Fish Granzyme A Shows a Greater Role Than Granzyme B in Fish Innate Cell-Mediated Cytotoxicity.

Granzymes (Gzm) are serine proteases, contained into the secretory granules of cytotoxic cells, responsible for the cell-mediated cytotoxicity (CMC) against tumor cells and intracellular pathogens such as virus and bacteria. In fish, they have received little attention to their existence, classification or functional characterization. Therefore, we aimed to identify and evaluate their functional and transcriptomic relevance in the innate CMC activity of two relevant teleost fish species, gilthead seabream and European sea bass. Afterwards, we wanted to focus on their regulation upon nodavirus (NNV) infection, a virus that causes great mortalities to sea bass specimens while seabream is resistant. In this study, we have identified genes encoding GzmA and GzmB in both seabream and sea bass, as well as GzmM in seabream, which showed good phylogenetic relation to their mammalian orthologs. In addition, we found enzymatic activity related to tryptase (GzmA and/or GzmK), aspartase (GzmB), metase (GzmM), or chymase (GzmH) in resting head-kidney leucocytes (HKLs), with the following order of activity: GzmA/K ~ GzmM >> GzmH >>> GzmB. In addition, during innate CMC assays consisting on HKLs exposed to either mock- or NNV-infected target cells, though all the granzyme transcripts were increased only the tryptase activity did. Thus, our data suggest a high functional activity of GzmA/K in the innate CMC and a marginal one for GzmB. Moreover, GzmB activity was detected into target cells during the CMC assays. However, the percentage of target cells with GzmB activity after the CMC assays was about 10-fold lower than the death target cells, demonstrating that GzmB is not the main inductor of cell death. Moreover, in in vivo infection with NNV, gzm transcription is differently regulated depending on the fish species, genes and tissues. However, the immunohistochemistry study revealed an increased number of GzmB stained cells and areas in the brain of seabream after NNV infection, which was mainly associated with the lesions detected. Further studies are needed to ascertain the molecular nature, biological function and implication of fish granzymes in the CMC activity, and in the antiviral defense in particular.

Complete genome sequence and phylogenetic analysis of megalocytivirus RSIV-Ku: A natural recombination infectious spleen and kidney necrosis virus.

Megalocytiviruses are classified into three genotypes, infectious spleen and kidney necrosis virus (ISKNV), red seabream virus (RSIV), and turbo reddish body iridovirus (TRBIV), based on the major capsid protein and ATPase genes. However, only a few complete genome sequences have been obtained. This paper reports the complete genome sequence and phylogenetic analysis of an RSIV-Ku strain megalocytivirus. The genome sequence comprises 111,154 bp, has 132 putative open reading frames, and is homologous mostly to ISKNV, except for the sequence in the region 58981-66830, which is more closely related to that of the RSIV genotype. The results imply that RSIV-Ku is actually a natural recombinant virus.

Application of a new real-time polymerase chain reaction assay for surveillance studies of lymphocystis disease virus in farmed gilthead seabream.

BACKGROUND: Lymphocystis disease (LCD) is the main viral infection reported to affect cultured gilthead seabream (Sparus aurata) in Europe. The existence of subclinical Lymphocystis disease virus (LCDV) infection in this fish species has been recognised by using polymerase chain reaction (PCR)-based methods. Nevertheless, these methods do not provide quantitative results that can be useful in epidemiological and pathological studies. Moreover, carrier fish have been involved in viral transmission, therefore the use of specific and sensitive diagnostic methods to detect LCDV will be relevant for LCD prevention. RESULTS: We have developed a real-time PCR (qPCR) assay to detect and quantify LCDV. The assay was evaluated for viral diagnosis in surveillance studies in gilthead seabream farms, and also to identify viral reservoirs in a hatchery. The prevalence of LCDV infection in the asymptomatic gilthead seabream populations tested varied from 30 to 100 %, including data from one farm without previous records of LCD. Estimated viral load in caudal fin of subclinically infected fish was two to five orders of magnitude lower than in diseased fish. The qPCR assay allowed the detection of carrier fish in broodstock from a farm with a history of clinical LCD in juvenile fish. In addition, the quantitative detection of LCDV was achieved in all samples collected in the hatchery, including fertilized eggs, larvae and fingerlings, and also rotifer cultures and artemia metanauplii and cysts used for larval rearing. CONCLUSIONS: The qPCR assay developed in this study has proved to be a rapid, sensitive, and reliable method for LCDV diagnosis, which could be valuable to identify LCDV reservoirs or to study viral replication in gilthead seabream.

