# Regulation and distribution of European sea bass perforins point to their role in the adaptive cytotoxic response against NNV

Miguel A García-Álvarez, Laura Cervera, Yulema Valero, Carmen González-Fernández, Luis Mercado, Elena Chaves-Pozo, Alberto Cuesta

<figure><img src="image_1.png" /></figure>

## To cite this version:

Miguel A García-Álvarez, Laura Cervera, Yulema Valero, Carmen González-Fernández, Luis Mercado, et al.. Regulation and distribution of European sea bass perforins point to their role in the adaptive cytotoxic response against NNV. Fish and Shellfish Immunology, 2024, 144, pp.109244. 10.1016/j.fsi.2023.109244. hal-04592064

HAL Id: hal-04592064

https://hal.inrae.fr/hal-04592064

Submitted on 29 May 2024

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Distributed under a Creative Commons Attribution 4.0 International License
# Regulation and distribution of European sea bass perforins point to their role in the adaptive cytotoxic response against NNV

Miguel A García-Álvarez, Laura Cervera, Yulema Valero, Carmen González-Fernández, Luis Mercado, Elena Chaves-Pozo, Alberto Cuesta

<figure><img src="image_1.png" /></figure>

## To cite this version:

Miguel A García-Álvarez, Laura Cervera, Yulema Valero, Carmen González-Fernández, Luis Mercado, et al.. Regulation and distribution of European sea bass perforins point to their role in the adaptive cytotoxic response against NNV. Fish and Shellfish Immunology, 2024, 144, pp.109244. 10.1016/j.fsi.2023.109244. hal-04592064

HAL Id: hal-04592064

https://hal.inrae.fr/hal-04592064v1

Submitted on 29 May 2024

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Full length article

# Regulation and distribution of European sea bass perforins point to their role in the adaptive cytotoxic response against NNV

Miguel A. García-Álvarez<sup>a,b</sup>, Laura Cervera<sup>a,b</sup>, Yulema Valero<sup>a</sup>, Carmen González-Fernández<sup>a,c</sup>, Luis Mercado<sup>d</sup>, Elena Chaves-Pozo<sup>b</sup>, Alberto Cuesta<sup>a,*</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

### Keywords:

Perforins

European sea bass

Nodavirus

Cell-mediated cytotoxicity

T cells

## ABSTRACT

Cell-mediated cytotoxicity is a complex immune mechanism that involves the release of several killing molecules, being perforin (PRF) one of the most important effector players. Perforin is synthesized by T lymphocytes and natural killer cells in mammals and responsible for the formation of pores on the target cell membrane during the killing process. Although perforin has been extensively studied in higher vertebrates, this knowledge is very limited in fish. Therefore, in this study we have identified four *prf* genes in European sea bass (*Dicentrarchus labrax*) and evaluated their mRNA levels. All sea bass *prf* genes showed the typical and conserved domains of its human orthologue and were closely clustered by the phylogenetic analysis. In addition, all genes showed constitutive and ubiquitous tissular expression, being *prf1.9* gene the most highly expressed in immune tissues. Subsequently, *in vitro* stimulation of head-kidney (HK) cells with phytohemagglutinin, a T-cell activator, showed an increase of all *prf* gene levels, except for *prf1.3* gene. European sea bass HK cells increased the transcription of *prf1.2* and *prf1.9* during the innate cell-mediated cytotoxic activity against xenogeneic target cells. In addition, sea bass infected with nodavirus (NNV) showed a similar expression pattern of all *prf* in HK and brain at 15 days post-infection, except for *prf1.3* gene and in the gonad. Finally, the use of a polyclonal antibody against PRF1.9 showed an increase of positive cells in HK, brain and gonad from NNV-infected fish. Taken together, the data seem to indicate that all *prf* genes, except *prf1.3*, appear to be involved in the European sea bass immunity, and probably in the cell-mediated cytotoxic response, with PRF1.9 playing the most important role against nodavirus. The involvement of the PRFs and the CMC activity in the vertical transmission success of the virus is also discussed.

## 1. Introduction

Perforin (PRF) is one of the main death effectors released, together with granzymes, during the cell-mediated cytotoxicity (CMC) immune response. PRF is mainly synthesized by cytotoxic CD8⁺ T lymphocytes (CTL) and natural killer (NK) cells and stored in their cytolytic granules [1,2]. In humans and mice, perforin is produced from a single-copy gene called *prf1*, which codes for a glycoprotein of about 70 kDa that belongs to the membrane attack complex/perforin (MACPF) superfamily. The mature protein consists of a MACPF domain at the N-terminal end, a

Ca²⁺ binding (C2) domain and an intermediate epidermal growth factor-like (EGF) domain [2,3]. Perforin is mainly involved in eliminating viruses-infected or tumour cells [4–7], although there is evidence about its role in clearing bacteria-infected cells [8]. The mechanism of action of perforin has been exhaustively studied. Briefly, inactivated perforin remains inside the cytotoxic granules of killer lymphocytes together with other molecules as granzymes. Once the recognition of the killer and the target cells takes place and the immunological synapse proceeds, the exocytosis of the lytic granules causes the release of their contents [6]. In this scenario, the high level of Ca²⁺ and the neutral pH

<footnote><sup>a</sup> Immunobiology for Aquaculture Group, Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, 30100, Murcia, Spain</footnote>

<footnote><sup>b</sup> Physiology and Welfare of Marine Species Group (PHYSIS), Centro Oceanográfico de Murcia, Instituto Español de Oceanografía (COMU-IEO), CSIC, Carretera de la Azohía s/n. 30860, Puerto de Mazarón, Murcia, Spain</footnote>

<footnote><sup>c</sup> INRAE, UR RiverLy, Laboratoire d'écotoxicologie, F-69625, Villeurbanne, France</footnote>

<footnote><sup>d</sup> Grupo de Marcadores Inmunológicos, Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile</footnote>

<footnote>* Corresponding author. Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, 30100, Murcia, Spain. E-mail address: alcuesta@um.es (A. Cuesta).</footnote>
<figure><img src="image_3.png" /></figure>

Full length article

# Regulation and distribution of European sea bass perforins point to their role in the adaptive cytotoxic response against NNV

Miguel A. García-Álvarez<sup>a,b</sup>, Laura Cervera<sup>a,b</sup>, Yulema Valero<sup>a</sup>, Carmen González-Fernández<sup>a,c</sup>, Luis Mercado<sup>d</sup>, Elena Chaves-Pozo<sup>b</sup>, Alberto Cuesta<sup>a,*</sup>

<figure><img src="image_4.png" /></figure>

## ARTICLE INFO

### Keywords:

Perforins

European sea bass

Nodavirus

Cell-mediated cytotoxicity

T cells

## ABSTRACT

Cell-mediated cytotoxicity is a complex immune mechanism that involves the release of several killing molecules, being perforin (PRF) one of the most important effector players. Perforin is synthesized by T lymphocytes and natural killer cells in mammals and responsible for the formation of pores on the target cell membrane during the killing process. Although perforin has been extensively studied in higher vertebrates, this knowledge is very limited in fish. Therefore, in this study we have identified four *prf* genes in European sea bass (*Dicentrarchus labrax*) and evaluated their mRNA levels. All sea bass *prf* genes showed the typical and conserved domains of its human orthologue and were closely clustered by the phylogenetic analysis. In addition, all genes showed constitutive and ubiquitous tissular expression, being *prf1.9* gene the most highly expressed in immune tissues. Subsequently, *in vitro* stimulation of head-kidney (HK) cells with phytohemagglutinin, a T-cell activator, showed an increase of all *prf* gene levels, except for *prf1.3* gene. European sea bass HK cells increased the transcription of *prf1.2* and *prf1.9* during the innate cell-mediated cytotoxic activity against xenogeneic target cells. In addition, sea bass infected with nodavirus (NNV) showed a similar expression pattern of all *prf* in HK and brain at 15 days post-infection, except for *prf1.3* gene and in the gonad. Finally, the use of a polyclonal antibody against PRF1.9 showed an increase of positive cells in HK, brain and gonad from NNV-infected fish. Taken together, the data seem to indicate that all *prf* genes, except *prf1.3*, appear to be involved in the European sea bass immunity, and probably in the cell-mediated cytotoxic response, with PRF1.9 playing the most important role against nodavirus. The involvement of the PRFs and the CMC activity in the vertical transmission success of the virus is also discussed.

## 1. Introduction

Perforin (PRF) is one of the main death effectors released, together with granzymes, during the cell-mediated cytotoxicity (CMC) immune response. PRF is mainly synthesized by cytotoxic CD8⁺ T lymphocytes (CTL) and natural killer (NK) cells and stored in their cytolytic granules [1,2]. In humans and mice, perforin is produced from a single-copy gene called *prf1*, which codes for a glycoprotein of about 70 kDa that belongs to the membrane attack complex/perforin (MACPF) superfamily. The mature protein consists of a MACPF domain at the N-terminal end, a

Ca²⁺ binding (C2) domain and an intermediate epidermal growth factor-like (EGF) domain [2,3]. Perforin is mainly involved in eliminating viruses-infected or tumour cells [4–7], although there is evidence about its role in clearing bacteria-infected cells [8]. The mechanism of action of perforin has been exhaustively studied. Briefly, inactivated perforin remains inside the cytotoxic granules of killer lymphocytes together with other molecules as granzymes. Once the recognition of the killer and the target cells takes place and the immunological synapse proceeds, the exocytosis of the lytic granules causes the release of their contents [6]. In this scenario, the high level of Ca²⁺ and the neutral pH

<footnote><sup>a</sup> Immunobiology for Aquaculture Group, Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, 30100, Murcia, Spain</footnote>

<footnote><sup>b</sup> Physiology and Welfare of Marine Species Group (PHYSIS), Centro Oceanográfico de Murcia, Instituto Español de Oceanografía (COMU-IEO), CSIC, Carretera de la Azohía s/n. 30860, Puerto de Mazarón, Murcia, Spain</footnote>

<footnote><sup>c</sup> INRAE, UR RiverLy, Laboratoire d'écotoxicologie, F-69625, Villeurbanne, France</footnote>

<footnote><sup>d</sup> Grupo de Marcadores Inmunológicos, Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile</footnote>

<footnote>* Corresponding author. Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, 30100, Murcia, Spain. E-mail address: alcuesta@um.es (A. Cuesta).</footnote>
of the extracellular medium favours the binding of the C2 domain of the monomeric perforins to the target cell membrane where they interact forming a transmembrane pore, through which granzymes will enter into the cytosol of the target cell triggering its death by either apoptosis or necrosis [9,10].

In teleost fish, the first group of vertebrates displaying CMC activity by both innate and adaptive effector cells [11,12], the knowledge on the presence and function of cytotoxic molecules, such as perforin [13], is very limited. *Perforin* coding genes have been identified in different species showing species-dependant differences. In zebrafish (*Danio rerio*), six *prf* genes were detected and showed tissue-dependant expression levels, being more highly expressed in immune-related tissues as spleen or gill [14]. In contrast, a unique *prf* gene was observed in Japanese flounder (*Paralichthys olivaceus*) [15] and rainbow trout (*Oncorhynchus mykiss*) [16], the expression of which was also linked to immune system tissues as head-kidney (HK), spleen or peripheral blood leukocytes (PBLs). Interestingly, *prf* transcription in trout was restricted to CD8α⁺ lymphocytes and greatly increased upon stimulation with phytohemagglutinin (PHA) [17]. Molecular characterization of *prf* from common carp (*Cyprinus carpio*) revealed the existence of three genes, which increased their expression in *in vitro* assays with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (Poly I:C) and *Aeromonas hydrophila*, providing evidence of their role against bacterial and viral pathogens [18]. In all previous studies, bioinformatic analysis validated the presence of a MACPF and a Ca²⁺-dependent binding C2 domain in the mature peptide of fish perforins [14-16,18]. In fact, the flounder recombinant perforin demonstrated a calcium-dependent haemolytic activity against rabbit erythrocytes [15]. Regarding the role of perforin during viral infections, several zebrafish *prf* genes increased their mRNA levels against Spring viraemia of carp virus (SVCV), especially *prf19b*, which increased 3,000-fold upon control levels [14]. In the case of rock bream (*Oplegnathus fasciatus*) infected with rock bream iridovirus (RBIV) an increase in *prf* genes, and also granzyme, was concomitant with low mortalities, suggesting a role for perforin-mediated cytotoxic response in the restraint of the infection [19]. Consistently, rainbow trout monocyte-macrophage RTS11 cell line, in response to viral haemorrhagic septicaemia virus (VHSV), increased the mRNA levels of *prf* and *granzyme*, eliciting cytotoxic activity [20]. Additionally, a reduction of the cytotoxic activity of CTLs of gibnuna crucian carp (*Carassius langsdorffii*) occurred in a dose-dependent manner after the addition of concanamycin A and EGTA, a well-known perforin inhibitors by means of depolymerization [21]. In general, there is little information on the role of perforin against viral infections in European sea bass (*Dicentrarchus labrax*), but particularly, against nervous necrosis virus (NNV). This virus is the most threatening pathogen for this species causing massive mortalities in larvae and juveniles [22], and it is also able to colonize the gonad for transmission purposes thanks to a weak anti-viral immune response [23]. A transcriptomic study revealed that *prf* transcription levels increased during the innate CMC response, as well as during intravitreal NNV infection, pointing to a possible activation of the CMC to clear NNV-infected cells in European sea bass [24,25]. Therefore, it is necessary to further study perforins in fish, particularly in European sea bass, to clarify their participation in the immune response, but especially in the CMC activity for viral removal.

