Viral disease pandemics are a major cause of economic losses in fish and crustacean farming worldwide. One of the prerequisites in studying viral pathogenesis and antiviral immunity in fish is to develop an infectious clone of the virus. Availability of an infectious clones enable genomic manipulation feasible and opens avenues to developing viral vaccine.
Recently, we engineered an infectious cDNA clone of a bi-segmented RNA virus of shrimp, Macrobrachium rosenbergii Nodavirus (MrNV), using the baculovirus expression system in insect cells, Sf9 (Gangnonngiw et al., 2020. Virology 2020, 540, 30–37). Subsequently, we modified the infectious MrNV cDNA clone by replacing RdRp gene with a marker gene, Green Fluorescent Protein (GFP) to generate a replication incompetent viral vector to determine the feasibility of the shrimp viral vector to deliver different payloads (antigen/therapeutic RNA) through diet in crustacean (Alenton, Mai & Dhar. 2023. PNAS-Nexus 2: 1-9). This opens a new frontier in developing antiviral therapy and gene delivery in crustaceans and opens an opportunity to engineer RNA viruses infecting fish.
To determine the feasibility of generating infectious fish virus in insect cells, we attempted to produce a Viral Nervous Necrosis (VNN) virus, Red-Spotted Grouper Nervous Necrosis Virus (RGNNV), in insect cells, Sf9. VNN causes large-scale mortalities and major economic losses to a wide range of marine and freshwater fish including striped bass, barramundi (also called Asian sea bass), European sea bass, grouper, Atlantic halibut, Atlantic cod, tilapia, and many others.
When full-length genomic RNA1 and RNA2 of RGNNV were cloned using a baculovirus expression system and recombinant baculovirus was used to infect Sf9 cells, mature virus particles of RGNNV were observed by transmission electron microscopy; and the full-length RNA1 and RNA2 of RGNNV were amplified by RT-PCR. This suggests both genomic segments of RGNNV were expressed and packaged within the capsid protein to form mature virion. When Sf9 cell-derived RGNNV was used to infect tilapia cell line Omb, cytopathic effects were observed. Additionally, using a TaqMan real-time RT-PCR assay, the viral copy number was found to increase over time in the study, which indicated successful replication of the insect cell derived RGNNV in fish cells. RGNNV produced in fish cells were then used to inject live hybrid sea bass. The virus was detected in target tissue and histopathological manifestation in brain and eye were recorded. The findings clearly demonstrated that infectious RGNNV can be produced in insect cells. The new ability to produce a fish virus in insect cells opens the possibility of using this RGNNV-based viral vector for gene delivery or editing and accelerates the development of antiviral vaccines.