Faculty of Applied Biological Science, Hiroshima University, Higashihiroshima, 739-8528, Japan1
Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan2
Kamiura Station, Japan Sea-Farming Association, Oita 879-2602, Japan3
Author for correspondence: Kazuyuki Mise. Fax +81 75 753 6131. e-mail kmise{at}kais.kyoto-u.ac.jp
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Abstract |
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Introduction |
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Nodaviruses have a bipartite genome of positive-sense RNA, with RNA1 encoding the RNA-dependent RNA polymerase and RNA2 encoding the coat protein (CP). Both RNAs are capped, but not polyadenylated. During RNA replication, a subgenomic RNA3, which is co-terminal with RNA1 and encodes small proteins, is synthesized. The family Nodaviridae comprise two genera: Alphanodavirus and Betanovirus, members of which primarily infect insects and fish, respectively (van Regenmortel et al., 2000 ). Striped jack nervous necrosis virus (SJNNV), which had been purified from diseased larvae of the striped jack Pseudocaranx dentex, was first identified as a betanodavirus (Mori et al., 1992
). RNA1 (3·1 kb) and RNA2 (1·4 kb) of SJNNV encode a 100 kDa protein (presumably RNA-dependent RNA polymerase) and a major CP of 42 kDa, respectively (Mori et al., 1992
; Nagai & Nishizawa, 1999
). The sequence similarities of RNA2, about 870 bases in open reading frame (ORF), were less than 29% at the nucleotide level and less than 11% at the amino acid level between SJNNV and four representative insect nodaviruses, Nodamura virus (NoV), Black beetle virus (BBV), Flock house virus (FHV) and Boolarra virus (Dasgupta et al., 1984
; Dasgupta & Sgro, 1989
), whereas they were 70% or higher among four piscine nodavirus isolates (Nishizawa et al., 1995
). On the other hand, the RNA1 sequence similarities between SJNNV and the alphanodaviruses were 28% at the nucleotide and amino acid levels (Nagai & Nishizawa, 1999
).
With the progress of recombinant DNA technology, single- or double-stranded RNA viruses have been genetically analysed and infectious RNA transcripts or cDNA clones have been produced from a variety of RNA viruses (Boyer & Haenni, 1994 ). The alphanodaviruses can be propagated in a wide range of cultured cells, such as insect, plant, vertebrate and yeast cells, and their infectious RNA transcripts have been used frequently for RNA transfection into these permissive cells. This has led to studies of their RNA replication, gene expression and virion assembly (reviewed by Ball & Johnson, 1998
). In contrast, the establishment of such a reverse genetics system for betanodaviruses has long been hampered by the lack of an appropriate cell culture system (Breuil et al., 1991
; Mori et al., 1991
; Munday et al., 1992
; Nguyen et al., 1994
; Grotmol et al., 1995
). The studies of Frerichs et al. (1996)
and our own group (Iwamoto et al., 1999
, 2000
) have revealed, however, that the striped snakehead cell line (SSN-1) (Frerichs et al., 1991
) and the clonal cell line E-11 from the SSN-1 cells are useful for qualitative and quantitative analyses for all of the betanodaviruses, including SJNNV. Furthermore, it has been confirmed that the SJNNV genomic RNA is infectious when transfected into E-11 cells and that the progeny virus recovered from the cells is virulent to striped jack larvae (Iwamoto et al., 2001
).
Recently, a reverse genetics system was developed for Infectious pancreatic necrosis virus, a double-stranded RNA virus of fish (Yao & Vakharia, 1998 ). To date, however, there is no reverse genetics system for positive-sense single-stranded RNA viruses that infect fish or aquatic animals. In general, cDNA containing entire viral genome sequences is required to obtain infectious in vitro RNA transcripts. Although the nucleotide sequences for RNA1 and RNA2 of SJNNV and other betanodaviruses have been published (Nishizawa et al., 1995
, 1997
; Delsert et al., 1997a
; Sideris, 1997
; Aspehaug et al., 1999
; Nagai & Nishizawa, 1999
; Thiery et al., 1999
; Grotmol et al., 2000
; Starkey et al., 2000
), precise sequences of their 5' and 3' non-coding regions have not been determined. In this report, we describe the construction and sequencing of full-length cDNA clones of SJNNV and the recovery of infectious SJNNV from E-11 cells transfected with RNA transcripts that, in turn, were synthesized in vitro from their cDNAs. This is the first report of the production of cDNA clones of a betanodavirus from which infectious genomic RNAs can be transcribed.
