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

Virulent parasites cannot persist in small host populations unless the parasite also has a reservoir host. We hypothesize that, in hosts with complex life histories, one stage may act as an intraspecific reservoir for another. In amphibians, for example, larvae often occur at high densities, but these densities are ephemeral and fixed in space, whereas metamorphs are long-lived and vagile but may be very sparse. Parasite persistence is unlikely in either stage alone, but transmission between stages could maintain virulent parasites in seasonally fluctuating amphibian populations.We examined this hypothesis with a lethal ranavirus, Ambystoma tigrinum virus (ATV), that causes recurrent epidemics in larval tiger salamander populations, but which has no reservoir host and degrades quickly in the environment. Although exposure to ATV is generally lethal, larvae and metamorphs maintained sublethal, transmissible infections for Ͼ5 mo. Field data corroborate the persistence of ATV between epidemics in sublethally infected metamorphs. Three-quarters of dispersing metamorphs during one epidemic were infected, and apparently healthy metamorphs returning to breed harbored ATV infections. Our results suggest that larval epidemics amplify virus prevalence and sublethally infected metamorphs (re)introduce the virus into uninfected larval populations. Intraspecific reservoirs may explain the persistence of parasites in and declines of small, isolated amphibian populations.

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.

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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.

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 ...

Genes for 51.4- and 41.9-kDa insecticidal proteins of Bacillus sphaericus were separately cloned and expressed in Escherichia coli. Both proteins were required for toxicity. Approximately equal numbers of cells containing the 51.4- and 41.9-kDa proteins produced the greatest toxicity; excess 41.9-kDa protein did not affect toxicity, whereas excess 51.4-kDa protein reduced activity. Larvae were killed when 41.9-kDa protein was fed up to 24 h after the 51.4-kDa protein, but not when the order of feeding was reversed. Radiolabelled toxins bound in approximately equal amounts to the gastric caecum and posterior midgut of Culex quinquefasciatus larvae. Radiolabelled 51.4-kDa protein was rapidly degraded by ca. 12-13 kDa in the larval gut, while 41.9-kDa protein was degraded by 1-2 kDa. Nonreduced toxin extracted from B. sphaericus produced a band on SDS-PAGE of ca. 68-74 kDa that contained both 51.4- and 41.9-kDa proteins based on sequence analysis, and a band of ca. 51 kDa that contained primarily 41.9-kDa protein. Escherichia coli containing 51.4-kDa protein enhanced toxicity of the latter eluted SDS-PAGE band. These proteins may associate very strongly, and trace amounts of 51.4-kDa protein in preparations of 41.9-kDa protein from B. sphaericus may be responsible for the previously reported toxicity of the latter.

Ambystoma tigrinum virus (ATV) is a lethal virus originally isolated from Sonora tiger salamanders Ambystoma tigrinum stebbinsi in the San Rafael Valley in southern Arizona. USA. ATV is implicated in several salamander epizootics. We attempted to transmit ATV experimentally to fish and amphibians by injection, water bath exposure, or feeding to test whether ATV can cause clinical signs of infection or be recovered from exposed individuals that do not show clinical signs. Cell culture and polymerase chain reaction of the viral major capsid protein gene were used for viral detection. Salamanders and newts became infected with ATV and the virus was recovered from these animals, but virus could not be recovered from any of the frogs or fish tested. These results suggest that ATV may only infect urodeles and that fish and frogs may not be susceptible to ATV infection.