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Spring viremia of carp virus (SVCV) is a rhabdovirus that primarily infects cyprinid finfishes and causes a disease notifiable to the World Organization for Animal Health. Amphibians, which are sympatric with cyprinids in freshwater ecosystems, are considered non-permissive hosts of rhabdoviruses. The potential host range expansion of SVCV in an atypical host species was evaluated by testing the susceptibility of amphibians native to the Pacific Northwest. Larval long-toed salamanders Ambystoma macrodactylum and Pacific tree frog Pseudacris regilla tadpoles were exposed to SVCV strains from genotypes Ia, Ib, Ic, or Id by either intraperitoneal injection, immersion, or cohabitation with virus-infected koi Cyprinus rubrofuscus . Cumulative mortality was 100% for salamanders injected with SVCV, 98-100% for tadpoles exposed to virus via immersion, and 0-100% for tadpoles cohabited with SVCV-infected koi. Many of the animals that died exhibited clinical signs of disease and SVCV RNA was found by in situ hybridization in tissue sections of immersion-exposed tadpoles, particularly in the cells of the gastrointestinal tract and liver. SVCV was also detected by plaque assay and RT-qPCR testing in both amphibian species regardless of the virus exposure method, and viable virus was detected up to 28 days after initial exposure. Recovery of infectious virus from naïve tadpoles cohabited with SVCV-infected koi further demonstrated that SVCV transmission can occur between classes of ectothermic vertebrates. Collectively, these results indicated that SVCV, a fish rhabdovirus, can be transmitted to and cause lethal disease in two amphibian species. Therefore, members of all five of the major vertebrate groups (mammals, birds, reptiles, fish, and amphibians) appear to be vulnerable to rhabdovirus infections. Future research studying potential spillover and spillback infections of aquatic rhabdoviruses between foreign and domestic amphibian and fish species will provide insights into the stressors driving novel interclass virus transmission events.

Ranaviruses can cause mass mortality events in amphibians, thereby becoming a threat to populations that are already facing dramatic declines. Ranaviruses affect all life stages and persist in multiple amphibian hosts. The detrimental effects of ranavirus infections to amphibian populations have already been observed in the UK and in North America. In Central and South America, the virus has been reported in several countries, but the presence of the genus Ranavirus (Rv) in Colombia is unknown. To help fill this knowledge gap, we surveyed for Rv in 60 species of frogs (including one invasive species) in Colombia. We also tested for co-infection with Batrachochytrium dendrobatidis (Bd) in a subset of individuals. For Rv, we sampled 274 vouchered liver tissue samples collected between 2014 and 2019 from 41 localities covering lowlands to mountaintop páramo habitat across the country. Using quantitative polymerase chain reaction (qPCR) and end-point PCR, we detected Rv in 14 individuals from 8 localities, representing 6 species, including 5 native frogs of the genera Osornophryne, Pristimantis and Leptodactylus, and the invasive American bullfrog Rana catesbeiana. Bd was detected in 7 of 140 individuals, with 1 co-infection of Rv and Bd in an R. catesbeiana specimen collected in 2018. This constitutes the first report of ranavirus in Colombia and should set off alarms about this new emerging threat to amphibian populations in the country. Our findings provide some preliminary clues about how and when Rv may have spread and contribute to understanding how the pathogen is distributed globally.

To cope with amphibian die-offs caused by ranavirus, it is important to know the underlying ranavirus prevalence in a region. We studied the ranavirus prevalence in tadpoles of two native and one introduced anuran species inhabiting agricultural and surrounding areas at 49 locations across eight provinces of South Korea by applying qPCR. The local ranavirus prevalence and the individual infection rates at infected locations were 32.6% and 16.1%, respectively, for Dryophytes japonicus (Japanese tree frog); 25.6% and 26.1% for Pelophylax nigromaculatus (Black-spotted pond frog); and 30.5% and 50.0% for Lithobates catesbeianus (American bullfrog). The individual infection rate of L. catesbeianus was significantly greater than that of D. japonicus . The individual infection rate of P. nigromaculatus was related to the site-specific precipitation and air temperature. The individual infection rate gradually increased from Gosner development stage 39, and intermittent infection was confirmed in the early and middle developmental stages. Our results show that ranavirus is widespread among wild amphibians living in agricultural areas of South Korea, and mass die-offs by ranavirus could occur at any time.

