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 founder Verasper moseri). 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 single 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 plasmid, while the detection limit of the RT-PCR assay was 100 to 1000 copies of in vitro transcribed viral RNA.
SourceAvailable from: Renate Johansen10/2004; Norwegian School of Veterinary Science., ISBN: 82-90550-41-3
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ABSTRACT: A new aquareovirus was isolated from cultured Atlantic halibut (Hippoglossus hippoglossus) fry at a facility where massive mortalities had occurred during the start-feeding phase. The same virus was also detected in juveniles (about 10 grams) of the 2013 generation at two other production sites, but not in larger fish from generations 2007-2012. The virus replicated in BF-2 and CHSE-214 cell cultures and produced syncytia and plaque-like cytopathic effects. This Atlantic halibut reovirus (AHRV) was associated with necrosis of the liver and pancreas, syncytium formation in these tissues, and distinct viroplasm areas within the syncytium in halibut fry. Transmission electron microscopy revealed that the viroplasm contained virions, non-enveloped, icosahedral particles approximately 70 nm in diameter with a double capsid layer, amorphous material, and tubular structures. The RNA-dependent RNA polymerase (RdRp) gene from the AHRV isolates showed the highest amino acid sequence identity (80 %) to an isolate belonging to the species Aquareovirus A, Atlantic salmon reovirus TS (ASRV-TS). A partial sequence from the putative fusion-associated small transmembrane (FAST) protein of AHRV was obtained, and this sequence showed the highest amino acid sequence identity (46.8 %) to Green River Chinook virus which is an unassigned member of the genus Aquareovirus, while a comparison with isolates belonging to the species Aquareovirus A showed <33 % identity. A proper assessment of the relationship of AHRV to all members of the genus Aquareovirus, however, is hampered by the absence of genetic data from members of several Aquareovirus species. AHRV is the first aquareovirus isolated from a marine coldwater fish species and the second reovirus detected in farmed fish in Norway. A similar disease of halibut fry, as described in this paper, has also been described in halibut production facilities in Canada and Scotland.Archives of Virology 10/2014; 160(1). DOI:10.1007/s00705-014-2235-8 · 2.28 Impact Factor
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ABSTRACT: The coat protein encoded by the nodavirus RNA2 gene originally isolated from greasy grouper, Epinephelus tauvina, was cloned, expressed as a recombinant polyhistidine-tailed fusion protein and characterized by immunoblot analysis. The purified recombinant protein was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) to detect body exudate and plasma antibodies against the coat protein in both experimentally infected and commercial barramundi. In addition, the nucleotide sequence was employed to develop a RT–PCR detection assay based on the T4 region. The results showed that the virus could be detected as early as 3 days post-infection by RT–PCR while antibodies against the recombinant coat protein were detectable on day 6 post-infection. Among 112 commercial barramundi samples collected from October 1999 to April 2000, 9% showed positive ELISA results which were further verified by Western blot.Journal of Fish Diseases 03/2001; 24(3). DOI:10.1046/j.1365-2761.2001.00270.x · 1.51 Impact Factor