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1.
Biol Lett ; 8(4): 481-3, 2012 Aug 23.
Article in English | MEDLINE | ID: mdl-22048891

ABSTRACT

Emerging infectious diseases are a significant threat to global biodiversity. While historically overlooked, a group of iridoviruses in the genus Ranavirus has been responsible for die-offs in captive and wild amphibian, reptile and fish populations around the globe over the past two decades. In order to share contemporary information on ranaviruses and identify critical research directions, the First International Symposium on Ranaviruses was held in July 2011 in Minneapolis, MN, USA. Twenty-three scientists and veterinarians from nine countries examined the ecology and evolution of ranavirus-host interactions, potential reservoirs, transmission dynamics, as well as immunological and histopathological responses to infection. In addition, speakers discussed possible mechanisms for die-offs, and conservation strategies to control outbreaks.


Subject(s)
DNA Virus Infections/transmission , DNA Virus Infections/veterinary , Host-Pathogen Interactions , Ranavirus/pathogenicity , Amphibians/virology , Animals , Communicable Diseases, Emerging/transmission , Congresses as Topic , DNA Virus Infections/virology , Disease Vectors , Ecosystem , Fish Diseases/transmission , Fish Diseases/virology , Minnesota , Reptiles/virology
2.
Dev Comp Immunol ; 35(9): 937-48, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21147160

ABSTRACT

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.


Subject(s)
Capsid Proteins/metabolism , DNA Modification Methylases/metabolism , DNA Virus Infections/virology , Ranavirus/physiology , Viral Proteins/metabolism , Animals , Capsid Proteins/genetics , Capsid Proteins/immunology , Cell Line , DNA Modification Methylases/genetics , DNA Modification Methylases/immunology , DNA Virus Infections/immunology , Fishes , Gene Knockdown Techniques , Host-Pathogen Interactions , Immune Evasion , Oligonucleotides, Antisense/genetics , RNA, Small Interfering/genetics , Ranavirus/pathogenicity , Viral Proteins/genetics , Viral Proteins/immunology , Virus Replication
3.
Virology ; 391(2): 293-303, 2009 Sep 01.
Article in English | MEDLINE | ID: mdl-19608212

ABSTRACT

Frog virus 3 is the best characterized species within the genus Ranavirus, family Iridoviridae. FV3's large ( approximately 105 kbp) dsDNA genome encodes 98 putative open reading frames (ORFs) that are expressed in a coordinated fashion leading to the sequential appearance of immediate early (IE), delayed early (DE) and late (L) viral transcripts. As a step toward elucidating molecular events in FV3 replication, we sought to identify the temporal class of viral messages. To accomplish this objective an oligonucleotide microarray containing 70-mer probes corresponding to each of the 98 FV3 ORFs was designed and used to examine viral gene expression. Viral transcription was initially monitored during the course of a productive replication cycle at 2, 4 and 9 h after infection. To confirm results of the time course assay, viral gene expression was also monitored in the presence of cycloheximide (CHX), which limits expression to only IE genes, and following infection with a temperature-sensitive (ts) mutant which at non-permissive temperatures is defective in viral DNA synthesis and blocked in late gene expression. Subsequently, microarray analyses were validated by RT-PCR and qRT-PCR. Using these approaches we identified 33 IE genes, 22 DE genes and 36 L viral genes. The temporal class of the 7 remaining genes could not be determined. Comparison of protein function with temporal class indicated that, in general, genes encoding putative regulatory factors, or proteins that played a part in nucleic acid metabolism and immune evasion, were classified as IE and DE genes, whereas those involved in DNA packaging and virion assembly were considered L genes. Information on temporal class will provide the basis for determining whether members of the same temporal class contain common upstream regulatory regions and perhaps allow us to identify virion-associated and virus-induced proteins that control viral gene expression.


Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Viral , Ranavirus/physiology , Animals , Cell Line , Cyprinidae , Genes, Viral , Oligonucleotide Array Sequence Analysis/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Time Factors
4.
Curr Top Microbiol Immunol ; 328: 123-70, 2009.
Article in English | MEDLINE | ID: mdl-19216437

ABSTRACT

Members of the family Iridoviridae infect a diverse array of invertebrate and cold-blooded vertebrate hosts and are currently viewed as emerging pathogens of fish and amphibians. Iridovirid replication is unique and involves both nuclear and cytoplasmic compartments, a circularly permuted, terminally redundant genome that, in the case of vertebrate iridoviruses, is also highly methylated, and the efficient shutoff of host macromolecular synthesis. Although initially neglected largely due to the perceived lack of health, environmental, and economic concerns, members of the genus Ranavirus, and the newly recognized genus Megalocytivirus, are rapidly attracting growing interest due to their involvement in amphibian population declines and their adverse impacts on aquaculture. Herein we describe the molecular and genetic basis of viral replication, pathogenesis, and immunity, and discuss viral ecology with reference to members from each of the invertebrate and vertebrate genera.


Subject(s)
DNA Virus Infections/veterinary , Iridoviridae/physiology , Amphibians/virology , Animals , DNA Virus Infections/virology , Fish Diseases/virology
5.
Dev Comp Immunol ; 33(2): 224-34, 2009 Feb.
Article in English | MEDLINE | ID: mdl-18824023

ABSTRACT

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.


Subject(s)
Catfishes/immunology , Catfishes/metabolism , Lymphocytes/immunology , Lymphocytes/metabolism , Animals , Catfishes/genetics , Cell Line , Cell Separation , Gene Expression Profiling , Lymphocyte Culture Test, Mixed , Oligonucleotide Array Sequence Analysis , Phenotype
6.
Immunogenetics ; 58(1): 70-80, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16467986

ABSTRACT

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-gamma genes are dissimilar in sequence but closely linked on the same chromosome. One of the genes (IFN-gamma2) 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-gamma genes contain the hallmarks of authentic IFN-gamma including: (1) a conserved nuclear localization site at the C terminus (CF IFN-gamma2 only), (2) an IFN-gamma 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-gamma transcripts. This is the first unequivocal demonstration in any lower vertebrate species that NK and T cells synthesize IFN-gamma 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-gamma genes and demonstrate that CF possess three key elements of the innate immune response: NK cells and types I and II IFN.


Subject(s)
Ictaluridae/genetics , Interferon-gamma/blood , Interferon-gamma/genetics , Lymphocytes/metabolism , RNA Splice Sites , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Ictaluridae/immunology , Interferon-gamma/chemistry , Interferon-gamma/immunology , Molecular Sequence Data , Phylogeny , Sequence Homology, Nucleic Acid
7.
Fish Shellfish Immunol ; 21(1): 42-59, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16426862

ABSTRACT

Previously a cDNA encoding a putative interferon gene, designated CF IFN-1, was identified from a catfish EST library. However, its constitutive expression, absence of a signal peptide, and apparently low level of biological activity suggested that this cDNA likely encoded an expressed pseudogene. Since Southern blot analysis suggested the presence of two to three IFN genes, additional cDNAs were generated from catfish fibroblast and lymphoid cell lines using primers designed to conserved regions of zebrafish and catfish interferon. Using this approach, three novel CF IFN genes, two of which likely encode functional interferon molecules, were identified. At the amino acid level, similarity among CF IFNs ranged from 71% to 82%, whereas similarity to other fish IFNs ranged from 15% to 35%. Although CF IFN-3, like CF IFN-1, lacks a signal peptide, CF IFN-2 and -4 appear to encode full-length, signal sequence-bearing genes. Consistent with their putative identification as functional genes, CF IFN-2 and -4 were not expressed in unstimulated cell lines, and CF IFN-2 was rapidly upregulated in CCO cells in response to virus infection or treatment with dsRNA. Moreover, as with salmon, fugu, and zebrafish interferon genes, CF IFN-1 contained four introns whose locations were conserved not only with respect to other fish IFNs, but also with respect to mammalian IFN-lambda. While it is likely that CF IFNs represent Type I IFNs, several characteristics preclude assigning these cytokines to any particular subfamily.


Subject(s)
DNA, Complementary/isolation & purification , Ictaluridae/genetics , Ictaluridae/immunology , Interferon Type I/genetics , Amino Acid Sequence , Animals , Base Sequence/genetics , Cell Line , DNA Primers/chemistry , DNA, Complementary/chemistry , DNA, Complementary/physiology , Exons , Gene Expression Profiling/veterinary , Gene Order , Interferon Type I/biosynthesis , Interferon Type I/chemistry , Interferon Type I/immunology , Introns , Molecular Sequence Data , Phylogeny , Reoviridae/immunology , Reoviridae/physiology , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Sequence Alignment/veterinary , Sequence Homology, Amino Acid
8.
Mol Ecol ; 14(1): 213-24, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15643965

ABSTRACT

Our understanding of origins and spread of emerging infectious diseases has increased dramatically because of recent applications of phylogenetic theory. Iridoviruses are emerging pathogens that cause global amphibian epizootics, including tiger salamander (Ambystoma tigrinum) die-offs throughout western North America. To explain phylogeographical relationships and potential causes for emergence of western North American salamander iridovirus strains, we sequenced major capsid protein and DNA methyltransferase genes, as well as two noncoding regions from 18 geographically widespread isolates. Phylogenetic analyses of sequence data from the capsid protein gene showed shallow genetic divergence (< 1%) among salamander iridovirus strains and monophyly relative to available fish, reptile, and other amphibian iridovirus strains from the genus Ranavirus, suggesting a single introduction and radiation. Analysis of capsid protein sequences also provided support for a closer relationship of tiger salamander virus strains to those isolated from sport fish (e.g. rainbow trout) than other amphibian isolates. Despite monophyly based on capsid protein sequences, there was low genetic divergence among all strains (< 1.1%) based on a supergene analysis of the capsid protein and the two noncoding regions. These analyses also showed polyphyly of strains from Arizona and Colorado, suggesting recent spread. Nested clade analyses indicated both range expansion and long-distance colonization in clades containing virus strains isolated from bait salamanders and the Indiana University axolotl (Ambystoma mexicanum) colony. Human enhancement of viral movement is a mechanism consistent with these results. These findings suggest North American salamander ranaviruses cause emerging disease, as evidenced by apparent recent spread over a broad geographical area.


Subject(s)
Iridoviridae/isolation & purification , Urodela/virology , Animals , Canada , Capsid Proteins/genetics , Demography , Humans , Iridoviridae/classification , Iridoviridae/genetics , Iridoviridae/pathogenicity , Phylogeny , United States , Virus Diseases/veterinary
9.
Virology ; 323(2): 268-75, 2004 Jun 01.
Article in English | MEDLINE | ID: mdl-15193922

ABSTRACT

The ability of five purified amphibian antimicrobial peptides (dermaseptin-1, temporin A, magainin I, and II, PGLa), crude peptide fractions isolated from the skin of Rana pipiens and R. catesbeiana, and four antimicrobial peptides (AMPs) from hybrid striped bass (piscidin-1N, -1H, -2, and -3) were examined for their ability to reduce the infectivity of channel catfish virus (CCV) and frog virus 3 (FV3). All compounds, with the exception of magainin I, markedly reduced the infectivity of CCV. In contrast to CCV, FV3 was 2- to 4-fold less sensitive to these agents. Similar to an earlier study employing two other amphibian peptides, the agents used here acted rapidly and over a wide, physiologically relevant, temperature range to reduce virus infectivity. These results extend our previous findings and strongly suggest that various amphibian and piscine AMPs may play important roles in protecting fish and amphibians from pathogenic viruses.