Neuraminidase (NA) inhibitors are one of the two major classes of antivirals available for the treatment and prevention of influenza. X-ray crystal structure determination of NA head domains and their complexes with various inhibitors are of importance for the design and optimization of anti-influenza drugs. However, the globular tetrameric properties of NA head domains may produce crystals with pathological imperfections, lattice-translocation defects, making structure determination no longer straightforward. In this report, using a crystal of the NA head domain from the Wuhan Asiatic toad influenza virus as an example, the identification and solution of this type of crystal pathology are presented. Furthermore, its underlying mechanism of formation is explored.

Amphibians are declining worldwide, in part because of large-scale degradation of habitat from agriculture and pervasive pathogens. Yet a common North American amphibian, the wood frog (Lithobates sylvaticus), ranges widely and persists in agricultural landscapes. Conventional survey techniques rely on visual encounters and dip-netting efforts, but detectability limits the ability to test for the effects of environmental variables on amphibian habitat suitability. We used environmental DNA to determine the presence of wood frogs and an amphibian pathogen (ranavirus) in Prairie Pothole wetlands and investigated the effects of 32 water quality, wetland habitat, and landscape-level variables on frog presence at sites representing different degrees of agricultural intensity. Several wetland variables influenced wood frog presence, the most influential being those associated with wetland productivity (i.e., nutrients), vegetation buffer width, and proportion of the surrounding landscape that is comprised of other water bodies. Wood frog presence was positively associated with higher dissolved phosphorus (>0.4 mg/L), moderate dissolved nitrogen (0.1-0.2 mg/L), lower chlorophyll a (≤15 µg/L), wider vegetation buffers (≥10 m), and more water on the landscape (≥0.25). These results highlight the effects of environmental factors at multiple scales on the presence of amphibians in this highly modified landscape-namely the importance of maintaining wetland water quality, vegetation buffers, and surrounding habitat heterogeneity. Environ Toxicol Chem 2019;38:2750-2763. © 2019 SETAC., (© 2019 SETAC.)

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.

The early detection and identification of pathogenic microorganisms is essential in order to deploy appropriate mitigation measures. Viruses in the Iridoviridae family, such as those in the Ranavirus genus, can infect amphibian species without resulting in mortality or clinical signs, and they can also infect other hosts than amphibian species. Diagnostic techniques allowing the detection of the pathogen outside the period of host die-off would thus be of particular use. In this study, we tested a method using environmental DNA (eDNA) on a population of common frogs ( Rana temporaria ) known to be affected by a Ranavirus in the southern Alps in France. In six sampling sessions between June and September (the species' activity period), we collected tissue samples from dead and live frogs (adults and tadpoles), as well as insects (aquatic and terrestrial), sediment, and water. At the beginning of the breeding season in June, one adult was found dead; at the end of July, a mass mortality of tadpoles was observed. The viral DNA was detected in both adults and tadpoles (dead or alive) and in water samples, but it was not detected in insects or sediment. In live frog specimens, the virus was detected from June to September and in water samples from August to September. Dead tadpoles that tested positive for Ranavirus were observed only on one date (at the end of July). Our results indicate that eDNA can be an effective alternative to tissue/specimen sampling and can detect Ranavirus presence outside die-offs. Another advantage is that the collection of water samples can be performed by most field technicians. This study confirms that the use of eDNA can increase the performance and accuracy of wildlife health status monitoring and thus contribute to more effective surveillance programs.

