S.K.GIRISHPUNEETH, T. G.,2023-12-222023-12-222022https://krishikosh.egranth.ac.in/handle/1/5810204240Reverse vaccinology (RV) is a modern technique that accelerates the discovery of potential vaccine candidates suitable in vaccine development. Red sea bream iridovirus (RSIV) is the etiological agent responsible for Red sea bream iridovirus disease (RSIVD) in fish leading to huge economic losses to the aquaculture industry. The present study was aimed to screen, isolate, characterize RSIV genome and develop a vaccine candidate using the RV approach to control the RSIV infection. In this study RSIV was isolated from a moribund Asian sea bass cultured in estuarine cages. The PCR amplification and sequencing results of the DNA polymerase gene showed similarity with the sequences of RSIV strains from other countries. Experimental infection studies revealed 100% mortality in the RSIV challenged Asian sea bass on 6th day post infection (dpi). Histopathogical observations in the spleen, kidney and liver tissues were similar to that of typical RSIV infection. Replication efficiency test in the fish cell lines revealed that the RSIV could infect eleven cell lines with viral titers ranging from 102.6 to 106.3 TCID50/ml. The Illumina Hiseq sequencing of RSIV revealed that the complete genome size of RSIV is 111,557 bp with GC content of 53%. The phylogenetic analysis of the 26 core genes including Major capsid protein indicated that the virus belong to the genus Megalocytivirus of the RSIV-Genotype II. The genome encoded 114 ORFs of which 38 ORFs were predicted as functional proteins while the rest were hypothetical proteins. Of the 114, two proteins (MCP and ORF036) were identified as potential vaccine candidates using the RV strategy. The vaccine targets were cloned and expressed in Escherichia coli BL21 (DE3) cells. Asian sea bass vaccinated (injection method) with purified recombinant proteins, MCP and ORF036 emulsified with ISA763 adjuvant showed protection against RSIV infection with relative percentage survival of 76.7% and 63%, respectively.EnglishThesis