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Methods |
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SJNNV purification.
Naturally infected striped jack larvae that had been collected at the Nagasaki prefecture in Japan in 1993 were used as the source of SJNNV. SJNNV was purified as described by Mori et al. (1992) and stored at -80 °C.
Confirmation of the 5' and 3' end structures of the SJNNV genome.
SJNNV virion RNA was extracted from the purified virus as described previously (Kroner & Ahlquist, 1992 ). The 5' terminus of the virion RNA was treated with bacterial alkaline phosphatase from Escherichia coli C75 (Takara) and then labelled with T4 polynucleotide kinase (Toyobo) in the presence of [
-32P]ATP (Amersham) either with or without prior decapping treatment with tobacco acid pyrophosphatase (TAP) (Nippon gene) under the conditions recommended by the manufacturer. The 3' terminus of each strand was treated with T4 RNA ligase (Takara) and poly(A) polymerase (Takara) in the presence of [32P]pCp (Amersham) and [
-32P]ATP (Amersham), respectively, according to the manufacturers recommendations. The treated RNAs were separated by electrophoresis in 1% agarose gels and the signal was then detected by autoradiography.
Determination of the 5'- and 3'-terminal sequences of the SJNNV genome.
The rapid amplification of cDNA ends (RACE) method (Frohman et al., 1988 ) was used to determine the complete nucleotide sequences of the 5' and 3' termini of the SJNNV genome. For 5' RACE, virion RNA was reverse-transcribed with SuperScript II (Gibco BRL) using the synthetic oligonucleotide primers SJ1R1 or SJ2R1 (Table 1
). The first-strand cDNAs were polyadenylated with terminal deoxynucleotidyltransferase (Takara) and then the second-strand cDNAs were synthesized using the primer ANCH (Table 1
), after purification through the GLASS MAX Column (Pharmacia), according to the manufacturers instructions. The double-stranded cDNAs were amplified using the primers AUAP and either SJ1R2 or SJ2R2 (Table 1
). Decapped RNA, prepared as described above, was also used in 5' RACE for the detection of cap structure. For 3' RACE, viral RNA was polyadenylated with poly(A) polymerase in the presence of ATP and reverse-transcribed using the primer ANCH, as described above, and then amplified with the primers AUAP and either SJ1F1 or SJ2F1 (Table 1
). These amplified products were purified by 1% low-melting-point (LMP) agarose gel electrophoresis and then further amplified by nested PCR using the primers AUAP and either SJ1F2 or SJ2F2. The nested PCR products were purified and used to determine the terminal sequences. Sequencing reactions were performed using the BigDye Deoxy Terminator Cycle Sequencing kit (PE Applied Biosystems), according to the manufacturers instruction, and nucleotide sequences were determined using the automated sequencer ABI Prism 310 (PE Applied Biosystems).
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In vitro transcription.
Plasmids containing full-length SJNNV cDNA were linearized with EcoRI and then used for in vitro transcription with T7 RNA polymerase (Takara) in the presence of synthetic cap analogue [m7G(5')ppp(5')G] (New England Biolabs), as described previously (Kroner & Ahlquist, 1992 ). After being treated with RQ1 DNase I (Promega), transcripts were purified through a Sephadex G-50 column (Pharmacia) and their concentrations were quantified spectrophotometrically before transfection into cells. The RNA products were analysed by agarose gel electrophoresis in TBE buffer (Sambrook et al., 1989
).
Inoculation of cultured cells and infection assay.
The infectivity of transcripts was examined by transfection with lipofectin reagent (Gibco BRL) into E-11 cells followed by 24 h of incubation at 25 °C, as described previously (Iwamoto et al., 2001 ). For infection analysis of progeny virus, media cultured in the same manner as above for 72 h were collected, inoculated onto fresh E-11 cells and incubated at 25 °C for 24 h. Cell infectivity of the transcripts and their progeny was examined by immunofluorescence staining using anti-SJNNV rabbit polyclonal antibody and FITC-conjugated swine immunoglobulin (Ig) to rabbit Ig (Dako), as described previously (Nguyen et al., 1996
). The fluorescent cells were then counted.
Northern blot analysis.
Total RNA was extracted from E-11 cells infected with progeny viruses using ISOGEN (Nippon gene), according to the manufacturers instructions. The RNA was subjected to Northern blot analysis, essentially as described by Damayanti et al. (1999) , except that the DIG Labelling and Detection kit (Roche) was used, according to the suppliers instructions.