Coinfections are increasingly recognized as important drivers of disease dynamics. Consequently, greater emphasis has been placed on integrating principles from community ecology with disease ecology to understand within-host interactions among parasites. Using larval amphibians and two amphibian parasites (ranaviruses and the trematode Echinoparyphium sp.), we examined the influence of coinfection on disease outcomes. Our first objective was to examine how priority effects (the timing and sequence of parasite exposure) influence infection and disease outcomes in the laboratory. We found that interactions between the parasites were asymmetric; prior infection with Echinoparyphium reduced ranaviral loads by 9% but there was no reciprocal effect of prior ranavirus infection on Echinoparyphium load. Additionally, survival rates of hosts (larval gray treefrogs; Hyla versicolor) infected with Echinoparyphium 10 days prior to virus exposure were 25% greater compared to hosts only exposed to virus. Our second objective was to determine whether these patterns were generalizable to multiple amphibian species under more natural conditions. We conducted a semi-natural mesocosm experiment consisting of four larval amphibian hosts [gray treefrogs, American toads (Anaxyrus americanus), leopard frogs (Lithobates pipiens) and spring peepers (Pseudacris crucifer)] to examine how prior Echinoparyphium infection influenced ranavirus transmission within the community, using ranavirus-infected larval wood frogs (Lithobates sylvaticus) as source of ranavirus. Consistent with the laboratory experiment, we found that prior Echinoparyphium infection reduced ranaviral loads by 19 to 28% in three of the four species. Collectively, these results suggest that macroparasite infection can reduce microparasite replication rates across multiple amphibian species, possibly through cross-reactive immunity. Although the immunological mechanisms driving this outcome are in need of further study, trematode infections appear to benefit hosts that are exposed to ranaviruses. Additionally, these results suggest that consideration of priority effects and timing of exposure are vital for understanding parasite interactions within hosts and disease outcomes., (© 2017 The Authors. Journal of Animal Ecology © 2017 British Ecological Society.)

Emerging diseases have been increasingly associated with population declines, with co-infections exhibiting many types of interactions. The chytrid fungus (Batrachochytrium dendrobatidis) and ranaviruses have extraordinarily broad host ranges, however co-infection dynamics have been largely overlooked. We investigated the pattern of co-occurrence of these two pathogens in an amphibian assemblage in Serra da Estrela (Portugal). The detection of chytridiomycosis in Portugal was linked to population declines of midwife-toads (Alytes obstetricans). The asynchronous and subsequent emergence of a second pathogen - ranavirus - caused episodes of lethal ranavirosis. Chytrid effects were limited to high altitudes and a single host, while ranavirus was highly pathogenic across multiple hosts, life-stages and altitudinal range. This new strain (Portuguese newt and toad ranavirus - member of the CMTV clade) caused annual mass die-offs, similar in host range and rapidity of declines to other locations in Iberia affected by CMTV-like ranaviruses. However, ranavirus was not always associated with disease, mortality and declines, contrasting with previous reports on Iberian CMTV-like ranavirosis. We found little evidence that pre-existing chytrid emergence was associated with ranavirus and the emergence of ranavirosis. Despite the lack of cumulative or amplified effects, ranavirus drove declines of host assemblages and changed host community composition and structure, posing a grave threat to all amphibian populations.

