Phylogenomic characterization of ranaviruses isolated from cultured fish and amphibians in Thailand

Globally, aquaculture is an essential food-producing sector and it has played a crucial role in the Thai economy for decades (Food and Agriculture Organization of the United Nations 2020). Between 1998 and 2017, Thailand’s average annual aquaculture production was approximately 1.1 million tons per year, with freshwater aquaculture production contributing nearly half of the annual production (Department of Fisheries Thailand 2019a). Of the total Thai freshwater aquaculture production in 2017, 4000 tons were contributed from ranaculture products including live frogs, chilled and frozen frog legs, and whole frogs (Department of Fisheries Thailand 2019b). However, diseases including those caused by ranaviruses have emerged as an impediment to the continued growth of ranaculture around the world including Asia (Zhang et al. 2001; Weng et al. 2002), North America (Miller et al. 2007), and South America (Mazzoni et al. 2009).

Members of the genus Ranavirus (family Iridoviridae; subfamily Alphairidovirinae) possess an enveloped nucleocapsid (150–200 nm in diameter) that encloses a double-stranded DNA genome (103–220 kbp) (Chinchar et al. 2017). The genus includes the following species recognized by the International Committee on Taxonomy of Viruses: Frog virus 3 (FV3), Ambystoma tigrinum virus, Common midwife toad virus, Epizootic haematopoietic necrosis virus (EHNV), Santee-Cooper ranavirus, and Singapore grouper iridovirus (International Committee on Taxonomy of Viruses 2020). Ranaviruses are emerging pathogens infecting at least 175 species and 52 families of ectothermic vertebrates (fish, amphibians, and reptiles) across six continents (Duffus et al. 2015). FV3 has the widest-known host and geographic range of all ranaviruses and includes Asian strains isolated from diseased fish, amphibians, and reptiles reared for food in China and Thailand. FV3 strains have negatively impacted the culture of tiger frogs (Hoplobatrachus tigerinus) and East Asian bullfrogs (H. rugulosus) in Thailand and China (Kanchanakhan et al. 1998, 1999; Weng et al. 2002), Chinese softshell turtles (Pelodiscus sinensis) in China (Chen et al. 1999), pig frogs (Lithobates grylio) in China (Zhang et al. 2001), and marbled sleeper goby (Oxyeleotris marmorata) in Thailand (Prasankok et al. 2005). Ranaviruses have been identified as a global threat to free-ranging amphibian populations as they have the potential to cause population declines and extinctions (Duffus and Cunningham 2010; Teacher et al. 2010; Miller et al. 2011; Earl and Gray 2014; Price et al. 2014; Peace et al. 2019). Ranaviral infections in amphibians and EHNV infection in rainbow trout (Oncorhynchus mykiss) and redfin perch (Perca fluviatilis) are notifiable to the World Organization for Animal Health (2019).

Over the last two decades, strains of FV3 have repeatedly been isolated during mortality events in ranaculture and aquaculture facilities in Thailand. From 1998 to 2002, ranaculture facilities in nine provinces in the central part of Thailand experienced moderate (20%–50%) to high (95%) mortalities in cultured tadpoles and juvenile and adult tiger frogs (Kanchanakhan 1998, 2011; Kanchanakhan et al. 1999). Diseased frogs displayed ulcerative lesions on the rostrum (lips and mouth) and dorsal part of the body and legs. Bacteria were not isolated from the liver, kidney, and spleen in the early stages of the disease; however, bacteria were cultured from frogs that experienced chronic high mortality infections. Skin and internal tissue homogenates from diseased frogs and tadpoles were processed for viral isolation in epithelioma papulosum cyprini (EPC) cells and resulted in the recovery of passageable agents in 65% (70/107) of the individuals tested from 1998 to 2002 (Kanchanakhan 2011). Physicochemical and ultrastructural features of the isolates supported them as members of the family Iridoviridae. In 2004, high mortality (>50%) in Asian grass frogs (Fejervarya limnocharis) was reported on a private fish farm in Northeast Thailand following importation from Cambodia (Kanchanakhan et al. 2004). The diseased frogs showed skin ulcerations on the rostrum and legs. A replicating agent was isolated in EPC cultures and determined to be an iridovirus based on its physicochemical and ultrastructural features. PCR amplification and sequencing of the iridoviral partial major capsid protein (MCP) and ATPase genes demonstrated it was nearly identical (98%–99%) to a strain of FV3 known as tiger frog virus (TFV), previously isolated from diseased tiger frog tadpoles cultured in China in 2000 (Weng et al. 2002) and hereafter referred to as Chinese TFV. In 2000, high mortalities in cultured marbled sleeper goby were reported in the Nakonpathom province of Central Thailand (Prasankok et al. 2005). Fish displayed ulcerative lesions on the body and around the mouth. No consistent parasites or bacteria were observed or cultured from the diseased fish. A replicating agent was isolated in EPC cultures and again determined to be an iridovirus based on its physicochemical and ultrastructural features. PCR amplification and sequencing of the iridoviral partial MCP gene demonstrated the isolate was nearly identical (98%–99%) to the aforementioned Chinese TFV and Thai ranaviruses isolated from imported Asian grass frogs and cultured tiger frogs.

