Ranaviruses in the genus Ranavirus, Family Iridoviridae, can infect ectothermic organisms, including fishes, amphibians, and reptiles (Duffus et al. 2015; Jancovich et al. 2015). Ranavirus has extirpated local populations of amphibians in many countries and is known to be among the major causes of global amphibian declines (Brunner et al. 2021; Robert 2010). Currently, there are seven species belong to the genus Ranavirus, and Frog virus 3 (FV3), Ambystoma tigrinum virus (ATV), and Common midwife toad virus (CMTV) are representative species (Jancovich et al. 2015; Walker et al. 2021). FV3 was first discovered in leopard frogs (Granoff et al. 1965) and is known to infect a variety of amphibians, fishes, and reptiles (Duffus et al. 2015; Jancovich et al. 2015; Price et al. 2017; Waltzek et al. 2014). The most widely distributed virus FV3 is reported in almost all parts of the world, including the United States, Canada, Central and South America, Europe, Asia, and Africa (reviewed in Duffus et al. 2015). An elevated response level is needed to cope with increasing amphibian population declines due to ranavirus infections. This should include rigorous reporting of ranavirus infections and thorough genetic characterization of identified ranaviruses to allow global tracking of infection situations.

Recently, ranavirus-related research has increased in Asia, where research has been needed. Although most studies have been conducted in China, ranavirus-induced mass amphibian mortalities have also been reported in Japan, Taiwan, Korea, Malaysia, India, and Russia (Hazeri et al. 2017; Herath et al. 2021; Huang et al. 2011; Hsieh et al. 2021; Lisachov and Lisachova 2022; Mu et al. 2018; Sivasankar et al. 2017; Une et al. 2009; Xu et al. 2010). The first South Korean ranavirus-induced mass mortality was confirmed in 2009 involving larval gold-spotted pond frogs (Pelophylax chosenicus) (Kim et al. 2009). Since then, a total of seven cases have been reported, including infections of Rana uenoi, R. huanrenensis, Kaloula borealis, P. nigromaculatus, Dryophytes japonicus, and Lithobates catesbeianus (Kwon et al. 2017; Park et al. 2017; Park et al. 2021; Roh et al. 2022). Among the seven cases, three were confirmed by verification of a partial ranavirus major capsid protein (MCP) gene sequence (Kim et al. 2009; Kwon et al. 2017; Park et al. 2021). A recent report regarding the level of ranavirus prevalence using qPCR targeted three amphibian species (P. nigromaculatus, D. japonicus, and L. catesbeianus) inhabiting agricultural areas (Roh et al. 2022). In this study, between 16.1% and 50.0% of sampled amphibians were infected, varying by species. Prior to conducting a comprehensive genome study of ranavirus in South Korea, it is necessary to analyze the MCP gene sequences of recently isolated ranaviruses to allow an evaluation of their genetic relationship with previously reported ranaviruses and of existence of multiple ranavirus strains across different amphibian species in South Korea.

The purposes of this study were to obtain the partial MCP sequences of the ranavirus that infects black-spotted pond frogs (P. nigromaculatus) and American bullfrogs (L. catesbeianus) inhabiting rice paddies, and to preliminarily evaluate their genetic relationship with ranaviruses previously reported in South Korea and abroad.

For this study, we selected larvae of P. nigromaculatus and L. catesbeianus, which were confirmed to be subjected to ranavirus infection through qPCR, with a low cycle threshold (CT) value of 32 or less in our previous study (Roh et al. 2022). For PCR, we used tadpole genomic DNA, which was extracted from the liver tissue, using a DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany), and stored at –80°C (Roh et al. 2022). Before PCR, we measured the concentration of extracted DNA using a Qubit3 Fluorometer (Invitrogen, Waltham, MA, USA) with a Qubit 1X dsDNA HS Assay Kit (Invitrogen) and adjusted it to a 1 ng/µL concentration with molecular biology grade water (Cytiva Korea, Incheon, Korea).

