Emergence of Reassortment between a New and Reported Types of Betanodavirus in Shellfish

Recently, three types of betanodavirus including red spotted grouper nervous necrosis virus (RGNNV), barfin flounder nervous necrosis virus (BFNNV), and Korean shellfish nervous necrosis virus (KSNNV) (proposed as a new fifth type) have been detected in shellfish in the marine environment around Korea. To investigate the presence of reassortment between betanodavirus types, the type based on the RNA2 segment of betanodaviruses carried in 420 domestic shellfish (n = 306) and finfish (n = 35), as well as imported shellfish (n = 79), was compared with the type identified by reverse-transcriptase polymerase chain reaction (RT-PCR) for RNA1 segment. Only five samples carrying reassortant betanodaviruses were found, appearing as RG/KSNNV (n = 2), KS/RGNNV (n = 1), and SJ/RGNNV (n = 2) types. From these samples, we successfully isolated two reassortant strains from Korean and Chinese shellfish in E-11 cells and called them KG1-reKS/RG and CM1-reRG/KS, respectively. In the full genome sequences, each RNA segment of the reassortant strains exhibited the same gene length and high sequence homology (≥98%) with the reference strains corresponding to the type of each segment. Both these reassortant strains induced high mortality to sevenband grouper (Epinephelus septemfasciatus) larvae with high viral concentrations in the body (109 viral particles/mg) and severe vacuolation in the retina and brain. These are the first results showing the involvement of the KSNNV type in the reassortment of RNA segments in the reported types of betanodavirus, which could represent a new potential risk in fish.

Secretory Production of Functional Grouper Type I Interferon from Epinephelus septemfasciatus in Escherichia coli and Bacillus subtilis

Nervous necrosis virus (NNV) results in high mortality rates of infected marine fish worldwide. Interferons (IFNs) are cytokines in vertebrates that suppress viral replication and regulate immune responses. Heterologous overexpression of fish IFN in bacteria could be problematic because of protein solubility and loss of function due to protein misfolding. In this study, a protein model of the IFN-α of Epinephelus septemfasciatus was built based on comparative modeling. In addition, PelB and SacB signal peptides were fused to the N-terminus of E. septemfasciatus IFN-α for overexpression of soluble, secreted IFN in Escherichia coli (E-IFN) and Bacillus subtilis (B-IFN). Cytotoxicity tests indicated that neither recombinant grouper IFN-α were cytotoxic to a grouper head kidney cell line (GK). The GK cells stimulated with E-IFN and B-IFN exhibited elevated expression of antiviral Mx genes when compared with the control group. The NNV challenge experiments demonstrated that GK cells pretreated or co-treated with E-IFN and B-IFN individually had three times the cell survival rates of untreated cells, indicating the cytoprotective ability of our recombinant IFNs. These data provide a protocol for the production of soluble, secreted, and functional grouper IFN of high purity, which may be applied to aquaculture fisheries for antiviral infection.

Development of species-specific PCR for the identification of three grouper fish species (Epinephelus septemfasciatus, E. bruneus and E. akaara)

The aim of the study was to develop rapid, precise and species-specific method of the identification of three grouper species (Red-spotted grouper, Epinephelus akaara; Longtooth grouper, E. bruneus; Seven-band grouper, E. septemfasciatus), by using polymerase chain reaction (PCR) mediated molecular method. A common forward primer and three different species-specific primers were designed for all the three species from the fragment of mitochondrial cytochrome c oxidase I (COI) gene. The species-specific amplification patterns showed three different bands of basepair length of had 344 bp (Red-spotted grouper), 262 bp (Longtooth grouper), and 130 bp (Seven-band grouper) which were available in the DNA samples isolated from each verified species, unknown collections, and even in mixed processed samples. Thus PCR assay provides reliable, simple, easy and low cost method for species identification of these three species of groupers.

Cell Culture Isolation of Piscine Nodavirus (Betanodavirus) in Fish-Rearing Seawater

ABSTRACTPiscine nodavirus (betanodavirus) is the causative agent of viral nervous necrosis (VNN) in a variety of cultured fish species, particularly marine fish. In the present study, we developed a sensitive method for cell culture isolation of the virus from seawater and applied the method to a spontaneous fish-rearing environment. The virus in seawater was concentrated by an iron-based flocculation method and subjected to isolation with E-11 cells. A real-time reverse transcriptase PCR (RT-PCR) assay was used to quantify the virus in water. After spiking into seawater was performed, a betanodavirus strain (redspotted grouper nervous necrosis virus [RGNNV] genotype) was effectively recovered in the E-11 cells at a detection limit of approximately 105copies (equivalent to 10250% tissue culture infective doses [TCID50])/liter seawater. In an experimental infection of juvenile sevenband grouper (Epinephelus septemfasciatus) with the virus, the virus was isolated from the drainage of a fish-rearing tank when the virus level in water was at least approximately 105copies/liter. The application of this method to sevenband grouper-rearing floating net pens, where VNN prevailed, resulted in the successful isolation of the virus from seawater. No differences were found in the partial sequences of the coat protein gene (RNA2) between the clinical virus isolates of dead fish and the cell-cultured virus isolates from seawater, and the viruses were identified as RGNNV. The infection experiment showed that the virus isolates from seawater were virulent to sevenband grouper. These results showed direct evidence of the horizontal transmission of betanodavirus via rearing water in marine aquaculture.