The main objective of this study was to identify *prf* genes and characterize their regulation in European sea bass, due to its relevance in the Mediterranean aquaculture sector. The sequences, obtained from genome databases, were analysed bioinformatically and the transcription levels studied in naïve organs, *in vitro* stimulated head-kidney cells, CMC and *in vivo* NNV-infected fish. A polyclonal antibody was obtained and used to identify and evaluate the PRF⁺ cells after NNV infection. This information might serve to develop antiviral strategies or as a marker of resistance, breeding fish more resistant to viral infections via perforin.

## 2. Material and methods

### 2.1. Animals

Six-week old female CF-1 mice (n = 3) coming from the Instituto de Salud Pública (ISP, Chile) were transported and maintained at the Pontificia Universidad Católica de Valparaíso (PUCV, Chile) facilities for generating polyclonal antibodies.

Healthy specimens of European sea bass (*Dicentrarchus labrax*) were bred and reared at the Oceanographic Centre of Murcia of the Spanish Institute of Oceanography (COMU-IEO, CSIC; *Mazarrón*, Spain) facilities. Animals were transported to the University of Murcia and maintained in marine recirculating aquaculture systems (RAS) (30% salinity, 22-25 °C, 12:12 light: dark photoperiod) with suitable aeration and filtration devices and fed *ad libitum* with a commercial pellet diet (Skretting). Specimens with a body weight (BW) of 300-500 g were used for specificity test of antibodies, immunohistochemistry (IHC), analysis in naïve fish and *in vitro* studies while fish with 125 ± 25 g were used for *in vivo* infection with NNV. Before sampling, all specimens were anesthetized with 40 µL/L of clove oil in sea water, exsanguinated and immediately decapitated. Animal handling, transport and experimental procedures were always performed in accordance with the Guidelines of the European Union Council (2010/63/UE) and the Bioethical Committees of the IEO (reference REGA ES300261040017 and Permit Number 2010/02), of the PUCV (Permit Number BIOEPUCV-A159-2014) and of the University of Murcia (reference REGA ES300305440012 and Permit Number A13170109).

### 2.2. Bioinformatic analysis

European sea bass perforin (*prf*) sequence (KY801204), now renamed to *prf1.9*, was already known and used in previous studies [24, 25]. Novel mRNA and putative protein sequences of sea bass *prf* were obtained from genome databases (Ensembl and http://seabass.mpipz.mpg.de/). Predicted protein sequences were used to retrieve fish and tetrapod orthologues from the NCBI databases by the basic local alignment tool (BLAST). The evolutionary history was inferred using the Neighbour-Joining method by Mega software v11.0 with the bootstrap value set to 1,000 replications [26]. Proteins belonging to the MACP Family, such as the complement proteins C6–C9 and macrophage-expressed gene 1 protein (MPEG1), were also included. Predicted proteins were used for multiple sequence alignments using the CLUSTALW program v2.1 and conserved domains were identified using ExPASy tools (http://us.expasy.org).

### 2.3. Experimental procedures and sampling

For the tissue expression study, tissues (brain, gill, liver, skin, gonad, gut, HK, spleen, thymus, and blood) from 3 naïve fish specimens were sampled, immediately snap-frozen in TRIzol® Reagent (Life Technologies) and stored at -80 °C for later use.

For the *in vitro* stimulation of HK cells, European sea bass were exsanguinated, and small fragment of HK was obtained and transferred to 10 mL of Leibovitz's L-15 (Gibco) supplemented with 10% foetal bovine serum (FBS; Gibco), 2 mM glutamine (Gibco), penicillin (100 IU/mL; Gibco), streptomycin (100 µg/mL; Gibco) and 20 mM HEPES (Gibco). HK cells were obtained by forcing fragments of the tissue through a nylon mesh (100 µm), washed twice (400 g, 10 min), counted in an automatic counting chamber (BioRad) and adjusted to 10⁷ HK cells/mL. Cell viability was determined by the trypan blue exclusion test. Subsequently, HK cells were incubated in flat-bottomed 48-well microtiter plates (Nunc) at 22 °C during 24 h with: culture medium alone (control), 5 µg/mL lipopolysaccharide (LPS; Sigma-Aldrich), 10 µg/mL phytohemagglutinin (PHA; Sigma-Aldrich), 5 µg/mL concanavalin A (ConA; Sigma-Aldrich), 50 µg/mL synthetic unmethylated cytosine-phosphodiesterguanosine oligodeoxynucleotide 1668 (CpG
of the extracellular medium favours the binding of the C2 domain of the
monomeric perforins to the target cell membrane where they interact
forming a transmembrane pore, through which granzymes will enter
into the cytosol of the target cell triggering its death by either apoptosis
or necrosis [9,10].

In teleost fish, the first group of vertebrates displaying CMC activity
by both innate and adaptive effector cells [11,12], the knowledge on
the presence and function of cytotoxic molecules, such as perforin [13],
is very limited. *Perforin* coding genes have been identified in different
species showing species-dependant differences. In zebrafish (*Danio
rerio*), six *prf* genes were detected and showed tissue-dependant
expression levels, being more highly expressed in immune-related tis-
sues as spleen or gill [14]. In contrast, a unique *prf* gene was observed in
Japanese flounder (*Paralichthys olivaceus*) [15] and rainbow trout
(*Oncorhynchus mykiss*) [16], the expression of which was also linked to
immune system tissues as head-kidney (HK), spleen or peripheral blood
leukocytes (PBLs). Interestingly, *prf* transcription in trout was restricted
to CD8α⁺ lymphocytes and greatly increased upon stimulation with
phytohemagglutinin (PHA) [17]. Molecular characterization of *prf* from
common carp (*Cyprinus carpio*) revealed the existence of three genes,
which increased their expression in *in vitro* assays with lipopolysac-
charide (LPS), polyinosinic-polycytidylic acid (Poly I:C) and *Aeromonas
hydrophila*, providing evidence of their role against bacterial and viral
pathogens [18]. In all previous studies, bioinformatic analysis validated
the presence of a MACPF and a Ca²⁺-dependent binding C2 domain in
the mature peptide of fish perforins [14-16,18]. In fact, the flounder
recombinant perforin demonstrated a calcium-dependent haemolytic
activity against rabbit erythrocytes [15]. Regarding the role of perforin
during viral infections, several zebrafish *prf* genes increased their mRNA
levels against Spring viraemia of carp virus (SVCV), especially *prf19b*,
which increased 3,000-fold upon control levels [14]. In the case of rock
bream (*Oplegnathus fasciatus*) infected with rock bream iridovirus (RBIV)
an increase in *prf* genes, and also granzyme, was concomitant with low
mortalities, suggesting a role for perforin-mediated cytotoxic response
in the restraint of the infection [19]. Consistently, rainbow trout
monocyte-macrophage RTS11 cell line, in response to viral haemor-
rhagic septicaemia virus (VHSV), increased the mRNA levels of *prf* and
*granzyme*, eliciting cytotoxic activity [20]. Additionally, a reduction of
the cytotoxic activity of CTLs of gibnuna crucian carp (*Carassius lang-
dorfi*) occurred in a dose-dependent manner after the addition of con-
canamycin A and EGTA, a well-known perforin inhibitors by means of
depolymerization [21]. In general, there is little information on the role
of perforin against viral infections in European sea bass (*Dicentrarchus
labrax*), but particularly, against nervous necrosis virus (NNV). This
virus is the most threatening pathogen for this species causing massive
mortalities in larvae and juveniles [22], and it is also able to colonize the
gonad for transmission purposes thanks to a weak anti-viral immune
response [23]. A transcriptomic study revealed that *prf* transcription
levels increased during the innate CMC response, as well as during
intravitreal NNV infection, pointing to a possible activation of the CMC
to clear NNV-infected cells in European sea bass [24,25]. Therefore, it is
necessary to further study perforins in fish, particularly in European sea
bass, to clarify their participation in the immune response, but especially
in the CMC activity for viral removal.

The main objective of this study was to identify *prf* genes and char-
acterize their regulation in European sea bass, due to its relevance in the
Mediterranean aquaculture sector. The sequences, obtained from
genome databases, were analysed bioinformatically and the transcrip-
tion levels studied in naïve organs, *in vitro* stimulated head-kidney cells,
CMC and *in vivo* NNV-infected fish. A polyclonal antibody was obtained
and used to identify and evaluate the PRF⁺ cells after NNV infection.
This information might serve to develop antiviral strategies or as a
marker of resistance, breeding fish more resistant to viral infections *via*
perforin.

## 2. Material and methods

2.1. *Animals*

Six-week old female CF-1 mice (n = 3) coming from the Instituto de
Salud Pública (ISP, Chile) were transported and maintained at the Pon-
tificia Universidad Católica de Valparaíso (PUCV, Chile) facilities for
generating polyclonal antibodies.

Healthy specimens of European sea bass (*Dicentrarchus labrax*) were
bred and reared at the Oceanographic Centre of Murcia of the Spanish
Institute of Oceanography (COMU-IEO, CSIC; *Mazarrón*, Spain) facil-
ities. Animals were transported to the University of Murcia and main-
tained in marine recirculating aquaculture systems (RAS) (30% salinity,
22-25 °C, 12:12 light: dark photoperiod) with suitable aeration and
filtration devices and fed *ad libitum* with a commercial pellet diet
(Skretting). Specimens with a body weight (BW) of 300-500 g were used
for specificity test of antibodies, immunohistochemistry (IHC), analysis
in naïve fish and *in vitro* studies while fish with 125 ± 25 g were used for
*in vivo* infection with NNV. Before sampling, all specimens were anes-
thetized with 40 µL/L of clove oil in sea water, exsanguinated and
immediately decapitated. Animal handling, transport and experimental
procedures were always performed in accordance with the Guidelines of
the European Union Council (2010/63/UE) and the Bioethical Com-
mittees of the IEO (reference REGA ES300261040017 and Permit
Number 2010/02), of the PUCV (Permit Number BIOEPUCV-A159-
2014) and of the University of Murcia (reference REGA
ES300305440012 and Permit Number A13170109).

## 2.2. Bioinformatic analysis

European sea bass perforin (*prf*) sequence (KY801204), now
renamed to *prf1.9*, was already known and used in previous studies [24,
25]. Novel mRNA and putative protein sequences of sea bass *prf* were
obtained from genome databases (Ensembl and http://seabass.mpipz.
mpg.de/). Predicted protein sequences were used to retrieve fish and
tetrapod orthologues from the NCBI databases by the basic local align-
ment tool (BLAST). The evolutionary history was inferred using the
Neighbour-Joining method by Mega software v11.0 with the bootstrap
value set to 1,000 replications [26]. Proteins belonging to the MACP
Family, such as the complement proteins C6–C9 and
macrophage-expressed gene 1 protein (MPEG1), were also included.
Predicted proteins were used for multiple sequence alignments using the
CLUSTALW program v2.1 and conserved domains were identified using
ExPASy tools (http://us.expasy.org).

2.3. *Experimental procedures and sampling*

For the tissue expression study, tissues (brain, gill, liver, skin, gonad,
gut, HK, spleen, thymus, and blood) from 3 naïve fish specimens were
sampled, immediately snap-frozen in TRIzol® Reagent (Life Technolo-
gies) and stored at -80 °C for later use.

