Comprehensive validation of T- and B-cell deficiency in rag1-null zebrafish: Implication for the robust innate defense mechanisms of teleosts.

rag1 -/- zebrafish have been employed in immunological research as a useful immunodeficient vertebrate model, but with only fragmentary evidence for the lack of functional adaptive immunity. rag1-null zebrafish exhibit differences from their human and murine counterparts in that they can be maintained without any specific pathogen-free conditions. To define the immunodeficient status of rag1 -/- zebrafish, we obtained further functional evidence on T- and B-cell deficiency in the fish at the protein, cellular, and organism levels. Our developed microscale assays provided evidence that rag1 -/- fish do not possess serum IgM protein, that they do not achieve specific protection even after vaccination, and that they cannot induce antigen-specific CTL activity. The mortality rate in non-vaccinated fish suggests that rag1 -/- fish possess innate protection equivalent to that of rag1 +/- fish. Furthermore, poly(I:C)-induced immune responses revealed that the organ that controls anti-viral immunity is shifted from the spleen to the hepatopancreas due to the absence of T- and B-cell function, implying that immune homeostasis may change to an underside mode in rag-null fish. These findings suggest that the teleost relies heavily on innate immunity. Thus, this model could better highlight innate immunity in animals that lack adaptive immunity than mouse models.

V-GAP: Viral genome assembly pipeline.

Next-generation sequencing technologies have allowed the rapid determination of the complete genomes of many organisms. Although shotgun sequences from large genome organisms are still difficult to reconstruct perfect contigs each of which represents a full chromosome, those from small genomes have been assembled successfully into a very small number of contigs. In this study, we show that shotgun reads from phage genomes can be reconstructed into a single contig by controlling the number of read sequences used in de novo assembly. We have developed a pipeline to assemble small viral genomes with good reliability using a resampling method from shotgun data. This pipeline, named V-GAP (Viral Genome Assembly Pipeline), will contribute to the rapid genome typing of viruses, which are highly divergent, and thus will meet the increasing need for viral genome comparisons in metagenomic studies.

Influences of diurnal sampling bias on fixed-point monitoring of plankton biodiversity determined using a massively parallel sequencing-based technique.

In this study, we investigated the influence of diurnal sampling bias on the community structure of plankton by comparing the biodiversity among seawater samples (n=9) obtained every 3h for 24h by using massively parallel sequencing (MPS)-based plankton monitoring at a fixed point conducted at Himedo seaport in Yatsushiro Sea, Japan. The number of raw operational taxonomy units (OTUs) and OTUs after re-sampling was 507-658 (558 ± 104, mean ± standard deviation) and 448-544 (467 ± 81), respectively, indicating high plankton biodiversity at the sampling location. The relative abundance of the top 20 OTUs in the samples from Himedo seaport was 48.8-67.7% (58.0 ± 5.8%), and the highest-ranked OTU was Pseudo-nitzschia species (Bacillariophyta) with a relative abundance of 17.3-39.2%, followed by Oithona sp. 1 and Oithona sp. 2 (Arthropoda). During seawater sampling, the semidiurnal tidal current having an amplitude of 0.3ms(-1) was dominant, and the westward residual current driven by the northeasterly wind was continuously observed during the 24-h monitoring. Therefore, the relative abundance of plankton species apparently fluctuated among the samples, but no significant difference was noted according to G-test (p>0.05). Significant differences were observed between the samples obtained from a different locality (Kusuura in Yatsushiro Sea) and at different dates, suggesting that the influence of diurnal sampling bias on plankton diversity, determined using the MPS-based survey, was not significant and acceptable.

A functional genomics tool for the Pacific bluefin tuna: Development of a 44K oligonucleotide microarray from whole-genome sequencing data for global transcriptome analysis.

