Influence of temperature on Ranavirus infection in larval salamanders Ambystoma tigrinum

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 degrees C, whereas all died at 18 degrees C and nearly all died at 10 degrees C. Some asymptomatic salamanders that survived 60 d at 10 or 26 degrees 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 degrees C than at 18 degrees C but little virus, if any, was present in the small number of salamanders that died at 26 degrees C. These results may help explain periodic viral epizootics in field populations of A. tigrinum where water temperatures fluctuate widely.     

Antimicrobial peptide defenses in the salamander, Ambystoma tigrinum, against emerging amphibian pathogens

Skin peptides were collected from living Ambystoma tigrinum larvae and adults and tested against two emerging pathogens, Batrachochytrium dendrobatidis and the Ambystoma tigrinum virus (ATV), as well as bacteria isolated from A. tigrinum. Natural mixtures of skin peptides were found to inhibit growth of B. dendrobatidis, Staphylococcus aureus, and Klebsiella sp., but activity against ATV was unpredictable. Skin peptides collected from salamanders held at three environmentally relevant temperatures differed in activity against B. dendrobatidis. Activity of the A. tigrinum skin peptides was found to be strongly influenced by pH.     

Transmission dynamics of the amphibian ranavirus Ambystoma tigrinum virus

Transmission is central to pathogen fitness and strongly influences the impact of pathogens on host populations. Particularly important to transmission dynamics is the distinction between direct transmission requiring close physical contact (e.g. bumping, fighting, or coughing) and indirect transmission from environmental sources such as contaminated substrates. We present data from 4 experiments addressing the form, routes, and timing of transmission of Ambystoma tigrinum virus (ATV) among tiger salamanders Ambystoma tigrinum nebulosum. Our data suggest that ATV is efficiently transmitted by direct interactions between live animals (bumping, biting and cannibalism) as well as by necrophagy and indirectly via water and fomites. Determining which form of transmission is most important in nature is essential for understanding transmission at the population level. Our experiments also revealed an important temporal aspect of infectiousness: larval salamanders become infectious soon after exposure to ATV and their propensity to infect others increases with time. These results begin to clarify the mechanisms and dynamics of ATV transmission and lead to key questions that need to be addressed in future research.     

Innate immune evasion mediated by the Ambystoma tigrinum virus eukaryotic translation initiation factor 2alpha homologue

Ranaviruses (family Iridoviridae, genus Ranavirus) are large, double-stranded DNA (dsDNA) viruses whose replication is restricted to ectothermic vertebrates. Many highly pathogenic members of the genus Ranavirus encode a homologue of the eukaryotic translation initiation factor 2α (eIF2α). Data in a heterologous vaccinia virus system suggest that the Ambystoma tigrinum virus (ATV) eIF2α homologue (vIF2αH; open reading frame [ORF] 57R) is involved in evading the host innate immune response by degrading the interferon-inducible, dsRNA-activated protein kinase, PKR. To test this hypothesis directly, the ATV vIF2αH gene (ORF 57R) was deleted by homologous recombination, and a selectable marker was inserted in its place. The ATVΔ57R virus has a small plaque phenotype and is 8-fold more sensitive to interferon than wild-type ATV (wtATV). Infection of fish cells with the ATVΔ57R virus leads to eIF2α phosphorylation, in contrast to infection with wtATV, which actively inhibits eIF2α phosphorylation. The inability of ATVΔ57R to prevent phosphorylation of eIF2α correlates with degradation of fish PKZ, an interferon-inducible enzyme that is closely related to mammalian PKR. In addition, salamanders infected with ATVΔ57R displayed an increased time to death compared to that of wtATV-infected salamanders. Therefore, in a biologically relevant system, the ATV vIF2αH gene acts as an innate immune evasion factor, thereby enhancing virus pathogenesis.     

