Genomic Sequencing of Ranavirus Isolates from a Three-Spined Stickleback () and a Red-Legged Frog ().

Two ranavirus isolates were recovered during a wildlife disease investigation in California in 1996. Preliminary testing at the time of sample collection indicated that the two isolates were identical. Phylogenetic analysis of the full genomes of these two isolates revealed that they are a single strain of frog virus 3.

Cloning and characterization of antiviral cytotoxic T lymphocytes in channel catfish, Ictalurus punctatus.

To determine the role of piscine anti-viral cytotoxic cells, we analyzed the response of channel catfish to Ictalurid herpesvirus 1, commonly designated channel catfish virus (CCV). Peripheral blood leukocytes (PBL) from catfish immunized with MHC-matched, CCV-infected G14D cells (G14D-CCV) showed marked lysis of G14D-CCV but little to no lysis of uninfected allogenic (3B11) or syngeneic (G14D) cells. Expansion of effectors by in vitro culture in the presence of irradiated G14D-CCV cells generated cultures with enhanced cytotoxicity and often broader target range.

Molecular and Ecological Studies of a Virus Family () Infecting Invertebrates and Ectothermic Vertebrates.

Research involving viruses within the family (generically designated iridovirids to distinguish members of the family from members of the genus ) has markedly increased in recent years [...

Genomic sequence of a Bohle iridovirus strain isolated from a diseased boreal toad (Anaxyrus boreas boreas) in a North American aquarium.

Genomic sequence analysis of zoo ranavirus (ZRV) suggests it is a strain of Bohle iridovirus (BIV), a virus that was first detected in, and thought to be confined to, Australia. Furthermore, marked sequence similarity and genomic co-linearity among ZRV, BIV, and German gecko ranavirus (GGRV) are consistent with the view that all three are strains  of Frog virus 3, the type species of the genus Ranavirus, family Iridoviridae.

Ranavirus phylogenomics: Signatures of recombination and inversions among bullfrog ranaculture isolates.

Ranaviruses are emerging pathogens of fish, amphibians, and reptiles that threaten aquatic animal industries and wildlife worldwide. Our objective was to genetically characterize ranaviruses isolated during separate bullfrog Lithobates catesbeianus die-offs that occurred eight years apart on the same North American farm. The earlier outbreak was due to a highly pathogenic strain of common midwife toad virus (CMTV) previously known only from Europe and China.

Ranaviruses and other members of the family Iridoviridae: Their place in the virosphere.

Members of the family Iridoviridae, collectively referred to as iridovirids, are large, double-stranded DNA-containing viruses that infect invertebrates and cold-blooded (ectothermic) vertebrates. Infections in the former often lead to massive levels of virus replication resulting in iridescence of the infected animal and ultimately death. Among the latter, infections target a variety of organs and are capable of causing high levels of morbidity and mortality among commercially and ecologically important fish and amphibian species.

ICTV Virus Taxonomy Profile: Iridoviridae.

The Iridoviridae is a family of large, icosahedral viruses with double-stranded DNA genomes ranging in size from 103 to 220 kbp. Members of the subfamily Alphairidovirinae infect ectothermic vertebrates (bony fish, amphibians and reptiles), whereas members of the subfamily Betairidovirinae mainly infect insects and crustaceans. Infections can be either covert or patent, and in vertebrates they can lead to high levels of mortality among commercially and ecologically important fish and amphibians.

Characterization of Frog Virus 3 knockout mutants lacking putative virulence genes.

To identify ranavirus virulence genes, we engineered Frog Virus 3 (FV3) knockout (KO) mutants defective for a putative viral caspase activation and recruitment domain-containing (CARD) protein (Δ64R-FV3) and a β-hydroxysteroid dehydrogenase homolog (Δ52L-FV3). Compared to wild type (WT) FV3, infection of Xenopus tadpoles with Δ64R- or Δ52L-FV3 resulted in significantly lower levels of mortality and viral replication. We further characterized these and two earlier KO mutants lacking the immediate-early18kDa protein (FV3-Δ18K) or the truncated viral homolog of eIF-2α (FV3-ΔvIF-2α).

Environmental dependency of amphibian-ranavirus genotypic interactions: evolutionary perspectives on infectious diseases.

The context-dependent investigations of host-pathogen genotypic interactions, where environmental factors are explicitly incorporated, allow the assessment of both coevolutionary history and contemporary ecological influences. Such a functional explanatory framework is particularly valuable for describing mortality trends and identifying drivers of disease risk more accurately. Using two common North American frog species (Lithobates pipiens and Lithobates sylvaticus) and three strains of frog virus 3 (FV3) at different temperatures, we conducted a laboratory experiment to investigate the influence of host species/genotype, ranavirus strains, temperature, and their interactions, in determining mortality and infection patterns.