Bluefin tunas are one of the most important fishery resources worldwide. Because of high market values, bluefin tuna farming has been rapidly growing during recent years. At present, the most common form of the tuna farming is based on the stocking of wild-caught fish. Therefore, concerns have been raised about the negative impact of the tuna farming on wild stocks. Recently, the Pacific bluefin tuna (PBT), Thunnus orientalis, has succeeded in completing the reproduction cycle under aquaculture conditions, but production bottlenecks remain to be solved because of very little biological information on bluefin tunas. Functional genomics approaches promise to rapidly increase our knowledge on biological processes in the bluefin tuna. Here, we describe the development of the first 44K PBT oligonucleotide microarray (oligo-array), based on whole-genome shotgun (WGS) sequencing and large-scale expressed sequence tags (ESTs) data. In addition, we also introduce an initial 44K PBT oligo-array experiment using in vitro grown peripheral blood leukocytes (PBLs) stimulated with immunostimulants such as lipopolysaccharide (LPS: a cell wall component of Gram-negative bacteria) or polyinosinic:polycytidylic acid (poly I:C: a synthetic mimic of viral infection). This pilot 44K PBT oligo-array analysis successfully addressed distinct immune processes between LPS- and poly I:C- stimulated PBLs. Thus, we expect that this oligo-array will provide an excellent opportunity to analyze global gene expression profiles for a better understanding of diseases and stress, as well as for reproduction, development and influence of nutrition on tuna aquaculture production.

Effective de novo assembly of fish genome using haploid larvae.

Recent improvements in next-generation sequencing technology have made it possible to do whole genome sequencing, on even non-model eukaryote species with no available reference genomes. However, de novo assembly of diploid genomes is still a big challenge because of allelic variation. The aim of this study was to determine the feasibility of utilizing the genome of haploid fish larvae for de novo assembly of whole-genome sequences. We compared the efficiency of assembly using the haploid genome of yellowtail (Seriola quinqueradiata) with that using the diploid genome obtained from the dam. De novo assembly from the haploid and the diploid sequence reads (100 million reads per each datasets) generated by the Ion Proton sequencer (200 bp) was done under two different assembly algorithms, namely overlap-layout-consensus (OLC) and de Bruijn graph (DBG). This revealed that the assembly of the haploid genome significantly reduced (approximately 22% for OLC, 9% for DBG) the total number of contigs (with longer average and N50 contig lengths) when compared to the diploid genome assembly. The haploid assembly also improved the quality of the scaffolds by reducing the number of regions with unassigned nucleotides (Ns) (total length of Ns; 45,331,916 bp for haploids and 67,724,360 bp for diploids) in OLC-based assemblies. It appears clear that the haploid genome assembly is better because the allelic variation in the diploid genome disrupts the extension of contigs during the assembly process. Our results indicate that utilizing the genome of haploid larvae leads to a significant improvement in the de novo assembly process, thus providing a novel strategy for the construction of reference genomes from non-model diploid organisms such as fish.

Full-genome sequence of a novel myovirus, GF-2, infecting Edwardsiella tarda: comparison with other Edwardsiella myoviral genomes.

Edwardsiellosis, which is caused by Edwardsiella tarda, a Gram-negative bacterium, is one of the most serious infectious diseases in both marine and freshwater fish farms worldwide. Previously, we reported the complete genome sequences of three E. tarda-lytic bacteriophages (two podoviruses and a myovirus), which were isolated from fish tissues and fish-rearing seawater. Further genomic information regarding E. tarda phages is important for understanding phage-host interactions as well as for applications of the phages for the control of disease. Here, we report the complete genome sequence of a novel E. tarda phage (GF-2) of myovirus morphology (family Myoviridae), isolated from tissue homogenates of a cultured Japanese flounder (Paralichthys olivaceus) that succumbed to edwardsiellosis in Japan. The size of the entire genome was 43,129 bp, with a GC content of 51.3 % and containing 82 open reading frames (ORFs). The GF-2 genome possesses lysogeny-related genes that have not been found in the reported Edwardsiella phage genomes. Comparative genomics of Edwardsiella myophages suggest that the C-terminal domains of the tail fiber proteins have relevance to their host specificity. Thus, GF-2 genome information provides a novel resource for our understanding of the molecular mechanisms involved in their host specificity and for detection of E. tarda in aquaculture environments.

