Programa de Doutoramento en Ciencias Mariñas, Tecnoloxía e Xestión

Advances in the knowledge of the antiviral immune response and resistance to Viral Haemorrhagic Septicaemia Virus (VHSV) in turbot (Scophthalmus maximus)

Memoria de tesis presentada por

Patricia Pereiro González para optar al grado de Doctor por la Universidad de Santiago de Compostela

con mención internacional y en modalidad de compendio de artículos

Santiago de Compostela, 2017

Santiago de Compostela, 2017

Dña. BEATRIZ NOVOA GARCÍA, Doctora en Biología y Profesora de Investigación del Consejo Superior de Investigaciones Científicas (CSIC), junto con D. ANTONIO FIGUERAS HUERTA, Doctor en Biología y Profesor de Investigación del Consejo Superior de Investigaciones Científico (CSIC), en calidad de directores de tesis, así como también D. MANUEL LUIS LEMOS RAMOS, catedrático de la Universidad de Santiago de Compostela en el departamento de Microbiología y Parasitología, en calidad de tutor,

INFORMAN:

Que la presente memoria adjunta, titulada “Advances in the knowledge of the antiviral immune response and resistance to Viral Haemorrhagic Septicaemia Virus (VHSV) in turbot (Scophthalmus maximus)”, presentada por Dña. PATRICIA PEREIRO GONZÁLEZ para optar al grado de Doctor por la Universidad de Santiago de Compostela, con mención internacional y en modalidad de compendio de artículos, ha sido realizada bajo nuestra dirección y reúne los requisitos necesarios para ser defendida ante el tribunal calificador.

Y para que así conste, se firma la presente en Vigo, a 10 de abril de 2017.

La Doctoranda El Tutor

Fdo: Patricia Pereiro González Fdo: Dr. Manuel Luis Lemos Ramos

Los Directores de Tesis

Fdo: Dra. Beatriz Novoa García Fdo: Dr. Antonio Figueras Huerta

Santiago de Compostela, 2017

Los directores de la presente tesis doctoral, presentada en formato de compendio de artículos, la Dra. BEATRIZ NOVOA GARCÍA y el Dr. ANTONIO FIGUERAS HUERTA,

DECLARAN:

Que todos los coautores de los artículos científicos incluidos en la presente tesis doctoral aceptan la presentación de los mismos como parte de la tesis doctoral presentada por Dña. PATRICIA PEREIRO GONZÁLEZ, y que todos los coautores no doctores renuncian a presentarlos en sus futuras tesis de doctorado.

Y para que así conste, se firma la presente en Vigo, a 10 de abril de 2017.

Los Directores de Tesis

Fdo: Dra. Beatriz Novoa García Fdo: Dr. Antonio Figueras Huerta

The work presented in this doctoral thesis has been supported by the projects:

- Project CSD2007-00002 ‘‘AQUAGENOMICS’’ of the program Consolider-Ingenio 2010 from the Spanish Ministerio de Ciencia e Innovación.

- Project AGL2011-28921-CO3 “IMTRA-VAC” from the from the Spanish Ministerio de Ciencia e Innovación.

- Project AGL2014-51773-C3 “TRAINEDFISH” from the Spanish Ministerio de Economía y Competitividad.

I also want to thank the Spanish Ministerio de Educación for the predoctoral grant FPU (AP2010-2408).

AGRADECIMIENTOS

Siempre estuve esperando a tener uno de esos días de inspiración divina para escribir los agradecimientos de mi tesis… pero como ese día no llegaba y mi directora de tesis me reñía por no presentarla de una vez… (ya va Bea!!)… ¡aquí están!

Lo primero que me gustaría decir es que durante este tiempo trabajando en el Instituto de Investigaciones Marinas (IIM) me he sentido como en mi segunda casa… he llegado a plantearme si algún día me presentaba por allí como a mí me gusta estar en mi casa… ¡pantuflas y pijama como uniforme indiscutible! Es lo que tiene estar tan agustito (como diría Ortega Cano con unas copas de más)… Por ello, tengo que agradecer infinitamente a mis directores de tesis, Beatriz Novoa y Antonio Figueras, por haberme dado la oportunidad de “instalarme” y ser una más en esa gran casa que es el IIM! Yo era una inexperta total en esto de la investigación… Por ello, llamé a su puerta para hacer unas prácticas de verano durante mis estudios de máster, y ya me quedé! Deciros que os estoy infinitamente agradecida se queda corto. Ya no es sólo todo aquello que me habéis aportado en lo relativo a la ciencia, que es muchísimo más de lo que hubiese imaginado, sino el apoyo incondicional y la confianza que habéis depositado en mí. Y para que nos vamos a engañar… nuestras charlas de café arreglando el mundo, el intercambio de conocimientos seriófilos y cinéfilos, y demás temas que también cultivan el espíritu.

Qué decir de mis compañeros… No he podido tener unos mejores! Mis grandes maestros durante mis comienzos fueron sin lugar a dudas Pablo (iniciándome en el mundo de la ciencia “informática”) y Laura (mi maestra de bata y pipeta). Creo que ambos habéis influido enormemente en eso de que le pillase el gustillo a hacer una tesis doctoral. Os debo mucho, y aunque ambos estéis haciendo vuestras vidas en el extranjero, no me olvidaré nunca de lo que habéis hecho por mí. Otras dos personas que ya no están y que han sido muy importantes tanto en lo personal como en lo profesional son Marimar (AKA MM) y Patri Díaz (AKA PDR). Sois dos tías geniales, y trabajar con vosotras era como coser y cantar… PDR, el día que nos salió la purificación de la WAP65 creo que fue el toque de gracia a lo agradable que fue para mí trabajar a tu lado mano a mano. Quería resaltar también la inestimable ayuda de dos de mis compis que todavía están en el laboratorio… y esos son Sonia y Álex. Sonia, te tengo que dar las gracias por mil cosas, por ser una persona que sabe escuchar, por tu irónico sentido del humor, por ayudarme siempre, y (todos mis compañeros estarán de acuerdo conmigo) por no dejar que el laboratorio se convirtiese en la fiesta del desorden! A veces puede que bromeásemos incluso con eso (“coloca bien eso que viene Sonia y nos riñe!!” jijiji), pero todo el trabajo se hacía más organizado y llevadero gracias a ti. Álex, tú eres un fenómeno… Me encanta de ti la cara de felicidad que se te dibuja cuándo están haciendo ciencia… si a alguien he visto disfrutar con lo que hacía es a ti. Te debo muchísimo también por toda tu ayuda en el lab, y como no, por las risas que nos echamos tantas y tantas veces (chic chic para tiii chic para mi… claro que sí, guapi!! jajaja)…. No me puedo olvidar tampoco de las chicas del Laboratorio Nacional de Referencia de Enfermedades de Moluscos (esto suena a serio…). Raquel, definitivamente quiero ser como tú. Tienes esa actitud alegre y amable que me encanta! Da gusto estar trabajando y tenerle al lado… Bego, muchas gracias por ser tan riquiña siempre conmigo, eres un amor. Un enorme gracias también a nuestro aquaman Rubén Chamorro… un gran profesional y una bellísima persona, que siempre me ha ayudado en todo lo que he necesitado (a pesar de que lo he “vacunado” más de una vez como si de un rodaballo más se tratase…)

Y qué decir de mis compis de despachito!!!!! Que sois los mejores, los que estáis, y los que ya no estáis! Rebe, eres una de las personas más inteligentes que he conocido, y una de las más resilientes también (de hecho he aprendido esta palabra y su significado en persona gracias a ti). No cambies nunca. Mónica, me ha encantado que después de nuestra andadura juntas por el máster, la hayamos continuado por la tesis! Hemos resoplado juntas por los exámenes y también con los experimentos fallidos! Ojalá a partir de ahora resoplemos un poco menos, que ya nos toca! Paolo, il ragazzo italiano, grazie mille por ser como eres. Tienes un corazón enorme… el hecho de que siempre me trajeses algún detallito de tus viajes me demuestra que el aprecio que sentimos es mutuo, ya que con palabras no eras mucho de demostrar, eh!!!  Y bueno… dejo para el final de mi recorrido de despacho a mi amigo Gabriel Forn (AKA Fornitos…). Que decirte que no sepas… que contigo no he ganado a un colega de trabajo, he ganado a un amigo… Echo taaaaannnnnto de menos nuestras coñas! Sé, porque lo sé, que te irá genial en todo lo que te propongas. Y… quiero dar la bienvenida a la última adquisición del despachito, a Marga. Ánimo guapa!, que parece duro al empezar pero luego da mucha pena terminar… Muchas gracias a todos aquellos que han pasado por el laboratorio, tanto antiguos compañeros, como gente de prácticas… todos habéis sido importantes y hemos compartido momentos geniales.

Y no me puedo olvidar del resto del personal del IIM!! Mis chicos de centralita (Manuel y Bárbara), los chicos de informática siempre dispuestos a ayudar, y un largo etcétera!

Escribir esta parte que llega se me hace más difícil… porque es cuando me toca agradecer a mis padres. Difícil, porque uno de los pilares fundamentales en mi vida, mi padre, no llegó a ver mis comienzos en esto de la ciencia… por unas tres semanas nada más… Sé que estarías muy orgulloso de mí, tanto como yo lo estuve siempre de ti. Y qué decir de mi madre… sin ella sí que no estaría dónde estoy… a pesar de que ella hubiese preferido que estudiase fisioterapia!  Gracias mamá, por ser la mejor, por apoyarme siempre, por celebrar conmigo mis logros y consolarme en los fracasos. Gracias a los dos por haberme dado una educación a pesar del esfuerzo que para una familia humilde suponía. Os quiero. Y a mi hermano Gustavo también!, que no me olvido de ti… Sé que tú también, aunque no lo digas, estás muy orgulloso de tu hermana la bióloga.

Quiero agradecer también a Gabriel por haberme aguantado cuando estaba insoportable porque no me salían las cosas, por ayudarme con el inglés ya que yo siempre fui un poco nula, y por todo su apoyo durante mis primeros años en el IIM, ya que no tuve que ser muy fácil de aguantar (stress is in the air…) Mil gracias a mis amig@s, sobre todo a Aia, Bea, Eva, y Manolo por recordarme una y otra vez lo orgullosos que estáis de mí. Y a todos mis amigos, porque aunque fuese tomando una caña o viendo una peli, habéis contribuido durante estos años a que todo haya sido más llevadero.

GRACIAS A TODOS, POR TODO….

A mi padre…

RESUMO

Avances no coñecemento da resposta inmune antiviral e resistencia ao Virus da Septicemia Hemorráxica Viral (VHSV) en rodaballo (Scophthalmus maximus)

O rodaballo é un peixe cun alto valor comercial principalmente en Europa e na China. Aínda que o seu cultivo a día de hoxe está ben establecido, diversos patóxenos poden afectar ó seu estado sanitario, ocasionando importantes perdas económicas no sector. O virus da septicemia hemorráxica viral (VHSV) é unha das principais ameazas no cultivo do rodaballo, xa que non existen tratamentos nin vacinas comercialmente dispoñibles para este patóxeno. O obxectivo da presente tese doutoral foi, en primeiro lugar, incrementar a información dispoñible nas bases de datos no que respecta a secuencias de transcritos de rodaballos relacionadas coa resposta inmune fronte a virus. Grazas á enorme cantidade de secuencias obtidas púidose deseñar un microarray altamente enriquecido nestas secuencias inmunes, o que nos permitiu levar a cabo un amplo análise transcriptómico da resposta a unha infección con VHSV, así como avaliar a actividade dunha vacina de ADN fronte a VHSV tamén deseñada durante a presente tese doutoral. Esta gran cantidade de información permitiunos centrar a nosa atención en certas moléculas ou procesos que estaban a ser afectados pola vacina/infección. Este foi o caso de dous interferóns (IFNs) de tipo I, que foron caracterizados e estudados por primeira vez en rodaballo. Os IFNs de tipo I son as principais moléculas antivirais en vertebrados porque inducen a expresión de numerosos xenes capaces de bloquear a proliferación do virus. Para ampliar o coñecemento sobre este tipo de xenes tamén quixemos indagar na función do IFN de tipo II (ou IFN-gamma), o cal intervén tamén na defensa fronte aos virus pero actúa máis coma un inmunomodulador.

Palabras chave: rodaballo, VHSV, resposta inmune, interferóns de tipo I, interferón-gamma

RESUMEN Avances en el conocimiento de la respuesta inmune antiviral y resistencia al Virus de la Septicemia Hemorrágica Viral (VHSV) en rodaballo (Scophthalmus maximus) El rodaballo (Scophthalmus maximus) es un pez con un alto valor comercial principalmente en Europa y China. Aunque actualmente su cultivo está bien establecido, diversos patógenos pueden afectar a su estado sanitario, ocasionando importantes pérdidas económicas en el sector. El virus de la septicemia hemorrágica viral (VHSV) es una de las principales amenazas en su cultivo, ya que no existen tratamientos ni vacunas comerciales disponibles para este patógeno. El primer objetivo de la presente tesis doctoral fue incrementar la información disponible en las bases de datos en lo que respecta a secuencias de transcritos de rodaballo relacionadas con la respuesta inmune antiviral. Gracias a la enorme cantidad de secuencias obtenidas se pudo diseñar un microarray altamente enriquecido en estas secuencias inmunes, lo que nos permitió llevar a cabo un amplio análisis transcriptómico de la respuesta a una infección con VHSV, así como también evaluar la actividad de una vacuna de ADN frente a VHSV diseñada durante la presente tesis doctoral. Esta gran cantidad de información nos permitió centrar nuestra atención en ciertas moléculas o procesos que estaban siendo afectados por la vacuna/infección. Este fue el caso de dos Interferones (IFNs) de tipo I, que fueron caracterizados y estudiados por primera vez en rodaballo. Los IFNs de tipo I son las principales moléculas antivirales en vertebrados porque inducen la expresión de numerosos genes capaces de bloquear la proliferación del virus. Para ampliar el conocimiento sobre este tipo de genes también quisimos indagar en la función del IFN de tipo II (o IFN-gamma), el cual interviene también en la defensa frente a virus pero actúa más como un inmunomodulador. Finalmente otro gen que llamó nuestra atención fue la Nk-lisina, por lo que lo caracterizamos y analizamos su expresión en rodaballo, encontrando una interesante correlación entre su expresión y la resistencia a VHSV. Palabras clave: rodaballo, VHSV, respuesta inmune, interferones de tipo I, interferón-gamma

ABSTRACT

Advances in the knowledge of the antiviral immune response and resistance to Viral Haemorrhagic Septicaemia Virus (VHSV) in turbot (Scophthalmus maximus)

Turbot (Scophthalmus maximus) is an economically valuable fish in Europe and China. Currently, the culture of this fish is well established, although several pathogens can affect its health status, causing important economic losses in the sector. Viral Haemorrhagic Septicaemia Virus (VHSV) is one of the main threats in turbot farms due to the absence of commercially available treatments and vaccines for VHSV. The first goal of this doctoral thesis was to increase the amount of information available in public databases regarding the transcriptome sequences associated with the antiviral immune response of turbot. Due to the large number of sequences obtained, a microarray highly enriched in antiviral sequences was constructed. This microarray allowed us to conduct a broad transcriptome analysis of the response to VHSV infection and evaluate the activity of a DNA vaccine against VHSV, which was also designed during this doctoral thesis. This information led us to focus our attention on certain molecules or processes affected by the vaccine/infection. This was the case for two type I interferons (IFNs), which were characterized and studied for the first time in turbot. Type I IFNs are the main antiviral molecules in vertebrates, as they induce the expression of numerous molecules with the ability to block viral proliferation. To increase our knowledge about these genes, we also sought to investigate the role of the type II IFN (or IFN-gamma), which also acts in the defence against viruses but mainly functions as an immunomodulatory molecule.

Keywords: turbot, VHSV, immune response, type I interferons, interferon-gamma

LIST OF SCIENTIFIC PUBLICATIONS

AND QUALITY CRITERIA

LIST OF PUBLICATIONS

Scientific publications included in this doctoral thesis:

Pereiro P, Balseiro P, Romero A, Dios S, Forn-Cuni G, Fuste B, Planas J V, Beltran S, Novoa B, Figueras A (2012) High-Throughput Sequence Analysis of Turbot (Scophthalmus maximus) Transcriptome Using 454-Pyrosequencing for the Discovery of Antiviral Immune Genes. PLOS ONE, 7: e35369.

Pereiro P, Martinez-Lopez A, Falco A, Dios S, Figueras A, Coll JM, Novoa B, Estepa A (2012) Protection and antibody response induced by intramuscular DNA vaccine encoding for viral haemorrhagic septicaemia virus (VHSV) G glycoprotein in turbot (Scophthalmus maximus). Fish & Shellfish Immunology, 32: 1088–1094.

Pereiro P, Dios S, Boltaña S, Coll JM, Estepa A, MacKenzie S, Novoa B, Figueras A (2014) Transcriptome Profiles Associated to VHSV Infection or DNA Vaccination in Turbot (Scophthalmus maximus). PLOS ONE, 9: e104509.

Pereiro P, Costa MM, Díaz-Rosales P, Dios S, Figueras A, Novoa B (2014) The first characterization of two type I interferons in turbot (Scophthalmus maximus) reveals their differential role, expression pattern and gene induction. Developmental & Comparative Immunology, 45: 233-244.

Pereiro P, Forn-Cuní G, Figueras A, Novoa B. (2016) Pathogen-dependent role of turbot (Scophthalmus maximus) interferon-gamma. Fish & Shellfish Immunology, 59: 25-35.

Quality criteria of the journals:

IF 2015 5-YEAR IF Q1 Ranking ISSN PLOS ONE 3.057 3.535 Multidisciplinary Sciences 11/63 1932-6203 Veterinary Sciences 2/38 Fish & Shellfish Immunology 3.025 3.277 Marine and Freshwater Biology 8/104 1050-4648 Fisheries 4/52 Developmental & Comparative Immunology 3.620 3.543 Zoology 6/161 0145-305X

Other scientific publications:

Pereiro P, Figueras A, Novoa B (2012) A novel hepcidin-like in turbot (Scophthalmus maximus L.) highly expressed after pathogen challenge but not after iron overload. Fish & Shellfish Immunology, 32: 879–889.

Costa MM, Pereiro P, Wang T, Secombes CJ, Figueras A, Novoa B (2012) Characterization and gene expression analysis of the two main Th17 cytokines (IL- 17A/F and IL-22) in turbot, Scophthalmus maximus. Developmental & Comparative Immunology, 38: 505-516.

Rodríguez-Ramilo ST, De La Herrán R, Ruiz-Rejón C, Hermida M, Fernández C, Pereiro P, Figueras A, Bouza C, Toro MA, Martínez P, Fernández J (2014) Identification of Quantitative Trait Loci Associated with Resistance to Viral Haemorrhagic Septicaemia (VHS) in Turbot (Scophthalmus maximus): A Comparison Between Bacterium, Parasite and Virus Diseases. Marine Biotechnology, 16: 265-276.

Diaz-Rosales P*, Pereiro P*, Figueras A, Novoa B, Dios S (2014) The warm temperature acclimation protein (Wap65) has an important role in the inflammatory response of turbot (Scophthalmus maximus). Fish & Shellfish Immunology, 41: 80-92. (*) Equal contribution.

Varela M*, Diaz-Rosales P*, Pereiro P, Forn-Cuni G, Costa MM, Dios S, Romero A, Figueras A, Novoa B (2014). Interferon-Induced Genes of the Expanded IFIT Family Show Conserved Antiviral Activities in Non-Mammalian Species. PLoS ONE, 9: e100015. (*) Equal contribution.

Moreira R, Pereiro P, Costa MM, Figueras A, Novoa B (2014) Evaluation of reference genes of Mytilus galloprovincialis and Ruditapes philippinarum infected with three bacteria strains for gene expression analysis. Aquatic Living Resources, 27: 147–152.

Pereiro P*, Varela M*, Diaz-Rosales P, Romero A, Dios S, Figueras A, Novoa B (2015) Zebrafish Nk-lysins: First insights about their cellular and functional diversification. Developmental & Comparative Immunology, 51: 148–159. (*) Equal contribution.

Moreira R, Pereiro P, Canchaya C, Posada D, Figueras A, Novoa B (2015) RNA-Seq in Mytilus galloprovincialis: comparative transcriptomics and expression profiles among different tissues. BMC Genomics, 16: 728.

Smith LC, Barela Hudgell MA, Deiss T, Golconda P, Krasnec K, Lun CM, Neely H, Pereiro P, Priyam M, Semple SL, Skokal U, Tacchi L, Takizawa F, Yadav S, Xu Z (2016) Conference Report: The 13th Congress of the International Society of Developmental and Comparative Immunology. Developmental & Comparative Immunology, 55: 56-64.

Figueras A, Robledo D, Corvelo A, Hermida M, Pereiro P, Rubiolo JA, Gómez- Garrido J, Carreté L, Bello X, Gut M, Gut IG, Marcet-Houben M, Forn-Cuní G, Galán B, García JL, Abal-Fabeiro JL, Pardo BG, Taboada X, Fernández C, Vlasova A, Hermoso- Pulido A, Guigó R, Álvarez-Dios JA, Gómez-Tato A, Viñas A, Maside X, Gabaldón T, Novoa B, Bouza C, Alioto T, Martínez P (2016) Whole genome sequencing of turbot (Scophthalmus maximus; Pleuronectiformes): a fish adapted to demersal life. DNA Research, 23: 181-192.

Pereiro P, Figueras A, Novoa B (2016) Turbot (Scophthalmus maximus) vs VHSV (Viral Hemorrhagic Septicemia Virus): a review. Frontiers in Physiology, 7: 192.

Novoa B, Romero A, Álvarez ÁL, Moreira R, Pereiro P, Costa MM, Dios S, Estepa A, Parra F, Figueras A. (2016) Antiviral activity of myticin C peptide from mussel: an ancient defence against herpesviruses. Journal of Virology, pii: JVI.00591-16.

Piazzon MC, Galindo-Villegas J, Pereiro P, Estensoro I, Calduch-Giner JA, Gómez- Casado E, Novoa B, Mulero V, Sitjà-Bobadilla A, Pérez-Sánchez J (2016) Differential modulation of IgT and IgM upon parasitic, bacterial, viral and dietary challenges in a perciform fish. Frontiers in Immunology, 7: 637.

Forn-Cuní G, Varela M, Pereiro P, Novoa B, Figueras A (2017) Conserved gene regulation during acute inflammation between zebrafish and mammals. Scientific Reports, 7: 41905.

Book chapter:

Martínez P, Robledo D, Rodríguez-Ramilo ST, Hermida M, Taboada X, Pereiro P, Rubiolo JA, Ribas L, Gómez Tato A, Álvarez-Dios JA, Piferrer F, Novoa B, Figueras A, Pardo BG, Fernández J, Viñas A, Bouza C (2016) Turbot (Scophthalmus maximus) genomic resources: application for boosting aquaculture production. In: Genomics in Aquaculture, MacKenzie SA, Jentoft S (Eds.). Elsevier. pp. 131-163. ISBN: 978-0- 12-801418-9

INDEX

CHAPTER 1: GENERAL INTRODUCTION AND OBJECTIVES 1

1. General Introduction 3

1.1. Aquaculture 3 1.2. Turbot production 5 1.3. Diseases affecting turbot culture 7 1.3.1. Bacterial diseases 7 1.3.2. Parasitic diseases 8 1.3.3. Viral diseases 9 1.3.3.1. Viral Haemorrhagic Septicaemia Virus (VHSV) 10 1.3.3.2. Control and prevention of VHSV 13 1.4. Teleost Immune System 15 1.4.1. Overview 15 1.4.2. Antiviral immune mechanisms 16 1.4.2.1. Virus sensors 16 1.4.2.2. The interferon system 20 1.4.2.3. Inflammation 23 1.4.2.4. Antiviral strategies of cytotoxic T lymphocytes and natural killer cells 24 1.5. References 26

2. Objectives 44

CHAPTER 2: High-throughput sequence analysis of the turbot (Scophthalmus maximus) transcriptome using 454 pyrosequencing for the discovery of antiviral immune genes 45

Abstract 47

Introduction 47

Results and Discussion 48

Conclusions 58

Materials and Methods 60

References 62

Supporting Information 65

CHAPTER 3: Protection and antibody response induced by intramuscular DNA vaccine encoding for viral haemorrhagic septicaemia virus (VHSV) G glycoprotein in turbot 67 (Scophthalmus maximus)

Abstract 69

Introduction 69

Materials and Methods 70

Results 71

Discussion 73

References 74

CHAPTER 4: Transcriptome profiles associated to VHSV infection or DNA vaccination in turbot (Scophthalmus maximus) 77

Abstract 79

Introduction 79

Materials and Methods 80

Results and Discussion 84

Conclusions 96

References 96

Supporting Information 99

CHAPTER 5: The first characterization of two type I interferons in turbot (Scophthalmus maximus) reveals their differential role, expression pattern and gene induction 101

Abstract 103

Introduction 103

Material and Methods 104

Results 105

Discussion 110

Conclusion 113

References 113

Supplementary data 115

CHAPTER 6: Pathogen-dependent role of turbot (Scophthalmus maximus) interferon-gamma 117

Abstract 119

Introduction 119

Material and Methods 120 Results 122

Discussion 125

Conclusions 127

References 127

Supplementary data 131

CHAPTER 7: GENERAL DISCUSSION AND CONCLUSIONS 133

1. General Discussion 135

2. References 138

3. Conclusions 141

RESUMEN Y CONCLUSIONES EN ESPAÑOL 143

1. Resumen 145

2. Conclusiones 154

CHAPTER 1 GENERAL INTRODUCTION AND OBJECTIVES

1

2

1. GENERAL INTRODUCTION

1.1. AQUACULTURE

Following the FAO (Food and Agriculture Organization of the United Nations) definition, the term aquaculture refers to the farming of aquatic organisms, including fish, molluscs, crustaceans and aquatic plants. Farming implies some sort of intervention in the rearing process to enhance production, such as regular stocking, feeding, protection from predators, etc. Farming also implies individual or corporate ownership of the stock being cultivated, the planning, development and operation of aquaculture systems, sites, facilities and practices, and production and transport.

Currently, overfishing is an important problem mainly due to the on-going increase in the size of the human population. According to the most recent United Nations estimates, the human population of the world is expected to reach 8 billion people in 2024. Fish is a food with excellent nutritional value, providing high quality protein and a wide variety of vitamins, minerals and essential fatty acids. Globally, fish accounts for approximately 17% of animal protein intake, even exceeding 50% in many countries (Thilsted et al., 2014). Moreover, there is increasing interest, especially in developed countries, in having a healthier lifestyle, and fish represent one of the best choices for maintaining a balanced and optimal diet. For these reasons, fish consumption has increased extraordinarily during the last decades. Fortunately, aquaculture has emerged as an alternative supply of fish and shellfish. In recent years, although capture fishery production has been flat at approximately 90 million tonnes per year, aquaculture has continued to show sustained growth, amounting to 63.6 million tonnes in 2011 (Figure 1). A total of 154 million tonnes of fish were produced from all sources in 2011, of which 126 million tonnes were available for direct human consumption (Thilsted et al., 2014; FAO 2014).

3

Figure 1. World capture fisheries and aquaculture production since 1950 to 2012 (FAO, 2014) The contribution of aquaculture to global total fish production reached 43.1% in 2013, and it was only 30.6% a decade ago in 2003 (Figure 2). The FAO estimates that this percentage will reach 65% in 2030.

Figure 2. Share of aquaculture in total fish production (FAO, 2014)

The main producer of aquaculture products is China, followed by Indonesia, India, Vietnam, Filipinas, Bangladesh and Korea; the first European country is ranked 8th and corresponded to Norway. Fish represents almost 50% of the aquaculture in the world, although some shellfish species have very high commercial value. 4

In Europe, the most prominent aquaculture products are highly commercially valuable fish and molluscs. In 2012, European aquaculture accounted for 4.32% of worldwide production (excluding aquatic plants and non- food products) (FAO, 2014), but it is a leader in the culture of some species, such as Atlantic salmon, rainbow trout, sea bass, sea bream, turbot and Mediterranean mussel.

The main cultured fish species in Spain are listed in the next table (Table 1):

Table 1. Spain production (tons) evolution by species (2005-2014) (FEAP, 2015)

1.2. TURBOT PRODUCTION

Turbot (Scophthalmus maximus) is an economically important flatfish species belonging to the family Scophthalmidae (order Pleuronectiformes) that is widely distributed from Norway to the Mediterranean and the Black Sea (Nielsen, 1986). The first steps in the production of this fish were undertaken in Scotland (United Kingdom) during the 1970s, but then turbot aquaculture was quickly expanded to Spain and France (FAO). After numerous technical and biological improvements, production was also initiated in other European countries (Portugal, Denmark, Germany, Iceland, Ireland, Italy, Norway and Wales). Currently, the culture of this fish is well established, and the complete farm-raising cycle is conducted in land-based aquaculture facilities (Figure 3). In addition to great improvements in the facilities, other decisive factors have contributed to the development of turbot aquaculture. These have included the production of dry feeds and the development of vaccines for some of the most important bacterial diseases affecting turbot (FAO).

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Figure 3. Production cycle of Scophthalmus maximus (http://www.fao.org/fishery/culturedspecies/Psetta_maxima/en)

In Europe, turbot aquaculture production was approximately 11,000 tonnes in 2014, 38.3% higher than production in 2013, with Spain (particularly the Galicia region, with 99% of national production) being the main European producer (APROMAR, 2015). Indeed, 7,808 tonnes were produced in Spain in 2014. This species, which is native to Europe, is also cultured in Chile (approximately 107 tonnes per year) but especially in China, which reached an annual level of 50,000– 60,000 tonnes in recent years and is the largest producer of turbot in the world (FAO, 2010). Currently, one-third of the turbot we find in the markets in Spain comes from fisheries (APROMAR, 2015).

Nevertheless, there are currently some limitations affecting the culture of this flatfish, such as low genetic renewal and specific diseases that cause increases in mortality and morbidity, with subsequent economic losses.

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1.3. DISEASES AFFECTING TURBOT CULTURE

The development of turbot aquaculture caused a parallel increase in pathological conditions affecting the culture of this flatfish. Several pathogens, including bacteria (Toranzo el at., 2005), viruses (Walker & Winton, 2010) and parasites (Álvarez-Pellitero, 2008) affect the health status of farmed fish, causing important economic losses. Despite the relevance of turbot culture and the associated pathological processes, our knowledge of its immune system is still fragmented, and little is known about host-pathogen interactions. The pathways implicated in the response against pathogens remain incomplete in fish, and understanding how these defence mechanisms act is a relevant factor in enhancing the resistance of cultured fish to diseases. Although there are currently effective treatments or vaccines available against a variety of pathogens affecting turbot culture, other diseases, especially those induced by viral agents, do not have an easy solution. Neither vaccines nor therapeutic treatments are commercially available for the most of the viral diseases affecting fish.

1.3.1. Bacterial diseases

Several bacterial pathogens can be found in turbot facilities, with four of them representing important threats to the industry. Tenacibaculum maritimum, the causative agent of tenacibaculosis, is a filamentous bacterium responsible for severe mortality episodes. Fortunately, a specific turbot vaccine has been developed and shows a high protection rate, but the use of antibiotics is still necessary in some cases (Avendaño-Herrera et al., 2006). The gram-positive bacterium Streptococcus parauberis is associated with lesions and signs of streptococcosis in cultured turbot (Domenech et al., 1996). Good protection rates were also achieved with a bacterin against this disease in turbot (Romalde et al., 1996; Toranzo et al., 1995). Two gram-negative bacteria were also implicated in disease and mortality outbreaks, Vibrio (Listonella) anguillarum and Aeromonas salmonicida subsp. salmonicida. Currently, vibriosis is prevented via immersion vaccination with inactivated bacteria in small turbot (0.5-2 g). Furunculosis due to A. salmonicida was an extreme challenge for investigators for several years due to

7 dramatic mortality episodes in the salmon industry. In European turbot farms, several epizootic outbreaks of acute furunculosis have been reported (Lillehaug et al., 2003; Nougayrede et al., 1990; Pedersen et al., 1996; Toranzo & Barja, 1992). Although the application of highly effective vaccines in salmon is now a fact, a good vaccine against furunculosis is not yet commercially available for turbot. For this reason, antibiotics are needed to combat furunculosis episodes.

1.3.2. Parasitic diseases

The main parasitic agents affecting turbot culture include Neoparamoeba pemaquidensis (causing amoebic gill disease (AGD)), Trichodina spp. (trichodiniasis), Philasterides dicentrarchi (scuticociliatosis), Tetramicra brevifilum (microsporidiosis) and Enteromyxum scophthalmi (myxosporidiosis). The amoeba N. pemaquidensis, which causes severe gill tissue damage, was determined to be a causative agent of mortality in turbot cultures during the 1990s (Dyková et al., 1995, 1998). Freshwater baths are the main treatment to combat AGD. A high density of the ciliated protozoan Trichodina spp. can also produce skin and gill damage. It has been shown that natural infection with this parasite in cultured turbot could significantly reduce the growth rate (Sanmartín Durán et al., 1991), as was also observed in other fish species. Trichodiniasis is mainly combated using formalin baths. The first episodes of infection by histophagous scuticociliates in farmed turbot were reported in 1994 and 2000 (Dyková & Figueras 1994; Sterud et al. 2000), although it was not until 2001 that it was determined that P. dicentrarchi was the species responsible for these outbreaks (Iglesias et al., 2001). External signs include haemorrhagic skin ulcers and darkened skin, but when the parasite invades the internal tissues, the organs suffer important damage due to the histophagous activity of P. dicentrarchi. Erratic swimming, equilibrium loss, lethargy, anorexia, exophthalmia, and abdominal distension due to the accumulation of ascitic fluid in the body cavity are some of the signs observed under severe infection (Iglesias et al., 2001). Mortality can reach 100% in many cases, and it is therefore urgently necessary to develop efficient prevention and control strategies. Some laboratories are trying to find an efficient vaccine against this parasite, and some encouraging results were achieved (Palenzuela et al., 2009;

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Sanmartín et al., 2008). Although T. brevifilum does not cause severe mortality episodes, infection by this microsporidian species can affect the growth rate and probably susceptibility to other secondary infections (Figueras et al., 1992). Finally, E. scophthalmi was described by Palenzuela et al. (2002) as a species causing severe catarrhal enteritis and death in cultured turbot. The mortality rate can reach up to 100%, and the absence of effective drugs against this myxosporean also represents a new challenge that needs to be solved.

1.3.3. Viral Diseases

Viruses are probably the most destructive pathogens encountered in aquaculture and are a serious concern, since no specific chemotherapies are available. Illustrating the impact of fish viruses, 8 of the 10 notifiable fish diseases (diseases with great social and economic and/or public health repercussions or present or potential risk for the aquaculture industry) appearing at the 2014 Aquatic Animal Health Code of the OIE (Office International des Epizooties, now the World Organization for Animal Health; http://www.oie.int) are caused by viruses. The most relevant viruses affecting turbot farms are recorded in this section.

Nodavirus, causing viral encephalopathy and retinopathy (VER), produces important economic losses in the larval culture of a great number of marine fish species, but only sporadic cases have been reported in turbot (Barja, 2004). In these isolated cases, turbot developed the classical signs of VER, and high mortality levels were detected (Johansen et al., 2004). Nevertheless, the susceptibility of this flatfish to nodavirus is elevated, as was demonstrated in experimental infections (Húsgağ et al., 2001; Montes et al., 2010), and therefore this disease should be taken into consideration.

IPN virus shows a similar perspective because, although it mainly causes infectious pancreatic necrosis (IPN) in salmonids, punctual cases of infection were detected in turbot (Barja, 2004). Although very different degrees of mortality were observed depending on the IPNV serotype, infected turbot do not show the typical pancreatic necrosis symptoms (Novoa et al., 1995). These investigations suggest

9 that turbot could act principally as an asymptomatic carrier, transferring the infection to other susceptible species.

Finally, VHSV causes an important viral disease (viral haemorrhagic septicaemia (VHS)) affecting salmonids, but VHSV outbreaks have been detected in other farmed fish species such as turbot (Ross et al., 1994; Schlotfeldt et al., 1991). The infected individuals develop the characteristic symptoms of VHS. Although the mortality rate in natural infection cases is relatively low, this rhabdovirus is included within the OIE list of notifiable diseases.

In addition to these main viral diseases, other viruses can affect turbot, although due to the lower incidence or severity in the culture of this flatfish, these are not discussed in this introduction. Some of these viruses are Herpesvirus scophthalmi (Hellberg et al., 2002) and erythrocytic virus (Lamas et al., 1996).

1.3.3.1 Viral Haemorrhagic Septicaemia virus (VHSV)

This aetiological agent causes an important viral disease affecting rainbow trout (Oncorhynchus mykiss) and other salmonids (Castric & de Kinkelin, 1980; Hørlyck et al., 1984; Wolf, 1988), but VHSV outbreaks have been detected in other farmed fish species such as turbot (Ross et al., 1994; Schlotfeldt et al., 1991). Turbot (Scophthalmus maximus) is a high-value farmed marine fish with growing demand and production levels in Europe and Asia. In recent years, due to intensive farming conditions, disease outbreaks caused by turbot-specific strains have frequently become severe problems faced by the turbot industry.

VHSV is a fish pathogen belonging to the genus Novirhabdovirus within the family Rhabdoviridae (Trdo et al., 2005; Walker et al., 2000). Rhabdoviruses are bullet shaped enveloped viruses 170-180 nm in length and 60-70 nm in width (Elsayed et al. 2006), with a simple negative-sense, single-stranded RNA (ssRNA) genome of approximately 11 kb (Schutze et al., 1999). The typical rhabdoviral genome encodes five basic structural proteins: nucleoprotein (N), polymerase- associated phosphoprotein (P), matrix protein (M), glycoprotein (G), and large RNA-dependent RNA polymerase (L). Members of the genus Novirhabdovirus are distinguished by the presence of a sixth gene encoding a non-structural or non- virion (NV) protein located between the G and the L genes in the genome (Kurath

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& Leong, 1985; Schutze et al., 1999); this gene has been implicated in pathogenesis (Ammayappan & Vakharia, 2011; Choi et al, 2011) (Figure 4). All rhabdoviruses possess non-coding 3' leader and 5' trailer sequences, which are also known as 3’ and 5’ untranslated regions (UTRs).

Figure 4. Genetic organization of the VHSV genome. The gene order of VHSV is 3'- leader-N-P-M-G-NV-L-trailer-5' (Pereiro et al., 2016)

Structurally, all rhabdoviruses have two major structural components: a helical ribonucleoprotein core (RNP) and a surrounding envelope (Figure 5). In the RNP, genomic RNA is tightly encased by the nucleoprotein. The phosphoprotein and the large protein (L-protein or polymerase) are also associated with the RNP. The glycoprotein (G) forms trimeric spikes that are tightly inserted into the lipid bilayer (typical of enveloped viruses and derived from portions of the host cell membrane). Beneath and associated with the membrane by hydrophobic and electrostatic interactions is a layer formed by the matrix protein (M), which condenses the RNP. Moreover, the M protein is also associated with the lipid bilayer and the glycoprotein, forming a link between the ribonucleocapsid and glycoproteins in the viral envelope (Assenberg et al., 2010).

Figure 5. Schematic representation of the morphology and structural components of rhabdoviruses (Pereiro et al., 2016) Phylogenetic analysis has allowed the identification of four major, geographically distinct VHSV genogroups based on N- and G-gene nucleotide variations (Einer-Jensen et al, 2004; Snow et al. 1999, 2004). Genotype I is composed of rainbow trout freshwater isolates (Genotype Ia) and marine isolates

11 from the Baltic Sea (Ib) closely related to those belonging to Ia (Snow et al., 1999). European marine strains are divided into 2 groups: Baltic Sea isolates (Genotype II) and isolates from the North Sea and European Atlantic (Genotype III). Finally, Genotype IV is composed of North American strains. In this regard, genotypes Ia and II revealed low mortality in experimentally infected turbot, while Ib showed an intermediate effect, and the highest mortality levels were obtained in turbot infected with isolates from Genotype III (Snow et al., 2005). The outbreaks detected in turbot farms were mainly caused by the UK-860/94 strain (Genotype III). Indeed, this strain was isolated from an outbreak at the Gigha turbot farm (Scotland) (Ross et al, 1994) and, although it showed low overall mortality (approximately 6%), approximately 14 tonnes of fish were consequently collected and sacrificed as a part of a contingency plan (Hastein et al., 1999), generating subsequent relevant economic losses.

Diseased fish may display nonspecific clinical signs in the early stages of infection, including the rapid onset of mortality (which can reach up to 100% in fry), lethargy, darkening of the skin, exophthalmia, anaemia (pale gills), haemorrhages at the base of the fins, gills, mouth, eyes and skin, a distended abdomen due to oedema in the peritoneal cavity, and severe abnormal swimming behaviour. Some of the symptoms we observed after the intraperitoneal injection of VHSV strain UK-860/94 in juvenile turbot are reflected in Figure 6.

Figure 6. Clinical signs in juvenile turbot infected with VHSV strain UK- 860/94. External hemorrhages are observed around the eyes, mouth and fins. Internal organs also show a severe hemorrhage, especially noticeable in the liver when is compared with a healthy one (Pereiro et al., 2016).

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1.3.3.2. Control and prevention of VHSV

Due to the absence of effective antiviral treatments, prevention is a critical point in the eradication of this disease. Nevertheless, no vaccines are commercially available for VHSV. For more than 30 years, increased effort has been made to produce an efficient, safe and cost-effective vaccine against VHSV using subunits or single viral proteins as well as killed or attenuated viruses (Adelmann et al, 2008; Bernard et al, 1983; de Kinkelin et al, 1980, 1995; Lecocq-Xhonneux et al, 1994; Leong & Fryer, 1993). Although some of these vaccines induced good protection levels in laboratory conditions, sometimes they are unsafe for field use, production might be very expensive or high doses may be required. Deoxyribonucleic acid (DNA) vaccination is based on the administration of a plasmid DNA vector containing the gene encoding a specific antigen. This technology is a powerful tool for the design of effective vaccines against fish pathogens. It has become clear that one of the most efficient methods for inducing a protective immune response against VHS and other Rhabdoviruses in rainbow trout under experimental conditions is DNA vaccination, with vaccines encoding viral membrane glycoproteins being remarkably efficacious (Anderson et al, 1996; LaPatra et al, 2001; Lorenzen et al, 1998, 2000; Winton, 1997). Rhabdoviruses possess a surface glycoprotein (G protein) that serves as the target of virus-neutralizing antibodies (Lorenzen et al, 1990), and the more successful DNA vaccines against these viruses are based on the G glycoprotein gene under the control of the cytomegalovirus promoter (CMV). Intramuscular administration of microgram amounts of plasmid is sufficient for the expression of the viral G glycoprotein on the surface of muscular cells, and this triggers the immune response (Lorenzen et al, 2005; Lorenzen & LaPatra, 2005).

To our knowledge, previous studies on DNA vaccination in S. maximus are scarce and based on protection against nodavirus infection (Sommerset et al, 2003; 2005) and the bacteria Streptococcus iniae (Sun et al, 2010), Vibrio parahaemolyticus (Liu et al, 2011) and Vibrio harveyi (Wang et al, 2011). During this doctoral thesis, a highly protective DNA vaccine against VHSV was developed, reflecting the potential of these vaccines in fish aquaculture.

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Another way to prevent, or at least to reduce the prevalence of one disease, is genetic improvement. Marker-assisted selection (MAS) in fish breeding schemes has become a very promising strategy for obtaining individuals with a certain trait of interest. In fish aquaculture, these traits are specifically focused on growth, sex determination, and resistance to diseases. Although these markers can be morphological, biochemical or cytological, currently, most MAS work uses DNA- based markers, especially after the proliferation of genome-wide studies due to the lower cost of the genome sequencing strategies. These DNA markers can be used to detect allelic variation in the genes underlying a certain trait (Collard et al., 2005). Therefore, selection is not based on the trait itself, but on the marker linked to it. Thus, resistance to fish diseases could be improved by using DNA markers to assist in turbot breeding; this consists of the selection of allelic variations that are linked to disease resistance. The traits are usually controlled by several genes and are known as quantitative traits (Collard et al., 2005). Quantitative trait loci (QTLs) are those regions of the genome containing genes related to a quantitative trait, and the construction of physical linkage maps makes it possible to identify these chromosomal regions (Mohan et al., 1997). The marker used for selection is associated at a high frequency with the QTL of interest due to proximity on the chromosome, and therefore they should co-segregate (genetic linkage) (Mohan et al., 1997).

Numerous QTLs associated with resistance to VHSV have been identified in S. maximus (Rodriguez-Ramilo et al., 2014). Prior to this, QTL analyses were also used to identify those regions associated with resistance to the bacterium Aeromonas salmonicida (Rodríguez-Ramilo et al., 2011) and the parasite Philasterides dicentrarchi (Rodríguez-Ramilo et al., 2013). The existence of an accurate linkage map in turbot (Bouza et al., 2008) was crucial in the detection of these QTLs. Some QTLs were found to be related with resistance to more than one pathogen (Rodriguez-Ramilo et al., 2014), which is very interesting for designing selective breeding programmes. Until the sequencing of the turbot genome (Figueras et al., 2016), the identification of candidate genes associated with genetic markers was mainly based on comparative mapping of the turbot genetic map and the genome of model teleost species by analysing syntenic areas (Rodriguez- Ramilo et al., 2014). Currently, the whole genome sequencing of turbot has led to 14 the identification of numerous candidate genes associated with resistance to VHSV in a reliable and robust manner (Figueras et al., 2016). These genetic markers were located in the genome, and gene mining analysis around the QTLs was conducted using a ± 1 Mb window. Among the more than 200 candidate genes identified for VHSV resistance, some of the most remarkable findings were numerous genes implicated in T-cell activity, the blood coagulation cascade (probably due to the haemorrhagic activity of this virus) and genes related to iron homeostasis and scavenging (such as some transferrin-related genes and hepcidin) (Figueras et al., 2016) With this new information available for studies, our knowledge about the genes implicated in defence against this viral disease will probably grow in the next years.

1.4. TELEOST IMMUNE SYSTEM

1.4.1. Overview

The immune system of teleost fish is physiologically similar to that of mammals, as it consists of innate and adaptive immunity, although some differences are observed. Phylogenomic studies have suggested that, during the evolution of vertebrates, an additional genome duplication event (3R duplication) occurred in ray-finned fish (actinopterygian) 350 mya (Meyer & Van de Peer, 2005). Additionally, single gene duplication events are very common in organisms. Although most duplicated genes become non-functional (pseudogenes) after duplication, some of them evolve to acquire new functions, and the genomic complexity of the teleost is therefore generally higher than that in mammals (Meyer and Van de Peer, 2005). This is reflected in the higher number of paralog genes in fish. The existence of paralog genes in vertebrate species seems to be especially persistent in the case of immune-related genes (Flajnik & Kasahara, 2010; Ota & Nei, 1994; Piontkivska & Nei, 2003; Sarrias et al., 2004). As an example, zebrafish (Danio rerio) possesses four Nk-lysin genes (Pereiro et al., 2015), six perforins (Varela et al., 2016), and eight C3 complement components (Forn-Cuní, 2014). Only one copy of these genes is found in the genome of

15 tetrapods. Nevertheless, it is well known that paralog genes are especially abundant in zebrafish compared with other teleosts.

With the exception of lymphatic nodules and bone marrow, the remaining secondary lymphoid organs present in mammals are also found in fish (Press & Evensen, 1999). The main immune tissue is the head/anterior kidney and, together with the spleen, thymus and mucosa-associated lymphoid tissues (MALT), represents the lymphoid framework in teleosts (Press & Evensen, 1999). Moreover, fish also possess the main immune-related cell populations observed in mammals: monocytes, macrophages, granulocytes, dendritic cells, and T and B lymphocytes. The existence of natural killer cells in fish is not yet clear, although some investigations in the last decade have suggested the existence of these cells in teleost fish based on the presence of novel immune-type receptors (NITRs), the “functional orthologs” of mammalian natural killer receptors (NKRs) (Yoder, 2009).

Classically, the immune system in vertebrate organisms in divided into two main categories: innate, or non-specific, immunity and adaptive, or specific, immunity. Currently, it is known that these two systems work together to destroy microorganisms or trigger defence processes. The innate immune system is the first line of defence and can be divided into physical barriers, cellular and humoral components. Depending on the type of pathogen (bacterium, virus, parasite or fungus), and even depending on the particular species of microorganism, the immune response presents some specific components aimed at eradicating the disease. Although numerous molecules and processes are implicated in the response against viral agents, only the main components and pathways of antiviral defence in teleost fish are summarized in the next section.

1.4.2. Antiviral immune mechanisms

1.4.2.1. Virus sensors

The innate immune system’s recognition of pathogens is mediated by pattern recognition receptors (PRRs). After the detection of pathogen-associated molecular patterns (PAMPs), such as bacterial and fungal glycoproteins and lipopolysaccharides or viral components, through PRRs, intracellular signalling

16 cascades are activated to induce the expression of numerous immune and inflammatory mediators that coordinate the elimination of pathogens and infected cells (Takeuchi & Akira, 2010).

The virus sensors, or viral PRRs, can be classified into Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and nucleotide-binding oligomerization domain-containing (NOD)-like receptors (NLRs) (Jensen & Thomsen, 2012). Whereas TLRs are associated with the cell membrane or endosomal compartments, RLRs and NLRs patrol the cytoplasm for the presence of double- stranded viral RNA (Jensen & Thomsen, 2012) (Figure 7).

Figure 7. Viral pattern recognition receptors (PRRs).

In mammals, thirteen TLRs have been identified to date, of which 10 members are present in the human genome (TLR1-10) and thirteen are present in rodents (TLR1-13) (Areal et al., 2011). Each TLR recognizes specific PAMPs representing different components of pathogens (Takeda & Akira, 2004). Thus, human TLRs can be classified into non-viral (TLR1, 2, 4, 5, 6, 10) and viral TLRs (TLR3, 7, 8, 9) according to their ligand recognition (Areal et al., 2011). TLRs are composed of an ectodomain containing a variable number of leucine-rich repeats (LRRs), which is the responsible for binding PAMPs, a transmembrane segment, and a highly conserved cytoplasmic domain (TIR domain) that bind adapter

17 molecules (e.g., MyD88, TRIF) and triggers the intracellular cascades that finally result in the induction of numerous immune-related genes (Areal et al., 2011). The recognition of PAMPs by TLRs triggers the transcriptional up-regulation of distinct genes depending on the TLR, but mainly including type I interferons (IFNs), inflammatory cytokines and chemokines, and other molecules affecting the initiation of adaptive immune responses (Takeuchi & Akira, 2010).

In teleosts, numerous “fish-specific” TLRs have been identified in addition to those TLRs showing homology to mammals; 17 TLR types (TLR1, 2, 3, 4, 5, 5S, 7, 8, 9, 13, 14, 18, 19, 20, 21, 22, 23) were identified in more than a dozen teleost species, being in some cases duplicated in piscine genomes (Rebl et al., 2010). Orthologues of mammalian TLR6 and TLR10 were not identified in teleost species (Rebl et al., 2010), and TLR4 receptors are only present in cyprinids, although these genes do not seem to be functional receptors for bacterial lipopolysaccharide (LPS) as they are in mammals (Sullivan et al., 2009). In mammals, TLR3, 7, 8 and 9 (all of which are located on the surface of endosomes) are the main receptors responsible for virus detection; these receptors function by recognizing nucleic acids derived from viruses (Takeuchi & Akira, 2010), and this seems to be similar in fish (Rebl et al., 2010). TLR3 can detect viral replication by binding to double- stranded RNA (dsRNA), whereas TLR7 and TLR8 recognize single-stranded RNA (ssRNA), and TLR9 senses unmethylated DNA with CpG motifs (Takeuchi & Akira, 2010). In the case of teleosts, TLR22 also seems to be implicated in viral recognition by recognizing long-sized dsRNA on the cell surface (Matsuo et al., 2008). Moreover, TLR2 detects viral invasion by recognizing viral glycoproteins (Jensen and Thomsen, 2012). Until the completion of this doctoral thesis, there was only evidence for the presence of two TLRs in turbot, TLR3 (GenBank accession FJ009111) and TLR11 (Pardo et al., 2008).

The RLRs include three different cytoplasmic receptors: retinoic acid- inducible gene I (RIG-I, or DDX58), melanoma differentiation-associated gene 5 (MDA5, or IFIH1), and laboratory of genetics and physiology 2 (LGP2, or DHX58) (Onoguchi et al., 2011). The RLRs detect viral RNA ligands in the cytoplasm to trigger innate immunity and inflammation to control the infection, especially by activating the interferon (IFN) system (Loo & Gale, 2011). RIG-I and MDA5 possess

18 three domains: an N-terminal region consisting of tandem caspase activation and recruitment domains (CARDs), a central DExD/H box RNA helicase domain with the ability to hydrolyse ATP and bind to and possibly unwind RNA, and a C- terminal repressor domain (RD) embedded within the C-terminal domain (CTD) (Loo & Gale, 2011). On the other hand, LGP2 lacks the N-terminal CARDs and is currently thought to function as a regulator of RIG-I and MDA5 antiviral signalling (Rothenfusser et al., 2005; Venkataraman et al., 2007; Satoh et al., 2010; Yoneyama et al. 2004). It seems that RIG-I is preferentially activated by viral RNA bearing a triphosphate at the 5′ end and requires a short, blunt double-stranded structure for binding, whereas MDA5 has more affinity for long, double-stranded replication intermediates (Chen et al., 2015). The expression of RLRs is low in resting cells, but a great induction is generally observed after IFN exposure and viral infection (Loo & Gale, 2011). Numerous RLRs have been identified in teleost fish (Chang et al., 2011; Chen et al., 2012; Huang et al., 2010; Nie et al., 2015; Ohtani et al., 2010, 2011; Rajendran et al., 2012; Yang et al., 2011; Zou et al., 2009), although no information was available for turbot before the undertaking of this thesis. Their activity seems to be similar to that in mammals, being activated upon viral infection, polyinosine-polycytidylic acid (poly I:C) treatment and by the ubiquitin- like ISG15 protein (Langevin et al., 2013). Moreover, the downstream pathway of RLRs that includes mitochondrial antiviral signalling protein (MAVS) seems to be conserved (Biacchesi et al., 2009).

The last family of PRRs, NLRs, is also composed of cytosolic proteins sensing viruses. These proteins contain an LRR motif at the C terminus that functions as the sensor region, a central NACHT domain mediating oligomerization and activation, and an effector-binding domain at the N terminus (most often a CARD or PYD domain) that functions in downstream signalling (Jensen & Thomsen, 2012). A number of pathways, including the nuclear factor κB (NF-κB), mitogen- activated protein kinase (MAPK), inflammasome, and type I IFN signalling pathways, are activated after pathogen recognition by NLRs (Lupfer & Kanneganti, 2013). NLRP3 is the NLR most extensively studied with respect to viral infections, with a clear implication for both innate and adaptive immunity, mainly through inflammasome activation (Chakrabarti et al., 2015; Rajan et al., 2011; Thomas et al., 2009; Wang et al., 2014;). Inflammasome activation is one of the most well- 19 studied roles of NLRs, although not all NLRs are pro-inflammatory. The inflammasome is activated after the recognition of pathogens, mainly by NLRP1, NLRP3, and NLRC4, and it connects to caspase 1 via the adaptor molecule ASC (Latz et al., 2013). ASC forms large protein specks consisting mainly of multimers of ASC dimers and attracts monomers of pro-caspase 1 to initiate its cleavage and the formation of active caspase 1 (Latz et al., 2013). Caspase 1 mediates the cleavage of pro-interleukin-1β (pro-IL-1β) and pro-interleukin-18 (pro-IL-18) into their active forms and is able to trigger a pro-inflammatory form of cell death known as pyroptosis (Latz et al., 2013).

Other NLRs are also involved in viral infection. These include NLRC2, which was found to reduce inflammation during viral infection via autophagy (Sabbah et al., 2009), NLRX1, which has immunomodulatory properties and the ability to produce reactive oxygen species (Allen et al., 2011; Lei et al., 2012; Moore et al., 2008), and NLRC5, a hypothetical transactivator of major histocompatibility complex (MHC) class I and antigen presentation that has contradictory results about its implication in antiviral defence mechanisms (Kuenzel et al., 2010; Kumar et al., 2011; Neerincx et al., 2010)

In addition to these virus sensors, a new family of PRRs involved in DNA sensing, termed the AIM2-like receptors (ALRs), was recently described, and other DNA sensors have also been discovered (Keating et al., 2011). These DNA sensors are able to discriminate between self and non-self DNA. For example, the ALRs can mediate type I IFN induction (IFI16 and p204), inflammasome activation (AIM2 and IFI16) and negative regulation (p202) (Keating et al., 2011).

1.4.2.2. The interferon system

Interferons (IFNs) are a family of multifunctional cytokines that represent the first line of defence against viral infections; these cytokines are produced in response to different PAMPs via the activation of different signalling pathways (Honda et al., 2005). In mammals, three subfamilies of IFNs were established based on differences in structural and functional properties (type I, type II and type III) (González-Navajas et al., 2012; Platanias, 2005). The type I IFN subfamily comprises a broad group of typically antiviral proteins, with interferon alpha and

20 interferon beta being the most studied. In contrast, the type II IFN subfamily includes only one cytokine, interferon gamma, and the third type of IFNs is the interferon lambda subfamily, which is composed of three members, none of which have been identified in fish.

Type I IFNs are the main cytokines responsible for orchestrating the antiviral response in vertebrates, but, whereas these cytokines have been largely studied in mammalian species, the knowledge of these cytokines in teleosts is more recent and limited. The first reports regarding the cloning of type I IFNs in fish were published in 2003 for zebrafish (Danio rerio) (Altmann et al., 2003), Atlantic salmon (Salmo salar) (Robertsen et al., 2003) and pufferfish (Takifugu rubripes) (Lutfalla et al., 2003) and, to date, type I IFNs have been reported in several teleost species (revised in Zou & Secombes, 2011). Nevertheless, no sequence for a turbot IFN, complete or partial, or numerous other interferon- related genes was available in the public databases before the investigations conducted in this PhD project.

As mentioned above, viral recognition by PRRs culminates in, among other processes, the production of type I IFNs through different downstream pathways. The activation of latent transcription factors such as NF-κB and interferon regulatory factors (IRFs) via post-translational modifications, mainly phosphorylation events, leads to the recruitment of these factors to type I IFN promoters to induce the transcription of these genes (Hiscott, 2007). When IFNs are released, they activate other cells and induce an antiviral state by interacting with the corresponding receptor (interferon alpha/beta receptor in mammals) (Samuel, 2001). This interaction induces the activation of the JAK (Janus-activated kinase)/STAT (signal transducer and activator of transcription) signalling pathway and leads to the formation of the ISGF3 (IFN-stimulated gene factor 3) complex (Samuel, 2001). This complex translocates to the nucleus and binds IFN-stimulated response elements (ISREs) in DNA to initiate the transcription of those genes known as IFN-stimulated genes (ISGs) (Platanias, 2005). These ISGs (including PKR kinase, OAS synthetase and RNase L nuclease, the family of Mx protein GTPases and ISG15, among others) reduce viral replication and dissemination through different blocking mechanisms (Sadler & Williams, 2008; Samuel, 2001).

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These mechanisms for controlling all steps of viral replication include inhibition of viral transcription, degradation of viral RNA, inhibition of translation, or modification of protein function (Sadler & Williams, 2008).

IFN-gamma (type II IFN) is a markedly different IFN, possessing some ability to interfere with viral infections but mainly functioning as an immunomodulator (Boehm et al., 1997; Samuel, 2001). This cytokine is produced by different immune-related cell types, although T cells and NK cells are the major sources, and it is implicated in several aspects of immunity, such as activation of macrophages, stimulation of antigen presentation, orchestration of leukocyte- endothelium interactions, controlling cell proliferation and apoptosis, among others (Schroder et al., 2004). Regarding inflammation, IFN-gamma is typically described as a pro-inflammatory protein, although this categorization does not seem to be absolute because, in some cases, protective anti-inflammatory functions were associated with this cytokine (Mühl & Pfeilschifter, 2003; Zhang, 2007).

Unlike mammals, some bony fish, especially cyprinids, have two type II interferon genes, IFN-gamma (ifng) and IFN-gamma related (ifngrel) (Chen et al., 2010; Grayfer & Belosevic, 2009; Igawa et al., 2006; Milev-Milovanovic et al., 2006; Stolte et al., 2008). This additional gene is not a clear homologue of mammalian IFN-gamma, and it is believed than it originated after the duplication of the ifng gene (Zou & Secombes, 2011). The inflammatory functions of teleost type II IFNs have not been fully characterized, especially in the case of those species possessing two genes. Some studies have revealed that Ifng has the ability to induce the expression of pro-inflammatory cytokines (Arts et al., 2010; Grayfer et al., 2010; Sieger et al., 2009), whereas other investigations indicated that although zebrafish Ifng lacks the powerful pro-inflammatory activity of its mammalian counterpart, it helps to potentiate the induction of antiviral and pro-inflammatory genes by type I IFNs (López-Muñoz et al., 2009). It was observed that, as occurs in mammals, fish Ifng induces the activation of phagocytic cells by increasing the production of reactive oxygen intermediates (ROIs) and nitric oxide (NO), enhancing phagocytosis, and up-regulating the expression of different immune genes in this cell type (Arts et al., 2010; Grayfer & Belosevic, 2009; Grayfer et al., 2010; Zou et al., 2005).

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1.4.2.3. Inflammation

Inflammation is a key non-specific process in viral clearance. It consists of vascular, metabolic, and cellular changes triggered by harmful stimuli in healthy tissues of the body. During the earliest stages of a viral infection, cytokines are produced when innate immune defences are activated. The activation of viral PRRs results in the production of type I IFNs and inflammatory cytokines through the activation of nuclear factor κB (NFκB) (Kawai & Akira, 2006). The main pro- inflammatory cytokines are tumour necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and interferon gamma (IFN-γ), which increase the synthesis of vasoactive substances, such as platelet-activating factor, leukotrienes, prostaglandins, and nitric oxide (NO) (Dinarello, 2000). Moreover, they are inducers of endothelial adhesion molecules, which are essential for the adhesion of leukocytes to the endothelial surface prior to emigration into tissues, and induce the synthesis of chemokines (e.g., IL-8), which are cell chemoattractants that facilitate the passage of leukocytes from the circulation to the site of inflammation (Dinarello, 2000). These cells, especially neutrophils, are critically involved in the initiation and maintenance of inflammation. Neutrophils are crucial in controlling bacterial and fungal infections via phagocytosis, degranulation (mainly reactive oxygen species and antimicrobial peptides) and neutrophil extracellular traps (NETs) (Drescher & Bai, 2013; Galani & Andreakos, 2015). Although their relevance in viral diseases seems to be critical, the exact mechanisms by which neutrophils control viral infections are still under investigation, although these mechanisms likely are similar to those implicated in bacterial infections (Drescher & Bai, 2013; Galani & Andreakos, 2015). Pro-inflammatory cytokines can also promote their own production, exhibiting autocrine, paracrine, and self-propelling effects on the inflammatory process (Wojdasiewicz et al., 2014). This positive feedback loop amplifies the response and, consequently, excessive and uncontrolled inflammation is controlled through the production of anti- inflammatory molecules (such as interleukin 10 (IL-10) or transforming growth factor beta (TGF-β)), which mainly inhibit the synthesis of inflammatory cytokines to maintain homeostasis (Dinarello, 2000; Wojdasiewicz et al., 2014).

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Numerous pro-inflammatory and anti-inflammatory cytokines have been identified in teleosts. There is evidence that the main immune components implicated in inflammatory responses are present in fish (Grayfer & Belosevic, 2012). Nevertheless, as mentioned in section 4.3.1, numerous immune-related genes were found to be duplicated in teleosts. Regarding to the inflammation components, the existence of two IFNγ isoforms was confirmed in cypriniformes, which differ in their ability to modulate the inflammatory response (Grayfer & Belosevic, 2012); duplications of the IL-1β gene were also found in some fish species, although only one form seems to be functional in this case (Grayfer & Belosevic, 2012). Moreover, the presence of additional novel chemokines (Alejo & Tafalla, 2011) and PRRs (Poynter et al., 2015) in teleosts could reveal additional inflammatory pathways or mechanisms.

1.4.2.4. Antiviral strategies of cytotoxic T lymphocytes and natural killer cells

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are effector lymphocytes with the ability to eliminate tumours or virus-infected cells (Trapani & Smyth, 2002), and this mechanism seems to be highly conserved in vertebrates, including teleost fish (Nakanishi et al., 2011; Somamoto et al., 2014). Nevertheless, as mentioned above, the presence of NK cells in fish is not completely clear. There are two mechanisms for inducing the apoptosis of infected cells during a viral disease: the secretory (perforin/granzyme) pathway and the non-secretory (Fas- FasL) pathway (Trapani & Smyth, 2002). It seems that both mechanisms are indispensable for the efficient control of viral infections, as was observed through the disruption of the different pathways using murine models (Kagi et al., 1994; Parra et al., 2000; Rossi et al., 1998; Shrestha et al., 2007).

- Perforin/granzyme pathway

In the first pathway, the cytotoxic granules contained in the cytoplasm of these cell types are the protagonists. CTLs recognize viral antigens presented by major histocompatibility complex class I (MHC-I) through the T-cell receptor (TCR), and this ligation induces the activation of signalling cascades that result in polarization of the Golgi apparatus and microtubule-organizing centre and the docking and release of lytic granules (Berke, 1994). On the other hand, NK cells

24 recognize other signals associated with aberrant or virus-infected cells (Topham & Hewitt, 2009). The granules contain a membrane-disrupting protein known as perforin and a family of serine proteases (granzymes) implicated in the induction of apoptosis in target cells (Trapani and Smyth, 2002). Additionally, another protein, granulysin (or Nk-lysin), is present together with perforin and granzymes (Peña & Krensky, 1997). Perforin is a pore-forming member of the membrane- attack complex ⁄ PRF (MACPF) protein family. The monomers bind to the target cell membrane and polymerize in the presence of calcium to form transmembrane channels ranging from 5 to 20 nm in internal diameter and cause osmotic lysis of the target cells (Liu et al., 1995; Masson & Tschopp, 1985; Tschopp & Nabholz, 1990). Moreover, perforin allows the entry of the other cytotoxic components (mainly granzymes) into the target cell to induce apoptosis (Cullen et al., 2010; Hoves et al., 2012). To date, five different granzymes have been described in humans: granzymes A, B, H, K and M (Grossman et al., 2003). Granzyme B is the most extensively studied granzyme, and its activity is mediated by the induction of caspase-dependent apoptosis (Bots & Medema, 2006). This enzyme acts in two different ways. First, it cleaves the pro-apoptotic protein Bid, and consequently, Bid translocates to the mitochondria and together with Bax and/or Bak results in the release of pro-apoptotic proteins (such as cytochrome c) and mitochondrial outer membrane permeabilization. Granzyme B can also induce cytochrome c release through the cleavage and inactivation of the anti-apoptotic Bcl-2 family member Mcl-1. Cytochrome c is pivotal in the activation of caspase-9, which activates effector caspases. Second, granzyme B can directly process several caspases, including the effector caspase-3 and initiator caspase-8 (Bots & Medema, 2006). The other granzymes have a different a modus operandi but, in many cases, cell death is independent of caspase activation (Bots & Medema, 2006).

Regarding human granulysin (or Nk-lysin in other vertebrates), its function in antiviral mechanisms is not clear. This antimicrobial peptide was isolated from several vertebrate species and showed a broad antibacterial spectrum (Andersson et al., 1995; Andreu et al., 1999; Lee et al., 2014; Linde et al., 2005; Stenger et al., 1998; Zhang et al., 2014) and even antifungal (Andrä & Leippe, 1999) and antiparasitic activity (Jacobs et al., 2003; Gelhaus et al., 2008). This is due to its ability to alter membrane integrity, as occurs with the other members of the 25

SAPLIP family (Ruysschaert et al., 1998). Nevertheless, information about the role of this peptide in the antiviral response is very scarce. Previous works have attempted to elucidate the function of Nk-lysin/granulysin in viral diseases, but the results were contradictory in many cases. Interestingly, a microarray analysis of four turbot families showing different susceptibilities to Viral Haemorrhagic Septicaemia Virus (VHSV) revealed that Nk-lysin could be associated with resistance to the virus, as it was found to be differentially overexpressed in the highly resistant families compared with those more susceptible to viral challenge (Díaz-Rosales et al., 2012).

- Fas-FasL pathway

The second mechanism involves the engagement and aggregation of target cell death receptors (Fas) by their cognate ligand (FasL) on the killer-cell membrane, resulting in the caspase-dependent apoptosis of Fas-bearing cells (Lowin et al., 1994; Nagata & Golstein, 1995; Trapani & Smyth, 2002). Activation of CTLs through TCR interaction with viral antigens induces the expression of the FasL gene. FasL expressed on the surface of the effector cells binds to Fas on the target cell and causes apoptosis by activating caspases (Nagata, 1997). Both Fas and FasL belong to the tumour necrosis factor (TNF) family, and each contains a single transmembrane domain (Nagata, 1997). The binding of FasL with Fas instigates receptor oligomerization, which engages Fas-associated death domain (FADD); FADD activates caspase-8 through self-cleavage, which activates the effector caspases, initiating the process of apoptosis (Rauf et al., 2012).

1.5. REFERENCES

Adelmann M, Köllner BK, Bergmann SM, Fischer U, Lange B, Weitschies W, Enzmann PJ, Fichtner D (2008) Development of an oral vaccine for immunisation of rainbow trout (Oncorhynchus mykiss) against viral haemorrhagic septicaemia. Vaccine, 26: 837–844.

Alejo A, Tafalla C (2011) Chemokines in teleost fish species. Dev Comp Immunol, 35: 1215–1222.

26

Allen IC, Moore CB, Schneider M, Lei Y, Davis BK, Scull MA, Gris D, Roney KE, Zimmermann AG, Bowzard JB, Ranjan P, Monroe KM, Pickles RJ, Sambhara S, Ting JP (2011) NLRX1 protein attenuates inflammatory responses to infection by interfering with the RIG-I-MAVS and TRAF6-NF-kappaB signaling pathways. Immunity, 34: 854–865.

Altmann SM, Mellon MT, Distel DL, Kim CH (2003) Molecular and functional analysis of an interferon gene from the zebrafish, Danio rerio. J Virol, 77: 1992– 2002.

Álvarez-Pellitero P (2008) Fish immunity and parasite infections: from innate immunity to immunoprophylactic prospects. Vet Immunol Immunopathol, 126: 171–198.

Ammayappan A, Vakharia VN (2011) Nonvirion protein of novirhabdovirus suppresses apoptosis at the early stage of virus infection. J Virol, 85: 8393–8402.

Andersson M, Gunne H, Agerberth B, Boman A, Bergman T, Sillard R, Jornvall H, Mutt V, Olsson B, Wigzell H (1995) NK-lysin, a novel effector peptide of cytotoxic T and NK cells. Structure and cDNA cloning of the porcine form, induction by interleukin 2, antibacterial and antitumour activity. EMBO J, 14: 1615–1625.

Anderson ED, Mourich DV, Fahrenkrug SC, LaPatra S, Shepherd J, Leong JA (1996) Genetic immunization of rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis virus. Mol Mar Biol Biotechnol, 5: 114–122.

Andrä J, Leippe M (1999) Candidacidal activity of shortened synthetic analogs of amoebapores and NK-lysin. Med Microbiol Immunol, 188: 117–124.

Andreu D, Carreno C, Linde C, Boman HG, Andersson M (1999) Identification of an anti-mycobacterial domain in NK-lysin and granulysin. Biochem J, 344: 845–849.

APROMAR: Asociación Empresarial de Productores de Cultivos Marinos (2015) La Acuicultura en España.

Areal H, Abrantes J, Esteves PJ (2011) Signatures of positive selection in Toll-like receptor (TLR) genes in mammals. BMC Evol Biol, 11: 368.

Arts JA, Tijhaar EJ, Chadzinska M, Savelkoul HF, Verburg-van Kemenade BM (2010) Functional analysis of carp interferon-gamma: evolutionary conservation of classical phagocyte activation. Fish Shellfish Immunol, 29: 793–802.

Assenberg R, Delmas O, Morin B, Graham SC, De Lamballerie X, Laubert C, Coutard B, Grimes JM, Neyts J, Owens RJ, Brandt BW, Gorbalenya A, Tucker P, Stuart DI, Canard B,

27

Bourhy H (2010) Genomics and structure/function studies of Rhabdoviridae proteins involved in replication and transcription. Antiviral Res, 87: 149–161.

Avendaño-Herrera R, Toranzo AE, Magariños B (2006) Tenacibaculosis infection in marine fish caused by Tenacibaculum maritimum: a review. Dis Aquat Org, 71: 255–266.

Barja JL (2004) Report about fish viral diseases. In: Alvarez-Pellitero P, Barja JL, Basurco B, Berthe F, Toranzo AE, eds. Mediterranean aquaculture diagnostic laboratories. Zaragoza: CIHEAM. pp 91–102.

Berke G (1994). The binding and lysis of target cells by cytotoxic lymphocytes: molecular and cellular aspects. Annu Rev Immunol, 12: 735–773.

Bernard J, de Kinkelin P, Bearzotti-Le Berre M (1983) Viral hemorrhagic septicemia of rainbow trout: relation between the G polypeptide and antibody production in protection of the fish after infection with the F25 attenuated variant. Infect Immun, 39: 7–14.

Biacchesi S, LeBerre M, Lamoureux A, Louise Y, Lauret E, Boudinot P, Brémont M (2009) Mitochondrial antiviral signaling protein plays a major role in induction of the fish innate immune response against RNA and DNA viruses. J Virol, 83: 7815–7827.

Boehm U, Klamp T, Groot M, Howard JC (1997) Cellular responses to interferon- gamma. Annu Rev Immunol, 15: 749–795.

Bots M, Medema JP (2006) Granzymes at a glance. J Cell Sci, 119: 5011–5014.

Bouza C, Hermida M, Millán A, Vilas R, Vera M, Fernández C, Calaza M, Pardo BG, Martínez P (2008) Characterization of EST-derived microsatellites for gene mapping and evolutionary genomics in turbot. Anim Genet, 39: 666–670.

Castric J, de Kinkelin P (1980) Occurrence of viral haemorrhagic septicaemia in rainbow trout Salmo gairdneri Richardson reared in sea water. J Fish Dis, 3: 21–27.

Chakrabarti A, Banerjee S, Franchi L, Loo YM, Gale M Jr, Núñez G, Silverman RH (2015) RNase L activates the NLRP3 inflammasome during viral infections. Cell Host Microbe, 17: 466–477.

Chang M, Collet B, Nie P, Lester K, Campbell S, Secombes CJ, Zou J (2011) Expression and functional characterization of the RIG-I-like receptors MDA5 and LGP2 in Rainbow trout (Oncorhynchus mykiss). J Virol, 85: 8403–8412.

28

Chen HY, Liu W, Wu SY, Chiou PP, Li YH, Chen YC, Lin GH, Lu MW, Wu JL (2015) RIG-I specifically mediates group II type I IFN activation in nervous necrosis virus infected zebrafish cells. Fish Shellfish Immunol, 43: 427–435.

Chen L, Su J, Yang C, Peng L, Wan Q, Wang L (2012) Functional Characterizations of RIG-I to GCRV and Viral/Bacterial PAMPs in Grass Carp Ctenopharyngodon idella. PLOS ONE, 7: e42182.

Chen WQ, Xu QQ, Chang MX, Zou J, Secombes CJ, Peng KM, Nie P (2010) Molecular characterization and expression analysis of the IFN-gamma related gene (IFN-gammarel) in grass carp Ctenopharyngodon idella. Vet Immunol Immunopathol, 134: 199–207.

Choi MK, Moon CH, Ko MS, Lee UH, Cho WJ, Cha SJ, Do JW, Heo GJ, Jeong SG, Hahm YS, Harmache A, Bremont M, Kurath G, Park JW (2011) A nuclear localization of the infectious haematopoietic necrosis virus NV protein is necessary for optimal viral growth. PLOS ONE, 6: e22362.

Collard BCY, Jahufer MZZ, Brouwer JB, Pang ECK (2005) An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts. Euphytica, 142: 169-196.

Cullen SP, Brunet M, Martin SJ (2010) Granzymes in cancer and immunity. Cell Death Differ, 17: 616–623.

de Kinkelin P, Bearzotti-Le Berre M, Bernard J (1980) Viral hemorrhagic septicemia of rainbow trout: selection of a thermoresistant virus variant and comparison of polypeptide synthesis with the wild-type virus strain. J Virol, 36: 652–658.

de Kinkelin P, Béarzotti M, Castric J, Nougayrède P, Lecocq-Xhonneux F, Thiry M (1995) Eighteen years of vaccination against viral haemorrhagic septicaemia in France. Vet Res, 26: 379–387.

Díaz-Rosales P, Romero A, Balseiro P, Dios S, Novoa B, Figueras A (2012). Microarray-based identification of differentially expressed genes in families of turbot (Scophthalmus maximus) after infection with viral haemorrhagic septicaemia virus (VHSV). Mar Biotechnol, 14: 515–529.

Dinarello CA (2000) Proinflammatory cytokines. Chest, 118: 503–508.

29

Domenech A, Fernandez-Garayzabal JF, Pascual C, Garcia JA, Cutuli MT, Moreno MA, Collins MD, Dominguez L (1996) Streptococcosis in cultured turbot, Scophthalmus maximus (L.), associated with Streptococcus parauberis. J Fish Dis, 19: 33–38.

Drescher B, Bai F (2013) Neutrophil in viral infections, friend or foe? Virus Res, 171: 1–7.

Dyková I, Figueras A (1994) Histopathological changes in turbot Scophthalmus maximus due to a histophagous ciliate. Dis Aquat Org, 18: 5–9.

Dyková I, Figueras A, Novoa B (1995) Amoebic gill infection of turbot, Scophthalmus maximus. Folia Parasitol, 42: 91–96.

Dyková I, Figueras A, Novoa B, Casal JF (1998) Paramoeba sp., an agent of amoebic gill disease of turbot Scophthalmus maximus. Dis Aquat Org, 33: 137–141.

Einer-Jensen K, Ahrens P, Forsberg R, Lorenzen N (2004) Evolution of the fish rhabdovirus viral haemorrhagic septicaemia virus. J Gen Virol, 85: 1167–1179.

Elsayed E, Faisal M, Thomas M, Whelan G, Batts W, Winton J (2006) Isolation of viral haemorrhagic septicaemia virus from muskellunge, Esox masquinongy (Mitchill), in Lake St Clair, Michigan, USA reveals a new sublineage of the North American genotype. J Fish Dis, 29: 611–619.

FAO: Food and Agriculture Organization of the United Nations (2010) The state of world fisheries and aquaculture. Rome: FAO.

FAO: Food and Agriculture Organization of the United Nations (2014) The state of world fisheries and aquaculture. Rome: FAO.

FEAP: Federation of European Aquaculture Producers (2015) European Aquaculture Production Report 2005-2014.

Figueras A, Novoa B, Santarem M, Martinez E, Alvarez JM, Toranzo AE, Dyková I (1992) Tetramicra brevifilum, a potential threat to farmed turbot Scophthalmus maximus. Dis Aquat Org, 14:127–135.

Figueras A, Robledo D, Corvelo A, Hermida M, Pereiro P, Rubiolo JA, Gómez- Garrido J, Carreté L, Bello X, Gut M, Gut IG, Marcet-Houben M, Forn-Cuní G, Galán B, García JL, Abal-Fabeiro JL, Pardo BG, Taboada X, Fernández C, Vlasova A, Hermoso-Pulido A, Guigó R, Álvarez-Dios JA, Gómez-Tato A, Viñas A, Maside X, Gabaldón T, Novoa B, Bouza C,

30

Alioto T, Martínez P (2016) Whole genome sequencing of turbot (Scophthalmus maximus; Pleuronectiformes): a fish adapted to demersal life. DNA Res, 23: 181–192.

Flajnik MF, Kasahara M (2010) Origin and evolution of the adaptive immune system: genetic events and selective pressures. Nat Rev Genet, 11: 47–59.

Forn-Cuní G, Reis ES, Dios S, Posada D, Lambris JD, Figueras A, Novoa B (2014) The evolution and appearance of C3 duplications in fish originate an exclusive teleost c3 gene form with anti-inflammatory activity. PLOS ONE, 9: e99673.

Galani IE, Andreakos E (2015) Neutrophils in viral infections: Current concepts and caveats. J Leukoc Biol, 98: 557–564.

Gelhaus C, Jacobs T, Andra J, Leippe M (2008) The antimicrobial peptide NK-2, the core region of mammalian NK-lysin, kills intraerythrocytic Plasmodium falciparum. Antimicrob Agents Chemother, 52: 1713–1720.

Gonzalez-Navajas JM, Lee J, David M, Raz E (2012) Immunomodulatory functions of type I interferons. Nat Rev Immunol, 12: 125–135.

Grayfer L, Belosevic M (2009) Molecular characterization, expression and functional analysis of goldfish (Carassius auratus L.) interferon gamma. Dev Comp Immunol, 33: 235–246.

Grayfer L, Belosevic M (2012) Chapter 2: Cytokine Regulation of Teleost Inflammatory Responses. In: Türker H (ed.). New Advances and Contributions to Fish Biology. InTech.

Grayfer L, Garcia EG, Belosevic M (2010) Comparison of macrophage antimicrobial responses induced by type II interferons of the goldfish (Carassius auratus L.). J Biol Chem. 285: 23537–23547.

Grossman WJ, Revell PA, Lu ZH, Johnson H, Bredemeyer AJ, Ley TJ (2003) The orphan granzymes of humans and mice. Curr Opin Immunol, 15: 544–552.

Hastein T, Hill BJ, Winton JR (1999) Successful aquatic animal disease emergency programmes. Rev Sci Tech Off Int Epiz, 18: 214–227.

Hellberg H, Koppang EO, Tørud B, Bjerkås I (2002) Subclinical herpesvirus infection in farmed turbot Scophthalmus maximus. Dis Aquat Organ, 49: 27–31.

31

Hiscott J (2007) Triggering the innate antiviral response through IRF-3 activation. J Biol Chem, 282: 15325–15329.

Honda K, Yanai H, Takaoka A, Taniguchi T (2005) Regulation of the type I IFN induction: a current view. Int Immunol, 17: 1367–1378.

Hørlyck V, Mellergård S, Dalsgaard I, Jørgensen PEV (1984) Occurrence of VHSV in Danish maricultured rainbowtrout. Bull Eur Fish Pathol, 4: 11–13.

Hoves S, Sutton VR, Trapani JA (2012) A novel role for granzymes in anti-tumor immunity. Oncoimmunology 1: 219–221.

Huang T, Su J, Heng J, Dong J, Zhang R, Zhu H (2010) Identification and expression profiling analysis of grass carp Ctenopharyngodon idella LGP2 cDNA. Fish Shellfish Immunol, 29: 349–355.

Húsgağ S, Grotmol S, Hjeltnes BK, Rødseth OM, Biering E (2001) Immune response to a recombinant capsid protein of striped jack nervous necrosis virus (SJNNV) in turbot Scophthalmus maximus and Atlantic halibut Hippoglossus hippoglossus, and evaluation of a vaccine against SJNNV. Dis Aquat Organ, 45: 33–44.

Igawa D, Sakai M, Savan R (2006) An unexpected discovery of two interferon gamma-like genes along with interleukin (IL)-22 and -26 from teleost: IL-22 and -26 genes have been described for the first time outside mammals. Mol Immunol, 43: 999–1009.

Iglesias R, Paramá A, Álvarez MF, Leiro J, Fernández J, Sanmartín ML (2001) Philasterides dicentrarchi (Ciliophora, Scuticociliatida) as the causative agent of scuticociliatosis in farmed turbot Scophthalmus maximus in Galicia (NW Spain). Dis Aquat Org, 46: 47–55.

Jacobs T, Bruhn H, Gaworski I, Fleischer B, Leippe M (2003) NK-lysin and its shortened analog NK-2 exhibit potent activities against Trypanosoma cruzi. Antimicrob Agents Chemother, 47: 607–613.

Jensen S, Thomsen AR (2012) Sensing of RNA viruses: a review of innate immune receptors involved in recognizing RNA virus invasion. J Virol, 86: 2900–2910.

Johansen R, Sommerset I, Tørud B, Korsnes K, Hjortaas MJ, Nilsen F, Nerland AH, Dannevig BH (2004) Characterization of nodavirus and viral encephalopathy and retinopathy in farmed turbot, Scophthalmus maximus (L.). J Fish Dis, 27: 591–601.

32

Kagi D, Vignaux F, Ledermann B, Burki K, Depraetere V, Nagata S, Hengartner H, Golstein P (1994) Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science, 265: 528–530.

Kawai T, Akira S (2006) Innate immune recognition of viral infection. Nat Immunol, 7: 131–137.

Keating SE, Baran M, Bowie AG (2011) Cytosolic DNA sensors regulating type I interferon induction. Trends Immunol, 32: 574–581.

Kuenzel S, Till A, Winkler M, Häsler R, Lipinski S, Jung S, Grötzinger J, Fickenscher H, Schreiber S, Rosenstiel P (2010) The nucleotide-binding oligomerization domain-like receptor NLRC5 is involved in IFN-dependent antiviral immune responses. J Immunol, 184: 1990–2000.

Kumar H, Pandey S, Zou J, Kumagai Y, Takahashi K, Akira S, Kawai T (2011) NLRC5 deficiency does not influence cytokine induction by virus and bacteria infections. J Immunol, 186: 994–1000.

Kurath G, Leong JC (1985) Characterization of infectious hematopoietic necrosis virus mRNA species reveals a nonvirion rhabdovirus protein. J Virol, 53: 462–468.

Lamas J, Cepeda C, Dopazo C,Toranzo AE, Anadon R, Barja JL (1996) Occurrence of an erythrocytic virus infection in cultured turbot Scophthalmus maximus. Dis Aquat Organ, 24: 159–167.

Langevin C, Aleksejeva E, Passoni G, Palha N, Levraud JP, Boudinot P (2013) The antiviral innate immune response in fish: evolution and conservation of the IFN system. J Mol Biol, 425: 4904–4920.

LaPatra SE, Corbeil S, Jones GR, Shewmaker WD, Lorenzen N, Anderson ED, Kurath G (2001) Protection of rainbow trout against infectious hematopoietic necrosis virus four days after specific or semi-specific DNA vaccination. Vaccine, 19: 4011–4019.

Latz E, Xiao TS, Stutz A (2013) Activation and regulation of the inflammasomes. Nat Rev Immunol, 13: 397–411.

Lecocq-Xhonneux F, Thiry M, Dheur I, Rossius M, Vanderheijden N, Martial J, de Kinkelin P (1994) A recombinant viral haemorrhagic septicaemia virus glycoprotein expressed in insect cells induces protective immunity in rainbow trout. J Gen Virol, 75: 1579–1587.

33

Lee MO, Jang HJ, Han JY, Womack JE (2014) Chicken NK-lysin is an alpha-helical cationic peptide that exerts its antibacterial activity through damage of bacterial cell membranes. Poult Sci, 93: 864–870.

Lei Y, Wen H, Yu Y, Taxman DJ, Zhang L, Widman DG, Swanson KV, Wen KW, Damania B, Moore CB, Giguère PM, Siderovski DP, Hiscott J, Razani B, Semenkovich CF, Chen X, Ting JP (2012) The mitochondrial proteins NLRX1 and TUFM form a complex that regulates type I interferon and autophagy. Immunity, 36: 933–946.

Leong JC, Fryer JL (1993) Viral vaccines for aquaculture. Annu Rev Fish Dis, 3: 225– 240.

Lillehaug A, Lunestad BT, Grave K (2003) Epidemiology of bacterial diseases in Norwegian aquaculture—a description based on antibiotic prescription data for the ten- year period 1991 to 2000. Dis Aquat Org, 53: 115–125.

Linde CMA, Grundström S, Nordling E, Refai E, Brennan PJ, Andersson M (2005) Conserved structure and function in the granulysin and NK-lysin peptide family. Infect Immun, 73: 6332–6339.

Liu CC, Walsh CM, Young JDE (1995) Perforin: structure and function. Immunology Today, 16: 194–201.

Liu R, Chen J, Kesheng L, Zhang X (2011) Identification and evaluation as a DNA vaccine candidate of a virulence-associated serine protease from a pathogenic Vibrio parahaemolyticus isolate. Fish Shellfish Immunol, 30: 1241–1248.

Loo YM, Gale M Jr (2011) Immune signaling by RIG-I-like receptors. Immunity, 34: 680–692.

López-Muñoz A, Roca FJ, Meseguer J, Mulero V (2009) New insights into the evolution of IFNs: zebrafish group II IFNs induce a rapid and transient expression of IFN- dependent genes and display powerful antiviral activities. J Immunol, 182: 3440–3449.

Lorenzen E, Einer-Jensen K, Martinussen T, LaPatra SE, Lorenzen N (2000) DNA vaccination of rainbow trout against Viral Hemorrhagic Septicemia Virus: A dose-response and time-course study. J Aquat Anim Health, 12: 167–180.

Lorenzen E, Lorenzen N, Einer-Jensen K, Brudeseth B, Evensen O (2005) Time course study of in situ expression of antigens following DNA-vaccination against VHS in rainbow trout (Oncorhynchus mykiss Walbaum) fry. Fish Shellfish Immunol, 19: 27–41.

34

Lorenzen N, LaPatra SE (2005) DNA vaccines for aquacultured fish. Rev Sci Tech Off Int Epiz, 24: 201–213.

Lorenzen N, Lorenzen E, Einer-Jensen K, Heppell J, Wu T, Davis H (1998) Protective immunity to VHS in rainbow trout (Oncorhynchus mykiss, Walbaum) following DNA vaccination. Fish Shellfish Immunol, 8: 261–270.

Lorenzen N, Olesen NJ, Jorgensen PEV (1990) Neutralization of Egtved virus pathogenicity to cell cultures and fish by monoclonal antibodies to the viral G protein. J Gen Virol, 71: 561–567.

Lowin B, Hahne M, Mattmann C, Tschopp J (1994) Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways. Nature, 370: 650–652.

Lupfer C, Kanneganti TD (2013) The expanding role of NLRs in antiviral immunity. Immunol Rev, 255: 13–24.

Lutfalla G, Crollius HR, Stange-Thomann N, Jaillon O, Mogensen K, Monneron D (2003) Comparative genomic analysis reveals independent expansion of a lineage-specific gene family in vertebrates: the class II cytokine receptors and their ligands in mammals and fish. BMC Genomics, 4: 29.

Masson D, Tschopp J (1985) Isolation of a lytic, pore-forming protein (perforin) from cytolytic T-lymphocytes. J Biol Chem, 260: 9069–9072.

Matsuo A, Oshiumi H, Tsujita T, Mitani H, Kasai H, Yoshimizu M, Matsumoto M, Seya T (2008) Teleost TLR22 recognizes RNA duplex to induce IFN and protect cells from birnaviruses. J Immunol, 181: 3474–3485.

Meyer A, Van de Peer Y (2005) From 2R to 3R: evidence for a fish-specific genome duplication (FSGD). Bioessays, 27: 937–945.

Milev-Milovanovic I, Long S, Wilson M, Bengten E, Miller NW, Chinchar VG (2006) Identification and expression analysis of interferon gamma genes in channel catfish. Immunogenetics, 58: 70–80.

Mohan M, Nair S, Bhagwat A, Krishna T.G, Yano M, Bhatia CR, Sasaki T (1997) Genome mapping, molecular markers and marker-assisted selection in crop plants. Mol Breed, 3: 87–103.

35

Montes A, Figueras A, Novoa B (2010) Nodavirus encephalopathy in turbot (Scophthalmus maximus): inflammation, nitric oxide production and effect of anti- inflammatory compounds. Fish Shellfish Immunol, 28: 281–288.

Moore CB, Bergstralh DT, Duncan JA, Lei Y, Morrison TE, Zimmermann AG, Accavitti-Loper MA, Madden VJ, Sun L, Ye Z, Lich JD, Heise MT, Chen Z, Ting JP (2008) NLRX1 is a regulator of mitochondrial antiviral immunity. Nature, 451: 573–577.

Mühl H, Pfeilschifter J (2003) Anti-inflammatory properties of pro-inflammatory interferon-gamma. Int Immunopharmacol. 3: 1247–1255.

Nagata S (1997) Apoptosis by death factor. Cell, 88: 355–365.

Nagata S, Golstein P (1995) The Fas death factor. Science, 267: 1449–1456.

Nakanishi T, Toda H, Shibasaki Y, Somamoto T (2011) Cytotoxic T cells in teleost fish. Dev Comp Immunol, 35: 1317–1323.

Neerincx A, Lautz K, Menning M, Kremmer E, Zigrino P, Hösel M, Büning H, Schwarzenbacher R, Kufer TA (2010) A role for the human nucleotide-binding domain, leucine-rich repeat-containing family member NLRC5 in antiviral responses. J Biol Chem, 285: 26223–26232.

Nie L, Zhang YS, Dong WR, Xiang LX, Shao JZ (2015) Involvement of zebrafish RIG-I in NF-κB and IFN signaling pathways: insights into functional conservation of RIG-I in antiviral innate immunity. Dev Comp Immunol, 48: 95–101.

Nielsen JG (1986) Scophtalmidae. In Whitehead PJP, Bauchot ML, Hureau JC, Nielsen J, Tortonese E, eds. Fishes of the North-eastern Atlantic and the Mediterranean. Paris: Unesco. pp 1287–1293.

Nougayrede P, Sochon E, Vuillaume A (1990) Isolation of Aeromonas salmonicida subspecies salmonicida in farmed turbot (Psetta maxima) in France. Bull Eur Assoc Fish Pathol, 10: 139–140.

Novoa B, Rivas C, Toranzo AE, Figueras A (1995) Pathogenicity of birnaviruses isolated from turbot (Scophthalmus maximus): comparison with reference serotypes of IPNV. Aquaculture, 130: 7–14.

36

Ohtani M, Hikima J, Kondo H, Hirono I, Jung TS, Aoki T (2010) Evolutional conservation of molecular structure and antiviral function of a viral RNA receptor, LGP2, in Japanese flounder, Paralichthys olivaceus. J Immunol, 185: 7507–7517.

Ohtani M, Hikima J, Kondo H, Hirono I, Jung TS, Aoki T (2011) Characterization and antiviral function of a cytosolic sensor gene, MDA5, in Japanese flounder, Paralichthys olivaceus. Dev Comp Immunol, 35: 554–562.

Onoguchi K, Yoneyama M, Fujita T (2011) Retinoic acid-inducible gene-I-like receptors. J Interferon Cytokine Res, 31: 27–31.

Ota T, Nei M (1994). Divergent evolution and evolution by the birth-and-death process in the immunoglobulin V-H gene family. Mol Biol Evol, 11: 469–482.

Palenzuela O, Redondo MJ, Álvarez-Pellitero P (2002) Description of Enteromyxum scophthalmi gen. nov., sp. nov. (Myxozoa), an intestinal parasite of turbot (Scophthalmus maximus L.) using morphological and ribosomal RNA sequence data. Parasitology, 124: 369–379.

Palenzuela O, Sitjà-Bobadilla A, Riaza A, Silva R, Arán J, Álvarez-Pellitero P (2009) Antibody responses of turbot (Psetta maxima) against different antigen formulations of scuticociliates (Ciliophora). Dis Aquat Org, 86: 123–134.

Pardo BG, Fernández C, Millán A, Bouza C, Vázquez-López A, Vera M, Alvarez-Dios JA, Calaza M, Gómez-Tato A, Vázquez M, Cabaleiro S, Magariños B, Lemos ML, Leiro JM, Martínez P (2008) Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens. BMC Vet Res, 4: 37.

Parra B, Lin MT, Stohlman SA, Bergmann CC, Atkinson R, Hinton DR (2000) Contributions of Fas–Fas ligand interactions to the pathogenesis of mouse hepatitis virus in the central nervous system. J Virol, 74: 2447–2450.

Pedersen K, Larsen JL (1996) First report on outbreak of furunculosis in turbot Scophthalmus maximus caused by Aeromonas salmonicida subsp. salmonicida in Denmark. Bull Eur Assoc Fish Pathol, 16: 129–133.

Peña SV, Krensky AM (1997) Granulysin, a new human cytolytic granule- associated protein with possible involvement in cell-mediated cytotoxicity. Semin Immunol, 9: 117–125.

37

Pereiro P, Figueras A, Novoa B (2016) Turbot (Scophthalmus maximus) vs. VHSV (Viral Hemorrhagic Septicemia Virus): a review. Front Physiol, 7: 192.

Piontkivska H, Nei M (2003) Birth-and-death evolution in primate MHC class I genes: divergence time estimates. Mol Biol Evol, 20: 601–609.

Platanias LC (2005) Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol, 5: 375–386.

Poynter S, Lisser G, Monjo A, DeWitte-Orr S (2015) Sensors of infection: viral nucleic acid PRRs in fish. Biology, 4: 460–493.

Press CMCL, Evensen O (1999) The morphology of the immune system in teleost fishes. Fish Shellfish Immunol, 9: 309–318.

Rajan JV, Rodriguez D, Miao EA, Aderem A (2011) The NLRP3 inflammasome detects encephalomyocarditis virus and vesicular stomatitis virus infection. J Virol, 85: 4167–4172.

Rajendran KV, Zhang J, Liu S, Peatman E, Kucuktas H, Wang X, Liu H, Wood T, Terhune J, Liu Z (2012) Pathogen recognition receptors in channel catfish: II. Identification, phylogeny and expression of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs). Dev Comp Immunol, 37: 381–389.

Rauf A, Khatri M, Murgia MV, Saif YM (2012) Fas/FasL and perforin-granzyme pathways mediated T cell cytotoxic responses in infectious bursal disease virus infected chickens. Results Immunol, 2: 112–119.

Rebl A, Goldammer T, Seyfert HM (2010) Toll-like receptor signaling in bony fish. Vet Immunol Immunopathol, 134: 139–150.

Robertsen B, Bergan V, Rokenes T, Larsen R, Albuquerque A (2003) Atlantic salmon interferon genes: cloning, sequence analysis, expression, and biological activity. J Interferon Cytokine Res, 23: 601–612.

Rodríguez-Ramilo ST, De La Herrán R, Ruiz-Rejón C, Hermida M, Fernández C, Pereiro P, Figueras A, Bouza C, Toro MA, Martínez P, Fernández J (2014) Identification of quantitative trait loci associated with resistance to viral haemorrhagic septicaemia (VHS) in turbot (Scophthalmus maximus): a comparison between bacterium, parasite and virus diseases. Mar Biotechnol, 16: 256–276.

38

Rodríguez-Ramilo ST, Fernández J, Toro MA, Bouza C, Hermida M, Fernández C, Pardo BG, Cabaleiro S, Martínez P (2013) Uncovering QTL for resistance and survival time to Philasterides dicentrarchi in turbot (Scophthalmus maximus). Anim Genet, 44: 149–157.

Rodríguez-Ramilo ST, Toro MA, Bouza C, Hermida M, Pardo BG, Cabaleiro S, Martínez P, Fernández J (2011) QTL detection for Aeromonas salmonicida resistance related traits in turbot (Scophthalmus maximus). BMC Genomics, 12: 541.

Romalde JL, Silva R, Riaza A, Toranzo AE (1996) Long-lasting protection against turbot streptococcosis obtained with a toxoid-enriched bacterin. Bull Eur Assoc Fish Pathol, 16: 169–171.

Ross, K., McCarthy, U., Huntly, P.J., Wood, B.P., Stuart, D., Rough, E.I., Smail. D.A., Bruno, D.W. (1994) An outbreak of viral haemorrhagic septicaemia (VHS) in turbot (Scophthalmus maximus) in Scotland. Bull Eur Assoc Fish Pathol, 14: 213–214.

Rossi CP, McAllister A, Tanguy M, Kagi D, Brahic M (1998) Theiler's virus infection of perforin-deficient mice. J Virol, 72: 4515–4519.

Rothenfusser S, Goutagny N, DiPerna G, Gong M, Monks BG, Schoenemeyer A, Yamamoto M, Akira S, Fitzgerald KA (2005) The RNA helicase Lgp2 inhibits TLR- independent sensing of viral replication by retinoic acid-inducible gene-I. J Immunol, 175: 5260-5268.

Ruysschaert JM, Goormaghtigh E, Homblé F, Andersson M, Liepinsh E, Otting G (1998) Lipid membrane binding of NK-lysin. FEBS Lett, 425: 341–344.

Sabbah A, Chang TH, Harnack R, Frohlich V, Tominaga K, Dube PH, Xiang Y, Bose S (2009) Activation of innate immune antiviral responses by Nod2. Nat Immunol, 10: 1073– 1080.

Sadler AJ, Williams BRG (2008) Interferon-inducible antiviral effectors. Nat Rev Immunol, 8: 559–568.

Samuel CE (2001) Antiviral actions of interferons. Clin Microbiol Rev, 14: 778–809.

Sanmartín Durán ML, Fernandez Casal J, Tojo JL, Santamarina MT, Estevez J, Urbeira F (1991) Trichodina sp.: effect on the growth of farmed turbot (Scophthalmus maximus). Bull Eur Assoc Fish Pathol, 11: 89–91.

39

Sanmartín ML, Paramá A, Castro R, Cabaleiro S, Leiro J, Lamas J, Barja JL (2008) Vaccination of turbot, Psetta maxima (L.), against the protozoan parasite Philasterides dicentrarchi: effects on antibody production and protection. J Fish Dis, 31: 135–140.

Sarrias MR, Grønlund J, Padilla O, Madsen J, Holmskov U, Lozano F (2004) The scavenger receptor cysteine-rich (SRCR) domain: an ancient and highly conserved protein module of the innate immune system. Crit Rev Immunol, 24: 1–37.

Satoh T, Kato H, Kumagai Y, Yoneyama M, Sato S, Matsushita K, Tsujimura T, Fujita T, Akira S, Takeuchi O (2010) LGP2 is a positive regulator of RIG-I- and MDA5-mediated antiviral responses. Proc Natl Acad Sci USA, 107: 1512–1517.

Schlotfeldt HJ, Ahne W, Vestergård-Jørgensen PE, Glende W (1991) Occurrence of viral haemorrhagic septicaemia virus in turbot (Scophthalmus maximus) - a natural outbreak. Bull Eur Assoc Fish Pathol, 11: 105–107.

Schroder K, Hertzog PJ, Ravasi T, Hume DA (2004) Interferon-gamma: an overview of signals, mechanisms and functions. J Leukoc Biol, 75: 163–189.

Schutze H, Mundt E, Mettenleiter TC (1999) Complete genomic sequence of viral hemorrhagic septicemia virus, a fish rhabdovirus. Virus Genes, 19: 59–65.

Shrestha B, Diamond MS (2007) Fas ligand interactions contribute to CD8+ T-cell- mediated control of West Nile virus infection in the central nervous system. J Virol, 81: 11749–11757.

Sieger D, Stein C, Neifer D, van der Sar AM, Leptin M (2009) The role of gamma interferon in innate immunity in the zebrafish embryo. Dis Model Mech, 2: 571–581.

Snow M, Cunningham CO, Melvin WT, Kurath G (1999) Analysis of the nucleoprotein identifies distinct lineages of viral haemorrhagic septicaemia virus within the European marine environment. Virus Res, 63: 35–44.

Snow M, King JA, Garden A, Shanks AM, Raynard RS (2005) Comparative susceptibility of turbot Scophthalmus maximus to different genotypes of viral haemorrhagic septicaemia virus. Dis Aquat Org, 67: 31–38.

Somamoto T, Koppang EO, Fischer U (2014) Antiviral functions of CD8(+) cytotoxic T cells in teleost fish. Dev Comp Immunol, 43: 197–204.

40

Sommerset I, Lorenzen E, Lorenzen N, Bleie H, Nerland AH (2003) A DNA vaccine directed against a rainbow trout rhabdovirus induces early protection against a nodavirus challenge in turbot. Vaccine, 21: 4661–4667.

Sommerset I, Skern R, Biering E, Bleie H, Fiksdal IU, Grove S, Nerland AH (2005) Protection against Atlantic halibut nodavirus in turbot is induced by recombinant capsid protein vaccination but not following DNA vaccination. Fish Shellfish Immunol, 18: 13–29.

Sterud E, Hansen MK, Mo TA (2000) Systemic infection with Uronema-like ciliates in farmed turbot, Scophthalmus maximus (L.). J Fish Dis, 23: 33–37.

Stolte EH, Savelkoul HF, Wiegertjes G, Flik G, Lidy Verburg-van Kemenade BM (2008) Differential expression of two interferon-gamma genes in common carp (Cyprinus carpio L.). Dev Comp Immunol, 32: 1467–1481.

Sullivan C, Charette J, Catchen J, Lage CR, Giasson G, Postlethwait JH, Millard PJ, Kim CH (2009) The gene history of zebrafish tlr4a and tlr4b is predictive of their divergent functions. J Immunol, 183: 5896–5908.

Sun Y, Hu YH, Liu CS, Sun L (2010) Construction and analysis of an experimental Streptococcus iniae DNA vaccine. Vaccine, 28: 3905–3912.

Takeda K, Akira S (2004) TLR signaling pathways. Sem Immunol, 16: 3–9.

Takeuchi O, Akira S (2010) Pattern recognition receptors and inflammation. Cell, 140: 805–820.

Thilsted SH, James D, Toppe J, Subasinghe R, Karunasagar I (2014) Maximizing the contribution of fish to human nutrition. ICN2 Second International Conference on Nutrition. FAO and World Health Organisation.

Thomas PG, Dash P, Aldridge JR Jr, Ellebedy AH, Reynolds C, Funk AJ, Martin WJ, Lamkanfi M, Webby RJ, Boyd KL, Doherty PC, Kanneganti TD (2009) The intracellular sensor NLRP3 mediates key innate and healing responses to influenza A virus via the regulation of caspase-1. Immunity, 30: 566–575.

Topham NJ, Hewitt EW (2009) Natural killer cell cytotoxicity: how do they pull the trigger? Immunology, 128: 7–15.

Toranzo AE, Barja JL (1992) First report of furunculosis in turbot reared in floating cages in northwest of Spain. Bull Eur Assoc Fish Pathol, 12: 147–149.

