Genome analysis of Ranavirus frog virus 3 isolated from American Bullfrog (Lithobates catesbeianus) in South America.

Ranaviruses (family Iridoviridae) cause important diseases in cold-blooded vertebrates. In addition, some occurrences indicate that, in this genus, the same virus can infect animals from different taxonomic groups. A strain isolated from a Ranavirus outbreak (2012) in the state of Sao Paulo, Brazil, had its genome sequenced and presented 99.26% and 36.85% identity with samples of Frog virus 3 (FV3) and Singapore grouper iridovirus (SGIV) ranaviruses, respectively. Eight potential recombination events among the analyzed sample and reference FV3 samples were identified, including a recombination with Bohle iridovirus (BIV) sample from Oceania. The analyzed sample presented several rearrangements compared to FV3 reference samples from North America and European continent. We report for the first time the complete genome of Ranavirus FV3 isolated from South America, these results contribute to a greater knowledge related to evolutionary events of potentially lethal infectious agent for cold-blooded animals.

Discovery of Wild Amphibians Infected with Ranavirus in Brazil.

Ranavirus is a double-stranded DNA virus associated with amphibian, fish and reptile die-offs worldwide. International trade of live animals farmed for human consumption, such as the American bullfrog (Lithobates catesbeianus), plays a key role in spreading the pathogen. In Brazil, ranavirus has only been reported in captive bullfrog farms. We found infected tadpoles of both native species and the American bullfrog in the wild, and a case of mass mortality of amphibians and fish potentially associated with ranavirus. Dead animals presented skin ulcerations, hemorrhages, and edemas. We also found an overall prevalence of 37% of the amphibian chytrid in the area, and two bullfrog tadpoles were co-infected with both pathogens. We suggest that the interaction between the two pathogens should be investigated to improve global conservation of ectothermic vertebrates.

Pathogen Risk Analysis for Wild Amphibian Populations Following the First Report of a Ranavirus Outbreak in Farmed American Bullfrogs (Lithobates catesbeianus) from Northern Mexico.

Ranaviruses are the second deadliest pathogens for amphibian populations throughout the world. Despite their wide distribution in America, these viruses have never been reported in Mexico, the country with the fifth highest amphibian diversity in the world. This paper is the first to address an outbreak of ranavirus in captive American bullfrogs (Lithobates catesbeianus) from Sinaloa, Mexico. The farm experienced high mortality in an undetermined number of juveniles and sub-adult bullfrogs. Affected animals displayed clinical signs and gross lesions such as lethargy, edema, skin ulcers, and hemorrhages consistent with ranavirus infection. The main microscopic lesions included mild renal tubular necrosis and moderate congestion in several organs. Immunohistochemical analyses revealed scant infected hepatocytes and renal tubular epithelial cells. Phylogenetic analysis of five partial ranavirus genes showed that the causative agent clustered within the Frog virus 3 clade. Risk assessment with the Pandora⁺ protocol demonstrated a high risk for the pathogen to affect amphibians from neighboring regions (overall Pandora risk score: 0.619). Given the risk of American bullfrogs escaping and spreading the disease to wild amphibians, efforts should focus on implementing effective containment strategies and surveillance programs for ranavirus at facilities undertaking intensive farming of amphibians.

Infection and co-infection by the amphibian chytrid fungus and ranavirus in wild Costa Rican frogs.

Amphibian populations are globally threatened by emerging infectious diseases, and 2 pathogens in particular are recognized as major threats: the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) and ranaviruses. Here, we evaluated the prevalence of infection by Bd and ranavirus in an assemblage of frogs from a lowland wet forest in Costa Rica. We found an overall prevalence of 21.3% for Bd and 16.6% for ranavirus, and detected both pathogens widely among our 20 sampled species. We found a positive association between ranavirus and Bd infection in one of our 4 most commonly sampled species. We also found a positive but non-significant association between infection by ranavirus and infection by Bd among species overall. Our study is among the first detailed evaluations of ranavirus prevalence in the American tropics, and to our knowledge is the first to detect a positive association between Bd and ranavirus in any species. Considerable research attention has focused on the ecology of Bd in tropical regions, yet we argue that greater research focus is necessary to understand the ecology and conservation impact of ranaviruses on amphibian populations already decimated by the emergence of Bd.

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.

Isolation and characterization of iridoviruses from the giant toad Bufo marinus in Venezuela.

In this communication we describe for the first time the isolation of 7 iridoviruses from the toad Bufo marinus and an unknown species of frog Leptodactylus in Venezuela, South America. The viruses are icosahedral with electron-dense cores, each of which is surrounded by an inner membrane, capsid and a cell-derived envelope. The virus(es) have an average vertex to vertex diameter of 160 nm and replicate in the cytoplasm of a range of cell lines. Within the cytoplasm of infected cells, rarefied areas could be observed; structures lacked cellular organelles and contained complete, empty and developing viruses. Results from antigen-capture enzyme-linked immunosorbent assays (ELISA) with polyclonal antibody raised against epizootic haematopoietic necrosis virus (EHNV) indicated cross-reactivity between these isolates, Bohle iridovirus (BIV) and frog virus 3 (FV3). Comparison of polypeptide and genomic profiles indicated that the Venezuelan viruses shared many polypeptides of equivalent molecular weight with type species FV3. There were, however, differences between the group of Venezuelan viruses and FV3 and BIV. The viruses belongs to the family Iridoviridae and the genus Ranavirus.