Complete Genome Sequence of the Edwardsiella ictaluri-Specific Bacteriophage PEi21, Isolated from River Water in Japan.

We present the complete genome sequence for a novel Edwardsiella ictaluri-specific bacteriophage, PEi21, isolated from river water in Japan. An initial comparative genome analysis revealed that the phage was closely related to the previously reported Edwardsiella tarda phage MSW-3 isolated from a red sea bream farm in Japan.

A ddRAD-based genetic map and its integration with the genome assembly of Japanese eel (Anguilla japonica) provides insights into genome evolution after the teleost-specific genome duplication.

BACKGROUND: Recent advancements in next-generation sequencing technology have enabled cost-effective sequencing of whole or partial genomes, permitting the discovery and characterization of molecular polymorphisms. Double-digest restriction-site associated DNA sequencing (ddRAD-seq) is a powerful and inexpensive approach to developing numerous single nucleotide polymorphism (SNP) markers and constructing a high-density genetic map. To enrich genomic resources for Japanese eel (Anguilla japonica), we constructed a ddRAD-based genetic map using an Ion Torrent Personal Genome Machine and anchored scaffolds of the current genome assembly to 19 linkage groups of the Japanese eel. Furthermore, we compared the Japanese eel genome with genomes of model fishes to infer the history of genome evolution after the teleost-specific genome duplication. RESULTS: We generated the ddRAD-based linkage map of the Japanese eel, where the maps for female and male spanned 1748.8 cM and 1294.5 cM, respectively, and were arranged into 19 linkage groups. A total of 2,672 SNP markers and 115 Simple Sequence Repeat markers provide anchor points to 1,252 scaffolds covering 151 Mb (13%) of the current genome assembly of the Japanese eel. Comparisons among the Japanese eel, medaka, zebrafish and spotted gar genomes showed highly conserved synteny among teleosts and revealed part of the eight major chromosomal rearrangement events that occurred soon after the teleost-specific genome duplication. CONCLUSIONS: The ddRAD-seq approach combined with the Ion Torrent Personal Genome Machine sequencing allowed us to conduct efficient and flexible SNP genotyping. The integration of the genetic map and the assembled sequence provides a valuable resource for fine mapping and positional cloning of quantitative trait loci associated with economically important traits and for investigating comparative genomics of the Japanese eel.

Transcription analysis of two Eomesodermin genes in lymphocyte subsets of two teleost species.

Eomesodermin (Eomes), a T-box transcription factor, is a key molecule associated with function and differentiation of CD8(+) T cells and NK cells. Previously, two teleost Eomes genes (Eomes-a and -b), which are located on different chromosomes, were identified and shown to be expressed in zebrafish lymphocytes. For the present study, we identified these genes in rainbow trout and ginbuna crucian carp. Deduced Eomes-a and -b amino acid sequences in both fish species contain a highly conserved T-box DNA binding domain. In RT-PCR, both Eomes transcripts were readily detectable in a variety of tissues in rainbow trout and ginbuna. The high expression of Eomes-a and -b in brain and ovary suggests involvement in neurogenesis and oogenesis, respectively, while their expression in lymphoid tissues presumably is associated with immune functions. Investigation of separated lymphocyte populations from pronephros indicated that both Eomes-a and -b transcripts were few or absent in IgM(+) lymphocytes, while relatively abundant in IgM(-)/CD8α(+) and IgM(-)/CD8α(-) populations. Moreover, we sorted trout CD8α(+) lymphocytes from mucosal and non-mucosal lymphoid tissues and compared the expression profiles of Eomes-a and -b with those of other T cell-related transcription factor genes (GATA-3, T-bet and Runx3), a Th1 cytokine gene (IFN-γ) and a Th2 cytokine gene (IL-4/13A). Interestingly, the tissue distribution of Eomes-a/b, T-bet, and Runx3 versus IFN-γ transcripts did not reveal simple correlations, suggesting tissue-specific properties of CD8α(+) lymphocytes and/or multiple modes that drive IFN-γ expressions.

Growth of cyprinid herpesvirus 2 (CyHV-2) in cell culture and experimental infection of goldfish Carassius auratus.

