Molecular Characterization of Megalocytiviruses from Diseased Fishes in Korean Aquatic Farms from 2013 to 2017

Ⅰ. Introduction

Members of the family Iridoviridae genus Megalocytivirus are icosahedral viruses that contain a double-stranded DNA genome (Rimmer et al., 2016; Subramaniam et al., 2012; Whittington et al., 2010). This genus is the major agent causing disease in >50 freshwater and marine fish species, resulting in significant economic losses to the aquaculture industry in East and Southeast Asia (Song et al., 2008; Jung et al., 2017a; Inouye et al., 1992; Shi et al., 2004).

The Iridoviridae comprises the following five genera: Megalocytivirus, Ranavirus, Lymophocystivirus, Iridovirus, and Choloriridovirus (Chinchar et al., 2005). Megalocytiviruses have been divided into three types based on phylogenetic analysis of the major capsid protein (MCP) gene: the red sea bream iridovirus (RSIV) type cluster, including RSIV subgroup1 and rock bream iridovirus (RBIV) subgroup2, reported as the major epizootic viral strains affecting aquaculture worldwide (Kim et al. 2018, Jung and Oh, 2000; Zhang et al., 2012. 2013); infectious spleen and kidney necrosis virus (ISKNV) type that generally causes diseases in freshwater fish species (He et al. 2000); and turbot reddish body iridovirus (TRBIV) type (Shi et al., 2004), which infects flatfish species.

Megalocytiviruses mainly occur in summer when the water temperature reaches 23°C and above (Jung et al., 2015, 2016, 2017a, 2017b; Jun et al., 2009). Diseased fish show signs of lethargy, exhibit severe anemia and petechiae in the gills, and have enlarged spleens. Histopathology is characterized by development of enlarged cells with basophilic inclusion bodies in the spleen (Do et al., 2004; Do et al., 2005; OIE 2018). In Korea, megalocytiviruses have caused massive damage to aquacultures of various fish species such as rock bream (Oplegnathus fasciatus), rockfish (Sebastes schlegelii), red sea bream (Pagrus major), and olive flounder (Paralichthys olivaceus) (Jeong et al., 2003, 2006; Jin et al., 2018; Won et al., 2013; Kim et al., 2018).

Megalocytiviruses are notable for their variability in infecting a broad host range. Genomic analysis of RSIVs can be useful in identifying the introduction of exotic viruses due to the importation of live fish or natural fish migration from the sea of other countries. Thus, gaining a better understanding of the genetic diversity of megalocytiviruses would provide critical information of their biology and evolution to guide strategies for controlling these diseases. To contribute such information, in this study, we investigated the genetic relationships of the MCP and DNA polymerase (DPOL) genes of megalocytiviruses, especially RSIV type-subgroup2, isolated from diseased aquacultured fish in Korea from 2013 to 2017 and compared these findings with previous reports.

Ⅱ. Materials and Methods

1. Samples

Moribund or dead fishes showing typical clinical symptoms of iridoviral disease were collected from 15 domestic aquatic farms by surveillance, disease identification, or an investigation into damage of the National Institute of Fisheries Science (NIFS) and institutions specializing in the disease identification of aquatic organism diseases from 2013 to 2017. The aquatic disease control division of the NIFS confirmed the RBIVD outbreak by verification with additional experiments. Specifically, rock bream was sampled in Tongyeong (four farms), Yeosu (five farms), Wando (one farm), Sacheon (one farm), Namhae (one farm), and Seogwipo of Jeju Island (one farm) and red sea bream and rock fish were sampled from one farm each at Tongyeong <Table 1>. Pooled samples including spleen and kidney tissue from approximately ~30 fishes collected at each farm were used for virus detection.

<Table 1>

Samples infected with megalocytivirus used in this study

2. Total nucleic acid extraction and megalocytivirus identification

Total nucleic acids (50 μL) were isolated from 200 μL of the tissue homogenate in phosphate buffered saline (pH 7.4, 1:10 w/v) using a Viral NA Extraction Kit on a SPRI-TE^TM Nucleic Acid Extractor (Beckman Coulter Inc., USA) according to the manufacturer's instructions. Polymerase chain reactions (PCRs) were performed in a final volume of 25 μL with 2 μL of DNA template using Takara EX Taq^TM (Takara) in a Mastercycler (Eppendorf, Germany). The primer sets 1-F/1-R and 4-F/4-R (Kurita et al. 1998) were used for megalocytivirus detection, and reaction conditions were determined according to the Manual of Diagnostic Tests for Aquatic Animals of the World Organization for Animal Health (OIE, 2018). The PCR conditions for sequence analysis were as follows: pre-denaturation at 94°C for 5 min; 30 cycles at 94°C for 30 s, 58°C for 60s, and 72°C for 60 s; followed by an extension period at 72°C for 5 min. The amplified PCR products were subjected to QIAxcel Advanced System (Qiagen). For identification of megalocytivirus, nucleotide sequences were determined by the ABI 3500xl genetic analyzer (Thermo Fisher Scientific, UK)using the PCR products purified from a PCR Purification Kit (GeneAll, Korea).