We performed a rapid response investigation to evaluate the presence and distribution of amphibian pathogens in Madagascar following our identification of amphibian chytrid fungus (Batrachochytrium dendrobatidis, Bd) and ranavirus in commercially exported amphibians. This targeted risk-based field surveillance program was conducted from February to April 2014 encompassing 12 regions and 47 survey sites. We simultaneously collected amphibian and environmental samples to increase survey sensitivity and performed sampling both in wilderness areas and commercial amphibian trade facilities. Bd was not detected in any of 508 amphibian skin swabs or 68 water filter samples, suggesting pathogen prevalence was below 0.8%, with 95% confidence during our visit. Ranavirus was detected in 5 of 97 amphibians, including one adult Mantidactylus cowanii and three unidentified larvae from Ranomafana National Park, and one adult Mantidactylus mocquardi from Ankaratra. Ranavirus was also detected in water samples collected from two commercial amphibian export facilities. We also provide the first report of an amphibian mass-mortality event observed in wild amphibians in Madagascar. Although neither Bd nor ranavirus appeared widespread in Madagascar during this investigation, additional health surveys are required to disentangle potential seasonal variations in pathogen abundance and detectability from actual changes in pathogen distribution and rates of spread. Accordingly, our results should be conservatively interpreted until a comparable survey effort during winter months has been performed. It is imperative that biosecurity practices be immediately adopted to limit the unintentional increased spread of disease through the movement of contaminated equipment or direct disposal of contaminated material from wildlife trade facilities. The presence of potentially introduced strains of ranaviruses suggests that Madagascar's reptile species might also be threatened by disease. Standardized population monitoring of key amphibian and reptile species should be established with urgency to enable early detection of potential impacts of disease emergence in this global biodiversity hotspot.

Gross and histopathological examination was performed on seven captive magnificent tree frogs (Litoria splendida) and one green tree frog (Litoria caerulea) that had died or been humanely destroyed while naturally infected with Mahaffey Road virus, a Bohle iridovirus-like ranavirus. Necropsy examination revealed skin lesions consisting of multiple small pale or haemorrhagic papules and ulcers in most frogs. Other common gross findings were perineural haemorrhage affecting the spinal nerves, hydrocoelom, hepatomegaly and splenomegaly with pinpoint pale foci throughout the parenchyma. On histological examination, vasculitis with prominent endothelial necrosis was found in a wide range of tissues. Widespread lymphoid necrosis and fibroblast necrosis were usual findings. Multifocal epithelial cell necrosis in the epidermis, liver and pancreas was found commonly. Non-suppurative meningoencephalitis, myelitis and ganglioneuritis were present variably. Intracytoplasmic basophilic inclusion bodies were found variably in hepatocytes, renal tubular epithelium and keratinocytes. Immunohistochemistry demonstrated ranavirus antigen in endothelial cells, fibroblasts, macrophages, lymphocytes and epithelial cells in a wide range of tissues. The finding of widespread venous and lymphatic endothelial necrosis and demonstration of abundant endothelial antigen suggests that endothelial tropism of the virus plays a significant role in the pathogenesis of the infection., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)

Background: LTR retroelements (LTR REs) constitute a major group of transposable elements widely distributed in eukaryotic genomes. Through their own mechanism of retrotranscription LTR REs enrich the genomic landscape by providing genetic variability, thus contributing to genome structure and organization. Nonetheless, transcriptomic activity of LTR REs still remains an obscure domain within cell, developmental, and organism biology., Results: Here we present a first comparative analysis of LTR REs for anuran amphibians based on a full depth coverage transcriptome of the European pool frog, Pelophylax lessonae, the genome of the African clawed frog, Silurana tropicalis (release v7.1), and additional transcriptomes of S. tropicalis and Cyclorana alboguttata. We identified over 1000 copies of LTR REs from all four families (Bel/Pao, Ty1/Copia, Ty3/Gypsy, Retroviridae) in the genome of S. tropicalis and discovered transcripts of several of these elements in all RNA-seq datasets analyzed. Elements of the Ty3/Gypsy family were most active, especially Amn-san elements, which accounted for approximately 0.27% of the genome in Silurana. Some elements exhibited tissue specific expression patterns, for example Hydra1.1 and MuERV-like elements in Pelophylax. In S. tropicalis considerable transcription of LTR REs was observed during embryogenesis as soon as the embryonic genome became activated, i.e. at midblastula transition. In the course of embryonic development the spectrum of transcribed LTR REs changed; during gastrulation and neurulation MuERV-like and SnRV like retroviruses were abundantly transcribed while during organogenesis transcripts of the XEN1 retroviruses became much more active., Conclusions: The differential expression of LTR REs during embryogenesis in concert with their tissue-specificity and the protein domains they encode are evidence for the functional roles these elements play as integrative parts of complex regulatory networks. Our results support the meanwhile widely accepted concept that retroelements are not simple "junk DNA" or "harmful genomic parasites" but essential components of the transcriptomic machinery in vertebrates.

