Prevalence of Ranavirus Infection in Three Anuran Species across South Korea

Abstract

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.

Comparison of detective ranavirus with major capsid protein gene from infected frogs (Pelophylax nigromaculatus and Lithobates catesbeianus) in South Korea

“…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.…”

“…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).…”

“…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).…”

“…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).…”

“…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).…”