Immunological responses of turbot (Psetta maxima) to nodavirus infection or polyriboinosinic polyribocytidylic acid (pIC) stimulation, using expressed sequence tags (ESTs) analysis and cDNA microarrays.

To investigate the immunological responses of turbot to nodavirus infection or pIC stimulation, we constructed cDNA libraries from liver, kidney and gill tissues of nodavirus-infected fish and examined the differential gene expression within turbot kidney in response to nodavirus infection or pIC stimulation using a turbot cDNA microarray. Turbot were experimentally infected with nodavirus and samples of each tissue were collected at selected time points post-infection. Using equal amount of total RNA at each sampling time, we made three tissue-specific cDNA libraries. After sequencing 3230 clones we obtained 3173 (98.2%) high quality sequences from our liver, kidney and gill libraries. Of these 2568 (80.9%) were identified as known genes and 605 (19.1%) as unknown genes. A total of 768 unique genes were identified. The two largest groups resulting from the classification of ESTs according to function were the cell/organism defense genes (71 uni-genes) and apoptosis-related process (23 uni-genes). Using these clones, a 1920 element cDNA microarray was constructed and used to investigate the differential gene expression within turbot in response to experimental nodavirus infection or pIC stimulation. Kidney tissue was collected at selected times post-infection (HPI) or stimulation (HPS), and total RNA was isolated for microarray analysis. Of the 1920 genes studied on the microarray, we identified a total of 121 differentially expressed genes in the kidney: 94 genes from nodavirus-infected animals and 79 genes from those stimulated with pIC. Within the nodavirus-infected fish we observed the highest number of differentially expressed genes at 24 HPI. Our results indicate that certain genes in turbot have important roles in immune responses to nodavirus infection and dsRNA stimulation.

Identification of a transcript encoding a soluble form of toll-like receptor 5 (TLR5) in Atlantic salmon during Aeromonas salmonicida infection.

Toll-like receptors (TLRs) are involved in the innate immune response against microbial pathogens in vertebrates and insects. The extracellular region of a TLR recognizes pathogen-associated molecules, while the intracellular region initiates the signaling pathway leading to immune response. Membrane-bound TLRs have been found in most vertebrates, but few soluble forms have been reported. A novel transcript corresponding to a portion of a soluble TLR was identified in liver of infected Atlantic salmon. The complete coding sequence of this TLR was obtained and BLASTN analysis showed the highest sequence identity to a recently described full-length cDNA sequence of a soluble TLR5 from rainbow trout (GenBank Accession No.: ). The deduced protein is 40% identical to the mammalian counterpart of the leucine-rich repeat (LRR)/LRR-like motifs of TLR5. Based on the structure of human TLRs, it contains 21 LRRs with conserved LxxLxLxxNx*xx*xxxxFxxL pattern. Since TLR5 is essential for the recognition of bacterial flagellins, we hypothesize that flagellin and perhaps some other pathogen-derived factors from Aeromonas salmonicida bind to this soluble TLR through an unknown binding domain within the LRR.

Characteristics of inhibition of infectious pancreatic necrosis virus (IPNV) by normal rainbow trout Oncorhynchus mykiss serum.

We studied the characteristics of rainbow trout serum (RTS) inhibitory activity against infectious pancreatic necrosis virus (IPNV). Serum inhibition was related to the serum source and host cell in which the virus had been propagated. IPNV was more efficiently inhibited by RTS in salmonid cell lines than in non-salmonid cell lines, with inhibition highest in rainbow trout gonad (RTG)-2 cells. The RTS sensitivity of the virus was modified by the cell line through which the virus passed, with multiple passages through Chinook salmon embryo (CHSE)-214 cells producing a virus that was less sensitive to RTS. The RTS inhibition level was dependent on cell density: at a cell density of < or = 2 x 10(5) cells ml(-1), inhibition was insignificant (tissue culture infective dose 50% = 10(-1.1) TCID50 ml(-1) reduction); however, above a density of 3 x 10(5) cells ml(-1), the inhibition level was very high (> or = 10(-6.3) TCID50 ml(-1) reduction). The salmonid sera tested showed high inhibition, except for brook trout serum (BTS), while non-salmonid sera did not inhibit IPNV, replication on RTG-2 cells. Pretreatment of cultured cells with RTS prior to exposure did not affect inhibition of IPNV and thus did not mask a viral receptor. The RTS inhibition level was dependent on the time of serum addition, with inhibition being maintained for at least 16 h postinfection. Pretreatment of IPNV revealed that the virus is directly inhibited by RTS, and more strongly so when RTS is present during viral replication.

Viral skin infections: diagnosis and treatment considerations.

Skin lesions are prominent features of many viral diseases. In some instances, characteristic skin lesions suggest a specific viral illness, the diagnosis of which can be quickly established by appropriate procedures. In addition to clinical manifestations, laboratory methods including virus isolation are used to diagnose viral infections. In viral diseases, prophylaxis has proved more successful than the specific treatment of established infection. However, recent progress in molecular biology has facilitated the development of new vaccines and new drugs to treat viral infections.

Expressed sequence tags analysis of Atlantic halibut (Hippoglossus hippoglossus) liver, kidney and spleen tissues following vaccination against Vibrio anguillarum and Aeromonas salmonicida.

To investigate the response of Atlantic halibut to vaccination and pathogen exposure, a cDNA library was constructed from liver, kidney and spleen mRNA collected following vaccination against Vibrio anguillarum and Aeromonas salmonicida. After sequencing 1114 clones 1072 (96.23%) readable sequences were obtained of which 106 sequences are the first reported from the fish. Of these, 182 clones (16.98%) contained cell/organism defence genes including immunoglobulin light chain, MHC class I and II, interferon consensus sequence binding protein, B-cell receptor-associated protein, early B-cell factor, 10 complement components, heat shock protein 70 and 90, antimicrobial peptides hepcidin type 1 and 2, and CC chemokine (macrophage inflammatory protein-1 beta-like chemokine, MIP-1beta). Expression of MIP-1beta-like was elevated in the kidney and spleen at 1, 2, 7 and 14 days post vaccination. Functional genes involved in cellular processes of hematopoietic tissues were also identified. These results indicate that this cDNA library contains many important genes involved in the immune response, making it an important resource for studying the response of Atlantic halibut to vaccination or pathogen exposure.