Rana grylio virus (RGV) is a pathogenic iridovirus that has resulted in high mortality in cultured frog. Here, an envelope protein gene, 2L, was identified from RGV and its possible role in virus infection was investigated. Database searches found that RGV 2L had homologues in all sequenced iridoviruses and is a core gene of iridoviruses. Western blotting detection of purified RGV virions confirmed that 2L protein was associated with virion membrane. Fluorescence localization revealed that 2L protein co-localized with viral factories in RGV infected cells. In co-transfected cells, 2L protein co-localized with two other viral envelope proteins, 22R and 53R. However, 2L protein did not co-localize with the major capsid protein of RGV in co-transfected cells. Meanwhile, fluorescence observation showed that 2L protein co-localized with endoplasmic reticulum, but did not co-localize with mitochondria and Golgi apparatus. Moreover, a conditional lethal mutant virus containing the lac repressor/operator system was constructed to investigate the role of RGV 2L in virus infection. The ability to form plaques and the virus titres were strongly reduced when expression of 2L was repressed. Therefore, the current data showed that 2L protein is essential for virus infection. Our study is the first report, to our knowledge, of co-localization between envelope proteins in iridovirus and provides new insights into the understanding of envelope proteins in iridovirus.

Twelve captive magnificent tree frogs Litoria splendida and 2 green tree frogs L. caerulea on a property in the Darwin rural area (Northern Territory, Australia) either died or were euthanased after becoming lethargic or developing skin lesions. Samples from both species of frog were submitted for histopathology and virus isolation. An irido-like virus was cultured from tissue samples taken from both species and was characterised using electron microscopy, restriction enzyme digests and nucleic acid amplification and sequencing. The isolates were determined to belong to the genus Ranavirus, were indistinguishable from each other and shared a 98.62% nucleotide similarity and a 97.32% deduced amino acid homology with the Bohle iridovirus over a 1161 bp region of the major capsid gene. This is the first isolation of a ranavirus from amphibians in the Northern Territory and the first report of natural infection in these 2 species of native frog. The virus is tentatively named Mahaffey Road virus (MHRV).

Research that identifies the potential host range of generalist pathogens as well as variation in host susceptibility is critical for understanding and predicting the dynamics of infectious diseases within ecological communities. Ranaviruses have been linked to amphibian die-off events worldwide with the greatest number of reported mortality events occurring in the United States. While reports of ranavirus-associated mortality events continue to accumulate, few data exist comparing the relative susceptibility of different species. Using a series of laboratory exposure experiments and comparative phylogenetics, we compared the susceptibilities of 19 amphibian species from two salamander families and five anurans families for two ranavirus isolates: frog virus 3 (FV3) and an FV3-like isolate from an American bullfrog culture facility. We discovered that ranaviruses were capable of infecting 17 of the 19 larval amphibian species tested with mortality ranging from 0 to 100%. Phylogenetic comparative methods demonstrated that species within the anuran family Ranidae were generally more susceptible to ranavirus infection compared to species from the other five families. We also found that susceptibility to infection was associated with species that breed in semi-permanent ponds, develop rapidly as larvae, and have limited range sizes. Collectively, these results suggest that phylogeny, life history characteristics, and habitat associations of amphibians have the potential to impact susceptibility to ranaviruses.

