Risk assessment of wild fish as environmental sources of red sea bream iridovirus (RSIV) outbreaks in aquaculture.

Red sea bream iridovirus (RSIV) causes substantial economic damage to aquaculture. In the present study, RSIV in wild fish near aquaculture installations was surveyed to evaluate the risk of wild fish being an infection source for RSIV outbreaks in cultured fish. In total, 1102 wild fish, consisting of 44 species, were captured from 2 aquaculture areas in western Japan using fishing, gill nets, and fishing baskets between 2019 and 2022. Eleven fish from 7 species were confirmed to harbor the RSIV genome using a probe-based real-time PCR assay. The mean viral load of the RSIV-positive wild fish was 101.1 ± 0.4 copies mg-1 DNA, which was significantly lower than that of seemingly healthy red sea bream Pagrus major in a net pen during an RSIV outbreak (103.3 ± 1.5 copies mg-1 DNA) that occurred in 2021. Sequencing analysis of a partial region of the major capsid protein gene demonstrated that the RSIV genome detected in the wild fish was identical to that of the diseased fish in a fish farm located in the same area in which the wild fish were captured. Based on the diagnostic records of RSIV in the sampled area, the RSIV-infected wild fish appeared during or after the RSIV outbreak in cultured fish, suggesting that RSIV detected in wild fish was derived from the RSIV outbreak in cultured fish. Therefore, wild fish populations near aquaculture installations may not be a significant risk factor for RSIV outbreaks in cultured fish.

Application of Environmental DNA for Monitoring Red Sea Bream Iridovirus at a Fish Farm.

Red sea bream iridoviral disease (RSIVD) causes high economic damage in mariculture in Asian countries. However, there is little information on the source of infection and viral dynamics in fish farms. In the present study, the dynamics of RSIV in a fish farm that mainly reared juveniles and broodstocks of red sea bream (Pagrus major) were monitored over 3 years (2016 to 2018) by targeting environmental DNA (eDNA) of seawater. Our monitoring demonstrated that red sea bream iridovirus (RSIV) was detected from the eDNA at least 5 days before an RSIVD outbreak in the juveniles. The viral loads of eDNA during the outbreak were highly associated with the numbers for daily mortality, and they reached a peak of 106 copies/liter seawater in late July in 2017, when daily mortality exceeded 20,000 fish. In contrast, neither clinical signs nor mortality was observed in the broodstocks during the monitoring periods, whereas the broodstocks were confirmed to be virus carriers by an inspection in October 2017. Interestingly, the viral load of eDNA in the broodstock net pens (105 copies/liter seawater) was higher than that in the juvenile net pens (104 copies/liter seawater) just before the RSIVD outbreak in late June 2017. After elimination of all RSIV-infected surviving juveniles and 90% of broodstocks, few RSIV copies were detected in the eDNA in the fish farm from April 2018 onward (fewer than 102 copies/liter seawater). These results imply that the virus shed from the asymptomatically RSIV-infected broodstock was transmitted horizontally to the juveniles and caused further RSIVD outbreaks in the fish farm. IMPORTANCE Environmental DNA (eDNA) could be applied in monitoring waterborne viruses of aquatic animals. However, there are few data for practical application of eDNA in fish farms for the control of disease outbreaks. The results of our field research over 3 years targeting eDNA in a red sea bream (Pagrus major) fish farm implied that red sea bream iridoviral disease (RSIVD) outbreaks in juveniles originated from virus shedding from asymptomatically virus-infected broodstocks. Our work identifies an infection source of RSIVD in a fish farm via eDNA monitoring, and it could be applied as a tool for application in aquaculture to control fish diseases.

Scale Drop Disease Virus Associated Yellowfin Seabream (Acanthopagrus latus) Ascites Diseases, Zhuhai, Guangdong, Southern China: The First Description.

