The Sonora tlger salamander Ambystoma tignnuni stebb~nsi Lowe is a genetically d~stinct race restricted to about 30 small ponds in the San Rafael Valley 111 southern Arizona, USA, which was added recently to the USA Federal List of Endangered Species Populations of these salamanders periodically expenence decimating epizootics. Virus was ~solated from diseased salamanders using fish cell cultures, injected into healthy laboratory-reared salamanders, and then reisolated in cell culture. Electron microscopy of thin sections from dying salamanders revealed abundant enveloped and nonenveloped icosahedral virus particles approximately 160 to 180 nm in diameter in the cytoplasm of skin and liver cells and free in the intercellular spaces. This virus, believed to be an iridovirus based on viral morphology and host pathology, was demonstrated to be the primary pathogen in these epizootics, and is the first lethal epizootic virus reported from salamanders. We have named the virus Ambystoma tigrinum Virus (ATV). Hemolytic bacteria were isolated from sick individuals, but we were unable to induce the disease by exposing salamanders to isolated bacteria at concentrations up to 10' ml-' KEY WORDS: Salamander. Ambystoma tigrinum stebbinsi. Virus. Indovirus. Amphibian decline

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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. This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Iridoviridae, which is available at www.ictv.global/report/iridoviridae.

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.

Members of the genus Ranavirus (family Iridoviridae) have been recognized as major viral pathogens of cold-blooded vertebrates. Ranaviruses have been associated with amphibians, fish, and reptiles. At this time, the relationships between ranavirus species are still unclear. Previous studies suggested that ranaviruses from salamanders are more closely related to ranaviruses from fish than they are to ranaviruses from other amphibians, such as frogs. Therefore, to gain a better understanding of the relationships among ranavirus isolates, the genome of epizootic hematopoietic necrosis virus (EHNV), an Australian fish pathogen, was sequenced. Our findings suggest that the ancestral ranavirus was a fish virus and that several recent host shifts have taken place, with subsequent speciation of viruses in their new hosts. The data suggesting several recent host shifts among ranavirus species increase concern that these pathogens of cold-blooded vertebrates may have the capacity to cross numerous poikilothermic species barriers and the potential to cause devastating disease in their new hosts.

Disease is among the suspected causes of amphibian population declines, and an iridovirus and a chytrid fungus are the primary pathogens associated with amphibian mortalities. Ambystoma tigrinum virus (ATV) and a closely related strain, Regina ranavirus (RRV), are implicated in salamander die-offs in Arizona and Canada, respectively. We report the complete sequence of the ATV genome and partial sequence of the RRV genome. Sequence analysis of the ATV/RRV genomes showed marked similarity to other ranaviruses, including tiger frog virus (TFV) and frog virus 3 (FV3), the type virus of the genus Ranavirus (family Iridoviridae), as well as more distant relationships to lymphocystis disease virus, Chilo iridescent virus, and infectious spleen and kidney necrosis virus. Putative open reading frames (ORFs) in the ATV sequence identified 24 genes that appear to control virus replication and block antiviral responses. In addition, >50 other putative genes, homologous to ORFs in other iridoviral genomes but of unknown function, were also identified. Sequence comparison performed by dot plot analysis between ATV and itself revealed a conserved 14-bp palindromic repeat within most intragenic regions. Dot plot analysis of ATV vs RRV sequences identified several polymorphisms between the two isolates. Finally, a comparison of ATV and TFV genomic sequences identified genomic rearrangements consistent with the high recombination frequency of iridoviruses. Given the adverse effects that ranavirus infections have on amphibian and fish populations, ATV/RRV sequence information will allow the design of better diagnostic probes for identifying ranavirus infections and extend our understanding of molecular events in ranavirus-infected cells.

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. Current studies using knock down (KD) mediated by antisense morpholino oligonucleotides (asMO) and small, interfering RNAs (siRNA), knock out (KO) following replacement of the targeted gene with a selectable marker by homologous recombination, ectopic viral gene expression, and recombinant viral proteins have enabled researchers to systematically ascertain replicative- and virulence-related gene functions. In addition, the application of molecular tools to ecological studies is providing novel ways for field biologists to identify potential pathogens, quantify infections, and trace the evolution of ecologically important viral species. In this review, we summarize current studies using not only FV3, but also other iridoviruses infecting ectotherms. As described below, general principles ascertained using FV3 served as a model for the family, and studies utilizing other ranaviruses and megalocytiviruses have confirmed and extended our understanding of iridovirus replication. Collectively, these and future efforts will elucidate molecular events in viral replication, intrinsic and extrinsic factors that contribute to disease outbreaks, and the role of the host immune system in protection from disease.

Temperature strongly influenced percent mortality and time to death of salamanders exposed to the Ambystoma tigrinum virus (iridovirus) (ATV). Most salamanders survived when exposed at 26°C, whereas all died at 18°C and nearly all died at 10°C. Some asymptomatic salamanders that survived 60 d at 10 or 26°C were found to be carrying virus. Polymerase chain reaction (PCR) confirmed the presence of virus in ATV-exposed salamanders but was found to be less sensitive than cell culture in detecting ATV at low concentrations. PCR products were 100% identical to ATV in the major capsid protein sequence. Virus titer was higher in salamanders held at 10°C than at 18°C but little virus, if any, was present in the small number of salamanders that died at 26°C. These results may help explain periodic viral epizootics in field populations of A. tigrinum where water temperatures fluctuate widely. KEY WORDS: Epizootic · Cell culture · Iridovirus · Mortality Resale or republication not permitted without written consent of the publisherDis Aquat Org 63: [95][96][97][98][99][100] 2005 MATERIALS AND METHODSGeneral methods. ATV was cultured using epithelioma papilloma cyprini (EPC) cells (Fijan et al. 1983). Plaque and TCID 50 (tissue culture infectious dose 50 ; Reed & Muench 1938) assays employing EPC cells were used to estimate the concentration of virus used in each experiment. Three to 4 mo old Ambystoma tigrinum nebulosum larvae originated from stock held in the laboratory for at least 2 generations. No mortality due to possible virus infection was observed in the rearing facility during this period. During the experiments, each salamander was held individually in 300 ml of water in a Zip Lock ® (S. C. Johnson) container and water was changed weekly. Salamanders were fed brine shrimp Artemia sp. 3 times a week and observed daily. Salamanders that died were stored at -70°C. At the end of each experiment, body wall samples were taken from all dead larvae and tail clips from surviving salamanders for virus detection. Each sample was placed in a Stomacher ® (Seward) bag containing 2 ml of Eagle's Minimum Essential Medium (MEM) + 2% Fetal Bovine Serum (FBS) + penicillin-streptomycin-neomycin (PSN; Sigma) + diatomaceous earth powder, and homogenized in a Stomacher device. The homogenate was centrifuged at 9000 × g for 10 min and the supernatant was stored at -80°C. Supernatant (100 µl) was inoculated onto EPC cells in each well of 12-or 24-well plates, the preparation was rocked for 1 h and then 1 ml of MEM + 10% FBS + PSN was added. Cells were incubated at 22°C under 5% CO 2 and observed for 2 wk for cytopathic effect (CPE). Samples showing no CPE were passed through 2 further sets of EPC cells to confirm the presence or absence of virus.To determine the multiplication of virus at the 3 experimental temperatures, replicate EPC cultures in 24-well plates were inoculated with dilutions of ATV and held at 10, 18 or 26°C for 10 to 16 d. Development of CPE was observed and TCID 50 was calculated during incubation.In order ...