Herpesviral haematopoietic necrosis has caused great economic damage to goldfish Carassius auratus aquaculture in Japan. The existence of cyprinid herpesvirus 2 (CyHV-2), the causative agent, has also been reported from several other countries. To prevent spread to other areas, basic virological information such as viral kinetics in infected fish is essential. Experimental infection trials using reliably prepared CyHV-2 for defining viral kinetics are difficult to carry out because successful and sustainable propagation of this virus in cell culture has previously been limited. Here we describe a method for sustainable propagation of CyHV-2 in cell culture, and the results of fish infection experiments using the propagated virus. We found that goldfish fin (GFF) cells and standard Ryukin Takafumi (SRTF) cells established from goldfish fin can be used for continuous propagation of CyHV-2. Experimental infections using 2 varieties of goldfish, Ryukin and Edonishiki, were performed with the virus passaged 7 times in GFF cells. In transmission experiments with water temperature at 20°C, cumulative mortality was 30% in Ryukin infected by immersion, and 90 and 100% in Edonishiki and Ryukin intraperitoneally injected with the virus, respectively. In an experiment carried out at 25°C, 90% of Edonishiki challenged by immersion died. PCR detection of viral DNA from the organs of infected fish showed that systemic infection occurs and also that the kidney is a main viral multiplication site. Moreover, CyHV-2 was successfully re-isolated in GFF cells from the dead fish.

Conservation of characteristics and functions of CD4 positive lymphocytes in a teleost fish.

The presence of helper and cytotoxic T cells in fish has been suggested, although T cell subsets have yet to be identified at the cellular level. In order to investigate the functions of CD4 and CD8α positive T cells we attempted to produce and characterize monoclonal antibodies (mAbs) against teleost CD4 and CD8α. Here we report the successful production of mAbs against CD4 and CD8α in clonal ginbuna crucian carp Carassius auratus langsdorfii and the function of CD4 positive T cells. In this study we demonstrate the presence of teleost CD4- and CD8α-positive T cell subsets with morphology, tissue distribution and gene expression similar to those of mammalian CD4- and CD8-positive T lymphocytes. Using mAbs we found that CD4/CD8 double positive T cells are only present in the thymus, suggesting that it is the site of T cell development. We further demonstrated in vitro proliferation of CD4 positive T cells by allogeneic combination of mixed leukocyte culture and antigen-specific proliferation of CD4 positive T cells after in vitro sensitization with OVA. In our previous study we showed that CD8α-positive lymphocytes are the primary cell type showing specific cytotoxicity against allogeneic targets. Collectively, these findings suggest that CD4 and CD8α positive T cells in ginbuna are equivalent to helper and cytotoxic T lymphocytes (CTL) in mammals, respectively. This is the first report to show the characteristics and functions of CD4 positive T cells in fish and these findings shed light into the evolutionary origins and primordial functions of helper T cells.

In vitro generation of viral-antigen dependent cytotoxic T-cells from ginbuna crucian carp, Carassius auratus langsdorfii.

Little is known about antigen-specific T-cell responses to viruses in teleosts due to a lack of a suitable experimental system using inbred or clonal animals. In the present study we have successfully induced an in vitro generation of virus-specific cytotoxic T-cells (CTLs) from isogeneic ginbuna crucian carp. Responder cells (primarily lymphocytes) from crucian carp haematopoietic necrosis virus (CHNV)-infected fish were capable of proliferating after stimulation in vitro with CHNV-infected syngeneic stimulator cells (primarily lymphocytes and macrophages). The effector cells collected 8 and 12 days after the in vitro stimulation efficiently lysed CHNV-infected syngeneic cells, but not CHNV-infected allogeneic cells or different virus (EVA)-infected syngeneic cells. Furthermore, in situ hybridization analysis showed that some effector cells binding to a CHNV-infected target were TCRbeta or CD8alpha positive. These results provide evidence that the teleost effector cells generated in vitro correspond to virus-specific CTL and they recognize virus-infected target cells in a similar manner of mammalian counterparts.

Alloantigen-specific killing is mediated by CD8-positive T cells in fish.

CD8-positive (CD8(+)) cytotoxic T lymphocytes (CTL) have antigen-specific cytotoxic activity. In fish, however, CTL expressing CD8 on their cell surface have not been identified. In order to characterize the cells involved in specific cell-mediated cytotoxicity in teleosts, we separated and sorted ginbuna kidney leucocytes into CD8alpha(+), CD4(+) and surface IgM (sIgM)(+) cells by magnetic activated cell sorting using monoclonal antibodies and examined their cytotoxic activities. Effector donor ginbuna (OB1 clone) were sensitized by allografting scales from S3N clone fish followed by injection of an allogeneic cell line (CFS) derived from S3N fish. In cytotoxic assays, target cells were labeled with CFSE and cytotoxicity was calculated based on the number of viable target cells using flow cytometry. CD8alpha(+) cells from sensitized OB1 fish showed relatively high cytotoxicity against CFS cells (immunogen) but not against allogeneic CFK cells (third party) nor isogeneic CFO cells. Pre-sensitized sIgM(+) cells exhibited cytotoxicity against not only CFS cells but also CFK cells. However, CD4(+) or CD8alpha(-) CD4(-)sIgM(-) cells as well as cells from non-sensitized fish did not show any significant cytotoxic activity. These results suggest that CD8alpha(+) cells in fish have characteristics similar to those of CTL in mammals, and that the sIgM(+) cells include NK-like cells which non-specifically killed the target cells.

GATA3 mRNA in ginbuna crucian carp (Carassius auratus langsdorfii): cDNA cloning, splice variants and expression analysis.

GATA3, a transcriptional activator, plays a critical role in the development of T-cells and differentiation to T helper type 2 cells. To date, no information is available on the role of GATA3 in the teleost immune system. We identified full-length cDNA and alternatively spliced variants of ginbuna crucian carp GATA3 (gbGATA3). The gbGATA3 gene is transcribed into multiple splice variants lacking either one or both zinc finger domains, although the sequences of both domains are fully conserved between ginbuna and other vertebrates. We found that alternative splice site and stop codon in gbGATA3 intron 3, located between exons that separately encode the two zinc finger domains, are conserved among teleosts, suggesting that teleost GATA3 gene can be translated into multiple isoforms. RT-PCR analysis revealed that the gbGATA3 is strongly expressed in the brain, thymus and gill of unstimulated fish. Moreover, gbGATA3 expression was detected in surface-IgM-negative lymphocytes among kidney cells sorted by FACS. Real-time PCR demonstrated that expression levels of full-length gbGATA3 and the splice variants differed with tissue type, but full length was always the predominantly expressed form. These results suggest that gbGATA3, including its splice variants, is involved in teleost T-cell function.

Macrophage migration inhibitory factor (MIF) is essential for development of zebrafish, Danio rerio.

Macrophage migration inhibitory factor (MIF) was discovered as the first cytokine that inhibited the random migration of macrophages. Recently, MIF has been reported to be involved in embryonic development in higher vertebrates. In fish, however, nothing is known about the function of MIF at early life stages, although immunological functions of MIF have been reported in adult fish. To elucidate the function of MIF during embryonic development in fish, we examined expression patterns and function of the zebrafish MIF gene using antisense morpholino-mediated knockdown (morpholino oligonucleotide-MO). In whole-mount in situ hybridization analysis, zebrafish MIF mRNA was detected in developing eyes, tectum, branchial arches, pectoral fin buds, liver and gut. The onset of MIF mRNA expression coincided with the beginning of tissue differentiation during embryogenesis. MIF-MO-injected embryos (morphants) displayed malformed eyes, abnormal swelling in the tectum and fourth ventricle region, and undeveloped jaw cartilage and pectoral fins. An increased number of apoptotic cells in the eye and neural tissues were observed in MIF morphants by histological analysis and acridine orange staining. Moreover, proliferating cell nuclear antigen (PCNA)-positive cells were reduced in morphant eyes. These results suggest that MIF is essential for normal embryonic development even at the level of teleosts and that it functions as a growth factor for the proliferation and differentiation of embryonic tissues.