Scale drop disease virus (SDDV), an emerging piscine iridovirus prevalent in farmed Asian seabass Lates calcarifer in Southeast Asia, was firstly scientifically descripted in Singapore in 2015. Here, an SDDV isolate ZH-06/20 was isolated by inoculating filtered ascites from diseased juvenile yellowfin seabream into MFF-1 cell. Advanced cytopathic effects were observed 6 days post-inoculation. A transmission electron microscopy examination confirmed that numerous virion particles, about 140 nm in diameter, were observed in infected MFF-1 cell. ZH-06/20 was further purified and both whole genome and virion proteome were determined. The results showed that ZH-06/20 was composed of 131,122 bp with 135 putative viral proteins and 113 of them were further detected by virion proteome. Western blot analysis showed that no (or weak) cross-reaction was observed among several major viral proteins between ZH-06/20 and ISKNV-like megalocytivirus. An artificial challenge showed that ZH-06/20 could cause 100% death to juvenile yellowfin seabream. A typical sign was characterized by severe ascites, but not scale drop, which was considerably different from SDD syndrome in Asian seabass. Collectively, SDDV was confirmed, for the first time, as the causative agent of ascites diseases in farmed yellowfin seabream. Our study offers useful information to better understanding SDDV-associated diseases in farmed fish.

Transcriptome analysis reveals a complex response to the RGNNV/SJNNV reassortant Nervous Necrosis Virus strain in sea bream larvae.

The gilthead sea bream (Sparus aurata) is a marine fish of great importance for Mediterranean aquaculture. This species has long been considered resistant to Nervous Necrosis Virus (NNV), an RNA virus that causes massive mortalities in several farmed fish animals. However, the recent appearance of RGNNV/SJNNV reassortant strains started to pose a serious threat to sea bream hatcheries, as it is able to infect larvae and juveniles of this species. While host response to NNV has been extensively studied in adult fish, little attention has been devoted to early life history stages, which are generally the most sensitive ones. Here we report for the first time a time-course RNA-seq analysis on 21-day old fish gilthead sea bream larvae experimentally infected with a RGNNV/SJNNV strain. NNV-infected and mock-infected samples were collected at four time points (6 h, 12 h, 24 h, and 48 h post infection). Four biological replicates, each consisting of five pooled larvae, were analysed for each time point and group. A large set of genes were found to be significantly regulated, especially at early time points (6 h and 12 h), with several heat shock protein encoding transcripts being up-regulated (e.g. hspa5, dnaj4, hspa9, hsc70), while many immune genes were down-regulated (e.g. myd88 and irf5 at T06, pik3r1, stat3, jak1, il12b and il6st at T12). A gene set enrichment analysis (GSEA) identified several altered pathways/processes. For instance, the formation of peroxisomes, which are important anti-viral components as well as essential for nervous system homeostasis, and the autophagy pathway were down-regulated at 6 h and 24 h post infection (hpi). Finally, two custom "reactomes" (i.e. significant gene sets observed in other studies) were defined and used. The first reactome integrated the transcriptomic response to NNV in different fish species, while the second one included all genes found to be stimulated either by interferon (IFN) or by IFN and Chikungunya virus in zebrafish. Genes in both reactomes showed predominant up-regulation at 6hpi and 12hpi and a general down-regulation at 24hpi. Such evidence suggest a certain degree of similarity between the response of sea bream and that of other fish species to NNV, while the observed down-regulation of IFN- and viral-stimulated pathways argues for a possible interference of NNV against the host response.

Immune gene expression in gilthead seabream (Sparus aurata) after Lymphocystis disease virus (LCDV-Sa) challenge resulting in asymptomatic infection.

AIMS: To determine the immune gene expression response of gilthead seabream (Sparus aurata) that is experimentally infected with the lymphocystivirus LCDV-Sa. METHODS AND RESULTS: Viral DNA and transcripts were detected by qPCR in all samples from fish injected with LCDV-Sa, demonstrating that the virus establish a systemic and asymptomatic infection. The expression of 23 immune-related genes was also analysed by RT-qPCR in the head kidney (HK) and intestine at several times post-infection (dpi). In HK, the expression of five type I interferon (IFN)-related genes (ifn, irf3, mx2, mx3 and isg15), il10 and ck10 was upregulated at 1-3 dpi, while genes related to the inflammation process (tnfα, il1ß, il6, casp1) were not differentially expressed or even downregulated. The expression profile in the intestine was different regarding type I INF-related genes. An upregulated c3 and ighm expression was observed in both HK and intestine at 3-8 dpi. Finally, the transcription of nccrp1 and mhcIIα was induced in HK, whereas tcrß expression was downregulated in both organs. CONCLUSIONS: LCDV-Sa seems to trigger an immune response in gilthead seabream characterized by a partial activation of type I IFN system and a lack of systemic inflammatory response which may be related to viral persistence. SIGNIFICANCE AND IMPACT OF THE STUDY: The immune response observed in gilthead seabream infected by LCDV-Sa could be implicated in the establishment of an asymptomatic persistent infection.