Subject(s)
Amphibian Proteins/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Antiviral Agents/pharmacology , Herpesviridae/pathogenicity , Ranavirus/pathogenicity , Virus Inactivation , Amino Acid Sequence , Amphibian Proteins/chemistry , Animals , Antimicrobial Cationic Peptides/chemistry , Antiviral Agents/chemistry , Bass , Cells, Cultured , Drug Synergism , Herpesviridae/drug effects , Ictaluridae/virology , Molecular Sequence Data , Rana catesbeiana , Rana pipiens , Ranavirus/drug effects , Skin/chemistry
10.
Dev Comp Immunol ; 28(2): 97-111, 2004 Feb.
Article in English | MEDLINE | ID: mdl-12969796

ABSTRACT

Despite considerable advances in our understanding of teleost immunity, relatively few cytokine genes, including those for interferon (IFN), have been identified at the molecular level. In contrast, numerous studies have shown that following virus infection or exposure to double-stranded RNA, fish or fish cells produce a soluble factor that is functionally similar to mammalian IFN. A putative catfish (CF) IFN cDNA was identified by BLASTX screening of a catfish EST library generated from a mixed lymphocyte culture enriched for NK-like cells. Consistent with its designation as a putative cytokine cDNA, the 3' non-translated region contained multiple copies of an RNA instability motif. Analysis of the deduced amino acid sequence of CF IFN showed low levels of identity/similarity to a panel of mammalian and avian IFN proteins, and markedly higher similarity to a recently identified zebrafish IFN. To determine if the identified cDNA encoded CF IFN, expression was monitored following infection of channel catfish ovary (CCO) cells with UV-inactivated catfish reovirus or exposure to double-stranded RNA, treatments which induce IFN or IFN-like activity in catfish and other species. In both cases, upregulation of putative CF IFN mRNA was detected. Moreover, upregulation of CF IFN mRNA was accompanied by the appearance of an antiviral factor in the culture medium. To confirm these results, recombinant CF IFN was synthesized in COS-7 cells and shown to have antiviral activity in CCO cells. Collectively, these results argue strongly that the identified catfish cDNA is an IFN homolog.


Subject(s)
DNA, Complementary/genetics , Ictaluridae/genetics , Interferons/genetics , Phylogeny , Amino Acid Sequence , Animals , Base Sequence , COS Cells , Cells, Cultured , Chlorocebus aethiops , Expressed Sequence Tags , Female , Ictaluridae/immunology , Interferons/immunology , Molecular Sequence Data , Ovary/metabolism , RNA, Double-Stranded/immunology , Reoviridae Infections/immunology , Sequence Homology, Amino Acid , T-Lymphocytes/immunology , Up-Regulation
11.
Dev Comp Immunol ; 27(10): 845-58, 2003 Dec.
Article in English | MEDLINE | ID: mdl-12880635

ABSTRACT

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.


Subject(s)
Gene Expression Profiling , Ictaluridae/genetics , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cloning, Molecular , Humans , Ictaluridae/metabolism , Molecular Sequence Data , Phylogeny , RNA/genetics , RNA/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Tumor Necrosis Factor-alpha/chemistry
12.
Virology ; 306(2): 303-12, 2003 Feb 15.
Article in English | MEDLINE | ID: mdl-12642103

ABSTRACT

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.


Subject(s)
Apoptosis , RNA Virus Infections/pathology , Ranavirus/pathogenicity , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Apoptosis/drug effects , Apoptosis/physiology , Caspase Inhibitors , Cell Line , Cricetinae , Cyprinidae , Cysteine Proteinase Inhibitors/pharmacology , DNA Fragmentation , Gene Expression , Genes, Viral , Models, Biological , Osmotic Pressure , RNA Virus Infections/metabolism , RNA Virus Infections/virology , Ranavirus/genetics , Ranavirus/physiology , Virus Replication
13.
Dev Comp Immunol ; 26(9): 775-84, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12377217

ABSTRACT

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.


Subject(s)
DNA-Binding Proteins/immunology , Ictaluridae/immunology , Nuclear Proteins , T-Lymphocytes/immunology , Transcription Factors/immunology , Animals , Blotting, Western , Calcimycin/immunology , Concanavalin A/immunology , Cyclosporine/pharmacology , DNA-Binding Proteins/antagonists & inhibitors , Ictaluridae/metabolism , Immunosuppressive Agents/pharmacology , Lipopolysaccharides/immunology , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , NFATC Transcription Factors , Phosphoric Monoester Hydrolases/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Tetradecanoylphorbol Acetate/immunology , Thymidine/metabolism , Transcription Factors/antagonists & inhibitors
14.
Arch Virol ; 147(3): 447-70, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11958449

ABSTRACT

Although possessing novel replicative and structural features, the family Iridoviridae has not been as extensively studied as other families of large, DNA-containing viruses (e.g., poxviridae and herpesviridae). This oversight most likely reflects the inability of iridoviruses to infect mammals and birds, and their heretofore low pathogenicity among cold-blooded animals and invertebrates. In fact, the original frog virus isolates (e.g., frog viruses 1-3) would likely have been considered orphan viruses since they were isolated from apparently healthy frogs. However, recent disease outbreaks among commercially and recreationally important fish, cultured and wild frogs, and endangered salamanders has challenged this benign view and have implicated several members of the genus Ranavirus as pathogens. This review explores three facets of ranavirus biology. In the first the salient features of ranavirus replication are summarized using frog virus 3 as a model. Secondly, criteria for characterizing new ranavirus isolates, based on biochemical (viral protein profiles, DNA restriction fragment length polymorphisms, and nucleotide sequence analysis), ecological (host range, tissue tropism), and clinical considerations, are detailed. Lastly, the principal agents of ranavirus-mediated disease and immune responses to these viruses are discussed. In light of the above, it is clear that ranaviruses are no longer orphan viruses, and that they have a significant impact on diverse populations of ectothermic animals.


Subject(s)
Amphibians/virology , Fish Diseases/virology , RNA Virus Infections/veterinary , Ranavirus , Animals , RNA Virus Infections/virology , Ranavirus/genetics , Ranavirus/pathogenicity , Ranavirus/physiology
15.
Virology ; 288(2): 351-7, 2001 Sep 30.
Article in English | MEDLINE | ID: mdl-11601906

ABSTRACT

While it is clear that some amphibian populations have recently experienced precipitous declines, the causes of those die-offs are complex and likely involve multiple variables. One theory suggests that environmental factors may trigger events that result in depressed immune function and increased susceptibility to infectious disease. Here we examine one aspect of innate immunity in amphibians and show that esculentin-2P (E2P) and ranatuerin-2P (R2P), two antimicrobial peptides isolated from Rana pipiens, inactivate frog virus 3, a potentially pathogenic iridovirus infecting anurans, and channel catfish herpesvirus. In contrast to mammalian antimicrobial peptides, E2P and R2P act within minutes, at temperatures as low as 0 degrees C, to inhibit viral infectivity. Moreover, these compounds appear to inactivate the virus directly and do not act by inhibiting replication in infected cells. This is the first report linking amphibian antimicrobial peptides with protection from an amphibian viral pathogen and suggests that these compounds may play a role in maintaining amphibian health.


Subject(s)
Amphibian Proteins , Antimicrobial Cationic Peptides/pharmacology , Antiviral Agents/pharmacology , Herpesviridae/drug effects , Ictaluridae/virology , Proteins/pharmacology , Ranavirus/drug effects , Animals , Cell Line , Herpesviridae/growth & development , Herpesviridae/ultrastructure , Microscopy, Electron , Peptides/pharmacology , Rana pipiens , Ranavirus/growth & development , Ranavirus/ultrastructure , Skin/metabolism
16.
J Immunol ; 167(3): 1325-32, 2001 Aug 01.
Article in English | MEDLINE | ID: mdl-11466349

ABSTRACT

Two types of catfish alloantigen-dependent cytotoxic T cells were cloned from PBL from a fish immunized in vivo and stimulated in vitro with the allogeneic B cell line 3B11. Because these are the first clonal cytotoxic T cell lines derived from an ectothermic vertebrate, studies were undertaken to characterize their recognition and cytotoxic mechanisms. The first type of CTL (group I) shows strict alloantigen specificity, i.e., they specifically kill and proliferate only in response to 3B11 cells. The second type (group II) shows broad allogeneic specificity, i.e., they kill and proliferate in response to several different allogeneic cells in addition to 3B11. "Cold" target-inhibition studies suggest that group II CTL recognize their targets via a single receptor, because the killing of one allotarget can be inhibited by a different allotarget. Both types of catfish CTL form conjugates with and kill targets by apoptosis. Killing by Ag-specific cytotoxic T cells (group I) was completely inhibited by treatment with EGTA or concanamycin A, and this killing is sensitive to PMSF inhibition, suggesting that killing was mediated exclusively by the secretory perforin/granzyme mechanism. In contrast, killing by the broadly specific T cytotoxic cells (group II) was only partially inhibited by either EGTA or concanamycin A, suggesting that these cells use a cytotoxic mechanism in addition to that involving perforin/granzyme. Consistent with the presumed use of a secretory pathway, both groups of CTL possess putative lytic granules. These results suggest that catfish CTL show heterogeneity with respect to target recognition and cytotoxic mechanisms.