For the *in vitro* stimulation of HK cells, European sea bass were
exsanguinated, and small fragment of HK was obtained and transferred
to 10 mL of Leibovitz's L-15 (Gibco) supplemented with 10% foetal
bovine serum (FBS; Gibco), 2 mM glutamine (Gibco), penicillin (100 IU/
mL; Gibco), streptomycin (100 µg/mL; Gibco) and 20 mM HEPES
(Gibco). HK cells were obtained by forcing fragments of the tissue
through a nylon mesh (100 µm), washed twice (400 g, 10 min), counted
in an automatic counting chamber (BioRad) and adjusted to 10⁷ HK
cells/mL. Cell viability was determined by the trypan blue exclusion
test. Subsequently, HK cells were incubated in flat-bottomed 48-well
microtiter plates (Nunc) at 22 °C during 24 h with: culture medium
alone (control), 5 µg/mL lipopolysaccharide (LPS; Sigma-Aldrich), 10
µg/mL phytohemagglutinin (PHA; Sigma-Aldrich), 5 µg/mL concanav-
alin A (ConA; Sigma-Aldrich), 50 µg/mL synthetic unmethylated
cytosine-phosphodiesterguanosine oligodeoxynucleotide 1668 (CpG
ODN 1668; sequence 5'-TCCATGACGTTCCTGATGCT-3'; Eurogentec), 25 µg/mL polyinosinic: polycytidylic acid (Poly I:C; Sigma-Aldrich), $10^8$ bacteria/mL (Vibrio anguillarum or Photobacterium damselae) or $10^6$ TCID$_{50}$/mL of NNV (strain 411/96, genotype RGNNV). Afterwards, HK cells were washed with 0.01 M phosphate buffer saline (PBS) and stored in TRIzol® Reagent at -80 °C for later isolation of RNA.

For the CMC assays, they were performed as elsewhere [24]. In brief, growing cultures of SAF-1 (ECACC 00122301) fish cell line were detached by standard trypsinization, washed, seeded in flat-bottomed 96 well plates (Nunc) and incubated without (mock-infected) or with $10^6$ TCID$_{50}$ NNV/mL (NNV-infected) for 24 h. These cells were used as targets in the CMC assays. Then, target cells were washed and 100 µL of freshly isolated HK cells added as effectors (50 HK cells per target cell). Samples were centrifuged (400 g, 1 min) and incubated at 25 °C for 4 h. Finally, samples were centrifuged, and cell pellets stored in TRIzol® Reagent at -80 °C for later RNA isolation. CMC samples at 0 h of incubation were used as controls. Samples with targets alone or HK cells alone were also used as technique controls. Infection of the target cells with NNV was confirmed by real-time PCR (qPCR) [24].

Finally, *in vivo* NNV-infection was performed by intramuscular injection. For this, fish received a 100 µL injection of culture medium alone (mock-infected) or containing $10^6$ TCID$_{50}$/fish of NNV. Fish ($n = 5$ fish/group and time) were sampled 1, 7 or 15 days post-infection (dpi) and brain, HK and gonad tissues were removed and immediately frozen in TRIzol® Reagent and stored at -80 °C for later RNA isolation or in Bouin's solution for IHC analysis. We have selected these tissues because i) brain is the main target tissue for NNV replication and infection, ii) HK is the main lymphohematopoietic tissue in fish, and iii) gonad is used by NNV to hide, evade the immune response, and be spread to the progeny by vertical transmission.

## 2.4. Analysis of gene expression by qPCR

Total RNA was isolated from TRIzol® Reagent frozen samples following the manufacturer's instructions and quality and quantity evaluated using the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). One µg of total RNA was treated with DNAse I (Promega) to remove genomic DNA and the first strand of cDNA synthesized by reverse transcription using the Superscript IV (Life Technologies). The qPCR was performed with an ABI PRISM 7500 instrument (Applied Biosystems) using SYBR Green PCR Core Reagents (Applied Biosystems). Reaction mixtures were incubated at 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C, 1 min at 60 °C, and finally 15 s at 95 °C, 1 min at 60 °C and 15 s at 95 °C. For each mRNA, gene expression was corrected by the geometric average of the elongation factor 1 alpha (*ef1a*) and ribosomal protein 18 (*rps18*) gene expressions in each sample and expressed as either $2^{-\Delta Ct}$ or $2^{-\Delta\Delta Ct}$ [27]. Stability of the housekeeping genes was routinely confirmed by the BestKeeper® software. The primers used were designed specifically using the Oligo Perfect software tool (Thermo Fisher Scientific) and are shown in Supplementary Table 1. Prior the experiment, the specificity of each primer pair was studied using positive and negative samples. A melting curve analysis of the amplified products validated the primer for specificity at each use. Negative controls with no template were always included in the reactions.

## 2.5. Generation and validation of polyclonal antibodies

Polyclonal antibodies against the putative European sea bass PRF1.9 sequence (Uniprot acc. number A0A8C4EQ87) were produced in mice by using the chemically synthesized CETSPQRNQSLTTQSPKYDEIC peptide as described elsewhere [28]. The designed peptide was only present in the carboxyl-terminal end of PRF1.9. The recognition of the corresponding synthetic epitopes or the complete molecule by the serum was tested by using an indirect ELISA as hitherto defined [28]. To check the ability to recognize the complete molecule, European sea bass whole

spleen was homogenized in lysis buffer (50 mM Tris-HCl, pH 7.6, 0.3 M NaCl, 0.5% Triton X-100, 0.5 M EDTA and 0.2% protease inhibitor cocktail) and protein concentration determined (BCA protein assay kit; Pierce). In addition, the specificity of the mouse serum was evaluated by the pre-absorption (1:5,000) with its corresponding specific synthetic peptide (4 µg) for 2 h and then tested in the indirect ELISA.

## 2.6. Immunohistochemistry (IHC)

Fragments of HK, brain and gonad from mock- and NNV-infected fish from the *in vivo* infection described above were fixed in Bouin's solution for 24 h at 4 °C, embedded in paraffin (Paraplast Plus; Sherwood Medical) and sectioned at 5 µm. After dewaxing and rehydration, the sections were subjected to an streptavidin-biotin complex technique as previously described [29]. The anti-PRF1.9 sera was used at its optimal dilution of 1:100. The secondary antibody was a commercial mix of biotinylated secondary antibodies goat anti-mouse IgG and goat anti-rabbit IgG (Sigma-Aldrich) conjugated with streptavidin (Cytiva). The reaction was revealed using the horseradish peroxidase-streptavidin complex (Invitrogen) and 0.5 mg/mL 3,3'-diaminobenzidine tetrahydrochloride hydrate (Sigma-Aldrich) in PBS containing 25 µL H$_2$O$_2$ as substrate. Some sections were slightly counterstained with hematoxylin. The specificity of the reaction was determined by using the pre-immune serum instead of the first antiserum.

## 2.7. Statistical analysis

All data are presented as mean ± standard error of the mean (SEM). *Perforin* transcription was presented as relative expression in naïve tissues or as fold change respect to the respective controls in the HK cells, CMC and NNV-infection assays. Results were analysed by a Student-t test to denote statistical differences between treated/infected and control groups. Normality was evaluated by the Shapiro-Wilk test. Non-parametric Pearson correlation test was applied to identify potential relations among gene expression. Statistical analyses were conducted using SPSS 20 software. Minimum level of significance was fixed in 0.05.

# 3. Results

## 3.1. Perforins of European sea bass belong to the MACPF superfamily

Within the European sea bass genome, we clearly identified four *prf* genes and named *prf1.2* (ENSLAT00005031934.2), *prf1.3* (ENS-LAT00005009634.2), *prf1.5* (ENSLAT00005052249.2) and *prf1.9* (ENSLAT00005024231.2), which generated predicted putative proteins of 583, 566, 523 and 610 amino acids (aa), respectively. Alignment of identified European sea bass PRF sequences with the PRF1 human orthologue, together with protein domain architecture prediction, pointed to a relatively high conservation within the MACPF and C2 domains, including important Cys residues (Fig. 1). However, the main differences among them were present in the leader peptide and in the C-terminus whereas MACPF domain showed slight differences among the sequences. The phylogenetic analysis confirmed the similarities of the four perforins found in European sea bass within others from multiple fish species, since all of them differed from the clade formed by complement proteins C6–C9 as well as MPEG1 families, both members of the MACPF superfamily (Fig. 2). Nevertheless, within the clade of perforins, the four European sea bass proteins were distributed along different branches, distant from each other, indicating a relative diversification, which could lead to functional specialization. Interestingly, the sea bass PRF1.3 was the closest to the mammalian perforins.

## 3.2. *Perforin* transcription shows different basal expression patterns

The data obtained in this study demonstrated constitutive expression of sea bass perforins across all sampled tissues. However, differential
ODN 1668; sequence 5'-TCCATGACGTTCCTGATGCT-3'; Eurogentec), 25 µg/mL polyinosinic: polycytidylic acid (Poly I:C; Sigma-Aldrich), $10^8$ bacteria/mL (Vibrio anguillarum or Photobacterium damselae) or $10^6$ TCID$_{50}$/mL of NNV (strain 411/96, genotype RGNNV). Afterwards, HK cells were washed with 0.01 M phosphate buffer saline (PBS) and stored in TRIzol® Reagent at -80 °C for later isolation of RNA.

For the CMC assays, they were performed as elsewhere [24]. In brief, growing cultures of SAF-1 (ECACC 00122301) fish cell line were detached by standard trypsinization, washed, seeded in flat-bottomed 96 well plates (Nunc) and incubated without (mock-infected) or with $10^6$ TCID$_{50}$ NNV/mL (NNV-infected) for 24 h. These cells were used as targets in the CMC assays. Then, target cells were washed and 100 µL of freshly isolated HK cells added as effectors (50 HK cells per target cell). Samples were centrifuged (400 g, 1 min) and incubated at 25 °C for 4 h. Finally, samples were centrifuged, and cell pellets stored in TRIzol® Reagent at -80 °C for later RNA isolation. CMC samples at 0 h of incubation were used as controls. Samples with targets alone or HK cells alone were also used as technique controls. Infection of the target cells with NNV was confirmed by real-time PCR (qPCR) [24].

Finally, *in vivo* NNV-infection was performed by intramuscular injection. For this, fish received a 100 µL injection of culture medium alone (mock-infected) or containing $10^6$ TCID$_{50}$/fish of NNV. Fish ($n = 5$ fish/group and time) were sampled 1, 7 or 15 days post-infection (dpi) and brain, HK and gonad tissues were removed and immediately frozen in TRIzol® Reagent and stored at -80 °C for later RNA isolation or in Bouin's solution for IHC analysis. We have selected these tissues because i) brain is the main target tissue for NNV replication and infection, ii) HK is the main lymphohematopoietic tissue in fish, and iii) gonad is used by NNV to hide, evade the immune response, and be spread to the progeny by vertical transmission.

## 2.4. Analysis of gene expression by qPCR

Total RNA was isolated from TRIzol® Reagent frozen samples following the manufacturer's instructions and quality and quantity evaluated using the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). One µg of total RNA was treated with DNAse I (Promega) to remove genomic DNA and the first strand of cDNA synthesized by reverse transcription using the Superscript IV (Life Technologies). The qPCR was performed with an ABI PRISM 7500 instrument (Applied Biosystems) using SYBR Green PCR Core Reagents (Applied Biosystems). Reaction mixtures were incubated at 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C, 1 min at 60 °C, and finally 15 s at 95 °C, 1 min at 60 °C and 15 s at 95 °C. For each mRNA, gene expression was corrected by the geometric average of the elongation factor 1 alpha (*ef1a*) and ribosomal protein 18 (*rps18*) gene expressions in each sample and expressed as either $2^{-\Delta Ct}$ or $2^{-\Delta\Delta Ct}$ [27]. Stability of the housekeeping genes was routinely confirmed by the BestKeeper® software. The primers used were designed specifically using the Oligo Perfect software tool (Thermo Fisher Scientific) and are shown in Supplementary Table 1. Prior the experiment, the specificity of each primer pair was studied using positive and negative samples. A melting curve analysis of the amplified products validated the primer for specificity at each use. Negative controls with no template were always included in the reactions.

## 2.5. Generation and validation of polyclonal antibodies

Polyclonal antibodies against the putative European sea bass PRF1.9 sequence (Uniprot acc. number A0A8C4EQ87) were produced in mice by using the chemically synthesized CETSPQRNQSLTTQSPKYDEIC peptide as described elsewhere [28]. The designed peptide was only present in the carboxyl-terminal end of PRF1.9. The recognition of the corresponding synthetic epitopes or the complete molecule by the serum was tested by using an indirect ELISA as hitherto defined [28]. To check the ability to recognize the complete molecule, European sea bass whole

spleen was homogenized in lysis buffer (50 mM Tris-HCl, pH 7.6, 0.3 M NaCl, 0.5% Triton X-100, 0.5 M EDTA and 0.2% protease inhibitor cocktail) and protein concentration determined (BCA protein assay kit; Pierce). In addition, the specificity of the mouse serum was evaluated by the pre-absorption (1:5,000) with its corresponding specific synthetic peptide (4 µg) for 2 h and then tested in the indirect ELISA.