Molecular cloning and expression analysis of T-bet in ginbuna crucian carp (Carassius auratus langsdorfii).

In the adaptive immune system of mammals, naive helper T (Th) cells differentiate into Th1 or Th2 cells. The T-box expressed in T cells (T-bet) is a member of a family of T-box transcription factors that regulates the expression of IFN-gamma and plays a crucial role in Th1 cell differentiation and cell-mediated immunity. We cloned and sequenced T-bet cDNA for the first time from non-mammalian species, ginbuna crucian carp. Ginbuna T-bet was composed of 608 predicted amino acids and showed 41.5% identity with human T-bet (Tbx21), and human and ginbuna T-bet share 77.3% identity in their T-box regions. Comparative genomic analysis showed conserved synteny in these regions between zebrafish, fugu, medaka and human T-bet. Phylogenetic analysis indicated that ginbuna T-bet is closely related to that of mouse and human. In unstimulated fish, ginbuna T-bet mRNA was strongly expressed in peripheral blood leukocytes (PBL), head kidney (HK) and spleen. RT-PCR analysis in kidney cells sorted by FACS revealed that T-bet was strongly expressed in surface-IgM-negative lymphocytes in comparison to IgM-positive lymphocytes. These results suggest that ginbuna T-bet is involved in the immune system, especially in T-cell function, and is an important tool to analyze teleost cell-mediated immunity.

Cell-mediated immune responses in rainbow trout after DNA immunization against the viral hemorrhagic septicemia virus.

To identify viral proteins that induce cell-mediated cytotoxicity (CMC) against viral hemorrhagic septicemia virus (VHSV)-infected cells, rainbow trout were immunized with DNA vectors encoding the glycoprotein G or the nucleocapsid protein N of VHSV. The G protein was a more potent trigger of cytotoxic cells than the N protein. Peripheral blood leukocytes (PBL) isolated from trout immunized against the G protein killed both VHSV-infected MHC class I matched (RTG-2) and VHSV-infected xenogeneic (EPC) target cells, suggesting the involvement of both cytotoxic T lymphocytes (CTL) and NK cells, respectively. In contrast, PBL from trout that were immunized against the N protein only killed VHSV-infected RTG-2 cells, indicating that this protein only elicits a CTL response. Further, a significant killing capacity of these PBL was only observed during summer months. PBL from fish that were immunized against the VHSV G protein significantly killed VHSV-infected but not infectious hematopoietic necrosis virus (IHNV)-infected targets indicating antigen specificity. Thus, this is the first report on cytotoxic immune responses after DNA vaccination in fish. Furthermore, cells isolated from the inflamed site of DNA injection were stained and transferred to isogeneic DNA-vaccinated recipients. Most of the stained donor leukocytes accumulated at the recipients' DNA injection site showing, for the first time, leukocyte homing in fish. Transferred donor leukocytes mainly migrated to the homologous vaccine injection site rather than to injection sites of heterologous vaccines, suggesting the antigen specificity of homing. By demonstrating CMC responses to distinct viral proteins and homing in rainbow trout, these results substantially contribute to the understanding of the teleost immune system.

The C3 subtypes are differentially regulated after immunostimulation in rainbow trout, but head kidney macrophages do not contribute to C3 transcription.

The complement system plays key roles in innate and adaptive immunity through mediating phagocytosis, chemotaxis and cell lysis. Complement component C3 is a central component in the complement cascade and belongs to the acute-phase proteins whose synthesis increase immediately upon inflammatory stimuli. The liver is the main producer of C3 and it is a well-known fact that the mammalian monocyte-macrophage lineage is a major contributor to extrahepatic C3. Immunomodulators, such as LPS and beta-glucan, can stimulate complement, lysozyme, natural killer cells and antibody responses in fish, thus enhancing the resistance to bacterial pathogens and parasitic infections. The aim of this study was to assess the effects of LPS and beta-glucan on the expression of interleukins (IL-1beta1, IL-1beta2 and IL-6) and the modulated expression of C3 subtypes (C3-1, C3-3 and C3-4) in the rainbow trout (Oncorhynchus mykiss) using real-time RT-PCR. From in vitro studies, we demonstrated that head kidney macrophages from rainbow trout and Atlantic salmon showed no basal transcription of C3. After immunostimulation, the cells responded by increased levels of ILs, but transcription of C3 was not induced. In contrast to the in vitro findings, the rainbow trout complement C3 subtypes were differentially regulated 48 h after in vivo stimulation with LPS and beta-glucan. These results support the previous findings of absence of C3 in macrophages of the spotted wolffish (Anarhichas minor) and is the first functional study showing differential regulation of the C3 subtypes in any vertebrate.

