Characterization 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: Antonio Terlizzi04/2012; , ISBN: 978-953-51-0497-1
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ABSTRACT: The family Nodaviridae include the genera Alphanodavirus and the Betanodavirus which are non-enveloped, single stranded RNA viruses. Alphanodavirus include the insect viruses while betanodavirus include species that are responsible for causing disease outbreaks in hatchery-reared larvae and juveniles of a wide variety of marine and freshwater fish throughout the world and has impacted fish culture over the last decade. According to International Committee on Taxonomy of Viruses, the genus Betanodavirus comprises four recognized species viz barfin flounder nervous necrosis virus, red-spotted grouper nervous necrosis virus (RGNNV), striped jack nervous necrosis virus and tiger puffer nervous necrosis virus with the RGNNV being the most common. The viruses are distributed worldwide having been recorded in Southeast Asia, Mediterranean countries, United Kingdom, North America and Australia. The disease has been reported by different names such as viral nervous necrosis, fish encephalitis, viral encephalopathy and retinopathy by various investigators. The virus is composed of two segments designated RNA1 and RNA2 and sometimes possesses an additional segment designated RNA3. However, genome arrangement of the virus can vary from strain to strain. The virus is diagnosed by microscopy and other rapid and sensitive molecular methods as well as immunological assays. Several cell lines have been developed for the virus propagation and study of infection mechanism. Control of nodavirus infection is a serious issue in aquaculture industry since it is responsible for huge economic losses. In combination with other management practices, vaccination of fish would be a useful strategy to control the disease.Indian Journal of Virology 09/2012; 23(2):114-23. · 0.36 Impact Factor
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ABSTRACT: A nodavirus isolated from red-spotted grouper (Epinephelus akaara) larvae in China has been subjected to genome analysis. The full-length genome sequences of RNA1 and RNA2 were determined, and the 5'-non-coding region (NCR) and 3'NCR sequences were determined by 5' rapid amplification of cDNA ends (RACE) and 3'RACE. RNA1 is 3,103 nt in length and contains a 982-amino-acid open reading frame (ORF) encoding protein A with a calculated molecular mass of 110.74 kDa. RNA2 is 1,433 nt long and contains a 338-amino-acid major ORF encoding coat protein with a calculated molecular mass of 37.059 kDa. Multiple alignment and phylogenetic analysis clearly supported including this virus in the species Redspotted grouper nervous necrosis virus, genus Betanodavirus, family Nodaviridae.Archives of Virology 01/2012; 157(4):777-82. · 2.03 Impact Factor