Estudo in vivo e in vitro da infecção viral por IMNV e desenvolvimento de um sistema de ensaio em hemócitos de camarão marinho Litopenaeus vannamei visando a prospecção de agentes antivirais.

Abstract

Shrimp farming has become a very representative industry worldwide, dealing with over millions of tons of products. It is an important economic activity in tropical countries like Brazil. However, the activity has been facing recurrent outbreaks of epizootics, causing heavy losses for the sector. The problems have been mainly caused by the spread of viruses. Infectious myonecrosis virus (IMNV) is one of these epizootic agents that affect shrimp production in Brazil. In the main Brazilian region of shrimp production, it is known that a reduction in the salinities of cultured farms causes viral diseases, that is, when the rainy season of each year begins. However, there are no drugs on the market available to treat or prevent recurrent outbreaks of farm viruses. Therefore, the objective was to study a replication of the IMNV in vivo and in vitro and to test the potential antiviral activity of the Ctn [15-34]. In view of this, a study of the replication of IMNV at controlled salinity levels during the first 12 hours of in vivo infection was carried out, as well as the development of an in vitro assay system using Litopenaeus vannamei hemocytes in culture with the objective to obtain the inhibitory concentration (IC50) of the virus in these cells, together with the search for antiviral substances. Using real-time quantitative PCR and statistical analysis, we found that low salinity facilitates the replication and proliferation of IMNV in vivo, reducing the generation time from 57.4 min (at 35 g L-1, ideal salinity) to 25.2 min to (5 g L-1, stress concentration). Likewise, a positive correlation between the decrease in salinity and the reduction in the time of generation of another virus, hypodermic and hematopoietic infectious necrosis virus (IHHNV) was demonstrated. The IHHNV has a high prevalence rate and normally co-infects shrimps in nurseries where the IMNV emerges as outbreaks. With respect to in vitro replication, fluorescence-based cytotoxicity assays, in combination with quantitative PCR, demonstrated in this work that the IC50 of the IMNV was 227 copies of virus transcripts in hemocytes. Theses assays were then used to test an eicosapeptide, named Ctn[15-34], derived from a cathelicidin, a class of antimicrobial peptides. The peptide Ctn[15-34] inhibited IMNV cytotoxicity (IC50) with concentrations between 0.75 and 12.5 μM. Thus, the present study reports for the first time the replication of the IMNV in vitro and the use of a direct methodology to evaluate the cellular viability of hemocytes that, in turn, support viral replication, as well as apply a system for screening of substances with antiviral and cytoprotective activity. In addition, it demonstrates the antiviral activity of the eicosapeptide Ctn[15-34] for the development of analogues to compose formulations against epizootic diseases of viral nature that affect marine shrimp and aquaculture.