DIG-labelled RNA probes specific for the positive- and negative-sense strand of SJNNV RNA1 and RNA2 were prepared as follows. For RNA1, a PCR product was amplified from pSJ1TK19 using the M13 primers M4 and RV (Takara) and digested with ClaI/EcoRI and the resulting 0·3 kb fragment was inserted into the transcription vector pBluescript II KS(-) (Stratagene) to create pSJ1BS1. For RNA2, pSJ2TK30 was digested with BamHI/EcoRI and the resulting 0·4 kb fragment was ligated into pBluescript II SK(-) to create pSJ2BS2. To prepare probes for positive-sense RNA1 and RNA2, pSJ1BS1 and pSJ2BS2 were linearized with SalI and BamHI, respectively, and transcribed with T7 RNA polymerase (Takara). To prepare probes for negative-sense RNA1 or RNA2, either pSJ1BS1 or pSJ2BS2 was linearized with EcoRI and then transcribed with T3 RNA polymerase (Gibco BRL).
Western blot analysis.
Infected fish cells were suspended in Laemmlis sample buffer and subjected to SDSPAGE. Western blot analysis was carried out as described previously (Damayanti et al., 1999 ) using an Immobilon-P transfer membrane (Millipore). SJNNV CP was detected using anti-SJNNV rabbit polyclonal antibody and alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (Bio-Rad) followed by incubation with BCIP/NBT for colour development.
Electron microscopy of progeny.
Cell culture supernatants containing progeny virus were layered onto 1040% sucrose gradients in TE buffer (10 mM TrisHCl, 1 mM EDTA, pH 7·2) and centrifuged at 80000 g for 2 h at 16 °C. Each fraction was collected, analysed by Western blotting and concentrated using the Centricon centrifugal filter unit (Millipore), according to the manufacturers instructions. Virus suspensions were stained with 1% uranyl acetate. Simultaneously, progeny virus was inoculated onto freshly prepared E-11 cells and incubated at 25 °C. After incubation for 3 days, the cells were fixed in 2·5% glutaraldehyde and post-fixed with 1% osmium tetroxide. Ultra-thin sections were stained with 1% uranyl acetate and 1% lead citrate. These samples were examined under a Hitachi (Model H-7100FA) electron microscope.
Virulence assay for fish larvae.
One-day-old striped jack larvae reared at Kamiura Station of the Japan Sea-Farming Association were used for a virulence assay of the progeny virus produced in cells infected with the in vitro RNA transcripts. Before the infection experiment, these larvae were confirmed to be SJNNV-free by RTPCR (Nishizawa et al., 1994 ). Duplicate groups of 250 larvae were kept at 23 °C in glass beakers containing 1 litre of sea water to which kanamycin was added to a final concentration of 5 µg/ml. The 96 h culture supernatant of E-11 cells (50 µl) infected with progeny viruses was inoculated into the beakers and moribund fish were collected daily. The homogenate (progenitor virus) of striped jack larvae naturally infected with SJNNV and the culture supernatant of uninfected E-11 cells were used as positive and negative controls, respectively. Virus titres of the inocula used were determined, as described previously (Iwamoto et al., 2000
), to be 109·6 TCID50/ml for the progeny and 1010·6 TCID50/ml for the progenitor. Fish were fixed with 10% formalin and embedded in paraffin. Sections were subjected to immunofluorescence staining as described above.
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Results |
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We then used the sample containing a small quantity of in vitro polyadenylated virion RNAs for the determination of the 3'-terminal sequence. Both the 5' and 3' termini of SJNNV RNA1 or RNA2 were amplified by RACE using SJNNV-specific primers that were synthesized according to the known sequence (Nishizawa et al., 1995 ; Nagai & Nishizawa, 1999
). We found 14 additional bases at the 5' terminus and 12 additional bases at the 3' terminus on RNA1 (Fig. 1A
). RNA2 had another 11 bases at the 5' terminus and the 3'-terminal sequence corresponded to the published sequence, although one nucleotide had been substituted (Fig. 1A
).
