Isolation and initial characterization of new betanodaviruses in shellfish

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 (10² -10⁵ copies/mg brain) was much lower compared to that found in sevenband grouper (Epinephelus septemfasciatus Thunberg) sampled in the moribund stage with RGNNV infection (10⁶ -10⁷ 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.