Sensitivity of a diagnostic test for amphibian Ranavirus varies with sampling protocol

Field samples are commonly used to estimate disease prevalence in wild populations. Our confidence in these estimates requires understanding the sensitivity and specificity of the diagnostic tests. We assessed the sensitivity of the most commonly used diagnostic tests for amphibian Ranavirus by infecting salamanders (Ambystoma tigrinum; Amphibia, Caudata) with Ambystoma tigrinum virus (ATV) and then sampling euthanized animals (whole animal) and noneuthanized animals (tail clip) at five time intervals after exposure. We used a standard polymerase chain reaction (PCR) protocol to screen for ATV. Agreement between test results from whole-animal and tail-clip samples increased with time postexposure. This indicates that the ability to identify infected animals increases following exposure, leading to a more accurate estimate of prevalence in a population. Our results indicate that tail-clip sampling can underestimate the true prevalence of ATV in wild amphibian populations.     

Susceptibility of the endangered California tiger salamander, Ambystoma californiense, to ranavirus infection

Emerging infectious diseases are implicated in the declines and extinctions of amphibians worldwide. Ranaviruses in the family Iridoviridae are a global concern and have caused amphibian die-offs in wild populations in North America, Europe, South America, and in commercial populations in Asia and South America. The movement of amphibians for bait, food, pets, and research provides a route for the introduction of ranaviruses into naive and potentially endangered species. In this report, we demonstrate that the California tiger salamander, Ambystoma californiense, is susceptible to Ambystoma tigrinum virus (ATV). This virus has not been previously reported in California tiger salamander, but observed mortality in experimentally infected animals suggests that California tiger salamander populations could be adversely affected by an ATV introduction.     

First case of ranavirus-associated morbidity and mortality in natural populations of the South American frog Atelognathus patagonicus

Atelognathus patagonicus is an endangered leptodactylid frog endemic to a small region in and around Laguna Blanca National Park in northern Patagonia, Argentina. All of the lakes and small ponds of the region (except Laguna Blanca itself) contain A. patagonicus and in all but one of these lakes the species shows clinical signs of a previously undiagnosed disease, the characteristics of which suggested a ranavirus. We collected symptomatic and asymptomatic A. patagonicus frogs and tadpoles from 4 small lakes and analyzed tissues for ranavirus and the chytrid fungus Batrachochytrium dendrobatidis using PCR amplification of pathogen DNA. Of the 32 specimens tested, 25 were positive for ranavirus major capsid protein (MCP). Sequence alignments of the ranavirus MCP from these specimens showed 100% similarity with published FV3 and FV3-like viruses from anurans, 98 to 99 % similarity with Bohle iridovirus, and 95 % similarity with Ambystoma tigrinum virus (ATV) and Regina ranavirus (RRV). A search of the NCBI Blast nucleotide database using the 500 base pair MCP sequence obtained from these samples did not suggest any homology to any other pathogen. In addition, 1 sample (3 pooled individuals) from 1 lake tested positive for B. dendrobatidis. The clinical signs observed primarily in late-stage tadpoles and recent metamorphs, which have reoccurred each year since at least 2001, are consistent with ranaviral disease, but until histopathology of diseased individuals is carried out, chytridiomycosis or other diseases cannot be ruled out.     

中国淋巴囊肿病毒胸苷酸合酶基因结构特点及分析

胸苷酸合酶(Thymidylate synthase,TS)是进行DNA合成所必需的酶类,与细胞分化及肿瘤发生密切相关。淋巴囊肿病毒属于虹彩病毒科成员,是能引起百余种淡、海水鱼感染,并产生肿瘤的病毒病原。在已完成中国淋巴囊肿病毒株(Lymphocystis disease virus-China,LCDV-C)基因组序列测定的基础上,本文对位于LCDV-C基因组开放阅读框ORF011L的TS基因结构、及其推定蛋白结构进行了分析。该基因全长858bp,GC含量为28．2％,编码一个长为286aa、分子量为32．7kD、等电点为7．1的推定蛋白,称之为中国淋巴囊肿病毒胸苷酸合酶(LCDV-CTS)。该酶所具有的叶酸结合区在第44和70位氨基酸之间,dUMP结合区在第163和206位氨基酸之间,24个必需氨基酸具有高度保守性。二级结构预测结果显示LCDV-C TS含8个a螺旋,6个β折叠和23个环,表明其具备酶活性分子所有的柔性和可变性结构特征。这是迄今所知在脊椎动物虹彩病毒中唯一含两个完整结合区的TS。对来自包括LCDV在内二十个物种的TS结构进行同源性分析,显示LCDV-C TS被单独分为一枝。进一步对LCDV-C TS可能的起源途径及与宿主的作用等进行了探讨。     