## 2.6. Immunohistochemistry (IHC)

Fragments of HK, brain and gonad from mock- and NNV-infected fish from the *in vivo* infection described above were fixed in Bouin's solution for 24 h at 4 °C, embedded in paraffin (Paraplast Plus; Sherwood Medical) and sectioned at 5 µm. After dewaxing and rehydration, the sections were subjected to an streptavidin-biotin complex technique as previously described [29]. The anti-PRF1.9 sera was used at its optimal dilution of 1:100. The secondary antibody was a commercial mix of biotinylated secondary antibodies goat anti-mouse IgG and goat anti-rabbit IgG (Sigma-Aldrich) conjugated with streptavidin (Cytiva). The reaction was revealed using the horseradish peroxidase-streptavidin complex (Invitrogen) and 0.5 mg/mL 3,3'-diaminobenzidine tetrahydrochloride hydrate (Sigma-Aldrich) in PBS containing 25 µL $H_2O_2$ as substrate. Some sections were slightly counterstained with hematoxylin. The specificity of the reaction was determined by using the pre-immune serum instead of the first antiserum.

## 2.7. Statistical analysis

All data are presented as mean ± standard error of the mean (SEM). *Perforin* transcription was presented as relative expression in naïve tissues or as fold change respect to the respective controls in the HK cells, CMC and NNV-infection assays. Results were analysed by a Student-t test to denote statistical differences between treated/infected and control groups. Normality was evaluated by the Shapiro-Wilk test. Non-parametric Pearson correlation test was applied to identify potential relations among gene expression. Statistical analyses were conducted using SPSS 20 software. Minimum level of significance was fixed in 0.05.

# 3. Results

## 3.1. Perforins of European sea bass belong to the MACPF superfamily

Within the European sea bass genome, we clearly identified four *prf* genes and named *prf1.2* (ENSLAT00005031934.2), *prf1.3* (ENS-LAT00005009634.2), *prf1.5* (ENSLAT00005052249.2) and *prf1.9* (ENSLAT00005024231.2), which generated predicted putative proteins of 583, 566, 523 and 610 amino acids (aa), respectively. Alignment of identified European sea bass PRF sequences with the PRF1 human orthologue, together with protein domain architecture prediction, pointed to a relatively high conservation within the MACPF and C2 domains, including important Cys residues (Fig. 1). However, the main differences among them were present in the leader peptide and in the C-terminus whereas MACPF domain showed slight differences among the sequences. The phylogenetic analysis confirmed the similarities of the four perforins found in European sea bass within others from multiple fish species, since all of them differed from the clade formed by complement proteins C6–C9 as well as MPEG1 families, both members of the MACPF superfamily (Fig. 2). Nevertheless, within the clade of perforins, the four European sea bass proteins were distributed along different branches, distant from each other, indicating a relative diversification, which could lead to functional specialization. Interestingly, the sea bass PRF1.3 was the closest to the mammalian perforins.

## 3.2. *Perforin* transcription shows different basal expression patterns

The data obtained in this study demonstrated constitutive expression of sea bass perforins across all sampled tissues. However, differential
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Alignment of the predicted putative European sea bass perforins (PRFs) with their human orthologue using ClustalW. Numbering was according to the sea bass mature protein PRF1.9. Gaps are introduced to strengthen the alignment. Similar residues are shown in different colours. Different regions in the mature protein are denoted.</figcaption></figure>

expression patterns were observed among the genes. Specifically, *prf1.2*, *prf1.3* and *prf1.5* exhibited relative higher expression levels in the brain and gut (Fig. 3). In contrast, *prf1.9* displayed the highest expression levels in the liver and in immune tissues, where T cells can be found, such as the spleen and thymus.

### 3.3. Perforins are differentially regulated and related to the CMC

We aimed to investigate the transcriptional regulation of the four *prf* genes in European sea bass. Firstly, we evidenced an up-regulation of the relative expression of *prf1.2*, *prf1.5* and *prf1.9* upon HK cells stimulation with PHA, whereas other T cell mitogen as ConA failed to regulate their transcription levels (Fig. 4). Interestingly, LPS, the major constituent of the outer leaflet of the Gram-negative bacterial membrane, and *Vibrio anguillarum*, down-regulated the relative expression of *prf1.9* in European sea bass HK cells. Notably, the mRNA levels of *prf1.3* remained unchanged under all treatments. Subsequently, we evaluated the levels of expression of European sea bass *prf* genes in HK cells during the innate CMC response against SAF-1 cell line as target cells, either mock- or NNV-infected. The data obtained evidenced that the transcription of *prf1.2* and *prf1.9* was significantly increased during the CMC response against SAF-1 target cells (Fig. 5), but significantly down-regulated when the target cells were infected with NNV. All *prf* genes showed lower expression levels in the response against virus-infected cells than against mock-infected cells; nevertheless, *prf1.2* and *prf1.9* displayed statistically significant down-regulations.

Finally, we conducted an analysis to assess their expression pattern during an *in vivo* NNV infection. The results revealed that only *prf1.2*, *prf1.5*, and *prf1.9* exhibited significant alterations in their mRNA levels upon infection. In the HK (Fig. 6A), all genes, except *prf1.3*, displayed an up-regulation of their expression levels at 15 dpi. Similarly, in the brain, *prf1.2* and *prf1.9* showed increased expression levels at 15 dpi (Fig. 6B), while *prf1.3* and *prf1.5* did not exhibit modulation. Furthermore, in the gonad, only the *prf1.9* transcription significantly increased at 1 dpi, whereas the other genes did not exhibit statistically significant changes

(Fig. 6C).

A correlation study (Table 1) was performed comparing the transcription levels of *prf* genes between them and with other genes related to the CMC response [28,30,31]. We found that *prf1.2*, *prf1.3* and *prf1.5* showed positive correlations among them but not with *prf1.9*, as expected by their differential expression pattern observed in the *in vitro* and *in vivo* experiments presented herein. Strikingly, all *prf* expression levels showed positive and strong correlations with *granzyme B* (gzmb) gene expression, while only *prf1.2* and *prf1.5* weakly correlated with *granzyme A* (gzma). Interestingly, *prf1.9* showed a positive and strong correlation with T cell receptor beta chain (tcrb) (Table 1).

### 3.4. PRF1.9⁺ cells are increased in the head-kidney of NNV-infected fish

We generated an anti-PRF1.9 polyclonal antiserum in mice using a synthetic peptide and validated its specificity by the indirect ELISA test (Fig. 7). Absorbance data reveal that the antiserum was able to recognize the pure synthetic peptide and the perforin present in the spleen tissue in a specific manner as the signal is completely blocked by the pre-absorption control.

Then, we decided to perform IHC on head-kidney, brain and gonad to study the presence of PRF1.9 in cells upon an *in vivo* NNV infection. PRF1.9⁺ cells were identified throughout the tissues studied, presenting rounded morphology with stained cytoplasm. The results demonstrated that the number of PRF1.9⁺ cells are increased upon infection in all studied tissues compared to uninfected fish at 15 dpi (Fig. 8). Interestingly, scarce PRF1.9⁺ cells were observed in HK and gonad but not in brain of mock-infected fish.

## 4. Discussion

CMC plays a crucial role in eradicating tumours and virus-infected cells in animals. This mechanism involves various effector cells, including T lymphocytes and NK cells, as well as a cytotoxic pathway mediated by the killing effector proteins, namely perforins and
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Alignment of the predicted putative European sea bass perforins (PRFs) with their human orthologue using ClustalW. Numbering was according to the sea bass mature protein PRF1.9. Gaps are introduced to strengthen the alignment. Similar residues are shown in different colours. Different regions in the mature protein are denoted.</figcaption></figure>

expression patterns were observed among the genes. Specifically, *prf1.2*, *prf1.3* and *prf1.5* exhibited relative higher expression levels in the brain and gut (Fig. 3). In contrast, *prf1.9* displayed the highest expression levels in the liver and in immune tissues, where T cells can be found, such as the spleen and thymus.

### 3.3. Perforins are differentially regulated and related to the CMC

We aimed to investigate the transcriptional regulation of the four *prf* genes in European sea bass. Firstly, we evidenced an up-regulation of the relative expression of *prf1.2*, *prf1.5* and *prf1.9* upon HK cells stimulation with PHA, whereas other T cell mitogen as ConA failed to regulate their transcription levels (Fig. 4). Interestingly, LPS, the major constituent of the outer leaflet of the Gram-negative bacterial membrane, and *Vibrio anguillarum*, down-regulated the relative expression of *prf1.9* in European sea bass HK cells. Notably, the mRNA levels of *prf1.3* remained unchanged under all treatments. Subsequently, we evaluated the levels of expression of European sea bass *prf* genes in HK cells during the innate CMC response against SAF-1 cell line as target cells, either mock- or NNV-infected. The data obtained evidenced that the transcription of *prf1.2* and *prf1.9* was significantly increased during the CMC response against SAF-1 target cells (Fig. 5), but significantly down-regulated when the target cells were infected with NNV. All *prf* genes showed lower expression levels in the response against virus-infected cells than against mock-infected cells; nevertheless, *prf1.2* and *prf1.9* displayed statistically significant down-regulations.

Finally, we conducted an analysis to assess their expression pattern during an *in vivo* NNV infection. The results revealed that only *prf1.2*, *prf1.5*, and *prf1.9* exhibited significant alterations in their mRNA levels upon infection. In the HK (Fig. 6A), all genes, except *prf1.3*, displayed an up-regulation of their expression levels at 15 dpi. Similarly, in the brain, *prf1.2* and *prf1.9* showed increased expression levels at 15 dpi (Fig. 6B), while *prf1.3* and *prf1.5* did not exhibit modulation. Furthermore, in the gonad, only the *prf1.9* transcription significantly increased at 1 dpi, whereas the other genes did not exhibit statistically significant changes

(Fig. 6C).

A correlation study (Table 1) was performed comparing the transcription levels of *prf* genes between them and with other genes related to the CMC response [28,30,31]. We found that *prf1.2*, *prf1.3* and *prf1.5* showed positive correlations among them but not with *prf1.9*, as expected by their differential expression pattern observed in the *in vitro* and *in vivo* experiments presented herein. Strikingly, all *prf* expression levels showed positive and strong correlations with *granzyme B* (gzmb) gene expression, while only *prf1.2* and *prf1.5* weakly correlated with *granzyme A* (gzma). Interestingly, *prf1.9* showed a positive and strong correlation with T cell receptor beta chain (tcrb) (Table 1).

### 3.4. PRF1.9⁺ cells are increased in the head-kidney of NNV-infected fish

We generated an anti-PRF1.9 polyclonal antiserum in mice using a synthetic peptide and validated its specificity by the indirect ELISA test (Fig. 7). Absorbance data reveal that the antiserum was able to recognize the pure synthetic peptide and the perforin present in the spleen tissue in a specific manner as the signal is completely blocked by the pre-absorption control.

Then, we decided to perform IHC on head-kidney, brain and gonad to study the presence of PRF1.9 in cells upon an *in vivo* NNV infection. PRF1.9⁺ cells were identified throughout the tissues studied, presenting rounded morphology with stained cytoplasm. The results demonstrated that the number of PRF1.9⁺ cells are increased upon infection in all studied tissues compared to uninfected fish at 15 dpi (Fig. 8). Interestingly, scarce PRF1.9⁺ cells were observed in HK and gonad but not in brain of mock-infected fish.

## 4. Discussion

CMC plays a crucial role in eradicating tumours and virus-infected cells in animals. This mechanism involves various effector cells, including T lymphocytes and NK cells, as well as a cytotoxic pathway mediated by the killing effector proteins, namely perforins and
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Phylogenetic tree including the European sea bass perforin (PRF) putative proteins was constructed using the Neighbour-Joining method, where genetic distances were calculated based on protein differences (p-distance) with pairwise deletion. The number of each node represents the percentage of bootstrapping after 1,000 replications. Species and accession numbers are shown. European sea bass putative proteins are highlighted. Protein clades are indicated in different colours. Proteins containing the membrane attack complex/Perforin (MACPF) domains, MPEG1 (macrophage-expressed gene 1 protein) and C6–C9 complement proteins, are also included.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** mRNA levels of *perforin* (*prf*) genes in different organs in naïve European sea bass tissues. Data are shown as mean ± SEM (n = 3) target gene expression relative to the expression of the geometric average of the endogenous control *ef1a* and *rps18* genes.</figcaption></figure>

granzymes. Despite the comprehensive understanding of the CMC in mammals [32] our knowledge in fish is still limited. In an endeavour to shed light on this subject, we searched *prf* genes in the teleost European

sea bass genome databases to evaluate their presence and functional characteristics.

