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

Abstract

A 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.

Betanodavirus of Marine and Freshwater Fish: Distribution, Genomic Organization, Diagnosis and Control Measures

“…Reverse transcriptase PCR has been applied to amplify a portion of the coat protein gene (RNA2) of betanodavirus and is a powerful and sensitive method for detecting the infection [6,10,50,90]. Till date, PCR is the main diagnostic test that is being used by most of laboratories.…”

Immune response to a recombinant capsid protein of striped jack nervous necrosis virus (SJNNV) in turbot Scophthalmus maximus and Atlantic halibut Hippoglossus hippoglossus, and evaluation of a vaccine against SJNNV

“…The rT2 with a predicted molecular weight of 32 kDa was prepared as described in Grotmol et al (2000). In short, total RNA was isolated from diseased striped jack larvae using the Trizol reagent (Gibco BRL, Life Technologies Inc., Rockville, Maryland, USA).…”

Infection competition against grouper nervous necrosis virus by virus-like particles produced in Escherichia coli

“…The nucleotide sequences of both genomic RNAs of striped jack nervous necrosis virus (SJNNV) (Nishizawa et al, 1995;Nagai & Nishizawa, 1999) and greasy grouper nervous necrosis virus (GGNNV) (Tan et al, 2001) have been determined. The sequences of the RNA2 of Dicentrarchus labrax encephalitis virus (DlEV) (Delsert et al, 1997), Atlantic halibut virus (AHV) (Grotmol et al, 2000) and two grouper viruses [malabaricus grouper (MGNNV) and dragon grouper (DGNNV)] (Lin et al, 2001) have also been determined. The larger genome strand, RNA1, presumably encodes the viral replicase, while the smaller genome strand, RNA2, has been demonstrated to encode the capsid protein (Lin et al, 2001).…”