Low-temperature immunization attenuates the residual virulence of orf074r gene-deleted infectious spleen and kidney necrosis virus: a candidate immersion vaccine.

The development of immersion vaccines for preventing viral aquatic infectious diseases is important for Asian aquaculture. Gene-deletion-attenuated vaccines have good potential for administration via immersion. However, most gene-deletion viruses still have residual virulence, and thus, gene-deletion immersion vaccines for aquatic viruses are rarely developed. Herein, an orf074r deletion strain (Δ orf074r ) of infectious spleen and kidney necrosis virus (ISKNV) with residual virulence was constructed, and an immunization process was developed to further attenuate Δ orf074r at low temperature (22°C), making it a candidate immersion vaccine for ISKNV. Under conventional aquaculture temperatures (28°C), Δ orf074r was attenuated but still had a mortality rate of 46.7% in the mandarin fish Siniperca chuatsi , indicating that Δ orf074r was not suitable for development as an immersion vaccine. However, over 95% of the surviving fish were protected against ISKNV challenge, and the surviving fish had an increased innate response along with an increase in antiviral antibody levels. To further reduce the mortality rate of Δ orf074r , we carried out immersion experiments at 22°C. The results showed that the residual virulence of Δ orf074r was further reduced, and all fish survived after immersion in Δ orf074r at 22°C. Then, the immunized fish were maintained at 28°C, and 80% of them were protected against ISKNV challenge; their immune responses were also induced. These results suggested that Δ orf074r could be a candidate immersion vaccine against ISKNV at 22°C and has good application prospects in the fish farming industry. Our work will aid in the development of an aquatic gene-deletion live attenuated immersion vaccine.IMPORTANCEGlobal aquaculture production yielded a record of 122.9 million tons in 2022. However, ~10% of farmed aquatic animal production is lost each year due to various infectious diseases, resulting in substantial economic waste. Therefore, the development of vaccines is important for the prevention and control of aquatic infectious diseases. Gene-deletion live attenuated vaccines are efficacious because they mimic natural pathogen infection and generate a strong antibody response, thus showing good potential for administration via immersion. However, most gene-deletion viruses still have residual virulence, and thus, gene-deletion immersion vaccines for aquatic viruses are rarely developed. In this study, an orf074r deletion strain (Δ orf074r ) of ISKNV with residual virulence was constructed, and an immunization process was developed to reduce its residual virulence at 22°C, thereby making it a potential immersion vaccine against ISKNV. Our work will aid in the development of an aquatic gene-deletion live-attenuated immersion vaccine.