First, we performed an *in silico* search of the *prf* gene sequences in the
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Phylogenetic tree including the European sea bass perforin (PRF) putative proteins was constructed using the Neighbour-Joining method, where genetic distances were calculated based on protein differences (p-distance) with pairwise deletion. The number of each node represents the percentage of bootstrapping after 1,000 replications. Species and accession numbers are shown. European sea bass putative proteins are highlighted. Protein clades are indicated in different colours. Proteins containing the membrane attack complex/Perforin (MACPF) domains, MPEG1 (macrophage-expressed gene 1 protein) and C6–C9 complement proteins, are also included.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** mRNA levels of *perforin* (*prf*) genes in different organs in naïve European sea bass tissues. Data are shown as mean ± SEM (n = 3) target gene expression relative to the expression of the geometric average of the endogenous control *ef1a* and *rps18* genes.</figcaption></figure>

granzymes. Despite the comprehensive understanding of the CMC in mammals [32] our knowledge in fish is still limited. In an endeavour to shed light on this subject, we searched *prf* genes in the teleost European

sea bass genome databases to evaluate their presence and functional characteristics.

First, we performed an *in silico* search of the *prf* gene sequences in the
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Heatmap presenting the transcription of *perforin* (*prf*) genes in European sea bass head-kidney cells under different treatments for 24 h (n = 5). Data are presented as fold change respect to the control. Asterisks denote significant differences between control and treated groups (*p* < 0.05). LPS, lipopolysaccharide; PHA, phytohemagglutinin; ConA, concanavalin A; CpG ODN, synthetic unmethylated cytosine-phosphodiester-guanosine oligodeoxynucleotide; NNV, nodavirus.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Expression of *perforin* (*prf*) genes in European sea bass isolated head-kidney cells after the cell-mediated cytotoxic (CMC) response against mock-(SAF-1) and NNV-infected (SAF-1-NNV) SAF-1 cells for 4 h. Data are presented as mean fold change ± SEM (n = 3) respect to the controls. Asterisks denote significant differences between control and NNV-infected fish according to a t-Student test (*p* < 0.05) while symbol # denotes differences between the mock-and NNV-infected target cells.</figcaption></figure>

European sea bass genome, obtaining four genes, named as *prf1.2*, *prf1.3*, *prf1.5* and *prf1.9* with similar sizes in the predicted mature proteins compared to other fish [13] and human orthologues. Mammalian

and certain teleost fish species, including Japanese flounder [15], rainbow trout [16] and half smooth tongue sole [33], have a single copy of *prf* gene. However, several *pfr* genes have been identified in other fish species such as zebrafish [14] and common carp [18], and this study also does for European sea bass. This fact is explained by the genomic duplication events occurred across the fish evolutionary lineage that have caused them to possess several gene copies [34]. The alignment of the putative European sea bass perforins against the human orthologue showed a relatively high degree of conservation of the MACPF and C2 domains, suggesting that fish perforins might have a similar function as in humans [13]. In addition, the phylogenetic analysis showed the high conservation grade of European sea bass PRF proteins compared to other teleost fish, all of them being separated from other pore-forming proteins, such as MPEG1 and C6–C9 complement proteins, providing further evidence for a possible functional conservation of European sea bass PRFs. In fact, it is remarkable how the PRF and C6–C9 branches arise from the MPEG1 branch, which is considered to be the ancestor for perforins [35]. However, it is noteworthy that the different European sea bass perforins do not cluster in the same branch, which may indicate a sub-functionalization among them.

This hypothesis is firstly supported by the gene expression patterns through different tissues. We found basal expression of the four *prf* genes in all tissues tested; however, *prf1.2*, *prf1.3* and *prf1.5* showed a high expression in brain and intestine, while *prf1.9* was more highly expressed in immune-related tissues such as spleen and thymus. Thus, the pattern of expression of European sea bass *prf1.9* was similar to mammalian *prf1*, whose expression levels were high in thymus, spleen or lymph nodes, since they contain high numbers of T lymphocytes and NK cells [10]. Interestingly, the expression pattern of *prf1.9* and *tcrb* genes strongly correlated supporting the idea that T lymphocytes are involved in PRF1.9 production in European sea bass. On the other hand, the presence of tissue-specific expression profiles between *prf* genes in European sea bass is a phenomenon that occurs equally in other fish species [14,16,36] and implies differences at cell subpopulations or functional level. So, in teleost fish, different cell types could be involved in perforin-mediated cytotoxicity as the rainbow trout monocyte-macrophage RTS11 cell line [20] or the non-specific cytotoxic cells of tilapia [37]. In that sense, further studies are still needed to clarify the tissue and cell distribution, and possible physiological functions, of the identified perforins in European sea bass.

We also wanted to evaluate their regulation upon different immune-stimulation conditions. In HK cells stimulated *in vitro*, all *prf* genes, except *prf1.3*, were up-regulated by PHA, a well-known T cell mitogen, indicating that the main perforin-producing cells might be T lymphocytes, as also demonstrated in rainbow trout CD8α⁺ lymphocytes [17] and in mammals [1]. Indeed, PHA causes enhanced expression in European sea bass HK cells of other T lymphocyte-related genes such as *cd4*, *cd28*, *ctla4* and *crtam* [30,38] as in trout occurred with *perforin*, *granulysin*, *interferon gamma* or *IL-4/13A* genes [17]. Surprisingly, LPS, the main virulence factor of gram-negative bacteria, and the bacteria *V. anguillarum* decreased the levels of expression of *prf1.9*. It seems that both stimuli suppressed B lymphocyte activation pathway at 24 h of exposition. These data point to a possible mechanism triggered by bacteria to evade the CMC response. In fact, *V. anguillarum* causes a decrease in the respiratory burst of European sea bass HK leucocytes, which allows the evasion of the bacteria from the immune system [39]. By contrast to our data, increased *prf* transcription levels upon LPS treatment of carp HK leucocytes and PBLs [18] and after an *in vivo* challenge with LPS in zebrafish [14] were observed in previous studies. Regarding NNV *in vivo* infection, we found that the transcription of all *prf* genes, except *prf1.3* again, were increased in HK at 15 dpi, as well as *prf1.2* and *prf1.9* expression levels did in the brain at the same time. In rock bream, however, kidney *prf* transcription increased from 8 to 28 dpi while in zebrafish some *prf* genes increased at shorter times in both the kidney and whole larvae [14,19]. All these data suggest that perforins, except *prf1.3*, are stimulated at different times upon a viral infection,
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Heatmap presenting the transcription of *perforin* (*prf*) genes in European sea bass head-kidney cells under different treatments for 24 h (n = 5). Data are presented as fold change respect to the control. Asterisks denote significant differences between control and treated groups (*p* < 0.05). LPS, lipopolysaccharide; PHA, phytohemagglutinin; ConA, concanavalin A; CpG ODN, synthetic unmethylated cytosine-phosphodiester-guanosine oligodeoxynucleotide; NNV, nodavirus.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Expression of *perforin* (*prf*) genes in European sea bass isolated head-kidney cells after the cell-mediated cytotoxic (CMC) response against mock-(SAF-1) and NNV-infected (SAF-1-NNV) SAF-1 cells for 4 h. Data are presented as mean fold change ± SEM (n = 3) respect to the controls. Asterisks denote significant differences between control and NNV-infected fish according to a t-Student test (*p* < 0.05) while symbol # denotes differences between the mock-and NNV-infected target cells.</figcaption></figure>

European sea bass genome, obtaining four genes, named as *prf1.2*, *prf1.3*, *prf1.5* and *prf1.9* with similar sizes in the predicted mature proteins compared to other fish [13] and human orthologues. Mammalian

and certain teleost fish species, including Japanese flounder [15], rainbow trout [16] and half smooth tongue sole [33], have a single copy of *prf* gene. However, several *pfr* genes have been identified in other fish species such as zebrafish [14] and common carp [18], and this study also does for European sea bass. This fact is explained by the genomic duplication events occurred across the fish evolutionary lineage that have caused them to possess several gene copies [34]. The alignment of the putative European sea bass perforins against the human orthologue showed a relatively high degree of conservation of the MACPF and C2 domains, suggesting that fish perforins might have a similar function as in humans [13]. In addition, the phylogenetic analysis showed the high conservation grade of European sea bass PRF proteins compared to other teleost fish, all of them being separated from other pore-forming proteins, such as MPEG1 and C6–C9 complement proteins, providing further evidence for a possible functional conservation of European sea bass PRFs. In fact, it is remarkable how the PRF and C6–C9 branches arise from the MPEG1 branch, which is considered to be the ancestor for perforins [35]. However, it is noteworthy that the different European sea bass perforins do not cluster in the same branch, which may indicate a sub-functionalization among them.

This hypothesis is firstly supported by the gene expression patterns through different tissues. We found basal expression of the four *prf* genes in all tissues tested; however, *prf1.2*, *prf1.3* and *prf1.5* showed a high expression in brain and intestine, while *prf1.9* was more highly expressed in immune-related tissues such as spleen and thymus. Thus, the pattern of expression of European sea bass *prf1.9* was similar to mammalian *prf1*, whose expression levels were high in thymus, spleen or lymph nodes, since they contain high numbers of T lymphocytes and NK cells [10]. Interestingly, the expression pattern of *prf1.9* and *tcrb* genes strongly correlated supporting the idea that T lymphocytes are involved in PRF1.9 production in European sea bass. On the other hand, the presence of tissue-specific expression profiles between *prf* genes in European sea bass is a phenomenon that occurs equally in other fish species [14,16,36] and implies differences at cell subpopulations or functional level. So, in teleost fish, different cell types could be involved in perforin-mediated cytotoxicity as the rainbow trout monocyte-macrophage RTS11 cell line [20] or the non-specific cytotoxic cells of tilapia [37]. In that sense, further studies are still needed to clarify the tissue and cell distribution, and possible physiological functions, of the identified perforins in European sea bass.

We also wanted to evaluate their regulation upon different immune-stimulation conditions. In HK cells stimulated *in vitro*, all *prf* genes, except *prf1.3*, were up-regulated by PHA, a well-known T cell mitogen, indicating that the main perforin-producing cells might be T lymphocytes, as also demonstrated in rainbow trout CD8α⁺ lymphocytes [17] and in mammals [1]. Indeed, PHA causes enhanced expression in European sea bass HK cells of other T lymphocyte-related genes such as *cd4*, *cd28*, *ctla4* and *crtam* [30,38] as in trout occurred with *perforin*, *granulysin*, *interferon gamma* or *IL-4/13A* genes [17]. Surprisingly, LPS, the main virulence factor of gram-negative bacteria, and the bacteria *V. anguillarum* decreased the levels of expression of *prf1.9*. It seems that both stimuli suppressed B lymphocyte activation pathway at 24 h of exposition. These data point to a possible mechanism triggered by bacteria to evade the CMC response. In fact, *V. anguillarum* causes a decrease in the respiratory burst of European sea bass HK leucocytes, which allows the evasion of the bacteria from the immune system [39]. By contrast to our data, increased *prf* transcription levels upon LPS treatment of carp HK leucocytes and PBLs [18] and after an *in vivo* challenge with LPS in zebrafish [14] were observed in previous studies. Regarding NNV *in vivo* infection, we found that the transcription of all *prf* genes, except *prf1.3* again, were increased in HK at 15 dpi, as well as *prf1.2* and *prf1.9* expression levels did in the brain at the same time. In rock bream, however, kidney *prf* transcription increased from 8 to 28 dpi while in zebrafish some *prf* genes increased at shorter times in both the kidney and whole larvae [14,19]. All these data suggest that perforins, except *prf1.3*, are stimulated at different times upon a viral infection,
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Transcription of *perforin* (*prf*) genes in European sea bass brain (A), head-kidney (B) and gonad (C) tissues upon infection with 10⁶ TCID₅₀ of NNV/fish after 1, 7 and 15 days of infection (dpi). Data are presented as mean fold change ± SEM (n = 4–6) in infected groups compared with the controls. Asterisks denote significant differences between control and NNV-infected fish at each infection time according to a t-Student test (*p* < 0.05).</figcaption></figure>

**Table 1**

Correlations in the transcription levels of *prf* with other genes related to the cell-mediated cytotoxicity [26,28,29], where Pearson index and *P* values are indicated. Parameters that correlate are highlighted in bold. *prf*, perforin; *gzma*, granzyme A; *gzmb*, granzyme B; *nkl*, NK-lysin; *crtam*, cytotoxic and regulatory T cell coreceptor; *tcrb*, T cell receptor beta chain.

<table><thead><tr><th></th><th></th><th>prf1.3</th><th>prf1.5</th><th>prf1.9</th><th>gzma</th><th>gzmb</th><th>nkl</th><th>crtam</th><th>tcrb</th></tr></thead><tbody><tr><td rowspan="2">prf1.2</td><td>Pearson</td><td><strong>0.729</strong></td><td><strong>0.986</strong></td><td>-0.026</td><td><strong>0.573</strong></td><td><strong>0.947</strong></td><td>0.012</td><td>-0.01</td><td>-0.096</td></tr><tr><td><em>P</em> value</td><td><strong>0.000</strong></td><td><strong>0.000</strong></td><td><strong>0.805</strong></td><td><strong>0.000</strong></td><td><strong>0.000</strong></td><td><strong>0.904</strong></td><td><strong>0.989</strong></td><td><strong>0.494</strong></td></tr><tr><td rowspan="2">prf1.3</td><td>Pearson</td><td></td><td><strong>0.723</strong></td><td>0.219</td><td>0.333</td><td><strong>0.657</strong></td><td>-0.085</td><td>0.108</td><td>-0.101</td></tr><tr><td><em>P</em> value</td><td><strong>0.000</strong></td><td><strong>0.000</strong></td><td><strong>0.033</strong></td><td><strong>0.01</strong></td><td><strong>0.000</strong></td><td><strong>0.405</strong></td><td><strong>0.296</strong></td><td><strong>0.473</strong></td></tr><tr><td rowspan="2">prf1.5</td><td>Pearson</td><td></td><td></td><td>-0.051</td><td><strong>0.573</strong></td><td><strong>0.953</strong></td><td>-0.044</td><td>0.011</td><td>-0.104</td></tr><tr><td><em>P</em> value</td><td></td><td></td><td><strong>0.625</strong></td><td><strong>0.000</strong></td><td><strong>0.000</strong></td><td><strong>0.667</strong></td><td><strong>0.913</strong></td><td><strong>0.460</strong></td></tr><tr><td rowspan="2">prf1.9</td><td>Pearson</td><td></td><td></td><td></td><td>0.033</td><td><strong>0.893</strong></td><td>0.112</td><td>-0.066</td><td><strong>0.883</strong></td></tr><tr><td><em>P</em> value</td><td></td><td></td><td></td><td><strong>0.757</strong></td><td><strong>0.000</strong></td><td><strong>0.284</strong></td><td><strong>0.560</strong></td><td><strong>0.000</strong></td></tr></tbody></table>

Specificity test of polyclonal anti-PRF1.9 by indirect ELISA

<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Specificity test of the polyclonal antibody serum specific against PRF1.9 by indirect ELISA using synthetic peptide or European sea bass spleen homogenates. Pre-absorbed antiserum was used as controls.</figcaption></figure>

depending on the species, and possibly related to the cell type where perforin is expressed. In addition, the lack of modulation of the *prf1.3* gene expression, both *in vitro* or *in vivo*, could be attributed to a functional redundancy, where the other copies of perforin genes might be compensating *prf1.3* gene to preserve the biological process. In European sea bass, the expression of *prf* upon NNV infection follows the same pattern identified in other cytotoxic-related genes as *cd8a*, *gzma*, *nkl* and *crtam* [30,31,38,40]. The timing of expression of all these genes and of the increase of the PRF1.9⁺ cells number in HK and other tissues upon

infection, suggest that the adaptive immune response of European sea bass occurs at later stages of the infection through the generation and activation of CTLs in the HK and their subsequent mobilization to other tissues in an effort to maintain vital functions and fight the infection. In addition, our data showed that the expression profiles of most *prf* genes and *granzyme A* and *B* genes showed a positive, and sometimes very strong, correlation between them, allowing us to hypothesize that CTLs and the perforin/granzyme pathway might be involved in the clearance of NNV-infected cells in European sea bass, as seen in other species [20,
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Transcription of *perforin* (*prf*) genes in European sea bass brain (A), head-kidney (B) and gonad (C) tissues upon infection with $10^6$ TCID$_{50}$ of NNV/fish after 1, 7 and 15 days of infection (dpi). Data are presented as mean fold change ± SEM (n = 4–6) in infected groups compared with the controls. Asterisks denote significant differences between control and NNV-infected fish at each infection time according to a t-Student test (*p* < 0.05).</figcaption></figure>

**Table 1**

Correlations in the transcription levels of *prf* with other genes related to the cell-mediated cytotoxicity [26,28,29], where Pearson index and *P* values are indicated. Parameters that correlate are highlighted in bold. *prf*, perforin; *gzma*, granzyme A; *gzmb*, granzyme B; *nkl*, NK-lysin; *crtam*, cytotoxic and regulatory T cell coreceptor; *tcrb*, T cell receptor beta chain.

<table><thead><tr><th></th><th></th><th>prf1.3</th><th>prf1.5</th><th>prf1.9</th><th>gzma</th><th>gzmb</th><th>nkl</th><th>crtam</th><th>tcrb</th></tr></thead><tbody><tr><td rowspan="2">prf1.2</td><td>Pearson</td><td><strong>0.729</strong></td><td><strong>0.986</strong></td><td>-0.026</td><td><strong>0.573</strong></td><td><strong>0.947</strong></td><td>0.012</td><td>-0.01</td><td>-0.096</td></tr><tr><td><em>P</em> value</td><td><strong>0.000</strong></td><td><strong>0.000</strong></td><td><strong>0.805</strong></td><td><strong>0.000</strong></td><td><strong>0.000</strong></td><td><strong>0.904</strong></td><td><strong>0.989</strong></td><td><strong>0.494</strong></td></tr><tr><td rowspan="2">prf1.3</td><td>Pearson</td><td></td><td><strong>0.723</strong></td><td>0.219</td><td>0.333</td><td><strong>0.657</strong></td><td>-0.085</td><td>0.108</td><td>-0.101</td></tr><tr><td><em>P</em> value</td><td><strong>0.000</strong></td><td><strong>0.000</strong></td><td><strong>0.033</strong></td><td><strong>0.01</strong></td><td><strong>0.000</strong></td><td><strong>0.405</strong></td><td><strong>0.296</strong></td><td><strong>0.473</strong></td></tr><tr><td rowspan="2">prf1.5</td><td>Pearson</td><td></td><td></td><td>-0.051</td><td><strong>0.573</strong></td><td><strong>0.953</strong></td><td>-0.044</td><td>0.011</td><td>-0.104</td></tr><tr><td><em>P</em> value</td><td></td><td></td><td><strong>0.625</strong></td><td><strong>0.000</strong></td><td><strong>0.000</strong></td><td><strong>0.667</strong></td><td><strong>0.913</strong></td><td><strong>0.460</strong></td></tr><tr><td rowspan="2">prf1.9</td><td>Pearson</td><td></td><td></td><td></td><td>0.033</td><td><strong>0.893</strong></td><td>0.112</td><td>-0.066</td><td><strong>0.883</strong></td></tr><tr><td><em>P</em> value</td><td></td><td></td><td></td><td><strong>0.757</strong></td><td><strong>0.000</strong></td><td><strong>0.284</strong></td><td><strong>0.560</strong></td><td><strong>0.000</strong></td></tr></tbody></table>

Specificity test of polyclonal anti-PRF1.9 by indirect ELISA

<figure><img src="image_11.png" /><figcaption>**Fig. 7.** Specificity test of the polyclonal antibody serum specific against PRF1.9 by indirect ELISA using synthetic peptide or European sea bass spleen homogenates. Pre-absorbed antiserum was used as controls.</figcaption></figure>

depending on the species, and possibly related to the cell type where perforin is expressed. In addition, the lack of modulation of the *prf1.3* gene expression, both *in vitro* or *in vivo*, could be attributed to a functional redundancy, where the other copies of perforin genes might be compensating *prf1.3* gene to preserve the biological process. In European sea bass, the expression of *prf* upon NNV infection follows the same pattern identified in other cytotoxic-related genes as *cd8a*, *gzma*, *nkl* and *crtam* [30,31,38,40]. The timing of expression of all these genes and of the increase of the PRF1.9⁺ cells number in HK and other tissues upon

infection, suggest that the adaptive immune response of European sea bass occurs at later stages of the infection through the generation and activation of CTLs in the HK and their subsequent mobilization to other tissues in an effort to maintain vital functions and fight the infection. In addition, our data showed that the expression profiles of most *prf* genes and *granzyme A* and *B* genes showed a positive, and sometimes very strong, correlation between them, allowing us to hypothesize that CTLs and the perforin/granzyme pathway might be involved in the clearance of NNV-infected cells in European sea bass, as seen in other species [20,
<figure><img src="image_11.png" /><figcaption>**Fig. 8.** Head-kidney (A–C), brain (D–F) and gonad (G–I) sections of European sea bass mock- (B,E,H) and NNV-infected (A,C,D,F,G,I) at 15 days post infection (dpi) immunostained with the anti-PRF1.9 serum at optimal dilution 1:100 (B–C,E,F,H–I) or with the preimmune serum (A,D,G) and counterstained with hematoxylin (D–I) or not (A–C). HK, head-kidney; NNV, nodavirus. Green arrows denote PRF1.9⁺ cells in all tissues and blue arrows denote melanomacrophages in the head-kidney. Scale bars = 50 µm.</figcaption></figure>

[41].

The gonad tissue has gained interest in the studies of NNV-infected specimens due to the ability of the virus to be vertically transmitted and disseminated through the gonad [42]. In addition, the inflammatory, antimicrobial and interferon immune responses in NNV infected European sea bass gonads showed clear differences between *in vivo* and *in vitro* infections [25,43,44], suggesting that some responses observed in the gonad *in vivo* are due to a systemic immune regulation or interaction between different cell types, among others, but not due to a local response against the virus. In our study, only *prf1.9* gene expression was up-regulated in the gonad at the beginning of the *in vivo* infection, 1 dpi, but not later on. Moreover, *prf1.2* and *prf1.9* were activated during the CMC against mock-cells, but not against NNV-infected target cells, as also occurs with the innate CMC activity [24]. This would result in the intracellular survival of the virus during early infection times and its spread throughout different tissues, being able to reach the gonad. Later on, those specimens capable of surpassing the initial infection stage might be able to mount a specific CMC response (15 dpi) through the involvement of CTLs and the up-regulation of the expression of *prf1.2*, *prf1.5* and *prf1.9* genes in HK and brain, but not in gonad. Our hypothesis states that this CMC response pattern in HK and brain tissues promotes fish survival and the persistence of the virus in the gonad and its subsequent transmission. All this data taken together shed light in the cellular and molecular mechanisms used by the virus to reach the gonad and success to be vertically transmitted in a very susceptible fish species. Unfortunately, little is found in the literature in this regard and further studies are welcomed.

In conclusion, our *in silico* study confirms the presence of four *prf* genes in the European sea bass genome, called *prf1.2*, *prf1.3*, *prf1.5* and *prf1.9*, exhibiting the characteristic MACPF and C2 domains of its mammalian orthologue. All *prf* genes are constitutively expressed with distinct expression profiles, with *prf1.9* being the most highly expressed in lymphoid tissues such as the spleen and thymus. Moreover, mRNA levels in the *in vitro* assay and the correlations observed between CMC-related genes suggest that European sea bass *prf* are predominantly expressed in T lymphocytes, except *prf1.3*, which remained unmodified under any treatment. All *prf* gene expressions, except *prf1.3*, increased upon NNV infection, mainly at 15 dpi in most tissues, concomitantly with the increase of PRF1.9⁺ cells in head-kidney, brain and gonad. The correlation analysis shows a strong and positive correlation between *perforins* and *granzymes*, as the main CMC mediators, and between *prf1.9* and *tcrb*, pointing to a greater role for PRF1.9 in the specific CMC response against NNV infection mediated by T cells.

CRediT authorship contribution statement

**Miguel A. García-Álvarez:** Methodology, Investigation, Software, Data curation, Writing – original draft, Writing – review & editing.
**Laura Cervera:** Methodology, Investigation, Writing – review & editing.
**Yulema Valero:** Methodology, Investigation, Writing – review & editing.
**Carmen González-Fernández:** Methodology, Investigation, Writing – review & editing.
**Luis Mercado:** Investigation, Writing – review & editing.
**Elena Chaves-Pozo:** Supervision, Writing – review & editing, Funding acquisition.
**Alberto Cuesta:** Conceptualization,
<figure><img src="image_12.png" /><figcaption>**Fig. 8.** Head-kidney (A–C), brain (D–F) and gonad (G–I) sections of European sea bass mock- (B,E,H) and NNV-infected (A,C,D,F,G,I) at 15 days post infection (dpi) immunostained with the anti-PRF1.9 serum at optimal dilution 1:100 (B–C,E,F,H–I) or with the preimmune serum (A,D,G) and counterstained with hematoxylin (D–I) or not (A–C). HK, head-kidney; NNV, nodavirus. Green arrows denote PRF1.9⁺ cells in all tissues and blue arrows denote melanomacrophages in the head-kidney. Scale bars = 50 µm.</figcaption></figure>

[41].

The gonad tissue has gained interest in the studies of NNV-infected specimens due to the ability of the virus to be vertically transmitted and disseminated through the gonad [42]. In addition, the inflammatory, antimicrobial and interferon immune responses in NNV infected European sea bass gonads showed clear differences between *in vivo* and *in vitro* infections [25,43,44], suggesting that some responses observed in the gonad *in vivo* are due to a systemic immune regulation or interaction between different cell types, among others, but not due to a local response against the virus. In our study, only *prf1.9* gene expression was up-regulated in the gonad at the beginning of the *in vivo* infection, 1 dpi, but not later on. Moreover, *prf1.2* and *prf1.9* were activated during the CMC against mock-cells, but not against NNV-infected target cells, as also occurs with the innate CMC activity [24]. This would result in the intracellular survival of the virus during early infection times and its spread throughout different tissues, being able to reach the gonad. Later on, those specimens capable of surpassing the initial infection stage might be able to mount a specific CMC response (15 dpi) through the involvement of CTLs and the up-regulation of the expression of *prf1.2*, *prf1.5* and *prf1.9* genes in HK and brain, but not in gonad. Our hypothesis states that this CMC response pattern in HK and brain tissues promotes fish survival and the persistence of the virus in the gonad and its subsequent transmission. All this data taken together shed light in the cellular and molecular mechanisms used by the virus to reach the gonad and success to be vertically transmitted in a very susceptible fish species. Unfortunately, little is found in the literature in this regard and further studies are welcomed.

In conclusion, our *in silico* study confirms the presence of four *prf* genes in the European sea bass genome, called *prf1.2*, *prf1.3*, *prf1.5* and *prf1.9*, exhibiting the characteristic MACPF and C2 domains of its mammalian orthologue. All *prf* genes are constitutively expressed with distinct expression profiles, with *prf1.9* being the most highly expressed in lymphoid tissues such as the spleen and thymus. Moreover, mRNA levels in the *in vitro* assay and the correlations observed between CMC-related genes suggest that European sea bass *prf* are predominantly expressed in T lymphocytes, except *prf1.3*, which remained unmodified under any treatment. All *prf* gene expressions, except *prf1.3*, increased upon NNV infection, mainly at 15 dpi in most tissues, concomitantly with the increase of PRF1.9⁺ cells in head-kidney, brain and gonad. The correlation analysis shows a strong and positive correlation between *perforins* and *granzymes*, as the main CMC mediators, and between *prf1.9* and *tcrb*, pointing to a greater role for PRF1.9 in the specific CMC response against NNV infection mediated by T cells.

### CRediT authorship contribution statement

**Miguel A. García-Álvarez:** Methodology, Investigation, Software, Data curation, Writing – original draft, Writing – review & editing.
**Laura Cervera:** Methodology, Investigation, Writing – review & editing.
**Yulema Valero:** Methodology, Investigation, Writing – review & editing.
**Carmen González-Fernández:** Methodology, Investigation, Writing – review & editing.
**Luis Mercado:** Investigation, Writing – review & editing.
**Elena Chaves-Pozo:** Supervision, Writing – review & editing, Funding acquisition.
**Alberto Cuesta:** Conceptualization,
Supervision, Writing – review & editing, Project administration, Fund-
ing acquisition, All authors have read and agreed to the published
version of the manuscript.

Declaration of competing interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Data availability

Data will be made available on request.

Acknowledgements

This work was funded by Ministerio de Ciencia e Innovación-Agenzia Estatal de Investigación (MCIN/AEI/10.13039/501100011033, grant PID2019-105522 GB-100 to A.C. and FPI contract PRE2020-093771 to L. C.) and by the ThinkInAzul programme cofounded by the European Union NextGenerationEU/PRTR, MCIN/AEI 10.13039/501100011033 and Fundación Séneca. Ministerio de Ciencia e Innovación is also acknowledged by the Juan de la Cierva Incorporación contract to YV (IJC2020-042733-I).

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.fsi.2023.109244.

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[7] A.J. Brennan, J. Chia, J.A. Trapani, I. Voskoboinik, Perforin deficiency and susceptibility to cancer, Cell Death Differ. 17 (2010) 607-615, https://doi.org/10.1038/cdd.2009.212.

[8] C.-C. Lu, T.-S. Wu, Y.-J. Hsu, C.-J. Chang, C.-S. Lin, J.-H. Chia, T.-L. Wu, T.-T. Huang, J. Martel, D.M. Ojcius, J.D. Young, H.-C. Lai, NK cells kill mycobacteria directly by releasing perforin and granulysin, J. Leukoc. Biol. 96 (2014) 1119-1129, https://doi.org/10.1189/jlb.4a.0713-363rr.

[9] R.H.P. Law, N. Lukoyanova, I. Voskoboinik, T.T. Caradoc-Davies, K. Baran, M. A. Dunstone, M.E. D'Angelo, E.V. Orlova, F. Coulibaly, S. Verschoor, K.A. Browne, A. Ciccone, M.J. Kuiper, P.I. Bird, J.A. Trapani, H.R. Saibil, J.C. Whisstock, The structural basis for membrane binding and pore formation by lymphocyte perforin, Nature 468 (2010) 447-451, https://doi.org/10.1038/nature09518.

[10] I. Voskoboinik, J.C. Whisstock, J.A. Trapani, Perforin and granzymes: function, dysfunction and human pathology, Nat. Rev. Immunol. 15 (2015) 388-400, https://doi.org/10.1038/nri3839.

[11] T. Nakanishi, Y. Shibasaki, Y. Matsuura, T cells in fish, Biology (Basel) 4 (2015) 640-663, https://doi.org/10.3390/biology4040640.

[12] D.L. Evans, L. Jaso-Friedmann, Nonspecific cytotoxic cells as effectors of immunity in fish, Annu. Rev. Fish Dis. 2 (1992) 109-121, https://doi.org/10.1016/0959-8030(92)90059-7.

[13] J. Xu, N. Yang, T. Xie, G. Yang, L. Chang, D. Yan, T. Li, Summary and comparison of the perforin in teleosts and mammals: a review, Scand. J. Immunol. 94 (2021) 1-13, https://doi.org/10.1111/sji.13047.

[14] M. Varela, G. Forn-Cuni, S. Dios, A. Figueras, B. Novoa, 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 (2016) 43-56, https://doi.org/10.1159/000431287.

[15] Y.H. Lee, T. Ohira, I. Hirono, T. Aoki, A pore-forming protein, perforin, from a non-mammalian organism, Japanese flounder, *Paralichthys olivaceus*, Immunogenetics 56 (2004) 360-367, https://doi.org/10.1007/s00251-004-0688-8.

[16] S. Athanasopoulou, D. Marioli, A. Mikrou, A.D. Papanastasiou, I.K. Zarkadis, Cloning and characterization of the trout perforin, Fish Shellfish Immunol. 26 (2009) 908-912, https://doi.org/10.1016/j.fsi.2009.03.021.

[17] F. Takizawa, J.M. Dijkstra, P. Kotterba, T. Korytár, H. Kock, B. Köllner, B. Jaureguiberry, T. Nakanishi, U. Fischer, The expression of CD8α discriminates distinct T cell subsets in teleost fish, Dev. Comp. Immunol. 35 (2011) 752-763, https://doi.org/10.1016/j.dci.2011.02.008.

[18] T. Li, L. Wang, Y. Zhang, X. Guo, X. Chen, F. Zhang, G. Yang, W. Wen, H. Li, Molecular characterization of three novel perforins in common carp (*Cyprinus carpio* L.) and their expression patterns during larvae ontogeny and in response to immune challenges, BMC Vet. Res. 14 (2018) 1-15, https://doi.org/10.1186/s12917-018-1613-y.

[19] M.H. Jung, C. Nikapitiya, J.Y. Song, J.H. Lee, J. Lee, M.J. Oh, S.J. Jung, Gene expression of pro- and anti-apoptotic proteins in rock bream (*Oplegnathus fasciatus*) infected with megalocytivirus (family *Iridoviridae*), Fish Shellfish Immunol. 37 (2014) 122-130, https://doi.org/10.1016/j.fsi.2014.01.012.

[20] M.C. Ordás, A. Cuesta, L. Mercado, N.C. Bols, C. Tafalla, Viral hemorrhagic septicemia virus (VHSV) up-regulates the cytotoxic activity and the perforin/granzyme pathway in the rainbow trout RTS11 cell line, Fish Shellfish Immunol. 31 (2011) 252-259, https://doi.org/10.1016/j.fsi.2011.05.010.

[21] H. Toda, K. Araki, T. Moritomo, T. Nakanishi, Perforin-dependent cytotoxic mechanism in killing by CD8 positive T cells in gribuna crucian carp, Carassius auratus langsdorfii, Dev. Comp. Immunol. 35 (2011) 88-93, https://doi.org/10.1016/j.dci.2010.08.010.

[22] P. Moreno, B. Lopez-Jimena, E. Randelli, G. Scapigliati, F. Buonocore, E. Garcia-Rosado, J.J. Borrego, M.C. Alonso, Immuno-related gene transcription and antibody response in nodavirus (RGNNV and SJNNV)-infected European sea bass (Dicentrarchus labrax L.), Fish Shellfish Immunol. 78 (2018) 270-278, https://doi.org/10.1016/j.fsi.2018.04.054.

[23] Y. Valero, P. Morcillo, J. Meseguer, F. Buonocore, M.A. Esteban, E. Chaves-Pozo, A. Cuesta, Characterization of the IFN pathway in the teleost fish gonad against vertically transmitted viral nervous necrosis virus, J. Gen. Virol. 96 (2015) 2176-2187, https://doi.org/10.1099/vir.0.000164.

[24] E. Chaves-Pozo, Y. Valero, A. Esteve-Codina, J. Gómez-Garrido, M. Dabad, T. Alioto, J. Meseguer, M.Á. Esteban, A. Cuesta, Innate cell-mediated cytotoxic activity of European sea bass leucocytes against nodavirus-infected cells: a functional and RNA-seq Study, Sci. Rep. 7 (2017) 1-15, https://doi.org/10.1038/s41598-017-15629-6.

[25] Y. Valero, B. Boughlala, M. Arizcun, S. Patel, I.U. Fiksdal, M.Á. Esteban, J. De Juan, J. Meseguer, E. Chaves-Pozo, A. Cuesta, Genes related to cell-mediated cytotoxicity and interferon response are induced in the retina of European sea bass upon intravitreal infection with nodavirus, Fish Shellfish Immunol. 74 (2018) 627-636, https://doi.org/10.1016/j.fsi.2018.01.034.

[26] K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, MEGA6: molecular evolutionary genetics analysis version 6.0, Mol. Biol. Evol. 30 (2013) 2725-2729, https://doi.org/10.1093/molbev/mst197.

[27] M.W. Pfaffl, A new mathematical model for relative quantification in real-time RT-PCR, Nucleic Acids Res. 29 (2001) 45e-45, https://doi.org/10.1093/nar/29.9.e45.

[28] Y. Valero, M. Arizcun, J. Cortés, F. Ramírez-Cepeda, F. Guzmán, L. Mercado, M.A. Esteban, E. Chaves-Pozo, A. Cuesta, Nk-lysin, Dicentracin and hepcidin antimicrobial peptides in European sea bass. Ontogenetic development and modulation in juveniles by nodavirus, Dev. Comp. Immunol. 103 (2020), 10356, https://doi.org/10.1016/j.dci.2019.103516.

[29] A. Garca-Ayala, E. Chaves-Pozo, Immunocytochemical tools reveal a new research field between the boundaries of immunology and reproductive biology in teleosts, Appl. Immunocytochem. (2012), https://doi.org/10.5772/35231.

[30] M.Á. García-Álvarez, C. González-Fernández, M.Á. Esteban, A. Cuesta, Molecular characterization of the cytotoxic and regulatory T cell coreceptor (CRTAM), and its ligand CADM1, in the European seabass and gilthead seabream, Fish Shellfish Immunol. 134 (2023), https://doi.org/10.1016/j.fsi.2023.108569.

[31] E. Chaves-Pozo, Y. Valero, M.T. Lozano, P. Rodríguez-Cerezo, L. Miao, V. Campo, M.A. Esteban, A. Cuesta, Fish granzyme a shows a greater role than granzyme b in fish innate cell-mediated cytotoxicity, Front. Immunol. 10 (2019) 2579, https://doi.org/10.3389/fimmu.2019.02579.

[32] P. Golstein, G.M. Griffiths, An early history of T cell-mediated cytotoxicity, Nat. Rev. Immunol. 18 (2018) 527-535, https://doi.org/10.1038/s41577-018-0009-3.