41

Toranzo AE, Devesa S, Romalde JL, Lamas J, Riaza A, Leiro J, Barja JL (1995) Efficacy of intraperitoneal and immersion vaccination against Enterococcus sp. infection in turbot. Aquaculture, 134: 17–27.

Toranzo AE, Magariños B, Romalde JL (2005) A review of the main bacterial fish diseases in mariculture systems. Aquaculture, 246: 37–61.

Trapani JA, Smyth MJ (2002) Functional significance of the perforin/granzyme cell death pathway. Nat Rev Immunol, 2: 735–747.

Trdo N, Benmansour A, Calisher C, Dietzgen RG, Fang RX, Jackson AO, Kurath G, Nadin-Davis S, Tesh RB, Walker PJ (2005) Rhabdoviridae. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA (eds). Virus Taxonomy. Eighth Report of the International Committee on Taxonomy of Viruses. London: Elsevier Academic Press. pp 623–644.

Tschopp J, Nabholz M (1990) Perforin-mediated target cell lysis by cytolytic T lymphocytes. Annu Rev Immunol, 8: 279–302.

Varela M, Forn-Cuní G, Dios S, Figueras A, Novoa B (2016) Proinflammatory caspase A activation and an antiviral state are induced by a zebrafish perforin after possible cellular and functional diversification from a myeloid ancestor. J Innate Immun. 8: 43–56.

Venkataraman T, Valdes M, Elsby R, Kakuta S, Caceres G, Saijo S, Iwakura Y, Barber GN (2007) Loss of DExD/H box RNA helicase LGP2 manifests disparate antiviral responses. J Immunol, 178: 6444–6455.

Walker PJ, Benmansour A, Dietzgen R, Fang RX, Jackson AO, Kurath G, Leong JC, Nadin-Davies S, Tesh RB, Trdo N (2000) Family Rhabdoviridae. In: van Regenmortel MHV, Fauquet CM, Bishop DHL, Carstens EB, Estes MK, Lemon SM, Maniloff J, Mayo MA, McGeoch DJ, Pringle CR, Wickner RB, eds. Virus Taxonomy. Classification and Nomenclature of Viruses. Seventh Report of the International Committee on Taxonomy of Viruses. San Diego: Elsevier Academic Press. pp 563–583.

Walker PJ, Winton JR (2010) Emerging viral diseases of fish and shrimp. Vet Res, 41: 51–75.

42

Wang Q, Chen J, Liu R, Jia J (2011) Identification and evaluation of an outer membrane protein OmpU from a pathogenic Vibrio harveyi isolate as vaccine candidate in turbot (Scophthalmus maximus). Lett Appl Microbiol, 53: 22–29.

Wang X, Jiang W, Yan Y, Gong T, Han J, Tian Z, Zhou R (2014) RNA viruses promote activation of the NLRP3 inflammasome through a RIP1-RIP3-DRP1 signaling pathway. Nat Immunol, 15: 1126–1133.

Winton JR (1997) Immunization with viral antigens: infectious haematopoietic necrosis. Dev Biol Stand, 90: 211–220.

Wojdasiewicz P, Poniatowski LA, Szukiewicz D (2014) The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of osteoarthritis. Mediators Inflamm, 2014: 561459.

Wolf K (1988) Viral hemorrhagic septicemia. In: Wolf K (ed). Fish Viruses and Fish Viral Diseases. Ithaca, New York, USA: Cornell University Press. pp 217–249.

Yang C, Su J, Huang T, Zhang R, Peng L (2011) Identification of a retinoic acid- inducible gene I from grass carp (Ctenopharyngodon idella) and expression analysis in vivo and in vitro. Fish Shellfish Immunol, 30: 936–943.

Yoder JA (2009) Form, function and phylogenetics of NITRs in bony fish. Dev Comp Immunol, 33: 135–144.

Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, Taira K, Akira S, Fujita T (2004) The RNA helicase RIG-I has an essential function in double- stranded RNA-induced innate antiviral responses. Nat Immunol, 5: 730–737.

Zhang J (2007) Yin and yang interplay of IFN-gamma in inflammation and autoimmune disease. J Clin Invest, 117: 871–873.

Zou J, Carrington A, Collet B, Dijkstra JM, Yoshiura Y, Bols N, Secombes C (2005) Identification and bioactivities of IFN-gamma in rainbow trout Oncorhynchus mykiss: the first Th1-type cytokine characterized functionally in fish. J Immunol, 175: 2484–2494.

Zou J, Chang M, Nie P, Secombes CJ (2009) Origin and evolution of the RIG-I like RNA helicase gene family. BMC Evol Biol, 9: 85.

Zou J, Secombes CJ (2011) Teleost fish interferons and their role in immunity. Dev Comp Immunol, 35: 1376–1387.

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2. OBJECTIVES OF THIS THESIS

1. Although turbot (S. maximus) is a very valuable fish species both in Europe and in Asia, the gene sequence information available in public databases was very scarce until the publication of the first work that forms part of this doctoral thesis. Currently, mortality and morbidity episodes due to several pathogens, especially viral diseases, represent one of the main problems affecting the culture of this flatfish. Therefore, the first objective was to increase the transcriptome information for this species using high-throughput sequencing (454 pyrosequencing - Roche) with a special enrichment in antiviral sequences, which provided a rich source of data for further studies.

2. One of the most threatening viral diseases affecting turbot aquaculture is Viral Haemorrhagic Septicaemia (VHS). Currently, neither vaccines nor therapeutic treatments are commercially available to control the effects of VHSV. DNA vaccines have proven to be highly effective in combating salmonid fish novirhabdoviruses (VHSV and IHNV). Thus, we wanted to design a DNA vaccine encoding the viral glycoprotein in order to obtain a high protection level against VHSV in turbot.

3. The third objective of this thesis was to design a microarray (based on the transcriptome information obtained in the high-throughput sequencing of the turbot transcriptome) to analyse the transcriptome profiles after administration of the DNA vaccine against VHSV and the effect of viral infection in vaccinated and non-vaccinated fish.

4. Microarray analysis provided a large amount of transcriptomic information. The overall analysis of these data revealed interesting information about the genes implicated in the defence mechanisms against viral diseases and led us to focus our attention on some specific genes to be further studied in detail. Therefore, the last objective of the present doctoral thesis was to characterize and study the main group of antiviral cytokines, the interferon (IFN) system:

4.1. Type I IFNs (ifn1 and ifn2)

4.2. Type II IFNs (ifng)

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CHAPTER 2 High-throughput sequence analysis of the turbot (Scophthalmus maximus ) transcriptome using 454 pyrosequencing for the discovery of antiviral immune genes

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High-Throughput Sequence Analysis of Turbot (Scophthalmus maximus) Transcriptome Using 454- Pyrosequencing for the Discovery of Antiviral Immune Genes

Patricia Pereiro1, Pablo Balseiro1, Alejandro Romero1, Sonia Dios1, Gabriel Forn-Cuni1, Berta Fuste3, Josep V. Planas2, Sergi Beltran3, Beatriz Novoa1, Antonio Figueras1* 1 Instituto de Investigaciones Marinas, IIM, CSIC, Vigo, Spain, 2 Departament de Fisiologia i Immunologia, Facultat de Biologia, Universitat de Barcelona i Institut de Biomedicina de la Universitat de Barcelona, IBUB, Barcelona, Spain, 3 Centros Cientı´ficos y Tecnolo´gicos de la UB, CCiT-UB, Universitat de Barcelona, Edifici Clu´ster, Parc Cientı´fic de Barcelona, Barcelona, Spain

Abstract

Background: Turbot (Scophthalmus maximus L.) is an important aquacultural resource both in Europe and Asia. However, there is little information on gene sequences available in public databases. Currently, one of the main problems affecting the culture of this flatfish is mortality due to several pathogens, especially viral diseases which are not treatable. In order to identify new genes involved in immune defense, we conducted 454-pyrosequencing of the turbot transcriptome after different immune stimulations.

Methodology/Principal Findings: Turbot were injected with viral stimuli to increase the expression level of immune-related genes. High-throughput deep sequencing using 454-pyrosequencing technology yielded 915,256 high-quality reads. These sequences were assembled into 55,404 contigs that were subjected to annotation steps. Intriguingly, 55.16% of the deduced protein was not significantly similar to any sequences in the databases used for the annotation and only 0.85% of the BLASTx top-hits matched S. maximus protein sequences. This relatively low level of annotation is possibly due to the limited information for this specie and other flatfish in the database. These results suggest the identification of a large number of new genes in turbot and in fish in general. A more detailed analysis showed the presence of putative members of several innate and specific immune pathways.

Conclusions/Significance: To our knowledge, this study is the first transcriptome analysis using 454-pyrosequencing for turbot. Previously, there were only 12,471 EST and less of 1,500 nucleotide sequences for S. maximus in NCBI database. Our results provide a rich source of data (55,404 contigs and 181,845 singletons) for discovering and identifying new genes, which will serve as a basis for microarray construction, gene expression characterization and for identification of genetic markers to be used in several applications. Immune stimulation in turbot was very effective, obtaining an enormous variety of sequences belonging to genes involved in the defense mechanisms.

Citation: Pereiro P, Balseiro P, Romero A, Dios S, Forn-Cuni G, et al. (2012) High-Throughput Sequence Analysis of Turbot (Scophthalmus maximus) Transcriptome Using 454-Pyrosequencing for the Discovery of Antiviral Immune Genes. PLoS ONE 7(5): e35369. doi:10.1371/journal.pone.0035369 Editor: Zhanjiang Liu, Auburn University, United States of America Received December 9, 2011; Accepted March 16, 2012; Published May 18, 2012 Copyright: ß 2012 Pereiro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work has been supported by the funding from the project CSD2007-00002 ‘‘Aquagenomics’’ of the program Consolider-Ingenio 2010 from the Spanish Ministerio de Ciencia e Innovacio´n. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]

Introduction in recent years and being the largest producer of turbot in the world [3]. Turbot (Scophthalmus maximus) is an economically important This development caused a parallel increase in pathological flatfish species belonging to the family Scophthalmidae (order conditions affecting this culture. Several pathogens, including Pleuronectiformes) widely distributed from Norway to the Mediter- bacteria [4], virus [5] and parasites [6] affect the health status of ranean and the Black Sea [1]. Nowadays, the culture of this fish is the farmed fish causing important economic losses. In spite of the well-established and a full-cycle aquaculture is in place. In Europe, relevance of turbot culture and the associated pathological process, turbot aquaculture production was 9,067 tons in 2008, represent- knowledge about its immune system is still fragmentary and little is ing almost 90% of total flatfish production, being Spain the main known about host-pathogen interactions. The pathways implicated European producer [2]. This species, native to Europe, is also in the response against pathogens remain incomplete in fish and cultured in China, reaching an annual level of 50,000–60,000 tons the understanding of how those defense mechanisms act is a

PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e35369 454-Pyrosequencing of Turbot Antiviral Genes relevant factor in order to enhance resistance of cultured fish to to increase the knowledge on the turbot immune transcriptome diseases. The identification of immune-relevant genes, that could had been previously conducted using strategies based in Sanger be potential markers for disease resistance, is a step to establish a sequencing. Wang et al. [17] obtained 49 ESTs from kidney and successful genetic breeding program. Moreover, several immune spleen of turbot following challenge with Vibrio harveyi. Pardo et al. molecules (antimicrobial peptides and cytokines) could be used as [18] achieved 1,073 contigs and 2,409 singletons from 9,256 ESTs novel anti-infective drugs and also as adjuvants in vaccination from liver, spleen and head kidney of turbot infected with processes due to their immunomodulatory properties [7–14]. In Aeromonas salmonicida, Philasterides dicentrarchi and from healthy fish. addition, increase in immune-related genes and pathways in turbot Park et al. [19] obtained 3,173 ESTs from liver, kidney and gill could help us to improve the microarray tools available for tissues of nodavirus-infected turbot. Pyrosequencing represents a research in fish immunology. step forward compared to classical Sanger sequencing strategies Expressed sequence tags (ESTs) are short sequence reads and allows to generate great amounts of genomic and transcrip- derived from cDNA libraries that represent a useful tool for tomic information at relatively low cost and in short periods of discovery and identification of transcripts mainly in species time. The present work increases dramatically the number of without a fully sequenced genome [15,16]. All published putative transcripts by providing 55,404 contigs for further approaches to the turbot immune transcriptome are based on genomic studies in turbot and represents the most effective classical cloning and Sanger sequencing strategies [17–19]. attempt to improve the knowledge of S. maximus transcriptome. Pyrosequencing is a relatively new sequencing technology in fish Moreover, it was possible to annotated 24,845 of these contigs biology, which provides a fast and cost-effective way to generate (44.84%) with an E value cut off of 1e-3 after Blastx to selected large amounts of data from non-model species such as turbot. databases. This relatively low value of annotation is almost The aim of this work was to increase the genomic resources of certainly due to the scarce information available in the database turbot, particularly the transcriptome in response to viral for pleuronectiform fish. stimulations and to identify the main components of the immune pathways. For this, we have used 454-pyrosequencing on several turbot tissues stimulated with two different viruses (viral haemor- Table 1. Summary statistics of Scophthalmus maximus 454- rhagic septicaemia virus and nodavirus), plasmid constructs used pyrosequencing. as DNA vaccines and polyriboinosinic polyribocytidylic acid (poly I:C). The data generated in this high-throughput sequence analysis will serve as a basis for microarray construction, which will also be Data generation and filtering useful for further research on this important aquaculture species. Number of total reads 915,782 Total Megabases 291.04 Results and Discussion Average read length (bp) 317.8 Transcriptomic Analysis N50 read length (bp) 396 Table 1 shows the summarized statistics for Roche 454- Number of high quality reads (after filtering) 915,256 (99.94%) pyrosequencing of turbot normalized library. Sequencing of turbot Total Megabases (after filtering) 271.95 transcriptome yielded a total of 915,782 reads totaling 291.04 Average read length (after filtering) 297.14 megabases with an average read length of 318 bp. After processing N50 read length (after filtering) 381 the reads and filtering the low-quality and small ones, 915,256 high quality reads with an average size of 297 bp were submitted Assembly statistics to MIRA V3.2.0 for assembly. Reads obtained in this high- Number of reads assembled 733,411 (80.13%) throughput sequence analysis were submitted to the NCBI Short Number of contigs 55,404 Read Archive (http://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi) Total consensus Megabases 37.2 under accession number SRA046015.2. 733,411 reads were Average contig coverage 4.4 assembled into 55,404 contigs, with an average contig length of 671 bp. Moreover, contigs generated in this work are available on Average contig length 671.3 the NCBI Transcriptome Shotgun Assembly Sequence Database N50 contig length 756 (http://www.ncbi.nlm.nih.gov/genbank/tsa): BioProjectID Range contig length 40–6,821 PRJNA89091 (Genbank accessions JU351362 to JU404686). In Number of contigs .500 pb 31,764 (57.33%) order to minimize redundancy, contigs were grouped into a total Number of contigs with 2 reads 21,270 (38.39%) of 41,870 clusters according to their sequence similarity. Of 55,404 Number of contigs with .2 reads 34,134 (61.61%) contigs, 24,845 (44.84%) had a significant BLASTx hit to proteins present in Swissprot/Metazoan Refseq/nr databases and matched Number of clusters 41,870 12,591 unique protein accessions. The descriptive analysis Number of clusters with 1 contig 39,578 (94.53%) presented here utilized only the contigs produced by the assembly. Number of clusters with .1 contig 2,292 (5.47%) Contig and clustering construction are summarized in Figure 1. Number of singletons 181,845 Singletons (181,845 sequences) were separately analyzed and Annotation annotated for the search of new sequences showing homology to proteins involved in the main immune signaling cascades but were Total number of annotated contigs 24,845 (44.84%) not included in the other analysis in order to obtain more reliable SwissProt+Metazoan RefSeq hits 20,175 (81.2%) results. Only 13,722 singletons were successfully annotated. An nr hits 4,670 (18.8%) additional file containing the singletons selected for their Unique protein hits 12,591 annotation is also provided (Table S1). Before starting our analysis Total number of annotated singletons 13,722 there were only 12,471 EST and less of 1,500 nucleotide sequences for S. maximus in NCBI database. Other approaches doi:10.1371/journal.pone.0035369.t001

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Figure 1. Scophthalmus maximus transcriptome assembly statistics. (A) Distribution of number of reads per contig in the normalized library. (B) Size distribution of 454 sequences after contig construction. (C) Distribution of cluster composition by contigs. doi:10.1371/journal.pone.0035369.g001

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A top-25 showing the most commonly detected proteins terms reported for mammalians and fish, including antiviral, antibacte- in the annotation process represented different functional groups rial and anti-inflammatory activities [26–32]. including an elevated amount of immune-related proteins Both precursor of alpha-1 microglycoprotein (365 hits) and (Figure 2). The precursor of type 2 ice structuring protein was precursor of homolog to alpha-1-antitrypsin (326 hits) possess an surprisingly the more represented BLAST hit (654 hits). Antifreeze immunomodulatory role regulating the inflammation process [33– proteins (AFPs) have in common the ability to bind to ice and 35]. Precursor of trypsin-1 (83 hits) and precursor of trypsin-2 inhibit its growth [20]. Type II antifreeze proteins found in smelt (45 hits) are serine proteases presenting a proinflammatory role (Osmerus mordax) and herring (Clupea harengus harengus) belong to the during the pathogenesis [36,37] besides their digestive function. C-type lectin superfamily, although these ice-binding proteins do Other group widely represented was related with the comple- not appear to function as lectins [21]. Milla´n et al. [22] using an ment cascade (complement component C3 with 114 hits) and oligo-microarray specific for turbot, found a great up-regulation of coagulation (precursor of fibrinogen beta chain with 90 hits and antifreeze polypeptide precursor in fish challenged intraperitone- fibrinogen-gamma chain with 60 hits). Fibrinogen is important not ally with the bacteria Aeromonas salmonicida. It could be interesting only for its pro-coagulatory functions but also for its pro- to study whether this molecule possesses any antiviral function. inflammatory activities [38,39]. Precursor of Hyaluronan-binding Lectins are recognition molecules capable to adhere to carbohy- protein 2 (56 hits) and other hyaluronan related proteins regulate drates that are implicated in direct first-line defense against inflammation and tissue damage through the cell recruitment and pathogens [23]. L-rhamnose-binding lectin CSL2 (476 hits) and release of inflammatory cytokines [40]. lectin (137 hits) also appear represented in the BLASTx top-hit Precursor of serotransferrin (280 hits) is a glycoprotein with sequences. iron-chelating properties that transports and scavenges extracel- Other immune-related proteins appearing in the top-hit lular iron [41]. Precursor of haptoglobin (56 hits) and precursor of sequences are 14 kDa apolipoprotein (591 hits), precursor of hemopexin (47 hits) are acute phase proteins that recycle apolipoprotein A1 (480 hits) and apolipoprotein A1 (52 hits). hemoglobin from senescent erythrocytes [42,43]. Moreover, Apolipoproteins with a molecular weight of 14 kDa (apo-14 kDa) Hemoglobin subunit alpha-1 (174 hits) was another sequence are associated with fish plasma high density lipoproteins (HDL) belonging to the top-hit sequences. The high transcriptional level and phylogenetic analysis indicates that fish apo-14 kDa can be of these genes could be due to the anemia and hemorrhaging the homologue of mammalian apoA-II [24]. Although the main caused by viral haemorrhagic septicaemia virus (VHSV) [44] and role of HDL/apolipoproteins seems to be its participation in also to the ability of the viruses to maintain an iron-replete host for cholesterol transport [25], multiple immune functions have been replication [45].

Figure 2. BLASTx top-hit sequence distribution of gene annotations. Y-axis represents the most abundant annotation terms for the contigs obtained in the 454-pyrosequencing. X-axis represents the number of contigs presenting each annotation term. doi:10.1371/journal.pone.0035369.g002

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Furthermore, Precursor of Perforin-1 (45 hits) is a molecule Perciformes (18.03%), Salmoniformes (14.7%) and Tetraodontiformes implicated in the cellular lysis through the formation of pores in (13.92%) (Figure 5B). These orders include model fish species and biological membranes [46]. The other sequences represented in organisms with economic relevance and, consequently, with the top-hit BLASTx are mainly implicated in transport, motor abundant transcriptomic information available. The order Pleur- activity and transcription. The large presence of immune-related onectiformes represented only 9.62% of the total hits from fish, terms in this ranking revealed a very effective stimulation of turbot highlighting once again the scarce information available for this and a successful transcriptome sequencing. group in the databases and reaffirming the need to improve and increase the information. The present massive transcriptome Gene Ontology Analysis analysis has helped to solve this problem. The kingdom Plantae Gene ontology (GO) is commonly used to categorize gene represented 0.6% of annotated sequences, Fungi 0.446%, Protista products and standardize their representation across species [47]. 0.43%, Eubacteria 0.462%, Archaebacteria 0.045% and Viruses In order to eliminate redundancy and given that the contigs 0.118%. Despite water treatment at our facilities with filters and belonging to the same cluster present the same annotation, only UV light it is possible that certain microorganisms such as algae, the largest contig of each cluster was submitted to Blast2GO suite fungi, protozoa and bacteria have survived these conditions and [48] for the assignment to three functional groups based on GO have contaminated the samples. Moreover, the normal microbiota terminology: Cellular Component, Biological Process and Molec- of turbot tissues could be present. However, these contigs showing ular Function. 12,534 contigs (29.9%) were assigned to a GO homology to non-animal species represent a negligible percentage category. Figure 3 summarizes GO terms at 2nd level. Cellular of the total annotated contigs. Among bacteria, some sequences component terms (Figure 3A) showed a significant percentage of seemed to show homology to proteins from genera that include clusters assigned to cell (24.95%) and cell part (24.95%), whereas relevant pathogens for fish species: Vibrio, Pseudomonas and 19.27% were related to organelle and 12.3% to organelle part. Mycobacterium. Among viruses, there was also homology to proteins The most represented biological process terms (Figure 3B) were from viral families that can affect fish: Herpesviridae, Retroviridae and related to cellular process (15.57%), metabolic process (12.05%) Rhabdoviridae. The Rhabdoviridae VHSV, which was used to and biological regulation (10.12%), suggesting a high degree of inoculate turbot in our study, appeared represented in the metabolic activity of the sampled tissues. Immune-related proteins annotation. could be included within cellular process category (which includes the molecules implicated in cell activation), death (1.68%), Comparative Analysis immune system process (2%), multicellular organismal process The turbot transcriptomic sequences were compared to ESTs (8.49%) (which includes proteins related to the coagulation and nucleotide sequences from zebrafish (D. rerio, order Cyprini- process), response to stimulus (8.47%) and signaling (4.9%). formes) and Japanese flounder (Paralichthys olivaceous, order Pleur- Finally, within the molecular function category (Figure 3C), onectiformes) present in the NCBI database in order to detect the 48.93% were related to binding (including several immune- presence of novel proteins in turbot remaining unknown in other processes) followed by 27.15% related to catalytic activity. Our fish species more studied so far. The results are displayed as a GO analysis followed a similar pattern to that obtained in previous Venn diagram in Figure 6. Zebrafish was chosen because it pyrosequencing efforts performed in other fish species [49–51]. In represents a model fish and its genome has been sequenced. order to provide a more detailed analysis of the Molecular Japanese flounder was chosen because it is a species phylogenet- Function category an additional file showing GO terms at the 3rd ically close to turbot and with abundant genetic information level is also supplied (Table S2). compared to other flatfish. The comparison of our pyrosequencing results with the information available in databases for two species Taxonomic Analysis presenting a better genomic and transcriptomic knowledge can A BLASTx top-hit species distribution of gene annotations give an idea of the new contributions to the fish transcriptome showed highest homology to Homo sapiens, followed by Mus musculus study. Of the total 41,870 contigs representing each cluster, 21,536 (Figure 4). These results could be explained by the elevated (51.44%) had no significant similarity to any protein identified presence of human and murine sequences in the databases. The within the sequences of D. rerio and P. olivaceus. This high third position was for Danio rerio, which has been widely used as a percentage of unique transcripts can be attributed to the presence model organism for biomedical and fish studies. Other species with of novel genes but also it can be derived from untranslated regions known genome sequences appearing in the BLASTx top-hit were (59 and 39 UTR) and non-conserved areas of proteins [52,53]. the mammals Bos taurus and Rattus norvergicus, the chicken Gallus 10,326 contigs (24.66%) were shared between zebrafish and turbot gallus and the fruit fly Drosophila melanogaster. There were 13 fish but not with Japanese flounder, possibly due to the high number of species among the top-hits, but only 3 species belonging to the sequences present in the NCBI database for this model species. order Pleuronectiformes. Turbot appeared in position 24 and only 3,440 sequences (8.22%) were shared between Japanese flounder 0.85% of the BLASTx top-hits matched Scophthalmus maximus and turbot but not with zebrafish, suggesting that they are protein sequences. This may be explained on the basis of the potential flatfish-specific sequences. However, 6,568 contigs limited number of turbot proteins (605) currently available in the (15.69%) had homology with both D. rerio and P. olivaceus NCBI database compared to other fish species. sequences and probably represent well conserved genes across A more detailed taxonomic analysis was performed in order to the species. further characterize our pyrosequencing data. The immense majority of the annotated sequences matched organisms from Immune Transcriptome Analysis the kingdom Animalia (97.8%), but there was representation from Our high-throughput sequencing effort revealed the presence of all other kingdoms, as well as from viruses. In Animalia, mammals an elevated number of relevant molecules for the immune represented 48.7% of the hits, whereas fish represented 38.79% response, but hitherto remaining unknown in S. maximus. There (Figure 5A). This suggests that we have identified a large number were no evidences of existence of the majority of the detected of genes poorly described in fish. The teleost orders most immune-related proteins in turbot before performing this pyrose- represented in the annotation results were Cypriniformes (27.18%), quencing analysis. Here, we describe the main components of the

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Figure 3. Gene ontology (GO) assignment (2nd level GO terms) for the transcriptomic sequences (contigs) of Scophthalmus maximus. (A) Cellular component, (B) Biological process and (C) Molecular function. Numbers refer to percentage of occurrences. doi:10.1371/journal.pone.0035369.g003

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Figure 4. BLASTx top-hit species distribution of gene annotations. Y-axis represents the species presenting more match hits with the contigs obtained in the 454-pyrosequencing. X-axis represents the number of contigs presenting protein match hits to these species. doi:10.1371/journal.pone.0035369.g004 immune pathways identified in the Scophthalmus maximus transcrip- activation of nuclear factor–kB (NF-kB) inducing pro-inflammatory tome (contigs and singletons). cytokines through MyD88 dependent or independent pathways Complement pathway. The complement system is a [56,57]. We have found numerous contigs presenting high similarity biochemical cascade involving more than 35 soluble plasma to several members of the TLR cascade (Figure 8). So far in turbot, proteins. This defense mechanism is essential in the innate there was only evidence for the presence of two Toll-like receptors: immunity, but it exhibits also the ability to stimulate the specific TLR3 (GenBank accession FJ009111) and TLR11 [18]. We have immune response [54]. In vertebrates, the central component C3 identified several sequences with similarity to four new TLRs in S. is proteolytically activated by a C3 convertase through the classic, maximus: TLR2, TLR5, TLR6, TLR7, TLR8, TLR13 and lectins and alternative routes [55]. In the present study, we TLR21B. Moreover, most of the central proteins belonging to the identified the main putative central molecules in the complement turbot TLR signaling pathway have been discovered for the first system (C1 to C9) (Figure 7). Only the C6 component appeared time in this transcriptome analysis: MyD88, interleukin-1 receptor- not to be represented. C2, C4 and C5 were identified for the first associated kinase 4 (IRAK4), IkB kinase (IKK), NF-kB, and several time in turbot, as well as the receptors for C3a (C3aR1), C5a mitogen-activated protein kinases (MAPKs) amongst others. An (C5aR1), the complement receptor type 1 or C3b/C4b receptor additional table containing information about contigs and singletons (CR1) and the complement receptor type 2 (CR2 or CD21). showing homology to molecules implicated in the Toll-like receptor Moreover, other important proteins intervening in the comple- signaling pathway is provided (Table S4). ment cascade were annotated, including the complement factors H B cell receptor signaling pathway. B lymphocytes and (Hf), B (Bf), D (Df), I (If) and P (Pf) or properdin. Only one of these, T lymphocytes are the main cells involved in antigen-specific Bf, had been reported previously in S. maximus [19]. The elevated defense. B cells are responsible for the production of specific number of complement components detected in turbot exposed to antibodies that can neutralize pathogens [58]. The activation of viral stimulation could reflect the pivotal importance of these B lymphocytes is achieved through the binding of the antigen to molecules in protection during viral infections. An additional table B-cell receptors [59]. Prior to the present study, there was only containing information about contigs and singletons showing genetic and bibliographic information available for turbot homology to molecules implicated in the complement pathway is immunoglobulins (Ig) (GenBank accessions FJ617006, provided (Table S3). DQ848956, DQ848958, AJ296096; [19]), Ras-related C3 Toll-like receptor signaling pathway. Recognition of botulinum toxin substrate 1 (Rac1) (FJ361904), Ras GTPase pathogen-associated molecular patterns (PAMPs) is essential for (EU711052), extracellular signal-regulated kinase (ERK) [19] the activation of innate immunity and it is carried out by Toll-like and interferon induced transmembrane protein (Leu13 or receptors (TLRs). Cell activation triggered by TLR entails the CD225) (DQ848974). We have detected in S. maximus

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Figure 5. Pie chart showing species distribution of the BLAST hits of the Scophthalmus maximus sequences. (A) Distribution of the BLAST hits within the kingdom Animalia. (B) Distribution of the BLAST hits among teleost orders. doi:10.1371/journal.pone.0035369.g005 transcriptome the majority of the proteins implicated in the B showing homology to molecules involved in the B cell receptor cell signaling pathway in mammals (Figure 9). An additional signaling pathway is also provided (Table S5). table containing information about contigs and singletons T cell receptor signaling pathway. During their develop- ment, T-cells differentiate into either CD4+ helper or CD8+ cytolytic T cells [60]. Antigen-presenting cells expose antigenic peptides to T lymphocytes through the major histocompatibility complex (MHC) molecules [61]. CD4+ lymphocytes have the function of collaborating on the activation of other immune system cells, as well as recruiting them to sites of infection [62]. CD8+ T lymphocytes present cytotoxic capability, eliminating cells infected with viruses, intracellular bacteria and parasites as well as tumor cells [63]. The present turbot pyrosequencing effort provided several contigs similar to proteins intervening in the cascade of T lymphocyte activation (Figure 10). As occurred with the B cell receptor signaling pathway, the previous information for this activation cascade was very scarce in S. maximus and was limited to few proteins. An additional table containing information about contigs and singletons showing homology to molecules involved in the T cell receptor signaling pathway is provided (Table S6). Apoptosis or programmed cell death. Apoptosis is an Figure 6. Venn diagram showing the comparison among essential mechanism for the development and survival of Scophthalmus maximus transcriptomic sequences with the known sequences from Danio rerio and Paralichthys olivaceus organisms. Unwanted, damaged or infected cells are removed deposited in the NCBI database. involving numerous molecules [64]. Caspases are proteases with a doi:10.1371/journal.pone.0035369.g006 pivotal role in apoptosis among other functions [65]. Our

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Figure 7. Complement pathway representing the present and absent proteins in the pyrosequencing results. Proteins appearing in the turbot transcriptome are represented in reddish colour and absent proteins in grey color. C1q: complement C1q subcomponent; C1r: complement C1r subcomponent; C1s: complement C1s subcomponent; C2: complement component 2; C3: complement component 3; C4: complement component 4; C5: complement component 5; C6: complement component 6; C7: complement component 7; C8: complement component 8; C9: complement component 9; C4BP: complement component 4 binding protein; MBL: mannose-binding lectin; MASP1/2: mannan-binding lectin serine protease 1/2; Bf: component factor B; Hf1: complement factor H; Df: component factor D; If: complement factor I; C3aR1: C3a anaphylatoxin chemotactic receptor; CR1: complement component receptor 1; CR2: complement component receptor 2; C5aR1: C5a anaphylatoxin chemotactic receptor. doi:10.1371/journal.pone.0035369.g007 transcriptome analysis showed for the first time that the vast TNFa, a pleiotropic pro-inflammatory cytokine produced by majority of central molecules that control programmed cell death numerous immune cells during inflammation [69], appeared also in higher vertebrates, presented homologues in the flatfish in our collection, as well as TNFb. Among the CSFs, which S. maximus, suggesting the existence of a phylogenetically con- regulate the production and functional activities of hematopoietic served apoptotic machinery (Figure 11). An additional table cells [70], we have found four types: macrophage colony- containing information about contigs and singletons showing stimulating factor 1 (MCSF-1) and 2 (MCSF-2), granulocyte homology to molecules implicated in the apoptosis is also provided colony-stimulating factor 1 (GCSF-1) and the megakaryocyte (Table S7). colony-stimulating factor thrombopoietin. Some growth factors Cytokines. Cytokines are cell-signalling proteins involved in appearing in the turbot transcriptome were fibroblast growth haematopoiesis, inflammation and defense against pathogens [66]. factor (FGF), transforming growth factor (TGF), bone morphoge- Major cytokines include interleukins (ILs), chemokines, interferons netic protein (BMP), epidermal growth factor (EGF), platelet- (IFNs), tumor necrosis factors (TNFs), colony stimulating factors derived growth factor (PDGF), vascular endothelial growth factor (CSFs) and growth factors (GFs) [67]. Among the interleukins, our (VEGF), nerve growth factor (NGF) and hepatocyte growth factor results showed the presence of putative contigs for IL-1b, IL-8, IL- or hematopoietin (HGF). Moreover, we found several contigs 11, IL-12b, IL-15, IL-16, IL-18 and IL-25. Chemokines are presenting homology to cytokine receptors and cytokine-related characterized by cysteine residues and separated into two groups proteins. depending on the presence (C-X-C family) or absence (CC family) Other immune molecules. Concerning other pattern rec- of an intervening amino acid between cysteine residues [68]. ognition receptors (PRRs) different to TLRs, lectins are a family of Several contigs and singletons were identified as CC and C-X-C carbohydrate-recognition proteins and they have been implicated motif chemokines. For the IFNs group, there was a unique contig in the direct first-line defense against pathogens and immune presenting homology to D. rerio IFN phi 2 (IFNW2) and a singleton regulation [23]. The turbot transcriptome exhibited abundant annotated as IFN interferon alpha 2 precursor [Salmo salar]. lectins, especially C-type lectins. However, other lectins were also

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Figure 8. Toll-like receptor signaling pathway representing the present and absent proteins in the pyrosequencing results. Proteins appearing in the turbot transcriptome are represented in reddish color and absent proteins in grey color. LBP: lipopolysaccharide-binding protein; MD2: myeloid differentiation factor 2; TLR1: toll-like receptor 1; TLR2: toll-like receptor 2; TLR3: toll-like receptor 3; TLR4: toll-like receptor 4; TLR5: toll- like receptor 5; TLR6: toll-like receptor 6; TLR7: toll-like receptor 7; TLR8: toll-like receptor 8; TLR9: toll-like receptor 9; TLR13: toll-like receptor 13; CD14: cluster of differentiation antigen 14; MyD88: myeloid differentiation primary response protein MyD88; TIRAP: toll-interleukin 1 receptor domain-containing adaptor protein; TOLLIP: toll-interacting protein; Rac1: Ras-related C3 botulinum toxin substrate 1; PI3K: phosphatidylinositol-4,5- bisphosphate 3-kinase; AKT: Rac serine/threonine-protein kinase; FADD: FAS-associated via death domain; IRAK1: interleukin-1 receptor-associated kinase 1; IRAK4: interleukin-1 receptor-associated kinase 4; TRAM: TRIF-related adaptor molecule; BinCARD: BCL-10-interacting protein with CARD; BCL-10: B cell lymphoma 10; MALT1: mucosa associated lymphoid tissue lymphoma translocation gene; CASP8: caspase 8; TRAF: TNF receptor- associated factor 3; TRAF6: TNF receptor-associated factor 6; TAB1: TAK1-binding protein 1; TAB2: TAK1-binding protein 2; TAK1: TGF-beta activated kinase 1; RIP1: receptor-interacting serine/threonine-protein kinase 1; MKK3/6: mitogen-activated protein kinase kinase 3/6; MKK4: mitogen-activated protein kinase kinase 4; JNK: c-Jun N-terminal kinase; p38: p38 MAP kinase; NF-kB: nuclear factor NF-kappa-B; IKKa: inhibitor of nuclear factor kappa-B kinase subunit alpha; IKKb: inhibitor of nuclear factor kappa-B kinase subunit beta; IKKc: inhibitor of nuclear factor kappa-B kinase subunit gamma; IKKe: inhibitor of nuclear factor kappa-B kinase subunit epsilon; IkBa: NF-kappa-B inhibitor alpha; p105: nuclear factor NF-kappa-B p105 subunit; Tpl2: tumor progression locus 2; MEK2: mitogen-activated protein kinase kinase 2; ERK: extracellular signal-regulated kinase; TRIF: toll-like receptor adapter molecule 1; TBK1: TANK-binding kinase 1; mTOR: mechanistic target of rapamycin; Raptor: regulatory associated protein of mTOR; IRF3: interferon regulatory factor 3; IRF5: interferon regulatory factor 5; IRF7: interferon regulatory factor 7; AP-1: activator protein-1; TNFa: tumor necrosis factor alpha; IL-1b: interleukin 1 beta; IL-6: interleukin 6; IL-12: interleukin 12; IL-8: interleukin 8; RANTES: regulated on Activation. Normal T-cell Expressed and Secreted; MIP-1a: macrophage inflammatory protein-1a; MIP-1b: macrophage inflammatory protein-1b; IFN: interferon; CD86: cluster of differentiation antigen 86; CD80: cluster of differentiation antigen 80; CD40: cluster of differentiation antigen 40; MIG: monokine induced by gamma interferon; I-TAC: interferon-inducible T-cell alpha chemoattractant; IP-10: interferon-gamma-inducible protein 10. doi:10.1371/journal.pone.0035369.g008

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Figure 9. B cell receptor signaling pathway representing the present and absent proteins in the pyrosequencing results. Proteins appearing in the turbot transcriptome are represented in reddish color and absent proteins in grey color. PIR-B: paired-immunoglobulin like receptor- B; CD22: cluster of differentiation antigen 22; CD72: cluster of differentiation antigen 72; BCR: B cell receptor; FcgRIIB: Fc fragment of IgG, low affinity IIb, receptor; LEU13: leukocyte surface antigen LEU-13; CD81: cluster of differentiation antigen 81; CD19: cluster of differentiation antigen 19; CD21: cluster of differentiation antigen 21; SHP1: protein-tyrosine phosphatase-1; LYN: tyrosine-protein kinase Lyn; BCAP: B-cell phosphoinositide 3-kinase adapter protein; SHIP: SH-2-containing Inositol 5’ Phosphatase; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; VAV: vav guanine nucleotide exchange factor; SYK: spleen tyrosine kinase; BTK: Bruton agammaglobulinemia tyrosine kinase; BLNK: B-cell linker; GRB2: growth factor receptor- binding protein 2; SOS: son of sevenless; PCL-c2: phospholipase C gamma 2; PKCb: protein kinase C beta; AKT: Rac serine/threonine-protein kinase, GSK3b: glycogen synthase kinase 3 beta; Bam32: B-cell adapter molecule of 32 kDa; Rac: Ras-related C3 botulinum toxin substrate 1; Ras: GTPase HRas; RasGRP3: Ras guanyl-releasing protein 3; CARMA1: caspase recruitment domain family, member 11; BCL-10: B cell lymphoma 10; MALT1: mucosa associated lymphoid tissue lymphoma translocation gene; IKKa: inhibitor of nuclear factor kappa-B kinase subunit alpha; IKKb: inhibitor of nuclear factor kappa-B kinase subunit beta; IKKc: inhibitor of nuclear factor kappa-B kinase subunit gamma; Raf: Raf proto-oncogene serine/ threonine-protein kinase; MEK2: mitogen-activated protein kinase kinase 2; NFAT: nuclear factor of activated T-cells; ERK: extracellular signal- regulated kinase; IkB: NF-kappa-B inhibitor beta; NF-kB: nuclear factor NF-kappa-B; AP1: activator protein-1. doi:10.1371/journal.pone.0035369.g009 detected, such as S-type lectins (Galectins 1, 2, 4, 8 and 9) and L- Among other immune-related proteins, it is worth pointing rhamnose binding lectins. With regards to b-glucan recognition out the identification of several protease inhibitors, antimicrobial proteins, only one contig was found with homology to b-1,3- peptides and other proteins with cytolytic activity (hepcidin, glucan-binding protein 1 (BGBP-1). Moreover, peptidoglycan defensin, antimicrobial peptide precursor, Nk-lysin, lysozyme G recognition proteins (PGRPs) and only one contig for lipopoly- and C, perforin-1, granzyme and other serine proteases) and saccharide-binding protein (LBP) were detected. also several heat shock proteins (Hsp10, Hsp16, Hsp20, Hsp27,

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Figure 10. T cell receptor signaling pathway representing the present and absent proteins in the pyrosequencing results. Proteins appearing in turbot the transcriptome are represented in reddish color and absent proteins in grey color. PD-1: programmed cell death protein 1; CTLA4: cytotoxic T-lymphocyte-associated protein 4; CD45: cluster of differentiation antigen 45; CD8: cluster of differentiation antigen 8; CD4: cluster of differentiation antigen 4; CD3e: cluster of differentiation antigen 3 epsilon chain; CD3c: cluster of differentiation antigen 3 gamma chain; CD3d: cluster of differentiation antigen 3 delta chain; Cd3f: cluster of differentiation antigen 3 zeta chain; TCRa: T cell receptor alpha chain; TCRb: T cell receptor beta chain; CD40L: cluster of differentiation antigen 40 ligand; ICOS: inducible T-cell co-stimulator; CD28: cluster of differentiation antigen 28; SHP1: protein-tyrosine phosphatase-1; LCK: lymphocyte-specific protein tyrosine kinase; FYN: proto-oncogene tyrosine-protein kinase Fyn; LAT: linker for activation of T cells; GRB2: growth factor receptor-binding protein 2; SOS: son of sevenless; PI3K: phosphatidylinositol-4,5-bisphosphate 3- kinase; CBL: Casitas B-lineage lymphoma; ZAP70: zeta-chain (TCR) associated protein kinase; GADS: GRB2-related adaptor protein 2; PCL-c1: phospholipase C gamma 1; PKCh: protein kinase C theta; PDK1: pyruvate dehydrogenase kinase isoform 1; AKT: Rac serine/threonine-protein kinase; SLP-76: SH2 domain-containing leukocyte protein of 76 kDa; IKT: IL2-inducible T-cell kinase; RasGRP1: Ras guanyl-releasing protein 1; TAK1: TGF-beta activated kinase 1; CARMA1: caspase recruitment domain family, member 11; BCL-10: B cell lymphoma 10; MALT1: mucosa associated lymphoid tissue lymphoma translocation gene; COT: cancer Osaka thyroid oncogene; GSK-3b: glycogen synthase kinase 3 beta; NCK: NCK adaptor protein 1; VAV: vav guanine nucleotide exchange factor; Ras: GTPase HRas; MKK7: mitogen-activated protein kinase kinase 7; Dlgh1: disks large protein 1; NIK: NF-kappa- beta-inducing kinase; P38: p38 MAP kinase; PAK: p21-activated kinase 1; Rho/Cdc42: cell division control protein 42; Raf: Raf proto-oncogene serine/ threonine-protein kinase; JNK2: c-Jun N-terminal kinase 2; IKKa: inhibitor of nuclear factor kappa-B kinase subunit alpha; IKKb: inhibitor of nuclear factor kappa-B kinase subunit beta; IKKc: inhibitor of nuclear factor kappa-B kinase subunit gamma; MEK2: mitogen-activated protein kinase kinase 2; IkB: NF-kappa-B inhibitor; NF-kB: nuclear factor NF-kappa-B; ERK: extracellular signal-regulated kinase; NFAT: nuclear factor of activated T-cells; AP1: activator protein-1. doi:10.1371/journal.pone.0035369.g010

Hsp40, Hsp60, Hsp68, Hsp70, Hsp71, Hsp75, Hsp83 and produced by VHSV, could influence the expression of these Hsp90). molecules. Furthermore, there was a large representation of iron-binding proteins. Hemoglobin, ferritin, transferrin, serotransferrin, hapto- Conclusions globin and hemopexin were the most represented. As was mentioned Comparison of our results with previous transcriptomic studies above, viruses need an iron-replete host for an efficient replication from S. maximus using conventional Sanger sequencing confirms and they can alter the levels of proteins involved in iron homeostasis. the efficacy of 454-pyrosequencing to improve the genomic Moreover, hemorrhages generated during infections, especially knowledge on this flatfish, revealing a large number of contigs. We have obtained 55,404 contigs containing at least 2 reads and

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Figure 11. Apoptosis cascade representing the present and absent proteins in the pyrosequencing results. Proteins appearing in the turbot transcriptome are represented in reddish color and absent proteins in grey color. Fas-L: Fas ligand; TRAIL: TNF-related apoptosis-inducing ligand; TNFa: Tumor necrosis factor alpha; IL-1: interleukin 1; NGF: nerve growth factor; IL-3: interleukin 3; Fas: tumor necrosis factor receptor superfamily member 6; FAIM2: Fas apoptotic inhibitory molecule 2; TRAIL-R: tumor necrosis factor receptor superfamily member 10; TRAIL-R Decoy: TRAIL receptor with a truncated death domain; TNF-R1: tumor necrosis factor receptor superfamily member 1A; IL-1R: interleukin 1 receptor; TrkA: neurotrophic tyrosine kinase receptor type 1; IL-3R: interleukin 3 receptor; FADD: FAS-associated via death domain; TRADD: TNF receptor superfamily 1alpha-associated via death domain; MyD88: myeloid differentiation primary response protein MyD88; PTEN: Phosphatase and tensin homolog; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; RIP1: receptor-interacting serine/threonine-protein kinase 1; IRAK: interleukin-1 receptor-associated kinase; TRAF2: TNF receptor-associated factor 2; AKT: TNF receptor-associated factor; PKB: protein kinase B; PKA: protein kinase A; NIK: NF-kappa-beta- inducing kinase; FLIP: FADD-like apoptosis regulator; BCL-2: B cell lymphoma 2; IKK: inhibitor of nuclear factor kappa-B kinase; Bad: BCL2-antagonist of cell death; CASP3: caspase 3; CASP6: caspase 6; CASP7: caspase 7; CASP8: caspase 8; CASP9: caspase 9; CASP10: caspase 10; CASP12: caspase 12; Bid: BH3 interacting domain death agonist; CyC: cytochrome c; Apaf-1: apoptotic protease-activating factor; IAP: inhibitor of apoptosis protein; SMAC: second mitochondria-derived activator of caspases; IkBa: NF-kappa-B inhibitor alpha; NF-kB: nuclear factor NF-kappa-B; DFF45: DNA fragmentation factor of 45 kD; DFF40: DNA fragmentation factor of 40 kD; BI-1: Bax inhibitor-1; AIF: apoptosis-inducing factor; ENDO-G: endonuclease G; Bcl-XL: B- cell lymphoma-extra large; Bax: Bcl-2-associated X protein; p53: tumor protein p53; ATM: ataxia telangectasia mutated family protein. doi:10.1371/journal.pone.0035369.g011

181,845 singletons. Approximately 45% of the contigs were ing in S. maximus provides a new resource for future investigations successfully annotated and 21.21% of the singletons submitted to in this economically important species, including improvement of Blast2GO presented homology to proteins deposited in the microarray tools and identification of genetic markers. Moreover, databases. The wealth of information generated by pyrosequenc- we have identified several thousand contigs and singletons with a

PLoS ONE | www.plosone.org 13 May 2012 | Volume 7 | Issue 5 | e35369 454-Pyrosequencing of Turbot Antiviral Genes potential immune function and observed that the majority of denaturing agarose gel (1X) in MOPS buffer in order to visualize the proteins involved in the main immune-pathways in humans have 18S and 28S ribosomal RNA bands. RNA integrity was furthermore also its putative homologues in turbot. investigated using the Bioanalyzer 2100 (Agilent Technologies). RNA extracted from stimulated/infected S. maximus was used as Materials and Methods the source of starting material for cDNA synthesis Full-length- enriched double stranded cDNA was synthesized from 1,5 mgof Turbot Sampling pooled total RNA using MINT cDNA synthesis kit (Evrogen, Fifty-two juvenile turbots (average weight 36 g) were obtained Moscow, Russia) according to manufacturer’s protocol, and was from a commercial fish farm in Galicia (NW Spain). Animals were subsequently purified using the QIAquick PCR Purification Kit acclimated to laboratory conditions for 2 weeks, maintained at (Qiagen USA, Valencia, CA). The amplified cDNA was normal- 18uC and fed daily with a commercial diet. The fish were divided ized using Trimmer kit (Evrogen, Moscow, Russia) to minimize into one batch of 4 individuals and four batches of 12 individuals differences in representation of transcripts. The method involves each one. Turbots from the batch with 4 individuals were denaturation-reassociation of cDNA, followed by a digestion with challenged by intramuscular injection with 100 ml of a Nodavirus a Duplex-Specific Nuclease (DSN) enzyme [75,76]. The enzymat- 7 (10 TCID50/fish) suspension (strain AH95-NorA) [71]. The first ic degradation occurs primarily on the highly abundant cDNA batch of 12 individuals was challenged by intraperitoneal injection fraction. The single-stranded cDNA fraction was then amplified 9 with 100 ml of VHSV (4 x 10 TCID50/fish) suspension (strain twice by sequential PCR reactions according to the manufacturer’s UK-860/94) [72]. The second batch was intramusculary injected protocol. Normalized cDNA was purified using the QIAquick with pMCV 1.4 (Ready-Vector, Madrid, Spain) (2 mg/fish). PCR Purification Kit (Qiagen USA, Valencia, CA). pMCV 1.4 is an expression vector typically used for the 500 ng of normalized cDNA were used to generate a 454 construction of DNA vaccines and empty pMCV 1.4 served as a library. cDNA was fractionated into small, 300- to 800-basepair double-stranded DNA viral stimulus. The third batch was fragments and the specific A and B adaptors were ligated to both challenged with pMCV 1.4-G860 (2 mg/fish), which codes the G the 3’ and 5’ ends of the fragments. The A and B adaptors were glycoprotein from VHSV UK-860/94 strain (GenBank accession used for purification, amplification, and sequencing steps. One AY546628) and was used as a DNA vaccine. The fourth batch was sequencing run was performed on the GS-FLX using Titanium intraperitoneally stimulated with poly I:C (0.1 mg/fish) (SIGMA) chemistry. 454 Sequencing is based on sequencing-by-synthesis, mimicking a viral infection. Viral suspensions were prepared using addition of one (or more) nucleotide(s) complementary to the minimal essential medium (MEM, GIBCO) supplemented with template strand results in a chemiluminescent signal recorded by 2% fetal bovine serum (FBS), penicillin (100 IU/ml) (Invitrogen) the CCD camera within the instrument. The signal strength is and streptomycin (100 mg/ml) (Invitrogen). Poly I:C and plasmid proportional to the number of nucleotides incorporated in a single constructs were inoculated in 1x Phosphate Buffered Saline nucleotide flow. All reagents and protocols used were from Roche (PBS 1x). 454 Life Sciences, USA. RNA was normalized, processed and In order to obtain mRNA representative of both innate and sequenced by the Unitat de Geno`mica (CCiT-UB, Barcelona, adaptive immune systems comprising the full spectrum of the Spain). immune response, gill, liver, spleen and head kidney tissues were removed from 2 individuals for each sampling point (1 h, 6 h, Assembly, Functional Annotation and Comparative 24 h, 3 days, 7 days and 14 days post-inoculation) for the batches Analysis with 12 individuals. For the batch of fish infected with Nodavirus, Pyrosequencing raw data has been processed with the Roche the brain, as target organ, was removed from 2 individuals at 24 h quality control pipeline using default settings. Seqclean software and 3 days after injection. We have used only 2 time points (1 and (http://compbio.dfci.harvard.edu/tgi/software/) was used in or- 3 days) for Nodavirus infection since previous works in other fish der to process all sequences prior to assembly. Sequences shorter species [73,74] showed that the up-regulation of several immune- than 50 bp were discarded and reads with at least 18 bp and 95% relevant genes begins at 24 hours post-infection and reached their identity with a contaminant vector were considered invalid. Then highest levels at 3 days after intramuscular infection. This could A homopolymers and adapters from cDNA library construction suggest that immune responses are activated when the viruses were trimmed. Sequences were submitted to MIRA V3.2.0 [77] reach their target organ (brain) and start their replication [73]. For for assembling the transcriptome ensuring a minimum left clip on each sampling time and treatment, equal amounts of the same each read for the first 4 nucleotides in order to eliminate possible tissue from the two fish were pooled. biases introduced by RNA normalization that could not be fully Healthy turbot larvae at early developmental stages (until 1 removed by the cleaning process and only those clean reads month) were also taken in order to detect immune-related genes presenting a minimum length of 40 bp were assembled. Blastclust highly expressed during the ontogenic development. Turbot larvae program [78], one part of BLAST package from NCBI, was used were pooled in one sample. All samples were stored at 280 uCin on the full fasta contig file to group similar contigs into clusters 500 mlofTRIzolH (Invitrogen) until RNA isolation. (groups of transcripts from the same gene) if at least 60% of the positions is equal or over 95% identity. Singletons shorter than RNA Isolation, cDNA Production and Pyrosequencing 100 bp were removed and the remaining sequences (about Total RNA was extracted from pooled tissues using TRIzolH 100,000) were submitted together with the contigs to Blastclust (Invitrogen) in accordance with instructions provided by the for identifying the singletons which do not cluster with the contigs. manufacturer in combination with the RNeasy mini kit (Qiagen) A total of 64,704 singletons were selected for the annotation step. for RNA purification after DNase treatment (RNase-free DNase set, The annotation of S. maximus consensus contigs and singletons Qiagen). Quantity of total RNA purified from each pool was not clustering with contigs was carried out by Blastx search [78] determined using the spectrophotometer Nanodrop ND-100. RNA against the Swissprot and Metazoan RefSeqs databases. The non- from each organ and sampling point was pooled by mixing 1 mgof annotated sequences using these databases were submitted to RNA from each type of stimuli. The final concentration was adjusted GenBank non-redundant (nr) database. In both cases the E-value to 1 mg/ml. RNA quality was assessed by electrophoresis on a cut-off was 1e203. The largest contig belonging to each cluster was

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Figure 12. Flowchart representing the data processing pipeline for de novo transcriptome assembly and annotation of Scophthalmus maximus. doi:10.1371/journal.pone.0035369.g012 submitted to Blast2GO software [49,50] in order to predict the Supporting Information functions of the sequences and assign Gene Ontology terms. The same sequences of turbot were compared to the ESTs and Table S1 Sequence of the singletons submitted to nucleotide sequences of zebrafish (D. rerio) and Japanese flounder Blast2GO for annotation. (P. olivaceus) deposited in the NCBI databases using BLASTn (XLSX) algorithm. For D. rerio there were 1,488,275 ESTs and 72,554 Table S2 Molecular function assignment (3rd level GO unique nucleotide sequences (based on GeneInfo identifiers) and terms) for the transcriptomic sequences (contigs) of for P. olivaceus there were 15,234 and 1,936, respectively. The E- Scophthalmus maximus. 205 value cut-off was 1e . With the aim of illustrating the outline (DOCX) flow for the transcriptome annotation procedure a diagram is shown in Figure 12. Table S3 Information of the contigs and singletons identified as homologous to molecules involved in the Identification of Immune-related Proteins Complement pathway. (XLSX) GO terms at level 2, 3 and 4 having a direct relationship with immunity were used for selecting putative immune-related Table S4 Information of the contigs and singletons proteins together with a thorough analysis conducted in our identified as homologous to molecules involved in the laboratory based on an extensive list of immune terms and a Toll-like receptor signaling pathway. comprehensive literature review. (XLSX) In order to establish the presence and absence of proteins Table S5 Information of the contigs and singletons belonging to the more relevant immune-pathways in our identified as homologous to molecules involved in the pyrosequencing results, we have used the KEGG reference B cell receptor signaling pathway. pathways [79] as a template for constructing the following (XLSX) immune-cascades by hand: Complement pathway, Toll-like receptor signaling pathway, B cell receptor signaling pathway, T Table S6 Information of the contigs and singletons cell receptor signaling pathway and Apoptosis cascade. Additional identified as homologous to molecules involved in the molecules were included in some cases after bibliographic review. T cell receptor signaling pathway. (XLSX)

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Table S7 Information of the contigs and singletons Author Contributions identified as homologous to molecules involved in the Conceived and designed the experiments: BN AF. Performed the Apoptosis or programmed cell death. experiments: PP. Wrote the paper: PP. Conducted the 454 sequencing (XLSX) and the bioinformatic work: SB BF GF-C. Performed functional annotation analyses: PP PB. Deposited the sequences in databases: PP Acknowledgments GF-C. Involved in the construction and annotation of the immune cascades: AR SD. Revised the study and manuscript: JP PB BN AF. Turbot larvae were provided by Dr. J. Rotllant, IIM, CSIC and the Edited, read and approved the manuscript: AF PP PB AR SD GF-C BF JP reagents used for VHSV vaccination/stimulation were obtained from Dra. SB BN. A. Estepa, Universidad Miguel Herna´ndez.