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Infectivity test of transcripts and progeny virions
To identify infectious transcripts of RNA2, individual transcripts were combined with a preparation of cognate RNA1 (purified twice by 1% LMP agarose gel electrophoresis) derived from virions and the mixtures were tested for their infectivity to cells. The results of indirect immunofluorescence staining showed that all transcripts except for pSJ2TK10 were infectious when transfected into E-11 cells with virion RNA1. From these plasmids, including the full-length cDNA of RNA2, pSJ2TK30 was selected because the transcript showed the highest infectivity in this trial (data not shown). Equimolar mixtures of transcripts from pSJ1TK19 and pSJ2TK30 at two different concentrations were transfected into E-11 cells (ca. 4x104 cells). After 24 h of incubation at 25 °C, fluorescent cells (28·7±8·6 cells) were observed in the transfections with the higher concentration (1·5 µg) of transcripts, but not with the lower one (0·15 µg) and the infectivity of the transcripts was significantly less than that following transfection with authentic virion RNAs (1914·0±99·7 fluorescent cells per 0·15 µg of inoculum RNA) (Fig. 2A, B
). However, progeny viruses (recombinant SJNNV, rSJ) in the culture supernatants of transfected cells were highly infectious to fresh E-11 cells (Fig. 2D
).
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Northern blot analysis of total RNA extracted from infected E-11 cells was performed using DIG-labelled riboprobes specific for positive- or negative-sense RNA. In the sample taken 24 h post-inoculation (p.i.), we detected strong signals that hybridized to SJNNV positive-sense-specific probes and co-migrated with original virion RNAs (Fig. 3A). Negative-sense RNA1 and RNA2 were also detected from cells at 24 h p.i. (Fig. 3B
). However, the signals for RNA2, especially for the negative-sense one, were significantly lower than those of RNA1 (Fig. 3A
, B
). These differences were due to the difference in hybridization efficiency between the probe for (+) RNA1 and that for (+) RNA2 as well as between the probe for (-) RNA1 and that for (-) RNA2, which was confirmed by hybridizing those probes with known amounts of RNAs transcribed in vitro (data not shown). In addition to RNA1 and RNA2, bands showing faster migration were also detected in both positive- and negative-sense hybridizations (Fig. 3A
, B
). In parallel hybridization analyses using each segment-specific probe, these extra bands reacted with the RNA1-specific probes, but not with the RNA2-specific probes (data not shown). Meanwhile, Western blot analysis of the sample of cells at 24 h p.i. showed an obvious increase in CP accumulation when compared with that of cells at 1 h p.i. (Fig. 3C
).
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Discussion |
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Single-stranded RNA genomes of many plant and animal viruses have unique structures at their 5' and 3' termini: the cap or the VPg structure at the 5' end and the tRNA-like structure or the poly(A) tract at the 3' end (Goldbach, 1987 ). The 3' ends of alphanodavirus RNAs lack a poly(A) tail (Newman & Brown, 1976
; Scotti et al., 1983
) and are not reactive with RNA ligase or poly(A) polymerase (Dasgupta et al., 1984
; Guarino et al., 1984
; Dasmahapatra et al., 1985
; Kaesberg et al., 1990
), suggesting that the 3' end is modified by an unidentified blocking group. Our results suggest that SJNNV also has the cap structure at the 5' end and the blocking group at the 3' end. Because the 3' end of alphanodavirus RNA is blocked in, the double-stranded RNAs or head-to-tail homodimers in infected cultured cells have been used to determine the 3'-terminal sequences (Guarino et al., 1984
; Johnson et al., 2000
). Although this was a significant obstacle to conquer for the sequencing of the 3' end of SJNNV, a small quantity of viral RNAs were, fortunately, polyadenylated in vitro and hence we were able to obtain the 3'-terminal cDNA fragments of SJNNV genomic RNAs by 3' RACE. Those minor unmodified RNAs might exist because nascent viral RNAs were packaged before modification and/or because the blocking structure was eventually removed from the 3' end during the RNA extraction procedure. Consistent with our results, the 3'-terminal sequences of genomic RNAs of the betanodaviruses Greasy grouper nervous necrosis virus (GGNNV) and Dicentrarchus labrax encephalitis virus (DlEV) have been determined after in vitro genomic RNA self-ligation (Tan et al., 2001
) and in vitro polyadenylation (Delsert et al., 1997a
), respectively. Consequently, we completely determined the 5' and 3' non-coding sequences of both RNAs in which 1114 nucleotides were found at both termini for RNA1 and at the 5' terminus for RNA2, in addition to the published sequences of SJNNV (Nishizawa et al., 1995
; Nagai & Nishizawa, 1999
). Comparison of the 5'- and 3'-terminal sequences between both RNA species demonstrated that each 5' terminus begins with 5' UAA... 3' and each 3' terminus ends with 5'...UCGGCG 3'. The identical sequences are also present at the 5' end of RNA1 and RNA2 of GGNNV (accession nos AF318942 and AF319555, respectively) (Tan et al., 2001
) and at the 3' end of RNA2 of DlEV (accession no. U39876) (Delsert et al., 1997a
). Such sequences, conserved between two genomic RNAs but different from those of SJNNV, were found among the alphanodaviruses BBV [accession nos K02560 (Dasmahapatra et al., 1985
) and X00956 (Dasgupta et al., 1984
)], FHV (accession nos X77156 and X15959) (Dasgupta & Sgro, 1989
) and NoV (accession nos AF174533 and AF174534), in which the sequence starts with 5' GU... 3' and ends with 5'...GGU 3'. These results might suggest that other betanodaviruses also have consensus sequences such as those found in SJNNV.
