Detection and characterization of current strains of ISKNV (Infectious necrosis virus of spleen and kidney), through phenotypic and molecular methods

• Grant number: 21/13051-9
• Support Opportunities: Regular Research Grants
• Start date: June 01, 2022
• End date: December 31, 2024
• Field of knowledge: Agronomical Sciences - Fishery Resources and Fishery Engineering - Aquaculture
• Principal Investigator: Cláudia Maris Ferreira Mostério
• Grantee: Cláudia Maris Ferreira Mostério
• Host Institution: Instituto de Pesca. Agência Paulista de Tecnologia dos Agronegócios (APTA). Secretaria de Agricultura e Abastecimento (São Paulo - Estado). São Paulo , SP, Brazil
• City of the host institution: São Paulo
• Associated researchers: Danielle de Carla Dias ; Leonardo Tachibana ; Ricardo Harakava ; Ricardo Luiz Moro de Sousa ; Vander Bruno dos Santos
• Associated research grant(s): 23/14812-9 - Genomic sequencing of ISKNV (infectious spleen and kidney necrosis virus) isolated from tilapia (Oreochromis niloticus) from fish farms in the state of São Paulo - Brazil, AP.R SPRINT
• Associated scholarship(s): 24/08424-9 - Isolation in cell culture of circulating strains of ISKNV (infectious spleen and kidney necrosis virus) in cell lines for aquatic organisms, BP.TT; 23/07957-0 - Isolation in cell culture of circulating strains of ISKNV (infectious spleen and kidney necrosis virus) in two different cell lines for aquatic organisms, BP.TT; 23/00425-3 - Isolation in cell culture of circulating strains of ISKNV (infectious spleen and kidney necrosis virus) in two different cell lines for aquatic organisms, BP.TT; 22/12321-5 - Isolation in cell culture of circulating strains of ISKNV (infectious spleen and kidney necrosis virus) in two different cell lines for aquatic organisms, BP.TT

Abstract

Emerging disease cases, mainly viral ones, are vanguard and concern themes in the last years, for the academic environment as well as the productive sector. Among the emerging diseases in aquaculture that have a severe economic impact, there is the infectious spleen and kidney necrosis virus disease (ISKNV). This viral disease is not a zoonosis, and it is not a required notification disease either, but it brings serious losses to fish farming. The infection is characterized by cellular spleen, kidney, conjunctival cranial tissue and endocardium hypertrophy. Main clinic symptoms are: anorexia, lethargy, rapid opercular movement, body colour change, exophthalmos and abdominal distension. Diagnosis can be done by clinic symptoms, to be confirmed by histopathological tests or PCR. However, to understand about the virulence of the strains and their phylogeny, the viruses need to be isolated in cell cultures. In vitro cell culture or culture is an alternative method that meets the ethical principles of replacing the use of animals in the laboratory. The exclusive use of this technique will allow us to be able to advance in the diagnosis and production of vaccines to control diseases, especially viral ones. Thus, this project aims to detect, through molecular diagnosis, circulating ISKNV strains in the State of São Paulo, characterize them by verifying their virulence, through total genomic sequencing of the virus and isolation in cell culture. Additionally, we will carry out the experimental infection in fingerlings, in order to follow the kinetics of this disease evolution. Fingerlings will be randomly sampled from the population of two fish farms that work with larviculture, in four different hydrographic basins in the State of São Paulo (n=1248). Preference will be given to symptomatic animals, whether native or exotic. Samples will be processed using conventional PCR. Those that are ISKNV positive will proceed to sequencing through the Sanger method for confirmation and isolation in cell culture in two distinct lines: BF-2 cells (Bluegill Fry) and GRF-1 cells (Grunt Fin cell). Samples with an effective cytopathogenic effect will be sent to the Wildlife and Aquatic Veterinary Disease Laboratory (WAVDL), at the University of Florida, for total genome sequencing and phylogenetic analysis. After ISKNV isolation, we will perform the experimental infection that will be monitored by in situ hybridization analyses. The results will help to increase knowledge of ISKNV virulence, with practical applications for the development of monoclonal antibody production, rapid tests, vaccines and other mechanisms for preventing and mitigating damage caused by these pathogens. (AU)