Diagnostic and molecular evaluation of three iridovirus-associated salamander mortality events

In 1998 viruses were isolated from tiger salamander larvae (Ambystoma tigrinum diaboli and A. tigrinum melanostictum) involved in North Dakota and Utah (USA) mortality events and spotted salamander (A. maculatum) larvae in a third event in Maine (USA). Although sympatric caudates and anurans were present at all three sites only ambystomid larvae appeared to be affected. Mortality at the North Dakota site was in the thousands while at the Utah and Maine sites mortality was in the hundreds. Sick larvae were lethargic and slow moving. They swam in circles with obvious buoyancy problems and were unable to remain upright. On the ventral surface, near the gills and hind limbs, red spots or swollen areas were noted. Necropsy findings included: hemorrhages and ulceration of the skin, subcutaneous and intramuscular edema, swollen and pale livers with multifocal hemorrhage, and distended fluid-filled intestines with areas of hemorrhage. Light microscopy revealed intracytoplasmic inclusions, suggestive of a viral infection, in a variety of organs. Electron microscopy of ultra thin sections of the same tissues revealed iridovirus-like particles within the inclusions. These viruses were isolated from a variety of organs, indicating a systemic infection. Representative viral isolates from the three mortality events were characterized using molecular assays. Characterization confirmed that the viral isolates were iridoviruses and that the two tiger salamander isolates were similar and could be distinguished from the spotted salamander isolate. The spotted salamander isolate was similar to frog virus 3, the type species of the genus Ranavirus, while the tiger salamander isolates were not. These data indicate that different species of salamanders can become infected and die in association with different iridoviruses. Challenge assays are required to determine the fish and amphibian host range of these isolates and to assess the susceptibility of tiger and spotted salamanders to heterologous virus isolates.     

Combined Effects of Atrazine and Chlorpyrifos on Susceptibility of the Tiger Salamander to Ambystoma tigrinum Virus

Several hypotheses have been examined as potential causes of global amphibian declines, including emerging infectious diseases and environmental contaminants. Although these factors are typically studied separately, animals are generally exposed to both stressors simultaneously. We examined the effects of the herbicide atrazine and the insecticide chlorpyrifos on the susceptibility of tiger salamander larvae, Ambystoma tigrinum, to a viral pathogen, Ambystoma tigrinum virus (ATV). Environmentally relevant concentrations of atrazine (0, 20, 200 μg/L) and chlorpyrifos (0, 2, 20, 200 μg/L) were used along with ATV in a fully factorial experimental design whereby individually housed, 4-week-old larvae were exposed for 2 weeks. Atrazine alone was not lethal to larvae, and chlorpyrifos alone was lethal only at the highest concentration. When combined with ATV, chlorpyrifos increased susceptibility to viral infection and resulted in increased larval mortality. A significant interactive effect between atrazine and ATV was detected. Atrazine treatments slightly decreased survival in virus-exposed treatments, yet slightly increased survival in the virus-free treatments. These findings corroborate earlier research on the impacts of atrazine, in particular, on disease susceptibility, but exhibit greater effects (i.e., reduced survival) when younger larvae were examined. This study is the first of its kind to demonstrate decreases in amphibian survival with the combination of pesticide and a viral disease. Further examination of these multiple stressors can provide key insights into potential significance of environmental cofactors, such as pesticides, in disease dynamics.     

