichia coli and silkworm-BEVS systems by mixing the cell or fat body lysates of 3C protein and 3C site containing target protein in vitro. Further verification has been performed in the fat body lysate from co-expression of both constructs, showing remarkable cleavage efficiency in vivo silkworm larvae. We also achieved the glutathione-S-transferase (GST) tag-cleaved product of the VP15 protein from the White spot syndrome virus after purification, suggesting that we successfully established a coinfection-based recognition-and-reaction BEVS platform for the tag-free protein engineering.

Identification of antigenic domains and peptides from VP15 of white spot syndrome virus and their antiviral effects in Marsupenaeus japonicus.

White spot syndrome virus (WSSV) is one of the most devastating pathogens in penaeid shrimp and can cause massive damage in shrimp aquaculture industries. Previously, the WSSV structural protein VP15 was identified as an antigenic reagent against WSSV infections. In this study, we truncated this protein into VP15(1-25), VP15(26-57), VP15(58-80), and VP15(1-25,58-80). The purified proteins from the E. coli expression system were assayed as potential protective agents in Kuruma shrimp (Marsupenaeus japonicus) using the prime-and-boost strategy. Among the four truncated constructs, VP15(26-57) provided a significant improvement in the shrimp survival rate after 20 days of viral infection. Subsequently, four peptides (KR11, SR11, SK10, and KK13) from VP15(26-57) were synthesized and applied in an in vivo assay. Our results showed that SR11 could significantly enhance the shrimp survival rate, as determined from the accumulated survival rate. Moreover, a multiligand binding protein with a role in the host immune response and a possible VP15-binding partner, MjgC1qR, from the host M. japonicus were employed to test its binding with the VP15 protein. GST pull-down assays revealed that MjgC1qR binds with VP15, VP15(26-57), and SR11. Taken together, we conclude that SR11 is a determinant antigenic peptide of VP15 conferring antiviral activity against WSSV.

Antigenic properties of VP15 from white spot syndrome virus in kuruma shrimp Marsupenaeus japonicus.

White spot syndrome virus (WSSV) is known as one of the most lethal pathogenic viruses in shrimp causing massive damage to shrimp aquaculture industries. To date, no effective treatment or prevention has been found. In this study, five recombinant viral proteins VP15, VP19, VP24, VP26, and VP28 were expressed and purified in E. coli, which were employed as candidates against WSSV in Kuruma shrimp Marsupenaeus japonicus. In vivo antiviral assay in this study newly revealed that VP15 of major nucleocapsid protein, being known as a DNA-binding protein provided the substantial protection against the viral infection when pre-injected into shrimps. Furthermore, we also verified the immunogenic effects of purified VP15 and VP19 proteins produced in a silkworm-bacmid expression system. Taken together, our study identified VP15 as an effective candidate against WSSV infection in the Kuruma shrimp. It is interesting to uncover why and how VP15 is involved in the immune memory in shrimp in the future study.