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.

Artemia spp., a Susceptible Host and Vector for Lymphocystis Disease Virus.

Different developmental stages of Artemia spp. (metanauplii, juveniles and adults) were bath-challenged with two isolates of the Lymphocystis disease virus (LCDV), namely, LCDV SA25 (belonging to the species Lymphocystis disease virus 3) and ATCC VR-342 (an unclassified member of the genus Lymphocystivirus). Viral quantification and gene expression were analyzed by qPCR at different times post-inoculation (pi). In addition, infectious titres were determined at 8 dpi by integrated cell culture (ICC)-RT-PCR, an assay that detects viral mRNA in inoculated cell cultures. In LCDV-challenged Artemia, the viral load increased by 2-3 orders of magnitude (depending on developmental stage and viral isolate) during the first 8-12 dpi, with viral titres up to 2.3 × 102 Most Probable Number of Infectious Units (MPNIU)/mg. Viral transcripts were detected in the infected Artemia, relative expression values showed a similar temporal evolution in the different experimental groups. Moreover, gilthead seabream (Sparus aurata) fingerlings were challenged by feeding on LCDV-infected metanauplii. Although no Lymphocystis symptoms were observed in the fish, the number of viral DNA copies was significantly higher at the end of the experimental trial and major capsid protein (mcp) gene expression was consistently detected. The results obtained support that LCDV infects Artemia spp., establishing an asymptomatic productive infection at least under the experimental conditions tested, and that the infected metanauplii are a vector for LCDV transmission to gilthead seabream.

Phylogenomic characterization of red seabream iridovirus from Florida pompano Trachinotus carolinus maricultured in the Caribbean Sea.

Between 2010 and 2016, six mortality events were observed in Florida pompano (Trachinotus carolinus) maricultured in the Dominican Republic. Histopathological examination and conventional PCR confirmed a megalocytivirus (MCV) infection in each case. Subsequently, next-generation sequencing and phylogenomic analyses confirmed that MCV DNA was present in the infected pompano tissue samples from 2010, 2014, and 2016, and each was determined to be red seabream iridovirus (RSIV). Annotation of the RSIV genome sequences identified 121 open reading frames, and BLASTN analysis revealed the highest nucleotide sequence identity (> 99%) to a RSIV clade 1 MCV isolated from a moribund red seabream (Pagrus major) maricultured in Japan. These cases represent the first fully sequenced RSIV genomes detected outside of Asia and are the earliest reports of MCV infections in Florida pompano. This recent geographical expansion of RSIV warrants further attention to determine its potential economic and ecological impact.

Red seabream iridovirus associated with cultured Florida pompano Trachinotus carolinus mortality in Central America.

Mariculture of Florida pompano Trachinotus carolinus in Central America has increased over the last few decades and it is now a highly valued food fish. High feed costs and infectious diseases are significant impediments to the expansion of mariculture. Members of the genus Megalocytivirus (MCV), subfamily Alphairidovirinae, within the family Iridoviridae, are emerging pathogens that negatively impact Asian mariculture. A significant mortality event in Florida pompano fingerlings cultured in Central America occurred in October 2014. Affected fish presented with abdominal distension, darkening of the skin, and periocular hemorrhages. Microscopic lesions included cytomegalic 'inclusion body-bearing cells' characterized by basophilic granular cytoplasmic inclusions in multiple organs. Transmission electron microscopy revealed arrays of hexagonal virions (155-180 nm in diameter) with electron-dense cores within the cytoplasm of cytomegalic cells. Pathological findings were suggestive of an MCV infection, and the diagnosis was later confirmed by partial PCR amplification and sequencing of the viral gene encoding the myristylated membrane protein. The viral sequence revealed that the fingerlings were infected with an MCV genotype, red seabream iridovirus (RSIV), previously reported only from epizootics in Asian mariculture. This case underscores the threat RSIV poses to global mariculture, including the production of Florida pompano in Central America.