References 1. Nielsen JG (1986) Scophtalmidae. In Whitehead PJP, Bauchot ML, Hureau JC, 27. Tada N, Sakamoto T, Kagami A, Mochizuki K, Kurosaka K (1993) Nielsen J, Tortonese E., eds. Fishes of the North-eastern Atlantic and the Antimicrobial activity of lipoprotein particles containing apolipoprotein Al. Mediterranean. Paris: Unesco. pp 1287–1293. Mol Cell Biochem 119: 171–178. 2. Federation of European Aquaculture Producers (FEAP) (2008) Production and 28. Motizuki M, Satoh T, Takey T, Itoh T, Yokota S, et al. (2002) Structure-activity price reports of member associations of the F.E.A.P. Lie`ge: FEAP. analysis of an antimicrobial peptide derived from bovine apolipoprotein A-II. 3. Food and Agriculture Organization of the United Nations (FAO) (2010) The J Biochem 132: 115–119. state of world fisheries and aquaculture. Rome: FAO. 29. Concha MI, Molina S, Oyarzu´n C, Villanueva J, Amthauer R (2003) Local 4. Toranzo AE, Magarin˜os B, Romalde JL (2005) A review of the main bacterial expression of apolipoprotein A-I gene and a possible role for HDL in primary fish diseases in mariculture systems. Aquaculture 246: 37–61. defence in the carp skin. Fish Shellfish Immunol 14: 259–273. 5. Walker PJ, Winton JR (2010) Emerging viral diseases of fish and shrimp. Vet 30. Concha MI, Smith VJ, Castro K, Bastias A, Romero A, et al. (2004) Res 41: 51–75. Apolipoproteins A-I and A-II are potentially important effectors of innate 6. A´ lvarez-Pellitero P (2008) Fish immunity and parasite infections: from innate immunity in the teleost fish Cyprinus carpio. Eur J Biochem 271: 2984–2990. immunity to immunoprophylactic prospects. Vet Immunol Immunopathol 126: 31. Chang MX, Nie P, Liu GY, Song Y, Gao Q (2005) Identification of immune 171–198. genes in grass carp Ctenopharyngodon idella in response to infection of the parasitic 7. Sarmasik A (2002) Antimicrobial peptides: a potential therapeutic alternative for copepod Sinergasilus major. Parasitol Res 96: 224–229. the treatment of fish diseases. Turk J Biol 26: 201–207. 32. Villarroel F, Bastias, A, Casado A, Amthauer R, Concha MI (2007) 8. Biragyn A (2005) Defensins – Non-antibiotic use for vaccine development. Curr Apolipoprotein A-I, an antimicrobial protein in Oncorhynchus mykiss: evaluation Protein Pept Sci 6: 53–60. of its expression in primary defence barriers and plasma levels in sick and 9. Bowdish DME, Davidson DJ, Hancock REW (2006) Immunomodulatory healthy fish. Fish Shellfish Immunol 23: 197–209. properties of defensins and cathelicidins. CTMI 306: 27–66. 33. Akerstro¨m B, Lo¨gdberg L, Berggard T, Osmark P, Lindqvist A (2000) a1- 10. Jenssen H, Hamill P, Hancock REW (2006) Peptide Antimicrobial Agents. Clin Microglobulin: a yellow-brown lipocalin. Biochim Biophys Acta 1482: 172–184. Microbiol Rev 19: 491–511. 34. Lo¨gdberg L, Wester L (2000) Immunocalins: a lipocalin subfamily that 11. Kornbluth RS, Stone GW (2006) Immunostimulatory combinations: designing modulates immune and inflammatory responses. Biochim Biophys Acta 1482: 284–297. the next generation of vaccine adjuvants. J Leuk Biol 80: 1084–1102. 35. Pott GB, Chan ED, Dinarello CA, Shapiro L (2009) a1-Antitrypsin is an 12. Mookherjee N, Hancock REW (2007) Cationic host defense peptides: innate endogenous inhibitor of proinflammatory cytokine production in whole blood. immune regulatory peptides as a novel approach for treating infections. Cell Mol J Leukoc Biol 85: 886–895. Life Sci 64: 922–933. 36. Steinhoff M, Buddenkotte J, Shpacovitch V, Rattenholl A, Moormann C, et al. 13. Smith VJ, Desbois AP, Dyrynda EA (2010) Conventional and unconventional (2005) Proteinase-activated receptors: transducers of proteinase-mediated antimicrobials from fish, marine invertebrates and micro-algae. Mar Drugs 8: signaling in inflammation and immune response. Endocr Rev 26: 1–43. 1213–1262. 37. Maeda K, Hirota M, Kimura Y, Ichihara A, Ohmuraya M, et al. (2005) 14. Nicholls EF, Madera L, Hancock REW (2010) Immunomodulators as adjuvants Proinflammatory role of trypsin and protease-activated receptor-2 in a rat model for vaccines and antimicrobial therapy. Ann N Y Acad Sci 1213: 46–61. of acute pancreatitis. Pancreas 31: 54–62. 15. Marra MA, Hillier L, Waterston RH (1998) Expressed sequence tags- 38. Jensen T, Kierulf P, Sandset PM, Klingenberg O, Joø GB, et al. (2007) ESTablishing bridges between genomes. Trends Genet 14: 4–7. Fibrinogen and fibrin induce synthesis of proinflammatory cytokines from 16. Nagaraj SH, Gasser RB, Ranganathan S (2007) A hitchhikers guide to expressed isolated peripheral blood mononuclear cells. Thromb Haemost 97: 822–829. sequence tag (EST) analysis. Brief Bioinform 8: 6–21. 39. Lu PP, Liu JT, Liu N, Guo F, Ji YY, et al. (2011) Pro-inflammatory effect of 17. Wang C, Zhang XH, Jia A, Chen J, Austin B (2008) Identification of immune- fibrinogen and FDP on vascular smooth muscle cells by IL-6, TNF-a and iNOS. related genes from kidney and spleen of turbot, Psetta maxima (L.), by suppression Life Sci 88: 839–845. subtractive hybridization following challenge with Vibrio harveyi. J Fish Dis 31: 40. Jiang D, Liang J, Noble PW (2011) Hyaluronan as an immune regulator in 505–514. human diseases. Physiol Rev 91: 221–264. 18. Pardo BG, Ferna´ndez C, Milla´n A, Bouza C, Va´zquez-Lo´pez A, et al. (2008) 41. Hallquist NA, Klasing KC (1994) Serotransferrin, ovotransferrin and metallo- Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus thionein levels during an immune response in chickens. Comp Biochem Physiol maximus) challenged with pathogens. BMC Vet Res 4: 37. Biochem Mol Biol 108: 375–384. 19. Park KC, Osborne JA, Montes A, Dios S, Nerland AH, et al. (2009) 42. Quaye IK (2008) Haptoglobin, inflammation and disease. Trans R Soc Trop Immunological responses of turbot (Psetta maxima) to nodavirus infection or Med Hyg 102: 735–742. polyriboinosinic polyribocytidylic acid (pIC) stimulation, using expressed 43. Tolosano E, Fagoonee S, Morello N, Vinchi F, Fiorito V (2010) Heme sequence tags (ESTs) analysis and cDNA microarrays. Fish Shellfish Immunol scavenging and the other facets of hemopexin. Antioxid Redox Signal 12: 26: 91–108. 305–320. 20. Yeh Y, Feeney RE (1996) Antifreeze proteins: structures and mechanisms of 44. Office International des Epizooties (OIE) (2009) Manual of Diagnostic test for function. Chem Rev 96: 601–617. Aquatic Animals. Paris: OIE. 21. Ewart KV, Fletcher GL (1993) Herring antifreeze protein: primary structure and 45. Drakesmith H, Prentice A (2008) Viral infection and iron metabolism. Nat Rev evidence for a C-type lectin evolutionary origin. Mol Mar Biol Biotechnol 2: Microbiol 6: 541–552. 20–27. 46. Law RHP, Lukoyanova N, Voskoboinik I, Caradoc-Davies TT, Baran K, et al. 22. Millan A, Gomez-Tato A, Fernandez C, Pardo BG, Alvarez-Dios JA, et al. (2010) The structural basis for membrane binding and pore formation by (2010) Design and performance of a turbot (Scophthalmus maximus) oligo- lymphocyte perforin. Nature 468: 447–453. microarray based on ESTs from immune tissues. Mar Biotechnol 12: 452–465. 47. Go¨tz S, Garcı´a-Go´mez JM, Terol J, Williams TD, Nagaraj SH, et al. (2008) 23. Kilpatrick DC (2002) Animal lectins: a historical introduction and overview. High-throughput functional annotation and data mining with the Blast2GO BBA-Gen Subjects 1572: 187–197. suite. Nucleic Acids Res 36: 3420–3435. 24. Choudhury M, Yamada S, Komatsu M, Kishimura H, Ando S (2009) 48. Conesa A, Go¨tz S, Garcı´a-Go´mez JM, Terol J, Talon M, et al. (2005) Blast2GO: Homologue of mammalian apolipoprotein A-II in non-mammalian vertebrates. a universal tool for annotation, visualization and analysis in functional genomics Acta Biochim Biophys Sin 41: 370–378. research. Bioinformatics 21: 3674–3676. 25. Bolanos-Garcı´a VM, Miguel RN (2003) On the structure and function of 49. Salem M, Rexroad III CE, Wang J, Thorgaard GH, Yao J (2010) apolipoproteins: more than a family of lipid-binding proteins. Prog Biophys Mol Characterization of the rainbow trout transcriptome using Sanger and 454- Biol 83: 47–68. pyrosequencing approaches. BMC Genomics 11: 564. 26. Srinivas RV, Venkatachalapathi YV, Rui Z, Owens RJ, Gupta KB, et al. (1991) 50. Zhang Z, Wang Y, Wang S, Liu J, Warren W, et al. (2011) Transcriptome Inhibition of virus-induced cell fusion by apolipoprotein A-I and its amphipathic analysis of female and male Xiphophorus maculatus Jp 163 A. PLoS ONE 6: peptide analogs. J Cell Biochem 45: 224–237. e18379.

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51. Coppe A, Pujolar JM, Maes GE, Larsen PF, Hansen MM, et al. (2010) 68. Miller MD, Krangel MS (1992) Biology and biochemistry of the chemokines: a Sequencing, de novo annotation and analysis of the first Anguilla anguilla family of chemotactic and inflammatory cytokines. Crit Rev Immunol 12: transcriptome: EeellBase opens new perspectives for the study of the critically 17–46. endangered european eel. BMC Genomics 11: 635. 69. Wang H, Czura CJ, Tracey KJ (2003) Tumor necrosis factor. In Thomson AW, 52. Wang JPZ, Lindsay BG, Leebens-Mack J, Cui L, Wall K, et al. (2004) EST Lotze MT, eds. The Cytokine Handbook. Amsterdam: Academic Press. pp clustering error evaluation and correction. Bioinformatics 20: 2973–2984. 837–860. 53. Serazin AC, Dana AN, Hillenmeyer ME, Lobo NF, Coulibaly MB, et al. (2009) 70. Clark SC, Kamen R (1987) The human hematopoietic colony stimulating Comparative analysis of the global transcriptome of Anopheles funestus from Mali, factors. Science 236: 1229–1237. West Africa. PLoS ONE 4: e7976. 71. Grotmol S, Nerland AH, Biering E, Totland GK, Nishizawa T (2000) 54. Carroll MC (2004) The complement system in regulation of adaptive immunity. Characterization of the capsid protein gene from a nodavirus strain affecting Nat Immunol 5: 981–986. the Atlantic halibut Hippoglossus hippoglossus and design of an optimal reverse- 55. Gasque P (2004) Complement: a unique innate immune sensor for danger transcriptase polymerase chain reaction (RT-PCR) detection assay. Dis Aquat signals. Mol Immunol 41: 1089–1098. Organ 39: 79–88. 56. Barton, GM, Medzhitov R (2003) Toll-Like Receptor Signaling Pathways. 72. Ross K, McCarthy U, Huntly PJ, Wood BP, Stuart D, et al. (1994) An outbreak Science 300: 1524–1525. of viral haemorrhagic septicaemia (VHS) in turbot (Scophthalmus maximus)in 57. Takeda K, Akira S (2004) TLR signaling pathways. Sem Immunol 16: 3–9. Scotland. Bull Eur Assoc Fish Pathol 14: 213–214. 58. Kurosaki T, Shinohara H, Baba Y (2010) B cell signaling and fate decision. 73. Dios S, Poisa-Beiro L, Figueras A, Novoa B (2007) Suppression subtraction Annu Rev Immunol 28: 21–55. 59. Batista FD, Neuberger MS (1998) Affinity dependence of the B cell response to hybridization (SSH) and macroarray techniques reveal differential gene antigen: a threshold, a ceiling, and the importance of off-rate. Immunity 8: expression profiles in brain of sea bream infected with nodavirus. Mol Immunol 751–759. 44: 2195–2204. 60. Starr TK, Jameson SC, Hogquist KA (2003) Positive and negative selection of T 74. Poisa-Beiro L, Dios S, Montes A, Aranguren R, Figueras A, Novoa B (2008) cells. Annu Rev Immunol 21: 139–176. Nodavirus increases the expression of Mx and inflammatory cytokines in fish 61. Germain RN (1994) MHC-dependent antigen processing and peptide brain. Mol Immunol 45: 218–225. presentation: providing ligands for T lymphocyte activation. Cell 76: 287–299. 75. Shagin DA, Rebrikov DV, Kozhemyako VB, Altshuler IM, Shcheglov AS, et al. 62. Mosmann TR, Coffman RL (1989) TH1 and TH2 cells: different patterns of (2002) A novel method for SNP detection using a new duplex-specific nuclease lymphokine secretion lead to different functional properties. Annu Rev Immunol from crab hepatopancreas. Genome Res 12: 1935–42. 7: 145–173. 76. Zhulidov PA, Bogdanova EA, Shcheglov AS, Vagner LL, Khaspekov GL, et al. 63. Chaplin DD (2010) Overview of the immune response. J Allergy Clin Immunol (2004) Simple cDNA normalization using kamchatka crab duplex-specific 125: S3–S23. nuclease. Nucleic Acids Res 32: e37. 64. Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 77. Chevreux B, Pfisterer T, Drescher B, Driesel AJ, Mu¨ller WE, et al. (2004) Using 35: 495–516. the miraEST Assembler for Reliable and Automated mRNA Transcript 65. Crawford ED, Wells JA (2011) Caspase substrates and cellular remodeling. Assembly and SNP Detection in Sequenced ESTs. Genome Res 14: 1147–1159. Annu Rev Biochem 80: 1055–1087. 78. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local 66. Townsend MJ, McKenzie AN (2000) Unravelling the net? cytokines and alignment search tool. J Mol Biol 215: 403–410. diseases. J Cell Sci 113: 3549–3550. 79. Kanehisa M, Goto S (2000) KEGG: Kyoto Encyclopedia of Genes and 67. Feliciani C, Gupta AK, Saucier DN (1996) Keratinocytes and Cytokine/Growth Genomes. Nucleic Acids Res 28: 27–30. Factors. CROBM 7: 300–318.

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Supporting information

Supporting information associated with this article can be found at: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.003 0035

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CHAPTER 3 Protection and antibody response induced by intramuscular DNA vaccine encoding for viral haemorrhagic septicaemia virus (VHSV) G glycoprotein in turbot (Scophthalmus maximus)

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Fish & Shellfish Immunology 32 (2012) 1088e1094

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Fish & Shellfish Immunology

journal homepage: www.elsevier.com/locate/fsi

Protection and antibody response induced by intramuscular DNA vaccine encoding for viral haemorrhagic septicaemia virus (VHSV) G glycoprotein in turbot (Scophthalmus maximus)

P. Pereiro a, A. Martinez-Lopez b, A. Falco b, S. Dios a, A. Figueras a, J.M. Coll c, B. Novoa a,*, A. Estepa b,** a Instituto de Investigaciones Marinas (IIM), CSIC, Vigo 36208, Spain b Instituto de Biología Molecular y Celular (IBMC), Miguel Hernández University, 03202 Elche, Spain c Dpto Biotecnología, INIA, 28040 Madrid, Spain article info abstract

Article history: Turbot (Scophthalmus maximus) is a high-value farmed marine flatfish with growing demand and Received 17 January 2012 production levels in Europe susceptible to turbot-specific viral haemorrhagic septicaemia virus (VHSV) Received in revised form strains. To evaluate the possibility of controlling the outbreaks of this infectious disease by means of DNA 1 March 2012 vaccination, the gpG of a VHSV isolated from farmed turbot (VHSV ) was cloned into an expression Accepted 5 March 2012 860 plasmid containing the human cytomegalovirus (CMV) promoter (pMCV1.4-G ). In our experimental Available online 13 March 2012 860 conditions, DNA immunised turbots were more than 85% protected against VHSV860 lethal challenge and showed both VHSV-gpG specific and neutralizing antibodies. To our knowledge this is the first report Keywords: fi DNA vaccine showing the ef cacy of turbot genetic immunisation against a VHSV. Work is in progress to determine the VHSV contribution of innate and adaptive immunity to the protective response elicited by the immunization. Turbot Ó 2012 Published by Elsevier Ltd. Glycoprotein G Rhabdovirus

1. Introduction against homologous challenges as well as short and unspecific protection against heterologous challenge [1e5]. In fact, a DNA Viruses are probably the most destructive pathogens in fish vaccine to IHNV (Apex-IHN, Aqua Health Ltd, Canada) has been aquaculture and they are a serious concern since no specific chemo- approved for commercialization in July 2005 by the Canadian Food therapies are available. Among the 9 notifiable fish diseases (diseases Inspection Agency, Veterinary Biologist and Biotechnology Division with great social and economic and/or public health repercussion or [6,7]. In spite of this, DNA vaccines still face several challenges mainly with present or potential risk for the aquaculture industry) registered concerning safety and delivery routes what constitutes an obstacle at the 2009 Aquatic Animal Health Code of the OIE (Office Interna- for their approval in many countries, as it occurs in European ones. tional des Epizooties, now the World Organization for Animal Health) Among the fish novirhabdoviruses, VHSV is indeed one of the (http://www.oie.int), 7 are caused by viruses. Furthermore, two of most important viral diseases of fish in European aquaculture [8,9]. them (viral hemorrhagic septicaemia, VHS, and infectious hemato- In addition, VHSV is spreading to wild ecosystems as it is being poietic necrosis, IHN) are caused by novirhabdoviruses hence isolated all over the world from an increasing number of free-living showing their high risk to worldwide aquaculture. marine fish species [10e13]. To date, phylogenetic studies have To date, rhabdoviral disease prevention by means of vaccination is grouped all these isolates into four genotypes (IeIV) [14,15]. considered a very efficient strategy for the control and prevention of Moreover, it has been also reported that susceptibility of the host these diseases. For the salmonid fish novirhabdoviruses VHSV and species to VHSV infections depends on the genotype [16]. IHNV, it has been demonstrated that DNA vaccines based on the Due to intensive farming conditions, disease outbreaks caused glycoprotein G (gpG) gene are able to confer long-term protection by VHSV have become a severe problem for global turbot industries [14,17]. For instance, after the outbreak at the Gigha turbot farm * Corresponding author. Instituto de Investigaciones Marinas (IIM), CSIC, Vigo (Scotland), caused by the UK-860/94 strain [17] 14 tonnes of fish 36208, Spain. were sacrificed (with subsequent relevant economic losses) [18]. ** Corresponding author. Miguel Hernandez University, Instituto de Biologia Therefore, the need for the development of effective means of Molecular y Celular (IBMC), Avenida de la Universidad s/n, 03202 Elche, Alicante, Spain. Tel.: þ34 966 658 436. disease control is urgent. However, it is still unknown whether or E-mail address: [email protected] (A. Estepa). not a DNA vaccine against VHSV based on its gpG gene can confer

1050-4648/$ e see front matter Ó 2012 Published by Elsevier Ltd. doi:10.1016/j.fsi.2012.03.004 P. Pereiro et al. / Fish & Shellfish Immunology 32 (2012) 1088e1094 1089 turbot protection against VHSV lethal challenge. In contrast, FuGene 6 (Roche, Barcelona, Spain) were incubated for 15 min in a vaccine for Japanese flounder (Paralichthys olivaceus) based on the 50 ml of RPMI-1640 containing 2 mM Cl2Ca and then added to each use of VHSV strains isolated in their farm outbreaks [19,20] has well in 200 ml of culture medium with 10% of FCS. As an additional been developed. Interestingly, a DNA vaccine to VHSV has been also control, EPC cells were transfected with FuGene 6 without DNA used to confer short-term unspecific protection to turbot against following the same procedure. The plates were further incubated at nodavirus infection [21,22]. 20 C for 2 days. As an additional control, the cells were also Since VHSV virulence in host species is genotype related [16],in transfected with the previously characterised pMCV1.4-G07.71 [29] this work a DNA vaccine encoding the gpG from a virulent VHSV encoding the cDNA sequence of the gpG of VHSV strain 07.71 strain isolated from a turbot outbreak (UK-860/94, genotype III) (genotype I) isolated from rainbow trout Oncorhynchus mykiss [30]. [14,17] was constructed, characterized and its protective role determined in juvenile turbots. The results showed that at least 2.4. Turbots over the time scale and conditions analysed, DNA vaccination of turbot with the gpG protein of VHSV confers resistance and elicits Juvenile turbot of approximately 2.5 g each, obtained from specific antibodies with neutralising activity. To our knowledge this a VHSV-free commercial farm (Insuiña S.L., Mougás, Galicia, Spain), is the first published report describing the successful immunisation were maintained in 500 L fibreglass tanks at the IIM (CSIC) facilities of turbot with a DNA vaccine against VHSV. at 18 C with a re-circulating saline-water system and fed daily with a commercial diet (LARVIVA-BioMar, France). Prior conducting 2. Materials and methods experiments, fish were acclimatised to laboratory conditions for 2 weeks and then it was confirmed that their sera were negative for 2.1. Cell cultures and virus the presence of antibodies against VHSV as below described.

The fish cell line EPC (Epithelioma papulosum cyprinid) [23] 2.5. DNA immunisation protocol purchased from the American Type Culture Collection (ATCC number CRL-2872) was used in this work. Recently, the ATCC has DNA immunisation of turbot was carried out following revealed that the EPC cell line, originally deposited as a carp procedures for fish genetic immunisation previously described (Cyprinus carpio) cell line, is in fact a fathead minnow (Pimephales [31].Briefly, turbots were anaesthetised by immersion in 50 mg/ml promelas) cell line. EPC cells were maintained at 28 C in a 5% CO2 buffered tricaine methanesulfonate (MS-222; Sigma) prior to atmosphere with RPMI-1640 Dutch modified (Gibco, Invitrogen handling and then divided into 6 tanks (52 fish/tank). Turbots corporation, UK) cell culture medium containing 10% fetal calf from two tanks were then intramuscularly injected (i.m.) with serum (FCS) (Sigma, Che. Co, St. Louis, Ms, USA), 1 mM Piruvate one of the following: 50 ml of PBS (non-immunised or control (Gibco, Invitrogen corporation, UK), 2 mM Glutamine (Gibco), fish), 50 ml of PBS containing 2 mg of pMCV1.4 or pMCV1.4-G860. 50 mg/ml gentamicin (Gibco) and 2 mg/ml fungizone. The replicate tanks were placed alternatively to minimize the Viral haemorrhagic septicaemia virus UK-860/94 (VHSV860)iso- influence of tank position. Three days after immunisation (Fig. 1), latedfromfarmedturbotinEngland[14,17] was propagated in EPC 3 fish from each group (a total of 6 fish/treatment) were sacrificed cells at 14 C as previously described for other VHSV isolates [24]. by overexposure to MS-222 and muscle (site of injection) was Supernatants from VHSV860-infected EPC cell monolayers were clar- removed and processed for real time quantitative PCR (RT-qPCR) ified by centrifugation at 4000 g during 30 min and kept in aliquots as indicated below. Moreover, at day 30 post-immunisation at 70 C. Viruses from clarified supernatants were concentrated by (Fig. 1) blood from the caudal vein was extracted (3 fish per ultracentrifugation at 100,000 g for 45 min [16]. The virus stock was group or 6 fish per treatment) in order to study antibody titrated in 96-well plates according to Reed and Muënch [25]. production. An additional untreated group composed by 23 fish was also added in order to control possible mortalities caused by 2.2. Plasmids maintenance conditions.

The expression vector pMCV1.4 (Ready-Vector, Madrid, Spain) 2.6. Analysis of VHSV-G protein expression in transfected cells and (2863 bp) [26] containing the CMV regulatory sequences was used. DNA immunised-fish muscle The regulatory sequences of pMCV1.4 vector promoter included an enhancer/promoter sequence (740 bp from the human cytomega- The expression of gpG860 in EPC transfected cells as well as in lovirus, CMV, major immediate-early gene) and an intron sequence turbot muscle tissue was analysed at transcriptional levels by RT- (w200 bp of a synthetic chimeric intron). qPCR. Cell RNA extraction and cDNA synthesis were performed as To obtain the pMCV1.4-G860 construct, the gpG cDNA sequence previously described [29]. RT-qPCR was carried out using the from VHSV860 (Gene bank accession number AY546628) was syn- gpG860 gene primers and probe designed to detect specifically the thesised by BioS&T (Montreal, Canada) and then cloned into the vector pMCV1.4 digested with the restriction enzymes KpnI and XbaI following standard procedures. Finally, the sequence of the gpG gene in the construct pMCV1.4-G860 was confirmed by DNA sequencing.

2.3. Cell transfection assays

Cell transfection assays were performed as previously described [27,28]. Briefly, EPC cell monolayers grown in culture flasks of 75 cm2 were detached using trypsine (Sigma), washed, resus- pended in culture medium supplemented with 10% of FCS and Fig. 1. Schematic overview of vaccination/challenge protocol and sampling procedure. dispensed into 24-well plates at a concentration of 2.5 105 cells Vaccination/challenge protocol carried out for determining the vaccine effectiveness. fi m per well in a nal volume of 250 l. The following day, the Moreover, muscle and sera samples were collected at the indicated time points in pMCV1.4-G860 or the pMCV 1.4 (0.5 mg) complexed with 0.7 mlof order to analyse the immune response. p.v., post-vaccination; p.c., post-challenge. 1090 P. Pereiro et al. / Fish & Shellfish Immunology 32 (2012) 1088e1094

0 gpG from the isolate VHSV860 (forward 5 -GGTCTTCCTTCTATTGG- antibody anti-protein N of VHSV07.71,2C9[36] and then the virus stock TACTTTGCT-30; reverse 50-GAAATCCCGTAGTTTGGA ATAGG-30; was titrated in 96-well plates using a previously developed immu- Probe 50-CTGGAGCCTCTGGCCGTCATTAGC A 3) by using the RT- nostaining focus assay (focus forming units, f.f.u.) [37]. qPCR conditions previously described [32,33]. The internal refer- For neutralisation tests, we used the same immunostaining ence to normalize data was the cellular 18S rRNA. Results are focus assay that for virus titration. Briefly, 10 ml of serial 5-fold D expressed as 2 Ct, where DCt is determined by subtracting the 18S dilutions of turbot serum (6 fish per group) in reduced-serum Ct value from the target (gpG860 gene) Ct. culture medium (Opti-Mem, Invitrogen), beginning with a dilu- tion ratio of 1:10, were mixed with 1.5 ml of turbot complement 2.7. Challenge with VHSV (fresh turbot pooled sera) in wells of 96-well plates (30 min at 4 14 C) and then 50 ml of a VHSV860 dilution adjusted to 1 10 f.f.u./ Four weeks after DNA immunisation, all the remaining turbots in ml were added and the plates incubated for 2 h at 14 C. After each tank (46 fish/tank) were divided in two batches (23 fish/batch). incubation, each serumecomplementevirus mixture was added to Fish from one batch were intraperitoneally injected (i.p.) with a dose EPC monolayer, grown in 96 well plates, in a final volume of 60 ml 5 of VHSV860 of 5 10 TCID50/fish and fish from the other batch with per well. Two hour post-infection at 14 C, infected cells were 6 5 10 TCID50/fish. Mortality was recorded daily over a 21 days washed, 100 ml/well of fresh cell culture medium (RPMI Dutch period. The Relative percentage of survival (RPS) was calculated by modified) supplemented with 2% FCS added and plates further the formula: RPS ¼ [1 (% mortality of immunized fish/% mortality incubated for 24 h at 14 C. To evaluate VHSV860 infectivity, the EPC of control fish)] 100. In addition, at day 21 post-challenge (Fig. 1), cell monolayers were fixed for 10 min in cold methanol and air- blood from the caudal vein of survival fish was also extracted (a total dried and the immunostaining focus assay was performed as of 30 fish were sampled) in order to study antibody production. previously described by Mas et al. (2006). Brown foci of DAB stained cells (VHSV-infected cell foci) were counted with an 2.8. IgM antibody response inverted microscope with a 10 ocular eye grid. The results were expressed as inhibition of VHSV860 infectivity calculated by the The presence of specific antibodies against gpG in turbot sera following formula, (100 (number of DAB positive foci in EPC was determined at day 30 post-vaccination through enzyme-linked cultures inoculated with turbot serum, complement and VHSV860/ immunosorbent assay (ELISA) using a synthetic peptide (aa number of DAB positive foci in EPC cultures inoculated with turbot 280e310) from the gpG of the VHSV07.71 [30], previously named serum and VHSV860 in the absence of complement) 100). peptide 31 LTPNKCVNTDIQMRGATDDFSYLNHLITNMA, as antigen [34]. There are only 2 amino acid differences between the VHSV 2.10. Statistical analysis strains (underlined N283K and D298E, single letter aminoacid in 07.71 position amino acid in 860 strain, respectively). Briefly, sera (6 Data were analysed using the computer software package SPSS fish per group) from turbot injected with PBS, pMCV1.4 or v.19.0. t-test was performed to determine the differences between pCMV1.4-gpG860 were analysed and ELISA assays were carried out each time and treatment group and its corresponding control. as previously described [29] but using the monoclonal antibody to Differences were considered significant when p < 0.05. turbot IgM, UR3, kindly donated by Dr. J.M Leiro and Dr. J. Lamas, University of Santiago de Compostela [35]. As a positive control 6 3. Results pools of sera (each containing serum from 5 different fish) obtained from turbots surviving to VHSV860 infection were used. 3.1. In vitro and in vivo gpG860 transcript expression

2.9. Serum neutralising activity Since the amino acid sequences of VHSV gpG07.71 isolated from rainbow trout (X59148) and the gpG860 isolated from turbot Before carrying out the neutralisation tests, we evaluated whether (AY546628) have variations in 20 positions, differences in virulence or not the isolate 860 of VHSV was recognised by the monoclonal and immunization might arise between the corresponding VHSV AB 800 600 400

0,0008

0,0006

0,0004

0,0002

0,0000

Fig. 2. In vitro (A) and in vivo (B) transcript expression of the gpG gene from VHSV860. A, EPC cells were grown in wells of 24-well plates and left untreated or transfected with 0.5 mg per well of the pMCV 1.4-G860, pMCV 1.4-G07.71 or the pMCV 1.4 (c-). The cells were then harvested for RNA isolation 2 days post-transfection. cDNAs were amplified with specific gpG860 gene primers and their corresponding TaqMan probe. The internal reference to normalize data was the cellular 18S rRNA. Bars represent the average values and standard deviations from 3 different experiments, each performed in triplicate. Black bars, cells transfected with pMCV 1.4-G860; grey bars, cells transfected with pMCV 1.4-G07.71; white bars, cells transfected with pMCV 1.4. B, gpG860 expression was examined in juvenile turbot intramuscularly (i.m.) injected with 2 mg of pMCV 1.4-gpG860. After 3 days the injected animals were sacrificed by anaesthetic overdose and muscle tissue at the injection site were removed and processed. Expression of gpG860 transcripts in muscle tissue was estimated by RT-qPCR. Bars represent the average values and standard deviations for 6 fish per group (fish 1e6). a.u., arbitrary units. P. Pereiro et al. / Fish & Shellfish Immunology 32 (2012) 1088e1094 1091

turbots intramuscularly injected with the gpG860 encoding plasmid. As in transfected cells, gpG860 transcripts were detectable in the muscle of all fish. Although a high variability was found among the expression levels of the different fish, all the injected fish expressed the gpG860 (Fig. 2B). As expected, expression of the gpG860 gene was not detected in any fish of the control groups.

3.2. gpG peptide-specific IgM response

Having established that the gpG860 gene transcripts were effi- ciently expressed both in vitro and in vivo, the capacity of pMCV1.4- G860 to induce specific antibodies in turbot against a peptide of the VHSV-gpG was studied. Thus groups of 6 turbots were intramus- cularly injected with PBS, pMCV1.4-G860 or pMCV1.4. Thirty days after immunisation, all the sera from turbot injected with pMCV1.4- G860 developed gpG peptide-specific antibodies (specific antibody titers >1/200 at 30 days post-vaccination), whereas no specific antibodies were detected in the sera from control turbot (PBS or pMCV1.4-injected fish) (Fig. 3). Furthermore, the titers in the sera from pMCV1.4-G860 immunised turbot were similar to those of sera from turbot surviving an infection with VHSV860 (Fig. 3). Fig. 3. Anti-VHSV-gpG specific antibodies in sera from turbots immunised with pMCV1.4-G . Juvenile turbots were i.m. injected with 2 mg pMCV 1.4-G (solid VHSV860 860 3.3. Protective immunity elicited by pMCV1.4-G860 immunisation fi circles) or pMCV 1.4 (open circles) or PBS (open stars). After 30 days, sh sera were against VHSV-860 lethal challenge collected and anti-VHSV-gpG protein specific antibodies were analysed by ELISA using a synthetic peptide of gpG07.71 as solid phase. Absorbance was measured at 492 and 620 nm. The results show the average values and standard deviations of sera from 6 Immunized and control turbots were challenged by i.p. injection fi 5 6 sh per group, each assay in triplicate. Open triangles, sera from turbots surviving an 30 days post-inmmunisation with 5 10 or 5 10 TCID50 of infection with VHSV . *Significantly different from respective controls as determined 860 VHSV860 per fish. The effect of pMCV1.4-G860 immunization on RPS by unpaired t-test (P < 0.05). 6 resulting from a challenge dose of 5 10 TCID50/fish is shown in strains/fish species. Therefore, to study the best possible vaccina- Fig. 4A. Significant protection was observed with a RPS of w80%. In fi tion of turbot, the gpG860 was cloned in an expression plasmid and contrast, the control sh that received either PBS or pMCV1.4 had their transcript expression studied. RPS of w0% (Fig. 4A). As expected, there was less mortality in the fi 5 fi EPC cells were thus transfected with pMCV1.4-G860 or pMCV1.4- challenged sh, which received 5 10 TCID50/ sh. In this case, the RPS for pMCV1.4-G immunised fish was w90% and for control G07.71, encoding the gpG sequence from the VHSV 860 and 07.71, 860 respectively. For detection of their transcripts, the primers and fish 10% (pMCV1.4-injected fish) and 0% (PBS-injected fish) probe specifically designed in this work to detect the gpG of (Fig. 4B). All the dead fish had massive haemorrhages in the VHSV860 were used. Cell transfection assays revealed an efficient abdominal cavity, which is a typical symptom of VHSV infection (data not shown). expression of gpG860 in transfected EPC cells as well as a specific detection of these transcripts with the primers and probe designed in this work (Fig. 2A). In contrast, only a negligible transcription of 3.4. Neutralising antibodies to VHSV in vaccinated turbot gpG07.71 was detected with the gpG860-specific primers, showing the specificity of the assay. To test the neutralising activity of sera collected in this work, we Since the in vivo expression of the antigenic proteins encoded by used an immunostaining focus assay previously developed for the the plasmids might be different from those in vitro [38], we also immunodetection of viral haemorrhagic septicaemia virus strain evaluated the expression of the gpG860 gene in the muscle of 07.71 [37,39,40]. Therefore to begin with, we tested whether or not A B RPS

Fig. 4. Relative percentage survival (RPS) of DNA vaccinated turbots infected with VHSV860. Juvenile turbots were intramuscularly (i.m.) injected with 2 mg pMCV 1.4-G860, pMCV 1.4 empty plasmid or PBS and VHSV860 challenge was performed 30 days post-immunisation by intraperitoneal injection. Cumulative mortality was monitored over 21 days. The 6 relative percent survival (RPS) was calculated by the formula: RPS ¼ [1 (% mortality of immunized fish/% mortality of control fish)] 100. A, challenge with 5 10 TCID50 5 VHSV860/fish. B, challenge with 5 10 TCID50 VHSV860/fish. Solid circles, turbots injected with pMCV1.4-G860; open circles, turbots injected with pMCV1.4; open stars, turbots injected with PBS. 1092 P. Pereiro et al. / Fish & Shellfish Immunology 32 (2012) 1088e1094 the monoclonal antibody (MAb) 2C9 directed towards the N protein susceptible to those viruses and amongst the most important fish of VHSV07.71 also recognised the N protein of VHSV860. After species in levels of production worldwide. However, it should be following the established protocols for this assay, the N protein of taking into account that fish rhabdoviruses, mainly VHSV, also VHSV860 was successful detected in EPC infected cell monolayers cause mortalities in other fish species. In fact, VHSV strains have (Fig. 5A). Next, fish sera were tested for neutralizing antibody been also isolated from an increasing number of marine fish species against VHSV860. Neutralizing activity was only observed in the sera [9] including turbot [11,17], a high-value farmed flatfish. Therefore, from pMCV1.4-G860-injected turbots since VHSV860 infectivity was the aim of this work has been the study of the feasibility of an inhibited w75 and 50% at sera dilutions of 1/10 and 1/50, respec- experimental turbot DNA vaccination protocol against a turbot- tively (Fig. 5B). specific VHSV strain. To carry out the work, we chose the virulent VHSV strain UK-860/ 4. Discussion 94 causing high mortalities in turbot farming [14,17], since only moderate mortalities were shown by the VHSV07.71 strain isolated DNA vaccines based on the fish novirhabdovirus glycoprotein G from trout [42]. Before immunising the fish, the ability of the (gpG) gene are the most efficient vaccines against viral diseases in resulting vaccination plasmid (pMCV1.4-G860) to express the gpG860 fish [29,41]. To date, most studies concerning rhabdovirus DNA was evaluated in cell culture. Since successful in vitro expression vaccines have been performed in salmonids since they are highly was achieved (Fig. 1A), as it has been previously reported for the A

B

Fig. 5. Serum neutralizing activity of immunised fish. VHSV860 aliquots were incubated with 5-fold serial dilutions of serum pools from turbots as described in material and methods. EPC cell monolayers were then infected with virus-serum mixtures (B) for 2 h at 14 C and then washed. Twenty four hours post-infection, the number of N protein positive foci was counted. A, staining of both non-infected (left panel) and VHSV860-infected (right panel) EPC cell monolayers with the anti-VHSV0771 N protein. B, inhibition of VHSV860 infectivity calculated by the following formula, (100 (number of DAB positive foci in EPC cultures inoculated with turbot serum, complement and VHSV860/number of DAB positive foci in EPC cultures inoculated with turbot serum and VHSV860 in the absence of complement) 100). Data are the average values and standard deviations of two independent experiments each performed in duplicate. Solid circles, turbots injected with pMCV1.4-G860; open circles, turbots injected with pMCV1.4; open stars, turbots injected with PBS. *Significantly different from respective controls as determined by unpaired t-test (P < 0.05). P. Pereiro et al. / Fish & Shellfish Immunology 32 (2012) 1088e1094 1093 expression of the gpG from the VHSV strain 07.71 in different cell [10] Hopper K. The isolation of VHSV from salmon at Glenwood Springs, Orcas lines [29,31], the pMCV 1.4-G was intramuscularly injected in Island, Washington. Am Fish Soc Fish Health Newslett 1989;17:1. 860 [11] Schlotfeldt HJ, Ahne W, Vestergard-Jorgensen PE, Glende W. Occurrence of turbot muscle. The presence of gpG860 transcripts in all the turbot viral haemorrhagic septicaemia in turbot (Scophthalmus maximus) e a natural analysed (Fig. 1B) confirmed the in vitro expression results and outbreak. Bull Eur Assoc Fish Pathol 1991;11:105e7. further reinforced the results obtained in previous reports [29,43] [12] Meyers TR, Sullivan J, Emmeneger E, Follet J, Short S, Batts WN, et al. Iden- tification of viral haemorrhagic septicemia virus isolated from pacific cod, showing the utility of the EPC cell line to test the expression of Gadus macrocephalus in prince William Sound, Alaska, USA. Dis Aquat Org gene candidates to develop fish rhabdoviral DNA vaccines. 1992;12:167e75. When the immunisation/infection-challenge assays were [13] Isshik T, Nishizawa T, Kobayashi T, Nagano T, Miyazaki T. An outbreak of VHSV (viral hemorrhagic septicemia virus) infection in farmed Japanese flounder carried out to study the protection conferred by the DNA vaccine, Paralichthys olivaceus in Japan. Dis Aquat Organ 2001;47:87e99. the high levels of gpG860 expression obtained with pMCV1.4-G860 at [14] Snow M, Smail DA. Experimental susceptibility of turbot Scophthalmus max- the primary site of antigen delivery (muscle) were translated into imus to viral haemorrhagic septicaemia virus isolated from cultivated turbot. fi Dis Aquat Organ 1999;38:163e8. a meaningful protective response, as well as a high speci c IgM [15] Einer-Jensen K, Ahrens P, Forsberg R, Lorenzen N. Evolution of the fish (Fig. 3) and neutralizing antibody response (Fig. 5B). In fact, the rhabdovirus viral haemorrhagic septicemia virus. J Gen Virol 2004;85: e titers of specific and neutralizing antibodies against gpG860 of 1167 79. pMCV1.4-G -injected turbot were similar to those found in the [16] Snow M, King JA, Garden A, Shanks AM, Raynard RS. Comparative suscepti- 860 bility of turbot Scophthalmus maximus to different genotypes of viral hae- sera of turbot surviving an infection with VHSV860. morrhagic septicaemia virus. Dis Aquat Organ 2005;67:31e8. The protective efficiency of the VHSV860 gpG gene DNA vaccine [17] Ross K, McCarthy U, Huntly PJ, Wood BP, Stuart D, Rough EI, et al. An outbreak of viral haemorrhagic septicemia (VHS) in turbot (Scophthalmus maximus)in (Fig. 3) was independent of the virus dose challenge and resulted in e 6 Scotland. Bull Eur Assoc Fish Pathol 1994;14:213 4. high RPS values (80 and 90% for challenge doses of 5 10 and [18] Hastein T, Hill BJ, Winton JR. Successful aquatic animal disease emergency 5 e 5 10 TCID50/fish, respectively). In contrast, the pMCV1.4 and PBS programmes. Rev Sci Tech 1999;18:214 27. control groups had very low survival rates (<10%). These RPS are [19] Byon JY, Ohira T, Hirono I, Aoki T. Comparative immune responses in Japanese flounder, Paralichthys olivaceus after vaccination with viral hemorrhagic similar to those found in the fresh water rainbow trout septicemia virus (VHSV) recombinant glycoprotein and DNA vaccine using [3,5,29,41,44] and in the marine Japanese flounder [19] with fish a microarray analysis. Vaccine 2006;24:921e30. specific VHSV strains. Therefore, although rainbow trout belongs to [20] Byon JY, Ohira T, Hirono I, Aoki T. Use of a cDNA microarray to study immunity against viral hemorrhagic septicemia (VHS) in Japanese flounder (Paralichthys the order Salmoniformes, which is evolutionary distant from the olivaceus) following DNA vaccination. Fish Shellfish Immunol 2005;18: turbot and flounder (flatfish belonging to the Pleuronectiformes, 135e47. which is a more recent order in evolutionary terms) [21], the [21] Sommerset I, Lorenzen E, Lorenzen N, Bleie H, Nerland AH. A DNA vaccine directed against a rainbow trout rhabdovirus induces early protection against protective mechanisms triggered by VHSV DNA vaccine seem to be a nodavirus challenge in turbot. Vaccine 2003;21:4661e7. an evolutionary conserved immune response in fish. [22] Sommerset I, Krossoy B, Biering E, Frost P. Vaccines for fish in aquaculture. In summary, this is the first report showing the successful Expert Rev Vaccines 2005;4:89e101. induction of neutralizing antibodies and protection to challenge of [23] Fijan N, Sulimanovic D, Bearzotti M, Mizinic D, Zwillenberg LO, Chilmonczyk S, et al. Some properties of the epithelioma papulosum cyprini (EPC) cell line turbot with a DNA vaccine against a highly virulent VHSV strain. from carp Cyprinus carpio. Ann Virol 1983;134:207e20. Work is in progress to further analyse the immune response elicited [24] Basurco B, Coll JM. Spanish isolates and reference strains of viral haemor- by VHSV DNA vaccine as well as the mechanism implicated in rhagic septicaemia virus shown similar protein size patterns. Bull Eur Assoc 860 Fish Pathol 1989;9:92e5. turbot protection by using high throughput genomic techniques. [25] Reed LJ, Muench H. Simple method of estimating fifty per cent endpoints. Am J Hyg 1938;27:493e7. [26] Rocha A, Ruiz S, Coll JM. Improvement of transfection efficiency of epithe- Acknowledgements lioma papulosum cyprini carp cells by modification of their cell cycle and using an optimal promoter. Marine Biotechnol 2005;6:401e10. [27] Brocal I, Falco A, Mas V, Rocha A, Perez L, Coll JM, et al. Stable expression of Thanks are due to Beatriz Bonmati and Rubén Chamorro for bioactive recombinant pleurocidin in a fish cell line. Appl Microbiol Bio- technical assistance. This work was supported by the projects technol 2006;72:1217e28. Consolider Ingenio 2010, CSD2007-00002 and AGL2011-28921-C03 [28] Lopez A, Fernandez-Alonso M, Rocha A, Estepa A, Coll JM. Transfection of epithelioma cyprini (EPC) carp cells. Biotechnol Lett 2001;23:481e7. both from the Spanish Ministerio de Ciencia e Innovación (MICINN). [29] Chico V, Ortega-Villaizan M, Falco A, Tafalla C, Perez L, Coll JM, et al. The immunogenicity of viral haemorragic septicaemia rhabdovirus (VHSV) DNA vaccines can depend on plasmid regulatory sequences. Vaccine 2009;27: References 1938e48. [30] LeBerre M, De Kinkelin P, Metzger A. Identifi cation sérologique des rhabdo- [1] Anderson ED, Mourich DV, Leong JC. Gene expression in rainbow trout virus des salmonidés. Bull Off Int Epizoot 1977;87:391e3. (Onchorynchus mykiss) following intramuscular injection of DNA. Mol Marine [31] Tafalla C, Chico V, Perez L, Coll JM, Estepa A. In vitro and in vivo differential Biol Biotechnol 1996;5:105e13. expression of rainbow trout (Oncorhynchus mykiss) Mx isoforms in response [2] Winton JR. Immunization with viral antigens: infectious haematopoietic to viral haemorrhagic septicaemia virus (VHSV) G gene, poly I: C and VHSV. necrosis. Dev Biol Stand 1997;90:211e20. Fish Shellfish Immunol 2007;23:210e21. [3] Lorenzen N, Lorenzen E, Einer-Jensen K, Heppell J, Wu T, Davis H. Protective [32] Chico V, Gomez N, Estepa A, Perez L. Rapid detection and quantitation of viral immunity to VHS in rainbow trout (Oncorhynchus mykiss, Walbaum) following hemorrhagic septicemia virus in experimentally challenged rainbow trout by DNA vaccination. Fish Shellfish Immunol 1998;8:261e70. real-time RT-PCR. J Virol Methods 2006;132:154e9. [4] LaPatra SE, Corbeil S, Jones GR, Shewmaker WD, Lorenzen N, Anderson ED, [33] Falco A, Chico V, Marroqui L, Perez L, Coll JM, Estepa A. Expression and et al. Protection of rainbow trout against infectious hematopoietic necrosis antiviral activity of a beta-defensin like peptide identified in the rainbow virus four days after specific or semi-specific DNA vaccination. Vaccine 2001; trout (Oncorhynchus mykiss) EST sequences. Mol Immunol 2008;45:757e65. 19:4011e9. doi:10.1016/j.molimm.2007.06.358. [5] Lorenzen N, Lorenzen E, Einer-Jensen K, LaPatra SE. DNA vaccines as a tool for [34] Chico V, Martinez-Lopez A, Ortega-Villaizan M, Falco A, Perez L, Coll JM, et al. analysing the protective immune response against rhabdoviruses in rainbow Pepscan mapping of viral hemorrhagic septicemia virus glycoprotein G major trout. Fish Shellfish Immunol 2002;12:439e53. lineal determinants implicated in triggering host cell antiviral responses [6] Salonius K, Simard N, Harland R, Ulmer JB. The road to licensure of a DNA mediated by type I interferon. J Virol 2010;84:7140e50. vaccine. Curr Opin Investig Drugs 2007;8(8):635e41. [35] Estevez J, Leiro J, Santamarina MT, Dominguez J, Ubeira FM. Monoclonal [7] Alonso M, Chiou PP, Leong JA. Development of a suicidal DNA vaccine for antibodies to turbot (Scophthalmus maximus) immunoglobulins: character- infectious hematopoietic necrosis virus (IHNV). Fish Shellfish Immunol 2011; ization and applicability in immunoassays. Vet Immunol Immunopathol 1994; 30:815e23. 41:353e66. [8] Olesen N. Sanitation of viral haemorrhagic septicaemia (VHS). J Appl Ichthyol [36] Sanz F, Coll JM. Detection of viral haemorrhagic septicemia virus by direct 1998;14:173e7. immunoperoxidase with selected anti-nucleoprotein monoclonal antibody. [9] Skall HF, Olesen NJ, Mellergaard S. Viral haemorrhagic septicaemia virus in Bull Eur Assoc Fish Pathol 1992;12:116e9. marine fish and its implications for fish farming e a review. J Fish Dis 2005;28: [37] Falco A, Mas V, Tafalla C, Perez L, Coll JM, Estepa A. Dual antiviral activity of 509e29. human alpha-defensin-1 against viral haemorrhagic septicaemia rhabdovirus 1094 P. Pereiro et al. / Fish & Shellfish Immunology 32 (2012) 1088e1094

(VHSV): inactivation of virus particles and induction of a type I interferon- [41] Lorenzen N, LaPatra SE. DNA vaccines for aquacultured fish. Rev Sci Tech Off related response. Antiviral Res 2007;76:111e23. Int Epiz 2005;24(1):201e13. [38] Xiang ZQ, Knowles BB, McCarrick JW, Erlt HCJ. Immune effector mech- [42] Brañas MV, Coll JM, Estepa A. A sandwhich ELISA to detect VHSV and IPNV in anisms required for protection to rabies virus. Virology 1995;214: turbot. Aquacult Int 1994;2:206e17. 398e404. [43] Ruiz S, Tafalla C, Cuesta A, Estepa A, Coll JM. In vitro search for alternative [39] Lorenzo G, Estepa A, Coll JM. Fast neutralization/immunoperoxidase assay for promoters to the human immediate early-cytomegalovirus (IE-CMV) to viral haemorrhagic septicemia with anti-nucleoprotein monoclonal antibody. express the G gene of viral haemorrhagic septicemia virus (VHSV) in fish J Virol Methods 1996;58:1e6. epithelial cells. Vaccine 2008;26:6620e9. [40] Mas V, Perez L, Encinar JA, Pastor MT, Rocha A, Perez-Paya E, et al. Salmonid [44] McLauchlan PE, Collet B, Ingerslev E, Secombes CJ, Lorenzen N, Ellis AE. DNA viral haemorrhagic septicaemia virus: fusion-related enhancement of virus vaccination against viral haemorrhagic septicaemia (VHS) in rainbow trout: infectivity by peptides derived from viral glycoprotein G or a combinatorial size, dose, route of injection and duration of protection-early protection library. J Gen Virol 2002;83:2671e81. correlates with Mx expression. Fish Shellfish Immunol 2003;15:39e50.

