DAO 72:225-239 (2006)  -  doi:10.3354/dao072225

ABSTRACT: I investigated the presence of DNA homologous to genome RNA1 and RNA2 (RNA1 DNA and RNA2 DNA) of betanodaviruses—the causative agent of viral nervous necrosis (VNN)—in eggs, sperm, ovarian cavity fluid, larvae, and juveniles of barfin flounder Verasper moseri and larvae and juveniles of Japanese flounder Paralichthys olivaceus collected at 6 sites in Hokkaido, Japan, from 1994 to 2001. RNA1 DNA and RNA2 DNA were detected by PCR in 13 and 33% of barfin flounder samples and 0 and 69% of Japanese flounder samples, respectively. No infectious virus was detected by cell culture or by successive immunoblot against coat protein (genome RNA2 product) using an E-11 cell line, except for a virus present in 1 dead fish collected during an outbreak of VNN in 1995. Nucleotide sequence analysis showed that RNA1 DNA had a 82 to 96% similarity to betanodavirus genome RNA1, and that RNA2 DNA had a 69 to 98% similarity to RNA2. The detection rate of RNA2 DNA after intraperitoneal injection of betanodavirus strain HCF-1 into larvae and juveniles of the 2 flounder species was higher in samples from surviving fish than in the uninfected controls, whereas the detection rate of RNA1 DNA did not show a clear trend. Infectious virus was only detected in samples from fish that died subsequent to injection. Transfection assays of the viral genome RNAs into the barfin flounder cell line MK-1 and Japanese flounder cell lines H-1 and H-2 resulted in production of RNA2 DNA in all 3 cell lines. Quantitative measurement by ELISA revealed reverse transcriptase (RTase) activity. These results suggest that the DNA forms are produced and persist in the 2 flounder species as both clinical and subclinical infections, and do not lead to virion production.

KEY WORDS: DNA form · Betanodavirus · Viral nervous necrosis · VNN · Barfin flounder · Japanese flounder