Isolation of a Bohle-like iridovirus from boreal toads housed within a cosmopolitan aquarium collection.

A captive 'survival assurance' population of 56 endangered boreal toads Anaxyrus boreas boreas, housed within a cosmopolitan collection of amphibians originating from Southeast Asia and other locations, experienced high mortality (91%) in April to July 2010. Histological examination demonstrated lesions consistent with ranaviral disease, including multicentric necrosis of skin, kidney, liver, spleen, and hematopoietic tissue, vasculitis, and myriad basophilic intracytoplasmic inclusion bodies. Initial confirmation of ranavirus infection was made by Taqman real-time PCR analysis of a portion of the major capsid protein (MCP) gene and detection of iridovirus-like particles by transmission electron microscopy.

Differential transcription of fathead minnow immune-related genes following infection with frog virus 3, an emerging pathogen of ectothermic vertebrates.

Frog virus 3 (FV3) and other ranaviruses are responsible for die-offs involving wild, farmed, and captive amphibians, fish, and reptiles. To ascertain which elements of the immune system respond to infection, we explored transcriptional responses following infection of fathead minnow cells with either wild type (wt) FV3 or a knock out (KO) mutant targeting the 18 kDa immediate early gene (18K). At 8h post infection we observed marked upregulation of multiple transcripts encoding proteins affecting innate and acquired immunity.

The molecular biology of frog virus 3 and other iridoviruses infecting cold-blooded vertebrates.

Frog virus 3 (FV3) is the best characterized member of the family Iridoviridae. FV3 study has provided insights into the replication of other family members, and has served as a model of viral transcription, genome replication, and virus-mediated host-shutoff. Although the broad outlines of FV3 replication have been elucidated, the precise roles of most viral proteins remain unknown.

Ranavirus: past, present and future.

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.

Improved knockout methodology reveals that frog virus 3 mutants lacking either the 18K immediate-early gene or the truncated vIF-2alpha gene are defective for replication and growth in vivo.

To better assess the roles of frog virus 3 (FV3; genus Ranavirus, family Iridoviridae) genes in virulence and immune evasion, we have developed a reliable and efficient method to systematically knock out (KO) putative virulence genes by site-specific integration into the FV3 genome. Our approach utilizes a dual selection marker consisting of the puromycin resistance gene fused in frame with the enhanced green fluorescent protein (EGFP) reporter (Puro-EGFP cassette) under the control of the FV3 immediate-early (IE) 18K promoter. By successive rounds of selection for puromycin resistance and GFP expression, we have successfully constructed three recombinant viruses.

Characterization of a ranavirus inhibitor of the antiviral protein kinase PKR.

Background: Ranaviruses (family Iridoviridae) are important pathogens of lower vertebrates. However, little is known about how they circumvent the immune response of their hosts. Many ranaviruses contain a predicted protein, designated vIF2α, which shows homology with the eukaryotic translation initiation factor 2α.

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.

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.

Transcriptome analysis of Frog virus 3, the type species of the genus Ranavirus, family Iridoviridae.

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.

Identification and expression analyses of poly [I:C]-stimulated genes in channel catfish (Ictalurus punctatus).

Channel catfish (Ictalurus punctatus) have proven to be an excellent model with which to study immune responses of lower vertebrates. Identification of anti-viral antibodies and cytotoxic cells, as well as both type I and II interferon (IFN), demonstrates that catfish likely mount a vigorous anti-viral immune response. In this report, we focus on other elements of the anti-viral response, and identify more than two dozen genes that are induced following treatment of catfish cells with poly [I:C].

Family Iridoviridae: poor viral relations no longer.

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.

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.

Rana catesbeiana virus Z (RCV-Z): a novel pathogenic ranavirus.

A virus, designated Rana catesbeiana virus Z (RCV-Z), was isolated from the visceral tissue of moribund tadpoles of the North American bullfrog Rana catesbeiana. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis of viral proteins and sequence analysis of the amino terminal end of the major capsid protein showed that RCV-Z was similar to frog virus 3 (FV3) and other ranaviruses isolated from anurans and fish. However, analysis of restriction fragment profiles following digestion of viral genomic DNA with XbaI and BamHI indicated that RCV-Z was markedly different from FV3.

Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II.

Frog virus 3 (FV3) is a large DNA virus that encodes approximately 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication.