Faculty of Applied Biological Science, Hiroshima University, Higashi-hiroshima 739-8528, Japan1
Author for correspondence: Toyohiko Nishizawa.Fax +81 824 22 7059. e- mail jjnishi{at}ipc.hiroshima-u.ac.jp
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Currently, the family Nodaviridae is made up of one genus, Nodavirus, which contains insect nodaviruses such as Nodamura virus (NOV), black beetle virus (BBV), Flock House virus (FHV) and Boolarra virus (Hendry et al., 1995 ). The genomic RNA2 of insect nodaviruses is a coat protein gene and encodes a protein
, the precursor of virion proteins ß and
, whereas the genomic RNA1 encodes two non-structural proteins, protein A [110 kDa; the RNA-dependent RNA polymerase (RDRP)] and protein B [11 kDa; the translation product of subgenomic RNA (RNA3) which is derived from RNA1 during replication] (Gallagher et al., 1983
; Dasmahapatra et al., 1985
; Kaesberg et al., 1990
; Ball, 1995
). Fish nodaviruses are clearly distinguished from insect nodaviruses based on a comparative study of the coat protein gene. Thus, a new genus has been proposed in the family Nodaviridae to include the fish nodaviruses (Nishizawa et al., 1995
).
Little is known about the primary structure of fish nodavirus RNA1 and the non-structural protein encoded by it. In this study, SJNNV RNA1 was cloned and sequenced. Analysis showed that the non-structural protein of SJNNV is RDRP and again emphasizes that fish nodaviruses can be clearly distinguished from insect nodaviruses.
Striped jack larvae infected with SJNNV were collected during an outbreak of VNN at a hatchery in Japan and stored at -20 °C. Whole bodies of infected larvae were homogenized with 9 volumes of Dulbecco's PBS (D-PBS) and centrifuged (10000 g, 10 min, 4 °C). The supernatant was filtered through a membrane (0·45 µm; Advantec) and centrifuged (150000 g, 40 min, 4 °C). The pellet was resuspended in D-PBS and centrifuged (100000 g, 60 min, 4 °C) through a discontinuous gradient composed of 20, 35 and 50% (w/v) sucrose in D-PBS. The virus was collected from the interface between 35 and 50% (w/v) sucrose and pelleted by centrifugation (150000 g, 40 min, 4 °C). The virus pellet was resuspended in CsCl (=1·32) and centrifuged (100000 g, 12 h, 4 °C). The virus was collected, diluted and then pelleted by centrifugation (150000 g, 40 min, 4 °C). The pelleted viral particles were resuspended in TE (10 mM TrisHCl, pH 8·0, 1 mM EDTA). After phenol and chloroform extraction, cDNAs were synthesized using a cDNA synthesis kit (Pharmacia). After addition of EcoRI adapters, the cDNAs were ligated into the plasmid vector, pBluescript KS(-) (Stratagene) and used to transform Escherichia coli DH5
(Toyobo). The recombinant plasmids were labelled with a digoxigenin labelling kit (Boehringer Mannheim) and hybridized with SJNNV RNAs to select recombinant plasmids with a cDNA specific for viral RNA1. Nucleotide sequences were determined with a dye terminator cycle sequencing kit (ABI) and analysed with the autosequencer A373-36 (ABI). Sequences were assembled and analysed with the computer program MacDNASISpro (Hitachi Software Engineering).
The schematic illustrations of the physical map of SJNNV RNA1 and recombinant plasmids used for the sequence analyses are shown in Fig. 1. A total of 13 plasmids was needed to perform the sequence analyses of SJNNV RNA1. The nucleotide and deduced amino acid sequences of SJNNV RNA1 are shown in Fig. 2
. The nucleotide sequence was 3081 bases in length containing an ORF at nt 653016. Thus, there are 65 bases of 5'-non-coding region and 65 bases of 3'-non- coding region. The molecular mass of SJNNV RNA1 calculated from the determined sequence was 1·01x106 Da, which corresponds to the estimated molecular mass of RNA1 obtained by PAGE (Mori et al., 1992
). A polypeptide of 983 aa encoded by the ORF of SJNNV RNA1 has a molecular mass of approximately 111 kDa, which agrees with the size of the polypeptide translated from SJNNV RNA1 (Mori et al., 1992
).