[Fig. 1]

PCR detection test for megalocytiviruses from diseased fishes of Korean aquatic farms from 2013 to 2017 based on the QIAxcel capillary electrophoresis system. Electrophoresis after PCR using 1-F/1-R (A) and 4-F/4-R primer sets (B). Lane 1, ADC-RSIV2016-2; lane 2, ADC-RSIV2017-18; lane 3, ADC-RSIV2017-22; lane 4, ADC-RSIV2013-24; lane 5, ADC-RSIV2015-24; lane 6, ADC-RSIV2017-27; lane 7, ADC-RSIV2016-29; lane 8, ADC-RSIV2014-35; lane 9, ADC-RSIV2016-38; lane 10, ADC-RSIV2016-41; lane 11, ADC-RSIV2014-44; lane 12, ADC-RSIV2014-46; lane 13, ADC-RSIV2017-49; lane 14, ADC-RSIV2017-68; lane 15, ADC-RSIV2016-56; N, negative control; P, positive control; M, 15 bp ~ 3 kb marker.

3. Cloning and sequencing of MCP and DPOL genes

For sequence analysis of MCP and DPOL genes, PCR was performed with the M1F/M1R (He et al., 2001) and DPO-F/DPO-R primer sets targeting the full-length sequence of the MCP gene and the partial sequence of the DPOL gene using extracted DNA samples as the template <Table 2>. The PCR conditions for sequence analysis were pre-denaturation at 95°C for 5 min; 35 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 90 s; followed by an extension period at 72°C for 7 min. The amplified PCR products were cloned to a TA vector (Invitrogen) and the nucleotide sequences were determined using the ABI 3500xl genetic analyzer (Thermo Fisher Scientific, UK).

<Table 2>

Primers used in this study

4. Multiple alignment and phylogenetic analysis of the MCP and DPOL genes

The sequences of both viral genome segments were compared by gene alignment using BioEdit software (ver. 7.0.6 Department of Microbiology, North Carolina State University, Raleigh, NC, USA) to determine the identity of the genotype sequences.

[Fig. 2]

Degree of identity (%) of the nucleotide and deduced amino acid sequence of complete major capsid protein (MCP) (A) and partial DNA polymerase (DPOL) (B) gene in megalocytiviruses of this study to those of other strains.

A phylogenetic tree was constructed using ClustalW software (http://www.ebi.ac.uk/clustalW) based on neighbor-joining analysis with MEGA4 (ver. 4.1) (Center for Evolutionary Functional Genomics, The Biodesign Institute, Tempe, AZ, USA), and nodes were confirmed from bootstrap analysis with 1,000 replicates. The complete sequences of MCP genes and the partial sequences of DPOL genes from megalocytiviruses in the GenBank database (http://www.ncbi.nlm.nih.gov/GenBank) [Fig. 3] were used to construct the phylogenetic tree for comparison.

[Fig. 3]

Molecular phylogenetic tree by neighbor-joining analysis constructed based on the complete sequences of megalocytiviruses MCP and DPOL genes. The trees were constituted to compare the phylogenetic relationship of MCP gene (A) and DNA polymerase gene (B) with other megalocytivirus genes from Entrez Database. Bootstrap values were obtained from 1,000 replicates and these values less than 90 were hidden in the tree. The scale bar represents 0.01 nucleotide substitution per site. We marked analyzed sequences of this study (◆).

Ⅲ. Results

4. Multiple alignments of MCP and DPOL gene sequences in megalocytiviruses

Sequence alignment of the MCP gene among viral strains showed that the majority of the deduced amino acid sequences were identical with only one strain (ADC-RSIV2017-49) showing a single amino acid substitution (N->D at position 107) compared with the others [Fig. 4]. By contrast, for the DPOL gene, three strains contained an amino acid substitution: ADC-RSIV2017-49 (C->R at position 661), ADC-RSIV2016-38 (C->R at position 661), and ADC-RSIV2014-35 (M->T at position 944). Moreover, the DPOL gene of five strains, ADC-RSIV2017-22 (aa 867), ADC-RSIV2017-27 (aa 871), ADC-RSIV2015-24 (aa 804), ADC-RSIV2014-44 (aa 909), and ADC-RSIV2014-35 (aa 945), showed a silent mutation in a single region, in which there was no amino acid substitution despite the nucleotide substitution.

