Tissue tropism of nervous necrosis virus (NNV) in Atlantic cod, Gadus morhua L., after intraperitoneal challenge with a virus isolate from diseased Atlantic halibut, Hippoglossus hippoglossus (L.).

Atlantic cod, Gadus morhua, averaging 100 g, were experimentally challenged by intraperitoneal injection of nervous necrosis virus (NNV) originating from Atlantic halibut. Cod tissues, including blood, gill, pectoral fin, barbel, ventricle, atrium, spleen, liver, lateral line (including muscle tissue), eye (retina) and brain, were sampled at day 25 and 130 and investigated by real-time RT-PCR for the presence of NNV. Relative quantifications at day 130 were calculated using the 2(-DeltaDeltaCt) method. Immunosuppression by injection of prednisolone-acetate was introduced for a 30-day period, and tissue sampled at day 180 and relative quantification estimated. No mortality or clinical signs of disease were observed in the challenged group. The challenge resulted in detection of NNV in blood, spleen, kidney, liver, heart atrium and heart ventricle at day 25, and by the end of the experiment NNV showed a clear increase in brain and retina, suggesting these to be the primary tissues for viral replication. There was no increase in the relative amount of NNV in blood, atrium, ventricle, spleen, liver and kidney. Corticosteroid implants resulted in a weak increase in virus RNA in spleen, kidney, liver and brain. These findings suggest that Atlantic cod is susceptible to infection with NNV from halibut. The observed tissue tropism patterns suggest an initial viraemic phase, followed by neurotrophy. Head-kidney is the best tissue identified for possible NNV detection by non-lethal biopsy, but detection was not possible in all injected fish.

Cytokine profile during latent and slowly progressive primary tuberculosis: a possible role for interleukin-15 in mediating clinical disease.

Recently, mouse models for latent (LTB) and slowly progressive primary tuberculosis (SPTB) have been established. However, cytokine profiles during the two models are not well established. Using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) we studied the expression levels of interleukin (IL)-2, IL-4, IL-10, IL-12, IL-15, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha during the course of LTB and SPTB in the lungs and spleens of B6D2F1Bom mice infected with the H37Rv strain of Mycobacterium tuberculosis (Mtb). The results show that, except for IL-4, cytokine expression levels were significantly higher during SPTB than LTB in both the lungs and spleens. During LTB, all the cytokines (except IL-2 in the lungs) had higher expression levels during the initial period of infection both in the lungs and spleens. During SPTB, the expression levels of IL-15 increased significantly from phases 1 to 3 in the lungs. The expression levels of IL-10, IL-12 and IFN-gamma increased significantly from 2 to 3 in the lungs. IL-10 and IL-15 increased significantly from phases 2 to 3, whereas that of TNF-alpha decreased significantly and progressively from phases 1 to 3 in the spleens. Over-expression of proinflammatory cytokines during active disease has been well documented, but factor(s) underlying such over-expression is not known. In the present study, there was a progressive and significant increase in the expression levels of IL-15, together with Th1 cytokines (IL-12 and IFN-gamma) during SPTB but a significant decrease during LTB. IL-15 is known to up-regulate the production of proinflammatory cytokines, IL-1beta, IL-8, IL-12, IL-17, IFN-gamma and TNF-alpha and has an inhibitory effect on activation-induced cell death. IL-15 is known to be involved in many proinflammatory disease states such as rheumatoid arthritis, sarcoidosis, inflammatory bowel diseases, autoimmune diabetes, etc. Our results, together with the above observations, suggest that IL-15 may play an important role in mediating active disease during Mtb infection.

Characterization of nodavirus and viral encephalopathy and retinopathy in farmed turbot, Scophthalmus maximus (L.).

An outbreak of nodavirus infection in turbot larvae is described with respect to histopathology, immunohistochemistry, cell culture cultivation, RT-PCR amplification and sequence analysis of the capsid protein gene RNA2. Affected turbot developed classical signs of viral encephalopathy and retinopathy (VER) with abnormal swimming behaviour and high mortality levels. In the acute stage of infection, light microscopy revealed vacuolation of the central nervous system (CNS), with positive immunohistochemical staining for nodavirus. Later in the infection, CNS lesions appeared more chronic and contained clusters of cells immunopositive for nodavirus. Bacterial overgrowth in the intestines of the fish may have provoked or influenced the course of the nodavirus infection. We were unable to propagate the virus in cell culture. While RT-PCR using primers designed to detect Atlantic halibut nodavirus gave negative results, further testing with primers complementary to a more conserved region of RNA2 resulted in amplification of a product of the expected size. The entire RNA2 segment was cloned and sequenced. Sequence alignment showed that the turbot nodavirus (TNV) was different from previously described fish nodaviruses. In addition, phylogenetic analysis based on an 823 nt region of the sequence indicated that TNV clustered outside the four established fish nodavirus genotypes, suggesting a fifth genotype within the betanodaviruses.

Associations of MHC class II alleles in Norwegian primary Sjögren's syndrome patients: implications for development of autoantibodies to the Ro52 autoantigen.

Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by dryness of the eyes and mouth. Currently, the highly polymorphic major histocompatibility complex (MHC) genes are the best documented genetic risk factor for the development of autoimmune disease. We examined the MHC class II alleles DRB1, DRB3, DRB4, DRB5, DQA1 and DQB1 in a group of Norwegian pSS patients and compared with a group of healthy controls. Because a number of studies have shown that some of the MHC class II alleles are not associated with the disease as a whole, but rather to the development of autoantibodies, anti-Ro52 autoantibodies in serum were measured and compared to MHC class II allele status. A clear association with pSS was detected for the DRB1*0301 and DRB3*0101 alleles, but these alleles were more closely associated with the presence of anti-Ro52 autoantibodies than with pSS itself. Moreover, the DQA1*0501 and DQB1*0201 alleles were only associated with the presence of anti-Ro52 autoantibodies. This study shows that the production of anti-Ro52 autoantibodies in pSS is associated with the DRB1*0301, DRB3*0101, DQA1*0501 and DQB1*0201 alleles which are in strong linkage disequilibrium.

Characterization of the capsid protein gene from a nodavirus strain affecting the Atlantic halibut Hippoglossus hippoglossus and design of an optimal reverse-transcriptase polymerase chain reaction (RT-PCR) detection assay.

A 1349 nucleotide fragment of the RNA2 from a nodavirus affecting Atlantic halibut Hippoglossus hippoglossus was characterised and the nuclotide sequence (accession no. AJ245641) was employed to develop an optimal reverse-transcriptase polymerase chain reaction (RT-PCR) detection assay. The sequenced part of the RNA2 of Atlantic halibut nodavirus (strain AH95NorA) was highly similar in organisation to that of the RNA2 of striped jack nervous necrosis virus (SJNNV), and comprised features common to all nodaviruses. These characteristics confirmed that the virus that causes viral encephalopathy and retinopathy (VER) in Atlantic halibut is a nodavirus. The nucleotide sequence of the 1349 nucleotide fragment of Atlantic halibut nodavirus RNA2 was 80% identical to the RNA2 of SJNNV. The T2 region (830 nucleotides) of the RNA2 of Atlantic halibut nodavirus shared 98% of the nucleotide sequence when compared with the homologous region of barfin flounder nervous necrosis virus (BFNNV), while the nucleotide sequence identity to SJNNV in this region was 76%. Phylogenetic analysis based on the nucleotide sequences of the T4 region (421 nucleotides) of Atlantic halibut nodavirus and of other fish nodaviruses revealed a close relationship to the nodaviruses of the barfin flounder clad that have been found in other cold-water species (Pacific cod Gadus macrocephalus and barfin founder Verasper moseri). The nucleotide sequence of the RNA2 of Atlantic halibut nodavirus included some features that differ from that of SJNNV. The ORF of the RNA2 of Atlantic halibut nodavirus lacked 6 nucleotides through a single deletion and a 5-nucleotide deletion, separated by 4 nucleotides. The 3'-non-encoding region contained a 21 nucleotide insert and a 3 nucleotide deletion when compared with SJNNV. In comparison with the RNA2 of SJNNV, the 3'-non-encoding region showed a nucleotide sequence identity of 84.5%. A primer set based on the Atlantic halibut nodavirus nucleotide sequence was employed in order to design an optimal RT-PCR. The detection limit of the PCR was 10 to 100 copies of plasmid, while the detection limit of the RT-PCR assay was 100 to 1000 copies of in vitro transcribed viral RNA.

Lymphoid cell accumulation in salivary glands of autoimmune MRL mice can be due to impaired apoptosis.

