Abstract
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.
Highlights
Betanodavirus is a causative pathogen of one of the most prevalent viral diseases in aquatic fish farmed around the world, causing viral encephalopathy, retinopathy (VER), and viral nervous necrosis (VNN)
420 samples were previously collected using two consecutive RTPCRs, RNA2-detection semi-nested reverse-transcriptase polymerase chain reaction (RT-PCR) (R2-DSN-2 RT-PCR), and RNA2-discriminative multiplex RT-PCR (R2-DMT RT-PCR) for the detection and discrimination of betanodaviruses in shellfish based on the nucleotide sequence of the RNA2 segment
The amplicon size used for type identification in R2-DMT RT-PCR was 211, 292, 347, 413, and 468 for Korean shellfish nervous necrosis virus (KSNNV), barfin flounder nervous necrosis virus (BFNNV), SJNNV, TPNNV, and red spotted grouper nervous necrosis virus (RGNNV), respectively [10,11]
Summary
Betanodavirus is a causative pathogen of one of the most prevalent viral diseases in aquatic fish farmed around the world, causing viral encephalopathy, retinopathy (VER), and viral nervous necrosis (VNN). The wide distribution of a new betanodavirus, Korean shellfish nervous necrosis virus (KSNNV), which was suggested to belong to a new fifth type, was reported through surveillance studies using shellfish species as viral reservoirs in the marine environments of Korea and China [10,11]. At least three betanodavirus types (RGNNV, BFNNV, and KSNNV) exist in the marine environment of East Asia, so it may strongly implicate the generation of new and diverse reassortant viruses by mutual genetic reassortment. We established a new detection and genotyping system based on types of RNA1 and RNA2 segments simultaneously and examined the pivotal role of the KSNNV type in the emergence of reassortment between types of betanodavirus in the marine environment
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