Molecular and functional characterization of the gilthead seabream ß-defensin demonstrate its chemotactic and antimicrobial activity.

Antimicrobial peptides (AMPs) are important mediators of the innate immune response against bacteria and viruses. We have found a ß-defensin (BD) gene searching the expressed sequence tags (ESTs) of the teleost fish gilthead seabream (Sparus aurata). The clone contains an open reading frame of 201 bp mRNA that encodes a putative seabream ß-defensin (saBD) propeptide of 66 amino acids containing the six conserved cysteines as the main signature of this AMP. The phylogenetic tree shows that saBD, and its fish orthologues, are closely related to the human BD-4. Transcripts of the saBD gene were mainly detected by real-time PCR in the skin, peritoneal leucocytes and head-kidney but scarcely expressed in the peripheral blood. Interestingly, head-kidney leucocytes incubation with synthetic unmethylated CpG oligodeoxynucleotides and bacterial DNA up-regulated the saBD gene expression. Recombinant protein (saBD-V5-His) was expressed in the HEK293 cell line and its functional activity determined. First, seabream head-kidney leucocytes showed chemotactic activity towards supernatants containing saBD-V5-His whilst failed to do so to human recombinant BD-1 y BD-4. Moreover, both cell lysates and supernatants containing saBD-V5-His showed strong antimicrobial activity against Vibrio anguillarum (a seabream pathogenic bacterium) and Bacillus subtilis whilst little on other fish pathogens such as Vibrio harvey and Photobacterium damselae. Further studies will elucidate the existence of other BD genes and their implications on the seabream defense against bacteria and virus.

Identification of six novel CC chemokines in gilthead seabream (Sparus aurata) implicated in the antiviral immune response.

Chemokines are key regulators of migration and consequent activation of migrating leukocytes. CC chemokines constitute the largest chemokine group with 24-28 members in mammalian species, and even more in teleost fish, with up to 81 members in zebrafish Danio rerio. Further studies concerning fish chemokine genes will help elucidate the complexity of this chemokine group which has considerably expanded in some teleosts. In the current work, we have identified six novel CC chemokine genes within previously generated gilthead seabream (Sparus aurata) EST databases. The six novel chemokine sequences all show characteristic features of CC chemokines, such as the 4 conserved cysteine residues and a signal peptide. The nomenclature for chemokines in different fish species is not in concordance to mammalian nomenclature as it is difficult to establish true mammalian orthologs, and therefore a different nomenclature has been established for each fish species. In this work, we have named the seabream genes according to the rainbow trout CC chemokine with which they have the highest identity, therefore we have designated the novel seabream CC chemokines as CK1, CK3, CK5, CK7, CK8 and CK10. Expression analysis have also been performed with these new chemokines, as well as with the previously identified seabream chemokine designated as CCL4, which according to our proposed nomenclature should be renamed CK5B. In this sense, we have determined the pattern of constitutive chemokine expression in different seabream tissues. The effect that different immune non-replicative stimuli had in the levels of expression of the chemokines in head kidney leukocytes showed many strong suppressive effects in their transcription levels, and up-regulations mainly in response to mitogens. In vivo, however, when non-replicative virus or heat-killed bacteria were injected, the viral particles up-regulated chemokine expression in the spleen and not in head kidney. Finally, in the context of a real infection such as that of nodavirus in the brain, all the CC chemokines studied were significantly induced. This study constitutes a further step towards the elucidation of an immunological and/or physiological role for fish chemokines.

