Characterisation of the capsid protein gene from a nodavirus strain affecting the Atlantic halibut Hippogossus hippogossus and design of an optimal RT-PCR detection assay

University of Bergen, Bergen, Hordaland, Norway
Diseases of Aquatic Organisms (Impact Factor: 1.75). 02/2000; 39(2):79-88. DOI: 10.3354/dao039079
Source: PubMed


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.


Available from: Audun Helge Nerland, Jan 05, 2016
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    • "The disease is highly contagious and capable of causing mass mortalities in several fish species, particularly during the earliest developmental stages (Munday, Kwang & Moody 2002). Due to its high infectivity and its adaptability to a wide range of hosts and environmental conditions, VNN represents a serious risk for economically valuable finfish species (Grotmol et al. 2000; Ucko, Colorni & Diamant 2004; Ch erif et al. 2009; Ransangan & Manin 2010) and wild fish stocks (Ciulli et al. 2007; Gomez et al. 2008; Sakamoto et al. 2008) including endangered fish species (Gomez et al. 2009; Vendramin et al. 2013; Kara et al. 2014). Up to 10 years ago, VNN was believed to be a purely marine fish disease. "
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    Full-text · Article · Apr 2015 · Journal of Fish Diseases
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    • "and yellow grouper Epinephelus awoara (Temminck et Schlegel) [AF283554], Sea bass Dicentrarchus labrax (L.) [Y08700] and Sea bass [AJ698093] and Nile tilapia [EU700416] (Nishizawa et al. 1995; Sideris 1997; Lai et al. 2001; Lin et al. 2001; Skliris et al. 2001; Tan et al. 2001; Hegde et al. 2003; Iwamoto et al. 2004; Thiery et al. 2004; Bigarre et al. 2009; Liu et al. 2012); BFNNV genotype: Atlantic cod Gadus morhua (L.) [AF445800], halibut Hippoglossus hippoglossus (L.) [AF160473 and AJ245641], barfin flounder Verasper moseri (Jordan et Gilbert) [D38635] (Nishizawa et al. 1995; Aspehaug, Devold & Nylund 1999; Grotmol et al. 2000; Johnson et al. 2002); TPNNV genotype: tiger puffer Takifugu rubripes (Temminck et Schlegel ) [D38637] (Nishizawa et al. 1995); SJNNV genotype: striped jack Pseudocaranx dentex (Bloch et Schneider) [D30814, AF175519, AB056572 and NC_003449] (Nishizawa et al. 1995; Iwamoto et al. 2001; Skliris et al. 2001); TNV genotype: turbot Scophthalmus maximus (L.) [AJ608266] (Johansen et al. 2004)). The phylogenetic relationship was generated by the maximum likelihood algorithm under the Kimura 2-parameter model using gamma distribution (MEGA version 5 software). "
    [Show abstract] [Hide abstract] ABSTRACT: Betanodavirus infection was diagnosed in larvae of farm-raised tilapia Oreochromis niloticus (L.), in central Thailand. Extensive vacuolar degeneration and neuronal necrosis were observed in histological sections with positive immunohistochemical staining for betanodavirus. Molecular phylogenetic analysis was performed based on the nucleotide sequences (1333 bases) of the capsid protein gene. The virus strain was highly homologous (93.07-93.88%) and closely related to red-spotted grouper nervous necrosis virus (RGNNV).
    Full-text · Article · Oct 2013 · Journal of Fish Diseases
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    • "from orange-spotted grouper, Epinephelus coioides (Ep. coioides) was identified to be of the red-spotted grouper nervous necrosis virus (RGNNV) genotype, the most frequently isolated betanodavirus in Asia (Grotmol et al., 2000). OGNNV is the main pathogen that reduces the quantity and quality of grouper larvae in China. "
    [Show abstract] [Hide abstract] ABSTRACT: Betanodaviruses are the causative agents of viral nervous necrosis (VNN), a serious disease of cultured marine fish worldwide. Virus-like particles (VLPs) are one of the good novel vaccine candidates to control this disease. Until now, betanodavirus vaccine studies mainly focused on the humoral immune response and mortality after virus challenge. However, little is known about the activation of genes responsible for cellular and innate immunity by vaccines. In the present study, VLPs of orange-spotted grouper nervous necrosis virus (OGNNV) were produced in prokaryotes and their ability to enter Asian sea bass cells was the same as native virus, suggesting that they possess a similar structure to OGNNV. VLPs immunogenicity was then determined by intramuscularly vaccinating Epinephelus coioides at different concentrations (1.5 or 15μgg(-1) fish body weight, FBW) and immunizing frequencies (administration once, twice and thrice). A single vaccination with the dosage of 1.5μgg(-1) FBW is enough to provoke high titer antibodies (average 3 fold higher than that of negative control) with strong neutralizing antibody titer as early as 1 week post immunization. Furthermore, quantitative PCR analysis revealed that eleven genes associated with humoral, cellular and innate immunities were up-regulated in the liver, spleen and head kidney at 12h post immunization, correlating with the early antibody response. In conclusion, we demonstrated that VLP vaccination induced humoral immune responses and activated genes associated with cellular and innate immunity against betanodavirus infection in orange-spotted grouper.
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