Habitat fragmentation as a result of biotic and abiotic factors controls pathogen transmission throughout a host population

1. The hypothesis that habitat fragmentation (biotic or abiotic) alters the transmission of disease within a population is explored using field data from a well-studied amphibian-pathogen system. 2. We used the Ambystoma tigrinum-A. tigrinum virus (ATV) model system to show how habitat fragmentation as a result of emergent vegetation and habitat management affects disease transmission dynamics in ponds across a landscape. 3. We quantified variation in ATV infection over time and across the landscape. ATV infection was significantly higher in ponds modified for livestock use (P = 0.032). Disease incidence decreased with increased amounts of emergent vegetation (P < 0.001). These factors appear to control disease transmission by altering the host contact rate and with it disease transmission. 4. A field experiment to test the effect of emergent vegetation on the distribution of larvae in ponds demonstrated a behavioural change in larvae found in sparsely vegetated ponds. Microhabitat choices resulted in larvae being concentrated at the pond edge resulting in a 'halo effect' in sparsely vegetated ponds, whereas larvae in heavily vegetated ponds were distributed more evenly throughout. Microhabitat choice affects the effective density that larvae experience. This 'halo effect' increases contact rates in the shallows of sparsely vegetated ponds and increases the transmission of a directly transmitted pathogen. 5. Despite recurrent epidemics of a lethal Ranavirus in tiger salamanders on the Kaibab Plateau, Arizona, USA, these populations persist. We discuss the implications of our results in the context of density-dependent transmission and homogeneous mixing, two increases key assumptions of epidemiological theory.     

Viral load of various tissues of rainbow trout challenged with viral haemorrhagic septicaemia virus at various stages of disease

Market-sized rainbow trout Oncorhynchus mykiss were challenged by waterborne exposure to viral haemorrhagic septicaemia virus (VHSV isolate of genogroup Ia). Fish were sampled at 4 stages of infection (before onset of clinical signs, clinically affected fish, mortalities and survivors) and the viral load determined in (1) internal organs, (2) muscle tissue and (3) brain and gill tissue. Virus levels were determined by virus titration and real-time RT-PCR. VHSV was detected by either method in the majority of fish before onset of clinical signs and in the survivor group as well as in all fish in the clinically affected fish and mortality groups. Mean virus amounts per mg of tissue determined by virus titration (TCID50) or real-time RT-PCR (copy number) were > 10(4) in preclinical fish, > 10(3.8) in clinically affected fish, > 10(3.9) in mortalities and > 10(1.2) in survivors. Virus levels tended to be highest in the internal organs of subclinical and clinically affected fish and in brain and gill tissue of survivors. The results demonstrate that significant levels of VHSV can be found in tissues of rainbow trout that may be marketed for human consumption, which may have relevance for the biosecurity of VHS-free areas.     

Geographically variable selection in Ambystoma tigrinum virus (Iridoviridae) throughout the western USA

We investigated spatially variable selection in Ambystoma tigrinum virus (ATV) which causes frequent and geographically widespread epizootics of the tiger salamander, Ambystoma tigrinum. To test for evidence of selection, we sequenced several coding and noncoding regions from virus strains isolated from epizootics throughout western North America. Three of the sequenced regions contained homologues for genes putatively involved in host immune evasion and virulence: eIF‐2α, caspase activation and recruitment domain (CARD) and β‐OH‐steroid oxidoreductase. Selection analysis showed evidence of very strong purifying selection on eIF‐2α, purifying selection within certain viral clades on CARD and positive selection on β‐OH‐steroid oxidoreductase within certain clades. Analysis using multidivtime and Tajima’s relative rate tests indicate accelerated rates of evolution within clades associated with anthropogenic movement. These clades also demonstrate greater spatial variability in selection, suggesting a lack of local adaptation (i.e. locally adapted populations should exhibit little to no selection because of absent or reduced variation in fitness once a fitness optimum is reached). Increased transfer of non‐native viral strains to naïve salamander populations, in conjunction with local maladaptation as a result of local selection pressures, may explain the spread and emergence of ATV epizootics in A. tigrinum in western North America.     

