Detection of DNAs homologous to betanodavirus genome RNAs in barfin flounder Verasper moseri and Japanese flounder Paralichthys olivaceus.
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.
Suzuki
Kunio
K
Eastern Hokkaido Inland Water Fisheries Section, The Hokkaido Fish Hatchery, Notorominato 1-1, Abashiri, Hokkaido 093-0131, Japan. suzukiku@fishexp.pref.hokkaido.jp
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000302:isolation & purification*; D005393:Fish Diseases / Q000235:genetics* / Q000821:virology; D005432:Flounder / Q000235:genetics / Q000821:virology*; D007564:Japan; D008969:Molecular Sequence Data; D030381:Nodaviridae / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012327:RNA Virus Infections / Q000235:genetics*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D016415:Sequence Alignment; D014162:Transfection / Q000662:veterinary
D036022:Capsid Proteins; D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins