P-194. Development of a recombinant vaccine against ISA virus based on synthetic genes S. Valenzuela 1, 2, * , E. Menares 1, 2 , J. Gajardo 3 , M. Rosemblatt 1, 2 , P. Valenzuela 1, 2 , V. Wilhelm 1, 2 . 1 Fundación Ciencia & Vida, Santiago, Chile; 2 Facultad de Ciencias Biológicas, Universidad Andrés Bello, Santiago, Chile; 3 Farmacología en Acuacultura Veterinaria FAV S.A., Santiago, Chile Abstract The infectious salmon anemia (ISA) is a viral disease that affects farmed fish, in particular the Atlantic salmon (Salmo salar). The etiological agent is the ISA virus (ISAV) that belongs to the genus Isavirus of the Orthomyx- oviridae family. It is considered an emergent pathogen that has caused significant economic losses to the Chilean salmon industry. In this work, a prototype recombinant vaccine was developed against the virus and different assays were performed to study its immunogenic and protective properties. To accomplish this, the sequences encoding the four major structural proteins of the virus were designed to optimize their expression in bacteria and were chemically synthesized. By this strategy it was possible to express in E. coli the matrix protein (M1), the amino-terminal half of the nucleoprotein (N-NP), the amino-terminal and carboxyl-ter- minal fragments of the hemagglutinin-esterase (N-HE and C-CHE, respectively), the amino-terminal half of the fusion protein (N-Fp), and another fragment (FP3) that includes domains of the amino and carboxyl regions of the fusion protein. The recombinant proteins were obtained in the form of inclusion bodies, with the exception of the matrix protein that was expressed in both, native and denatured forms. Different combina- tions of the recombinant proteins were formulated and their immunogenic properties were analyzed in rabbits and salmon. Rabbit immunized with the different recombinant proteins presented high titers of antibodies against N-NP and M1 as shown by ELISA. On the other hand, fish that survived to a challenge with live ISAV presented higher titers against NP in comparison to the rest of the recombinant proteins. This high immuno- genicity of NP and M1, made them interesting candidates for an ISAV vaccine. Based on these results, the efficacy of protection of vaccine for- mulations containing mixtures of the recombinant proteins were evalu- ated in salmon challenged with live ISAV. The results indicate that formulations with doses of 5 mg or 10 mg of each of the fragments of the four ISAV structural proteins have a protective effect against ISAV with a relative percent survival (RPS) of 60% to 64%. Moreover, we observed an enhancement of the efficacy of protection from 57% to 75% of a presently available multivalent vaccine containing inactivated ISAV particles when it was combined with the mixture of recombinant ISAV antigens reported here. * Corresponding author. E-mail address: sveterinario@yahoo.com (S. Valenzuela) P-270. Molecular characterization of IRAK-4 and Interleukin-17 genes in the California red abalone ( Haliotis rufescens ) exposed to Vibrio anguillarum V. Valenzuela-Muñoz x , C. Gallardo-Escárate *,x . Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for Aquaculture Research (INCAR), University of Concepción, PO Box 160-C, Concepcion, Chile Abstract Immune function gene expression in the California red abalone (Haliotis rufescens) hemocytes was evaluated following in vivo infection with Vibrio anguillarum. Herein, two candidate genes were characterized and assessed by real time qPCR: interleukin-1 receptor-associated kinase 4 (IRAK-4) and Interleukin 17 (IL-17). IRAK-4 is a mediator in the trans- duction of interleukin (IL) family and it is up-regulate in response a bacterial infection. On other hand IL-17 family consisted of six members, each of one have a different expression pattern and are up-regulate during the inflammatory response. For abalones, relationship between IRAK-4 and IL-17 in response to a bacterial infection has not yet been evaluated. In this report, IRAK-4 and IL-17 homologues were identified from a cDNA library pyrosequenced for H. rufescens and the UTRs for both genes were obtained by RACE-PCR technique. The IL-17 and IRAK-4 transcription were evaluated by qPCR in mantle, hemocytes, muscle, gill tissues. An in vivo infection with V. anguillarum was evaluated in in- dividuals of 60 mm at 0, 3, 6, 12 and 24 hours post-challenge. Our results showed a differential expression pattern between the tissues evaluated. After the challenge, mRNA transcription of both genes evidences an in- crease activity, being the IL-17 belatedly expressed. These results suggest a pivotal role of IL-17 and IRAK-4 in the immune response of the California red abalone. * Corresponding author. E-mail address: crisgallardo@udec.cl (C. Gallardo-Escárate) xThese authors have contributed equally to this work. P-216. Histones h1 and h2b might have a role in the innate immune response of European seabass gonad upon infection with nodavirus Y. Valero 1 , M. Arizcun 1 , C. Cuesta 2 , M.A. Esteban 2 , E. Chaves-Pozo 1,* . 1 Centro Oceanográfico de Murcia, Instituto Español de Oceanografía, Puerto de Mazarrón 30860, Spain; 2 Departamento de Biología Celular e Histología, Universidad de Murcia, Murcia 30100, Spain Abstract In vertebrates, the essential function of the immune system is the defence against infections. In case of fish, a strong innate immune sys- tem preponderates, which acts as the first line of defence against a broad spectrum of pathogens. Within the reproductive organs of vertebrates, the regulation of immune response is tightly regulated in order to avoid germ cell damage. Antimicrobial peptides (AMPs) are increasingly recognized as a critical first line of defence against many pathogens and have been extensively studied in invertebrates and vertebrates species, including fish. The main function described until the date is its action against bacteria; although there are some evidences about its anti-virus activity. Recent studies have determined that several fish AMPs display anti-viral activity against fish virus including nodavirus, a virus that cause encephalopathy and retinopathy (VER) disease in the European sea bass (Dicentrarchus labrax) and known to be vertically transmitted pro- ducing VER disease in the progeny of asymptomatic infected brood- stocks. Histones have been largely known as part of chromatin structure in the nucleosome, however, over last years, their presence and role as AMP has been identified in fish. In this study, we have evaluated their presence and gene regulation of in European sea bass. In this study we determined for the first time the expression pattern of gene histones h1 and h2b in gonads throughout the reproductive cycle of naïve male sea bass. The data related to the histones h1 and h2b gene expression in the male and female gonad upon in vitro challenge with bacteria (Vibrio anguillarum), virus (nodavirus) or poly I:C or in vivo challenge with nodavirus, suggested a role of those genes in the innate immune response of the gonad. Alberto Cuesta and Elena Chaves-Pozo want to thank the Ministerio de Economía y Competitividad for Ramón y Cajal research contract and Yulema Valero want to thank the Instituto Español de Oceanografía for a PhD student's grant. This work was supported by grants of the Ministerio de Economía y Competitividad (AGL2010-20801-C02-01 and AGL2010- 20801-C02-02) and Fundación Séneca (Grupo de Excelencia de la Región de Murcia 04538/GERM/06). Nodavirus strain and SSN-1 cells were kindly donated by Pilar Fernández Somalo (Laboratorio Central de Veterinaria de Algete, Ministerio de Medio Ambiente, Rural y Marino). * Corresponding author. E-mail address: elena.chaves@mu.ieo.es (E. Chaves-Pozo) Abstracts / Fish & Shellfish Immunology 34 (2013) 1692–1752 1743