Nodavirus increases the expression of Mx and inflammatory cytokines in fish brain.

Nodavirus has become a serious pathogen for a wide range of cultured marine fish species. In the present work, the expression of genes related to immune and inflammatory responses of sea bream (Sparus aurata L.), considered as non susceptible species, was studied both in vitro and in vivo. No replication of the virus was observed in head kidney macrophages and blood leukocytes. Moreover, the enhancement of expression of several immune genes (tumor necrosis factor alpha (TNFalpha), interleukin-1-beta (IL-1beta), interferon-induced Mx protein) was not detected in both head kidney macrophages and blood leucocytes in response to an in vitro infection with nodavirus. However, in vivo, nodavirus was detected 1 day post-infection (p.i.) by a reverse transcription-polymerase chain reaction (RT-PCR) in blood, liver, head kidney and brain of experimentally infected sea bream, while its presence clearly decreased in blood after 3 days p.i. Also, a transitory increment of the expression of TNFalpha and IL-1beta was detected in the brain of intramuscular (i.m.) infected sea bream 3 days p.i. In head kidney, the over expression of TNFalpha was only observed 1 day p.i. The expression of Mx, an interferon induced gene, was increased in brain and head kidney of infected sea bream, reaching values of 1300-fold compared to controls in brain three days post-infection. For comparative purposes, we analyzed the expression of the same genes on a susceptible species, such as sea bass (Dicentrarchus labrax) and, although the same pattern of expression was observed both in brain and kidney, the magnitude was different mainly in the case of brain, the key organ of the infection, where higher expression of TNFalpha and lower expression of Mx compared with control was observed.

Protein and glycoprotein content of lymphocystis disease virus (LCDV).

The polypeptide and glycoprotein composition of eight strains of the fish-pathogenic lymphocystis disease virus (LCDV) isolated from gilt-head seabream (Sparus aurata), blackspot seabream (Pagellus bogaraveo), and sole (Solea senegalensis) were determined. The protein electrophoretic patterns of all LCDV isolates were quite similar regardless of the host fish, showing two major proteins (79.9 and 55.6 kDa) and a variable number of minor proteins. Three groups of LCDV isolates were distinguished according to the number and molecular masses of the minor proteins. Eight glycoproteins were detected inside viral particles of LCDV 2, LCDV 3 and LCDV 5 isolates, but only seven glycoproteins were found inside viral particles of LCDV 1, LCDV 4, LCDV 6, LCDV 7, and LCDV 11 isolates and the reference virus ATCC VR 342 by using five lectins. LCDV glycoproteins were mainly composed of mannose and sialic acid. These glycoproteins could be part of an external viral envelope probably derived from the host cell membrane.

Morphological changes contribute to apoptotic cell death and are affected by caspase-3 and caspase-6 inhibitors during red sea bream iridovirus permissive replication.

Red sea bream iridovirus (RSIV) of the Iridoviridae family is a causative agent of lethal infections in many cultured marine fish species in southwestern Japan. RSIV-induced apoptosis was divided as follows: (1). cell shrinkage and rounding at the early apoptotic stage, (2). cell enlargement at the middle apoptotic stage, (3). formation of apoptotic body-like vesicles at the late apoptotic stage and phagocytosis by neighboring cells, and (4). loss of membrane integrity in apoptotic body-like vesicles without phagocytosis by neighboring cells. By affinity labeling, RSIV-induced apoptosis included caspase-dependent apoptosis. RSIV infection caused cell rounding but not cell enlargement or formation of apoptotic body-like vesicles and further restricted part of the structural protein synthesis in the presence of caspase-3 and -6 inhibitors. These findings showed the involvement of caspase-3 and -6 in the morphological changes at the middle and late apoptotic stages and viral protein synthesis in the late stage of RSIV infection.