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Transcriptome profiles associated to VHSV infection or DNA vaccination in turbot (Scophthalmus maximus)

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Transcriptome Profiles Associated to VHSV Infection or DNA Vaccination in Turbot (Scophthalmus maximus)

Patricia Pereiro1, Sonia Dios1, Sebastia´n Boltan˜ a2,5, Julio Coll3, Amparo Estepa4, Simon Mackenzie2,5, Beatriz Novoa1*, Antonio Figueras1 1 Instituto de Investigaciones Marinas (IIM), CSIC, Vigo, Spain, 2 Institute of Aquaculture, University of Stirling, Stirling, Scotland, United Kingdom, 3 Dpto Biotecnologı´a, Instituto Nacional Investigaciones Agrarias (INIA), Madrid, Spain, 4 Instituto de Biologı´a Molecular y Celular (IBMC), Miguel Herna´ndez University, Elche, Spain, 5 Institut de Biotecnologia i de Biomedicina, Universitat Auto`noma de Barcelona, Barcelona, Spain

Abstract DNA vaccines encoding the viral G glycoprotein show the most successful protection capability against fish rhabdoviruses. Nowadays, the molecular mechanisms underlying the protective response remain still poorly understood. With the aim of shedding light on the protection conferred by the DNA vaccines based in the G glycoprotein of viral haemorrhagic septicaemia virus (VHSV) in turbot (Scophthalmus maximus) we have used a specific microarray highly enriched in antiviral sequences to carry out the transcriptomic study associated to VHSV DNA vaccination/infection. The differential gene expression pattern in response to empty plasmid (pMCV1.4) and DNA vaccine (pMCV1.4-G860) intramuscular administration with regard to non-stimulated turbot was analyzed in head kidney at 8, 24 and 72 hours post-vaccination. Moreover, the effect of VHSV infection one month after immunization was also analyzed in vaccinated and non-vaccinated fish at the same time points. Genes implicated in the Toll-like receptor signalling pathway, IFN inducible/regulatory proteins, numerous sequences implicated in apoptosis and cytotoxic pathways, MHC class I antigens, as well as complement and coagulation cascades among others were analyzed in the different experimental groups. Fish receiving the pMCV1.4-G860 vaccine showed transcriptomic patterns very different to the ones observed in pMCV1.4-injected turbot after 72 h. On the other hand, VHSV challenge in vaccinated and non-vaccinated turbot induced a highly different response at the transcriptome level, indicating a very relevant role of the acquired immunity in vaccinated fish able to alter the typical innate immune response profile observed in non-vaccinated individuals. This exhaustive transcriptome study will serve as a complete overview for a better understanding of the crosstalk between the innate and adaptive immune response in fish after viral infection/vaccination. Moreover, it provides interesting clues about molecules with a potential use as vaccine adjuvants, antiviral treatments or markers for vaccine efficiency monitoring.

Citation: Pereiro P, Dios S, Boltan˜a S, Coll J, Estepa A, et al. (2014) Transcriptome Profiles Associated to VHSV Infection or DNA Vaccination in Turbot (Scophthalmus maximus). PLoS ONE 9(8): e104509. doi:10.1371/journal.pone.0104509 Editor: Ilhem Messaoudi, University of California Riverside, United States of America Received May 26, 2014; Accepted July 7, 2014; Published August 6, 2014 Copyright: ß 2014 Pereiro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The microarray platform TurbotV2_SSFN (ID041183 Agilent) has been submitted to the Gene Expression Omnibus (GEO) repository under accession number GPL16776. Funding: This work has been funded by the project CSD2007-00002 ‘‘Aquagenomics’’ and AGL2011-28921-C03 from the Spanish Ministerio de Ciencia e Innovacio´n. The authors’ laboratory is also funded by the project 201230E057 from the Agencia Estatal Consejo Superior de Investigaciones Cientı´ficas (CSIC). P. Pereiro gratefully acknowledges the Ministerio de Educacio´n for a FPU fellowship (AP2010-2408). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected]

Introduction VHSV using subunits or single viral proteins as well as killed or attenuated viruses [6–11]. Although some of those vaccines have Viral haemorrhagic septicaemia virus (VHSV) is a fish pathogen induced good protection levels in laboratory conditions, they can belonging to the genus Novirhabdovirus, within the family either be unsafe for field use, its production very expensive or Rhabdoviridae. This etiological agent causes an important viral require high doses. DNA vaccination is based on the administra- disease affecting rainbow trout Oncorhyncus mykiss and other tion of a plasmidic DNA vector containing the gene encoding a salmonids [1–3] but VHSV outbreaks have been detected in other specific antigen. This technology is a powerful tool for the design marine farmed fish species such as turbot (Scophthalmus maximus of effective vaccines against fish rhabdoviral pathogens. Rhabdo- L. 1758) [4], [5]. Nowadays, the culture of this flatfish is well- viruses possess a surface glycoprotein G that acts as the target of established being a very important commercial species for the virus neutralizing antibodies [12] and therefore, the most aquaculture industry in Europe and Asia. However, infectious successful DNA vaccines against these viruses are based on the diseases are one of the most relevant limiting factors, causing G glycoprotein gene under the control of the cytomegalovirus severe economic losses in many cases. Neither vaccines nor promoter (CMV). We have recently constructed a DNA vaccine therapeutic treatments are commercially available for this disease. encoding the G glycoprotein from VHSV strain UK-860/94 Increased efforts were performed for more than 30 years in order (isolated from infected turbot) and have demonstrated the high to produce an efficient, safe and cost-effective vaccine against degree of protection provided against this virus as well as the

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Figure 1. Diagram reflecting the experimental design and sampling points employed in the study of the transcriptomic profiles associated to the vaccination and VHSV infection. This experimental prodecure is explained in detail in the materials and methods section. doi:10.1371/journal.pone.0104509.g001 production of specific neutralizing antibodies one month after VHSV G protein DNA vaccination before and after VHSV vaccination [13]. However, the early immune mechanisms challenge. implicated in the success of that vaccination remain still unclear. Microarray technology is a very useful tool for the understand- Materials and Methods ing of the immune process implicated in the protective response provided by efficient DNA vaccines against fish rhabdoviral Ethical statement infection. Some studies have been previously performed using Experimental procedures followed Spanish Law (Royal Exec- microarrays, including the effect of a DNA vaccine encoding the utive Order, 53/2013) for Animal Experimentation, in accordance infectious hematopoietic necrosis virus (IHNV) G glycoprotein in with European Union directive 2010/63/UE. Fish care and trout [14], the effect of the expression of the G protein from challenge experiments were reviewed and approved by the CSIC VHSV in Japanese flounder [15], [16], as well as the differences in National Committee on Bioethics (approval number: the gene expression profile following hirame rhabdovirus 07_09032012). (HIRRV) G and N protein DNA vaccination in Japanese flounder [17] and the expression pattern after HIRRV challenge in Fish vaccinated and non-vaccinated fish [18]. However, the informa- Juvenile turbot (average weight 2.5 g) were obtained from a tion provided by these reports was, in some cases, limited due to VHSV-free commercial fish farm (Insuin˜a S.L., Mouga´s, Galicia, the relatively low number of annotated immune-related sequences Spain). Animals were maintained in 500 L fibreglass tanks at the included in the microarray. To our knowledge, this is the first IIM (CSIC) facilities with a re-circulating saline water system with global work in fish including both the analysis of the expression a light-dark cycle of 12:12 h at 18uC and fed daily with a profile after DNA vaccination and the analysis of the differential commercial diet (LARVIVA-BioMar). Prior to experiments, fish transcriptomic patterns in vaccinated and non-vaccinated fish after were acclimatized to laboratory conditions for 2 weeks. rhabdoviral infection and the first performed in turbot. Moreover, the microarray has been constructed using a high number of Plasmids annotated sequences obtained from an enriched 454-pyrosequenc- The expression vector pMCV1.4 (Ready-Vector, Madrid, ing of turbot transcriptome after viral stimulations [19], providing Spain) was used for the construction of the vaccine containing a higher quantity of information compared to previous similar the G glycoprotein cDNA sequence from VHSV strain UK-860/ publications in other fish species. The gene expression patterns of 04 (GeneBank accession number AY546628) as was previously several immune-relevant pathways were analyzed and allowed a described by Pereiro et al. [13]. High amounts of DNA vaccine better comprehension of the protective mechanisms underlying (pMCV1.4-G860) and the corresponding empty plasmid (pMCV1.4) were obtained by transformation in One Shot

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TOP10F’ chemically competent E. coli (Invitrogen) for its cloning exhibited clinical signs of disease (8, 24 and 72 hours post- following the protocol instructions. Bacteria were cultured on LB- infection). Kanamycin (50 mg/ml) agar plates during 24 h at 37uC and an isolated colony from each transformation was selected and Total RNA extraction, quality control and cDNA synthesis cultured in LB-Kanamycin (50 mg/ml) medium at 37uCin RNA was extracted from 68 samples using TRIzol (Invitrogen) agitation. Plasmid constructions were purified using the PureLink in accordance with instructions provided by the manufacturer in HiPure Plasmid Midiprep Kit (Invitrogen) following the manu- combination with the RNeasy mini kit (Qiagen) for RNA facturer’s instruction. purification after DNase treatment (RNase-free DNase set, Qiagen). RNA concentration was quantified using the spectro- VHSV strain UK-860/94 photometer Nanodrop ND-1000 (Thermo Scientific). RNA Viral haemorrhagic septicaemia virus UK-860/94 (VHSV860) integrity and quality were also assessed (Bioanalyzer 2100, Agilent isolated from farmed turbot in Scotland [5] was propagated in Technologies). The RNA integrity number (RIN) was calculated Epithelioma papulosum cyprini (EPC) cells at 14uC containing for each sample and only RNAs with a RIN number .7 were Eagle’s minimum essential medium (MEM, Gibco) supplemented processed. For qPCR validation of the microarray results, the with 2% fetal bovine serum (FBS), penicillin (100 IU/ml) cDNA synthesis was performed with SuperScript II Reverse (Invitrogen) and streptomycin (100 mg/ml) (Invitrogen). The Transcriptase (Invitrogen) using 0.5 mg of RNA by following the supernatants were clarified by centrifugation at 40006g during manufacturer indications. 30 min and viruses from these supernatants were concentrated by ultracentrifugation at 100,0006g for 45 min. VHSV860 aliquots Oligonucleotide microarray design were maintained at –80uC until use. Virus stock was titrated in 96- A specific turbot microarray enriched in immune-related genes well plates according to Reed & Mue¨nch [20] and the in vivo was designed by selecting the sequences obtained after a 454- infectivity was tested using juvenile turbot. pyrosequencing of several S. maximus tissues at different sampling points after viral stimulation (VHSV strain UK-860/94 and Immunization and viral infection protocols Nodavirus strain AH95-NorA) or using molecules mimicking viral A schematic overview of the vaccination/challenge protocol and infection (pMCV1.4, pMCV1.4-G860 and Poly I:C) [19]. This tool sampling procedure is shown in Fig. 1. A total number of 204 was used for analyzing the transcriptome profiles associated to the juvenile turbot were divided into 3 groups, two of them containing VHSV infection or DNA vaccination in turbot, as well as the 72 fish and the last one 60 fish. Turbot were anaesthetized by differences between vaccinated and non-vaccinated fish after viral immersion in 50 mg/ml buffered tricaine methanesulfonate (MS- infection. The five proteins encoded by the VHSV genome (RNA- 222; Sigma) and then, fish from the first two groups were directed RNA polymerase L, Nucleoprotein, Phosphoprotein, intramuscularly (i.m.) injected with 50 ml of PBS containing 2 mg Spike glycoprotein and Matrix protein), whose sequences were of pMCV1.4 or pMCV1.4-G860. Turbot from the last batch were obtained in the pyrosequencing, were also included in the i.m. inoculated with 50 ml of PBS. At 8, 24 and 72 h after microarray in order to analyze the evolution of the viral injection, 12 fish were removed from the first two tanks and, at 8 h replication in vaccinated and non-vaccinated fish. after PBS inoculation, other 12 fish were taken from the last tank. A total of 43,398 oligonucleotide probes (60-mer long each) These turbot were sacrificed by anaesthetic overdose and the head were used to construct a high-density turbot microarray based on kidney was removed. Equal amounts of tissue from three fish the Agilent 4644K design format using the Agilent eArray belonging to the same tank and sampling point were pooled, interface. Thus, 10,907 annotated contigs were spotted in obtaining 4 biological replicates for each treatment and time point triplicated into the slide (total probes 32,721), as well as 4,654 (3 turbot/replicate). The remaining fish (36 in the plasmid-injected spotted in duplicated (9,308). In addition, 18 selected singletons groups and 48 in the PBS-inoculated tank) were maintained with 2 replicated probes (total probes 36) were also spotted as well during one month and then, 12 fish from the PBS injected group as 1,417 internal control probes of Agilent (N = 43,398). The were separated to another tank. This new group of fish was microarray platform TurbotV2_SSFN (ID041183 Agilent) has intraperitoneally (i.p.) injected with 50 ml of MEM + penicillin and been submitted to the Gene Expression Omnibus (GEO) streptomycin+2% FBS (PBS - MEM group), whereas the other repository under accession number GPL16776. 5 turbot were i.p. infected with a dose of VHSV860 of 5610 TCID50/fish (pMCV1.4 - VHSV and pMCV1.4-G860 - VHSV RNA Labeling and Microarray Hybridization groups). At 8, 24 and 72 hours after infection, 12 fish were RNA labelling, hybridizations and scanning were performed removed from the VHSV-infected tanks, and at 8 h after MEM according to manufacturer’s instructions. Total RNA (500 ng) was injection the 12 fish were taken from the non-infected tank. The amplified and Cy3-labeled with Agilent’s One-Color Microarray- fish were sacrificed by anaesthetic overdose and the head kidney Based Gene Expression Analysis (Quick Amp Labelling kit) along was removed. Equal amounts of tissue from three fish belonging to with Agilent’s One-Color RNA SpikeIn Kit. Each amplified and the same tank and sampling point were pooled, obtaining 4 labelled sample was briefly hybridized at 65uC for 17 hours. biological replicates for treatment and time point (3 turbot/ Microarray slides were scanned with Agilent Technologies replicate). The whole experimental procedure was conducted in Scanner model G2505B. Spot intensities and other quality control parallel to the previously published work in which the high features were extracted with the Feature Extraction software protective effect induced by the pMCV 1.4-G860 vaccine was version 10.4.0.0 (Agilent). One-channel TIFF images were demonstrated, obtaining a relative percent survival (RPS) higher imported into the GeneSpring GX 12.0 software (Agilent). than 80% [13]. Mortality events were not recorded in the absolute control groups (non-immunized and non-infected fish) during the Microarray data analysis experiment. The health status of the fish was daily monitored and Fluorescence intensity data and quality measurements were no adverse health effects were observed in non-infected turbot. analyzed using the GeneSpring GX 12.0 software (Agilent). After The animals used in this work were sacrificed before they grouping the biological replicates (4 replicates for treatment and

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Figure 2. qPCR validation of the microarray data. Correlation between microarray (x-axis) and qPCR (y-axis) data (Log10 fold-change) from 5 genes at 3 different times after infection of vaccinated and unvaccinated turbots. Fold-change values of the selected genes were displayed in Table S1. The correlation between microarray and qPCR data analyzed by the Spearman’s Rho test was r = 0.969 and with a statistical significance of p, 0.01. doi:10.1371/journal.pone.0104509.g002 sampling point), data were filtered by flags and then by expression qPCR validation th between the 20 and 95 percentile in the raw data. Once the The expression profiles of five immune-related genes modulated analysis by probes was performed, the gene-level experiment was in the microarray (Tumor necrosis factor, Interferon phi 2, th conducted normalizing the data by percentile shifts at the 75 Interferon-induced GTP-binding protein Mx, IFI56 and Interfer- percentile and using as baseline transformation the median of all on-stimulated gene 15) were determined at 3 different times from samples. In order to identify differentially expressed genes, the vaccinated and non-vaccinated fish by using reverse transcriptase normalized data were analyzed by filtering on Volcano Plot in real-time quantitative qPCR. Specific PCR primers were designed order to compare the mean expression levels between treatments using the Primer3 program [22] and their amplification efficiency (pMCV1.4 and pMCV1.4-G860 treatments against PBS [8 h] - was calculated using seven serial five-fold dilutions of head kidney control group- and PBS - VHSV, pMCV1.4 - VHSV and cDNA from unstimulated turbot with the Threshold Cycle (CT) pMCV1.4-G860 - VHSV treatments against PBS - MEM [8 h] - slope method [23]. Primer sequences are listed in the Table S1. control group-). An unpaired t-test was conducted without Individual reactions were carried out in 25 ml reaction volume correction and data were considered significant at p,0.05. The using 12.5 ml of SYBR GREEN PCR Master Mix (Applied fold-change cut-off was set at 1.5. The data presented in this Biosystems), 10.5 ml of ultrapure water (Sigma-Aldrich), 0.5 mlof publication and the MIAME-compliant information has been each specific primer (10 mM) and 1 ml of five-fold diluted cDNA deposited in the NCBI’s Gene Expression Omnibus (GEO, template in MicroAmp optical 96-well reaction plates (Applied http://www.ncbi.nlm.nih.gov/geo/) and is available under the Biosystems). All reactions were performed using technical tripli- accession number GSE56487. cates in a 7300 Real-Time PCR System thermocycler (Applied The five viral proteins encoded by VHSV were excluded for Biosystems) with an initial denaturation (95uC, 10 min) followed subsequent analysis of the turbot transcriptomic profiles. Venn by 40 cycles of a denaturation step (95uC, 15 s) and one diagrams representing the number of shared and exclusive hybridization-elongation step (60uC, 1 min). An analysis of modulated genes among different experimental conditions were melting curves was performed for each reaction. Relative also constructed by using GeneSpring GX 12.0 software. This expression of each gene was normalized using the Elongation bioinformatic tool was also chosen for performing hierarchical Factor-1 alpha as reference gene, which was constitutively clustering using Euclidean distance metric of several groups of expressed and not affected by the experimental treatments, and selected genes. Blast2GO suite [21] was used for Gene Ontology calculated using the Pfaffl method [23]. The correlation between (GO) classification into biological process terms of the significantly microarray and qPCR data (Log10 fold-change) were analyzed by modulated genes from each comparison. the Spearman’s Rho test.

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Figure 3. Evolution of viral genes transcription in non-vaccinated and vaccinated turbot. Sequences for the five proteins encoded by VHSV were included in the microarray design for analyzing their evolution in non-vaccinated (PBS – VHSV and pMCV1.4– VHSV) and vaccinated (pMCV1.4-G860– VHSV) individuals at 8, 24 and 72 h post-challenge. doi:10.1371/journal.pone.0104509.g003

Results and Discussion was able to reduce the number of colony-forming units (CFU) of the bacteria Paracoccidioides brasiliensis in mice. Other publica- Validation of microarray data by qPCR tion showed some but modest protective effect against Mycobac- Quantitative real-time PCR (qPCR) is a commonly used terium tuberculosis in mice after vaccination with the empty validation tool for confirming gene expression results obtained plasmid pGX10 [31]. With regard to fish viruses, a protective from microarray analysis [24]. In order to validate the expression response against the Infectious Pancreatic Necrosis Virus (IPNV) profiles from microarray analysis, the relative mRNA level for 5 in Atlantic salmon was also observed one week after CpG immune-relevant genes (Tumor necrosis factor, Interferon phi 2, oligodeoxynucleotides stimulation, revealing the induction of a Interferon-induced GTP-binding protein Mx, IFI56 and Interfer- nonspecific immune response against virus [32]. Indeed, some on-stimulated gene 15) was measured by qPCR. The expression experiments have shown that the early immune response induced data obtained by microarray and qPCR for the selected genes are shortly after DNA vaccination against VHSV in trout is non- listed in the Table S2 and plotted in Fig. 2. Microarray and qPCR specific and cross-protective against other rhabdoviruses [33], [34] results were analyzed by Spearmans Rho test and a high and even against nodavirus in turbot [35]. Although in our turbot correlation (r = 0.969) and a statistical significance (p,0.01) were vaccination trials we did not observe any significant difference in observed. final mortalities between PBS and empty plasmid-injected fish groups, only a slight reduction and delay in the mortality [13], our Transcription of viral genes in non-vaccinated and results suggest the importance of the intrinsic adjuvant properties vaccinated turbot infected with VHSV of the plasmids used in DNA vaccination [28] and the persistence In order to assess the viral replication success of VHSV strain of the non-specific immune response. UK-860/94 in head kidney cells from vaccinated and non- vaccinated fish infected with VHSV, the expression of the five viral Overall effect of pMCV1.4-G860 immunization on the host genes was analyzed. The evolution of the VHSV replication gene expression throughout the tested sampling points (8, 24 and 72 hours after Total quantity and fold-changes of up and down-regulated infection) in the different experimental groups is shown in Fig. 3. genes in head kidney after pMCV1.4 or pMCV1.4-G860 Those fish that were previously inoculated intramuscularly with intramuscular injections are shown as stacked column charts in PBS or with the empty plasmid (pMCV1.4) and one month later Fig. 4. The number of modulated genes increased with time when infected with VHSV showed an increasing viral replication from 8 compared to the control group (PBS-injected fish) in both cases. In hours to 72 hours post-challenge, being the five proteins already agreement with the reduction of the viral genes expression, the detected at 8 hours. On the other hand, turbot previously injected empty plasmid was able to induce the modulation of several genes with the DNA vaccine encoding the G glycoprotein (pMCV1.4- and this induction increased from 8 to 72 h. Differences in the G860) showed a limited quantity of viral transcripts after VHSV number of modulated genes between pMCV1.4 and pMCV1.4- challenge. Thus, matrix protein and RNA-directed RNA poly- G860 were highest at 72 h, when the transcription of the G merase L were not significantly increased in vaccinated fish at any glycoprotein gene is on-going as was previously observed [13]. of the tested time points, whereas the other three genes were only Intramuscular administration of microgram amounts of DNA detected after 72 hours post-infection and in a lower level in vaccine is enough for the expression of the viral G glycoprotein on comparison with the other two groups of non-vaccinated fish. This the surface of muscular cells and this is the way to trigger the reduction in the number of viral transcripts in the host cells and, as orchestration of an adaptive immune response [25], [36]. A total consequence, the high survival rates obtained after vaccination, of 1,495 genes were found to be regulated at 72 h after vaccination might be directly related to the presence of specific neutralizing (630 up-regulated and 865 down-regulated), whereas the empty antibodies against VHSV (strain UK-860/94) one month after plasmid induced the expression of 382 genes at the same time immunization as was previously reported [13]. However, we point (140 up-regulated and 242 down-regulated). cannot rule out that some non-specific immune responses could A Gene Ontology (GO) classification of biological processes at also be contributing to the reduction of viral replication because it the 2nd level of the modulated genes after plasmids injection is is known that the specific protection provided by VHSV and provided in Fig. S1. Those GO categories containing a high IHNV G gene DNA vaccines in fish is preceded by a protective representation of genes with a direct implication in immunity nonspecific antiviral response, possibly related to interferon- (Viral reproduction, Signaling, Response to stimulus, Death, Cell induced mechanisms [25]. In fact, interestingly, turbot previously proliferation and Immune system process) showed, in general, receiving only the empty plasmid and then challenged with VHSV their highest representations at 24 h when the empty plasmid was one month later showed a significantly reduced expression of all administrated and at 72 h after pMCV1.4-G860 vaccination. VHSV genes when compared to the PBS-injected VHSV- Venn diagrams representing exclusive and common genes after challenged group. This reduction in the transcription of viral pMCV1.4 or pMCV1.4-G860 injection are provided in Fig. S2. genes could be related to the persistence of a non-specific immune The number of exclusive genes at 72 h after vaccination was 580 response probably due to the induction of several immune factors for the up-regulated sequences and 799 for the down-regulated by unmethylated CpG motifs present in the plasmid backbone sequences, whereas the empty plasmid administration induced the [26–28]. DNA vaccines are constructed from plasmids of bacterial up-regulation of 90 exclusive genes and the down-regulation of DNA that are able to induce the maturation, differentiation and 176 at 72 h. In order to identify those GO categories especially proliferation of the immune cells and, therefore, to increase the affected during the expression of the G glycoprotein, biological production of the several cytokines [29]. Ritter et al. [30] revealed process multilevel pie charts of exclusive and common regulated that immunization using the empty plasmids pcDNA3 and pORF sequences at 72 h after DNA vaccine or pMCV1.4 empty plasmid

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Figure 4. Stacked column chart reflecting the distribution of regulated genes through time after pMCV1.4 or pMCV1.4-G860 intramuscular injection. Statistically significant differential gene expressions are subdivided according to intensity (fold change) and sense (up and down-regulation). doi:10.1371/journal.pone.0104509.g004 injection were constructed and are displayed in Fig. S3. Several including the highest number of exclusive sequences both in the immune-related GO terms were found to be highly represented group of up and down-regulated genes (29 and 34 genes, among the pMCV1.4-G860 exclusive genes, such as I-kappaB respectively), which is a GO term containing several molecules kinase/NF-kappaB cascade, activation of immune response, implicated in the antigen processing and presentation. On the antigen processing and presentation, cytokine production, immune other hand, empty plasmid exclusive genes as well as common effector process, innate immune response and regulation of genes between both groups did not show a remarkable represen- apoptosis, among others. Proteolysis was found to be the category tation of these terms at the tested time point.

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Figure 5. Stacked column chart reflecting the distribution of regulated genes through time after VHSV challenge in non-vaccinated (PBS – VHSV or pMCV1.4– VHSV) and vaccinated (pMCV1.4-G860– VHSV) turbot. Statistically significant differential gene expressions are subdivided according to intensity (fold change) and sense (up and down-regulation). doi:10.1371/journal.pone.0104509.g005

Differential gene expression profile in vaccinated and Six different TLRs were found to be modulated by some non-vaccinated turbot after VHSV challenge treatment in the microarray analysis (TLR2, TLR3, TLR5, Stacked column charts reflecting the number and intensity of up TLR6, TLR8 and TLR13) as well as numerous molecules directly and down-modulated genes in non-vaccinated and vaccinated fish implicated in the signalling cascade downstream of the TLR- after VHSV challenge is represented in Fig. 5. The pattern of PAMP interaction. The immune response mediated by these regulated genes induced by VHSV challenge is quite different PRRs implies the activation of the transcription factors NF-kappa- from that observed after viral infection in vaccinated fish. The B, IRF3 and IRF7 [39], [40]. IRF3 and IRF7 are the main factors viral challenge induced a time-increasing modulation in the global responsible in the induction of antiviral innate immunity by gene expression, whereas vaccinated fish showed the highest inducing the expression of type I IFN genes [41]. The expression number of up and down-regulated genes at 24 h. Moreover, the level of these genes in the different experimental groups was number of genes appearing significantly modulated in vaccinated represented as a heat map (Fig. 6). individuals at 24 and 72 h after VHSV infection is quite lower in DNA vaccine administration significantly up-regulated the comparison with the other treatments. Another remarkable point expression of TLR8, a PRR directly implicated in the recognition is the difference observed between both non-vaccinated groups of viral nucleic acids, with a fold-change (FC) of 3.5 with regard to (PBS – VHSV and pMCV1.4– VHSV), where the fish previously the PBS control group at 72 h post-immunization. IRF3 and IRF7 receiving the empty plasmid showed a higher number of were also affected by the vaccine at the same time point (FC = 15 modulated genes at 8 h, suggesting once again a persistence of and FC = 3.6, respectively). Moreover, some genes related with the innate immune factors one month after empty plasmid injection. activity of the transcription factor NF-kappa-B, such as the activator IKKbeta or the NF-kappa-B inhibitor zeta, were A GO classification of biological processes at the 2nd level (Fig. significantly down-regulated by pMCV1.4-G . TLR2 recognize S4) revealed a very higher number of those sequences with direct 860 other viral components such as envelope glycoproteins [42] and, implication in immune defense in the pMCV1.4– VHSV group at although no significant up-regulations were observed for this gene 8 h compared to the PBS – VHSV fish. Unlike the other two after vaccination, the heat map reflects a slight induction. TLR2 treatments, where the number of immune genes increased from 8 activation induces apoptosis through a FADD/Caspase 8 pathway to 72 h, vaccinated individuals showed the peak of maximum [43] and both genes appeared overrepresented at 72 h, revealing a expression of these sequences at 24 h and a relevant reduction at possible stimulation of the TLR pathway via TLR2 and 72 h. But, interestingly, as it was illustrated in the Venn diagrams ultimately, inducing apoptosis. (Fig. S5) vaccinated fish presented the major number of exclusive With regard to the viral challenge (PBS – VHSV and modulated genes at 72 h. Therefore, although the number of pMCV1.4– VHSV groups), it is interesting to highlight that some regulated sequences decreased at 72 h and the total number of up Toll-like receptors with a typical role in bacterial component and down-regulated genes was lower in vaccinated fish with regard detection (TLR5 and TLR6) have been found to be strongly to the other groups, this exclusivity could be associated with genes regulated after VHSV infection, and this induction could suggest a directly related with the existence of adaptive immunity. novel role of these receptors in the recognition of viral components. A sequence annotated as TLR13 was also modulated Hierarchical clustering analysis of pathways or groups of at 24 h; there is little information about the function of this molecules involved in defense mechanisms receptor and the nature of their ligands remains still poorly In order to effectively combat viral infections and other diseases, understood, but there are evidences about the recognition of vertebrate organisms have developed an efficient, powerful and bacterial rRNA [44] as well as vesicular stomatitis virus [45] by integrated defense network comprising both innate and adaptive TLR13. As was expected, the typical viral-recognition receptors immune mechanisms. Numerous defensive processes or families of TLR3 and TLR8 were significantly up-regulated after VHSV molecules implicated in non-specific or specific responses against challenge. Focusing the attention in the downstream signalling VHSV were analyzed using hierarchical clustering in order to components, a pronounced induction of several proteins was define the transcriptomic profiles after pMCV1.4-G860 vaccination observed (MyD88, IRF3, IRF7, FADD, Caspase-8, etc). Some as well as after VHSV infection in vaccinated and non-vaccinated molecules were also found to be down-regulated, including turbot. Sequences directly related with the TLR pathway, IFN inhibitors of the transcription factor NF-kappa-B as well as system, apoptosis, MHC-I antigen presentation, and coagulation numerous molecules implicated in their activation, possibly due to among others were shown to be involved in the viral infection and the maintenance of equilibrium in the NF-kappa-B activity. also in the protection provided by the vaccine. On the other hand, VHSV infection in vaccinated fish TLR pathway. Virus detection by the innate immune system (pMCV1.4-G860– VHSV) revealed a completely different pattern, is carried out by a class of molecules known as pattern recognition even TLR2 and TLR5 were found to be significantly down- receptors (PRRs), which detect specific evolutionary conserved regulated at 24 h and the other TLRs were not affected by the structures on pathogens, termed pathogen-associated molecular viral infection. As a consequence, the induction of downstream patterns (PAMPs) [37]. Toll-like receptors (TLRs), a class of PRRs, proteins was practically suppressed or down-regulated and only in have been established to play a crucial role in the innate immune the case of IRF3 and IRF7 significant up-regulations were response to pathogens through the activation of intracellular detected at 24 h, with a return to the basal levels at 72 h. signalling pathways, which ultimately induce expression of a large The IFN system. The interferon (IFN) system is an early number of genes encoding type I interferons (IFNs), inflammatory antiviral immune process controlling most virus infections in the cytokines and chemokines, and other molecules affecting the absence of specific immunity, buying time for the generation of initiation of adaptive immune responses [38]. adaptive defense mechanisms [46]. Nowadays is well known that

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Figure 6. Heat map displaying hierarchical clustering results from microarray expression data of genes implicated in the Toll-like receptor signalling pathway. All the experimental groups, including the corresponding controls (PBS 8 h and PBS – MEM 8 h), were included in the analysis. Different genes are represented in different rows, and different experiments in different columns. Raw expression values are represented as a color scale from red for lower expressions to green for higher expressions. doi:10.1371/journal.pone.0104509.g006 fish type I IFNs induce the expression of a wide variety of IFN- No up-regulations were detected in the level of any of both IFNs stimulated genes (ISGs) after recognition of specific IFN receptors after vaccination, and even Interferon alpha 2 precursor (renamed [47]. These ISGs reduce the viral replication and dissemination as Ifn2) was significantly down-regulated in comparison with through different blocking mechanisms. Two type I IFNs, control fish (FC = 24.3). However, Interferon regulatory factors annotated as ‘‘Interferon phi 2’’ and ‘‘Interferon alpha 2 (IRFs 1, 3, 4, 7 and 8) and the majority of ISGs were up-regulated precursor’’, as well as some of the most relevant IFN-related after 72 h. The most induced ISG by pMCV1.4-G860 was Mx sequences modulated in the microarray were analyzed in the (FC = 66), followed by Interferon-inducible protein 56 (FC = 44.4) different groups (Fig. 7). Recently these two turbot type I IFNs and IFI56 (FC = 39.3), two genes or different parts of the same were characterized and renamed as Ifn1 and Ifn2, respectively gene belonging to the IFIT (IFN-induced protein with TPR [48]. repeats) family [49]. These up-regulations in downstream genes revealed an activation of the IFN signalling pathway after

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Figure 7. Heat map displaying hierarchical clustering results from microarray expression data of genes involved in the IFN system (IFNs, IRFs, ISGs…). All the experimental groups, including the corresponding controls (PBS 8 h and PBS – MEM 8 h), were included in the analysis. Different genes are represented in different rows, and different experiments in different columns. Raw expression values are represented as a color scale from red for lower expressions to green for higher expressions. doi:10.1371/journal.pone.0104509.g007

immunization with pMCV1.4-G860 even when no up-regulations Nevertheless, an overall and strong up-regulation of ISGs was in the expression of IFNs were observed. observed. There are evidences suggesting that different forms of After viral infection a remarkable induction of these genes was type I IFNs may have complementary antiviral activities in observed with some exception. Thus, Interferon phi 2 (Ifn1) was different cells, at different stages of infection or differ functionally highly up-regulated especially at 72 h (FC = 593.3 and FC = 177.7 [47], and this was also observed for turbot type I IFNs. Whereas in fish injected with PBS - VHSV and pMCV1.4 - VHSV, Ifn1 (‘‘Interferon phi 2’’) showed a typical antiviral activity (ISGs respectively), whereas Interferon alpha 2 precursor (Ifn2) tran- induction and protection against VHSV infection), Ifn2 was no scription was down-regulated. Interestingly, we previously ob- able to increase the expression of ISGs and therefore, did not show served that both turbot IFNs were overexpressed after VHSV any protective effect against VHSV, but was able to up-regulate challenge although at different level, being Ifn1 strongly induced the level of several immune-related genes, including pro-inflam- and Ifn2 slightly up-regulated and with a brief induction time [48]. matory cytokines [48].

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Figure 8. Heat map displaying hierarchical clustering results from microarray expression data of genes involved in the (A) apoptosis pathway and in the (B) cell death induced by cytotoxic cells. All the experimental groups, including the corresponding controls (PBS 8 h and PBS – MEM 8 h), were included in the analysis. Different genes are represented in different rows, and different experiments in different columns. Raw expression values are represented as a color scale from red for lower expressions to green for higher expressions. doi:10.1371/journal.pone.0104509.g008

When vaccinated fish were infected (pMCV1.4-G860– VHSV), Moreover, cross-presentation of antigens derived from apoptotic a weaker up-regulation of some IFN-related genes was observed at infected cells by professional antigen presenting cells, such as 24 h in comparison with both non-vaccinated groups. This dendritic cells, is another important way for initiating CD8 modulation diminished at 72 h, nearly returning to basal levels. cytotoxic lymphocyte responses to virus [53]. Therefore, a direct In fact, the high expression of Interferon phi 2 (Ifn1) after VHSV correlation between apoptosis induction and overexpression of challenge was not significantly affected in pMCV1.4-G860 antigen presentation molecules could be established. vaccinated turbot after infection at any of the sampling points. MHC class I antigen presentation. Protein ubiquitination Therefore, vaccinated turbot showed a limited IFN-response after is a mechanism that serves as a mark for the degradation of self viral challenge. and foreign proteins, such as viral molecules. The process of Apoptosis. One of the most important mechanisms prevent- ubiquitination allows the recognition of proteins by the 26S ing viral replication and dissemination is the apoptosis or proteasome, a complex that degrades ubiquitinated proteins to programmed cell death, in which infected cells are eradicated small peptides [54]. These peptides could be finally presented as through the activation of a group of proenzymes known as antigens on the plasma membrane, throughout the Major caspases [50]. The heat map containing several of the most Histocompatibility Complex class I (MHC-I) assembly and peptide relevant proteins implicated in apoptosis (Fig. 8A) revealed the binding process [55]. The expression pattern of several ubiquitin- induction of multitude of pro-apoptotic genes following related genes was analyzed for determining the effect of pMCV1.4-G860 vaccination, including the initiator caspases pMCV1.4-G860 vaccination as well as the response to VHSV Caspase-8 and Caspase-10 and the effector caspases Caspase-6 infection (Fig. 9A). At 72 h after pMCV1.4-G860 vaccine injection, and Caspase-7, as well as Caspase-1 or Caspase-1A. The injection several genes corresponding to ubiquitin-protein ligases were up- of the empty plasmid pMCV1.4 was also capable of inducing up- regulated, as well as some ubiquitin-conjugating enzymes. Slight regulation of some genes especially implicated in the apoptotic down-regulations were also observed in other ubiquitin-related intrinsic pathway (Diablo homolog mitochondrial, BCL2/adeno- genes. The viral challenge induced the expression of a broad range virus E1B 19 kDa protein-interacting protein 3, Calpain-2 of these genes, but strong inhibitions of some genes were also catalytic subunit, DNA-damage-inducible transcript 4-like protein, detected and, therefore, more investigations will be necessary in Apoptosis regulator BAX, Cytochrome c). order to determine the role of the different ubiquitin-related VHSV infection (PBS – VHSV and pMCV1.4– VHSV groups) proteins in the viral antigen presentation process. On the other revealed an extensive induction of genes implicated in apoptosis. hand, VHSV infection of vaccinated turbot showed only a Thus, all the caspases contained in the microarray were moderate induction at 24 h of some of the genes up-regulated in significantly and strongly up-regulated, indicating a powerful non-vaccinated fish and, interestingly, some ubiquitin-ligases up- activation of the programmed cell death. The profile reflecting the regulated in both groups of non-vaccinated fish were significantly apoptotic induction after viral challenge in pMCV1.4-G860 down-regulated in vaccinated individuals, especially E3 ubiquitin- vaccinated turbot was totally different. Thus, the existence of a protein ligase NEURL3 and Protein neuralized. The opposite specific immune response seems to reduce the viral transcription to response of these genes in non-immunized and immunized turbot a level that practically avoids the activation of the apoptotic after VHSV infection is an interesting point for further investiga- mechanisms. Indeed, the caspases analyzed in the microarray were tions. not affected in pMCV1.4-G860 vaccinated fish after VHSV Regarding the genes encoding the different subunits of the challenge at any of the sampling points. Significant but slight proteasome complex (Fig. 9B), the injection of pMCV1.4-G860 up-regulation in the expression of some specific apoptosis genes induced the transcription of multitude of them. More evident was was only detected at 24 h (Apoptosis regulator BAX, Apoptosis the up-regulation of all the analyzed genes after viral challenge in regulator BAX membrane isoform alpha, Apoptosis-associated both groups of non-vaccinated individuals, whereas vaccinated fish speck-like protein containing a CARD). showed only a modest up-regulation of some of these subunits at On the other hand, cytotoxic T lymphocytes (CTLs) and 24 h after VHSV challenge. natural killer (NK) cells are also able to induce cell death through As a consequence of the activation of ubiquitin and proteasome- the Perforin/Granzyme-induced apoptosis, in which Perforin and related genes, it was expected that pMCV1.4-G860 administration Granulysin (or NK-lysin) generate membrane disruption of virally and VHSV infection would have induced the up-regulation of the infected cells and a family of structurally related serine proteases main genes implicated in the MHC-I antigen presentation (Granzymes) induces apoptosis of the target cell activating the (Fig. 9C). pMCV1.4-G860 vaccination and VHSV infection up- caspases [51]. As it is shown in Fig. 8B, at 72 h after pMCV1.4- regulated the expression of MHC-I and related genes, although G860 injection the levels of Granzyme A (FC = 7.6), Perforin-1 some down-regulations were also observed in genes implicated also (FC = 3.1) and Antimicrobial peptide NK-lysin (FC = 1.8) were in other biological functions (e.g. Platelet glycoprotein 4). On the significantly up-regulated, indicating the activation of the cytotoxic other hand, turbot previously vaccinated with pMCV1.4-G860 cells after viral G glycoprotein expression. As expected, these genes presented only a modest induction of some of the above mediating the cytotoxic response were also overexpressed after mentioned genes after viral challenge probably as consequence VHSV administration but, once again, the pattern was very of the existence of an adaptive immune response limiting the different in previously vaccinated fish (pMCV1.4-G860– VHSV), proliferation success of the virus. where this effect was practically voided. Coagulation, platelet-related proteins and complement Activated cytotoxic cells induce apoptosis in virally infected cells cascade. The complement system and coagulation are two after the recognition of MHC-I-presented peptides as foreign [52]. closely related pathways belonging to a complex inflammatory

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Figure 9. Heat map displaying hierarchical clustering results from microarray expression data of genes involved in the MHC-I antigen presentation process. (A) Enzymes involved in the ubiquitination of target proteins. (B) Subunits belonging to the proteasome complex. (C) MHC class I related-proteins and molecules implicated in the antigen-MHC class I assembly. All the experimental groups, including the corresponding controls (PBS 8 h and PBS – MEM 8 h), were included in the analysis. Different genes are represented in different rows, and different experiments in different columns. Raw expression values are represented as a color scale from red for lower expressions to green for higher expressions. doi:10.1371/journal.pone.0104509.g009 network and showing an intense interaction between them [56]. Some coagulation-related genes were significantly modulated at Pro-inflammatory cytokines play a central role in the coagulation 3 days after pMCV1.4-G860 vaccine administration (Fig. 10A). and fibrinolysis pathways and, in an inverse way, the activation of Thus, the most up-regulated genes were Heparanase, Tetranectin- the coagulation system may affect the inflammatory responses like protein, Platelet basic protein, Vitamin K-dependent protein S [57]. and Myelin-associated protein. On the other hand, significant down-regulations were detected for example in Alpha-actinin-2,

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Figure 10. Heat map displaying hierarchical clustering results from microarray expression data of genes belonging to the (A) coagulation or (B) complement cascades. All the experimental groups, including the corresponding controls (PBS 8 h and PBS – MEM 8 h), were included in the analysis. Different genes are represented in different rows, and different experiments in different columns. Raw expression values are represented as a color scale from red for lower expressions to green for higher expressions. doi:10.1371/journal.pone.0104509.g010

Thrombospondin-1, Thombospondin-2, EGF-containing fibulin- subcomponent subunit C was observed. However, this transcript like extracellular matrix protein 2 and von Willebrand factor appeared slightly up-regulated in pMCV1.4-G860– VHSV turbot among others. Both coagulant and anticoagulant genes were up at 8, 24 and 72 h after infection. C1 complex is part of the classical and down-regulated after pMCV1.4-G860 vaccine injection and, pathway of the complement, which implicates the participation of therefore, it is difficult to establish a general pattern with regard to antibodies. The presence of specific antibodies against VHSV in this process. pMCV1.4-G860 vaccinated fish could be affecting the expression After VHSV infection an intense regulation of these genes was of Complement C1q subcomponent subunit C and therefore, also observed. Thus, some anticoagulatory and procoagulatory favouring the classical pathway of the complement system. genes were tightly up-regulated but others were down-regulated. Markers of activation and proliferation of immune- Viral Haemorrhagic Septicaemia (VHS) is a viral disease causing relevant cell types. The last hierarchical analysis was con- widespread haemorrhages (bleeding) in fish tissues, including ducted for determining the modulation of genes implicated in the internal organs. It is well known that tissue injury induces an maturation, proliferation or activation of different cell lineages. inflammatory response and the systemic inflammation is a potent Some membrane markers and two sequences with homology to prothombotic stimulus, up-regulating procoagulant factors, down- macrophage colony-stimulating factors were selected and their regulating natural anticoagulants and inhibiting fibrinolytic pattern of relative expression obtained (Fig. 11). Three days after activity [58]. Although the role of inflammation is to resolve vaccination down-regulations were observed for the gene corre- infection and injury, excessive or altered inflammation often leads sponding to the CD81 protein (FC = 23.6) and the Macrophage to a wide range of tissue injuries and diseases, such as multi-organ mannose receptor 1 (FC = 22.5). CD81 is a tetraspanin cell failure [59]. Therefore, the equilibrium between pro-inflammatory surface protein showing a broad expression on numerous immune and anti-inflammatory molecules is essential for the host survival. cells and it is known to play an important role in multiple cellular This could be the explanation for the alternative modulation of interactions [61]. Chang et al. [62] found that interferon-alpha these coagulation-related proteins during VHSV infection. As treatment is able to suppress the CD81 expression, possibly occurs with the previously analyzed groups of immune-related through the activity of the double-stranded RNA activated kinase; proteins, the VHSV infection of pMCV1.4-G860 vaccinated turbot our results revealed a significant up-regulation of this ISG after showed a more moderate modulation of these genes. The presence pMCV1.4-G860 vaccination in turbot. The other down-regulated of specific immunity would lead to a decrease of tissue injury and gene, Macrophage mannose receptor 1, is a cell surface inflammation through the reduction of the number of viral transmembrane glycoprotein expressed on macrophages that particles in the fish. serves as a phagocytic receptor mediating the binding and As mentioned above, the complement system is a cascade ingestion microorganisms with a mannose-rich surface [63]. closely related with the coagulation, with numerous interactions Harris et al. [64] observed that IFN-c administration decreased- between both pathways. Indeed, thrombin acts as a potent C3 and to-absent the cell-surface mannose receptor transcription in C5 convertase, leading the generation of the anaphylotoxins C3a murine macrophages. There are not sequences homologues to and C5a [56]. The central component of the complement system IFN-c in the turbot microarray and therefore, a negative is the component C3, which is proteolytically activated through correlation between the two proteins cannot be established. the classical, lectins and alternative routes [60], as well as by the However, Purcell et al. [14] found a high up-regulation of IFN- coagulation system. As it is observed in Fig. 10B, the administra- c in rainbow trout in the site of injection (muscle) 7 days after tion of the plasmid (both pMCV1.4 and pMCV1.4-G860) up- vaccination against the IHNV rhabdovirus after injecting a DNA regulated some complement components, especially at 24 h (for vaccine encoding its G glycoprotein. CD209 antigen-like protein instance, Complement component C8 gamma, CD59 glycopro- E, CD83 and CD9 were significantly up-regulated after vaccina- tein, Mannan-binding lectin serine protease 2, Complement tion. These three clusters of differentiation were also induced in component C9, Complement C2, Complement factor B and rainbow trout one week after injection of a similar DNA vaccine Complement C4). In contrast, Complement receptor type 1, C3a from IHNV [14]. CD209 antigen-like protein E and CD83 are anaphylatoxin chemotactic receptor and C5a anaphylatoxin mainly expressed in dendritic cells and these up-regulations could chemotactic receptor were down-regulated 24 h after any plasmid be indicating a proliferation and activation of this cell type, which injection. Therefore, the plasmid DNA backbone was able to forms a system of professional antigen-presenting cells. induce an immune response at the complement level. The VHSV infection especially induced the up-regulation of the modulation in the expression of these genes could be related to gene encoding the CD83 protein, indicating a possible activation/ the differences observed between both groups of non-vaccinated proliferation of dendritic cells. Interestingly, this dendritic cells fish after viral challenge (higher number of affected genes at 8 h marker was also found to be induced on monocytes [65] and post-infection, lower transcription of viral genes and reduction and polymorphonuclear neutrophils [66] under the influence of delay in the mortality of fish previously injected with pMCV1.4). specific cytokines, such as TNF-a, acquiring characteristics of In addition, pMCV1.4-G860 vaccination up-regulated the expres- dendritic cells. Slight up-regulations were also observed for the sion of Complement C4 and Complement C4-A, and down- CD166 antigen homolog, CD9 as well as for CD8 alpha chain and regulated the level of C5a anaphylatoxin chemotactic receptor at CD8 beta, reflecting a putative activation of CD8+ T lymphocytes 72 h. (cytotoxic cells). However, weak down-regulations were also On the other hand, VHSV challenge increased the expression obtained for several T-cell receptor (TCR) chain regions (fold- of the majority of the analyzed genes but, interestingly, a changes between 21.5 and 22 in both non-vaccinated groups). B- significant reduction in the transcription of Complement C1q cell receptor complex-associated protein alpha chain and B-cell

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Figure 11. Heat map displaying hierarchical clustering results from microarray expression data of gene markers of activation and proliferation of cell types. All the experimental groups, including the corresponding controls (PBS 8 h and PBS – MEM 8 h), were included in the analysis. Different genes are represented in different rows, and different experiments in different columns. Raw expression values are representedasa color scale from red for lower expressions to green for higher expressions. doi:10.1371/journal.pone.0104509.g011 receptor complex-associated protein beta chain (BCR chains) were stimulating factor, a reduction in the macrophage proliferation also down-modulated with a fold-change around 22. But the or differentiation could be happening. An alternative explanation more surprising down-regulations were observed for some would be a massive infection and destruction of these cells by the molecules with an exclusive/closely relation with macrophages. virus. It has been shown that VHSV is able to infect turbot blood Thus, the Macrophage receptor MARCO and the Macrophage leukocytes and kidney macrophages [67]. However, further mannose receptor 1, two phagocyte receptor molecules serving as investigations have reported that macrophages are heterogeneous a pattern-recognition receptor for bacterial components, were in their permissiveness for viral penetration and usually only a highly down-modulated by VHSV administration especially after subpopulation is infected [67–70]. Therefore, the drastic effect 72 h. In PBS - VHSV fish, MARCO showed a FC = 246 and induced by VHSV in the expression of macrophage-related Macrophage mannose receptor 1 a FC = 210.2, whereas in proteins is still poorly understood and more studies need to be pMCV 1.4 - VHSV turbot showed FC = 210.2 and 25.7, performed for understanding if VHSV massively replicates in this respectively. Taking this information into consideration, together cell type. It is also interesting to highlight that Interleukin-18 (IL- with the strong down-regulation of Macrophage colony-stimulat- 18) was down-regulated 72 h after VHSV challenge. IL-18 is a ing factor 1 and Precursor of second macrophage colony- potent pro-inflammatory cytokine essential to host defenses against

PLOS ONE | www.plosone.org 17 August 2014 | Volume 9 | Issue 8 | e104509 Transcriptome after VHSV Infection or Vaccination severe infections, taking part in the clearance of viruses [71]. head kidney at 8, 24 and 72 h after pMCV1.4 or Macrophages are the main IL-18 producers in response to stimuli pMCV1.4-G860 injection. of viral/bacterial origin [72] and the reduction in the mRNA level (PDF) of this cytokine after VHSV challenge was probably related with Figure S2 Venn diagrams reflecting the number of the inhibition of the macrophage markers described above. Little changes in the expression of the selected proteins were exclusive and common up- and down-regulated genes observed in vaccinated turbot after infection, but a slight tendency after pMCV1.4 and pMCV1.4-G860 administration. toward TCR sequences up-regulation was observed. Purcell et al. (PDF) [14] also suggested an enhanced T cell activation or proliferation Figure S3 Biological process multilevel pie chart re- serving as mechanism of protection in Japanese flounder flecting exclusive and common genes at 72 h after immunized using a DNA vaccine encoding the HIRRV G gene pMCV1.4 or pMCV1.4-G860 injection. when fish were infected with the virus. (PDF) nd Conclusions Figure S4 Gene Ontology (GO) assignment (2 level biological process terms) of sequences modulated in In summary, we have analyzed the importance of different head kidney at 8, 24 and 72 h after VHSV infection in immune processes implicated in the protection provided by vaccinated (pMCV1.4-G860 - VHSV) and non-vaccinated pMCV1.4-G860 in turbot before and after VHSV infection. This (PBS – VHSV and pMCV1.4– VHSV) turbot. work represents the most exhaustive transcriptomic study about (PDF) DNA vaccines against rhabdoviral pathogens in fish. The high- throughput screening provided will serve as a basis for a better Figure S5 Venn diagrams reflecting the number of understanding of the molecular processes implicated in the exclusive and common up- and down-regulated genes successful vaccination protocols against viral diseases in fish. The in PBS – VHSV, pMCV 1.4 - VHSV and pMCV1.4-G860– VHSV groups. plasmid pMCV1.4-G860 induces a powerful immune response at 3 days after vaccination affecting the main immune processes and (PDF) leading to an efficient antigen presentation and production of Table S1 List of primers used for qPCR validation of specific antibodies. After VHSV challenge, non-vaccinated fish the microarray data. revealed an uncontrolled immune response generating an intense (DOCX) pro-inflammatory status in the host. In contrast, vaccinated fish showed a moderate and controlled response due to the previous Table S2 Microarray and qPCR values obtained for the presence of specific immune factors. The analyses performed in 5 selected immune-related genes in the microarray this work provide interesting information about molecules with a validation. potential use as vaccine adjuvants, antiviral treatments or markers (DOCX) for vaccine efficiency monitoring as well. Moreover, some clues about the infectivity mechanisms of VHSV in fish are also Author Contributions proposed. Conceived and designed the experiments: AF BN AE JC. Performed the experiments: PP SB SM. Analyzed the data: PP SB SM SD BN AF. Supporting Information Contributed reagents/materials/analysis tools: SB SM AE JC AF BN. nd Contributed to the writing of the manuscript: PP SD SB JC AE SM BN Figure S1 Gene Ontology (GO) assignment (2 level AF. biological process terms) of sequences modulated in

References 1. Wolf K (1988) Viral hemorrhagic septicemia. In: Fish Viruses and Fish Viral 10. de Kinkelin P, Be´arzotti M, Castric J, Nougayre`de P, Lecocq-Xhonneux F, et al. Diseases, Ithaca, New York: Cornell University Press. pp. 217–249. (1995) Eighteen years of vaccination against viral haemorrhagic septicaemia in 2. Castric J, de Kinkelin P (1980) Occurrence of viral haemorrhagic septicaemia in France. Vet Res 26: 379–387. rainbow trout Salmo gairdneri Richardson reared in sea water. J Fish Dis 3: 21– 11. Adelmann M, Ko¨llner BK, Bergmann SM, Fischer U, Lange B, et al. (2008) 27. Development of an oral vaccine for immunisation of rainbow trout 3. Hørlyck V, Mellerga˚rd S, Dalsgaard I, Jørgensen PEV (1984) Occurrence of (Oncorhynchus mykiss) against viral haemorrhagic septicaemia. Vaccine 26: VHSV in Danish maricultured rainbowtrout. Bull Eur Fish Pathol 4: 11–13. 837–844. 4. Schlotfeldt HJ, Ahne W, Vesterga˚rd-Jørgensen PE, Glende W (1991) 12. Lorenzen N, Olesen NJ, Jorgensen PEV (1990) Neutralization of Egtved virus Occurrence of viral haemorrhagic septicaemia virus in turbot (Scophthalmus pathogenicity to cell cultures and fish by monoclonal antibodies to the viral G maximus) - a natural outbreak. Bull Eur Assoc Fish Pathol 11: 105–107. protein. J Gen Virol 71: 561–567. 5. Ross K, McCarthy U, Huntly PJ, Wood BP, Stuart D, et al. (1994) An outbreak 13. Pereiro P, Martinez-Lopez A, Falco A, Dios S, Figueras A, et al. (2012) of viral haemorrhagic septicemia (VHS) in turbot (Scophthalmus maximus)in Protection and antibody response induced by intramuscular DNA vaccine Scotland. Bull Eur Assoc Fish Pathol 14: 213–214. encoding for viral haemorrhagic septicaemia virus (VHSV) G glycoprotein in turbot ( ). Fish Shellfish Immunol 32: 1088–1094. 6. de Kinkelin, P., Bearzotti-Le Berre, M Bernard, J. (1980) Viral hemorrhagic Scophthalmus maximus 14. Purcell MK, Nichols KM, Winton JR, Kurath G, Thorgaard GH, et al. (2006) septicemia of rainbow trout: selection of a thermoresistant virus variant and Comprehensive gene expression profiling following DNA vaccination of rainbow comparison of polypeptide synthesis with the wild-type virus strain. J Virol 36: trout against infectious hematopoietic necrosis virus. Mol Immunol 43: 2089– 652–658. 2106. 7. Bernard J, de Kinkelin P, Bearzotti-Le Berre M (1983) Viral hemorrhagic 15. Byon JY, Ohira T, Hirono I, Aoki T (2005) Use of a cDNA microarray to study septicemia of rainbow trout: relation between the G polypeptide and antibody immunity against viral hemorrhagic septicemia (VHS) in Japanese flounder production in protection of the fish after infection with the F25 attenuated (Paralichthys olivaceus) following DNA vaccination. Fish Shellfish Immunol 18: variant. Infect Immun 39: 7–14. 135–147. 8. Leong JC, Fryer JL (1993) Viral vaccines for aquaculture. Annu Rev Fish Dis 3: 16. Byon JY, Ohira T, Hirono I, Aoki T (2006) Comparative immune responses in 225–240. Japanese flounder, Paralichthys olivaceus after vaccination with viral hemor- 9. Lecocq-Xhonneux F, Thiry M, Dheur I, Rossius M, Vanderheijden N, et al. rhagic septicemia virus (VHSV) recombinant glycoprotein and DNA vaccine (1994) A recombinant viral haemorrhagic septicaemia virus glycoprotein using a microarray analysis. Vaccine 24: 921–930. expressed in insect cells induces protective immunity in rainbow trout. J Gen 17. Yasuike M, Kondo H, Hirono I, Aoki T (2007) Difference in Japanese flounder, Virol 75: 1579–1587. Paralichthys olivaceus gene expression profile following hirame rhabdovirus

PLOS ONE | www.plosone.org 18 August 2014 | Volume 9 | Issue 8 | e104509 Transcriptome after VHSV Infection or Vaccination

(HIRRV) G and N protein DNA vaccination. Fish Shellfish Immunol 23: 531– 45. Shi Z, Cai Z, Sanchez A, Zhang T, Wen S, et al. (2011) A novel Toll-like 541. receptor that recognizes vesicular stomatitis virus. J Biol Chem 286: 4517–4524. 18. Yasuike M, Kondo H, Hirono I, Aoki T (2011) Gene expression profile of 46. Goodbourn S, Didcock L, Randall RE (2000) Interferons: cell signaling, immune HIRRV G and N protein gene vaccinated Japanese flounder, Paralichthys modulation, antiviral responses and virus countermeasures. J Gen Virol 81: olivaceus during HIRRV infection. Comp Immunol Microbiol Infect Dis. 34: 2341–2364. 103–110. 47. Zou J, Secombes CJ (2011) Teleost fish interferons and their role in immunity. 19. Pereiro P, Balseiro P, Romero A, Dios S, Forn-Cuni G, et al. (2012) High- Dev Comp Immunol 35: 1376–1387. throughput sequence analysis of turbot (Scophthalmus maximus) transcriptome 48. Pereiro P, Costa MM, Dı´az-Rosales P, Dios S, Figueras A, et al. (2014) The first using 454-pyrosequencing for the discovery of antiviral immune genes. PLoS characterization of two type I interferons in turbot (Scophthalmus maximus) ONE 7(5): e35369. reveals their differential role, expression pattern and gene induction. Dev Comp 20. Reed LJ, Muench H (1938) Simple method of estimating fifty per cent Immunol 45: 233–244. endpoints. Am J Hyg 27: 493–497. 49. Fensterl V, Sen GC (2011) The ISG56/IFIT1 gene family. J Interferon 21. Conesa A, Go¨tz S, Garcı´a-Go´mez JM, Terol J, Talon M, et al. (2005) Blast2GO: Cytokine Res 31: 71–78. a universal tool for annotation, visualization and analysis in functional genomics 50. Barber GN (2001) Host defense, viruses and apoptosis. Cell Death Differ 8: 113– research. Bioinformatics 21: 3674–3676. 126. 22. Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for 51. Trapani JA, Smyth MJ (2002) Functional significance of the perforin/granzyme biologist programmers. MIMB 132: 365–386. cell death pathway. Nat Rev Immunol 2: 735–747. 23. Pfaffl MW (2001) A new mathematical model for relative quantification in real- 52. Røder G, Geironson L, Bressendorff I, Paulsson K (2008) Viral proteins time RT-PCR. Nucleic Acids Res 29: e45. interfering with antigen presentation target the major histocompatibility 24. Morey JS, Ryan JC, Van Dolah FM (2006) Microarray validation: factors complex class I peptide-loading complex. J Virol 82: 8246–8252. influencing correlation between oligonucleotide microarrays and real-time PCR. Biol Proced Online 8: 175–193. 53. Brode S, Macary PA (2004) Cross-presentation: dendritic cells and macrophages 25. Lorenzen N, LaPatra SE (2005) DNA vaccines for aquacultured fish. Rev sci tech bite off more than they can chew! Immunology 112: 345–351. Off int Epi 24: 201–213. 54. Lecker SH, Goldberg AL, Mitch WE (2006) Protein degradation by the 26. Krieg AM, Yi AK, Schorr J, Davis HL (1998) The role of CpG dinucleotides in ubiquitin-proteasome pathway in normal and disease states. J Am Soc Nephrol DNA vaccines. Trends Microbiol 6: 23–27. 17: 1807–1819. 27. Manders P, Thomas R (2000) Immunology of DNA vaccines: CpG motifs and 55. Hewitt EW (2003) The MHC class I antigen presentation pathway: strategies for antigen presentation. Trends Microbiol49: 199–205. viral immune evasion. Immunology 110: 163–169. 28. Martinez-Alonso S, Martinez-Lopez A, Estepa A, Cuesta A, Tafalla C (2011) 56. Amara U, Flierl MA, Rittirsch D, Klos A, Chen H, et al. (2010) Molecular The introduction of multi-copy CpG motifs into an antiviral DNA vaccine intercommunication between the complement and coagulation system. J Im- strongly up-regulated its immunogenicity in fish. Vaccine 29: 1289–1296. munol 185: 5628–36. 29. Klinman DM, Currie D, Gursel I, Verthelyi D (2004) Use of CpG 57. Levi M, Keller TT, van Gorp E, ten Cate H (2003) Infection and inflammation oligodeoxynucleotides as immune adjuvants. Immunol Rev 199: 201–216. and the coagulation system. Cardiovasc Res 60: 26–39. 30. Ritter GMG, Mun˜oz JE, Marques AF, Nosanchuck JD, Taborda CP, et al. 58. Esmon CT (2003) Inflammation and thrombosis. J Thromb Haemost 1: 1343– (2012) Therapeutic DNA vaccine encoding peptide P10 against experimental 1348. paracoccidioidomycosis. PLoS Negl Trop Dis 6: e1519. 59. Glaros T, Larsen M, Li L (2009) Macrophages and fibroblast during 31. Ha SJ, Jeon BY, Kim SC, Kim DJ, Song MK, et al. (2003) Therapeutic effect of inflammation, tissue damage and organ injury. Front Biosci 14: 3988–3993. DNA vaccines combined with chemotherapy in a latent infection model after 60. Carroll MC (2004) The complement system in regulation of adaptive immunity. aerosol infection of mice with Mycobacterium tuberculosis. Gene Ther 10: 1592– Nat Immunol 5: 981–986. 1599. 61. Luo RF. Zhao S, Tibshirani R, Myklebust JH, Sanyal M, et al. (2010) CD81 32. Jørgensen JB, Johansen LH, Steiro K, Johansen A (2003) CpG DNA induces protein is expressed at high levels in normal germinal center B cells and in protective antiviral immune responses in Atlantic salmon (Salmo salar L.).J Virol subtypes of human lymphomas. Hum Pathol 41: 271–280. 77: 11471–11479. 62. Chang LL, Cheng PN, Chen JS, Young KC (2007) CD81 down-regulation on B 33. Kim CH, Johnson MC, Drennan JD, Simon BE, Thomann E, et al. (2000) DNA cells is associated with the response to interferon-alpha-based treatment for vaccines encoding viral glycoproteins induce nonspecific immunity and Mx chronic hepatitis C virus infection. Antiviral Res 75: 43–51. protein synthesis in fish. J Virol 74: 7048–7054. 63. Stein M, Keshav S, Harris N, Gordon S (1992) Interleukin 4 potently enhances 34. LaPatra SE, Corbeil S, Jones GR, Shewmaker WD, Lorenzen N, et al. (2001) murine macrophage mannose receptor activity: a marker of alternative Protection of rainbow trout against infectious hematopoietic necrosis virus four immunologic macrophage activation. J Exp Med 176: 287–292. days after specific or semi-specific DNA vaccination. Vaccine 19: 4011–4019. 64. Harris N, Super M, Rits M, Chang G, Ezekowitz RA (1992) Characterization of 35. Sommerset I, Lorenzen E, Lorenzen N, Bleie H, Nerland AH (2003) A DNA the murine macrophage mannose receptor: demonstration that the downreg- vaccine directed against a rainbow trout rhabdovirus induces early protection ulation of receptor expression mediated by interferon-gamma occurs at the level against a nodavirus challenge in turbot. Vaccine 21: 4661–4667. of transcription. Blood 80: 2363–2373. 36. Lorenzen E, Lorenzen N, Einer-Jensen K, Brudeseth B, Evensen O (2005) Time 65. Zhou LJ, Tedder TF (1996) CD14+ blood monocytes can differentiate into course study of in situ expression of antigens following DNA-vaccination against functionally mature CD83+ dendritic cells. Proc Natl Acad Sci USA 93: 2588– VHS in rainbow trout (Oncorhynchus mykiss Walbaum) fry. Fish Shellfish 2592. Immunol 19: 27–41. 66. Iking-Konert C, Wagner C, Denefleh B, Hug F, Schneider M, et al. (2002) Up- 37. Mogensen TH (2009) Pathogen recognition and inflammatory signaling in regulation of the dendritic cell marker CD83 on polymorphonuclear neutrophils innate immune defenses. Clin Microbiol Rev 22: 240–273. (PMN): divergent expression in acute bacterial infections and chronic 38. Kawai T, Akira S (2010) The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol 11: 373–384. inflammatory disease. Clin Exp Immunol 130: 501–508. 39. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, et al. (2006) 67. Tafalla C, Figueras A, Novoa B (1998) In vitro interaction of viral haemorrhagic Differential roles of MDA5 and RIG-I helicases in the recognition of RNA septicaemia virus and leukocytes from trout (Oncorhynchus mykiss) and turbot viruses. Nature 441: 101–105. (Scophthalmus maximus). Vet Immunol Immunopathol 62: 359–366. 40. Uematsu S, Akira S (2007) Toll-like receptors and type I interferons. J Biol 68. Morahan PS, Connor JR, Leary KR (1985) Viruses and the versatile Chem 282: 15319–15323. macrophage. Br Med Bull 41: 15–21. 41. Honda K, Taniguchi T (2006) IRFs: master regulators of signalling by Toll-like 69. Wu L, Morahan PS, Leary K (1990) Mechanisms of intrinsic macrophage-virus receptors and cytosolic pattern-recognition receptors. Nat Rev Immunol 6: 644– interactions in vitro. Microbiol Pathol 9: 293–301. 658. 70. Dakhama A, Kaan PM, Hegele RG (1998) Permissiveness of guinea pig alveolar 42. Xagorari A, Chlichlia K (2008) Toll-like receptors and viruses: induction of macrophage subpopulations to acute respiratory syncytial virus infection innate antiviral immune responses. Open Microbiol J 2: 49–59. in vitro. Chest 114: 1681–1608. 43. Aliprantis AO, Yang RB, Weiss DS, Godowski P, Zychlinsky A (2000) The 71. Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H (2001) Interleukin-18 is a apoptotic signaling pathway activated by Toll-like receptor-2. EMBO J 19: unique cytokine that stimulates both Th1 and Th2 responses depending on its 3325–3336. cytokine milieu. Cytokine Growth Factor Rev 12: 53–72. 44. Hidmark A, von Saint Paul A, Dalpke AH (2012) Cutting edge: TLR13 is a 72. Boraschi D, Dinarello CA (2006) IL-18 in autoimmunity: review. Eur Cytokine receptor for bacterial RNA. J Immunol 189: 2717–2721. Netw 17: 224–252.

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Developmental and Comparative Immunology 45 (2014) 233–244

Contents lists available at ScienceDirect

Developmental and Comparative Immunology

journal homepage: www.elsevier.com/locate/dci

The first characterization of two type I interferons in turbot (Scophthalmus maximus) reveals their differential role, expression pattern and gene induction ⇑ P. Pereiro, M.M. Costa, P. Díaz-Rosales, S. Dios, A. Figueras, B. Novoa

Instituto de Investigaciones Marinas (IIM), CSIC, Eduardo Cabello 6, 36208 Vigo, Spain article info abstract

Article history: Type I interferons (IFNs) are considered the main cytokines directing the antiviral immune response in Received 20 January 2014 vertebrates. These molecules are able to induce the transcription of interferon-stimulated genes (ISGs) Revised 10 March 2014 which, using different blocking mechanisms, reduce the viral proliferation in the host. In addition, a con- Accepted 12 March 2014 tradictory role of these IFNs in the protection against bacterial challenges using murine models has been Available online 27 March 2014 observed, increasing the survival or having a detrimental effect depending on the bacteria species. In tel- eosts, a variable number of type I IFNs has been described with different expression patterns, protective Keywords: capabilities or gene induction profiles even for the different IFNs belonging to the same species. In this Turbot work, two type I IFNs (ifn1 and ifn2) have been characterized for the first time in turbot (Scophthalmus Type I interferon Antiviral maximus), showing different properties. Whereas Ifn1 reflected a clear antiviral activity (over-expression ISG of ISGs and protection against viral haemorrhagic septicaemia virus), Ifn2 was not able to induce this Viral haemorrhagic septicaemia virus response, although both transcripts were up-regulated after viral challenge. On the other hand, turbot (VHSV) IFNs did not show any protective effect against the bacteria Aeromonas salmonicida, although they were Aeromonas salmonicida induced after bacterial challenge. Both IFNs induced the expression of several immune genes, but the effect of Ifn2 was mainly limited to the site of administration (intramuscular injection). Interestingly, Ifn2 but not Ifn1 induced an increase in the expression level of interleukin-1 beta (il1b). Therefore, the role of Ifn2 could be more related with the immune regulation, being involved mainly in the inflammation process. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Mammalian type I IFN genes do not contain introns and they all share a common receptor due to their significant structural homol- Interferons (IFNs) are a family of multifunctional cytokines rep- ogy (Platanias, 2005; Qi et al., 2010). On the other hand, type III resenting the first defensive line against viral infections among IFNs contain a gene structure composed of 5 exons and 4 introns, other immune relevant functions. These proteins are produced in but they possess similar antiviral functions to type I IFNs, despite response to different pathogen or pathogen-associated molecular binding to distinct receptors (Qi et al., 2010). Interferon gamma patterns (PAMPs) via the activation of different signaling pathways is a markedly different cytokine than the type I IFNs encoded by (Honda et al., 2005). In mammals, three subfamilies of IFNs were a gene which contains 4 exons and 3 introns (Taya et al., 1982), established in basis to different structural and functional proper- possessing some ability to interfere with viral infections but being ties (type I, type II and type III) (González-Navajas et al., 2012; mainly an immunomodulator (Samuel, 2001). Platanias, 2005). Type I IFN subfamily comprises a broad group of The antiviral activity of type I IFNs is mediated by the interac- typically antiviral proteins, being interferon alpha and interferon tion with the corresponding receptor (in mammals interferon al- beta the most studied. By contrast, type II IFN subfamily includes pha/beta receptor), which induces the activation of the JAK only one cytokine, the interferon gamma, and the third type of IFNs (Janus Activated Kinase)/STAT (Signal Transducer and Activator of is the interferon lambda subfamily, composed of three members, Transcription) signaling pathway and leads to the formation of which have only been characterized in higher vertebrates. the ISGF3 (IFN-Stimulated Gene Factor 3) complex (Samuel, 2001). This complex translocates to the nucleus and binds IFN-stimulated response elements (ISREs) in DNA to initiate the ⇑ Corresponding author. Tel.: +34 986 21 44 63; fax: +34 986 29 27 62. transcription of those genes known as IFN-stimulated genes (ISGs) E-mail address: [email protected] (B. Novoa). (Platanias, 2005). These ISGs (including the PKR kinase, OAS http://dx.doi.org/10.1016/j.dci.2014.03.006 0145-305X/Ó 2014 Elsevier Ltd. All rights reserved. 234 P. Pereiro et al. / Developmental and Comparative Immunology 45 (2014) 233–244 synthetase and RNase L nuclease, the family of Mx protein GTPases et al., unpublished) was performed in order to determine the num- or ISG15, among others) reduce the viral replication and dissemi- ber of exons/introns constituting each turbot IFN. nation through different blocking mechanisms (Sadler and Williams, 2008 and Samuel, 2001). Although typically considered 2.2. Proteins analysis and 3D structure to be antiviral proteins, type I IFNs are also induced by bacterial pathogens via Toll-like receptors or by cytosolic sensors recogniz- The presence of signal peptide was analysed with the SignalP 3.0 ing nucleic acid ligands, bacterial fragments or ligands released by Server (http://www.cbs.dtu.dk/services/signalp-3.0/)(Emanuels- the bacteria (González-Navajas et al., 2012; Monroe et al., 2010). It son et al., 2007) and putative N-glycosylation sites were predicted is interesting to highlight that the effect of the type I IFN results using the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/ contradictory depending of the bacterial type, playing in some NetNGlyc/). Molecular weight and isoelectric point were deter- cases a detrimental role in the host survival but resulting crucial mined using the Compute pI/Mw tool from ExPASy (Gasteiger for host resistance to some bacterial infection (González-Navajas et al., 2003). The potential disulphide bonds between cysteines et al., 2012; Monroe et al., 2010). were analysed using the server DiANNA 1.1 (Ferrè and Clote, The first reports about the cloning of type I IFNs in fish were 2005). The 3D-structures of turbot IFNs were predicted using published in 2003 for zebrafish (Danio rerio)(Altmann et al., I-TASSER server (Roy et al., 2010) selecting the model with the best 2003), Atlantic salmon (Salmo salar)(Robertsen et al., 2003) and C-score and viewed by PyMOL (http://www.pymol.org). The pufferfish (Takifugu rubripes)(Lutfalla et al., 2003) and, to date, Template Modelling Score (TM-score), a measure of structural type I IFNs have been reported in several teleost species (revised similarity between two proteins, was also considered in order to in Zou and Secombes, 2011). Surprisingly, all fish type I IFN genes identify those structural analogs with known crystal architecture contained 5 exons and 4 introns, with a genetic structure identical in the Protein Data Bank (PDB; http://www.rcsb.org/pdb/). to interferon lambda (Robertsen, 2006) but the higher sequence and structure similarity between these fish IFNs and mammalian 2.3. Phylogenetic analysis type I IFNs let us consider them as type I IFNs. Teleosts also possess multiple copies of the IFN genes in the genome, but the copy num- A comparison between both turbot IFNs and several IFN se- ber varies depending on the species (Zou and Secombes, 2011). quences from other fish and vertebrates was conducted using the Moreover, it has been shown that type I IFNs from the same teleost ClustalW server (Thompson et al., 1994). The phylogenetic tree can possess different properties and capabilities, suggesting in was drawn using Mega 6.0 software (Tamura et al., 2013). Neigh- some cases complementary or specialized roles (Aggad et al., bor-Joining algorithm (Saitou and Nei, 1987) was used as cluster- 2009; López-Muñoz et al., 2009; Zou et al., 2007). ing method, the distances matrix was computed using Poisson In the current work, we have characterized for the first time two correction method and partial deletion of the positions containing type I IFN genes in turbot, named ifn1 and ifn2. Due to the impor- alignment gaps and missing data was conducted. Statistical confi- tance of these genes as key modulators in the protection against dence of the inferred phylogenetic relationships was assessed by viral diseases and to the fact that turbot is a very valuable commer- performing 10,000 bootstrap replicates. Sequence similarity and cial species in Europe and Asia, the knowledge about the capabili- identity scores were calculated with the software MatGAT (Campa- ties and properties of each IFN is a very interesting issue. These nella et al., 2003) using the BLOSUM62 matrix. The GenBank acces- molecules could serve as antiviral therapeutic treatments or vac- sion numbers of the sequences used in this section are listed in cine adjuvants and therefore, the main purpose of this work was Supplementary data Table 2. trying to know the main immune properties of each turbot IFN. To get insights into their functions, we analysed their constitutive 2.4. Fish expression and gene modulation after viral and bacterial chal- lenges. Moreover, we tested the bioactivities of each IFN measuring Juvenile turbot (average weight 2.5 g) were obtained from a the induction of the immune gene expression profiles and the pro- commercial fish farm (Insuiña S.L., Galicia, Spain). Animals were tection capabilities against infection. The obtained results suggest maintained in 500 L fibreglass tanks with a re-circulating saline differential and non-redundant roles for both turbot IFNs. water system (total salinity about 35 g/L) with a light-dark cy- cle of 12:12 h at 18 °C and fed daily with a commercial dry diet (LARVIVA-BioMar). Prior to experiments, fish were acclimatized 2. Material and methods to laboratory conditions for 2 weeks. Fish care and challenge experiments were reviewed and approved by the CSIC National 2.1. Characterization of the turbot type I interferons Committee on Bioethics under approval number (07_09032012).

Two partial sequences annotated as ‘‘Interferon phi 2’’ and 2.5. Constitutive expression of turbot ifn1 and ifn2 ‘‘Interferon alpha 2 precursor’’ were obtained after a 454-pyrose- quencing of several turbot tissues following the treatment with Eight different tissues (kidney, spleen, gill, liver, intestine, heart, different viral stimuli (Pereiro et al., 2012). The contig annotated brain and muscle plus skin) were removed from 12 healthy fish as ‘‘Interferon phi 2’’ was named ifn1 whereas the singleton with after they were sacrificed via MS-222 overdose (500 mg L1) in or- homology to ‘‘Interferon alpha 2 precursor’’ was named ifn2. The der to examine the constitutive expression of both IFNs. Equal full-length cDNA of ifn1 and ifn2 was determined by means of amounts of the same tissue from four fish were pooled, obtaining the RACE technique (Rapid Amplification of cDNA Ends). The com- 3 biological replicates for each tissue (4 turbot/replicate) that were plete open reading frame (ORF) was confirmed by PCR using spe- processed for the analysis of gene expression (see below, cific primers and subsequent linking into pCR™2.1-TOPOÒ vector Section 2.9). (Invitrogen) for their cloning using One ShotÒ TOP10F0 competent cells (Invitrogen) following the protocol instructions. cDNA 2.6. Induction of turbot IFNs by viral haemorrhagic septicaemia virus sequencing was conducted using an automated ABI 3730 DNA Ana- (VHSV) or Aeromonas salmonicida subsp. salmonicida challenge lyzer (Applied Biosystems, Inc. Foster City, CA, USA). The primers used for RACE and ORF confirmation are listed in Supplementary A number of 144 turbot were divided into 4 groups, composed data Table 1. A local blast against the turbot genome draft (Figueras of 36 fish/each. Fish belonging to one group were intraperitoneally P. Pereiro et al. / Developmental and Comparative Immunology 45 (2014) 233–244 235

(i.p.) injected with 50 ll of a VHSV (strain UK-860/94) suspension 2.9. RNA extraction, cDNA synthesis and real-time quantitative PCR 5 (5 10 TCID50/fish), whereas other group was inoculated with analysis 50 llofanA. salmonicida subsp. salmonicida (strain VT 45.1 WT) suspension (5.5 105 CFU/fish). The other two groups were in- Total RNA from the different tissue samples was extracted using jected with 50 ll of viral medium (Eagle’s minimum essential the MaxwellÒ 16 LEV simplyRNA Tissue kit (Promega), including a medium supplemented with 2% fetal bovine serum, penicillin DNase treatment step for removing potential genomic DNA con- and streptomycin) or with 1 phosphate buffered saline (PBS tamination, with the automated MaxwellÒ 16 Instrument in accor- 1) and they served as the corresponding control groups for the dance with instructions provided by the manufacturer. The cDNA viral and bacterial challenges. For analysing the induction of synthesis was performed with the SuperScript II Reverse Transcrip- expression of ifn1 and ifn2 after viral or bacterial stimuli, head kid- tase (Invitrogen) using 0.5 lg of RNA and following the manufac- ney from 12 individuals belonging to each group was removed at turer indications. different sampling points (8, 24 and 72 h). For each sampling point The expression profiles of the immune genes ifn1, ifn2, myxovi- and treatment, equal amounts of each tissue from three turbot rus resistance protein (mx), interferon-induced 56 kDa protein (ifi56), were pooled, constituting 4 biological replicates (3 fish/replicate) interferon-stimulated 15 kDa protein (isg15), interferon regulatory that were processed for the analysis of gene expression (see below, factor 1 (irf1), caspase 7 (casp7), interleukin-1 beta (il1b), interleukin Section 2.9). 8 (il8), major histocompatibility complex class I (mhc1) and major his- tocompatibility complex class II (mhc2), as well as the quantification 2.7. Expression constructs encoding turbot IFNs of the VHSV glycoprotein or A. salmonicida in the different samples, were determined using real-time quantitative PCR (qPCR). Specific The expression plasmids pMCV1.4-ifn1 and pMCV1.4-ifn2 were qPCR primers were designed using the Primer3 program (Rozen synthesized by ShineGene Molecular Biotech, Inc. (Shanghai, and Skaletsky, 2000) with the exception of the oligonucleotides China) using the pMCV1.4 plasmid (Ready-Vector, Madrid, Spain) used in the A. salmonicida specific detection, which were designed containing the cytomegalovirus (CMV) promoter and using the in basis to the publication of Balcázar et al. (2007) but including nucleotide sequences encoding the IFNs mature peptides. Recom- some modifications. Their amplification efficiency was calculated binant or empty plasmids were obtained by transforming One using seven serial five-fold dilutions of head kidney cDNA from Ò 0 Shot TOP10F competent cells (Invitrogen) and the purification unstimulated turbot with the Threshold Cycle (CT) slope method was conducted using the PureLink™ HiPure Plasmid Midiprep Kit (Pfaffl, 2001). The identity of the amplicons was confirmed by (Invitrogen). sequencing using the same procedure described in Section 2.1. Pri- mer sequences are listed in Supplementary data Table 1. Individual 2.8. Analysis of the IFNs protective effect against VHSV or A. real-time PCR reactions were carried out in 25 ll reaction volume salmonicida subsp. salmonicida challenges using 12.5 ll of SYBRÒ GREEN PCR Master Mix (Applied Biosys- tems), 10.5 ll of ultrapure water (Sigma–Aldrich), 0.5 ll of each In order to determine the protective effect induced by turbot specific primer (10 lM) and 1 ll of five-fold diluted cDNA template Ifn1 and Ifn2 against VHSV (strain UK-860/94) infection, 200 fish in MicroAmpÒ optical 96-well reaction plates (Applied Biosys- were subdivided into 10 batches of 20 turbot each. Turbot from tems). All reactions were performed using technical triplicates in two tanks (two replicates per treatment) were then intramuscu- a 7300 Real-Time PCR System thermocycler (Applied Biosystems) larly injected (i.m.) with a volume of 50 ll of one of the following with an initial denaturation (95 °C, 10 min) followed by 40 cycles treatments: 2.5 lg of pMCV1.4-ifn1, 2.5 lg of pMCV1.4-ifn2, 2.5 lg of a denaturation step (95 °C, 15 s) and one hybridization-elonga- of pMCV1.4 (empty plasmid) and PBS 1. After 2 days, the individ- tion step (60 °C, 1 min). No-template controls were also included 5 uals were i.p. injected with a dose of VHSV of 5 10 TCID50/fish. on each plate to detect possible contamination or primer dimers The two remaining groups were first i.m. inoculated with PBS 1 formed during the reaction. An analysis of melting curves was per- and then i.p. with the viral medium and served as an absolute con- formed for each reaction. Relative expression of each gene was nor- trol (non-immunised and non-infected groups). The same experi- malized using the eukaryotic translation elongation factor 1 alpha mental procedure was conducted with the bacteria A. salmonicida (eef1a) as reference gene, which was constitutively expressed and using a dose of 5 106 CFU/ml and the corresponding control not affected by the experimental treatments, and calculated using batches were i.p. injected with PBS 1. Replicate batches were the Pfaffl method (Pfaffl, 2001). placed alternatively in order to minimize the influence of tank po- sition. Mortality was recorded over a period of 21 days. Cumulative 2.10. Statistical analysis mortality was represented as the mean of the two replicate batches (±standard deviation). Expression results were represented graphically as the mean + In parallel, 4 groups of 18 turbot were equally injected with the the standard deviation of the biological replicates. In order to expression plasmids or PBS 1. At 48 h, six individuals from each determine statistical differences, data were analysed with the com- batch were sacrificed and muscle (site of plasmid injection) and puter software package SPSS v.19.0 using the Student’s t-test. Dif- head kidney were sampled. The 12 remaining fish from each tank ferences were considered statistically significant at p < 0.05. were divided in batches of 6 turbot and one batch was i.p. chal- lenged with VHSV whereas the other 6 fish from each treatment were i.p. infected with A. salmonicida. At 24 h post-infection head 3. Results kidney samples were taken (6 individual samples) and were pro- cessed for the analysis of gene expression (see below, Section 2.9). 3.1. Cloning, sequencing and characterization of ifn1 and ifn2 Muscle samples were used for analysing the expression of the IFNs genes contained in the plasmids and the induction of several im- The complete coding regions of turbot ifn1 (GenBank accession mune-related genes. Head kidney samples before infection were number KJ150677) and ifn2 (GenBank accession number used for determining the expression of immune genes, and head KJ150678) were obtained by RACE and consisted of 546 and kidney samples after viral or bacterial challenge were used for ana- 468 nucleotides, respectively. Therefore, Ifn1 protein is composed lysing the proliferation of VHSV or A. salmonicida in the different of 181 amino acids, 21 corresponding to the signal peptide and fish groups. 160 to the mature protein, whereas Ifn2 protein is composed by 236 P. Pereiro et al. / Developmental and Comparative Immunology 45 (2014) 233–244

Fig. 1. (A) Nucleotide and amino acid sequence of turbot ifn1 (above) and ifn2 (below) open reading frames. The predicted signal peptides are underlined, whereas the N- glycosylation sites are boxed. (B) Protein multiple-alignment of selected type I IFNs from fish and Homo sapiens IFN-a and IFN-b. The GenBank accession numbers of the sequences used for the alignment are: Ifna1 Salmo salar: ACE75690, Ifnb1 S. salar: ACE75691, Ifnc1 S. salar: ACE75692, Ifnphi1 Danio rerio: NP_997523, Ifnphi2 D. rerio: NP_001104552, Ifnphi3 D. rerio: NP_001104553, IFN alpha 1 H. sapiens: AAH74929, IFN beta H. sapiens: NP_002167. (C) Predicted tertiary structure of turbot Ifn1 and Ifn2 using I-TASSER server.

155 amino acids, 19 belonging to the signal peptide and 136 to protein and 16.02 kDa and 9.08 for Ifn2, respectively. An the mature protein (Fig. 1A). Two putative N-glycosylation sites alignment between the ORFs and the corresponding genomic were predicted for Ifn1 and one for Ifn2. The molecular weight sequences showed that although the last intron of ifn2 contains and isoelectric point were 17.86 kDa and 5.41 for Ifn1 mature a region with multiple ambiguous bases (N´ s) possibly due to P. Pereiro et al. / Developmental and Comparative Immunology 45 (2014) 233–244 237

Fig. 2. Unrooted phylogenetic tree of type I, type II and type III IFNs from vertebrate organisms. The tree was constructed using the neighbour-joining method and node values represent the percentage of bootstrapping after 10,000 replicates. All positions containing alignment gaps and missing data were completely deleted. The turbot Ifn1 and Ifn2 molecules characterized in this study are underlined.

the high repetition frequency of the same motif in this intron, 3.2. Homology and phylogenetic analysis both turbot IFNs showed the typical gene structure observed in teleost type I IFNs, composed of 5 exons and 4 introns (Supple- The phylogenetic tree constructed using type I, type II and type mentary data Fig. 1). III IFNs from mammals and using type I and type II IFNs from other Multiple alignment with other fish type I IFNs and with human fish species showed the two turbot IFNs grouped in the cluster of IFN alpha and IFN beta showed four positions of relatively well type I IFNs (Fig. 2). Type I IFNs were divided into two main conserved cysteines (Fig. 1B) but nevertheless, the number of Cys branches, one of them corresponding to mammalian and Xenopus was variable among the different sequences. Turbot Ifn1 presents tropicalis IFNs but also including some fish IFNs (being turbot 6 Cys residues and Ifn2 contains 5. Three disulphide bounds were Ifn1 in this group) and the other branch grouping only fish IFNs predicted for Ifn1 mature protein (1–6, 2–5, 3–4) and two for (including turbot Ifn2). Gallus gallus IFN alpha and IFN beta formed Ifn2 (1–4, 3–5). The predicted 3D-structures of Ifn1 and Ifn2 were a separated cluster from the other type I IFNs. Whereas Ifn1 was constructed with a high confidence value using as a template more closely related to D. rerio Ifnphi2 and Ifnphi3, Ifn2 showed the zebrafish Ifnphi2 (TM-score = 0.854) and Ifnphi1 (TM-score = higher phylogenetic relation with Ifnphi1. On the other hand, type 0.899), respectively. Both turbot IFNs showed a tertiary structure III IFNs were more related with type I IFNs than with type II IFNs. composed by 5 alpha-helices (Fig. 1C). With regard to the identity/similarity matrix (Supplementary data 238 P. Pereiro et al. / Developmental and Comparative Immunology 45 (2014) 233–244

Fig. 3. Turbot ifn1 (A) and ifn2 (B) constitutive expression in kidney, liver, spleen, intestine, heart, brain, muscle + skin and gill obtained from healthy fish. The expression of transcripts was normalized to the expression of the housekeeping gene (eef1a) and expressed as arbitrary units. Data are represented as the mean + standard deviation (n = 3).

Table 3), Ifn1 showed the highest scores with S. salar Ifnc1 (33.7/ suspension was administrated, a significant up-regulation of the 57.2%) followed by D. rerio Ifnphi3 (26.8/48.6%) and Ifnphi2 ifn1 and ifn2 expression was observed at 72 h, with a FC = 150 for (24.0/46.4%). Ifn2 shared more identity and similarity with S. salar ifn1 and FC = 19 in the case of ifn2 (Fig. 4B). Ifna1 (29.5/53.7%), Oncorhynchus mykiss type I Ifn2 (28.5/50.3%) and D. rerio Ifnphi1 (26.2/44.1%). When compared with mamma- 3.5. Effect of the IFNs in the mortality induced by viral or bacterial lian sequences, turbot Ifn1 showed the highest scores with Mus infections musculus IFN alpha-1 (24.6/42.3%) followed by Homo sapiens IFN alpha-1 (22.6/41.3%). Interestingly, Ifn2 presented a higher similar- A significant delay and reduction in the mortality induced by ity with human IFN gamma (40.4%) than with type I IFNs (Mus VHSV administration was observed after 21 days in juvenile turbot musculus IFN alpha-1: 36.5%, H. sapiens IFN alpha-1: 34.4%) but, receiving the pMCV1.4-ifn1 injection, and no protective effect was its identity score was more similar to the sequences of G. gallus observed in the individuals inoculated with the empty plasmid or IFN beta (19.0%) and H. sapiens IFN lambda-1 (19.0%). pMCV1.4-ifn2 (Fig. 5A). The animals inoculated with the vector encoding Ifn1 reached a cumulative mortality of 47.5%, whereas 3.3. Tissue-specific expression of turbot IFNs genes in the other three groups a mortality rate of 100% was observed. Moreover, fish treated with pMCV1.4-ifn1 showed a reduction in The constitutive level of ifn1 and ifn2 transcripts was analysed the expression of the viral glycoprotein gene in head kidney, the le- in several tissues through qPCR (Fig. 3). Both IFNs were detected vel of which was significantly lower with regard to the other in all the tested tissues, being the liver the organ showing the low- groups of infected fish (Fig. 5B). On the other hand, the administra- er expression of both genes. The highest level of ifn1 was observed tion of the plasmids encoding the turbot IFNs did not significantly in gill whereas ifn2 was more expressed in muscle. The second tis- affect the survival of the individuals after a highly lethal dose of A. sue showing the highest transcription level of both genes was salmonicida, reaching for all the treatments a cumulative mortality intestine. On the other hand, ifn1 transcripts were more abundant rate higher than 90% (Fig. 5C). In this case, there were not differ- than ifn2 mRNA in kidney, liver, spleen and gill, and there was a ences between treatments with regard to the detection level of lower expression in heart, brain and muscle. the bacteria in head kidney samples (Fig. 5D). No mortality events were registered in the absolute control groups (non-treated and 3.4. Effect of the VHSV or A. salmonicida challenge in the expression of non-infected fish). turbot IFNs 3.6. Gene induction by Ifn1 or Ifn2 Turbot ifn1 gene showed an increasing expression after viral infection with time, revealing a fold-change (FC) of about 554 at In order to determine if differences in the IFN expression be- 24 h and a FC of almost 16,000 at 72 h; on the other hand, ifn2 tween both plasmids (pMCV1.4-ifn1 and pMCV1.4-ifn2) could af- was found to be significantly overexpressed at 24 h (FC = 7) and fect to the biological activity observed in the experimental returning to basal levels at 72 h (Fig. 4A). When the bacterial infections, the expression of ifn1 and ifn2 was analysed in muscle P. Pereiro et al. / Developmental and Comparative Immunology 45 (2014) 233–244 239

Fig. 4. Expression analysis of turbot ifn1 and ifn2 in head kidney after VHSV (A) or A. salmonicida subsp. salmonicida challenge (B). The expression of both cytokines was normalized to the expression of the housekeeping gene eef1a. Fold change was calculated by dividing the normalized expression values in the different samples by the normalized expression values obtained in the controls (non-infected individuals). Differences were considered statistically significant at p < 0.05 (⁄). Data are represented as the mean + standard deviation (n = 4). samples (site of injection) at 48 h after inoculation. The expression data Fig. 2). In these same samples, only Ifn1 was able to induce of ifn1 after pMCV1.4-ifn1 administration was similar to the the up-regulation of the typical antiviral genes mx (FC = 225), expression of ifn2 after pMCV1.4-ifn2 i.m. injection, and cross- ifi56 (FC = 63) and isg15 (FC = 165) (Fig. 6). With regard to the effect inductions between both IFNs were not observed (Supplementary of the plasmid injection in the induction of other immune-relevant

Fig. 5. Cumulative mortality (%) and pathogen detection in head kidney samples after VHSV (A and B) or A. salmonicida challenge (C and D) in individuals previously injected in muscle with PBS 1, pMCV1.4, pMCV1.4-ifn1 and pMCV1.4-ifn2. At 48 h after expression vector administration, the individuals were infected with the virus or the bacteria and head kidney samples from 6 individuals/treatment were taken at 24 h after challenge for analysing the replication of the pathogens. The transcription of the VHSV glycoprotein and the A. salmonicida specific probe were normalized to the expression of the housekeeping gene eef1a. Differences among treatments were considered statistically significant at p < 0.05 (⁄). qPCR data are represented as the mean + standard deviation (n = 6). 240 P. Pereiro et al. / Developmental and Comparative Immunology 45 (2014) 233–244

Fig. 6. Expression analysis of different immune genes in muscle (site of injection) at 48 h after pMCV1.4, pMCV1.4-ifn1 or pMCV1.4-ifn2 administration. The values were normalized to the expression of the housekeeping gene eef1a. Fold change was calculated by dividing the normalized expression values in the different samples by the normalized expression values obtained in the controls (PBS 1-injected individuals). Differences were considered statistically significant at p < 0.05 (⁄). Data are represented as the mean + standard deviation (n = 6). genes, both IFNs increased the mRNA level of irf1, casp7, il8, mhc1 overexpression of irf1, casp7, il8 and mhc1, whereas Ifn2 also in- and mhc2, whereas only Ifn2 affected the expression of the pro- duced an increase in the il8 transcription as well as in the level inflammatory cytokine il1b (Fig. 6). of il1b mRNA; the expression of mhc2 was not affected by any tur- In head kidney samples, the injection of the expression plasmid bot IFN in this tissue (Fig. 7). encoding Ifn1 induced a powerful up-regulation of the three ana- lysed ISGs after 48 h, with a FC higher than 160 for mx, a FC about 80 for ifi56 and a FC near to 200 for isg15; on the other hand, the 4. Discussion expression of ifn2 in muscular cells does not seem to affect the le- vel of expression of these genes (Fig. 7). Moreover, pMCV1.4-ifn1 Nowadays it is clear that type I IFNs are the main responsible of administration showed a relatively discrete but significant orchestrating the antiviral response in mammals and even in fish. P. Pereiro et al. / Developmental and Comparative Immunology 45 (2014) 233–244 241

Whereas this kind of cytokines has been largely studied in mam- higher vertebrates could have arisen due to a retroposition event malian species, the knowledge in fish is more recent and limited. that occurred during evolution after the separation of sarcoptery- Although turbot is an important and valuable species in aquacul- gians from actinopterygians (Lutfalla et al., 2003). ture industry, hitherto no IFN gene had been characterized in this Under basal conditions, ifn1 was more expressed than ifn2 in species. The first partial sequences with homology to IFN in turbot some tissues and vice versa, following a differential pattern as were obtained after a 454-pyrosequencing of several tissues after was previously reported for different type I IFNs in rainbow trout immune stimulation with virus or molecules mimicking viral (Zou et al., 2007). These observations, together with the differential infections (Pereiro et al., 2012) and, in this work, the complete induction of the Atlantic salmon IFNs in different organs after poly amplified coding sequences were named ifn1 and ifn2. Ifn1 was I:C or imidazoquinoline R848 treatment (Svingerud et al., 2012) similar in size to the type I IFNs described in fish (175–194 aa) could suggest a tissue-specific expression profile, being different (Zou et al., 2007) but Ifn2 was smaller and constituted of 155 aa. cells types specialized in the production of different IFNs. Differ- The predicted proteins showed a signal peptide suggesting, as ex- ences between the two turbot interferons were also detected in pected, that they are secreted, although recently, intracellular their expression after viral challenge. ifn1 was highly induced in interferons lacking a signal peptide were also identified in rainbow turbot after VHSV administration while ifn2 was very lowly over- trout (Chang et al., 2013). Both IFNs possessed potential glycosyla- expressed returning to basal levels at 72 h. This differential induc- tion sites, two in Ifn1 and one in Ifn2. Although IFN glycosylation tion after viral challenge also suggests a different and specific role does not seem to be essential for biological activity, higher antiviral of each turbot IFN in the immune system, as is discussed below. properties of the glycosylated molecules could be related with the Type I IFNs are key molecules in the protective immune re- protein stabilization/solubilization effect of the carbohydrate on sponse against viruses eliciting a subsequent specific response structure and to the higher half-life (Ceaglio et al., 2010; Dissing- involving the expression of a high number of ISGs, which reduce Olesen et al., 2008; Runkel et al., 1998). Potential glycosylation the viral proliferation. Myxovirus resistance protein (mx) is a well- sites (1–4) were detected in all the fish IFNs with some exception, studied ISG in fish (Abollo et al., 2005; Altmann et al., 2004; Jensen but the degree of glycosylation varies as in vertebrates, therefore, et al., 2002; Jensen and Robertsen, 2000; Larsen et al., 2004; this phenomenon seems not to be conserved during evolution Staeheli et al., 1989; Trobridge and Leong, 1995), but the knowl- (Robertsen, 2006). Differences were also observed in the isoelectric edge about other antiviral proteins, such as Isg15 or Ifi56 is more points (pI) of vertebrate IFNs and even between different IFNs limited (Langevin et al., 2013; Liu et al., 2013). When the expres- belonging to the same species, as occurs in turbot. sion plasmids containing the turbot IFNs were intramuscularly Fish type I IFNs were classically divided into two different injected to fish a high induction of the three antiviral genes was groups based on the number of cysteine residues present in the observed in the individuals injected with pMCV1.4-ifn1 after 48 h mature peptide: group I for the IFNs containing 2 Cys and group at the site of injection and in head kidney. Interestingly, the II for the members with 4 Cys (Zou et al., 2007; Zou and Secombes, expression of ifn2 in muscular cells did not seem to modulate the 2011). A multiple-alignment of some type I IFNs from vertebrates level of these transcripts in any of the tested tissues. Indeed, only showed four positions of relatively well conserved Cys but being pMCV1.4-ifn1 administration was able to protect the turbot against their number variable among the different sequences. Nowadays, a highly lethal dose of VHSV, significantly reducing the mortality the higher number of fish IFN-like sequences deposited in the dat- rate. This observation is correlated with the significant lower tran- abases revealed a higher variation in the number of Cys residues. scription of the viral glycoprotein gene in fish previously treated Indeed, turbot Ifn1 and Ifn2 contained 6 and 5 Cys respectively with the plasmid encoding Ifn1 but not in individuals inoculated and therefore, they cannot be classified according to the traditional with pMCV1.4-ifn2. Therefore, the antiviral state induced by Ifn1 groups described so far. These Cys residues could have an impor- reduces the viral replication and, as consequence, a protective ef- tant role in the 3D-structure of Ifn1 and Ifn2 due to the establish- fect against the infection is achieved with this treatment. Differ- ment of three and two potential disulphide bridges, respectively. ences in the induction pattern and protective capabilities among Both turbot IFNs showed a three-dimensional conformation com- the different type I IFNs belonging to the same fish species were posed of 5 alpha-helices as occurs with human IFN beta (Karpusas previously observed. Rainbow trout (O. mykiss) type I IFNs possess et al., 1997). The predicted 3D-models were constructed using the different properties and capabilities for inducing mx expression zebrafish Ifnphi2 (turbot Ifn1) and Ifnphi1 (turbot Ifn2) as a tem- and eliciting antiviral responses (Zou et al., 2007). On the other plate, which also show the typical structural basis of vertebrate hand, whereas zebrafish Ifnphi1 and Ifnphi2 are potent inducers type I IFNs (Hamming et al., 2011). Indeed, the phylogenetic anal- of the ISG viperin, Ifnphi4 is a poor inducer and this observation ysis revealed that Ifn1 grouped in the same clade that zebrafish Ifn- could be related with the absence of protective effect by zebrafish phi2 and Ifn2 was grouped closer to Ifnphi1. It is also interesting to Ifnphi4 against IHNV in early zebrafish larvae (Aggad et al., 2009). highlight that, although turbot IFNs do not share the pattern of 2 or Another publication revealed that zebrafish Ifnphi1, Ifnphi2 and 4 Cys, Ifn1 was more similar to group II of fish type I IFNs (4 Cys) Ifnphi3 were able to evoke a strong antiviral activity but with a dif- whereas Ifn2 was grouped with the members of group I (2 Cys), ferent pattern and kinetic, suggesting complementary roles as was also reflected by the identity/similarity scores. In zebrafish (López-Muñoz et al., 2009). the two subsets of type I IFNs (group I and II) do not share the same The implication of type I IFNs in the defence mechanisms receptor complex (Aggad et al., 2009). The higher similarity in se- against bacterial pathogens is still poorly understood. It has been quence and 3D-structure of Ifn1 with zebrafish IFNs belonging to shown that numerous bacteria are able to induce an increase in group II and of Ifn2 with those zebrafish IFNs belonging to group the expression of IFNs (Bogdan et al., 2004; Monroe et al., 2010) I could suggest also a different receptor complex for turbot IFNs. but there are contradictory results on their involvement on the Another interesting question is the closer phylogenetic relation- survival after a bacterial challenge depending on the species ship between turbot Ifn1 and mammalian type I IFNs. This could (González-Navajas et al., 2012; Monroe et al., 2010). In fish, some indicate a more conserved function of Ifn1, as was observed in this experiments have revealed an increase in the level of type I IFNs work. On the other hand, type I and type III IFNs formed a separate after LPS or bacteria administration (Chang et al., 2009; cluster from IFNs gamma. As was mentioned in the introduction, López-Muñoz et al., 2009; Wan et al., 2012) and even a protective fish type I IFNs have the same exon/intron structure as type III IFNs, effect of Ifnphi1 (but not of Ifnphi2 or Ifnphi3) against Streptococ- and this could suggest that these genes derived from a common cus iniae challenge in zebrafish (López-Muñoz et al., 2009). In tur- ancestral gene (Robertsen, 2006). The intronless type I IFNs from bot, however, although an overexpression of both IFNs was 242 P. Pereiro et al. / Developmental and Comparative Immunology 45 (2014) 233–244

Fig. 7. Expression analysis of different immune genes in head kidney samples at 48 h after pMCV1.4, pMCV1.4-ifn1 or pMCV1.4-ifn2 intramuscular administration. The values were normalized to the expression of the housekeeping gene eef1a. Fold change was calculated by dividing the normalized expression values in the different samples by the normalized expression values obtained in the controls (PBS 1-injected individuals). Differences were considered statistically significant at p < 0.05 (⁄). Data are represented as the mean + standard deviation (n = 6).

observed at 72 h in head kidney in response to A. salmonicida, the relevant genes was analysed in muscle and head kidney. Whereas administration of both IFNs did not seem to be able to protect ani- both turbot IFNs were able to induce an overexpression of irf1, mals against a highly lethal dose of bacteria. In this case, ifn2 casp7, and the complexes mhc1 and mhc2 in the site of the plasmid showed a higher expression in comparison with the up-regulation injection, only Ifn1 increased the mRNA level of these genes in observed after VHSV administration. head kidney with the exception of mhc2, which was not modulated Turbot Ifn1 seems clearly a typical antiviral IFN able to induce by any IFN in this tissue. All these genes are closely or directly re- ISGs and protecting against a viral infection but, what is the lated to the antigen presentation process. Indeed, IRF-1 plays a cru- implication of Ifn2 in the immunity? In order to investigate the cial role in the regulation of the antigen presentation, being needed hypothetical role of this gene the expression of other immune- for the expression of LMP-2, TAP-1, and MHC-I (Kröger et al., 2002). P. Pereiro et al. / Developmental and Comparative Immunology 45 (2014) 233–244 243

This molecule also induces the transcription of pro-apoptotic Ministerio de Ciencia e Innovación. P. Pereiro gratefully acknowl- genes, such as caspase-7 (Taniguchi et al., 2001), and apoptosis is edges the Spanish Ministerio de Educación for a FPU fellowship a mechanism facilitating the antigen presentation (Schaible et al., (AP2010-2408). 2003). While Ifn1 showed a broader effect in the regulation of these genes, the induction by Ifn2 was more limited to the injec- Appendix A. Supplementary data tion area. Another relevant point was the significantly higher up-regulation of mhc2 by Ifn2 with regard to Ifn1 in muscle. These Supplementary data associated with this article can be found, in results could indicate the potential of Ifn2 as vaccine adjuvant, the online version, at http://dx.doi.org/10.1016/jdci.2014.03.006. favouring the antigen presentation at the vaccine administration site. Unexpectedly, both turbot IFNs increased the transcription of References the pro-inflammatory chemokine il8 in the site of injection and head kidney cells. Numerous publications have reported the Abollo, E., Ordás, C., Dios, S., Figueras, A., Novoa, B., 2005. Molecular characterisation of a turbot Mx cDNA. Fish Shellfish Immunol. 19, 185–190. down-regulation of IL-8 transcription by type I IFNs in mammals Aggad, D., Mazel, M., Boudinot, P., Mogensen, K.E., Hamming, O.J., Hartmann, R., (Laver et al., 2008; Nozell et al., 2006; Oliveira et al., 1992). There- Kotenko, S., Herbomel, P., Lutfalla, G., Levraud, J.P., 2009. The two groups of fore, an opposite regulation of this gene by type I IFNs was ob- zebrafish virus-induced interferons signal via distinct receptors with specific and shared chains. J. Immunol. 183, 3924–3931. served in turbot with regard to mammals. To our knowledge, this Altmann, S.M., Mellon, M.T., Distel, D.L., Kim, C.H., 2003. Molecular and functional is the first work reporting the positive induction of il8 by type I analysis of an interferon gene from the zebrafish, Danio rerio. J. Virol. 77, 1992– IFNs in fish, and therefore, more investigations will be necessary 2002. Altmann, S.M., Mellon, M.T., Johnson, M.C., Paw, B.H., Trede, N.S., Zon, L.I., Kim, C.H., in order to clarify whether this observation could be attributed 2004. Cloning and characterization of an Mx gene and its corresponding to all teleost species. Another pro-inflammatory cytokine inhibited promoter from the zebrafish, Danio rerio. Dev. Comp. Immunol. 28, 295–306. by IFN-a/b in mammals is IL-1b (Guarda et al., 2011). However, Balcázar, J.L., Vendrell, D., de Blas, I., Ruiz-Zarzuela, I., Gironés, O., Múzquiz, J.L., 2007. Quantitative detection of Aeromonas salmonicida in fish tissue by real- zebrafish Ifnphi1, 2 and 3 induced a remarkable overexpression time PCR using self-quenched, fluorogenic primers. J. Med. Microbiol. 56, 323– of il1b (López-Muñoz et al., 2009), and this molecule was also sig- 328. nificantly up-regulated by turbot Ifn2 in both tested tissues but not Ben-Sasson, S.Z., Hu-Li, J., Quiel, J., Cauchetaux, S., Ratner, M., Shapira, I., Dinarello, by Ifn1. This interleukin has been also proposed as a key molecule C.A., Paul, W.E., 2009. IL-1 acts directly on CD4 T cells to enhance their antigen- driven expansion and differentiation. Proc. Natl. Acad. Sci. USA 106, 7119–7124. in the success of an adjuvant treatment in the vaccination process Bogdan, C., Mattner, J., Schleicher, U., 2004. The role of type I interferons in non-viral (Ben-Sasson et al., 2009) and even as a powerful adjuvant by itself infections. Immunol. Rev. 202, 33–48. (Tagliabue and Boraschi, 1993). Once again, our results reflect the Campanella, J.J., Bitincka, L., Smalley, J., 2003. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. BMC capability of Ifn2 for being used as an efficient adjuvant, eliciting Bioinformatics 4, 29. the antigen presentation process as well as inducing a pro- Ceaglio, N., Etcheverrigaray, M., Conradt, H.S., Grammel, N., Kratje, R., Oggero, M., inflammatory state at the vaccination site. 2010. Highly glycosylated human alpha interferon: an insight into a new therapeutic candidate. J. Biotechnol. 146, 74–83. Taking all this information into consideration, it seems clear Chang, M., Nie, P., Collet, B., Secombes, C.J., Zou, J., 2009. Identification of an that Ifn2 has the ability to affect the transcription of several im- additional two-cysteine containing type I interferon in rainbow trout mune-relevant genes with the exception of the ISGs mx, ifi56 and Oncorhynchus mykiss provides evidence of a major gene duplication event within this gene family in teleosts. Immunogenetics 61, 315–425. isg15, and also that this induction is mainly focused on the site of Chang, M.X., Zou, J., Nie, P., Huang, B., Yu, Z., Collet, B., Secombes, C.J., 2013. injection. Moreover, it is the only turbot IFN able to up-regulate Intracellular interferons in fish: a unique means to combat viral infection. PLoS the mRNA level of il-1b and, therefore, a direct role in the inflam- Pathog. 9, e1003736. Dissing-Olesen, L., Thaysen-Andersen, M., Meldgaard, M., Hojrup, P., Finsen, B., matory response could be attributed to this molecule. 2008. The function of the human interferon-beta 1a glycan determined in vivo.J. Pharmacol. Exp. Ther. 326, 338–347. Emanuelsson, O., Brunak, S., von Heijne, G., Nielsen, H., 2007. Locating proteins in 5. Conclusion the cell using TargetP, SignalP and related tools. Nat. Protoc. 2, 953–971. Ferrè, F., Clote, P., 2005. DiANNA: a web server for disulfide connectivity prediction. Nucleic Acids Res. 33, W230–W232. Two type I IFNs were characterized for the first time in turbot Gasteiger, E., Gattiker, A., Hoogland, C., Ivanyi, I., Appel, R.D., Bairoch, A., 2003. (Scophthalmus maximus) and their expression properties were ExPASy: the proteomics server for in-depth protein knowledge and analysis. analysed under constitutive conditions as well as after VHSV or Nucleic Acid Res. 31, 3784–3788. A. salmonicida challenge. Both genes were up-regulated after González-Navajas, J.M., Lee, J., David, M., Raz, E., 2012. Immunomodulatory functions of type I interferons. Nat. Rev. Immunol. 12, 125–135. viral or bacterial challenge, especially ifn1 after viral infection. Guarda, G., Braun, M., Staehli, F., Tardivel, A., Mattmann, C., Förster, I., Farlik, M., Indeed, only Ifn1 was able to induce the expression of ISGs Decker, T., Du Pasquier, R.A., Romero, P., Tschopp, J., 2011. Type I interferon (mx, ifi56 and isg15) and, as consequence, significantly reduced inhibits interleukin-1 production and inflammasome activation. Immunity. 34, 213–223. the mortality of turbot against VHSV challenge. Neither Ifn1 Hamming, O.J., Lutfalla, G., Levraud, J.P., Hartmann, R., 2011. Crystal structure of nor Ifn2 were able to increase the survival of the individuals zebrafish interferons I and II reveals conservation of type I interferon structure after an A. salmonicida infection. Therefore, an antiviral role of in vertebrates. J. Virol. 85, 8181–8187. Honda, K., Yanai, H., Takaoka, A., Taniguchi, T., 2005. Regulation of the type I IFN turbot Ifn1 may be clearly established. Ifn2 affected the expres- induction: a current view. Int. Immunol. 17, 1367–1378. sion of several immune genes mainly in the site of plasmid Jensen, V., Robertsen, B., 2000. Cloning of an Mx cDNA from Atlantic halibut injection and was the only turbot IFN with the ability to increase (Hippoglossus hippoglossus) and characterization of Mx mRNA expression in response to double-stranded RNA or infectious pancreatic necrosis virus. J. the transcription of il1b. A complementary pro-inflammatory Interferon Cytokine Res. 20, 701–710. function could be suggested for this type I IFN. This overexpres- Jensen, I., Albuquerque, A., Sommer, A.I., Robertsen, B., 2002. Effect of poly I: C on sion of il1b by Ifn2 as well as the overexpression of il8 by both the expression of Mx proteins and resistance against infection by infectious salmon anaemia virus in Atlantic salmon. Fish Shellfish Immunol. 13, 311–326. IFNs are very interesting points since these molecules are inhib- Karpusas, M., Nolte, M., Benton, C.B., Meier, W., Lipscomb, W.N., Goelz, S., 1997. The ited by type I IFNs in mammals. crystal structure of human interferon beta at 2.2-A resolution. Proc. Natl. Acad. Sci. USA 94, 11813–11818. Kröger, A., Köster, M., Schroeder, K., Hauser, H., Mueller, P.P., 2002. Activities of IRF- Acknowledgements 1. J. Interferon Cytokine Res. 22, 5–14. Langevin, C., van der Aa, L.M., Houel, A., Torhy, C., Briolat, V., Lunazzi, A., Harmache, A., Bremont, M., Levraud, J.P., Boudinot, P., 2013. Zebrafish ISG15 exerts a strong This work has been funded by the project CSD2007-00002 antiviral activity against RNA and DNA viruses and regulates the interferon ‘‘Aquagenomics’’ and AGL2011-28921-C03 from the Spanish response. J. Virol. 87, 10025–10036. 244 P. Pereiro et al. / Developmental and Comparative Immunology 45 (2014) 233–244

Larsen, R., Rokenes, T.P., Robertsen, B., 2004. Inhibition of infectious pancreatic functional differences between glycosylated and non-glycosylated forms of necrosis virus replication by Atlantic salmon Mx1 protein. J. Virol. 78, 7938– human interferon-beta (IFN-beta). Pharm. Res. 15, 641–649. 7944. Sadler, A.J., Williams, B.R.G., 2008. Interferon-inducible antiviral effectors. Nat. Rev. Laver, T., Nozell, S.E., Benveniste, E.N., 2008. IFN-beta-mediated inhibition of IL-8 Immunol. 8, 559–568. expression requires the ISGF3 components Stat1, Stat2, and IRF-9. J. Interferon Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for Cytokine Res. 28, 13–23. reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Liu, Y., Zhang, Y.B., Liu, T.K., Gui, J.F., 2013. Lineage-specific expansion of IFIT gene Samuel, C.E., 2001. Antiviral actions of interferons. Clin. Microbiol. Rev. 14, 778– family: an insight into coevolution with IFN gene family. PLoS One 8, e66859. 809. López-Muñoz, A., Roca, F.J., Meseguer, J., Mulero, V., 2009. New insights into the Schaible, U.E., Winau, F., Sieling, P.A., Fischer, K., Collins, H.L., Hagens, K., Modlin, evolution of IFNs: zebrafish group II IFNs induce a rapid and transient R.L., Brinkmann, V., Kaufmann, S.H., 2003. Apoptosis facilitates antigen expression of IFN-dependent genes and display powerful antiviral activities. J. presentation to T lymphocytes through MHC-I and CD1 in tuberculosis. Nat. Immunol. 182, 3440–3449. Med. 9, 1039–1046. Lutfalla, G., Crollius, H.R., Stange-Thomann, N., Jaillon, O., Mogensen, K., Monneron, Staeheli, P., Yu, Y.X., Grob, R., Haller, O., 1989. A double-stranded RNA-inducible fish D., 2003. Comparative genomic analysis reveals independent expansion of a gene homologous to the murine influenza virus resistance gene Mx. Mol. Cell lineage-specific gene family in vertebrates: the class II cytokine receptors and Biol. 9, 3117–3121. their ligands in mammals and fish. BMC Genomics 4, 29. Svingerud, T., Solstad, T., Sun, B., Nyrud, M.L., Kileng, O., Griner-Tollersrud, L., Monroe, K.M., McWhirter, S.M., Vance, R.E., 2010. Induction of type I interferons by Robertsen, B., 2012. Atlantic salmon type I IFN subtypes show differences in bacteria. Cell Microbiol. 12, 881–890. antiviral activity and cell-dependent expression: evidence for high IFNb/IFNc- Nozell, S., Laver, T., Patel, K., Benveniste, E.N., 2006. Mechanism of IFN-beta- producing cells in fish lymphoid tissues. J. Immunol. 189, 5912–5923. mediated inhibition of IL-8 gene expression in astroglioma cells. J. Immunol. Tagliabue, A., Boraschi, D., 1993. Cytokines as vaccine adjuvants: interleukin 1 and 177, 822–830. its synthetic peptide 163–171. Vaccine 11, 594–595. Oliveira, I.C., Sciavolino, P.J., Lee, T.H., Vilcek, J., 1992. Downregulation of interleukin Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular 8 gene expression in human fibroblasts: unique mechanism of transcriptional evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. inhibition by interferon. Proc. Natl. Acad. Sci. USA 89, 9049–9053. Taniguchi, T., Ogasawara, K., Takaoka, A., Tanaka, N., 2001. IRF family of Pereiro, P., Balseiro, P., Romero, A., Dios, S., Forn-Cuni, G., Fuste, B., Planas, J.V., transcription factors as regulators of host defense. Annu. Rev. Immunol. 19, Beltran, S., Novoa, B., Figueras, A., 2012. High-throughput sequence analysis of 623–655. turbot (Scophthalmus maximus) transcriptome using 454-pyrosequencing for Taya, Y., Devos, R., Tavernier, J., Cheroutre, H., Engler, G., Fiers, W., 1982. Cloning and the discovery of antiviral immune genes. PLoS One 7, e35369. structure of the human immune interferon-gamma chromosomal gene. EMBO J. Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real- 1, 953–958. time RT-PCR. Nucleic Acids Res. 29, e45. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the Platanias, L.C., 2005. Mechanisms of type-I- and type-II-interferon-mediated sensitivity of progressive multiple sequence alignments through sequence signalling. Nat. Rev. Immunol. 5, 375–386. weighting, position specific gap penalties and weight matrix choice. Nucleic Qi, Z., Nie, P., Secombes, C.J., Zou, J., 2010. Intron-containing type I and type III IFN Acid Res. 22, 4673–4680. coexist in amphibians: refuting the concept that a retroposition event gave rise Trobridge, G.D., Leong, J.A., 1995. Characterization of a rainbow trout Mx gene. J. to type I IFNs. J. Immunol. 184, 5038–5046. Interferon Cytokine Res. 15, 691–702. Robertsen, B., Bergan, V., Rokenes, T., Larsen, R., Albuquerque, A., 2003. Atlantic Wan, Q., Wicramaarachchi, W.D., Whang, I., Lim, B.S., Oh, M.J., Jung, S.J., Kim, H.C., salmon interferon genes: cloning, sequence analysis, expression, and biological Yeo, S.Y., Lee, J., 2012. Molecular cloning and functional characterization of two activity. J. Interferon Cytokine Res. 23, 601–612. duplicated two-cysteine containing type I interferon genes in rock bream Robertsen, B., 2006. The interferon system of teleost fish. Fish Shellfish Immunol. Oplegnathus fasciatus. Fish Shellfish Immunol. 33, 886–898. 20, 172–191. Zou, J., Tafalla, C., Truckle, J., Secombes, C.J., 2007. Identification of a second group of Roy, A., Kucukural, A., Zhang, Y., 2010. I-TASSER: a unified platform for automated type I IFNs in fish sheds light on IFN evolution in vertebrates. J. Immunol. 179, protein structure and function prediction. Nat. Protoc. 5, 725–738. 3859–3871. Rozen, S., Skaletsky, H.J., 2000. Primer3 on the WWW for general users and for Zou, J., Secombes, C.J., 2011. Teleost fish interferons and their role in immunity. Dev. biologist programmers. MIMB 132, 365–386. Comp. Immunol. 35, 1376–1387. Runkel, L., Meier, W., Pepinsky, R.B., Karpusas, M., Whitty, A., Kimball, K., Brickelmaier, M., Muldowney, C., Jones, W., Goelz, S.E., 1998. Structural and Supporting information

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CHAPTER 6

Pathogen -dependent role of turbot (Scophthalmus maximus)

interferon-gamma

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Fish & Shellfish Immunology 59 (2016) 25e35

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Fish & Shellfish Immunology

journal homepage: www.elsevier.com/locate/fsi

Full length article Pathogen-dependent role of turbot (Scophthalmus maximus) interferon-gamma

* Patricia Pereiro, Gabriel Forn-Cuni, Antonio Figueras, Beatriz Novoa

Instituto de Investigaciones Marinas (IIM), CSIC, Eduardo Cabello 6, 36208 Vigo, Spain article info abstract

Article history: Interferon-gamma has been typically described as a pro-inflammatory cytokine playing an important Received 22 July 2016 role in the resolution of both viral and bacterial diseases. Nevertheless, some anti-inflammatory func- Received in revised form tions are also attributed to this molecule. In this work we have characterized for the first time the turbot 3 October 2016 (Scophthalmus maximus) interferon-gamma gene (ifng) and its expression pattern under basal conditions, Accepted 10 October 2016 after type I IFNs administration, and viral and bacterial infection. The intramuscular injection of an Available online 11 October 2016 expression plasmid encoding turbot Ifng (pMCV1.4-ifng) was not able to affect the transcription of numerous immune genes directly related to the activity of IFN-gamma, with the exception of macro- Keywords: Interferon-gamma phage-colony stimulating factor (csf1). It was also unable to reduce the mortality caused by a Viral Turbot Hemorrhagic Septicemia Virus (VHSV) or Aeromonas salmonicida challenge. Interestingly, at 24 h post- Teleost infection, turbot previously inoculated with pMCV1.4-ifng and infected with VHSV showed an increase Virus in the expression of pro-inflammatory cytokines and type I IFNs compared to those fish not receiving Bacteria expression plasmid, indicating a synergic effect of Ifng and VHSV. On the other hand, some macrophage Inflammation markers, such as the macrophage receptor with collagenous structure (marco), were down-regulated by Type I interferons Ifng during the viral infection. Ifng had the opposite effect in those turbot infected with the bacteria, Macrophages activity showing a reduction in the transcription of pro-inflammatory and type I IFNs genes, and an increase in the expression of genes related to the activity of macrophages. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction molecule [5,6]. This cytokine is produced by diverse immune- related cell types, although T and NK cells are the major sources, Interferons (IFNs) are a family of multifunctional cytokines with and it is implicated in several aspects of the immunity, such as central importance against viral infections, among other immune activation of macrophages, stimulation of antigen presentation, functions [1]. These proteins are produced when a pathogen is orchestration of leukocyte-endothelium interactions, and effects on detected by the pattern recognition receptors (PRRs), which results cell proliferation and apoptosis, among others [7]. Regarding in the activation of different signaling pathways [2].Infish, two inflammation, IFN-gamma was typically described as a pro- subfamilies of IFNs were established in basis to differential struc- inflammatory protein, although this appreciation seems to not be tural and functional properties, type I and type II IFNs [3]. Type I IFN totally absolute because, in some cases, protective anti- subfamily comprises a group of typical antiviral proteins, and a inflammatory functions were related with this cytokine [8,9]. variable number of type I IFNs were described in several teleost Unlike mammals, it has been shown that some bony fish, species [3]. Indeed, it has been shown that type I IFNs from the especially cyprinids, possess two type II interferon genes [10e14].A same teleost can possess distinct properties and capabilities, sug- teleost-specific duplication of the ifng gene originated the addi- gesting in some cases complementary or specialized roles, as was tional IFN-gamma related (ifngrel) gene, although it is not a clear previously observed in turbot [4]. In contrast, IFN-gamma (type II homologue of mammalian IFN-gamma [3]. The inflammatory IFN) is a markedly different IFN, possessing some ability to interfere functions of teleost type II IFNs have not been fully characterized, with viral infections but being mainly an immunomodulatory especially in the case of those species possessing two genes. Some studies revealed that Ifng has the ability to induce the expression of pro-inflammatory cytokines [15e17]. Nevertheless, it was reported fi fl * Corresponding author. that zebra sh Ifng lacks the powerful pro-in ammatory activity of E-mail address: [email protected] (B. Novoa). its mammalian counterpart, although it helped to potentiate the http://dx.doi.org/10.1016/j.fsi.2016.10.021 1050-4648/© 2016 Elsevier Ltd. All rights reserved. 26 P. Pereiro et al. / Fish & Shellfish Immunology 59 (2016) 25e35 induction of antiviral and pro-inflammatory genes by type I IFNs 2.3. Fish, virus, bacteria [18]. It was observed that, as in mammals, fish Ifng induces the activation of phagocytic cells by increasing the production of Juvenile turbot (average weight 2.5 g) were obtained from a reactive oxygen intermediates (ROIs) and nitric oxide (NO), the commercial fish farm (Insuina~ S.L., Galicia, Spain). Animals were enhancement of phagocytosis and the up-regulation in the maintained in 500 L fibreglass tanks with a re-circulating saline expression of different immune genes in this cell type [13,16,17,19]. water system (total salinity about 35 g/L) with a light-dark cycle of Exploring the turbot genome [20], we found only one type II IFN 12:12 h at 18 C and fed daily with a commercial dry diet (LARVIVA- gene, corresponding to ifng. To get insights into its functions, we BioMar). Prior to experiments, fish were acclimatized to laboratory analyzed its constitutive expression and gene modulation after conditions for 2 weeks. Fish care and challenge experiments were viral and bacterial challenge, as well as its induction by type I IFNs. reviewed and approved by the CSIC National Committee on The bioactivity of turbot Ifng was measured by analyzing its pro- Bioethics under approval number 151-2014. tection capabilities against infections and the induction of specific Viral Hemorrhagic Septicemia Virus (VHSV) strain UK-860/94 immune-related genes in vivo. Although no protection was was propagated in EPC (Epithelioma Papulosum Cyprini) cells us- observed against bacterial or viral infections, our results showed ing Eagle's minimum essential medium (MEM) supplemented with the dual role of turbot Ifng in the expression of immune genes 2% fetal bovine serum (FBS), penicillin and streptomycin (P/S) at depending on the pathogen. Ifng seemed to synergistically induce 15 C. The second passage was stored at 80 C until use. inflammation and type I IFN synthesis upon viral infection, but a Aeromonas salmonicida subsp. salmonicida (strain VT 45.1 WT) reduction in macrophage-related molecules was observed. Strik- was cultured in tryptic soy agar (TSA) plates at 22 C during 24 h ingly, the opposite effect was observed during the infection with before being used. bacteria. 2.4. Expression plasmid pMCV1.4-ifng 2. Material and methods The expression plasmid encoding the turbot Ifng, pMCV1.4-ifng, 2.1. Characterization of turbot Ifng was produced using the nucleotide sequence encoding the Ifng mature peptide. The expression (pMCV1.4-ifng) and empty plasmid The complete sequence of the ifng gene was retrieved from the (pMCV1.4) were propagated using One Shot TOP10F' competent turbot genome project [20]. The open reading frame (ORF) was E. coli cells (Invitrogen) and the purification was conducted using confirmed by PCR using specific primers and subsequent linking the PureLinkTM HiPure Plasmid Midiprep Kit (Invitrogen). The ® into pCR™2.1-TOPO vector (Invitrogen) for its cloning using One expression plasmids encoding turbot type I IFNs (pMCV1.4-ifn1 and ® Shot TOP10F' competent cells (Invitrogen) following the protocol pMCV1.4-ifn2) were previously produced in the same way [4]. instructions. cDNA sequencing was conducted using an automated ABI 3730 DNA Analyzer (Applied Biosystems, Inc. Foster City, CA, 2.5. ifng constitutive expression, induction by type I IFNs and USA). The primers used for ORF confirmation are listed in modulation after viral and bacterial challenge Supplementary Data Table 1. The presence of interferon-gamma activated sites (GAS) and interferon-stimulated response ele- In order to examine the constitutive expression of ifng, eleven ments (ISRE) was analyzed in the promoter region using the ca- different tissues (peritoneal exudate cells e PECe, blood, head nonical sequences TT(C/A)CNNNAA(A/G) and (G/A)(G/A) kidney, trunk kidney, spleen, gill, liver, intestine, heart, brain, AANNGAAA(C/G), respectively. muscle) were obtained from 3 healthy fish (average weight 125 g). The presence of signal peptide was analyzed with the SignalP 3.0 After disinfecting the external surface of the fish with 70% ethyl Server (http://www.cbs.dtu.dk/services/signalp-3.0/) [21] and the alcohol, PEC were obtained by the injection of sterile PBS (500 ml) in nuclear localization signal (NLS) was determined using NLStrada- the peritoneal cavity by using 1 ml syringes with a 25 gauge needle mus (http://www.moseslab.csb.utoronto.ca/NLStradamus/) [22]. and, after massaging for 10 min, the cell suspensions were recov- Molecular weight and isoelectric point were calculated using the ered. Blood samples were taken from the caudal vein by using a Compute pI/Mw tool from ExPASy [23]. The 3D-structure of turbot heparinized syringe. In both cases the samples were centrifuged at Ifng was predicted using I-TASSER server [24] selecting the model 1500g for 10 min and the supernatants were discarded. All the with the best C-score and viewed by PyMOL (http://www.pymol. samples were processed for the analysis of ifng expression (Section org). The Template Modelling Score (TM-score), a measure of 2.7). structural similarity between two proteins, was also considered Stimulations with the plasmids encoding two turbot type I IFNs in order to identify those structural analogs with known crystal (ifn1 and ifn2) were conducted by intramuscular (i.m.) injection in architecture in the Protein Data Bank (PDB; http://www.rcsb.org/ the region near the caudal peduncle by using 1 ml insulin syringes pdb/). with a 30 gauge needle and positioned with a 45 insertion angle. Four groups of 6 turbot each were injected with a volume of 50 mlof 2.2. Phylogenetic analysis one of the following treatments: 2.5 mg of pMCV1.4-ifn1, 2.5 mgof pMCV1.4-ifn2, 2.5 mg of pMCV1.4 (empty plasmid), and phosphate IFN-gamma and IFN-gamma-related protein sequences from buffered saline (PBS) to the control group. At 48 h, the individuals representative species were retrieved from Genbank were sacrificed and muscle (site of plasmid injection) and head (Supplementary Data Table 2). MAFFT online server was used to kidney were sampled and processed for the analysis of ifng generate a starting alignment following the E-INS-i strategy [25], expression (Section 2.7). which was pruned using Gblocks server 0.91b [26]. The best-fitting Four groups, composed of 36 turbot/each, were intraperitone- amino acid replacement model was determined using ProtTest 3.2 ally (i.p.) injected with 50 ml of one of the following treatments: 5 [27] based on the Akaike Information Criterion (AIC) [28]. Finally, VHSV suspension (5 10 TCID50/fish), A. salmonicida subsp. sal- the maximum likelihood gene tree was estimated with PhyML 3.0 monicida suspension (5.5 105 CFU/fish), viral medium (MEM þ 2% [29]. Nodal confidence was calculated using 1000 non-parametric FBS þ P/S) or PBS. The last two groups served as the control groups bootstrap replicates and represented in a 100 scale in FigTree of the viral and bacterial infections, respectively. The head kidney v1.3.1 [30]. from 12 individuals belonging to each group was removed at P. Pereiro et al. / Fish & Shellfish Immunology 59 (2016) 25e35 27 different sampling points (8, 24 and 72 h), obtaining 4 pooled were i.p. injected with PBS. Replicate batches were placed alter- biological replicates (3 fish/replicate) that were processed for the natively in order to minimize the influence of tank position. Mor- analysis of ifng expression (Section 2.7). tality was recorded over a period of 21 days. In parallel, 2 groups of 18 turbot were also injected with 2.5 mgof 2.6. Effect of pMCV1.4-ifng injection before and after pathogen pMCV1.4-ifng or pMCV1.4. After 48 h, muscle (site of plasmid in- challenge jection) and head kidney from 6 fish were sampled. Then, 6 fish from each batch were i.p. infected with VHSV and other 6 with The protective ability of the intramuscular administration of the A. salmonicida. At 24 h post-challenge head kidney samples were plasmid encoding Ifng was tested against VHSV and A. salmonicida. taken (6 individual samples). All these samples were processed for For the viral challenge, 160 fish were subdivided into 8 batches of the analysis of gene expression (Section 2.7). 20 turbot each. Turbot from two tanks (two replicates per treat- ment) were then i.m. injected, in the same way as was done in the 2.7. RNA extraction, cDNA synthesis and quantitative PCR analysis previous section, with a volume of 50 ml of one of the following treatments: 2.5 mg of pMCV1.4-ifng, 2.5 mg of pMCV1.4 (empty Total RNA from the different tissue samples was extracted using plasmid) and PBS. After 2 days, the individuals were i.p. injected the Maxwell 16 LEV simplyRNA Tissue kit (Promega) with the 5 with a dose of VHSV of 5 10 TCID50/fish. The two remaining automated Maxwell 16 Instrument in accordance with instructions groups were first i.m. inoculated with PBS and then i.p. with the provided by the manufacturer. The cDNA synthesis was performed viral medium and served as an absolute control (non-immunised with the SuperScript II Reverse Transcriptase (Invitrogen) using and non-infected groups). The same experimental procedure was 0.5 mgofRNA. conducted with the Gram-negative bacterium A. salmonicida using The expression profiles of ifng and genes related with the ac- a dose of 5 106 CFU/ml and the corresponding control batches tivity of Ifng, as well as the quantification of the VHSV glycoprotein

Fig. 1. (A) Nucleotide and amino acid sequence of turbot ifng. The predicted signal peptide is underlined, whereas the nuclear localization signal (NLS) is boxed. (B) Comparison of the predicted tertiary structure of turbot Ifng (color cartoon view) with human IFN-gamma (blue ribbon diagram). (C) Promotor region of turbot ifng gene showing three potential GAS and one ISRE sequences. 28 P. Pereiro et al. / Fish & Shellfish Immunology 59 (2016) 25e35 or A. salmonicida, were determined using real-time quantitative 1 min). Relative expression data were normalized using the PCR (qPCR). Specific qPCR primers for turbot genes were designed eukaryotic translation elongation factor 1 alpha (eef1a) as reference based on sequences obtained in a 454-pyrosequencing of turbot gene, which is a good candidate for qPCR data normalization in fish tissues [31] or in the turbot genome [20] by using the Primer3 and fish cell lines under infection conditions [34e37], and calcu- program [32]. Primer sequences are listed in Supplementary data lated using the Pfaffl method [33]. Table 1. Their amplification efficiency was calculated using seven serial five-fold dilutions of head kidney cDNA from unstimulated 2.8. Statistical analysis turbot with the Threshold Cycle (CT) slope method [33] and the identity of the amplicons was confirmed by sequencing. Individual Expression results were represented graphically as the real-time PCR reactions were carried out in 25 ml reaction volume mean þ the standard deviation of the biological replicates. In order using 12.5 ml of SYBR GREEN PCR Master Mix (Applied Biosystems), to determine statistical differences, data were analyzed with the 10.5 ml of ultrapure water (Sigma-Aldrich), 0.5 ml of each specific computer software package SPSS v.19.0 using the Student's t-test. primer (10 mM) and 1 mloffive-fold diluted cDNA template in MicroAmp optical 96-well reaction plates (Applied Biosystems). All 3. Results reactions were performed using technical triplicates in a 7300 Real- Time PCR System thermocycler (Applied Biosystems) with an initial 3.1. Characterization of the turbot ifng gene and protein analysis denaturation (95 C,10 min) followed by 40 cycles of a denaturation step (95 C, 15 s) and one hybridization-elongation step (60 C, The complete coding region of turbot ifng, submitted to

Fig. 2. Phylogenetic tree showing the evolutionary relationships between vertebrate IFN-gamma and IFN-gamma related proteins. Only nodal bootstrap values lower than 95 are shown. P. Pereiro et al. / Fish & Shellfish Immunology 59 (2016) 25e35 29

Fig. 3. Turbot ifng constitutive expression in different tissues obtained from healthy fish.

GenBank under accession number KX360748, consists of 609 bp localization signal was identified in the protein sequence. We also encoding a protein of 202 amino acids (aa) (Fig. 1A). The first 22 aa examined the tridimensional structure of the turbot Ifng and, as belong to the signal peptide and therefore, the mature peptide is expected due to its high structural similarity with human IFN- composed by 180 residues, with a calculated molecular weight of gamma (TM-score ¼ 0.741), it is mainly alpha helical (Fig. 1B). 20.36 kDa and an isoelectric point of 10.25. Moreover, a nuclear As in other vertebrates, the genomic sequence turbot ifng is

Fig. 4. (A) Induction of the expression of turbot ifng 2 days after the administration of the expression plasmids encoding both type I IFNs (pMCV1.4-ifn1 or pMCV1.4-ifn2) in muscle (site of injection) and head kidney or (B) at different sampling points after a VHSV or A. salmonicida challenge in head kidney samples. Significant differences are displayed as ** (0.001 < p < 0.01), * (0.01 < p < 0.05). 30 P. Pereiro et al. / Fish & Shellfish Immunology 59 (2016) 25e35 composed of 4 exons and 3 introns. A detailed analysis of the promoter region (in a range of 1824 bp upstream) revealed that its expression is putatively regulated by three interferon-gamma activated sites (GAS) and one interferon-stimulated response element (ISRE) (Fig. 1C). Concurring with the taxonomic classification, the phylogenetic analysis revealed that turbot Ifng was closely related to Ifng pro- teins from other flatfish species (Fig. 2). As expected, Ifngrel pro- teins formed a separated cluster from the teleost and mammalian Ifng. Nevertheless, teleost Ifng and Ifngrel appear more closely related between them than to vertebrate IFN-gamma proteins (Fig. 2).

3.2. Constitutive expression of ifng in different tissues and induction of its transcription

The higher basal expression of turbot ifng was detected in PEC, followed by spleen (Fig. 3). A similar expression level was observed in gill, trunk kidney and head kidney. The remaining tissues showed a lower ifng transcription. The i.m. administration of the two expression plasmids encod- ing type I IFNs (pMCV1.4-ifn1 and pMCV1.4-ifn2), revealed that Ifn1 was able to significantly induce the expression of ifng both in muscle (Fold-Change ¼ 2.5) and in head kidney (FC ¼ 3.4), whereas Ifn2 induced a significant increase only in the site of injection (FC ¼ 1.4) (Fig. 4A). Notably, turbot ifng was highly modulated during viral and bacterial challenges (Fig. 4B). The i.p. injection of VHSV significantly induced the expression of this gene at 24 (FC ¼ 233) and 72 h post- infection (hpi) (FC ¼ 155). A. salmonicida challenge also induced significant modulation of ifng: higher expression was already detected early at 8 hpi (FC ¼ 20.5), which decreased at 24 hpi (FC ¼ 4.2), and rose again to its highest expression at 72 hpi (FC ¼ 131).

3.3. ifng expression plasmid (pMCV1.4-ifng) does not protect turbot against VHSV or A. salmonicida infection

Local overexpression of ifng was induced with the i.m. injection of 2.5 mg of pMCV1.4-ifng, and the increased expression was confirmed 48 h post-inoculation (Fig. 5A). Despite having a 470- fold higher expression of ifng than control individuals inoculated with the empty plasmid, pMCV1.4-ifng stimulated fish showed a similar pathogen proliferation during early infection stages and not significant differences were observed in the survival rates against VHSV (Fig. 5B) and A. salmonicida (Fig. 5C).

Fig. 5. (A) Expression of turbot ifng in muscle (site of injection) 2 days after the 3.4. Pathogen-dependent role of Ifng in macrophage-related administration of pMCV1.4-ifng compared to those individuals receiving the empty molecules expression, type I IFNs induction, and inflammation plasmid pMCV1.4. (B) Cumulative mortality (%) after a VHSV challenge in turbot pre- viously injected in muscle with PBS, pMCV1.4 or pMCV1.4-ifng, and VHSV detection by At 48 h after the administration of the expression plasmid qPCR in head kidney samples 24 h after infection. (C) Cumulative mortality (%) after an A. salmonicida challenge in turbot previously injected in muscle with PBS, pMCV1.4 or pMCV1.4-ifng, the modulation of sixteen immune-related genes pMCV1.4-ifng, and bacteria detection by qPCR in head kidney samples 24 h after typically related with the IFN-gamma activity was analyzed in head infection. kidney samples by qPCR. Interestingly, only macrophage-colony stimulating factor (csf1) was found to be significantly overexpressed (FC ¼ 3.33) compared to the pMCV1.4-injected individuals (Fig. 6). compared to pMCV1.4 e VHSV fish (Fig. 7A): il1b (from FC ¼ 6.5 Nevertheless, the most surprising results on the effect of ifng only with virus to FC ¼ 17.96 with Ifng and virus), il15 (from overexpression were observed during the viral and bacterial in- FC ¼ 1.75 to FC ¼ 4.53), interleukin-17 (il17) (from FC ¼ 1.64 to fections. VHSV challenge (pMCV1.4 e VHSV) significantly induced FC ¼ 14.15), ifn1 (from FC ¼ 29.43 to FC ¼ 3256) and ifn2 (from the expression of several immune-related genes compared to non- FC ¼ 5toFC¼ 52.36). On the other hand, ifng overexpression also infected fish: pyd and card domain containing (asc), caspase-1 had a detrimental effect in the expression of macrophage receptor (casp1), interleukin-1beta (il1b), interleukin-15 (il15), interferon with collagenous structure (marco), csf1, nadph oxidase 1 (nox1) and regulatory factor 1 (irf1), ifn1 and ifn2, and major histocompatibility mhc-II in VHSV-infected fish, significantly reducing their expression complex-class II (mhc-II)(Fig. 7A). Interestingly, ifng (pMCV1.4-ifng compared to the pMCV1.4 e VHSV individuals (Fig. 7A). In order to e VHSV fish) showed a synergic induction of immune-related genes discard the potential migration of macrophages to the site of P. Pereiro et al. / Fish & Shellfish Immunology 59 (2016) 25e35 31

Fig. 6. Expression analysis of several immune genes in head kidney samples 2 days after the i.m. injection of the expression plasmid pMCV1.4-ifng. Fold-changes were calculated by dividing the normalized expression values for the pMCV1.4-ifng inoculated turbot by the normalized expression values for the pMCV1.4- injected individuals (control group). Significant differences were displayed as * (p < 0.05). injection of the expression plasmid pMCV1.4-ifng, the transcription terminus, which is needed for the translocation into the nucleus of marco was analyzed in the muscle samples, but no significant and the bioactivity of this molecule [40]. differences were observed compared to the control individuals Three GAS and one ISRE sequences were found on the promotor (data not shown). region of the turbot ifng, which indicate that this gene can be After A. salmonicida challenge, numerous genes were also activated via the interferon-gamma- or type I IFNs-induced Jak-Stat overexpressed (csf1, asc, casp1, il1b, il17, irf1, and mhc-II)(Fig. 7B). signaling pathway [41]. Indeed, turbot ifng expression was signifi- Strikingly, in this case, the expression of some genes which were cantly induced by the i.m. administration of the two expression potentiated by Ifng during a viral infection was found to be down- plasmids encoding Ifn1 and Ifn2, two different type I IFNs. Agreeing regulated by ifng overexpression: il1b (from FC ¼ 28.56 only with with previous studies about the activity of turbot type I IFNs [4], bacteria to FC ¼ 8.09 with Ifng and bacteria), il17 (from FC ¼ 5.82 to Ifn1 showed a higher and broader activation of ifng transcription, FC ¼ 0.97) and ifn1 (from FC ¼ 2.3 to FC ¼ 0.54), together with other whereas Ifn2 activity was localized to the site of injection. ifng was genes such as casp1, irf1 and major histocompatibility complex-class I also overexpressed after pathogen challenge with both virus and (mhc-I)(Fig. 7B). On the contrary, marco and macrophage mannose bacteria. Type I IFNs are the main cytokines orchestrating the receptor 1 (mrc1) were significantly more expressed in those in- antiviral defense through the induction of numerous interferon- dividuals previously injected with pMCV1.4-ifng (Fig. 7B). stimulated genes (ISGs) [6]. However, whereas type I IFNs are In order to facilitate the comprehension of all these results, a pivotal in acute infections, IFN-gamma also contributes to the de- schematic representation is provided (Fig. 7C). The first boxes fense against viruses, but especially during long-term infections indicate the effect of a VHSV or A. salmonicida challenge in the [42]. Knockout murine models revealed that the absence of IFN- expression of the tested genes. The second part indicates the effect gamma or the corresponding receptors generates deficiencies in of the previous administration of Ifng in the gene modulation natural resistance to different viruses [43e47].Deficiencies in the during a viral or bacterial infection compared to those individuals immune response were also observed against bacterial infections not receiving the expression plasmid. [48e52]. Therefore, IFN-gamma seems to be important in the cor- rect resolution of both viral- and bacterial-caused diseases. 4. Discussion Nevertheless, the injection of an expression plasmid encoding turbot Ifng was not able to reduce the mortality induced by VHSV or Since the first teleost ifng gene was detected in the fugu (Taki- A. salmonicida infections, although we cannot rule out that lower fugu rubripes) genome several years ago [38] ifng genes were doses of pathogens could reveal any significant difference. More- characterized in numerous fish species. However, the absence of over, only csf1 gene was significantly up-regulated in head kidney 2 sequences for this gene in turbot hindered its identification until days after the administration of pMCV1.4-ifng. Similar results were now. The turbot (S. maximus) genome, recently sequenced [20], has observed in adult zebrafish injected with recombinant Ifng; this led us to the first-time identification of an ifng gene in this flatfish. molecule had a weak effect on the expression of immune genes and The additional fish type II IFN (ifngrel), mainly characterized in did not reduce the mortality caused by bacterial (Streptococcus numerous cyprinid species [10,12,14,39], appears to be absent in iniae) or viral (SVCV) infections [18]. Grayfer and Belosevic [53] turbot, as was observed in other teleosts. ifng gene encodes a hypothesized that this lack of response could be due to the fact protein composed of 202 aa, and contains a 22 aa signal peptide on that Ifng is bounded up by cells expressing only one interferon- the N-terminus and a nuclear localization signal (NLS) near its C- gamma receptor (Ifngr1) and not both receptors (Ifngr1 and 32 P. Pereiro et al. / Fish & Shellfish Immunology 59 (2016) 25e35

Fig. 7. Expression analysis of immune genes in head kidney samples 24 h after the infection with VHSV (A)orA. salmonicida (B)infish previously injected with the empty plasmid (pMCV1.4) or the expression plasmid (pMCV1.4-ifng). Fold-changes were calculated by dividing the normalized expression values for the infected turbot by the normalized expression values for the uninfected pMCV1.4-injected individuals (control group). ### (0.0001 < p < 0.001), ## (0.001 < p < 0.01) and # (0.01 < p < 0.05) represent gene modulations due to the effect of the infection itself (e.g. pMCV1.4-VHSV and pMCV1.4-A. salmonicida vs. pMCV1.4). *** (0.0001 < p < 0.001), ** (0.001 < p < 0.01) and * (0.01 < p < 0.05) represent differences between individuals previously receiving the expression plasmid (pMCV1.4-ifng) or the empty plasmid (pMCV1.4) and then infected (C) Schematic representation of the significant modulations observed in this experiment.