Herein, we describe the in vitro growth (i.e., observed cytopathic effects), ultrastructural, and phylogenomic characteristics of ranaviruses isolated from fish and amphibians cultured in Thailand for food or ornamental purposes. The determined complete genome sequences are the first to be publicly available for ranaviruses isolated from Thai ectothermic vertebrates. This genomic data was used in phylogenomic analyses that revealed TFV, a strain of FV3, has spread among facilities in Asia rearing fish and amphibians, and perhaps also in free-ranging reptiles in Oceania.

In this investigation, we report the complete genome sequences of eight ranaviruses isolated from cultured fish and amphibians in Thailand. The growth of the Thai ranaviruses in EPC cells facilitated their ultrastructural and phylogenomic characterization. The induction of CPE in EPC cultures (i.e., progression of coalescing round plaques) and the observed ultrastructural features of the viral particles (i.e., hexagonal viral particles within the cytoplasm and enveloped extracellular virions) were congruous with a previous report of TFV in tiger frogs in China (Weng et al. 2002) and expectations for ranaviruses (International Committee on Taxonomy of Viruses 2020). Phylogenomic analyses supported the Thai ranaviruses as a unique subclade (hereafter referred to as the TFV subclade) within the larger FV3 clade. To our knowledge, this study represents the only genetic characterization of ranaviruses in Thailand and adds to a growing body of literature on the expanding host range and potential threat ranaviruses pose to cultured fish and amphibians.

Our phylogenomic analyses strongly supported the TFV subclade as the sister group to a clade composed of three separate subclades: the Bohle iridovirus subclade (e.g., BIV, GGRV, ZRV), a second subclade with isolates from Chinese frogs (RGV, RNRV) and turtles (STIV), and a third subclade with strains from North American reptiles (TMRV1, TMRV2, TCCRV) and amphibians (FV3, SSME) and a European frog (FV3-Op). Further support for TFVs as strains of FV3 was evident by the fact that they all possess the previously defined FV3-like genome arrangement (Claytor et al. 2017). A shared derived feature (i.e., synapomorphy) that delineates TFVs from other ranaviruses, including other FV3 strains, is the acquisition of a putative gene (TFV ORF 28) that is predicted to encode a protein of unknown function. A second synapomorphy is the absence of FV3 ORF 30R (GenBank Accession No. NC005946) in TFV isolates. Our sequencing of eight Thai TFVs revealed a single nucleotide deletion resulting in the merger of two ORFs (FV3 ORFs 49L and 50L) as previously reported in the following FV3 strains: spotted salamander Maine (SSME), Rana grylio virus (RGV), and soft-shelled turtle iridovirus (STIV) (Morrison et al. 2014). In contrast, the Chinese TFV and FV3 lacked this deletion and resulting gene merger. The biological significance of these TFV gene gains, losses, and merger events remain to be determined.

The ranaviral-infected marbled sleeper goby, tadpoles, and frogs from farms in Thailand displayed cutaneous ulcers, edema (tadpoles), and no observable internal gross lesions (Kanchanakhan 1998, 2011; Kanchanakhan et al. 1999, 2004; Prasankok et al. 2005; J. Polchana, personal observation, 2017) (Fig. 1). In contrast, cutaneous ulcerative lesions were not observed in East Asian bullfrog tadpoles or frogs during TFV outbreaks on farms in China (Weng et al. 2002). The disease in cultured tadpoles in China was named “abdominal distension disease” as a result of the observed enlargement of the liver, kidney, and spleen. Petechial hemorrhages were also noted on the serosal surfaces of the enlarged internal organs. Although differences in gross lesions have been reported in cultured tadpoles and frogs on farms in China versus Thailand, the cutaneous ulceration (Thailand) and petechial hemorrhages along the serosal surfaces of enlarged internal organs (China) are all considered classic gross lesions associated with ranaviral-infected tadpoles and frogs (Miller et al. 2015). Furthermore, the tissue sections from diseased Chinese tadpoles and frogs displayed microscopic lesions consistent with ranaviral infections in amphibians including diffuse hepatocellular necrosis and focal necrosis in the kidney (Weng et al. 2002; Miller et al. 2015). Although histopathological examination of Thai TFV-infected tadpole and frog tissues has not been reported, the observation of cutaneous ulcerative lesions paired with the high prevalence of ranaviruses isolated from diseased tadpoles and frogs suggests TFV is likely a significant source of mortality in cultured amphibians in Thailand (Kanchanakhan 1998, 2011; Kanchanakhan et al. 1999, 2004; Prasankok et al. 2005). Controlled laboratory studies are needed to elucidate the role Thai TFV strains play in disease of cultured frog and fish species.

In this study, the TFV isolates from goldfish (F0207) and guppy (F2112) were recovered from overtly healthy fish during routine health surveys conducted at AAHRDD (S. Kanchanakhan, personal observation, 2001, 2002). Similarly, strains of FV3 have previously been reported from outwardly healthy fathead minnow (Pimephales promelas) and northern pike (Esox lucius) during surveillance efforts targeting viral haemorrhagic septicemia virus in the United States (Waltzek et al. 2014). Experimental challenge studies have resulted in little to no mortality in black bullhead (Ameiurus melas), bluegill (Lepomis macrochirus), channel catfish (Ictalurus punctatus), common carp (Cyprinus carpio), fathead minnow, goldfish (Carassius auratus), mosquito fish (Gambusia affinis), Nile tilapia (Oreochromis niloticus), northern pike (Esox Lucius), and pike-perch (Sander lucioperca) following exposure to FV3 isolates (Bang Jensen et al. 2009, 2011a, 2011b, Gobbo et al. 2010; Brenes et al. 2014a, 2014b). Similarly, experimental challenge studies resulted in no mortality in grass carp (Ctenopharyngodon Idella), goldfish, and mandarin fish (Siniperca chautsi) following injection of a Chinese TFV isolate (Weng et al. 2002). In contrast, farm-reared sturgeon species, including the highly endangered pallid sturgeon (Scaphirhynchus albus), appear highly susceptible to FV3 (Waltzek et al. 2014). Although few fish species appear highly susceptible to strains of FV3, at least some fish become infected and can transmit the virus to frog tadpoles under laboratory conditions (Brenes et al. 2014a).

Tiger frog virus was originally characterized from a disease episode in cultured tiger frog tadpoles and frogs in China in 2000 (He et al. 2002; Weng et al. 2002). The present study confirms that strains of TFV have been circulating in cultured fish and amphibian populations in Thailand from 1998 to 2017. Our analysis of the major capsid protein of the WV, isolated from diseased green pythons confiscated during their attempted importation into Australia from Papua New Guinea in 1998, suggests that TFVs may be circulating more widely, including in wild squamate reptiles in Oceania (Hyatt et al. 2002). Like other strains of FV3, TFVs are promiscuous pathogens capable of infecting three classes of vertebrates including bony fish, amphibians, and reptiles (Chinchar and Waltzek 2014). Experimental studies have demonstrated interclass transmission can occur by cohabitation of FV3-infected Cope’s treefrog tadpoles (Hyla chrysoscelis) with naïve red-eared sliders (Trachemys scripta elegans) and cohabitation of FV3-infected mosquitofish (Gambusia affinis) or FV3-infected red-eared sliders with naïve Cope’s treefrog tadpoles (Brenes et al. 2014a). In Thailand, opportunities for interclass transmission of TFVs may occur at large pet markets where tadpoles and frogs are housed in open containers next to ornamental fishes. The aquaculture practice of feeding juvenile frogs to increase the growth of large predatory ornamental fishes may have also contributed to the spread of TFVs in Thailand (S. Kanchanakhan, personal observation, 2016).

Continued TFV surveillance efforts are needed to monitor any impact these ranaviruses may have on Thai aquatic animal industries. Improved biosecurity programs should be implemented (e.g., discontinuing the practice of feeding live juvenile frogs to predatory fishes) to reduce the impact of infectious diseases on Thai farms and pet markets. Although TFVs have only been detected in Asia and likely in Oceania, their promiscuous nature coupled with the largely unregulated international trade in live animals raises concerns that they might emerge in more distant ranaculture and aquaculture markets or wildlife populations (Picco and Collins 2008; Schloegel et al. 2009; Kolby et al. 2014).

We would like to thank Yuwanda Yeeka at the Aquatic Animal Research and Development Division, Department of Fisheries, Thailand, for her assistance in cell culture.