Subject(s)
Cytotoxicity Tests, Immunologic/methods , Cytotoxicity, Immunologic/immunology , Ictaluridae/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Apoptosis/immunology , Cell Line , Clone Cells , Exocytosis/immunology , Lymphocyte Activation/immunology , T-Lymphocytes, Cytotoxic/enzymology , T-Lymphocytes, Cytotoxic/metabolism , T-Lymphocytes, Cytotoxic/ultrastructure
17.
Dev Comp Immunol ; 25(7): 579-95, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11472780

ABSTRACT

Two clones, designated Icpu-UA/3 and Icpu-UA/26, were isolated from a genomic library prepared from a single homozygous gynogenetic channel catfish. Sequence analysis showed that each clone encoded a gene product containing features conserved among MHC class I molecules. The genomic organization of both clones indicated that each domain, with the exception of the cytoplasmic, was encoded by a separate exon. Moreover, like mammals, catfish cytoplasmic regions were encoded by three exons rather than two as previously described for other teleost MHC class I genes. Analysis of nucleotide sequences upstream of catfish class I genes revealed the presence of several regulatory motifs similar to those seen in mammalian class I genes. These included a TATA box, Enhancer B, Site alpha, ISRE, and GAS elements. To determine the functional significance of these elements, EMSAs and tissue expression assays were performed. EMSAs demonstrated that an Enhancer B element within Icpu-UA/26, and an imperfect Enhancer B element and/or a GC-rich region within Icpu-UA/3 were responsible for formation of specific DNA/protein complexes. Expression studies detected Icpu-UA/26 transcripts in all tissues tested, whereas Icpu-UA/3 encoded messages were seen in a limited number of tissues. These results define the intron/exon organization of catfish MHC class I genes, suggest that Icpu-UA/3 encodes a nonclassical gene, and provide the first functional evidence that upstream sequences, similar to those seen in mammalian class I genes, play important roles in regulating teleost MHC gene expression.


Subject(s)
Genes, MHC Class I , Ictaluridae/genetics , Ictaluridae/immunology , Amino Acid Sequence , Animals , Base Sequence , DNA-Binding Proteins/isolation & purification , Gene Expression Regulation , Genomic Library , Molecular Sequence Data , Nuclear Proteins/isolation & purification , Regulatory Sequences, Nucleic Acid , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Tissue Distribution
18.
Dis Aquat Organ ; 48(1): 27-36, 2001 Dec 20.
Article in English | MEDLINE | ID: mdl-11843137

ABSTRACT

Three virus isolates, RGV-9506, RGV-9807 and RGV-9808, were obtained from cultured pig frogs Rana grylio undergoing lethal infections. Previously, the first isolate, RGV-9506, was shown to be an iridovirus based on ultrastructural and morphological studies. In the present study, the original isolate, along with 2 recent ones, were more extensively characterized by experimental infection studies, histopathology, electron microscopy, serological reactivity, gel electrophoresis of viral polypeptides and DNA restriction fragments, PCR amplification, and nucleic acid sequence analysis of the major capsid protein (MCP) gene. The 3 isolates were shown to be identical to each other, and very similar to FV3, the type species of the genus Ranavirus (family Iridoviridae). These results suggest that RGV should be considered a strain of FV3, and indicate that FV3-like iridoviruses are capable of causing widespread, severe disease among cultured frogs.