Testing a key assumption of host‐pathogen theory: density and disease transmission

Conventional disease theory suggests that extinction with density‐dependent transmission is unlikely as the threshold host density (KT) is greater than zero. Extinction may result if transmission is frequency dependent or the pathogen has an environmental reservoir. Given the importance of understanding how pathogens affect species richness and diversity there are few empirical tests of these conclusions. We used an Ambystoma tigrinum–Ambystoma tigrinum virus (ATV) model system in the laboratory to examine disease transmission dynamics. Susceptible A. tigrinum larvae were exposed to three different densities and proportions of infected larvae for 24 h. We then housed susceptible hosts individually for 28 days and monitored them for infection. The density of infected hosts to which susceptible hosts were exposed was the best predictor of infection (p=0.037). There was no effect of host clutch on the probability of becoming infected (p=0.67). Larvae in the highest density treatments died sooner than larvae in lower density treatments (p<0.001). Asymptomatic but infected hosts shed sufficient virus into the water in a 24‐h period to infect susceptible hosts without any direct contact between individuals. ATV transmission was best described by a power function, leading to the prediction that extinction of A. tigrinum as a result of this pathogen is unlikely. Indeed, field observations show that larval salamander populations that experience ATV‐driven epidemics may decrease, but not to extinction, and then recover. Disease is proposed as a possible explanation for the global decline of amphibians. Ranaviruses infect many amphibian populations, but based on our results may not be a general cause of declines to extinction. In contrast, frequency dependent transmission, environmental reservoirs and alternative hosts may be the most likely explanation for the enigmatic decline, at times to extinction, of some amphibian populations as a result of emerging infectious diseases, like the chytrid fungus Batrachochytrium dendrobatidis.     

Susceptibility of juvenile Atlantic cod Gadus morhua to viral haemorrhagic septicaemia virus isolated from wild-caught Atlantic cod

A European strain of viral haemorrhagic septicaemia virus (VHSV) isolated from wild-caught cod Gadus morhua (H16/7/95) was shown to cause clinical disease and mortality in excess of 80% in juvenile Atlantic cod when administered by the intra-peritoneal (i.p.) route. No virus was recovered from cod cohabiting with experimentally infected fish at a ratio of 1:1, and no VHSV-associated mortality was demonstrated following immersion infection. External signs of disease in cod were the presence of exophthalmia and ascites. Virus was identified as VHSV by enzyme-linked immunosorbent assay (ELISA) and was recovered from both brain and organ pools (kidney, liver and spleen) of 100% of i.p. infected cod mortalities. Virus was also detected using an indirect immunofluorescence test on tissue imprints of kidney, liver, spleen and brain taken from moribund fish. The fact that cod were not susceptible to VHSV following waterborne exposure raises important questions surrounding the propagation, maintenance and impact of a naturally occurring reservoir of virus in the marine environment.     

Evidence for emergence of an amphibian iridoviral disease because of human-enhanced spread

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.     

Effect of Food and Density on Development of Typical and Cannibalistic Salamander Larvae in Ambystoma tigrinum nebulosum

Two subspecies of the tiger salamander, Ambystoma tigrinum,have a distinctive polymorphic life history that can includefour adult morphs as well as typical and cannibalistic larvalmorphs. We evaluated the effect of environment on developmentof larval morphology in two laboratory experiments. In ExperimentI, 180 larvae were raised in individual 3-liter containers andfed one of three food levels. Larvae in Experiment II receivedone of two levels of food, and were raised at three densities:one larva per 3 liters of water (50 containers), three larvaeper 22 liters of water (18 containers), or seven larvae per22 liters of water (18 containers). Cannibalistic morphs developedonly in nine containers at the highest density, and their occurrencewas independent of the two food levels. Our results suggestthe typical and cannibalistic larvae which occur in some populationsof Ambystoma tigrinum nebulosum is an environmentally induceddevelopmental polymorphism that results from some individualsresponding to the environment differently than others. Thisdifference in response may or may not be associated with geneticdifferences between these morphs. Based on our results we cannotdiscriminate between two models that differ in their assumptionsabout the genetic background of individual larvae.     