[33] X. Fu, Y. Chen, L. Wang, Q. Zhou, M. Li, Y. Song, Y. Li, F. Zhao, S. Chen, Identification and functional analysis of the perforin-1 like gene in disease resistance in half smooth tongue sole (*Cynoglossus semilaevis*), Dev. Comp. Immunol. 122 (2021), 104135, https://doi.org/10.1016/j.dci.2021.104135.

[34] S.M.K. Glasauer, S.C.F. Neuhaus, Whole-genome duplication in teleost fishes and its evolutionary consequences, Mol. Genet. Genom. 289 (2014) 1045-1060, https://doi.org/10.1007/s00438-014-0889-2.

[35] M.E. Dangelo, M.A. Dunstone, J.C. Whisstock, J.A. Trapani, P.I. Bird, Perforin evolved from a gene duplication of MPEG1, followed by a complex pattern of gene gain and loss within *Euteleostomi*, BMC Evol. Biol. 12 (2012), https://doi.org/10.1186/1471-2148-12-59.

[36] H. Yang, Y.M. Lee, J.-H. Lee, J.K. Noh, H.C. Kim, C.-J. Park, J.-W. Park, I.J. Hwang, S.Y. Kim, Expression of perforin gene for early development of nephrons in olive
Supervision, Writing – review & editing, Project administration, Fund-
ing acquisition, All authors have read and agreed to the published
version of the manuscript.

Declaration of competing interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Data availability

Data will be made available on request.

Acknowledgements

This work was funded by Ministerio de Ciencia e Innovación-Agenzia
Estatal de Investigación (MCIN/AEI/10.13039/501100011033, grant
PID2019-105522 GB-100 to A.C. and FPI contract PRE2020-093771 to L.
C.) and by the ThinkInAzul programme cofounded by the European
Union NextGenerationEU/PRTR, MCIN/AEI 10.13039/501100011033
and Fundación Séneca. Ministerio de Ciencia e Innovación is also
acknowledged by the Juan de la Cierva Incorporación contract to YV
(IJC2020-042733-I).

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.fsi.2023.109244.

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[12] D.L. Evans, L. Jaso-Friedmann, Nonspecific cytotoxic cells as effectors of immunity in fish, Annu. Rev. Fish Dis. 2 (1992) 109-121, https://doi.org/10.1016/0959-8030(92)90059-7.

[13] J. Xu, N. Yang, T. Xie, G. Yang, L. Chang, D. Yan, T. Li, Summary and comparison of the perforin in teleosts and mammals: a review, Scand. J. Immunol. 94 (2021) 1-13, https://doi.org/10.1111/sji.13047.

[14] M. Varela, G. Forn-Cuni, S. Dios, A. Figueras, B. Novoa, 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 (2016) 43-56, https://doi.org/10.1159/000431287.

[15] Y.H. Lee, T. Ohira, I. Hirono, T. Aoki, A pore-forming protein, perforin, from a non-mammalian organism, Japanese flounder, *Paralichthys olivaceus*, Immunogenetics 56 (2004) 360-367, https://doi.org/10.1007/s00251-004-0688-8.

[16] S. Athanasopoulou, D. Marioli, A. Mikrou, A.D. Papanastasiou, I.K. Zarkadis, Cloning and characterization of the trout perforin, Fish Shellfish Immunol. 26 (2009) 908-912, https://doi.org/10.1016/j.fsi.2009.03.021.

[17] F. Takizawa, J.M. Dijkstra, P. Kotterba, T. Korytár, H. Kock, B. Köllner, B. Jaureguiberry, T. Nakanishi, U. Fischer, The expression of CD8α discriminates distinct T cell subsets in teleost fish, Dev. Comp. Immunol. 35 (2011) 752-763, https://doi.org/10.1016/j.dci.2011.02.008.

[18] T. Li, L. Wang, Y. Zhang, X. Guo, X. Chen, F. Zhang, G. Yang, W. Wen, H. Li, Molecular characterization of three novel perforins in common carp (*Cyprinus carpio* L.) and their expression patterns during larvae ontogeny and in response to immune challenges, BMC Vet. Res. 14 (2018) 1-15, https://doi.org/10.1186/s12917-018-1613-y.

[19] M.H. Jung, C. Nikapitiya, J.Y. Song, J.H. Lee, J. Lee, M.J. Oh, S.J. Jung, Gene expression of pro- and anti-apoptotic proteins in rock bream (*Oplegnathus fasciatus*) infected with megalocytivirus (family *Iridoviridae*), Fish Shellfish Immunol. 37 (2014) 122-130, https://doi.org/10.1016/j.fsi.2014.01.012.

[20] M.C. Ordás, A. Cuesta, L. Mercado, N.C. Bols, C. Tafalla, Viral hemorrhagic septicemia virus (VHSV) up-regulates the cytotoxic activity and the perforin/granzyme pathway in the rainbow trout RTS11 cell line, Fish Shellfish Immunol. 31 (2011) 252-259, https://doi.org/10.1016/j.fsi.2011.05.010.

[21] H. Toda, K. Araki, T. Moritomo, T. Nakanishi, Perforin-dependent cytotoxic mechanism in killing by CD8 positive T cells in gنبuna crucian carp, Carassius auratus langsdorfii, Dev. Comp. Immunol. 35 (2011) 88-93, https://doi.org/10.1016/j.dci.2010.08.010.

[22] P. Moreno, B. Lopez-Jimena, E. Randelli, G. Scapigliati, F. Buonocore, E. Garcia-Rosado, J.J. Borrego, M.C. Alonso, Immuno-related gene transcription and antibody response in nodavirus (RGNNV and SJNNV)-infected European sea bass (Dicentrarchus labrax L.), Fish Shellfish Immunol. 78 (2018) 270-278, https://doi.org/10.1016/j.fsi.2018.04.054.

[23] Y. Valero, P. Morcillo, J. Meseguer, F. Buonocore, M.A. Esteban, E. Chaves-Pozo, A. Cuesta, Characterization of the IFN pathway in the teleost fish gonad against vertically transmitted viral nervous necrosis virus, J. Gen. Virol. 96 (2015) 2176-2187, https://doi.org/10.1099/vir.0.000164.

[24] E. Chaves-Pozo, Y. Valero, A. Esteve-Codina, J. Gómez-Garrido, M. Dabad, T. Alioto, J. Meseguer, M.Á. Esteban, A. Cuesta, Innate cell-mediated cytotoxic activity of European sea bass leucocytes against nodavirus-infected cells: a functional and RNA-seq Study, Sci. Rep. 7 (2017) 1-15, https://doi.org/10.1038/s41598-017-15629-6.

[25] Y. Valero, B. Boughlala, M. Arizcun, S. Patel, I.U. Fiksdal, M.Á. Esteban, J. De Juan, J. Meseguer, E. Chaves-Pozo, A. Cuesta, Genes related to cell-mediated cytotoxicity and interferon response are induced in the retina of European sea bass upon intravitreal infection with nodavirus, Fish Shellfish Immunol. 74 (2018) 627-636, https://doi.org/10.1016/j.fsi.2018.01.034.

[26] K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, MEGA6: molecular evolutionary genetics analysis version 6.0, Mol. Biol. Evol. 30 (2013) 2725-2729, https://doi.org/10.1093/molbev/mst197.

[27] M.W. Pfaffl, A new mathematical model for relative quantification in real-time RT-PCR, Nucleic Acids Res. 29 (2001) 45e-45, https://doi.org/10.1093/nar/29.9.e45.

[28] Y. Valero, M. Arizcun, J. Cortés, F. Ramírez-Cepeda, F. Guzmán, L. Mercado, M.Á. Esteban, E. Chaves-Pozo, A. Cuesta, Nk-lysin, Dicentracin and hepcidin antimicrobial peptides in European sea bass. Ontogenetic development and modulation in juveniles by nodavirus, Dev. Comp. Immunol. 103 (2020), 10356, https://doi.org/10.1016/j.dci.2019.103516.

[29] A. Garca-Ayala, E. Chaves-Pozo, Immunocytochemical tools reveal a new research field between the boundaries of immunology and reproductive biology in teleosts, Appl. Immunocytochem. (2012), https://doi.org/10.5772/35231.

[30] M.Á. García-Álvarez, C. González-Fernández, M.Á. Esteban, A. Cuesta, Molecular characterization of the cytotoxic and regulatory T cell coreceptor (CRTAM), and its ligand CADM1, in the European seabass and gilthead seabream, Fish Shellfish Immunol. 134 (2023), https://doi.org/10.1016/j.fsi.2023.108569.

[31] E. Chaves-Pozo, Y. Valero, M.T. Lozano, P. Rodríguez-Cerezo, L. Miao, V. Campo, M.A. Esteban, A. Cuesta, Fish granzyme a shows a greater role than granzyme b in fish innate cell-mediated cytotoxicity, Front. Immunol. 10 (2019) 2579, https://doi.org/10.3389/fimmu.2019.02579.

[32] P. Golstein, G.M. Griffiths, An early history of T cell-mediated cytotoxicity, Nat. Rev. Immunol. 18 (2018) 527-535, https://doi.org/10.1038/s41577-018-0009-3.

[33] X. Fu, Y. Chen, L. Wang, Q. Zhou, M. Li, Y. Song, Y. Li, F. Zhao, S. Chen, Identification and functional analysis of the perforin-1 like gene in disease resistance in half smooth tongue sole (*Cynoglossus semilaevis*), Dev. Comp. Immunol. 122 (2021), 104135, https://doi.org/10.1016/j.dci.2021.104135.

[34] S.M.K. Glasauer, S.C.F. Neuhaus, Whole-genome duplication in teleost fishes and its evolutionary consequences, Mol. Genet. Genom. 289 (2014) 1045-1060, https://doi.org/10.1007/s00438-014-0889-2.

[35] M.E. Dangelo, M.A. Dunstone, J.C. Whisstock, J.A. Trapani, P.I. Bird, Perforin evolved from a gene duplication of MPEG1, followed by a complex pattern of gene gain and loss within *Euteleostomi*, BMC Evol. Biol. 12 (2012), https://doi.org/10.1186/1471-2148-12-59.

[36] H. Yang, Y.M. Lee, J.-H. Lee, J.K. Noh, H.C. Kim, C.-J. Park, J.-W. Park, I.J. Hwang, S.Y. Kim, Expression of perforin gene for early development of nephrons in olive
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[38] C. González-Fernandez, M.A. Esteban, A. Cuesta, Molecular characterization of the T cell costimulatory receptors 1 CD28 and CTLA4 in the European sea bass, Fish Shellfish Immunol. 109 (2021) 106–115.

[39] M.P. Sepulcre, E. Sarropoulou, G. Kotoulas, J. Meseguer, V. Mulero, Vibrio anguillarum evades the immune response of the bony fish sea bass (*Dicentrarchus labrax* L.) through the inhibition of leukocyte respiratory burst and down-regulation of apoptotic caspases, Mol. Immunol. 44 (2007) 3751–3757, https://doi.org/10.1016/j.molimm.2007.03.021.

[40] Y. Valero, E. Chaves-Pozo, A. Cuesta, NK-lysin is highly conserved in European sea bass and gilthead seabream but differentially modulated during the immune response, Fish Shellfish Immunol. 99 (2020) 435–441, https://doi.org/10.1016/j.fsi.2020.02.049.

[41] A. Companjen, B. Heinhuis, K. Aspers, J. Rombout, In Vivo Evoked Specific Cell Mediated Cytotoxicity in Carp (*Cyprinus carpio* L.) Uses Mainly a Perforin/ Granzyme-like Pathway, vol. 20, 2006, pp. 113–117, https://doi.org/10.1016/j.fsi.2005.03.009.

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[37] L. Jaso-Friedmann, J.H. Leary, D.L. Evans, Role of nonspecific cytotoxic cells in the induction of programmed cell death of pathogenic protozoans: participation of the Fas ligand-Fas receptor system, Exp. Parasitol. 96 (2000) 75–88, https://doi.org/10.1006/expr.2000.4561.

[38] C. González-Fernandez, M.A. Esteban, A. Cuesta, Molecular characterization of the T cell costimulatory receptors 1 CD28 and CTLA4 in the European sea bass, Fish Shellfish Immunol. 109 (2021) 106–115.

[39] M.P. Sepulcre, E. Sarropoulou, G. Kotoulas, J. Meseguer, V. Mulero, Vibrio anguillarum evades the immune response of the bony fish sea bass (*Dicentrarchus labrax* L.) through the inhibition of leukocyte respiratory burst and down-regulation of apoptotic caspases, Mol. Immunol. 44 (2007) 3751–3757, https://doi.org/10.1016/j.molimm.2007.03.021.

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