Subject(s)
DNA Virus Infections/veterinary , Iridovirus/classification , Ranidae/virology , Amino Acid Sequence , Animals , Aquaculture , Base Sequence , DNA Restriction Enzymes , DNA Virus Infections/mortality , DNA Virus Infections/pathology , Immunodiffusion/veterinary , Iridovirus/genetics , Iridovirus/isolation & purification , Iridovirus/pathogenicity , Molecular Sequence Data , Neutralization Tests/veterinary , Phylogeny , Polymerase Chain Reaction/veterinary , Sequence Alignment , Sequence Analysis, DNA
19.
Virus Res ; 63(1-2): 45-52, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10509715

ABSTRACT

Iridoviruses infect invertebrates (primarily insects and crustaceans) and ectothermic vertebrates (fish, amphibians, and reptiles). Identical, or nearly identical viruses, have been isolated from different animals within the same taxonomic class, indicating that infection by a given virus is not limited to a single species. Although inter-class infections have been documented following experimental infection with vertebrate iridoviruses, it is not clear whether such infections occur in nature. Here we report the isolation of apparently identical iridoviruses from wild sympatric fish (the threespine stickleback, Gasterostelus aculeatus) and amphibians (the red-legged frog, Rana aurora). Viruses isolated from sticklebacks (stickleback virus, SBV) and from a red-legged frog tadpole (tadpole virus 2, TV2) replicated in fathead minnow (FHM) cells and synthesized proteins which co-migrated with those of frog virus 3 (FV3). Following restriction endonuclease digestion of viral DNA with Hind III and Xba I, gel analysis showed that the profiles of SBV and TV2 were identical to each other and distinct from FV3. Using oligonucleotide primers specific for a highly conserved region of the iridovirus major capsid protein, an approximately 500 nucleotide DNA fragment was amplified from SBV and TV2. Sequence analysis showed that within this 500 nucleotide region SBV and TV2 were identical to each other and to FV3. Taken together these results provide the first evidence that iridoviruses naturally infect animals belonging to different taxonomic classes, and strengthen the suggestion that fish may serve as a reservoir for amphibian viruses or vice versa.


Subject(s)
Fish Diseases/virology , Fishes/virology , Iridoviridae/genetics , Nucleocapsid Proteins/chemistry , RNA Virus Infections/veterinary , Ranavirus/genetics , Ranidae/virology , Animals , California , Cell Line , Iridoviridae/chemistry , Iridoviridae/isolation & purification , Nucleocapsid Proteins/biosynthesis , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , RNA Virus Infections/virology , Ranavirus/isolation & purification , Ranavirus/metabolism , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid
20.
Dis Aquat Organ ; 37(2): 107-14, 1999 Jul 30.
Article in English | MEDLINE | ID: mdl-10494500

ABSTRACT

An iridovirus, isolated from largemouth bass Micropterus salmoides following a die-off among adult fish and provisionally designated largemouth bass virus (LMBV), was characterized by analysis of viral protein synthesis in infected cells, viral DNA restriction fragment length polymorphisms (RFLP), and sequence determination of the major capsid protein and viral DNA methyltransferase genes. All 3 approaches yielded results consistent with the suggestion that LMBV was a member of the genus Ranavirus. Moreover, LMBV was nearly identical to 2 isolates from Southeast Asia which had been previously detected in imported ornamental fish. It remains to be determined whether infection of largemouth bass resulted from exposure to an imported virus, or whether the presence of similar viruses in southeast Asia and the southeastern United States indicates that iridovirus species are not geographically limited as suggested earlier, but rather globally distributed.


Subject(s)
Bass , DNA, Viral/analysis , Fish Diseases/virology , RNA Virus Infections/veterinary , Ranavirus/genetics , Amino Acid Sequence , Animals , Base Sequence , Capsid/chemistry , Capsid/genetics , Cell Line , DNA Modification Methylases/genetics , DNA, Viral/chemistry , Fishes , Molecular Sequence Data , Polymorphism, Restriction Fragment Length , RNA Virus Infections/virology , Ranavirus/classification , Ranavirus/isolation & purification , Sequence Alignment/veterinary , Viral Proteins/biosynthesis , Viral Proteins/chemistry
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