Ranaviruses are emerging pathogens associated with worldwide epizootics in farmed and wild ectothermic vertebrates. In this study, we determined the full genomes of eight ranaviruses isolated from marbled sleeper goby ( Oxyeleotris marmorata), goldfish ( Carassius auratus), guppy ( Poecilia reticulata), tiger frog ( Hoplobatrachus tigerinus), Asian grass frog ( Fejervarya limnocharis), and East Asian bullfrog ( H. rugulosus) cultured or imported into Thailand. These ranaviral isolates induced the same cytopathic effects (i.e., progression of coalescing round plaques) in epithelioma papulosum cyprini (EPC) cell cultures. Transmission electron microscopy of infected EPC cells revealed cytoplasmic viral particles with ultrastructural features typical for ranaviruses. Pairwise genetic comparisons of the complete major capsid protein coding sequences from the Thai ranaviruses displayed the highest identity (99.8%–100%) to a ranavirus (tiger frog virus; TFV) isolated from diseased tiger frogs cultured in China, a slightly lower identity (99.3%–99.4%) to a ranavirus (Wamena virus; WV) isolated from diseased green tree pythons ( Morelia viridis) illegally exported from Papua New Guinea, and a lower identity to 35 other ranaviruses (93.7%–98.6%). Phylogenomic analyses supported the eight Thai ranaviruses, Chinese TFV, and WV as a subclade within a larger frog virus 3 clade. Our findings confirm the spread of TFV among cultured fish and amphibians in Asia and likely in reptiles in Oceania. Biosecurity measures are needed to ensure TFV does not continue to spread throughout Southeast Asia and to other parts of the world via international trade.

Over the last decade, a number of USA aquaculture facilities have experienced periodic mortality events of unknown aetiology in their clownfish (Amphiprion ocellaris). Clinical signs of affected individuals included lethargy, altered body coloration, reduced body condition, tachypnea, and abnormal positioning in the water column. Samples from outbreaks were processed for routine parasitological, bacteriological, and virological diagnostic testing, but no consistent parasitic or bacterial infections were observed. Histopathological evaluation revealed individual cell necrosis and mononuclear cell inflammation in the branchial cavity, pharynx, oesophagus and/or stomach of four examined clownfish, and large basophilic inclusions within the pharyngeal mucosal epithelium of one fish. Homogenates from pooled external and internal tissues from these outbreaks were inoculated onto striped snakehead (SSN-1) cells for virus isolation and cytopathic effects were observed, resulting in monolayer lysis in the initial inoculation and upon repassage. Transmission electron microscopy of infected SSN-1 cells revealed small round particles (mean diameter=20.0-21.7 nm) within the cytoplasm, consistent with the ultrastructure of a picornavirus. Full-genome sequencing of the purified virus revealed a novel picornavirus most closely related to the bluegill picornavirus and other members of the genus Limnipivirus. Additionally, pairwise protein alignments between the clownfish picornavirus (CFPV) and other known members of the genus Limnipivirus yielded results in accordance with the current International Committee on Taxonomy of Viruses criteria for members of the same genus. Thus, CFPV represents a proposed new limnipivirus species. Future experimental challenge studies are needed to determine the role of CFPV in disease.

Ranaviruses are large double-stranded DNA viruses within the genus Ranavirus (family Iridoviridae) that are being detected with increasing frequency among aquacultured and wild fishes. In the USA, multiple sturgeon hatcheries have experienced ranavirus epizootics resulting in significant morbidity and mortality in young-of-year (YOY). Significant economic losses have resulted from repeated outbreaks of frog virus 3 (FV3), the type species for the genus Ranavirus, in YOY pallid sturgeon Scaphirhynchus albus reared at a hatchery within the Missouri River Basin. Water temperature and stocking density are known to influence the severity of ranavirus disease in ectothermic vertebrates. To determine the effect of water temperature on ranavirus disease in hatchery-raised S. albus, we conducted FV3 challenges at 2 temperatures (17 and 23°C) and compared cumulative survival over a 28 d study period. A mean (±SE) survival rate of 57.5 ± 13.2% was observed in replicate tanks of sturgeon maintained at 23°C, whereas no mortality was observed among sturgeon maintained at 17°C. In a second challenge study, we compared the effect of water temperature on disease progression by regularly sampling fish over the study period and evaluating lesions by histopathology and in situ hybridization, and by assessing viral titer and load in external and internal tissues using virus isolation and qPCR, respectively. Results suggest that temperature manipulation may be an effective mitigation strategy that sturgeon hatcheries can employ to minimize ranavirus-associated disease.

Tilapia lake virus disease (TiLVD) is an emerging viral disease associated with high morbidity and mortality in cultured tilapia worldwide. In this study, we have developed and validated a TaqMan quantitative reverse transcription PCR (RT-qPCR) assay for TiLV, targeting a conserved region within segment 10 of the genome. The RT-qPCR assay was efficient (mean ± SD: 96.71 ± 3.20%), sensitive with a limit of detection of 10 RNA viral copies per reaction, and detected TiLV strains from different geographic regions including North America, South America, Africa, and Asia. The intra- and inter-assay variability ranged over 0.18-1.41% and 0.21-2.21%, respectively. The TaqMan RT-qPCR assay did not cross-react with other RNA viruses of fish, including an orthomyxovirus, a betanodavirus, a picornavirus, and a rhabdovirus. Analysis of 91 proven-positive and 185 proven-negative samples yielded a diagnostic sensitivity of 96.7% and a diagnostic specificity of 100%. The TaqMan RT-qPCR assay also detected TiLV RNA in infected Nile tilapia liver tissue extracts following an experimental challenge study, and it successfully detected TiLV RNA in SSN-1 (E-11 clone) cell cultures displaying cytopathic effects following their inoculation with TiLV-infected tissue homogenates. Thus, the validated TaqMan RT-qPCR assay should be useful for both research and diagnostic purposes. Additionally, the TiLV qPCR assay returns the clinically relevant viral load of a sample which can assist health professionals in determining the role of TiLV during disease investigations. This RT-qPCR assay could be integrated into surveillance programs aimed at mitigating the effects of TiLVD on global tilapia production.

Ranaviruses were isolated from wild edible frogs (Pelophylax esculentus) during epizootics in Denmark and Italy. Phylogenomic analyses revealed that these isolates are closely related and belong to a clade of ranaviruses that includes the Andrias davidianus ranavirus (ADRV), common midwife toad ranavirus (CMTV), Testudo hermanni ranavirus (THRV), and pike-perch iridovirus (PPIV).