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. Moreover, in contrast to FV3, RCV-Z contained a full-length copy of the viral homolog of eukaryotic initiation factor 2 alpha (eIF-2alpha). Experimental infection of bullfrog tadpoles with FV3 and RCV-Z demonstrated that RCV-Z was much more pathogenic than FV3, and that prior infection with FV3 protected them from subsequent RCV-Z induced mortality. Collectively, these results suggest that RCV-Z may represent a novel species of ranavirus capable of infecting frogs and that possession of a viral eIF-2alpha homolog (vIF-2alpha) correlates with enhanced virulence.     

Mortality Rates Differ Among Amphibian Populations Exposed to Three Strains of a Lethal Ranavirus

Infectious diseases are a growing threat to biodiversity, in many cases because of synergistic effects with habitat loss, environmental contamination, and climate change. Emergence of pathogens as new threats to host populations can also arise when novel combinations of hosts and pathogens are unintentionally brought together, for example, via commercial trade or wildlife relocations and reintroductions. Chytrid fungus (Batrachochytrium dendrobatidis) and amphibian ranaviruses (family Iridoviridae) are pathogens implicated in global amphibian declines. The emergence of disease associated with these pathogens appears to be at least partly related to recent translocations over large geographic distances. We experimentally examined the outcomes of novel combinations of host populations and pathogen strains using the amphibian ranavirus Ambystoma tigrinum virus (ATV) and barred tiger salamanders (Ambystoma mavortium, formerly considered part of the Ambystoma tigrinum complex). One salamander population was highly resistant to lethal infections by all ATV strains, including its own strain, and mortality rates differed among ATV strains according to salamander population. Mortality rates in novel pairings of salamander population and ATV strain were not predictable based on knowledge of mortality rates when salamander populations were exposed to their own ATV strain. The underlying cause(s) for the differences in mortality rates are unknown, but local selection pressures on salamanders, viruses, or both, across the range of this widespread host–pathogen system are a plausible hypothesis. Our study highlights the need to minimize translocations of amphibian ranaviruses, even among conspecifc host populations, and the importance of considering intraspecific variation in endeavors to manage wildlife diseases.     

Description of an as yet unclassified DNA virus from diseased Cyprinus carpio species

Numerous deaths of koi and common carp (Cyprinus carpio) were observed on many farms throughout Israel, resulting in severe financial losses. The lethal viral disease observed is highly contagious and extremely virulent, but morbidity and mortality are restricted to koi and common carp populations. Diseased fish exhibit fatigue and gasping movements in shallow water. Infected fish had interstitial nephritis and gill necrosis as well as petechial hemorrhages in the liver and other symptoms that were not consistent with viral disease, suggesting a secondary infection. Here we report the isolation of carp nephritis and gill necrosis virus (CNGV), which is the etiologic agent of this disease. The virus propagates and induces severe cytopathic effects by 5 days postinfection in fresh koi or carp fin cell cultures (KFC and CFC, respectively), but not in epithelioma papillosum cyprini cells. The virus harvested from KFC cultures induced the same clinical signs, with a mortality of 75 to 95%, upon inoculation into naive koi and common carp. Using PCR, we provide final proof that the isolated virus is indeed the etiologic agent of food and ornamental carp mortalities in fish husbandry. Electron microscopy revealed viral cores with icosahedral morphology of 100 to 110 nm that resembled herpesviruses. Electron micrographs of purified pelleted CNGV sections, together with viral sensitivities to ether and Triton X-100, suggested that it is an enveloped virus. However, the genome of the isolated virus is a double-stranded DNA (dsDNA) molecule of 270 to 290 kbp, which is larger than known herpesviruses. The viral DNA seems highly divergent and bears only small fragments (16 to 45 bp) that are similar to the genomes of several DNA viruses. Nevertheless, amino acid sequences encoded by CNGV DNA fragments bear similarities primarily to members of the Poxviridae and Herpesviridae and to other large dsDNA viruses. We suggest, therefore, that the etiologic agent of this disease may represent an as yet unclassified virus species that is endemic in C. carpio (carp).