Characterisation of the capsid protein gene from a nodavirus strain affecting the Atlantic halibut Hippoglossus hippoglossus and design of an optimal reverse-transcriptase polymerase chain reaction (RT-PCR) detection assay
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Date
2000-01-14Metadata
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Diseases of aquatic organisms, 39(2), 2000:79-88Abstract
1349 nucleotide fragment of the RNA2 from a nodavirus affecting Atlantic halibut Hippoglossus
hippoglossus was characterised and the nuclotide sequence (accession no, AJ245641) was
employed to develop an optimal reverse-transcriptase polymerase chain reaction (RT-PCR) detection
assay. The sequenced part of the RNA2 of Atlantic halibut nodavirus (strain AH95NorA) was highly
similar in organisation to that of the RNA2 of striped jack nervous necrosis virus (SJNNV), and comprised
features common to all nodaviruses. These characteristics confirmed that the virus that causes
viral encephalopathy and retinopathy (VER) in Atlantic halibut is a nodavirus. The nucleotide
sequence of the 1349 nucleotide fragment of Atlantic halibut nodavirus RNA2 was 80% identical to the
RNA2 of SJNNV. The T2 region (830 nucleotides) of the RNA2 of Atlantic halibut nodavirus shared
98% of the nucleotide sequence when compared with the homologous region of barfin flounder nervous
necrosis virus (BFNNV), while the nucleotide sequence identity to SJNNV in this region was 76 %.
Phylogenetic analysis based on the nucleotide sequences of the T4 region (421 nucleotides) of Atlantic
halibut nodavirus and of other fish nodaviruses revealed a close relationship to the nodaviruses of the
barfin flounder clad that have been found in other cold-water species (Pacific cod Gadus macrocephalus
and barfin flounder Verasper mosen). The nucleotide sequence of the RNA2 of Atlantic halibut
nodavirus included some features that differ from that of SJNNV. The ORF of the RNA2 of Atlantic
halibut nodavirus lacked 6 nucleotides through a slngle deletion and a 5-nucleotide deletion, separated
by 4 nucleotides. The 3'-non-encoding region contained a 21 nucleotide insert and a 3 nucleotide deletion
when compared with SJNNV. In comparison with the RNA2 of SJNNV, the 3'-non-encoding region
showed a nucleotide sequence identity of 84.5%. A primer set based on the Atlantic halibut nodavirus
nucleotide sequence was employed in order to design an optimal RT-PCR. The detection limit of the
PCR was 10 to 100 copies of plasrnid, while the detection limit of the RT-PCR assay was 100 to 1000
copies of in vitro transcribed viral RNA.
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