A third broad lineage of major histocompatibility complex (MHC) class I in teleost fish; MHC class II linkage and processed genes.

Most of the previously studied teleost MHC class I molecules can be classified into two broad lineages: "U" and "Z/ZE." However, database reports on genes in cyprinid and salmonid fishes show that there is a third major lineage, which lacks detailed analysis so far. We designated this lineage "L" because of an intriguing linkage characteristic. Namely, one zebrafish L locus is closely linked with MHC class II loci, despite the extensively documented nonlinkage of teleost class I with class II. The L lineage consists of highly variable, nonclassical MHC class I genes, and has no apparent orthologues outside teleost fishes. Characteristics that distinguish the L lineage from most other MHC class I are (1) absence of two otherwise highly conserved tryptophan residues W51 and W60 in the alpha1 domain, (2) a low GC content of the alpha1 and alpha2 exons, and (3) an HINLTL motif including a possible glycosylation site in the alpha3 domain. In rainbow trout (Oncorhynchus mykiss) we analyzed several intact L genes in detail, including their genomic organization and transcription pattern. The gene Onmy-LAA is quite different from the genes Onmy-LBA, Onmy-LCA, Onmy-LDA, and Onmy-LEA, while the latter four are similar and categorized as "Onmy-LBA-like." Whereas the Onmy-LAA gene is organized like a canonical MHC class I gene, the Onmy-LBA-like genes are processed and lack all introns except intron 1. Onmy-LAA is predominantly expressed in the intestine, while the Onmy-LBA-like transcripts display a rather homogeneous tissue distribution. To our knowledge, this is the first description of an MHC class I lineage with multiple copies of processed genes, which are intact and transcribed. The present study significantly improves the knowledge of MHC class I variation in teleosts.

Cell-mediated cytotoxicity in rainbow trout, Oncorhynchus mykiss, infected with viral haemorrhagic septicaemia virus.

Mammalian cytotoxic T cells as part of the adaptive immune system recognize virus-infected target cells by binding of their T-cell receptors (TCR) to classical MHC class I molecules loaded with viral peptides. Our previous studies have shown that the allele of the single dominant polymorphic classical MHC class I locus Onmy-UBA is identical in the rainbow trout clone C25 and in the permanent rainbow trout cell line RTG-2. This enabled us to develop an assay to measure antiviral cytotoxicity in rainbow trout using a system of MHC class I-matched effector and target cells. Peripheral blood leucocytes (PBL) isolated from low dose viral haemorrhagic septicaemia virus (VHSV)-infected rainbow trout killed MHC class I-matched and later also xenogeneic MHC class I-mismatched VHSV-infected cells. When compared to PBL from uninfected control fish PBL from infected fish showed a higher transcriptional level of the CD8alpha gene which is a typical marker for mammalian cytotoxic T cells. Concurrently, the expression of the natural killer cell enhancement factor (NKEF)-like gene was enhanced as measured by real-time RT - PCR. Taken together, these results suggest that both innate and adaptive cell-mediated immune responses represented by NK and cytotoxic T cells, respectively, are triggered after VHSV infection. PBL that were able to kill VHSV-infected MHC class I-mismatched xenogeneic cells were generated later during infection than PBL capable of lysing VHSV-infected MHC class I-matched targets. This is contradictory to the generally accepted rule that innate immune mechanisms represent the first line of defence after viral infections.