Ifngr2), indicating a localized nature of the zebrafish Ifng. The empty plasmid. These genes corresponded to pro-inflammatory majority of experiments performed in mammals and fish using cytokines (il1b, il15, il17) and both type I IFNs (ifn1 and ifn2). Sur- recombinant IFN-gamma were conducted in vitro and, in these prisingly, on the contrary, il1b, il17 and ifn1 (together with casp1, cases, significant immune effects were observed, especially in irf1 and mhc-I) were lower expressed after an A. salmonicida chal- macrophages and neutrophils [13,16,17,19,54e57]. Probably the lenge in those individuals previously stimulated with pMCV1.4- immune effects of recombinant Ifng in vivo are more subtle and ifng. Therefore, although pMCV1.4-ifng did not elicit a protective dependent on the doses and protocols of inoculation, among effect in VHSV- or Aeromonas-infected turbot, its i.m. injection was others. able to specifically modulate the expression of different pro- After infection with VHSV some immune genes were signifi- inflammatory cytokines and type I IFNs in an opposite way cantly higher expressed in those individuals previously inoculated depending on the pathogen. with the ifng expression plasmid than in those injected with the Classically, IFN-gamma has been regarded as a pivotal pro- P. Pereiro et al. / Fish & Shellfish Immunology 59 (2016) 25e35 33 inflammatory cytokine in inflammation and autoimmune diseases ligand, PRRs initiate intracellular signaling cascades with the [9]. However, during the last two decades numerous evidences objective to produce an adequate response to the stimulus that supported the idea of a dual role of IFN-gamma genes in inflam- activated them [72]. The specificinflammatory response is both mation [8,9], highlighting the complexity of the immune mecha- dependent on the PAMP that was detected and the cell type that nisms in which this molecule is implicated. Although type I IFNS are was activated, that is, viral PAMPs are recognized by different PRRs the main anti-viral signaling molecules, IFN-gamma also seems to than those recognizing bacterial components and use different be essential in the resolution of viral diseases [43e47]. The activity intracellular pathways that lead to the transcription of different of IFN-gamma includes the activation of a subset of IFN-gamma inflammatory genes and the activation of different immune cell inducible genes, such as the transcription factor IRF1 [58], which subsets [2]. This differential activation of the cellular immune is able to activate type I IFN gene promoters and induce type I IFN response, together with the fact that each pathogen possesses genes [59]. Therefore, in the case of VHSV infection, the effect of ifng preferences for its own target cells, culminates in a pathogen- overexpression could be expected, acting in synergy with the virus specific immune response [73]. Although more research is and potentiating the inflammation and expression of type I IFNs. needed, the dual role of Ifng that we demonstrate in this article Regarding the effect of IFN-gamma in bacterial infections, the ob- could be due to the differential effect that this molecule exerts on servations conducted in murine models are contradictory, attrib- the different already activated immune cell populations. uting to this protein a pro-inflammatory [60,61] or anti- inflammatory role [62,63] depending on the bacteria and experi- 5. Conclusions mental design. MARCO and MRC1 are receptors typically expressed in macro- We can resume all these results into three main points: I) Ifng phages and implicated in the clearance of pathogens [64e66]. The potentiated inflammation during viral infection, but it had anti- transcription of marco and mrc1 was significantly down-regulated inflammatory effects during bacterial disease; II) Ifng administra- after bacterial infection but restored in those fish also receiving tion had a synergic or detrimental effect in the transcription of type the expression plasmid encoding Ifng. The increase in the tran- I IFNs depending on the pathogen; III) Ifng seemed to favor the scription of marco and mrc1 by Ifng seems to indicate a positive expression of those genes directly related with the activity of effect in the macrophages activity during bacterial infection. In macrophages in A. salmonicida-infected turbot, but the opposite contrast, the opposite behavior was observed during VHSV infec- effect was observed in VHSV-infected individuals. tion, in which Ifng reduced the transcription of marco, csf1 and Further research is needed in order to elucidate these gene nox1. csf1 is implicated in the proliferation of macrophages [67], regulations, probably by using a model organism such as the and nox1 is an enzyme mediating the respiratory burst in macro- zebrafish. Given that in this study viral and bacterial infections phages and neutrophils, a potent microbicidal mechanism [68]. were conducted in parallel, using the same experimental design Therefore, the reduction in the expression of these genes could be and cohort of fish, this is the first time that the dual role of IFN- reflecting that Ifng exerts a suppressive effect in the macrophages gamma has been clearly exposed. activity when the individuals are suffering a viral infection. It has been reported that macrophages are probably the target Acknowledgements cells of the rhabdovirus infections, at least during the first stages of infection, and that they are destroyed through virus-induced This work has been funded by the projects CSD2007-00002 pyroptosis [69,70]. Due to the effect of Ifng during VHSV infection “Aquagenomics”, 201230E057 (CSIC) and AGL2014-51773-C3 from (down-regulation of marco and csf1, and overexpression of pro- the Spanish Ministerio de Economía y Competitividad. P. Pereiro inflammatory genes), we thought about the possibility that Ifng received a predoctoral grant from the gs3:Ministerio de Educacion was favoring the death of the macrophages. For that reason, we [F.P.U. fellowship AP2010-2408]. checked the transcriptomic modulation of asc and casp1, two genes encoding for key components of the pyroptosis process. However, Appendix A. Supplementary data no significant increases in the expression of both genes were observed in those turbot pre-treated with pMCV1.4-ifng compared Supplementary data related to this article can be found at http:// to the individuals injected with the empty plasmid. Therefore, Ifng dx.doi.org/10.1016/j.fsi.2016.10.021. could be directly modulating the expression of marco and csf1.It fi was reported that mutant mice de cient in MARCO (MARCO / ) References had lower morbidity and mortality caused by influenza pneumonia than wild-type mice because MARCO suppresses the early inflam- [1] V. Fensterl, G.C. Sen, Interferons and viral infections, Biofactors 35 (2009) matory response against the virus [71]. Therefore, if Ifng reduces 14e20. [2] O. Takeuchi, S. Akira, Pattern recognition receptors and inflammation, Cell 140 the expression of marco in turbot during a viral infection, the higher (2010) 805e820. level of pro-inflammatory cytokines could be due to the effect of [3] J. Zou, C.J. Secombes, Teleost fish interferons and their role in immunity, Dev. Marco in the inflammatory response. This is also in accordance with Comp. Immunol. 35 (2011) 1376e1387. [4] P. Pereiro, M.M. Costa, P. Díaz-Rosales, S. Dios, A. Figueras, B. Novoa, The first the activity of Ifng during the bacterial challenge, because marco characterization of two type I interferons in turbot (Scophthalmus maximus) was overexpressed in pre-treated fish compared to the individuals reveals their differential role, expression pattern and gene induction, Dev. only inoculated with the bacteria, and the pro-inflammatory cy- Comp. Immunol. 45 (2014) 233e244. tokines were down-regulated in this case. We hypothesize that [5] U. Boehm, T. Klamp, M. Groot, J.C. Howard, Cellular responses to interferon- gamma, Annu. Rev. Immunol. 15 (1997) 749e795. marco could be directly modulated by Ifng and, as consequence, this [6] C.E. Samuel, Antiviral actions of interferons, Clin. Microbiol. Rev. 14 (2001) can have an effect in the inflammation. 778e809. The innate immune system's recognition of pathogens is pre- [7] K. Schroder, P.J. Hertzog, T. Ravasi, D.A. Hume, Interferon-gamma: an over- view of signals, mechanisms and functions, J. Leukoc. Biol. 75 (2004) 163e189. dominantly mediated by a limited range of genome-coded pattern [8] H. Mühl, J. Pfeilschifter, Anti-inflammatory properties of pro-inflammatory recognition receptors (PRRs). PRRs are activated by the recognition interferon-gamma, Int. Immunopharmacol. 3 (2003) 1247e1255. of pathogen-associated molecular patterns (PAMPs), such as bac- [9] J. Zhang, Yin and yang interplay of IFN-gamma in inflammation and autoim- mune disease, J. Clin. Invest. 117 (2007) 871e873. terial and fungal glycoproteins and lipopolysaccharides or viral [10] D. Igawa, M. Sakai, R. Savan, An unexpected discovery of two interferon components [2]. Once activated by the recognition of its specific gamma-like genes along with interleukin (IL)-22 and -26 from teleost: IL-22 34 P. Pereiro et al. / Fish & Shellfish Immunology 59 (2016) 25e35

and -26 genes have been described for the first time outside mammals, Mol. anterior kidney leucocytes, BMC Mol. Biol. 11 (2010) 36. Immunol. 43 (2006) 999e1009. [37] C.G. Yang, X.L. Wang, J. Tian, W. Liu, F. Wu, M. Jiang, H. Wen, Evaluation of [11] I. Milev-Milovanovic, S. Long, M. Wilson, E. Bengten, N.W. Miller, reference genes for quantitative real-time RT-PCR analysis of gene expression V.G. Chinchar, Identification and expression analysis of interferon gamma in Nile tilapia (Oreochromis niloticus), Gene 527 (2013) 183e192. genes in channel catfish, Immunogenetics 58 (2006) 70e80. [38] J. Zou, Y. Yoshiura, J.M. Dijkstra, M. Sakai, M. Ototake, C. Secombes, Identifi- [12] E.H. Stolte, H.F. Savelkoul, G. Wiegertjes, G. Flik, B.M. Lidy Verburg-van cation of an interferon gamma homologue in Fugu, Takifugu rubripes, Fish Kemenade, Differential expression of two interferon-gamma genes in com- Shellfish Immunol. 17 (2004) 403e409. mon carp (Cyprinus carpio L.), Dev. Comp. Immunol. 32 (2008) 1467e1481. [39] Y. Shibasaki, T. Yabu, K. Araki, N. Mano, H. Shiba, T. Moritomo, T.Nakanishi, [13] L. Grayfer, M. Belosevic, Molecular characterization, expression and functional Peculiar monomeric interferon gammas, IFNgrel 1 and IFNgrel 2, in ginbuna analysis of goldfish (Carassius auratus L.) interferon gamma, Dev. Comp. crucian carp, FEBS J. 281 (2014) 1046e1056. Immunol. 33 (2009) 235e246. [40] H.M. Johnson, B.A. Torres, M.M. Green, B.E. Szente, K.I. Siler, J. Larkin 3rd, [14] W.Q. Chen, Q.Q. Xu, M.X. Chang, J. Zou, C.J. Secombes, K.M. Peng, P. Nie, P.S. Subramaniam, Cytokine-receptor complexes as chaperones for nuclear Molecular characterization and expression analysis of the IFN-gamma related translocation of signal transducers, Biochem. Biophys. Res. Commun. 244 gene (IFN-gammarel) in grass carp Ctenopharyngodon idella, Vet. Immunol. (1998) 607e614. Immunopathol. 134 (2010) 199e207. [41] C. Schindler, D.E. Levy, T. Decker, JAK-STAT signaling: from interferons to [15] D. Sieger, C. Stein, D. Neifer, A.M. van der Sar, M. Leptin, The role of gamma cytokines, J. Biol. Chem. 282 (2007) 20059e20063. interferon in innate immunity in the zebrafish embryo, Dis. Model Mech. 2 [42] R. Shtrichman, C.E. Samuel, The role of gamma interferon in antimicrobial (2009) 571e581. immunity, Curr. Opin. Microbiol. 4 (2001) 251e259. [16] L. Grayfer, E.G. Garcia, M. Belosevic, Comparison of macrophage antimicrobial [43] M.F. van den Broek, U. Muller, S. Huang, R.M. Zinkernagel, M. Aguet, Immune responses induced by type II interferons of the goldfish (Carassius auratus L.), defence in mice lacking type I and/or type II interferon receptors, Immunol. J. Biol. Chem. 285 (2010) 23537e23547. Rev. 148 (1995) 5e18. [17] J.A. Arts, E.J. Tijhaar, M. Chadzinska, H.F. Savelkoul, B.M. Verburg-van Keme- [44] E.B. Cantin, B. Tanamachi, H. Openshaw, Role for gamma interferon in control nade, Functional analysis of carp interferon-gamma: evolutionary conserva- of herpes simplex virus type 1 reactivation, J. Virol. 73 (1999) 3418e3423. tion of classical phagocyte activation, Fish Shellfish Immunol. 29 (2010) [45] A. Nansen, T. Jensen, J.P. Christensen, S.O. Andreasen, C. Ropke,€ O. Marker, 793e802. A.R. Thomsen, Compromised virus control and augmented perforin-mediated [18] A. Lopez-Mu noz,~ F.J. Roca, J. Meseguer, V. Mulero, New insights into the immunopathology in IFN-gamma-deficient mice infected with lymphocytic evolution of IFNs: zebrafish group II IFNs induce a rapid and transient choriomeningitis virus, J. Immunol. 163 (1999) 6114e6122. expression of IFN-dependent genes and display powerful antiviral activities, [46] S.M. van Schaik, N. Obot, G. Enhorning, K. Hintz, K. Gross, G.E. Hancock, J. Immunol. 182 (2009) 3440e3449. A.M. Stack, R.C. Welliver, Role of interferon gamma in the pathogenesis of [19] J. Zou, A. Carrington, B. Collet, J.M. Dijkstra, Y. Yoshiura, N. Bols, C. Secombes, primary respiratory syncytial virus infection in BALB/c mice, J. Med. Virol. 62 Identification and bioactivities of IFN-gamma in rainbow trout Oncorhynchus (2000) 257e266. mykiss: the first Th1-type cytokine characterized functionally in fish, [47] U. Dittmer, K.E. Peterson, R. Messer, I.M. Stromnes, B. Race, K.J. Hasenkrug, J. Immunol. 175 (2005) 2484e2494. Role of interleukin-4 (IL-4), IL-12, and gamma interferon in primary and [20] A. Figueras, D. Robledo, A. Corvelo, M. Hermida, P. Pereiro, J.A. Rubiolo, vaccine-primed immune response to Friend retrovirus infection, J. Virol. 75 J. Gomez-Garrido, L. Carrete, X. Bello, M. Gut, I.G. Gut, M. Marcet-Houben, (2001) 654e660. G. Forn-Cuní, B. Galan, J.L. García, J.L. Abal-Fabeiro, B.G. Pardo, X. Taboada, [48] N.A. Buchmeier, R.D. Schreiber, Requirement of endogenous interferon- C. Fernandez, A. Vlasova, A. Hermoso-Pulido, R. Guigo, J.A. Alvarez-Dios, gamma production for resolution of Listeria monocytogenes infection, Proc. A. Gomez-Tato, A. Vinas,~ X. Maside, T. Gabaldon, B. Novoa, C. Bouza, T. Alioto, Natl. Acad. Sci. U. S. A. 82 (1985) 7404e7408. P. Martínez, Whole genome sequencing of turbot (Scophthalmus maximus; [49] S. Huang, W. Hendriks, A. Althage, S. Hemmi, H. Bluethmann, R. Kamijo, Pleuronectiformes): a fish adapted to demersal life, DNA Res. 23 (2016) J. Vilcek, R.M. Zinkernagel, M. Aguet, Immune response in mice that lack the 181e192. interferon-gamma receptor, Science 259 (1993) 1742e1745. [21] O. Emanuelsson, S. Brunak, G. von Heijne, H. Nielsen, Locating proteins in the [50] J.L. Flynn, J. Chan, K.J. Triebold, D.K. Dalton, T.A. Stewart, B.R. Bloom, An cell using TargetP, SignalP and related tools, Nat. Protoc. 2 (2007) 953e971. essential role for interferon gamma in resistance to Mycobacterium tubercu- [22] A.N. Nguyen Ba, A. Pogoutse, N. Provart, A.M. Moses, NLStradamus: a simple losis infection, J. Exp. Med. 178 (1993) 2249e2254. Hidden Markov Model for nuclear localization signal prediction, BMC Bio- [51] R. Kamijo, J. Le, D. Shapiro, E.A. Havell, S. Huang, M. Aguet, M. Bosland, inform. 10 (2009) 202. J. Vilcek, Mice that lack the interferon-gamma receptor have profoundly [23] E. Gasteiger, A. Gattiker, C. Hoogland, I. Ivanyi, R.D. Appel, A. Bairoch, ExPASy: altered responses to infection with Bacillus Calmette-Guerin and subsequent the proteomics server for in-depth protein knowledge and analysis, Nucleic challenge with lipopolysaccharide, J. Exp. Med. 178 (1993) 1435e1440. Acids Res. 31 (2003) 3784e3788. [52] J.E. Pearl, B. Saunders, S. Ehlers, I.M. Orme, A.M. Cooper, Inflammation and [24] A. Roy, A. Kucukural, Y. Zhang, I-TASSER: a unified platform for automated lymphocyte activation during mycobacterial infection in the interferon- protein structure and function prediction, Nat. Protoc. 5 (2010) 725e738. gamma-deficient mouse, Cell. Immunol. 211 (2001) 43e50. [25] K. Katoh, K.I. Kuma, H. Toh, T. Miyata, MAFFT version 5: improvement in [53] L. Grayfer, M. Belosevic, Cytokine regulation of teleost inflammatory re- accuracy of multiple sequence alignment, Nucleic Acids Res. 33 (2005) sponses, in: H. Turker (Ed.), New Advances and Contributions to Fish Biology, 511e518. InTech, Rijeka, Croatia, 2012, pp. 59e96. [26] G. Talavera, J. Castresana, Improvement of phylogenies after removing [54] C.F. Nathan, H.W. Murray, M.E. Wiebe, B.Y. Rubin, Identification of interferon- divergent and ambiguously aligned blocks from protein sequence alignments, gamma as the lymphokine that activates human macrophage oxidative Syst. Biol. 56 (2007) 564e577. metabolism and antimicrobial activity, J. Exp. Med. 158 (1983) 670e689. [27] D. Darriba, G.L. Taboada, R. Doallo, D. Posada, ProtTest 3: fast selection of best- [55] K. Kagaya, K. Watanabe, Y. Fukazawa, Capacity of recombinant gamma fit models of protein evolution, Bioinformatics 27 (2011) 1164e1165. interferon to activate macrophages for Salmonella-killing activity, Infect. [28] H. Akaike, A new look at the statistical model identification, IEEE Trans. Immun. 57 (1989) 609e615. Autom. Contr. 19 (1974) 716e723. [56] A. Jeevan, C.T. McFarland, T. Yoshimura, T. Skwor, H. Cho, T. Lasco, [29] S. Guindon, J.F. Dufayard, V. Lefort, M. Anisimova, W. Hordijk, O. Gascuel, New D.N. McMurray, Production and characterization of Guinea pig recombinant algorithms and methods to estimate maximum-likelihood phylogenies: gamma interferon and its effect on macrophage activation, Infect. Immun. 74 assessing the performance of PhyML 3.0, Syst. Biol. 59 (2010) 307e321. (2006) 213e224. [30] A. Rambaut, A. Drummond, FigTree v1.3.1, Institute of Evolutionary Biology, [57] L.F. Marchi, R. Sesti-Costa, M.D. Ignacchiti, S. Chedraoui-Silva, B. Mantovani, University of Edinburgh, Edinburgh, United Kingdom, 2010. http://tree.bio.ed. In vitro activation of mouse neutrophils by recombinant human interferon- ac.uk/software/figtree/. gamma: increased phagocytosis and release of reactive oxygen species and [31] P. Pereiro, P. Balseiro, A. Romero, S. Dios, G. Forn-Cuni, B. Fuste, J.V. Planas, pro-inflammatory cytokines, Int. Immunopharmacol. 18 (2014) 228e235. S. Beltran, B. Novoa, A. Figueras, High-throughput sequence analysis of turbot [58] D.A. Chesler, C.S. Reiss, The role of IFN-gamma in immune responses to viral (Scophthalmus maximus) transcriptome using 454-pyrosequencing for the infections of the central nervous system, Cytokine Growth Factor Rev. 13 discovery of antiviral immune genes, PLos One 7 (2012) e35369. (2002) 441e454. [32] S. Rozen, H.J. Skaletsky, Primer3 on the WWW for general users and for [59] M. Miyamoto, T. Fujita, Y. Kimura, M. Maruyama, H. Harada, Y. Sudo, T. Miyata, biologist programmers, MIMB 132 (2000) 365e386. T. Taniguchi, Regulated expression of a gene encoding a nuclear factor, IRF-1, [33] M.W. Pfaffl, A new mathematical model for relative quantification in real time that specifically binds to IFN-beta gene regulatory elements, Cell 54 (1988) RT-PCR, Nucleic Acids Res. 29 (2001) e45. 903e913. [34] H.C. Ingerslev, E.F. Pettersen, R.A. Jakobsen, C.B. Petersen, H.I. Wergeland, [60] Y. Shinozawa, T. Matsumoto, K. Uchida, S. Tsujimoto, Y. Iwakura, Expression profiling and validation of reference gene candidates in immune K. Yamaguchi, Role of interferon-gamma in inflammatory responses in murine relevant tissues and cells from Atlantic salmon (Salmo salar L.), Mol. Immunol. respiratory infection with Legionella pneumophila, J. Med. Microbiol. 51 (2002) 43 (2006) 1194e1201. 225e230. [35] A.A. Pena,~ N.C. Bols, S.H. Marshall, An evaluation of potential reference genes [61] N. Sawai, M. Kita, T. Kodama, T. Tanahashi, Y. Yamaoka, Y. Tagawa, Y. Iwakura, for stability of expression in two salmonid cell lines after infection with either J. Imanishi, Role of gamma interferon in Helicobacter pylori-induced gastric Piscirickettsia salmonis or IPNV, BMC Res. Notes 3 (2010) 101. inflammatory responses in a mouse model, Infect. Immun. 67 (1999) [36] A.C. Øvergård, A.H. Nerland, S. Patel, Evaluation of potential reference genes 279e285. for real time RT-PCR studies in Atlantic halibut (Hippoglossus hippoglossus L.); [62] H.K. Johansen, H.P. Hougen, J. Rygaard, N. Høiby, Interferon-gamma (IFN- during development, in tissues of healthy and NNV-injected fish, and in gamma) treatment decreases the inflammatory response in chronic P. Pereiro et al. / Fish & Shellfish Immunology 59 (2016) 25e35 35

Pseudomonas aeruginosa pneumonia in rats, Clin. Exp. Immunol. 103 (1996) like receptor 4-mediated NADPH oxidase 1 expression in macrophages, FEBS J. 212e218. 277 (2010) 2830e2837. [63] B. Nandi, S.M. Behar, Regulation of neutrophils by interferon-g limits lung [69] M. Varela, A. Romero, S. Dios, M. van der Vaart, A. Figueras, A.H. Meijer, inflammation during tuberculosis infection, J. Exp. Med. 208 (2011) B. Novoa, Cellular visualization of macrophage pyroptosis and interleukin-1b 2251e2262. release in a viral hemorrhagic infection in zebrafish larvae, J. Virol. 88 (2014) [64] T. Pikkarainen, A. Brannstr€ om,€ K. Tryggvason, Expression of macrophage 12026e12040. MARCO receptor induces formation of dendritic plasma membrane processes, [70] P. Pereiro, S. Dios, S. Boltana,~ J. Coll, A. Estepa, S. Mackenzie, B. Novoa, J. Biol. Chem. 274 (1999) 10975e10982. A. Figueras, Transcriptome profiles associated to VHSV infection or DNA [65] A. Palecanda, L. Kobzik, Receptors for unopsonized particles: the role of vaccination in turbot (Scophthalmus maximus), PLos One 9 (2014) e104509. alveolar macrophage scavenger receptors, Curr. Mol. Med. 1 (2001) 589e595. [71] S. Ghosh, D. Gregory, A. Smith, L. Kobzik, MARCO regulates early inflammatory [66] L. Martinez-Pomares, The mannose receptor, J. Leukoc. Biol. 92 (2012) responses against influenza: a useful macrophage function with adverse 1177e1186. outcome, Am. J. Respir. Cell Mol. Biol. 45 (2011) 1036e1044. [67] E.R. Stanley, L.T. Guilbert, R.J. Tushinski, S.H. Bartelmez, CSF-1 a mononuclear [72] K. Newton, V.M. Dixit, Signaling in innate immunity and inflammation, Cold phagocyte lineage-specific hemopoietic growth factor, J. Cell. Biochem. 21 Spring Harb, Perspect. Biol. 4 (2012) a006049. (1983) 151e159. [73] A. Rivera, M.C. Siracusa, G.S. Yap, W.C. Gause, Innate cell communication kick- [68] J.S. Kim, S. Yeo, D.G. Shin, Y.S. Bae, J.J. Lee, B.R. Chin, C.H. Lee, S.H. Baek, starts pathogen-specific immunity, Nat. Immunol. 17 (2016) 356e363. Glycogen synthase kinase 3beta and beta-catenin pathway is involved in toll-

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Supporting information

Supporting information associated with this article can be found at: http://www.sciencedirect.com/science/article/pii/S10504648163065 32

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CHAPTER 7 GENERAL DISCUSSION AND CONCLUSIONS

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1. GENERAL DISCUSSION

Turbot (S. maximus) is an economically important species extensively aquacultured in Europe and China. Although the production of this flatfish has undergone several improvements in recent years, one of the main threats to the sustainable growth of fish farming is infectious disease (Álvarez-Pellitero, 2008; Toranzo el at., 2005; Walker & Winton, 2010). Viral diseases are especially problematic in the finfish aquaculture industry due to the lack of antiviral therapies and difficulties in developing effective vaccines, among other questions (Dhar et al., 2014). Thus, viral disease outbreaks have caused serious economic losses all around the world. Therefore, it is not surprising that eight of the ten notifiable fish diseases appearing at the 2014 Aquatic Animal Health Code of the OIE (Office International des Epizooties, now the World Organization for Animal Health) (http://www.oie.int) are caused by viruses.

Viral Haemorrhagic Septicaemia Virus (VHSV), a virus affecting turbot production, is included on the OIE list. Knowledge about the immune response of turbot to this disease is of pivotal importance to reduce its prevalence in turbot farms. Unfortunately, until recently, the genomic or transcriptomic information available for this species in public databases was very scarce. Indeed, there were only 12,471 ESTs and fewer than 1,500 nucleotide sequences for S. maximus in NCBI database, most of them providing redundant information, until the publication of the first paper forming part of this thesis (Chapter 2: High- throughput sequence analysis of the turbot (Scophthalmus maximus) transcriptome using 454 pyrosequencing for the discovery of antiviral immune genes). Next-generation sequencing (NGS) technologies provide a fast and cost-effective way to generate a large amount of data from non-model species (Metzker, 2010). The aim of this work was to increase the genomic resources available for turbot, specifically the transcriptome in response to viral stimulation, and to identify the main components of the immune pathways. Our results provided a rich source of data (55,404 contigs and 181,845 singletons) for discovering and identifying new genes.

It is very important to know how turbot respond to infection with VHSV, but understanding what occurs after the vaccination process is also relevant in

135 identifying markers of vaccine efficacy. Due to the absence of effective antiviral treatments against VHSV, prevention is a critical point in eradication of this disease. No vaccines are commercially available for VHSV. During the last decades, several attempts were made to produce a commercially suitable vaccine against VHSV (Adelmann et al, 2008; Bernard et al, 1983; de Kinkelin et al, 1980, 1995; Lecocq-Xhonneux et al, 1994; Leong & Fryer, 1993); however, for different reasons, the transfer of these results to the industry did not occur. DNA vaccines are a powerful tool for designing effective vaccines against fish Rhabdoviruses, especially those encoding the viral membrane glycoprotein (Anderson et al, 1996; LaPatra et al, 2001; Lorenzen et al, 1998, 2000; Winton, 1997). As detailed in Chapter 3 (Protection and antibody response induced by an intramuscular DNA vaccine encoding viral haemorrhagic septicaemia virus (VHSV) G glycoprotein in turbot (Scophthalmus maximus)), we designed a DNA vaccine encoding the G glycoprotein from VHSV strain UK-860/94, and highly promising results were obtained (relative percentage of survival (RPS) over 80%). These results reflect the potential of DNA vaccination strategies in fish aquaculture. Nevertheless, fish immunization with an antigen-encoding DNA vaccine was only approved for commercial use in Canada for IHNV (also a Novirhabdovirus) in farmed salmon (Evensen & Leong, 2013). Currently, no DNA vaccines have been approved for use in aquaculture in Europe.

As described in Chapter 4 (Transcriptome profiles associated to VHSV infection or DNA vaccination in turbot (Scophthalmus maximus)), the transcriptome information obtained in Chapter 2 was used to construct a microarray highly enriched in antiviral sequences to analyse the transcriptome modulations in the head kidney after administration of the DNA vaccine specific for VHSV (Chapter 3) and the response to the virus in vaccinated and non- vaccinated fish. The overall analysis of these data provided interesting information about the genes implicated in the defence mechanisms against viral diseases and led us to focus our attention on some specific genes to be further characterized and studied in detail.

This was the case for type I interferons (IFNs). These cytokines induce the expression of numerous genes (interferon-stimulated genes (ISGs)) that are

136 growth inhibitors with the ability to reduce viral proliferation in the host through different blocking mechanisms or strategies (Sadler & Williams, 2008). For this reason, the IFN system is considered the main antiviral immune response in vertebrates. The microarray results revealed that the expression pattern of two different type I IFNs was quite different after infection with VHSV. Due to this fact and the fact that type I IFNs are the main cytokines orchestrating the antiviral immune response, Chapter 5 (The first characterization of two type I interferons in turbot (Scophthalmus maximus) reveals their differential role, expression pattern and gene induction) describes the first characterization of two type I IFNs in turbot and analysis of their expression and bioactivity. Interestingly, these IFNs (ifn1 and ifn2) showed very different activities. Ifn1 was able to induce the expression of several ISGs and, as a consequence, it induced protection against VHSV. On the other hand, Ifn2 did not induce the expression of ISGs, and it was not able to reduce viral proliferation; however, it had a function more related to immune regulation, as it was mainly involved in the inflammatory process. This is not the first time that different type I IFNs from the same fish species have shown differences in their expression patterns and protective capabilities (Aggad et al., 2009: López-Muñoz et al., 2009; Zou et al., 2007), suggesting complementary or specialized roles.

To complete the characterization of the turbot IFN repertoire, the type II IFN (or IFN-gamma) gene was also analysed in Chapter 6 (Pathogen-dependent role of turbot (Scophthalmus maximus) interferon-gamma). Although no sequences with homology to IFN-gamma were obtained in the high-throughput sequence analysis of the turbot transcriptome described in Chapter 2, the recent publication of the turbot genome (Figueras et al., 2016) provided us with the genomic sequence of the ifng gene. It is well known that IFN-gamma is a markedly different IFN from type I IFNs, possessing some ability to interfere with viral infections but functioning mainly as an immunomodulatory molecule (Boehm et al., 1997; Samuel, 2001). IFN-gamma has been classically described as a pro- inflammatory cytokine, although some anti-inflammatory functions are also associated with this cytokine (Mühl & Pfeilschifter, 2003; Zhang, 2007). The most surprising result obtained in this paper was the observation that, although administration of an expression plasmid encoding turbot Ifng was not able to

137 reduce mortality or pathogen proliferation after viral (VHSV) or bacterial (Aeromonas salmonicida) challenge, this cytokine showed a dual role depending on the type of infection. It potentiated the expression of pro-inflammatory cytokines and type I IFNs during VHSV challenge, but it reduced the transcription of macrophage-related molecules. The opposite effect was observed during infection with A. salmonicida.

2. REFERENCES

Adelmann M, Köllner BK, Bergmann SM, Fischer U, Lange B, Weitschies W, Enzmann PJ, Fichtner D (2008) Development of an oral vaccine for immunisation of rainbow trout (Oncorhynchus mykiss) against viral haemorrhagic septicaemia. Vaccine, 26: 837–844.

Aggad D, Mazel M, Boudinot P, Mogensen KE, Hamming OJ, Hartmann R, Kotenko S, Herbomel P, Lutfalla G, Levraud JP ( 2009) The two groups of zebrafish virus-induced interferons signal via distinct receptors with specific and shared chains. J Immunol, 183: 3924–3931.

Álvarez-Pellitero P (2008) Fish immunity and parasite infections: from innate immunity to immunoprophylactic prospects. Vet Immunol Immunopathol, 126: 171–198.

Anderson ED, Mourich DV, Fahrenkrug SC, LaPatra S, Shepherd J, Leong, JA (1996) Genetic immunization of rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis virus. Mol Mar Biol Biotechnol, 5: 114–122.

Bernard J, de Kinkelin P, Bearzotti-Le Berre M (1983) Viral hemorrhagic septicemia of rainbow trout: relation between the G polypeptide and antibody production in protection of the fish after infection with the F25 attenuated variant. Infect Immun, 39: 7–14.

Boehm U, Klamp T, Groot M, Howard JC (1997) Cellular responses to interferon- gamma, Annu Rev Immunol, 15: 749–795.

de Kinkelin P, Bearzotti-Le Berre M, Bernard J (1980) Viral hemorrhagic septicemia of rainbow trout: selection of a thermoresistant virus variant and comparison of polypeptide synthesis with the wild-type virus strain. J Virol, 36: 652–658.

138

de Kinkelin P, Bearzotti-Le Berre M, Castric J, Nougayrède P, Lecocq-Xhonneux F, Thiry M (1995) Eighteen years of vaccination against viral haemorrhagic septicaemia in France. Vet Res, 26: 379–387.

Dhar AK, Manna SK, Allnutt FCT (2014) Viral vaccines for farmed finfish. Virusdisease, 25: 1–17.

Evensen Ø, Leong JA (2013) DNA vaccines against viral diseases of farmed fish. Fish Shellfish Immunol, 35: 1751–1758.

Figueras A, Robledo D, Corvelo A, Hermida M, Pereiro P, Rubiolo JA, Gómez- Garrido J, Carreté L, Bello X, Gut M, Gut IG, Marcet-Houben M, Forn-Cuní G, Galán B, García JL, Abal-Fabeiro JL, Pardo BG, Taboada X, Fernández C, Vlasova A, Hermoso-Pulido A, Guigó R, Álvarez-Dios JA, Gómez-Tato A, Viñas A, Maside X, Gabaldón T, Novoa B, Bouza C, Alioto T, Martínez P (2016) Whole genome sequencing of turbot (Scophthalmus maximus; Pleuronectiformes): a fish adapted to demersal life. DNA Res, 23: 181–192.

LaPatra SE, Corbeil S, Jones GR, Shewmaker WD, Lorenzen N, Anderson E.D., Kurath G (2001) Protection of rainbow trout against infectious hematopoietic necrosis virus four days after specific or semi-specific DNA vaccination. Vaccine, 19: 4011–4019.

Lecocq-Xhonneux F, Thiry M, Dheur I, Rossius M, Vanderheijden N, Martial J, de Kinkelin P (1994) A recombinant viral haemorrhagic septicaemia virus glycoprotein expressed in insect cells induces protective immunity in rainbow trout. J Gen Virol, 75: 1579–1587.

Leong JC, Fryer JL (1993) Viral vaccines for aquaculture. Annu Rev Fish Dis, 3: 225– 240.

López-Muñoz A, Roca FJ, Meseguer J, Mulero V (2009) New insights into the evolution of IFNs: zebrafish group II IFNs induce a rapid and transient expression of IFN- dependent genes and display powerful antiviral activities. J Immunol, 182: 3440–3449.

Lorenzen E, Einer-Jensen K, Martinussen T, LaPatra SE, Lorenzen N (2000) DNA vaccination of rainbow trout against Viral Hemorrhagic Septicemia Virus: A dose-response and time-course study. J Aquat Anim Health, 12: 167–180.

Lorenzen N, Lorenzen E, Einer-Jensen K, Heppell J, Wu T, Davis H (1998) Protective immunity to VHS in rainbow trout (Oncorhynchus mykiss, Walbaum) following DNA vaccination. Fish Shellfish Immunol, 8: 261–270.

139

Metzker ML (2010) Sequencing technologies - the next generation. Nat Rev Genet, 11: 31–46.

Mühl H, Pfeilschifter J (2003) Anti-inflammatory properties of pro-inflammatory interferon-gamma. Int Immunopharmacol, 3: 1247–1255.

Sadler AJ, Williams BRG (2008) Interferon-inducible antiviral effectors. Nat Rev Immunol, 8: 559–568.

Samuel CE (2001) Antiviral actions of interferons. Clin Microbiol Rev, 14: 778–809.

Toranzo AE, Magariños B, Romalde JL (2005) A review of the main bacterial fish diseases in mariculture systems. Aquaculture, 246: 37–61.

Walker PJ, Winton JR (2010) Emerging viral diseases of fish and shrimp. Vet Res, 41: 51–75.

Winton JR (1997) Immunization with viral antigens: infectious haematopoietic necrosis. Dev Biol Stand, 90: 211–220.

Zhang J (2007) Yin and yang interplay of IFN-gamma in inflammation and autoimmune disease. J Clin Invest, 117: 871–873.

Zou J, Tafalla C, Truckle J, Secombes CJ (2007) Identification of a second group of type I IFNs in fish sheds light on IFN evolution in vertebrates. J Immunol, 179: 3859–3871.

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3. CONCLUSIONS

1. The transcriptome information for turbot (S. maximus) was enormously enriched, especially in those sequences related to the antiviral immune response. Most of the components implicated in the main immune pathways were identified for the first time in this fish species. All this information was used to design a microarray.

2. The DNA vaccine designed during this work (pMCV1.4-G) encodes the VHSV glycoprotein (G) and was found to induce a good level of protection against VHSV. Moreover, specific anti-G neutralizing antibodies were detected in fish serum one month after vaccination, indicating activation of the adaptive immune system.

3. Microarray analysis of head kidney samples from vaccinated and non-vaccinated turbot revealed strong activation of the main immune pathways three days after vaccine administration.

4. After VHSV challenge, the transcriptome profile was completely different between vaccinated and non-vaccinated fish. Whereas naïve fish showed an extended and uncontrolled immune response, generating an intense pro- inflammatory state in the host, those individuals previously receiving the vaccine exhibited a moderate and controlled response due to the previous presence of specific immune factors.

5. Two type I IFNs (ifn1 and ifn2), the main cytokines directing the antiviral immune response in vertebrates, were characterized for the first time in turbot. The results indicated non-redundant or complementary roles for these turbot IFNs. Only Ifn1 was able to induce the expression of ISGs and, as a consequence, significantly reduced the mortality of turbot upon VHSV challenge. Ifn2 seemed to act as a regulator of inflammation.

6. Turbot type II IFN (ifng) showed a surprising dual role depending on the type of pathogen (virus or bacteria):

a) Ifng potentiated inflammation during viral infection but had anti-inflammatory effects during bacterial disease.

141 b) Ifng administration had a synergistic effect on the transcription of type I IFNs during VHSV infection, but an inhibitory effect was observed when the animals were inoculated with bacteria. c) Ifng seemed to promote the expression of those genes directly related to the activity of macrophages in A. salmonicida-infected turbot, but the opposite effect was observed in VHSV-infected individuals.

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RESUMEN Y CONCLUSIONES EN ESPAÑOL

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Avances en el conocimiento de la respuesta inmune antiviral y resistencia al Virus de la Septicemia Hemorrágica Viral (VHSV) en rodaballo (Scophthalmus maximus)

1. RESUMEN

Capítulo 1: Introducción General

El rodaballo (Scophthalmus maximus) es un pez plano que posee un alto valor comercial especialmente en Europa y China. En la actualidad su cultivo está bien establecido, llevándose a cabo el ciclo completo principalmente en instalaciones en tierra. No obstante, existen todavía algunas limitaciones que afectan al cultivo de esta especie, como pueden ser ciertas enfermedades, las cuales causan severos episodios de mortalidad y/o morbilidad, con las subsecuentes pérdidas económicas millonarias para el sector acuícola.

El desarrollo de la acuicultura del rodaballo trajo consigo un incremento paralelo de las condiciones patológicas que afectan al cultivo de este pez. Numerosos patógenos, incluyendo bacterias, virus y parásitos pueden afectar al estado sanitario del rodaballo en mayor o menor grado. A pesar de la relevancia del cultivo de esta especie, el conocimiento sobre su sistema inmune es todavía fragmentario y poco se sabe sobre las interacciones patógeno-hospedador. Las rutas implicadas en la respuesta frente a patógenos permanecen incompletas en peces y el comprender cómo actúan estos mecanismos de defensa es un factor relevante a la hora de mejorar la resistencia a enfermedades de los peces cultivados.

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Aunque a día de hoy existen tratamientos efectivos y/o vacunas disponibles frente a una gran variedad de patógenos que afectan al rodaballo, otras enfermedades, especialmente aquellas producidas por virus y endoparásitos, no tienen una solución sencilla. Los virus son probablemente los patógenos más destructivos, ya que para la mayor parte de las enfermedades virales que afectan a peces no existen vacunas ni tratamientos terapéuticos comercialmente disponibles. Para ilustrar el impacto sanitario de los virus basta con mencionar que entre las diez enfermedades de peces de declaración obligatoria que aparecen en el Código Sanitario para los Animales Acuáticos 2014 de la Organización Mundial para la Salud Animal (http://www.oie.int), ocho son causadas por virus.

Entre estas enfermedades de declaración obligatoria se encuentra la producida por el VHSV (Virus de la Septicemia Hemorrágica Viral), el cual ocasiona una importante enfermedad que afecta principalmente a la trucha arcoíris (Oncorhyncus mykiss) y otros salmónidos, aunque también se han detectado brotes de VHSV en otras especies de peces cultivados, como el rodaballo. Los peces que padecen la enfermedad de la septicemia hemorrágica viral presentan una serie de signos clínicos no específicos en fases tempranas de la infección, incluyendo una rápida mortalidad (la cual puede alcanzar el 100% en alevines), letargia, oscurecimiento de la piel, exoftalmia, anemia (palidez branquial), hemorragias en la base de las aletas, branquias, boca, ojos y piel, abdomen distendido debido a la acumulación de líquido ascítico y una conducta natatoria anormal. Los rodaballos infectados desarrollan los signos característicos de la enfermedad y, aunque la tasa de mortalidad debida a infecciones naturales en granjas de rodaballo es relativamente baja, esta enfermedad es de declaración obligatoria, lo que implica tomar medidas especiales en el manejo de la enfermedad que pueden agravar el impacto económico.

VHSV pertenece al género Novirhabdovirus, incluido dentro de la familia Rhabdoviridae. Se trata de virus envueltos, con un genoma de ARN de cadena sencilla el cual codifica para cinco proteínas estructurales básicas – nucleoproteína (N), fosfoproteína asociada a la polimerasa (P), proteína de matriz (M), glicoproteína (G) y ARN polimerasa dependiente del ARN (L) – y una sexta proteína no estructural, la proteína “non-virion” (NV). Existen cuatro genotipos

146 principales de VHSV con distinta distribución geográfica. Los brotes detectados en rodaballo son causados principalmente por la cepa UK-860/94 (Genotipo III). De hecho, esta cepa fue aislada por primera vez de un brote acontecido en una granja de rodaballo en la isla de Gigha (Escocia).

Debido a la ausencia de tratamientos antivirales efectivos la prevención es el punto crítico en la erradicación de esta enfermedad. No obstante, no existen vacunas comerciales disponibles frente a VHSV. Durante las últimas décadas se ha tratado de producir una vacuna eficaz frente a este virus usando proteínas virales así como virus muertos o atenuados y, aunque en algunos casos esas vacunas han resultado ser efectivas en condiciones experimentales, pueden no ser seguras para su uso industrial, su producción puede ser excesivamente cara o se requieren dosis muy altas. Las vacunas de ADN, basadas en la administración de un vector plasmídico que contiene un gen que codifica para un antígeno específico, han demostrado ser una herramienta altamente efectiva para el diseño de vacunas frente a rhabdovirus de peces, especialmente aquellas que codifican para la glicoproteína que constituye la membrana viral.

Otra forma de prevenir, o al menos reducir la prevalencia de una enfermedad, es la mejora genética. La selección asistida por marcadores (MAS, “Marker-assisted selection”) en la cría de peces se ha convertido en una estrategia muy prometedora para obtener individuos con un cierto rasgo de interés, como puede ser la resistencia a enfermedades. En la actualidad la mayoría del trabajo en MAS usa marcadores basados en ADN, los cuales pueden ser usados para detectar variaciones alélicas en los genes que proporcionan un cierto rasgo. Estos rasgos son normalmente controlados por varios genes y las regiones del genoma que contienen los genes relacionados con estos rasgos se conocen como “quantitative trait loci” (QTLs). Los marcadores usados en la selección están asociados en una alta frecuencia con los QTLs de interés debido a la proximidad en el cromosoma, y por lo tanto son co-segregantes (ligamiento genético).

El sistema inmune de los peces teleósteos es fisiológicamente parecido al de los mamíferos, ya que poseen tanto inmunidad innata como adaptativa, aunque existen algunas diferencias. El principal órgano inmune en peces es el riñón anterior que, junto con el bazo, timo y el tejido linfoide asociado a mucosas,

147 representa la infraestructura linfoide en teleósteos. Además, los peces también poseen las principales poblaciones celulares presentes en mamíferos: monocitos, granulocitos, células dendríticas, y linfocitos T y B. En consecuencia, el grueso de la respuesta inmune antiviral en peces parece ser similar a la de los demás vertebrados.

El objetivo de la presente tesis doctoral fue profundizar en la respuesta frente a virus, especialmente el VHSV, en rodaballo. Dado que la principal limitación que existe a la hora de estudiar el mecanismo de defensa frente a patógenos en esta especie es la ausencia de secuencias genómicas o transcriptómicas en las bases de datos públicas, el primer objetivo fue incrementar la información transcriptómica en rodaballo, con especial enriquecimiento en secuencias que codifican para genes antivirales. Esta gran cantidad de información fue empleada en el diseño de un microarray, el cual nos permitió estudiar los principales rasgos de la respuesta frente a VHSV en rodaballo, así como también analizar el perfil transcriptómico tras la administración de una vacuna de ADN frente a este virus, diseñada también durante la realización de esta tesis, y comparar la respuesta diferencial al virus entre individuos vacunados y no vacunados. Este análisis global nos proporcionó valiosa información sobre la inmunidad frente a virus y nos permitió centrar nuestra atención en algunos genes específicos, los cuales fueron estudiados en más detalle. Este fue el caso de los interferones (IFNs) de tipo I, el principal grupo de citoquinas antivirales de los vertebrados, ya que los dos IFNs de tipo I presentes en el microarray respondían de forma diferencial frente a la infección con VHSV. Por ello fueron caracterizados y analizados de una forma más detallada con el fin de elucidar su papel concreto frente a la infección. Finalmente, tras la publicación del genoma del rodaballo en el año 2016, pudimos conocer la secuencia del interferón de tipo II (o IFN-gamma), lo que nos llevó a fijar como último objetivo de esta tesis la caracterización completa del repertorio de IFNs en rodaballo.

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Capítulo 2: Análisis de alto rendimiento del transcriptoma del rodaballo mediante pirosecuenciación 454 para el descubrimiento de genes inmunes antivirales

Como se ha mencionado arriba, el rodaballo es una especie con un importante valor comercial tanto en Europa como en Asia. Sin embargo, hasta la fecha existía poca información en lo que respecta a secuencias genómicas y/o transcriptómicas en las bases de datos públicas. En la actualidad uno de los principales problemas que afectan al cultivo de este pez plano son los episodios de mortalidad debidos a diversos patógenos, especialmente aquellos ocasionados por las enfermedades virales, las cuales no tienen tratamiento comercial disponible. Con el fin de identificar nuevos genes implicados en la defensa inmune, llevamos a cabo una pirosecuenciación 454 (Roche) del transcriptoma. Numerosos individuos fueron inoculados con distintos estímulos de carácter vírico con el fin de incrementar el nivel de expresión de genes relacionados con la respuesta inmune antiviral. Se tomaron muestras de distintos tejidos a distintos tiempos post- estimulación con el fin de enriquecer lo máximo posible la información deseada. Este análisis de alto rendimiento del transcriptoma nos proporcionó 915.256 lecturas (“reads”), que fueron ensambladas en 55.404 contigs, los cuales fueron sometidos a un paso de anotación. Curiosamente, el 55,16% de las proteínas deducidas no fueron anotadas al no encontrase similitud significativa (según los criterios estadísticos establecidos) con ninguna secuencia de las bases de datos usadas en el proceso de anotación. Además, sólo un 0,85% de las secuencias fueron anotadas frente a secuencias proteicas de rodaballo, lo que viene la reflejar la escasa presencia hasta el momento de secuencias de esta especie en las bases de datos. Estos resultados sugieren la identificación de una gran cantidad de nuevos genes en rodaballo, e incluso en peces en general. Un análisis más detallado de esta información nos reveló la presencia de secuencias para una gran parte de las moléculas implicadas en las principales rutas inmunes, tanto innatas como específicas (ruta del complemento, cascada de señalización de los receptores tipo toll, ruta de señalización activada por los receptores de los linfocitos B y T, y apoptosis).

Este estudio supuso el primer análisis transcriptómico en rodaballo usando secuenciación masiva. Antes de llevarlo a cabo había solamente 12.471 expressed

149 sequence tags (ESTs) y menos de 1.500 secuencias nucleotídicas para S. maximus en las bases de datos del NCBI. Nuestros resultados supusieron una rica fuente de información (55.404 contigs y 181.845 singletons) para el descubrimiento e identificación de nuevos genes, los cuales servirán como base para la construcción de un microarray, para la caracterización y análisis de expresión de genes puntuales, así como también para la identificación de marcadores genéticos, entre otras aplicaciones.

Capítulo 3: Inducción de protección y producción de anticuerpos por la inyección intramuscular de una vacuna de ADN que codifica para la glicoproteína G del virus de la septicemia hemorrágica vital (VHSV) en rodaballo (Scophthalmus maximus)

Las vacunas de ADN que codifican para la glicoproteína viral (G) han demostrado ser las más eficaces a la hora de inducir protección frente a Rhabdovirus. Con el fin de evaluar la posibilidad de controlar los brotes de VHSV por medio de una vacuna de ADN, la glicoproteína G de una cepa de VHSV aislada de una granja de rodaballo (UK-860/94) fue clonada en un plásmido de expresión (pMCV1.4) que contiene el promotor del citomegalovirus humano. Bajo nuestras condiciones experimentales, los rodaballos a los que se les administró intramuscularmente el plásmido de expresión (pMCV1.4-G860) mostraron una protección superior al 85% frente a una infección letal con VHSV. Además, se observó que al cabo de un mes, el suero de aquellos individuos que habían sido vacunados mostraba anticuerpos específicos frente a VHSV, los cuales fueron detectados mediante enzyme-linked immunosorbent assay (ELISA). Midiendo la actividad neutralizante del suero se vio que estos anticuerpos presentaban capacidad neutralizante. Este trabajo supuso la primera publicación que mostraba la eficacia de la inmunización genética frente a VHSV en rodaballo.

Capítulo 4: Perfiles transcriptómicos asociados a la infección con VHSV o a la vacunación con una vacuna de ADN en rodaballo

En la actualidad los mecanismos moleculares implicados en la protección frente a patógenos todavía no se comprenden en su totalidad. Con el fin de arrojar algo de luz sobre la protección conferida por la vacuna de ADN basada en la

150 glicoproteína G de VHSV en rodaballo, hemos construido un microarray altamente enriquecido en secuencias antivirales (usando los contigs y algunos singletons obtenidos en el trabajo presentado en el capítulo 2) para llevar a cabo el estudio transcriptómico asociado a la vacunación/infección. El patrón de expresión génica en riñón anterior en respuesta a la inyección intramuscular del plásmido vacío (pMCV1.4) y la vacuna de ADN (pMCV1.4-G860) con respecto a individuos no estimulados (controles inoculados con suero salino) se analizó a 8, 24 y 72 horas post-vacunación. Además, el efecto de la infección con VHSV al cabo de un mes tras la vacunación fue también estudiado en individuos vacunados y no vacunados a los mismos tiempos de muestreo. Los genes implicados en la ruta de señalización de los receptores tipo Toll (Toll-like receptors), los genes inducidos o reguladores de la ruta de los Interferones, numerosas secuencias implicadas en la apoptosis y cascadas citotóxicas, genes relacionados con la presentación de antígenos, así como también en las cascadas del complemento y la coagulación, entre otros, fueron analizados en los diferentes grupos experimentales. Los peces que recibieron la vacuna pMCV1.4-G860 mostraron unos patrones transcriptómicos muy diferentes a los observados en los individuos inyectados con el plásmido vacío tras 72 horas, y con alto grado de similitud a los detectados tras la infección con VHSV. Por otra parte, la infección con VHSV en rodaballos vacunados y no vacunados indujo una respuesta altamente diferente a nivel transcriptómico, indicando la gran relevancia de la inmunidad adquirida o específica en los peces vacunados, capaz de alterar el perfil de respuesta inmune innata típico observado en los individuos no vacunados. Este exhaustivo estudio transcriptómico sirvió pata tener una imagen global completa con el objetivo de comprender mejor las relaciones entre la inmunidad innata y adaptativa en peces tras la infección viral/vacunación. Además, proporciona interesantes pistas sobre moléculas con un uso potencial como adyuvantes de vacunas, tratamientos antivirales o marcadores para monitorizar la eficacia de las vacunas. Lo interesante de este tipo de estudios es que permiten analizar de forma simultánea miles de genes para, a posteriori, centrarse de forma más detallada en moléculas concretas. Uno de los numerosos hallazgos que llamaron nuestra atención tras el análisis de este microarray fue la modulación diferencial de dos interferones (IFNs) de tipo I en rodaballo, ya que mientras uno de ellos se encontraba sobreexpresado tras la infección con VHSV, la

151 expresión del otro estaba inhibida. Por ello, el siguiente paso fue caracterizar y estudiar en más detalle estos dos IFNs de tipo I.

Capítulo 5: La primera caracterización de dos Interferones de tipo I en rodaballo revela sus diferencias en función, patrón de expresión e inducción génica.

Los IFNs de tipo I son considerados las principales citoquinas que dirigen y coordinan la respuesta inmune antiviral en organismos vertebrados. Estas moléculas son capaces de inducir la transcripción de numerosos genes que se conocen como genes estimulados por interferón (interferon-stimulated genes – ISGs) los cuales, usando diferentes mecanismos de bloqueo, reducen la proliferación viral en el hospedador. Además, se ha observado un papel contradictorio de los IFNs en la protección frente a infecciones bacterianas en roedores, incrementando la supervivencia o teniendo un efecto perjudicial dependiendo de la especie de bacteria. En teleósteos se han descrito un número variable de IFNs de tipo I dentro de una misma especie con diferentes patrones de expresión, capacidades de protección, o perfiles de inducción génica, indicando en muchos casos un papel especializado o complementario de los distintos IFNs de tipo I en la inmunidad.

En este trabajo se han caracterizado por primera vez dos IFNs de tipo I (ifn1 e ifn2) en rodaballo, los cuales mostraron distintas propiedades. Su actividad fue estudiada gracias a la producción de plásmidos de expresión que codifican para estas moléculas (pMCV1.4-ifn1 y pMCV1.4-ifn2). Aunque la expresión de ambos IFNs fue inducida tras una infección con VHSV, solamente Ifn1 presentó una clara actividad antiviral (sobreexpresión de ISGs y protección frente a VHSV), mientras que Ifn2 no fue capaz de emular esta respuesta. Por otra parte, aunque ambos genes fueron también inducidos tras la infección con Aeromonas salmonicida subsp. salmonicida, ninguno de los dos IFNs presentó efecto protector frente a la bacteria. La inyección intramuscular de los plásmidos que codifican para estos IFNs indujo la expresión de numerosos genes inmunes tanto en riñón anterior como en músculo (lugar de inyección), aunque el efecto de Ifn2 se limitó principalmente al lugar de inyección y en ningún caso indujo la expresión de ISGs, como fue mencionado con anterioridad. Curiosamente, Ifn2 pero no Ifn1 indujeron

152 un incremento en el nivel de expresión de interleuquina-1 beta (il1b). Por lo tanto, el papel del Ifn2 podría estar más relacionado con la regulación inmune, estando principalmente involucrado en el proceso de inflamación. Así pues, ambos IFNs de rodaballo podrían actuar de forma complementaria y diferencial durante las infecciones virales. Además, otro punto a destacar es que la sobreexpresión de il1b por Ifn2 así como la de interleuquina-8 (il8) por parte de ambos IFNs es un proceso no observado en otros vertebrados, ya que ambas moléculas son inhibidas por IFNs de tipo I en mamíferos.

Capítulo 6: La función del IFN-gamma de rodaballo es dependiente del tipo de patógeno administrado.

El IFN-gamma ha sido típicamente descrito como una citoquina pro- inflamatoria que juega un importante papel en la resolución tanto de infecciones virales como bacterianas. No obstante, algunas funciones anti-inflamatorias han sido también atribuidas a esta molécula. Con el fin de completar el repertorio de IFNs de rodaballo, en este trabajo hemos caracterizado por primera vez el gen del IFN-gamma (ifng) en este pez plano, cuya secuencia fue obtenida gracias a la reciente publicación de su genoma. Su patrón de expresión bajo condiciones basales, tras la administración de plásmidos de expresión codificando para IFNs de tipo I y tras la infección con virus y bacteria ha sido estudiado. La inyección intramuscular de un plásmido de expresión que codifica para el Ifng de rodaballo (pMCV1.4-ifng) no fue capaz de reducir la mortalidad causada por una infección con VHSV o Aeromonas salmonicida subsp. salmonicida. Además, la inyección del plásmido de expresión no afectó a la transcripción de numerosos genes inmunes relacionados con la actividad del IFN-gamma, con la excepción del macrophage- colony stimulating factor (csf1). Curiosamente, a las 24 horas post-infección, aquellos individuos previamente inoculados con pMCV1.4-ifng e infectados con VHSV mostraron un incremento en la expresión de citoquinas pro-inflamatorias e IFNs de tipo I en comparación con aquellos peces que no recibieron el plásmido de expresión, indicando un efecto sinérgico de Ifng y VHSV en la inducción de estos genes. Por otra parte, algunos marcadores de macrófagos, como el macrophage receptor with collagenous structure (marco) fueron inhibidos por Ifng durante la infección viral. Ifng produjo el efecto totalmente opuesto en aquellos rodaballos

153 infectados con bacteria, en los cuales ocasionó una reducción de la transcripción de genes pro-inflamatorios y de IFNs de tipo I, pero indujo la sobreexpresión de genes relacionados con la actividad de los macrófagos. Así pues, la actividad el Ifng de rodaballo parece ser dependiente del tipo de patógeno que causa la infección, reflejándose en este caso un claro y marcado papel dual.

2. CONCLUSIONES

1. La información transcriptómica en rodaballo (S. maximus) fue enormemente enriquecida, especialmente en lo que respecta a aquellas secuencias relacionadas con la respuesta inmune antiviral. Muchos de los componentes implicados en las principales rutas inmunes fueron identificados por primera vez en esta especie. Toda esta información obtenida fue usada en el diseño de un microarray.

2. La vacuna de ADN diseñada durante este trabajo (pMCV1.4-G), que codifica la glicoproteína G de VHSV, ha demostrado que induce buenos niveles de protección frente a VHSV. Además, un mes después de la vacunación de detectaron anticuerpos específicos frente a la glicoproteína G y con capacidad neutralizante en el suero de los rodaballos vacunados, indicando la activación del sistema inmune adaptativo.

3. Mediante el uso de microarrays, el análisis de las muestras de riñón anterior obtenidas de rodaballos vacunados y no vacunados reveló una fuerte activación de las principales rutas inmunes a los tres días de la administración de la vacuna.

4. Tras la infección con VHSV el perfil transcriptómico observado entre peces vacunados y no vacunados fue totalmente diferente. Mientras que los individuos que no habían sido previamente inmunizados mostraron una extensa e incontrolada respuesta inmune, lo que genera un intenso estado pro-inflamatorio en el hospedador, aquellos individuos que fueron previamente vacunados exhibieron una respuesta moderada y controlada debido a la presencia previa de factores inmunes específicos.

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5. Dos IFNs de tipo I (ifn1 y ifn2), que son las principales citoquinas que controlan la respuesta inmune antiviral en vertebrados, fueron caracterizados por primera vez en rodaballo. Los resultados indicaron que ambos IFNs poseen papeles no redundantes y complementarios. Solo el Ifn1 fue capaz de inducir la expresión de ISGs y, como consecuencia, de reducir de forma significativa la mortalidad tras la infección con VHSV. Ifn2 mostró una actividad que parece estar más relacionada con la regulación de la inflamación.

6. El IFN de tipo II (ifng) de rodaballo mostró un sorprendente papel dual dependiendo del tipo de patógeno (virus o bacteria):

a) Ifng presentó un efecto potenciador de la inflamación durante una infección bacteriana.

b) La administración de Ifng tuvo un efecto sinérgico en la transcripción de los IFNs de tipo I durante una infección con VHSV, pero se observó un efecto inhibidor cuando los animales fueron inoculados con bacteria.

c) El Ifng promovió la expresión de aquellos genes directamente relacionados con la actividad de los macrófagos en los rodaballos infectados con A. salmonicida, pero en aquellos individuos infectados con VHSV se observó el efecto opuesto.

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