V.G. Chinchar

Molecular identification and expression analysis of tumor necrosis factor in channel catfish (Ictalurus punctatus).

A tumor necrosis factor (TNF) alpha-like gene, encoding a propeptide of 230 amino acids and a mature (soluble) peptide of 162 amino acids, was identified in channel catfish (Ictalurus punctatus). While the catfish protein shared features in common with both mammalian TNFalpha and TNFbeta homologs, overall sequence identity/similarity was slightly higher vs. TNFalpha genes when mature TNF sequences were compared. Phylogenetic analysis placed catfish and other fish TNF sequences within their own cluster apart from mammalian TNFalpha and beta genes, and supported the suggestion that TNFalpha and beta genes separated after the divergence of mammals and teleosts. In contrast to trout and carp, but similar to flounder, catfish TNF was present as a single copy gene. Expression studies demonstrated that catfish TNFalpha mRNA was present in all tested tissues (i.e. liver, spleen, head kidney, mesonephros, gill, thymus, and PBLs) from an unstimulated fish. Moreover, catfish TNF was constitutively expressed in actively proliferating, but otherwise unstimulated, macrophage (42TA) and T cell (G14D; TS32.17) lines, but not in B cell (1G8 or 3B11) or fibroblast lines. TNF expression was upregulated in PBLs, and in G14D and 42TA cells, but not in 3B11 cells, by PMA/calcium ionophore treatment. These results demonstrate that a catfish homolog of TNFalpha has been identified, and indicate that catfish TNFalpha is expressed in catfish in a manner similar to that seen in mammals.

Identification and expression analysis of interferon gamma genes in channel catfish

Multiple species of type I interferon (IFN) were recently identified in catfish (CF) (Ictalurus punctatus). Herein we extend these studies and report the existence of two distinct type II IFN genes in channel CF. As with zebrafish and the green spotted pufferfish, the two CF IFN-γ genes are dissimilar in sequence but closely linked on the same chromosome. One of the genes (IFN-γ2) encodes two distinct messages that likely arose via alternative splicing at two closely spaced splice donor sites within the first intron. Sequence analysis indicates that CF IFN-γ genes contain the hallmarks of authentic IFN-γ including: (1) a conserved nuclear localization site at the C terminus (CF IFN-γ2 only), (2) an IFN-γ signature sequence, (3) six putative helical regions within the mature protein, (4) one or more potential glycosylation sites, and (5) multiple mRNA instability motifs within the 3′ untranslated region. Moreover, well-characterized CF T and NK cell clones were shown to synthesize IFN-γ transcripts. This is the first unequivocal demonstration in any lower vertebrate species that NK and T cells synthesize IFN-γ and is consistent with results in mammalian systems where T cells and NK cells are the major sources of type II IFN production. Collectively, these studies indicate that Siluriformes possess two evolutionarily conserved IFN-γ genes and demonstrate that CF possess three key elements of the innate immune response: NK cells and types I and II IFN.

Fish immunology: the utility of immortalized lymphoid cells--a mini review.

Long term cell lines can be readily established at high frequency with PBLs from normal channel catfish. Depending upon the mode of stimulation, morphologically and functionally distinct catfish lymphoid cell lines resembling B cells, T cells and monocytes have been developed. These fish cell lines appear unique from their putative mammalian counterparts in that they are immortalized without the need for exogenous factors or overt attempts at transformation.

Induction of apoptosis in frog virus 3-infected cells.

The ability of frog virus 3 (FV3), the type species of the family Iridoviridae, to induce apoptosis was examined by monitoring DNA cleavage, chromatin condensation, and cell-surface expression of phosphotidylserine (PS) in fathead minnow (FHM) and baby hamster kidney (BHK) cells. In productively infected FHM cells, DNA fragmentation was first noted at 6-7 h postinfection and was clearly seen by 17 h postinfection, while chromatin condensation was detected at 8.5 h postinfection. As with some other viruses, FV3-induced apoptosis did not require de novo viral gene expression as both heat-inactivated and UV-inactivated virus readily triggered DNA fragmentation in FHM cells. Moreover, FV3-induced apoptosis was blocked in FHM cells by the pan-caspase inhibitor Z-VAD-FMK, suggesting that virus infection triggers programmed cell death through activation of the caspase cascade. FV3 infection also triggered apoptosis in BHK cells as monitored by TUNEL and annexin V binding assays. To determine whether FV3, similar to other large DNA viruses, encoded proteins that block or delay apoptosis, mock- and FV3-infected FHM cells were osmotically shocked and assayed for DNA fragmentation 3 hours later. DNA fragmentation was clearly seen whether or not shocked cells were previously infected with FV3, indicating that infection with FV3 did not block apoptosis induced by osmotic shock in FHM cells. The above results demonstrate that iridoviruses triggered apoptosis and that the induction of programmed cell death did not require viral gene expression. However, it remains to be determined if virion attachment to target cells is sufficient to induce cell death, or if apoptosis is triggered directly or indirectly by one or more virion-associated proteins.

MHC class I genes of the channel catfish: sequence analysis and expression

Abstract Four cDNAs encoding the major histocompatibility complex (MHC) class I α chain were isolated from a channel catfish clonal B-cell cDNA library. Sequence analysis suggests these cDNAs represent three different MHC class I loci. All cDNAs encoded conserved residues characteristic of the MHC class I α chain: namely, those involved in peptide binding, salt bridges, disulfide bond formation, and glycosylation. Southern blot analyses of individual outbred and second-generation gynogenetic fish indicated the existence of both polygenic and polymorphic loci. Northern blot studies demonstrated that catfish B, T, and macrophage cell lines transcribed markedly higher levels of class I α and β2-microglobulin (β2m) mRNA than fibroblast cell lines. In addition, immunoprecipitation data showed that a 41 000 Mr glycoprotein (presumably class I α) was associated with β2m on the surface of catfish B cells. This latter finding is the first direct evidence for the cell surface association of β2m with the MHC class I α chain on teleost cells and supports the notion that functional MHC class I proteins exist in teleosts.

Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro.

Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granular bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.

Antisense approaches for elucidating ranavirus gene function in an infected fish cell line.

Viral virulence/immune evasion strategies and host anti-viral responses represent different sides of the continuing struggle between virus and host survival. To identify virus-encoding molecules whose function is to subvert or blunt host immune responses, we have adapted anti-sense approaches to knock down the expression of specific viral gene products. Our intention is to correlate knock down with loss of function and thus infer the role of a given viral gene. As a starting point in this process we have targeted several structural and catalytic genes using antisense morpholino oligonucleotides (asMO) and small, interfering RNAs (siRNA). In proof of concept experiments we show the feasibility of this approach and describe recent work targeting five frog virus 3 genes. Our results indicate that both 46K and 32R, two immediate-early viral proteins, are essential for replication in vitro, and confirm earlier findings that the major capsid protein, the largest subunit of the viral homolog of RNA polymerase II, and the viral DNA methyltransferase are also essential for replication in cell culture.

Activation of channel catfish (Ictalurus punctatus) T cells involves NFAT-like transcription factors

Cyclosporin A (CsA) specifically inhibits mammalian T cells by preventing activation of transcription factors (termed nuclear factor of activated T cells (NFAT)) involved in cytokine gene expression. In this study, catfish peripheral blood lymphocytes (PBL) and antigen specific T cells were treated with CsA to gain insights into the intracellular processes involved in fish T cell activation. To this end, CsA was observed to inhibit the in vitro proliferation of Con A stimulated catfish PBL, and specific alloantigen stimulated T cells. However, the inhibitory effect of CsA on catfish T cells was obviated by treatment with Con A, antigen activation or culture supernatant from activated catfish T cells prior to the addition of CsA. The use of a phosphatase assay coupled with Western blot analysis employing a polyclonal antibody to mammalian NFAT indicated that CsA prevents the dephosphorylation and subsequent nuclear translocation of an NFAT-like molecule in catfish T cells. Finally, a nuclear protein selection protocol demonstrated that a catfish NFAT-like protein binds to a known murine IL-2 promoter sequence. These results suggest that cytokines are involved in the activation of teleost T cells, and argue that T cell activation processes are conserved over a wide phylogenetic distance.

Expression profiles of cloned channel catfish (Ictalurus punctatus) lymphoid cell lines and mixed lymphocyte cultures.

Clonal channel catfish lymphoid cell lines and mixed lymphocyte cultures (MLCs) have proven extremely useful in examining immune responses at the cellular and molecular levels. To date clonal catfish cell lines and MLCs have been biologically and phenotypically characterized using a variety of techniques including reverse transcription polymerase chain reaction (RT-PCR), as well as Northern and Southern blotting. To expand the molecular characterization of these cultures, microarray analysis was employed. Clonal B (3B11), macrophage (42TA), and cytotoxic T cell (TS32.15 and TS32.17) lines and MLCs were examined using a cDNA array containing approximately 2500 probes derived from EST libraries prepared from the 42TA macrophage cell line, a MLC, and 5-14-day-old catfish fry. Analysis showed that each cell line displayed a unique RNA expression profile that included a variety of immune-related genes. Pearson correlation analysis indicated that one cytotoxic T cell line (TS32.15) clustered with the MLC, whereas a second cytotoxic T cell line (TS32.17) was more closely associated with a second cluster containing B cells and macrophages. This study illustrates the utility of microarray analyses in profiling RNA expression patterns in catfish lymphoid cell lines and will serve as a platform for examining catfish immune responses following virus infection or poly [I:C] treatment.