Development and Evaluation of a Shrimp Virus (IHHNV)-Mediated Gene Transfer and Expression System for Shrimps

• Published in: International journal of molecular sciences Vol. 25; no. 16; p. 8999
• Main Authors: Tao, Yiwen, Wang, Jinwu, Xiao, Rui, Zhang, Qingli, Guo, Huarong
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 19.08.2024; MDPI
• Subjects: Animals; Baculoviridae - genetics; Cloning; Densovirinae - genetics; Efficiency; Gene Expression; Gene Transfer Techniques; Genes; Genetic Vectors - genetics; Glucuronidase - genetics; Glucuronidase - metabolism; Infections; infectious hypodermal and hematopoietic necrosis virus (IHHNV); insect Sf9 cells; Penaeidae - genetics; Penaeidae - virology; Promoter Regions, Genetic; Proteins; Sf9 Cells; shrimp; shrimp hemolymph cells; Spodoptera - virology; Viral Envelope Proteins - genetics; Viral Envelope Proteins - metabolism; viral packaging; virus-mediated gene transfer and expression system; Viruses; White spot syndrome virus 1 - genetics; infectious hypodermal and hematopoietic necrosis virus (IHHNV); shrimp; viral packaging; virus-mediated gene transfer and expression system; insect Sf9 cells; shrimp hemolymph cells
• ISSN: 1661-6596; 1422-0067; 1422-0067
• DOI: 10.3390/ijms25168999

An efficient gene transfer and expression tool is lacking for shrimps and shrimp cells. To solve this, this study has developed a shrimp DNA virus-mediated gene transfer and expression system, consisting of insect Sf9 cells for viral packaging, the shrimp viral vector of pUC19-IHHNV-PH-GUS and the baculoviral vector of Bacmid or Bacmid-VP28 encoding the shrimp WSSV envelope protein VP28. The pUC19-IHHNV-PH-GUS vector was constructed by assembling the genomic DNA of shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), which has shortened inverted terminal repeats, into a pUC19 backbone, and then an expression cassette of baculoviral polyhedron (PH) promoter-driven (β-glucuronidase) reporter gene was inserted immediately downstream of IHHNV for proof-of-concept. It was found that the viral vector of pUC19-IHHNV-PH-GUS could be successfully packaged into IHHNV-like infective virions in the Sf9 cells, and the gene transfer efficiency of this system was evaluated and verified in three systems of Sf9 cells, shrimp hemolymph cells and tissues of infected shrimps, but the GUS expression could only be detected in cases where the viral vector was co-transfected or co-infected with a baculovirus of Bacmid or Bacmid-VP28 due to the Bacmid-dependence of the PH promoter. Moreover, the packaging and infection efficiencies could be significantly improved when Bacmid-VP28 was used instead of Bacmid.