Isolation and initial characterization of new betanodaviruses in shellfish

Abstract

Betanodaviruses cause the disease viral nervous necrosis (VNN) in finfish. Using a novel approach with two consecutive PCRs, detection semi-nested two-step RT-PCR (DSN-2 RT-PCR) and discriminative multiplex two-step RT-PCR (DMT-2 RT-PCR), we have identified the presence of a new type of betanodavirus in shellfish and called it Korean shellfish nervous necrosis virus (KSNNV). Partial nucleotide sequences of the T4 region in RNA2 fragment of KSNNVs were 73%-75% homologous to those of other reported genotypes and formed a new cluster of betanodavirus in phylogenetic tree analysis. Successful isolation of KSNNV was achieved in two of six shellfish samples containing high concentrations of virus using the blind passage method, and the typical shapes of betanodavirus were confirmed in KSNNV-KOR1 by electron microscopy. In the experimental infection test, seven of 14 fish species showed susceptibility to KSNNV-KOR1 isolate but without clinical signs or death. Although the range of susceptible host species was not significantly different from the RGNNV type, the concentration of KSNNV in the brain of infected fish (10 -10 copies/mg brain) was much lower compared to that found in sevenband grouper (Epinephelus septemfasciatus Thunberg) sampled in the moribund stage with RGNNV infection (10 -10 copies/mg brain). However, histopathological analyses showed the presence of multiple vacuoles in brains of all KSNNV-infected fish at 14 days postinjection. In detection test, as a single or multiple type with the other genotype(s) (RGNNV or BFNNV), the prevalence of KSNNV was 8.4% and 8.7% in domestic (62 of 741 samples) and Chinese samples (12 of 138 samples), respectively, but not in finfish. We propose that KSNNVs obtained from shellfish be classified into a separate and new genotype of betanodavirus.

Complete genome sequence and pathogenic analysis of a new betanodavirus isolated from shellfish

“…Nevertheless, infected fishes showed dramatic mortalities up to 100%, with high viral replication in the fish body and clear pathogenic lesions (Figures ). These results indicate that KSNNV‐KOR1 exhibits strong pathogenicity against larval fishes compared with previous results involving juvenile fish showing only subclinical infection (Kim, Kwon, Min, et al, ). Thus, KSNNV‐KOR1 showed age‐dependent pathogenicity in fish species, similar to other betanodaviruses.…”

“…New nodaviruses, designated KSNNV, were detected and isolated from shellfish in previous studies (Kim, Kwon, Min, et al, ). In this study, we determined the complete genomic sequence of KSNNV‐KOR1 and characterized it by comparing with other betanodaviruses in various genotypes.…”

“…In the previous study (Kim, Kwon, Min, et al, ), partial nucleotide sequence of RNA2 of KSNNV‐KOR1 in Pacific oyster (GenBank accession no. ) has been determined.…”

“…KSNNV‐KOR1 (isolated from Pacific oyster in Korea in 2013, Kim, Kwon, Min, et al, ) and SFRG10/2012BGGa1 isolates (isolated from granular ark in Korea in 2012, Kim, Kwon, Kim, et al, ) representing KSNNV and RGNNV, respectively, were used. Isolates were cultured on E‐11 cell line (ECACC no.…”

“…Previously, a few studies detected NNVs in some of shellfishes such as mussel Mytilus galloprovincialis and clam Ruditapes philippinarum in Europe and Korea. Interestingly, in previous study (Kim, Kwon, Min, et al, ), we have identified the presence of a number of putative new betanodaviruses (Korea shellfish nervous necrosis virus, KSNNV) in various shellfishes as Korean domestics and Chinese imports, including Pacific oyster Crassostrea gigas , mussel Mytilus edulis , Manila clam R. phillippinarum , granular arc Tegillarca granosa and common orient clam Meretrix meretrix , by partial nucleotide sequencing of RNA2 (Kim, Kwon, Min, et al, ). In this study, we determined the complete nucleotide sequence of the isolated KSNNV‐KOR1 genome and the pathogenicity against fish species.…”