The transcript RNAs were less infectious than their SJNNV virion RNA counterparts. This may be caused by the absence of a peculiar, unknown blocking structure at the 3' end and/or by the presence of extra non-viral nucleotides at both termini of the transcripts. At the 5' end of the transcripts, a non-viral extra G residue that originated from the T7 promoter sequence was added. It has been reported that transcription efficiency is increased when G residues are inserted between the T7 promoter and viral cDNA sequences, although the amplification efficiency in cells is lost to some extent (Janda et al., 1987 ). Our previous experiments suggest that the poly(A) tract was not entirely present or was not long in SJNNV viral RNAs, because they were not trapped with an oligo(dT) column (Mori et al., 1992
). However, we could not rule out the possibility that the 3' ends contain an oligo(A) tract that was short enough for the RNAs to pass through the column and could not be distinguished from the poly(A) added in vitro with poly(A) polymerase during 3' RACE. Alternatively, if the 3' terminus ending with oligo(A) is important for the infectivity of SJNNV, UU residues within the extra non-viral sequence AAUU at the 3' end might have lowered the infectivity of these transcripts.
It has been known that subgenomic RNA3 (0·4 kb), derived from RNA1 of alphanodavirus, can only be detected from infected cells (Guarino et al., 1984 ; Dasmahapatra et al., 1985
; Johnson et al., 2000
). In this study, we also detected signals for RNA with faster migration by Northern blot analysis from cells infected with wild-type or rSJ. Although a similar phenomenon has been reported for the betanodavirus DlEV (Delsert et al., 1997b
), detailed analysis was not performed. In this study, we detected the extra bands only from infected cells and verified that the bands reacted with both positive- and negative-strand-specific probes for the 3'-proximal region of SJNNV RNA1. Furthermore, the molecular size for these bands was estimated to be approximately 0·4 kb. These results strongly suggest that RNA3 is generated from RNA1 during SJNNV RNA replication.
Previously, we demonstrated the pathogenicity to striped jack larvae of the progeny that was generated by transfection of SJNNV virion RNAs into E-11 cells (Iwamoto et al., 2001 ). rSJ obtained from in vitro transcripts in this study was pathogenic to striped jack larvae. In regard to fish viruses, this is the first known instance in which a recombinant virus has the ability to kill the original hosts. As mentioned before, betanodaviruses can be classified into four genotypes, designated SJNNV, Barfin flounder nervous necrosis virus (BFNNV), Tiger puffer nervous necrosis virus and Redspotted grouper nervous necrosis virus, based on the RNA2 partial sequences (Nishizawa et al., 1997
). We have reported recently that the optimal growth temperature for virus growth in cultured cells differs among the genotypes. Furthermore, because SJNNV genotype virus was not infectious to the Atlantic halibut Hippoglossus hippoglossus, which BFNNV genotype virus can infect, it has been suggested that host-specificity might be different among some betanodaviruses (Totland et al., 1999
). A reverse genetics system for SJNNV, as reported here, will open the way for molecular studies directed at virus multiplication and pathogenesis of the betanodaviruses. In particular, the relationship between genetic variations and host specificities in betanodaviruses and comparative studies with alphanodaviruses will be of great interest.
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Acknowledgments |
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Footnotes |
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b Present address: Kamiura Station, Japan Sea-Farming Association, Oita 879-2602, Japan.
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References |
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Received 22 May 2001;
accepted 4 July 2001.