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.

Innate Cell-Mediated Cytotoxic Activity of European Sea Bass Leucocytes Against Nodavirus-Infected Cells: A Functional and RNA-seq Study.

Nervous necrosis virus (NNV) causes high mortalities in several marine species. We aimed to evaluate the innate cell-mediated cytotoxic (CMC) activity of head-kidney leucocytes (HKLs) isolated from naïve European sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata), a very susceptible and resistant fish species to NNV, respectively, against fish cell lines infected with NNV. Seabream HKLs showed significantly increased innate CMC activity against NNV-infected cells, compared to those uninfected, while sea bass HKLs failed to do so. Thus, we performed a RNA-seq study to identify genes related to the CMC activity of sea bass leucocytes. Thus, we found that sea bass HKLs incubated with DLB-1 cells alone (CMC_DLB1) or with NNV-infected DLB-1 cells (CMC_DLB1-NNV) showed very similar transcriptomic profiles and the GO analysis revealed that most of the up-regulated genes were related to immunity. Strikingly, when the CMC samples with and without NNV were compared, GO analysis revealed that most of the up-regulated genes in CMC_DLB1-NNV samples were related to metabolism and very few to immunity. This is also in agreement with the functional data. These data point to the escape of CMC activity by NNV infection as an important factor involved in the high susceptibility to nodavirus infections of European sea bass.

Identification of Quantitative Trait Loci for Resistance to RSIVD in Red Sea Bream (Pagrus major).

Red sea bream iridoviral disease (RSIVD) is a major viral disease in red sea bream farming in Japan. Previously, we identified one candidate male individual of red sea bream that was significantly associated with convalescent individuals after RSIVD. The purpose of this study is to identify the quantitative trait loci (QTL) linked to the RSIVD-resistant trait for future marker-assisted selection (MAS). Two test families were developed using the candidate male in 2014 (Fam-2014) and 2015 (Fam-2015). These test families were challenged with RSIV, and phenotypes were evaluated. Then, de novo genome sequences of red sea bream were obtained through next-generation sequencing, and microsatellite markers were searched and selected for linkage map construction. One immune-related gene, MHC class IIß, was also used for linkage map construction. Of the microsatellite markers searched, 148 and 197 were mapped on 23 and 27 linkage groups in the female and male linkage maps, respectively, covering approximately 65% of genomes in both sexes. One QTL linked to an RSIVD-resistant trait was found in linkage group 2 of the candidate male in Fam-2014, and the phenotypic variance of the QTL was 31.1%. The QTL was closely linked to MHC class IIß. Moreover, the QTL observed in Fam-2014 was also significantly linked to an RSIVD-resistant trait in the candidate male of Fam-2015. Our results suggest that the RSIVD-resistant trait in the candidate male was controlled by one major QTL closely linked to the MHC class IIß gene and could be useful for MAS of red sea bream.

Viral nervous necrosis in gilthead sea bream (Sparus aurata) caused by reassortant betanodavirus RGNNV/SJNNV: an emerging threat for Mediterranean aquaculture.

Viral nervous necrosis (VNN) certainly represents the biggest challenge for the sustainability and the development of aquaculture. A large number of economically relevant fish species have proven to be susceptible to the disease. Conversely, gilthead sea bream has generally been considered resistant to VNN, although it has been possible to isolate the virus from apparently healthy sea bream and sporadically from affected larvae and postlarvae. Unexpectedly, in 2014-2016 an increasing number of hatcheries in Europe have experienced mass mortalities in sea bream larvae. Two clinical outbreaks were monitored over this time span and findings are reported in this paper. Despite showing no specific clinical signs, the affected fish displayed high mortality and histological lesions typical of VNN. Fish tested positive for betanodavirus by different laboratory techniques. The isolates were all genetically characterized as being reassortant strains RGNNV/SJNNV. A genetic characterization of all sea bream betanodaviruses which had been isolated in the past had revealed that the majority of the strains infecting sea bream are actually RGNNV/SJNNV. Taken together, this information strongly suggests that RGNNV/SJNNV betanodavirus possesses a particular tropism to sea bream, which can pose a new and unexpected threat to the Mediterranean aquaculture.

Target organs for lymphocystis disease virus replication in gilthead seabream (Sparus aurata).

The lymphocystis disease (LCD), the main viral pathology described in cultured gilthead seabream (Sparus aurata), is a self-limiting condition characterized by the appearance of hypertrophied fibroblasts (named lymphocysts) in the connective tissue of fish, primarily in the skin and fins. The causative agent of the disease is the Lymphocystis disease virus (LCDV), a member of the Iridoviridae family. In the present study, LCDV genome and transcripts were detected by real-time PCR in caudal fin, as well as in several internal organs, such as intestine, liver, spleen, kidney and brain, from asymptomatic, diseased and recovered gilthead seabream juveniles. These results indicate that the LCDV has a broad range tissue tropism, and can establish a systemic infection, even in subclinically infected fish. As showed by in situ hybridization, the permissive cells for LCDV infection seem to be fibroblasts, hepatocytes and cells of the mononuclear phagocyte system. Histopathological alterations associated with LCD were observed in all the organs analysed, including necrotic changes in liver and kidney, inflammatory response in the intestine submucosa or brain haemorrhage, although lymphocysts were only detected in the dermis of the caudal fin. Nevertheless, these histological changes were reverted in recovered animals.

Rapid and Sensitive Detection of Lymphocystis Disease Virus Genotype VII by Loop-Mediated Isothermal Amplification.

Lymphocystis disease virus (LCDV) infections have been described in gilthead seabream (Sparus aurata L.) and Senegalese sole (Solea senegalensis, Kaup), two of the most important marine fish species in the Mediterranean aquaculture. In this study, a rapid, specific, and sensitive detection method for LCDV genotype VII based on loop-mediated isothermal amplification (LAMP) was developed. The LAMP assay, performed using an apparatus with real-time amplification monitoring, was able to specifically detect LCDV genotype VII from clinically positive samples in less than 12 min. In addition, the assay allowed the detection of LCDV in all asymptomatic carrier fish analysed, identified by qPCR, showing an analytical sensitivity of ten copies of viral DNA per reaction. The LCDV LAMP assay has proven to be a promising diagnostic method that can be used easily in fish farms to detect the presence and spread of this iridovirus.

Evaluation of an integrated cell culture RT-PCR assay to detect and quantify infectious lymphocystis disease virus.

The lymphocystis disease virus (LCDV), a member of the Iridoviridae family, infects a wide range of fish species including gilthead seabream (Sparus aurata L.), the most important species cultured in the Mediterranean. LCDV is difficult to propagate in cell culture and does not produce clear and consistent cytopathic effects (CPE), especially in samples collected from subclinically infected fish. An integrated cell culture reverse transcription-polymerase chain reaction (ICC-RT-PCR) assay, followed by dot-blot hybridization of the RT-PCR products, was developed to improve the detection of infectious LCDV. The sensitivity of the ICC-RT-PCR assay, which can be performed in 7 d, was at least 100-fold higher than viral diagnosis obtained by CPE development. The developed assay thus allows the determination of infectious titres in samples with low viral loads, including those from asymptomatic carrier fish, in which no CPE was recorded after a 14-d incubation period. The ICC-RT-PCR assay enables rapid, specific and sensitive detection and quantification of infectious LCDV, and may be a valuable tool in the study of aspects of LCDV infection including transmission or epizootiology.

Establishment of rock bream Oplegnathus fasciatus embryo (RoBE-4) cells with cytolytic infection of red seabream iridovirus (RSIV).

