Dr. K. RIJI JOHNB. SIVARAMAN, M.F.Sc.Dr. M. ROSALIND GEORGEDr. A. SRINIVASANDr. K. VEERABHADRAN2020-07-302020-07-302018http://krishikosh.egranth.ac.in/handle/1/5810150096Infectious diseases have led to the most devastating problem in aquaculture sector. Viral diseases are one of the major challenges to aquaculturists because it is very difficult to control once they occur in the system. Outbreaks of the viral diseases due to various improper farm managements lead to a greater economic loss in aquaculture production. Prevention is the only way to control disease incidence caused by viral pathogens. Incorrect use of antibiotics and chemical drugs in aquaculture is associated with several deleterious side effects. Instead of using antibiotics and chemical drugs, vaccination is an effective tool in controlling, preventing, protecting and recovery of fish from virus diseases in cultured fish contributing to sustainability in the aquaculture sector. Therefore, the development of viral vaccines against emergent viral diseases is crucial for promoting successful aquaculture production. In this study, experimental inactivated similar damselfish virus (SRDV) vaccine and recombinant major capsid protein vaccine were prepared and immunogenicity and protection against ranavirus infection of the vaccine were investigated in koi carp (Cyprinus carpio), seabass (Lates calcarifer) and similar damselfish (Pomacentrus similis). Firstly, experimental inactivated SRDV vaccine was prepared and expression profiles of immune related genes against virus challenge of the vaccine were investigated in koi carp. Formalin-inactivated virus at 4°C for 2 days exhibited no typical clinical symptoms of SRDV infection and no mortality following challenge at 28-day post-vaccination. The immune response was induced by intraperitoneal injection of koi carp with formalin-inactivated vaccine with added Quil-A® adjuvant. The expression levels of genes including IRF-7 in spleen of koi immunized with vaccine added Quil-A® adjuvant was highly up-regulated (52.2 fold) at 24 h post infection of the virus but in kidney it showed down-regulation (0.4 fold). Interestingly, the highest level of up-regulation (16 fold) was recorded at 96 h post infection of SRDV. Like IRF-7, highest up-regulation of IL-10 gene was observed in spleen of koi immunized with vaccine added Quil-A® adjuvant at 24 post infection of the virus while kidney was showed down-regulation. This experiment provides strong evidence to show the expression of immune related genes (IRF-7 and IL-10) which is up-regulated during the ranavirus infection. It also suggests that Quil-A® adjuvant enhances the immune response of the vaccine candidates. In the next experiment, expression profiles of immune related genes against virus challenge against formalin-inactivated SRDV vaccine were investigated in seabass. The immune response was induced by intra-peritoneal injection with inactivated viral vaccine added Quil-A® adjuvant. The expression levels of IRF-7 in the kidney and spleen of seabass immunized with vaccine added Quil-A® adjuvant was highly up-regulated (3 and 4.8 fold) at 24 h post challenge of the virus and it continued to be up-regulated (7.23 and 7.49 fold) at 48 hpi. IL-10 was slightly up- regulated (1.09 and 1.87 fold) in kidney and spleen at 24 h post infection. Like IRF-7, the expression of IL-10 showed continued up-regulation (2.23 and 4.81 fold) in kidney and spleen at 48 h post virus challenge. Expression profiles of immune related genes (IRF-7 and IL-10) in the kidney and spleen of seabass immunized with vaccine added adjuvant were up-regulated at 48 hpi of the virus. In comparison, spleen of seabass immunized with vaccine added adjuvant showed highest expression profiles than kidney. This study also provided an evidence for the presence of expression profiles of immune-related genes during the SRDV infection. The study also strongly suggests that Quil-A® adjuvant enhances the immune response of the vaccine candidates. Development of recombinant MCP vaccine was carried out to assess its efficacy in fish. Recombinant MCP gene inserted into cloning vector pTZ57R/T was confirmed by colony PCR. Subsequently, the plasmid was extracted from MCP gene inserted vector pTZ57R/T and confirmed by agarose gel electrophoresis. The MCP gene of SRDV containing BamHI and XhoI as restriction sites was amplified from viral DNA with specific primer set and confirmed by agarose gel electrophoresis with the target size of 1416bp. The gel purified PCR product was double digested with the said enzymes and ligated into pTriEx1.1 vector followed by transformation in E.coli DH5α competent cell. Plasmids from two clones namely pTriEx-MCP-1416-1 and pTriEx-MCP-1416-3 were transformed into E. coli BL21 (DE-3) pLacI. Two separate colonies obtained after transformation of pTriEx-MCP-1416-1 and pTriEx- MCP-1416-3 E. coli BL21 (DE-3) pLacI were tested for the expression of MCP gene. The polyclonal antibody produced from rabbit against the purified protein of SRDV has neutralised the homologous virus infectivity in vitro. The crude protein mixture obtained from the colony of E. coli BL21 (DE-3) strain transformed with MCP inserts was used to assess the efficacy in similar damselfish and koi carp by intra-peritoneal injection. After challenge with SRDV, damselfish immunized with recombinant protein showed the low level of protection with of relative percentage survival (RPS) value of 18.8% while vaccine added Quil-A® adjuvant showed RPS of 26%. No specific mortality was found in all treated groups of koi including control group-B which had received virus alone. Therefore, further investigations are required for the development of effective recombinant protein vaccine and assess its efficacy against SRDV challenge in marine and freshwater fishes.ennullThesis