Eastern equine encephalitis virus (EEEV) is endemic throughout most of the eastern United States. Although it is transmitted year round in Florida, transmission elsewhere is seasonal. The mechanism that enables EEEV to overwinter in seasonal foci remains obscure. In previous field studies, early season EEEV activity was detected in mosquito species that feed primarily upon ectothermic hosts, suggesting that reptiles and amphibians might represent overwintering reservoir hosts for EEEV. To determine if this might be possible, two commonly fed upon amphibian and reptile species were evaluated as hosts for the North American subtype I strain of EEEV. Neither amphibian species was a competent host. However, circulating viremias were detected in both reptile species examined. Hibernating infected garter snakes remained viremic after exiting hibernation. These data suggest that snakes may represent an overwintering host for North American EEEV.

Ranaviruses have caused die-offs of amphibians across the globe. In North America, these pathogens cause more amphibian mortality events than any other pathogen. Field observations suggest that ranavirus epizootics in amphibian communities are common during metamorphosis, presumably due to changes in immune function. However, few controlled studies have compared the relative susceptibility of amphibians to ranaviruses across life stages. Our objectives were to measure differences in mortality and infection prevalence following exposure to ranavirus at four developmental stages and determine whether the differences were consistent among seven anuran species. Based on previous studies, we hypothesized that susceptibility to ranavirus would be greatest at metamorphosis. Our results did not support this hypothesis, as four of the species were most susceptible to ranavirus during the larval or hatchling stages. The embryo stage had the lowest susceptibility among species probably due to the protective membranous layers of the egg. Our results indicate that generalizations should be made cautiously about patterns of susceptibility to ranaviruses among amphibian developmental stages and species. Further, if early developmental stages of amphibians are susceptible to ranaviruses, the impact of ranavirus epizootic events may be greater than realized due to the greater difficulty of detecting morbid hatchlings and larvae compared to metamorphs.

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.

Ranaviruses are known to cause mortality in a variety of anuran species and have the potential to significantly impact wild and captive frog populations. In this study, 16 captive frogs and toads from the Louisville Zoological Garden were examined for the presence of ranavirus; this group included 14 Cope's grey tree frogs (Hyla chrysoscelis), an American toad (Bufo americanus), and a southern toad (Bufo terrestris). All animals were wild caught and were evaluated via polymerase chain reaction (PCR), while animals that died were also assessed via histologic study to understand the role of ranaviral disease in these specimens. Of the animals that died, 82% were positive for ranavirus via PCR. Multiple swab samples collected over time from live tree frogs were positive for ranavirus via PCR. These findings reveal that ranaviral infection in captive adult anurans may occur without clinical signs or consistent histopathologic lesions.

Serious systemic disease in fish and amphibians is associated with the ranaviruses, epizootic haematopoietic necrosis virus (EHNV) and Bohle iridovirus (BIV) in Australia, and European sheatfish virus (ESV) and European catfish virus (ECV) in Europe. EHNV, ESV and ECV are recognized causative agents of the OIE (Office International des Epizooties) notifiable systemic necrotizing iridovirus syndrome and are currently identified by protein-based assays, none of which are able to rapidly identify the specific agents. The aim of this study was to develop TaqMan real-time PCR assays that differentiated these viruses using nucleotide sequence variation in two ranavirus genes. A conserved probe representing 100% sequence homology was used as a reference for virus-specific probes. The virus-specific probes produced a similar signal level to the conserved probe while those probes binding to non-target viral DNA produced an altered fluorescent curve. The pattern of probe binding was characteristic for each virus. Sensitivity, specificity and dynamic range of the assay were assessed. The test is currently useful as a research and initial screening tool, with the potential to become a sensitive and specific method for detection and differentiation of ranaviruses with further development.

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.