MRL-lpr mice and the congenic strain MRL +/+ exhibit pathological abnormalities in the salivary glands similar to Sjögren's syndrome in humans. The lpr genotype has been identified as a mutation in the gene encoding Fas which is a cell surface protein that mediates apoptosis. The mutation is leaky, allowing for low levels of the APO-1/Fas (CD95) receptor and partial activity of Fas/Fas ligand-mediated programmed cell death in this strain. To examine the expression of Fas in situ, the authors analysed thymus, lymph node and salivary gland tissue from BALB/c, MRL +/+ and MRL-lpr mice by an immunohistochemical technique (ABC-immunoperoxidase) using an anti-Fas (Jo2) antibody. For detection of apoptotic cells the authors used the terminal deoxynucleotidyl-transferase-mediated dUTP-digoxigenin nick end labelling (TUNEL) method. Thymus from MRL +/+ and normal BALB/c mice showed a higher frequency of Fas expression than was seen in the lpr mice, but the +/+ mice had similar expression of Fas in lymph nodes as lpr mice. The Fas protein was detected among infiltrating mononuclear cells in the salivary glands of both lpr and +/+ mice. Apoptotic cells were found in the thymus with similar frequency in all three strains, while in the lymph nodes only BALB/c mice showed apoptosis. There was no, or very low, frequency of apoptosis among infiltrating mononuclear cells in salivary glands of both MRL strains. In conclusion, despite mutation of the Fas gene in the MRL-lpr strain, there was nevertheless an expression of the apoptosis-related Fas protein in lymphoid tissue and salivary glands of these mice. Based on analysis of apoptotic activity, the impaired Fas in autoimmune MRL mice seems to affect primarily the peripheral organs.

Expression of growth hormone genes in Atlantic salmon.

Atlantic salmon (Salmo salar) possess two genes encoding GH. We have investigated the expression of these two genes in the salmon pituitary. The transcriptional start site was localized 64 nucleotides upstream of the first methionyl codon using primer extension and 5' specific polymerase chain reaction (PCR) assays. Northern analysis revealed a major Atlantic salmon GH (salGH) transcript band of approximately 1400 nucleotides. As coexpression of the salGH genes is not discernible by transcript length, other techniques were used to assess gene activity; RNase protection analysis revealed GH transcript heterogeneity, while reverse transcription-PCR assays detected transcripts from both genes at approximately equivalent amounts. The encoded salGH protein, generated in vitro and by Escherichia coli, shares electrophoretic and immunoreactive identity with native pituitary salGH.

The complete nucleotide sequence of the Atlantic salmon growth hormone I gene.

Two closely related genes encoding growth hormone were isolated from Atlantic salmon by genomic cloning. From one of these genes a total of 6500 nucleotides were determined including 3900 nucleotides in exons and introns and about 600 and 2000 nucleotides in 5' and 3' flanking regions. The gene is organized in six exons and encodes a polypeptide of 210 amino acids including a 22 amino acids signal sequence. The promoter region contains a typical TATA box 21 nucleotides upstream from the transcription start site. At the 3' end, three putative poly(A) signal sequences are present. The last two are within a 121 nt inverted repeat.

Heterogeneity among human T cell clones recognizing an HLA-DR4,Dw4-restricted epitope from the 18-kDa antigen of Mycobacterium leprae defined by synthetic peptides.

Synthetic peptides have been used to exactly define a T cell epitope region from the immunogenic 18-kDa protein of Mycobacterium leprae. Four M. leprae reactive CD4+ T cell clones, isolated from two healthy individuals vaccinated with killed M. leprae, recognized a determinant initially defined by the peptide (38-50). However, fine mapping of the minimal sequence required for T cell recognition revealed heterogeneity among the T cell clones with regard to the N- and carboxyl-terminal boundaries of the epitopes recognized. MHC restriction analysis showed that the immunogenic peptides were presented to the T cells in an HLA-DR4,Dw4-restricted manner in all cases. The results suggest that a polyclonal T cell response representing different fine specificities is directed toward a possible immunodominant epitope from the M. leprae 18-kDa Ag in individuals carrying this MHC haplotype.

A protein antigen of Mycobacterium leprae is related to a family of small heat shock proteins.

The gene encoding an immunologically important 18-kilodalton protein antigen of Mycobacterium leprae has been sequenced, and the amino acid sequence of the antigen has been deduced. The 18-kilodalton antigen is strikingly similar in size and sequence to a family of eucaryotic heat shock proteins.