Immune response of DNA vaccinated-gilthead seabream (Sparus aurata) against LCDV-Sa infection: relevance of the inflammatory process.

Lymphocystis disease is one of the main viral pathologies affecting cultured gilthead seabream (Sparus aurata) in the Mediterranean region. Recently, we have developed a DNA vaccine based on the major capsid protein (MCP) of the Lymphocystis disease virus 3 (LCDV-Sa). The immune response triggered by either LCDV-Sa infection or vaccination have been previously studied and seem to be highly related to the modulation of the inflammatory and the IFN response. However, a comprehensive evaluation of immune-related gene expression in vaccinated fish after viral infection to identify immunogenes involved in vaccine-induced protection have not been carried out to date. The present study aimed to fulfill this objective by analyzing samples of head-kidney, spleen, intestine, and caudal fin from fish using an OpenArray® platform containing targets related to the immune response of gilthead seabream. The results obtained showed an increase of deregulated genes in the hematopoietic organs between vaccinated and non-vaccinated fish. However, in the intestine and fin, the results showed the opposite trend. The global effect of fish vaccination was a significant decrease (p<0.05) of viral replication in groups of fish previously vaccinated, and the expression of the following immune genes related to viral recognition (tlr9), humoral and cellular response (rag1 and cd48), inflammation (csf1r, elam, il1ß, and il6), antiviral response (isg15, mx1, mx2, mx3), cell-mediated cytotoxicity (nccrp1), and apoptosis (prf1). The exclusive modulation of the immune response provoked by the vaccination seems to control the progression of the infection in the experimentally challenged gilthead seabream.

Nervous Necrosis Virus (NNV) Booster Vaccination Increases Senegalese Sole Survival and Enhances Immunoprotection.

A re-immunization programme has been tested to improve the protective response elicited in sole by a previously developed BEI-inactivated betanodavirus vaccine. The vaccine was prepared using a reassortant RGNNV/SJNNV strain which is highly pathogenic for sole, and vaccination assays were performed by intraperitoneal injection. Experimental design included a prime- and a booster-vaccination group, which consisted of individuals that received a second vaccine injection at 30 days post vaccination), and their respective controls. A month after prime/booster vaccination, fish were challenged by intramuscular injection with the homologous NNV strain. Samples were collected at different times post vaccination and post challenge to assess the immune response and viral replication. Booster dose enhanced the protection against NNV infection because a significant increase in survival was recorded when compared with prime-vaccinated individuals (relative percent survival 77 vs. 55). In addition, a clear decrease in viral replication in the brain of challenged sole was observed. During the immune induction period, no differences in IgM production were observed between prime- and booster-vaccinated fish, and the expression of the antigen presenting cells (APC)-related molecule MHC class II antigen was the only differential stimulation recorded in the re-immunized individuals. However, a significant upregulation of mhcII and the lymphocytes T helper (Th) marker cd4 was observed after the challenge in the booster-vaccinated group, suggesting these cells play a role in the protection conferred by the booster injection. In addition, after viral infection, re-immunized fish showed specific and neutralizing antibody production and overexpression of other immune-related genes putatively involved in the control of NNV replication.

Evaluation of Gilthead Seabream (Sparus aurata) Immune Response after LCDV-Sa DNA Vaccination.

Lymphocystis disease is the main viral pathology reported in gilthead seabream. Its etiological agent is Lymphocystis disease virus 3 (LCDV-Sa), genus Lymphocystivirus, family Iridoviridae. There are no effective treatments or vaccines for LCDV control, thus the main aim of this study was to develop a DNA vaccine, and to evaluate both the protection conferred against LCDV-Sa infection and the immune response in vaccinated fish. The vaccine was constructed by cloning the mcp gene (ORF LCDVSa062R) into pcDNA3.1/NT-GFP-TOPO. Two independent vaccination trials were conducted. In the first one, 5-7 g fish were intramuscularly injected with the vaccine (pcDNA-MCP) or the empty-plasmid, and the distribution and expression of the vaccine was investigated. Furthermore, vaccinated fish were challenged with LCDV-Sa in order to access the protective capacity of the vaccine. In the second trial, 70-100 g fish were vaccinated as specified, and the immune response was evaluated analyzing the expression of 23 immune-related genes and the production of specific antibodies. The results showed that the vaccine triggers an immune response characterized by the overexpression of genes relating to the inflammatory process, but not the innate antiviral immunity relating to type I IFN (interferon), and also induces the production of specific neutralizing antibodies, which could explain the protection against LCDV-Sa in vaccinated fish.

Immunogene expression analysis in betanodavirus infected-Senegalese sole using an OpenArray® platform.

The transcriptomic response of Senegalese sole (Solea senegalensis) triggered by two betanodaviruses with different virulence to that fish species has been assessed using an OpenArray® platform based on TaqMan™ quantitative PCR. The transcription of 112 genes per sample has been evaluated at two sampling times in two organs (head kidney and eye/brain-pooled samples). Those genes were involved in several roles or pathways, such as viral recognition, regulation of type I (IFN-1)-dependent immune responses, JAK-STAT cascade, interferon stimulated genes, protein ubiquitination, virus responsive genes, complement system, inflammatory response, other immune system effectors, regulation of T-cell proliferation, and proteolysis and apoptosis. The highly virulent isolate, wSs160.3, a wild type reassortant containing a RGNNV-type RNA1 and a SJNNV-type RNA2 segments, induced the expression of a higher number of genes in both tested organs than the moderately virulent strain, a recombinant harbouring mutations in the protruding domain of the capsid protein. The number of differentially expressed genes was higher 2 days after the infection with the wild type isolate than at 3 days post-inoculation. The wild type isolate also elicited an exacerbated interferon 1 response, which, instead of protecting sole against the infection, increases the disease severity by the induction of apoptosis and inflammation-derived immunopathology, although inflammation seems to be modulated by the complement system. Furthermore, results derived from this study suggest a potential important role for some genes with high expression after infection with the highly virulent virus, such as rtp3, sacs and isg15. On the other hand, the infection with the mutant does not induce immune response, probably due to an altered recognition by the host, which is supported by a different viral recognition pathway, involving myd88 and tbkbp1.

Differential immunogene expression profile of European sea bass (Dicentrarchus labrax, L.) in response to highly and low virulent NNV.

European sea bass is highly susceptible to the nervous necrosis virus, RGNNV genotype, whereas natural outbreaks caused by the SJNNV genotype have not been recorded. The onset and severity of an infectious disease depend on pathogen virulence factors and the host immune response. The importance of RGNNV capsid protein amino acids 247 and 270 as virulence factors has been previously demonstrated in European sea bass; however, sea bass immune response against nodaviruses with different levels of virulence has been poorly characterized. Knowing the differences between the immune response against both kinds of isolates may be key to get more insight into the host mechanisms responsible for NNV virulence. For this reason, this study analyses the transcription of immunogenes differentially expressed in European sea bass inoculated with nodaviruses with different virulence: a RGNNV virus obtained by reverse genetics (rDl956), highly virulent to sea bass, and a mutated virus (Mut247+270Dl956, RGNNV virus displaying SJNNV-type amino acids at positions 247 and 270 of the capsid protein), presenting lower virulence. This study has been performed in brain and head kidney, and the main differences between the immunogene responses triggered by both viruses have been observed in brain. The immunogene response in this organ is stronger after inoculation with the most virulent virus, and the main differences involved genes related with IFN I system, inflammatory response, cell-mediated response, and apoptosis. The lower virulence of Mut247+270Dl956 to European sea bass can be associated with a delayed IFN I response, as well as an early and transitory inflammation and cell-mediated responses, suggesting that those can be pivotal elements in controlling the viral infection, and therefore, their functional activity could be analysed in future studies. In addition, this study supports the role of capsid amino acids at positions 247 and 270 as important determinants of RGNNV virulence to European sea bass.

Persistence of Lymphocystis Disease Virus (LCDV) in Seawater.

Lymphocystis disease virus (LCDV), the causative agent of lymphocystis disease (LCD), is a waterborne pathogen that uses the external surfaces, including the gills, as portals to gain access to fish host. However, there are no data on LCDV persistence in the aquatic environment. In this study, the persistence of LCDV in natural (raw), treated (autoclaved and filtered) and synthetic seawater held at 22 and 18 °C has been evaluated. The estimated T99 values for LCDV in seawater ranged from 2.7 to 242 days depending on seawater type and temperature, with the highest value recorded at 22 °C in autoclaved seawater. Microbiota and temperature seem to be the main factors affecting the persistence of LCDV in seawater. The results indicated that LCDV is more stable in treated seawater than most of the fish pathogenic viruses studied so far, supporting the relevance of this medium for the prevalence of LCD in fish farms.

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.

Lymphocystis disease virus (LCDV-Sa), polyomavirus 1 (SaPyV1) and papillomavirus 1 (SaPV1) in samples of Mediterranean gilthead seabream.

Lymphocystis disease, caused by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of tumour-like lesions on the skin of affected animals associated with several environmental factors and/or with stress due to the intensive culture conditions of fish farms. In a previous study, the genomes of a new LCDV species, LCDV-Sa, were detected, together with 2 previously unknown viruses, Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1). Gilthead seabream from 17 fish farms in Spain, Italy and Turkey were sampled between 2009 and 2015 to investigate the role of the newly described SaPV1 and SaPyV1 viruses in lymphocystis disease development. Our results show that in diseased fish, either or both of the new viruses are almost invariably detected together with LCDV (98%). In asymptomatic fish, these viruses were detected in a much lower percentage (28%) and mostly in concurrence with LCDV (24%). These data confirm the suspected association among the 3 different viruses during lymphocystis disease development in gilthead seabream and warrant future studies to establish their respective contributions.

Transcriptomic Profiles of Senegalese Sole Infected With Nervous Necrosis Virus Reassortants Presenting Different Degree of Virulence.

Betanodaviruses [nervous necrosis virus (NNV)] are the causative agent of the viral encephalopathy and retinopathy, a disease that affects cultured Senegalese sole (Solea senegalensis). NNV reassortants, combining genomic segments from redspotted grouper nervous necrosis virus (RGNNV) and striped jack nervous necrosis virus (SJNNV) genotypes, have been previously isolated from several fish species. The wild-type reassortant wSs160.03, isolated from Senegalese sole, has been proven to be more virulent to sole than the parental genotypes (RGNNV and SJNNV), causing 100% mortality. Mutations at amino acids 247 (serine to alanine) and 270 (serine to asparagine) in the wSs160.03 capsid protein have allowed us to obtain a mutant reassortant (rSs160.03247+270), which provokes a 40% mortality decrease. In this study, the RNA-Seq technology has been used to comparatively analyze Senegalese sole transcriptomes in two organs (head kidney and eye/brain) after infection with wild-type and mutant strains. A total of 633 genes were differentially expressed (DEGs) in animals infected with the wild-type isolate (with higher virulence), whereas 393 genes were differentially expressed in animals infected with the mutant strain (37.9% decrease in the number of DEGs). To study the biological functions of detected DEGs involved in NNV infection, a gene ontology (GO) enrichment analysis was performed. Different GO profiles were obtained in the following subclasses: (i) biological process; (ii) cellular component; and (iii) molecular function, for each viral strain tested. Immune response and proteolysis have been the predominant biological process after the infection with the wild-type isolate, whereas the infection with the mutant strain induces proteolysis in head kidney and inhibition of vasculogenesis in nervous tissue. Regarding the immune response, genes coding for proteins acting as mediators of type I IFN expression (DHX58, IRF3, IRF7) and IFN-stimulated genes (ISG15, Mx, PKR, Gig1, ISG12, IFI44, IFIT-1, to name a few) were upregulated in animals infected with the wild-type isolate, whereas no-differential expression of these genes was observed in samples inoculated with the mutant strain. The different transcriptomic profiles obtained could help to better understand the NNV pathogenesis in Senegalese sole, setting up the importance as virulence determinants of amino acids at positions 247 and 270 within the RNA2 segment.

Photobacterium malacitanum sp. nov., and Photobacterium andalusiense sp. nov., two new bacteria isolated from diseased farmed fish in Southern Spain.

Three strains, H01100409BT, H01100413B, and H27100402HT, were isolated from several internal organs of diseased redbanded seabream (Pagrus auriga) reared in Andalusia (Southern Spain). All strains were studied by phenotypic, including chemotaxonomy, and genomic characteristics. Phylogenetic analysis based on concatenated sequences of six housekeeping genes (gyrB, ftsZ, topA, mreB, gapA, and 16S rRNA) supported the inclusion of the strains within the clade Phosphoreum of the genus Photobacterium, and two of the strains (H27100402HT and H01100409BT) formed a tight group separated from the closest species P. aquimaris. Genomic analyses, including average nucleotide identity (ANIb and ANIm) and DNA-DNA hybridization (DDH), clearly separated strains H27100402HT and H01100409BT from the other species within the clade Phosphoreum with values below the thresholds for species delineation. The chemotaxonomic features (including FAME analysis and MALDI-TOF-MS) of H27100402HT and H01100409BT strains confirmed their differentiation from the related taxa. The results demonstrated that strain H01100413B was classified as P. aquimaris and the strains H27100402HT and H01100409BT represented a new species each in the genus Photobacterium, for which we propose the names Photobacterium malacitanum sp. nov., type strain H27100402HT (=CECT 9190T=LMG 29992T), and Photobacterium andalusiense sp. nov., type strain H01100409BT (=CECT 9192T=LMG 29994T).

Description of New and Amended Clades of the Genus Photobacterium.

Phylogenetic relationships between species in the genus Photobacterium have been poorly studied despite pathogenic and ecological relevance of some of its members. This is the first phylogenetic study that includes new species of Photobacterium (validated or not) that have not been included in any of the previously described clades, using 16S rRNA sequences and multilocus sequence analysis (MLSA) in concatenated sequences of gyrB, gapA, topA, ftsZ and mreB housekeeping genes. Sequence analysis has been implemented using Maximum-parsimony (MP), Neighbour-joining (NJ) and Maximum likelihood (ML) treeing methods and the predicted evolutionary relationship between the Photobacterium clades was established on the basis of bootstrap values of >75% for 16S rRNA sequences and MLSA. We have grouped 22 species of the genus Photobacterium into the following 5 clades: Phosphoreum (comprises P. aquimaris, "P. carnosum," P. iliopiscarium, P. kishitanii, P. phosphoreum, "P. piscicola" and "P. toruni"); clade Profundum (composed of P. aestuarii, P. alginatilyticum, P. frigidiphilum, P. indicum, P. jeanii, P. lipolyticum, "P. marinum," and P. profundum); clade Damselae (two subspecies of P. damselae, damselae and piscicida); and two new clades: clade Ganghwense (includes P. aphoticum, P. aquae, P. galatheae, P. ganghwense, P. halotolerans, P. panuliri and P. proteolyticum); and clade Leiognathi (composed by P. angustum, P. leiognathi subsp. leiognathi and "P. leiognathi subsp. mandapamensis"). Two additional clades, Rosenbergii and Swingsii, were formed using a phylogenetic method based on 16S rRNA gene, although they are not confirmed by any MLSA methods. Only P. aplysiae could not be included in none of the established clade, constituting an orphan clade.

Carbon Quantum Dot Surface-Chemistry-Dependent Ag Release Governs the High Antibacterial Activity of Ag-Metal-Organic Framework Composites.

Nanocomposites and hybrid materials of Ag-1,3,5-benzenetricarboxylic acid metal-organic frameworks (MOFs) with S- and N-carbon quantum dots (CQDs) were synthesized and evaluated for their antibacterial activity against representative Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacterial strains using the qualitative disk-diffusion approach and the quantitative minimum inhibitory concentration test. The composites and hybrids were found to be nontoxic to living cells. The composite formation fostered a synergistic effect that enhanced their antibacterial activity compared with those of their pristine components. Charge transfer from AgMOF to CQDs facilitated the electrostatic interactions of the composites and hybrids with the bacterial cell membranes. Enhanced bactericidal activity was linked to morphological features (a nanorod-like morphology) and specific surface chemistry. The latter affected the release of silver. Silver on the surface of the MOFs rather than silver in the bulk was found to be important. The destruction of the MOF component in the extracellular environment led to the release of silver ions, which have a high affinity to S compounds of the cell physiology. The formation of metallic silver (Ag°) and silver sulfides (Ag2S) was suggested as essential for the ability of the composites and hybrids to inhibit bacterial growth. To the best of our knowledge, this is the first study that introduces the bactericidal effect of AgMOF-CQDs composites and hybrids.

Photobacterium toruni sp. nov., a bacterium isolated from diseased farmed fish.

Three bacterial strains were isolated from liver and spleen of diseased farmed redbanded seabream (Pagrus auriga) in south-west Spain. Their partial 16S rRNA gene sequences clustered within those of the genus Photobacterium, showing high similarity (98.6-99.3 %) to the type strains of Photobacterium iliopiscarium, P. piscicola, P. kishitanii, P. aquimaris and P. phosphoreum. Multilocus sequence analysis using six housekeeping genes (gapA, topA, mreB, ftsZ, gyrB and 16S rRNA) confirmed the new strains as forming an independent branch with a bootstrap value of 100, likely to represent a novel species. To confirm this, we used whole genome sequencing and genomic analysis (ANIb, ANIm and in silico DNA-DNA hybridization) obtaining values well below the thresholds for species delineation. In addition, a phenotypic characterization was performed to support the description and differentiation of the novel strains from related taxa. Cells were Gram-stain-negative, motile bacilli, chemo-organotrophic and facultatively anaerobic. They fermented glucose, as well as galactose and d-mannose, without production of gas. Oxidase and catalase were positive. The predominant cellular fatty acids were C16 : 1ω7c/C16 : 1ω6c and C16  :  0. The predominant respiratory quinone (Q-8) and major polar lipids (phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol) were inferred from annotated genes in the genome of strain H01100410BT, which had a G+C content of 38.6 mol%. The results obtained demonstrate that the three strains represent a novel species, for which the name Photobacterium toruni sp. nov. is proposed. The type strain is H01100410BT (=CECT 9189T=LMG 29991T).

Genomic analysis of the marine fish pathogen Photobacterium damselae subsp. piscicida: Insertion sequences proliferation is associated with chromosomal reorganisations and rampant gene decay.

Photobacterium damselae subsp. piscicida (Pdp) is an intracellular fish pathogen that causes photobacteriosis, a disease proven deadly in farmed fish worldwide. This work focuses on the analysis of genome sequences, chromosomes structure and gene contents of two strains from Sparus aurata (DI21) and Solea senegalensis (L091106-03H), isolated on the Spanish Atlantic coast. The comparative genomic analysis revealed that DI21 and L091106-03H share 98% of their genomes, including two virulence plasmids: pPHDP70 encoding siderophore piscibactin synthesis and pPHDP10 encoding the apoptotic toxin AIP56. Both genomes harbour a surprisingly large number of IS elements accounting for 12-17% of the total genome, representing an IS density of 0.15 elements per kb, one of the highest IS density values in a bacterial pathogen. This massive proliferation of ISs is responsible for the generation of a high number of pseudogenes that caused extensive loss of biological functions. Pseudogene formation is one of the main features of Pdp genome that explains most of the ecological and phenotypic differences with respect to its sibling subspecies P. damselae subsp. damselae and to other Vibrionaceae. Evidence was also found proving the existence of two chromosomal configurations depending on the origin of the strains: an European and an Asian/American types of genome organisation, reinforcing the idea of the existence of two geographically-linked clonal lineages in Pdp. In short, our study suggests that the host-dependent lifestyle of Pdp allowed massive IS proliferation and gene decay processes, which are major evolutionary forces in the shaping of the Pdp genome.

Revisiting the genus Photobacterium: taxonomy, ecology and pathogenesis.

The genus Photobacterium, one of the eight genera included in the family Vibrionaceae, contains 27 species with valid names and it has received attention because of the bioluminescence and pathogenesis mechanisms that some of its species exhibit. However, the taxonomy and phylogeny of this genus are not completely elucidated; for example, P. logei and P. fischeri are now considered members of the genus Aliivibrio, and previously were included in the genus Vibrio. In addition, P. damselae subsp. piscicida was formed as a new combination for former Vibrio damsela and Pasteurella piscicida. Moreover, P. damselae subsp. damselae is an earlier heterotypic synonym of P. histaminum. To avoid these incovenences draft and complete genomic sequences of members of Photobacterium are increasingly becoming available and their use is now routine for many research laboratories to address diverse goals: species delineation with overall genomic indexes, phylogenetic analyses, comparative genomics, and phenotypic inference. The habitats and isolation source of the Photobacterium species include seawater, sea sediments, saline lake waters, and a variety of marine organisms with which the photobacteria establish different relationships, from symbiosis to pathogenic interactions. Several species of this genus contain bioluminescent strains in symbiosis with marine fish and cephalopods; in addition, other species enhance its growth at pressures above 1 atmosphere, by means of several high-pressure adaptation mechanisms and for this, they may be considered as piezophilic (former barophilic) bacteria. Until now, only P. jeanii, P. rosenbergii, P. sanctipauli, and the two subspecies of P. damselae have been reported as responsible agents of several pathologies on animal hosts, such as corals, sponges, fish and homeothermic animals. In this review we have revised and updated the taxonomy, ecology and pathogenicity of several members of this genus. [Int Microbiol 20(1): 1-10 (2017)].

Revisiting the genus Photobacterium: taxonomy, ecology and pathogenesis

The genus Photobacterium, one of the eight genera included in the family Vibrionaceae, contains 27 species with valid names and it has received attention because of the bioluminescence and pathogenesis mechanisms that some of its species exhibit. However, the taxonomy and phylogeny of this genus are not completely elucidated; for example, P. logei and P. fischeri are now considered members of the genus Aliivibrio, and previously were included in the genus Vibrio. In addition, P. damselae subsp. piscicida was formed as a new combination for former Vibrio damsela and Pasteurella piscicida. Moreover, P. damselae subsp. damselae is an earlier heterotypic synonym of P. histaminum. To avoid these incovenences draft and complete genomic sequences of members of Photobacterium are increasingly becoming available and their use is now routine for many research laboratories to address diverse goals: species delineation with overall genomic indexes, phylogenetic analyses, comparative genomics, and phenotypic inference. The habitats and isolation source of the Photobacterium species include seawater, sea sediments, saline lake waters, and a variety of marine organisms with which the photobacteria establish different relationships, from symbiosis to pathogenic interactions. Several species of this genus contain bioluminescent strains in symbiosis with marine fish and cephalopods; in addition, other species enhance its growth at pressures above 1 atmosphere, by means of several high-pressure adaptation mechanisms and for this, they may be considered as piezophilic (former barophilic) bacteria. Until now, only P. jeanii, P. rosenbergii, P. sanctipauli, and the two subspecies of P. damselae have been reported as responsible agents of several pathologies on animal hosts, such as corals, sponges, fish and homeothermic animals. In this review we have revised and updated the taxonomy, ecology and pathogenicity of several members of this genus (AU)

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Concurrence of Iridovirus, Polyomavirus, and a Unique Member of a New Group of Fish Papillomaviruses in Lymphocystis Disease-Affected Gilthead Sea Bream.

UNLABELLED: Lymphocystis disease is a geographically widespread disease affecting more than 150 different species of marine and freshwater fish. The disease, provoked by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of papillomalike lesions on the skin of affected animals that usually self-resolve over time. Development of the disease is usually associated with several environmental factors and, more frequently, with stress conditions provoked by the intensive culture conditions present in fish farms. In gilthead sea bream (Sparus aurata), an economically important cultured fish species in the Mediterranean area, a distinct LCDV has been identified but not yet completely characterized. We have used direct sequencing of the virome of lymphocystis lesions from affected S. aurata fish to obtain the complete genome of a new LCDV-Sa species that is the largest vertebrate iridovirus sequenced to date. Importantly, this approach allowed us to assemble the full-length circular genome sequence of two previously unknown viruses belonging to the papillomaviruses and polyomaviruses, termed Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1), respectively. Epidemiological surveys showed that lymphocystis disease was frequently associated with the concurrent appearance of one or both of the new viruses. SaPV1 has unique characteristics, such as an intron within the L1 gene, and as the first member of the Papillomaviridae family described in fish, provides evidence for a more ancient origin of this family than previously thought. IMPORTANCE: Lymphocystis disease affects marine and freshwater fish species worldwide. It is characterized by the appearance of papillomalike lesions on the skin that contain heavily enlarged cells (lymphocysts). The causative agent is the lymphocystis disease virus (LCDV), a large icosahedral virus of the family Iridoviridae In the Mediterranean area, the gilthead sea bream (Sparus aurata), an important farmed fish, is frequently affected. Using next-generation sequencing, we have identified within S. aurata lymphocystis lesions the concurrent presence of an additional LCDV species (LCDV-Sa) as well as two novel viruses. These are members of polyomavirus and papillomavirus families, and here we report them to be frequently associated with the presence of lymphocysts in affected fish. Because papillomaviruses have not been described in fish before, these findings support a more ancient origin of this virus family than previously thought and evolutionary implications are discussed.

Evidence for horizontal gene transfer, gene duplication and genetic variation as driving forces of the diversity of haemolytic phenotypes in Photobacterium damselae subsp. damselae.

Photobacterium damselae subsp. damselae, a marine bacterium that causes infections in marine animals and in humans, produces up to three different haemolysins involved in virulence, which include the pPHDD1 plasmid-encoded damselysin (Dly) and HlyApl , and the chromosome-encoded HlyAch . We screened 45 isolates from different origins, and found a correlation between their haemolytic phenotypes and the differential haemolysin gene content. All highly and medium haemolytic strains harboured pPHDD1, with amino acid substitutions in HlyApl and HlyAch being the cause of the medium haemolytic phenotypes in some pPHDD1-harbouring strains. Weakly haemolytic strains contained only hlyAch , whereas nonhaemolytic isolates, in addition to lacking pPHDD1, either lacked hlyAch or contained a hlyAch pseudogene. Sequence analysis of the genomic context of hlyAch uncovered an unexpected genetic diversity, suggesting that hlyAch is located in an unstable chromosomal region. Phylogenetic analysis suggested that hlyApl and hlyAch originated by gene duplication within P. damselae subsp. damselae following acquisition by horizontal transfer. These observations together with the differential distribution of pPHDD1 plasmid among strains suggest that horizontal gene transfer has played a main role in shaping the haemolysin gene baggage in this pathogen.