[Fig. 4]

Comparison of the deduced amino acid sequences of complete MCP (A) and partial DPOL (B) genes of megalocytiviruses of this study with known strains, each representing 3 subtypes of Genus Megalocytivirus. Amino acid sequences written in alphabet, instead of dots which are represented identical amino acid residues, refer to sequence substitution compared with genes of representative ADC-RSIV2016-2.

Ⅳ. Discussion

RSVID, one of the major pathogenic agents in Korean marine aquaculture, is listed as a notifiable viral disease by the OIE. RSIVD, including RBIVD, is listed as a third-class communicable disease, requiring movement control and disinfection in Korea. In this study, we cloned the MCP and DPOL genes of megalocytiviruses obtained from tissues of infected fish and performed phylogenetic analysis and comparisons of genetic variation to investigate the evolution of megalocytiviruses in Korea from 2013 to 2017. We detected megalocytiviruses in moribund or dead fishes showing typical clinical symptoms of iridoviral disease with PCR using two different primer sets of the OIE Manual of Diagnostic Tests for Aquatic Animals [Fig. 1] : the 1-F/1-R of PCR primer set can amplify partial gene of RSIV and ISKNV type, whereas 4-F/4-R can amplify only the RSIV type. In addition to RSIVD, TRBIVD has also been reported in Korea (Do et al., 2005; Won et al., 2013), and there is no specific detection method for TRBIVD in Manual of Diagnostic Tests for Aquatic Animals of OIE. Therefore, to determine the genetic subtype of megalocytivirus that caused the iridoviral diseases in our sample, we are now developing a duplex PCR method that can detect both the RSIV and TRBIV types so as to be able to quickly prevent spread of the disease through rapid diagnosis.

The MCP and DPOL genes represent structural and non-structural genomic regions of megalocytiviruses, respectively, that are highly conserved, and thus serve as useful targets for evolutionary investigations. Megalocytivirus was previously divided into three subtypes, including RSIV, ISKNV, and TRBIV, based on the MCP gene (Do et al., 2004; Do et al., 2005; Song et al., 2008; Jeong et al., 2006). RSIV type-subgroup2 was shown to be widely distributed in various fish species worldwide, especially in Korea, Japan, China, and other Asian countries (Song et al., 2008; Wang et al., 2009; Zhang et al., 2012; Inouye et al., 1992). Consistently, our phylogenetic analysis demonstrated that megalocytiviruses can be divided into three clusters represented by RSIV, ISKNV, and TRBIV [Fig. 3]. Moreover, even though the MCP and DPOL genes of these viruses that have been present in Korea for the last 5 years clustered more closely with RIE12-1 and OSGIV from Japan and China than with RBIV-KOR-TY1 from Korea [Fig. 3], all of these virus genes still showed high identities (>99.8%) with RBIV-KOR-TY1 [Fig. 2]. This pattern suggests that RSIV type-subgroup2 in Korea have evolved in relation to other RSIV strains existing in the marine environment throughout East Asia.

Because a large number of fishes are transported into domestic aquaculture for commercial and ornamental use, gaining an understanding of the viral host range is important for the control of introducing exotic types from other countries and for disease prevention. Although megalocytiviruses were detected in Korean rockfish as well as rock bream as the main hosts, and were even found over a broad area, including Jeju Island <Table 1>, their gene sequences were still found to be nearly identical. Thus, the clustering pattern of the viral genes of RSIV-subgroup2 is not dependent on the host species, geographical location, or isolation year.

Multiple alignment of the MCP and DPOL nucleotide sequences also demonstrated a lower rate of genetic variation compared with the other strains [Fig. 4]. Our findings support that DNA viruses can evolve slowly to develop genetic variations with distinct amino acid substitutions (Kurita and Nakajima, 2012; Huang et al., 2011). However, the greater variation of nucleotide sequences implies that evolution of megalocytiviruses is nevertheless underway, and thus mutant strains may emerge, including novel pathogenic types. Therefore, it is necessary to conduct functional analysis of the amino acid substitutions and their potential relation to pathogenicity.

Taken together, the present results based on phylogenetic analysis and genetic comparisons suggest that megalocytivirus isolates from different marine fish in Korea belong to RSIV type-subgroup2 with minimal genetic variation compared with genes of reported strains. This study could provide valuable insight and novel genetic information of recently isolated megalocytiviruses in Korea, laying a foundation for the development of effective vaccines for preventing the spread of megalocytivirus disease in the aquaculture industry.

Acknowledgments

This work was supported by a grant from the National Institute of Fisheries Science (R2018065).

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