The objective was to study Orthomyxovirus in heterothermic animals. Blood samples from snakes (genus Bothrops and Crotalus) and from toads and frogs (genus Bufo and Rana) were collected to evaluate the red cell receptors and antibodies specific to influenza virus by the hemagglutination and hemagglutination inhibition tests, respectively. Both snakes and toads kept in captivity presented receptors in their red cells and antibodies specific to either influenza virus type A (human and equine origin) or influenza type B. The same was observed with recently captured snakes. Concerning the influenza hemagglutination inhibition antibodies protective levels were observed in the reptiles' serum, against influenza type A and type B. Unlike the toads, 83.3% of the frogs presented mean levels of Ab 40HIU for some influenza strains. It was concluded that heterothermic animals could offer host conditions to the influenza virus and also susceptibility to the infection.

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.

The genome of frog adenovirus (FrAdV-1) was sequenced and found to be the smallest of all known adenovirus genomes. The sequence obtained was 26163 bp in size and contains a substantial direct repeat near the right terminus, implying that it was derived by recombination from a parental genome of only 25517 bp. The closest relative of FrAdV-1 proved to be turkey adenovirus 3, an avian adenovirus with no previously known near relative. Sequence comparisons showed that the two viruses have equivalent gene complements, including one gene the product of which is related to sialidases. Phylogenetic analyses supported the establishment of a fourth adenovirus genus containing these two viruses, in addition to the established genera Mastadenovirus: and Aviadenovirus: and the proposed genus Atadenovirus: Sixteen genes were identified as being conserved between these four lineages and were presumably inherited from an ancestral adenovirus.

Ten virus isolates were obtained from three species of marine mammals sampled on San Miguel Island (California, USA) and 1,200 km north on Rogue Reef (Oregon, USA) during tagging operations in 1986-87. Seven of these 10 were derived from 30 sampled Steller sea lion (Eumetopias jubatus pups, while two of 10 were isolated from one of 19 sampled California sea lion (Zalophus californianus californianus pups, and the remaining isolate was derived from 30 sampled northern fur seal (Callorhinus ursinus) pups. All 10 isolates were identified as belonging to a single serotype, reptilian calicivirus Crotalus type 1 (RCV Cro-1), previously isolated from both healthy and diseased snakes and frogs in a California zoologic collection. The marine samples also showed that nine of 30 Steller sea lion pups, one of 19 California sea lion pups and zero of 30 fur seal pups were producing type specific neutralizing antibodies to RCV Cro-1. This represents the first reported instance of the isolation from marine sources of calicivirus originally isolated from a terrestrial species.

We isolated and characterized three endogenous retroviral fragments from the dart-poison frog Dendrobates ventrimaculatus. These are the first retroviral sequences to be identified in amphibians, and consequently retroviruses have now been found in each of the five major vertebrate classes. Comparison of the amphibian retroviral fragments, termed DevI, DevII, and DevIII, with mammalian and avian isolates revealed significant differences between their nucleotide sequences. This suggested that they were only distantly related to the seven currently recognized retroviral genera. Additional analysis by phylogeny reconstruction showed that the amphibian retroviral fragments were approximately equally related to the Moloney leukemia-related viruses, the spumaviruses, and walleye dermal sarcoma virus. Hybridization experiments revealed that viruses closely related to DevI, DevII, and DevIII do not appear to be widespread in other vertebrates and that DevI, DevII, and DevIII are all present at high copy numbers within their amphibian hosts, typically at over 250 copies per genome. The viruses described here, along with two others which have recently been found in a fish and a reptile, indicate that there may be some major differences in the retroviruses harbored by different vertebrate classes. This suggests that further characterization of retroviruses of fish, reptiles, and amphibians will help in understanding the evolution of the whole retroviral family and may well lead to the discovery of retroviruses with novel biological properties.

Blood erythrocytes of Brazilian tree-frogs, Phrynohyas venulosa were found to frequently contain single, small, densely staining inclusions. Electron microscopy showed these to be icosahedral viral particles which measured from 250-280 nm in diameter: they were devoid of an envelope, and thus differed from previously described viruses of frog erythrocytes. The infected erythrocytes lacked a crystalline body.