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In the RNA1 sequences of the insect nodaviruses BBV and FHV, there are 38 bases of 5'-non-coding region (of which 55% is A) and 74 bases of 3'-non-coding region. There is a stemloop structure in the first 19 bases of the 5'-non-coding region and two stemloop structures at the 3' terminus (Dasmahapatra et al., 1985 ; Kaesberg et al., 1990
). The 64 bases of the 5'-non-coding region in SJNNV RNA1 contained 28% A. Stemloop structures corresponding to those of insect nodaviruses were not detected in the non-coding regions. These results indicate that SJNNV and insect nodaviruses are related but distinguishable from each other. We believe that a new genus, Piscinodavirus, should be created in the family Nodaviridae , as has been previously proposed based on analysis of RNA2 (Nishizawa et al., 1995
).
The sequence motif YXDD is highly conserved amongst RNA polymerase, DNA polymerase and reverse transcriptase. In positive-strand RNA viruses, GDD is the consensus sequence (Delarue et al., 1990 ; Poch et al., 1989
). In fact, the eight conserved motifs, including the GDD motif, are identified from amino acid sequence alignment of viral RDRPs, and six of them are found in the protein A of insect nodavirus: the acidic motif at aa 582587 (motif 1); the SG.T motif at aa 649654 (motif 2); the GDD motif at aa 691693 (motif 3); the basic motif at aa 716 (motif 4); motif 7 is a basic sequence preceded by an aromatic residue at aa 796; and motif 8 is an aromatic residue preceded by a basic sequence at aa 819 (Bruenn, 1991
). As described above, the non-structural protein of SJNNV and the protein A of insect nodaviruses showed only 27·5% amino acid sequence identity. However, all of the six conserved motifs observed in the protein A of insect nodaviruses were found at aa 585590 (motif 1), aa 646651 (motif 2), aa 686688 (motif 3), aa 712 (motif 4), aa 788 (motif 7) and aa 808 (motif 8) of the non-structural protein of SJNNV. Interestingly, each conserved motif was located at almost the same position in the SJNNV and insect nodavirus proteins. It seems highly likely that the non-structural protein encoded by SJNNV RNA1 is RDRP.
The RNA1 of insect nodaviruses encodes two non-structural proteins, protein A (RDRP) and protein B (whose function is unknown). Protein B, with a molecular mass of 11 kDa, is the translation product of subgenomic RNA3, which is derived from the 3' end of RNA1 during virus replication. The ORF for protein B is found at nt 27373057 in RNA1 (Gallagher et al., 1983 ; Guarino et al., 1984
; Ball, 1995
). No report has been made concerning a putative protein B of fish nodaviruses, although an RNA3, with approximately 400 bases, was detected in cells of sea bass larva (Dicentrarchus labrax) infected with the fish nodavirus Dicentrarchus labrax encephalitis virus (Delsert et al., 1997
). Although the existence of RNA3 has not yet been confirmed for SJNNV, a potential ORF for protein B is found at nt 27422969 in the determined nucleotide sequence of SJNNV RNA1 (Fig. 2
). The calculated molecular mass from the deduced amino acid sequence was approximately 8·4 kDa, which is a little smaller than that of the insect nodavirus protein B. However, further studies will be needed to show whether the peptide encoded by this ORF of SJNNV RNA1 corresponds to the protein B of insect nodaviruses. Mapping of a neutralizing epitope on the coat protein of SJNNV is discussed in the accompanying paper by Nishizawa et al. (1999)
.
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References |
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Bruenn, J. A. (1991). Relationships among the positive-strand and double-strand RNA viruses as viewed through their RNA-dependent RNA polymerases. Nucleic Acids Research 19, 217-226.[Abstract]
Comps, M. , Pepin, J. F. & Bonami, J. R. (1994). Purification and characterization of two fish encephalitis viruses (FEV) infecting Lates calcarifer and Dicentrarchus labrax. Aquaculture 123, 1-10.
Dasmahapatra, B. , Dasgupta, R. , Ghosh, A. & Kaesberg, P. (1985). Structure of the black beetle virus genome and its functional implications. Journal of Molecular Biology 182, 183-189.[Medline]
Delarue, M. , Poch, O. , Tordo, N. , Moras, D. & Argos, P. (1990). An attempt to unify the structure of polymerases. Protein Engineering 3, 461-467.[Abstract]
Delsert, C. , Morin, N. & Comps, M. (1997). Fish nodavirus lytic cycle and semipermissive expression in mammalian and fish cell cultures. Journal of Virology 71, 5673-5677 .[Abstract]
Gallagher, T. M. , Friesen, P. D. & Rueckert, R. R. (1983). Autonomous replication and expression of RNA1 from black beetle virus. Journal of Virology 46, 481-489.
Guarino, L. A. , Ghosh, A. , Dasmahapatra, B. , Dasgupta, R. & Kaesberg, P. (1984). Sequence of the black beetle virus subgenomic RNA and its location in the viral genome. Virology 139, 199-203.[Medline]
Hendry, D. A. , Johnson, J. E. , Rueckert, R. R. & Scotti, P. D. (1995). Family Nodaviridae . In Virus Taxonomy. Sixth Report of the International Committee on Taxonomy of Viruses, pp. 368-371. Edited by F. A. Murphy, C. M. Fauquet, D. H. L. Bishop, S. A. Ghabrial, A. W. Jarvis, G. P. Martelli, M. A. Mayo & M. D. Summers. Vienna & New York: Springer-Verlag.
Kaesberg, P. , Dasgupta, R. , Sgro, J.-Y. , Wery, J.-P. , Selling, B. H. , Hosur, M. V. & Johnson, J. E. (1990). Structural homology among four nodaviruses as deduced by sequencing and X-ray crystallography. Journal of Molecular Biology 214, 423-435.[Medline]
Mori, K. , Nakai, T. , Muroga, K. , Arimoto, M. , Mushiake, K. & Furusawa, I. (1992). Properties of a new virus belonging to Nodaviridae found in larval striped jack (Pseudocaranx dentex) with nervous necrosis. Virology 187, 368-371.[Medline]
Munday, B. L. & Nakai, T. (1997). Special topic review: nodaviruses as pathogens in larval and juvenile marine finfish. World Journal of Microbiology & Biotechnology 13, 375-381.
Muroga, K., Furusawa, T. & Furusawa, I. (1998). A review: viral nervous necrosis in striped jack, Pseudocaranx dentex. Suisanzoshoku 46, 473480 (in Japanese).
Nishizawa, T. , Mori, K. , Nakai, T. , Furusawa, I. & Muroga, K. (1994). Polymerase chain reaction (PCR) amplification of RNA of striped jack nervous necrosis virus (SJNNV). Diseases of Aquatic Organisms 18, 103-107.
Nishizawa, T. , Mori, K. , Furuhashi, M. , Nakai, T. , Furusawa, I. & Muroga, K. (1995). Comparison of the coat protein genes of five fish nodaviruses, the causative agents of viral nervous necrosis in marine fish. Journal of General Virology 76, 1563-1569 .[Abstract]
Nishizawa, T. , Furuhashi, M. , Nagai, T. , Nakai, T. & Muroga, K. (1997). Genomic classification of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene. Applied and Environmental Microbiology 63, 1633-1636 .[Abstract]
Nishizawa, T. , Takano, R. & Muroga, K. (1999). Mapping a neutralizing epitope on the coat protein of striped jack nervous necrosis virus. Journal of General Virology 80, 3023-3027 .
Poch, O. , Sauvaget, I. , Delarue, M. & Tordo, N. (1989). Identification of four conserved motifs among the RNA-dependent RNA polymerase encoding elements. EMBO Journal 8, 3867-3874.[Abstract]
Received 14 April 1999;
accepted 23 July 1999.