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 (Delta orf074r) of infectious spleen and kidney necrosis virus (ISKNV) with residual virulence was constructed, and an immunization process was developed to further attenuate Delta orf074r at low temperature (22 degrees C), making it a candidate immersion vaccine for ISKNV. Under conventional aquaculture temperatures (28 degrees C), Delta orf074r was attenuated but still had a mortality rate of 46.7% in the mandarin fish Siniperca chuatsi, indicating that Delta 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 Delta orf074r, we carried out immersion experiments at 22 degrees C. The results showed that the residual virulence of Delta orf074r was further reduced, and all fish survived after immersion in Delta orf074r at 22 degrees C. Then, the immunized fish were maintained at 28 degrees C, and 80% of them were protected against ISKNV challenge; their immune responses were also induced. These results suggested that Delta orf074r could be a candidate immersion vaccine against ISKNV at 22 degrees 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.