Betanodavirus virulence to european sea bass (dicentrarchus labrax)a new insight into virus-host interaction

Resumo

Viral diseases are the result of the pathogen virulence, the host immunity mechanisms, and the interaction between those components, which constitutes the so called host-pathogen interaction. This PhD Thesis is a comprehensive study on the interaction between European sea bass (Dicentrarchus labrax) and nervous necrosis virus (NNV), which is the main viral pathogen affecting this fish species, being responsible for high mortalities and economical losses in the farming industry. Therefore, the main factors involved in this interaction, including those determining the NNV virulence and the antiviral sea bass defence mechanisms have been analysed. This study has evaluated the role of the capsid protein (CP) amino acids 247 and 270 in the virulence of a red-spotted grouper (Epinephelus akaara) nervous necrosis virus (RGNNV) isolate (SpDl_IAusc965.09) to sea bass. This isolate has been sequenced and recombinant viruses, displaying mutations in the above mentioned positions, have been generated by the reverse genetics technique. The effect of these changes on viral replication and virulence has been assessed after experimental infection. In addition, the European sea bass immune response against betanodavirus infections has been deeply analysed. First of all, the sea bass isg15 gene and ISG15 protein have been characterized for the first time. In this regard, the structure of the sea bass isg15 gene, including the description of regulatory motifs, has been analysed, as well as the phylogenetic relationships among different ISG15 proteins, and the transcription profile following polyinosinic:polycytidylic acid (poly I:C) inoculation or RGNNV infection. Furthermore, the antiviral activity of the sea bass ISG15 protein (Dl_ISG15) has been evaluated in vitro. To fulfil this aim, a cellular system, which expresses constitutively Dl_ISG15, has been established and characterized. On the basis of the suitability of this system, several studies on the intracellular and extracellular Dl_ISG15 activity against RGNNV and striped jack (Pseudocaranx dentex) nervous necrosis virus (SJNNV) isolates have been carried out. Finally, an extensive in vivo study on the European sea bass immune response has been performed by analysing the transcriptional profiles of several immunogenes, involved in both, innate and adaptive responses, following infection with RGNNV, SJNNV or attenuated mutant RGNNV viruses.