To amplify the partial MCP gene (500 bp), we used MCP4 and MCP5 primers, as used in our previous studies (Kwon et al. 2017; Mao et al. 1997; Park et al. 2021). We conducted PCR in a total volume of 20 µL, which contained 10 µL 2X TOPsimple PreMix nTaq (Enzynomics, Daejeon, Korea), 0.5 µL forward primer, 0.5 µL reverse primer, and 9 µL DNA template, using a SimpliAmp thermal cycler (Applied Biosystems, Waltham, MA, USA). All PCRs were performed with negative control of molecular biology grade water (Cytiva Korea). Targeted PCR products were confirmed on a 1.0% agarose gel, and the partial MCP sequences were obtained using an ABI 3730xl System (Applied Biosystems) at Macrogen, Inc. (Seoul, Korea).

We checked the resulting MCP sequences using Geneious Prime (Biomatters Ltd, Auckland, New Zealand). To examine the genetic relationship between the verified ranavirus types in this study and other previously known ranaviruses, we performed Basic Local Alignment Search Tool (BLAST) analysis and constructed a phylogenetic tree. For the custom nested BLAST analysis, we downloaded 39 partial MCP sequences of representative ranaviruses from the GenBank, such as the viruses previously reported from South Korea, including FV3-like viruses, CMTV-like viruses, and ATV-like viruses, and aligned the sequences using the default setting of MUSCLE (multiple sequence comparison by log‐expectation) (Edgar 2004). We performed BLAST using Geneious Prime (Biomatters Ltd). For the lineage-based genetic relationship analysis, we constructed a Bayesian inference (BI) tree using the Markov chain Monte Carlo (MCMC) methods in MrBayes v3.2.4 (Ronquist et al. 2012). We ran 60 million tree generations, starting with a random tree, while saving every 1,000th tree into a file and discarding the first 5% of the sampled generations as burn-ins. For the analysis, we included 44 ranavirus MCP sequences, which were used for the BLAST, and the MCP sequence of short-finned eel ranavirus (FJ358612) was used as an outgroup.

Among the ten tadpoles that we previously analyzed to confirm ranavirus infection in a previous study (Roh et al. 2022), we successfully obtained partial sequences (506 bp) of ranavirus MCP gene from four of the P. nigromaculatus and two L. catesbeianus tadpoles. The MCP sequences of the ranavirus from three P. nigromaculatus (one collected from Pohang and two from Yeongam) and one L. catesbeianus (from Gimje) were identical (called Type A hereafter, accession number OP009373-009376). Ranavirus from one P. nigromaculatus (from Hongcheon, Type B, accession number OP009377) and from one L. catesbeianus (from Imja, Type C, accession number OP009378) had a unique MCP sequence (Table 1, Fig. 1).

Table 1 . Results of the custom nested BLAST using partial MCP DNA sequences (506 bp) of the three ranavirus types (Type A, B, and C) from Pelophylax nigromaculatus and Lithobates catesbeianus in this study.