Red seabream iridovirus (RSIV) is a member of genus Megalocytivirus in the family Iridoviridae. RSIV infection causes significant economic losses of marine-fishes in East Asian countries. Grunt fin (GF) cell line has been commonly used for culturing RSIV. However, it is not suitable for definite evaluation of infectivity titer of RSIV because cells infected with RSIV are not completely cytolysed. Thus, we established a new cell line, RoBE-4, from rock bream (Oplegnathus fasciatus) eyed-egg embryos in this study. Morphologically, RoBE-4 cells were fibroblastic-like. They have been stably grown over two-years with 60 passages using Leibovitz's L-15 medium containing 10% (v/v) fetal bovine serum. RoBE-4 cells infected with RSIV exhibited cytopathic effects (CPE) with cell rounding. They were cytolysed completely after ≥2 weeks of culture. Numerous RSIV particles with icosahedral morphology of approximately 122nm in diameter were observed in cytoplasmic area of infected RoBE-4 cells. The RSIV-suceptibility and amount of extracellular RSIV released by RoBE-4 cells were 100-fold higher than those by GF cells. RSIV cultured with RoBE-4 cells was highly virulent to rock bream in infection experiments. Therefore, using RoBE-4 cells instead of GF cells will enable accurate and sensitive measurement of RSIV infectivity. In addition, RoBE-4 cells might be used to produce RSIV vaccine in the future with significant reduction in cost.

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.

Mapping QTL for Resistance Against Viral Nervous Necrosis Disease in Asian Seabass.

Viral nervous necrosis disease (VNN), caused by nervous necrosis virus (NNV), leads to mass mortality in mariculture. However, phenotypic selection for resistance against VNN is very difficult. To facilitate marker-assisted selection (MAS) for resistance against VNN and understanding of the genetic architecture underlying the resistance against this disease, we mapped quantitative trait loci (QTL) for resistance against VNN in Asian seabass. We challenged fingerlings at 37 days post-hatching (dph), from a single back-cross family, with NNV at a concentration of 9 × 10(6) TCID50/ml for 2 h. Daily mortalities were recorded and collected. A panel of 330 mortalities and 190 surviving fingerlings was genotyped using 149 microsatellites with 145 successfully mapped markers covering 24 linkage groups (LGs). Analysis of QTL for both resistance against VNN and survival time was conducted using interval mapping. Five significant QTL located in four LGs and eight suggestive QTL in seven LGs were identified for resistance. Another five significant QTL in three LGs and five suggestive QTL in three LGs were detected for survival time. One significant QTL, spanning 3 cM in LG20, was identified for both resistance and survival time. These QTL explained 2.2-4.1% of the phenotypic variance for resistance and 2.2-3.3% of the phenotypic variance for survival time, respectively. Our results suggest that VNN resistance in Asian seabass is controlled by many loci with small effects. Our data provide information for fine mapping of QTL and identification of candidate genes for a better understanding of the mechanism of disease resistance.

Nodavirus Colonizes and Replicates in the Testis of Gilthead Seabream and European Sea Bass Modulating Its Immune and Reproductive Functions.

Viruses are threatening pathogens for fish aquaculture. Some of them are transmitted through gonad fluids or gametes as occurs with nervous necrosis virus (NNV). In order to be transmitted through the gonad, the virus should colonize and replicate inside some cell types of this tissue and avoid the subsequent immune response locally. However, whether NNV colonizes the gonad, the cell types that are infected, and how the immune response in the gonad is regulated has never been studied. We have demonstrated for the first time the presence and localization of NNV into the testis after an experimental infection in the European sea bass (Dicentrarchus labrax), and in the gilthead seabream (Sparus aurata), a very susceptible and an asymptomatic host fish species, respectively. Thus, we localized in the testis viral RNA in both species using in situ PCR and viral proteins in gilthead seabream by immunohistochemistry, suggesting that males might also transmit the virus. In addition, we were able to isolate infective particles from the testis of both species demonstrating that NNV colonizes and replicates into the testis of both species. Blood contamination of the tissues sampled was discarded by completely fish bleeding, furthermore the in situ PCR and immunocytochemistry techniques never showed staining in blood vessels or cells. Moreover, we also determined how the immune and reproductive functions are affected comparing the effects in the testis with those found in the brain, the main target tissue of the virus. Interestingly, NNV triggered the immune response in the European sea bass but not in the gilthead seabream testis. Regarding reproductive functions, NNV infection alters 17ß-estradiol and 11-ketotestosterone production and the potential sensitivity of brain and testis to these hormones, whereas there is no disruption of testicular functions according to several reproductive parameters. Moreover, we have also studied the NNV infection of the testis in vitro to assess local responses. Our in vitro results show that the changes observed on the expression of immune and reproductive genes in the testis of both species are different to those observed upon in vivo infections in most of the cases.