Sequence (accession number)	Host species	Country	Identical sites (%)
			Type A	Type B	Type C
Type A (OP009373-009376)	Pelophylax nigromaculatus, Lithobates catesbeianus	Korea		99.4	99.6
Type B (OP009377)	Pelophylax nigromaculatus	Korea	99.4		99.8
Type C (OP009378)	Lithobates catesbeianus	Korea	99.6	99.8
Rana catesbeiana virus (AB474588)	Rana catesbeiana	Japan	99.6	99.8	100.0
KRV-1 (HM133594)	Pelophylax chosenicus	Korea	99.6	99.8	100.0
Rana grylio iridovirus (JQ654586)	Rana grylio	China	99.6	99.8	100.0
Rana catesbeiana virus (KC756964)	Rana catesbeiana	Taiwan	99.4	99.6	99.8
Soft shelled turtle iridovirus (DQ335253)	Trionyx sinensis	China	99.4	99.6	99.8
Rana grylio iridovirus (KY264204)	Rana huanrenensis	Korea	99.4	99.6	99.8
Frog virus 3 (MH332773)	Lithobates sp.	Canada	99.4	100.0	99.8
Frog virus 3 (KF646249)	Scaphirhynchus albus	USA	99.4	100.0	99.8
Frog virus 3 (MF360246)	Oophaga pumilio	Netherlands	99.4	100.0	99.8
Frog virus 3 (MF411070)	Rana temporaria	UK	99.4	100.0	99.8
Lacerta monticola ranavirus (KM516719)	Lacerta monticola	Portugal	99.4	100.0	99.8
Rana grylio iridovirus (KY264205)	Rana dybowskii Rana huanrenensis	Korea	99.2	99.4	99.6
Frog virus 3 (DQ897669)	Rana catesbeiana	Brazil	99.0	99.6	99.4
Bohle iridovirus (FJ358613)	Limnodynastes ornatus	Australia	99.0	99.2	99.4
Zoo ranavirus (KF699143)	Anaxyrus boreas boreas	USA	98.8	99.0	99.2
German gecko ranavirus (KP266742)	Uroplatus fimbriatus	Germany	98.8	99.0	99.2
Common midwife toad virus (JQ231222)	Alytes obstetricians	Spain	98.6	98.8	99.0
Common midwife toad virus (MF125269)	Pelophylax esculentus	Netherlands	98.4	98.6	98.8
Tiger frog virus (MT512504)	Hoplobatrachus tigerinus	Thailand	98.4	98.6	98.8
Tortoise ranavirus (KM516713)	Testudo hermanni	Germany	98.2	98.4	98.6
Pike perch iridovirus (FJ358610)	Stizostedion lucioperca	Finland	98.2	98.4	98.6
Blood python ranavirus (KM516715)	Python brongersmai	Germany	98.0	98.2	98.4

BLAST: Basic Local Alignment Search Tool; MCP: major capsid protein.

Reference ranavirus sequences were downloaded from GenBank.

Figure 1. Bayesian inference (BI) tree (A) based on the partial major capsid protein (MCP) DNA sequences (506 bp) of the ranaviruses from Pelophylax nigromaculatus and Lithobates catesbeianus in this study and the 39 sequences from GenBank (accession number) and the detected location (B) of six different FV3-like ranavirus types, which reported in South Korea to date. In BI tree, bold indicates the ranavirus previously reported in South Korea, and blue indicates the ranavirus identified in this study. Each different ranavirus type is indicated on the map with distinct colors.

In the BLAST analysis, Type A sequence showed 99.6% similarity with KRV-1 from South Korea (HM133594), Rana catesbeiana virus from Japan (AB474588), and Rana grylio iridovirus from China (JQ654586), and Type C sequence showed 100% similarity with these three virus types. Type B sequence showed 100% similarity with FV3 (MH332773, KF646249, MF360246, and MF411070) reported in Canada, the United States, the Netherlands, and the United Kingdom and with Lacerta monticola ranavirus (KM516719) reported in Portugal. The similarity between the three MCP sequence types in this study and the two types (KY264204-5), which were previously reported from two mountain frog species (R. uenoi and R. huanrenensis) in South Korea (Kwon et al. 2017; Park et al. 2021), ranged from 99.2% to 99.8% (Table 1). In the genetic relationship analysis, the three types of partial MCP gene sequences obtained in this study were all placed within the FV3-like ranavirus group in the genus Ranavirus (Fig. 1).

In this study, we successfully obtained the partial MCP gene sequence of the ranavirus from four P. nigromaculatus and two L. catesbeianus. As a result, in South Korea, ranavirus infection was confirmed by MCP gene sequence verification in a total of five anuran species (P. chosenicus, R. uenoi, R. huanrenensis, P. nigromaculatus, L. catesbeianus) (Kim et al. 2009; Kwon et al. 2017; Park et al. 2021; Roh et al. 2022). In particular, this study is the first report on the detection of FV3-like ranavirus in domestic wild American bullfrogs through MCP sequence confirmation in South Korea. In addition, we confirmed, through comparison of partial MCP sequences, that all six types of ranavirus MCP sequence identified in South Korea thus far belong to the FV3-like virus group in the genus Ranavirus.

Ranaviruses belonging to the FV3-like virus group in the genus Ranavirus are dominantly responsible for infecting Korean anuran amphibians. In the genetic relationship analysis, three newly discovered ranavirus MCP types and four previously reported ranavirus MCP types in South Korea were all placed within the FV3-like virus group. In the genus Ranavirus, Family Iridoviridae, FV3, ATV, CMTV, etc., are known to infect amphibians (Granoff et al. 1965; Jancovich et al. 2015; Speare and Smith 1992). In particular, FV3 infects not only amphibians but also fishes (Price et al. 2017) and reptiles (Waltzek et al. 2014), but there have been no such reports in fishes and reptiles in South Korea (Do et al. 2005; Kim et al. 2013; Lee et al. 2019). FV3 has been detected in various wild and cultured amphibian populations throughout the United States and Canada (Duffus et al. 2015; Price et al. 2017) and from introduced American bullfrogs in Brazil, the United Kingdom, Japan, and Taiwan (Ferreira et al. 2021; Gray et al. 2007; Hsieh et al. 2021; Une et al. 2009). Our results show that FV3-like ranaviruses in the genus Ranavirus could be the dominant ranavirus species infecting anuran amphibians in South Korea.

All six types of ranavirus MCP sequence reported in Korean amphibians to date belong to the FV3-like virus group with genetic variations. According to previous studies, genetic variations in FV3-like viruses occur frequently (Stöhr et al. 2015), caused by either genetic recombination between viruses of different strains or the process of adapting to different hosts and environments over time (Cronin et al. 2010; Grant et al. 2019; Vilaça et al. 2019). Regarding the possibility of interspecies genetic recombination, evaluation is not currently possible in South Korea. Unfortunately, studies on iridoviruses have not been conducted in other ectothermic organisms that cohabitate with amphibians. The genetic variations in FV3-like viruses due to the adaptation process may be applicable to our results. In this study, various types of ranavirus MCP sequence, within the FV3-like virus group, tended to differ according to host species and collection region. For example, virus MCP sequence types identified from R. uenoi and R. huanrenensis in the mountain valleys were more closely related to each other. These results suggest that the observed MCP sequence diversity of the ranaviruses in South Korea may be caused by differences in host species and their habitats.

In this study, we confirmed ranavirus infection in two more anuran species (P. nigromaculatus and L. catesbeianus) in South Korea through MCP sequencing. Additionally, we showed the genetic variety and phylogenetic positions of the ranavirus MCP sequences identified in this study and previous studies from South Korea. Considering increasing cases of ranavirus-induced mass mortalities of amphibians in South Korea (Kim et al. 2009; Kwon et al. 2017; Park et al. 2017; Park et al. 2021), further infection reports and full genome analyses of the identified ranaviruses are urgently needed to better understand the origin and spread of ranavirus across Korean amphibian populations.

We thank Woo-Jin Choi, Il-Kook Park, Ji-Ho Park for their help during field sampling and MinWoo Park, Ho-Jun Jung for their help during the dissection.

ATV: Ambystoma tigrinum virus

BI: Bayesian inference

BLAST: Basic Local Alignment Search Tool

CMTV: Common midwife toad virus

CT: Cycle threshold

FV3: Frog virus 3

MCMC: Markov chain Monte Carlo

MCP: Major Capsid Protein

JK did data curation, formal analysis, investigation, and writing-original draft. NHR did data curation, investigation, formal analysis, and writing-review and editing. JP and DP did conceptualization, funding acquisition, supervision, writing-original draft, and writing-review and editing.

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2020R1A6A3A1306094911).

All data generated or analyzed during this study are included in this published article.

This study was reviewed and approved by the Institutional Animal Care and Use Committee of Kangwon National University (KW-200618-3).

Not applicable

The authors declare that they have no competing interests.