Co-operative Research Centre for Aquaculture, CSIRO Tropical Agriculture, Long Pocket Laboratories, PMB3, Indooroopilly 4068, Australia1
Author for correspondence: Jeff Cowley. Fax +61 7 32142881. e-mail Jeff.Cowley{at}tag.csiro.au
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Abstract |
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Introduction |
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There has been considerable confusion about the taxonomic classification of these viruses. Based primarily on its size and enveloped rod-shaped appearance, YHV was originally proposed to be a baculovirus (Chantanachookin et al., 1993 ). Subsequent evidence that the YHV genome comprised ssRNA led, however, to the suggestion that it may be a rhabdovirus or coronavirus (Wongteerasupaya et al., 1995
). P. C. Loh and colleagues identified four major structural proteins (~200, 135, 67 and 22 kDa) in purified YHV, and obtained evidence that the 135 kDa protein is glycosylated and that the ~22 kb ssRNA genome may have negative polarity (Loh et al., 1997
; Nadala et al., 1997
). This led them to classify YHV provisionally in the Rhabdoviridae. More recently, Tang & Lightner (1999)
used strand-specific probes and PCR to demonstrate that YHV virions derived from haemolymph contain a (+)-ssRNA genome. It is evident that these conclusions are drawn from relatively few data and that additional information on the genome sequence, organization and replication strategy is required to establish the appropriate taxonomic classification of YHV and GAV.
In this paper, we describe the 20089 nucleotide (nt) 5'-terminal sequence of the GAV RNA genome. The sequence comprises a single gene encoding two long overlapping open reading frames (ORFs). The proteins encoded in these ORFs display homologies to conserved functional domains of the ORF1a and ORF1ab polyproteins of members of the Coronaviridae and Arteriviridae. We also show that the ORF1a/1b overlap contains an RNA pseudoknot that functions in vitro as a -1 ribosomal frameshift site. The structural organization and expression strategy of this genome region, together with the close genetic relationship of GAV to YHV (Cowley et al., 1999 ), indicate that they represent the first invertebrate viruses of the Order Nidovirales.
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Methods |
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RNA isolation and analysis.
Total RNA was isolated from 0·4 g pooled lymphoid organs from uninfected and GAV-infected P. monodon using 8 ml TRIzol-LS (Gibco-BRL). RNA was resolved (10 µg/lane) in 0·8% agaroseTAE gels containing 0·5 µg/ml ethidium bromide. To determine the nature of a discrete ~22 kb band, 10 µg RNA was treated with 1 U RQ1-DNase (Promega) for 15 min at 37 °C. DNase digestion was monitored using 1 µg 1 kb DNA ladder (Gibco-BRL) and 0·5 µg HindIII-cut bacteriophage DNA. RNase sensitivity was determined by incubating the gel in NTE pH 7·4 containing 50 µg/ml RNase A until ribosomal RNA had been digested. To isolate the ~22 kb dsRNA, 400 µg total RNA was resolved in a 0·6% neutral LMPagarose (Gibco-BRL) gel, the band excised and dsRNA recovered by
-agarase (Boehringer Mannheim) digestion.
RTPCR and cloning.
Sequences representative of the GAV genome were amplified using a random RTPCR method (Froussard, 1992 ) modified as described previously (Cowley et al., 1999
). Briefly, cDNA was synthesized from 10 ng gel-purified ~22 kb dsRNA using 150 ng UNI-primer-dN6 [5' GCCGGAGCTCTGCAGAATTC(N)6 3'], 200 U Superscript II (Gibco-BRL) reverse transcriptase (RT) and incubation at 42 °C for 1 h followed by 99 °C for 5 min. Second-strand cDNA was synthesized using 8 U Klenow polymerase (Promega) and incubation at 37 °C for 30 min. Primer was removed using a Sephadex S400 HR spin column (Pharmacia). PCR amplification of cDNA (1 µl) utilized the UNI-primer 5' GCCGGAGCTCTGCAGAATTC 3' and Qiagen Taq DNA polymerase and buffer containing 1·5 mM MgCl2 (Froussard, 1992
). PCR products were purified using a QIAquick column (Qiagen), ligated into 25 ng pGEM-T (Promega) and transformed into competent E. coli DH5
cells (Gibco-BRL) by standard methods (Sambrook et al., 1989
).
Additional clones were generated similarly using lymphoid organ total RNA and GAV-specific primers, Superscript II RT and either RTPCR using various conditions or long distance RTPCR using the Expand Long Template PCR System (Boehringer Mannheim). Alternatively, clones were generated from cDNA synthesized using GAV-specific primers followed by second-strand synthesis randomly primed with UNI-primer-dN6 and extended using Klenow DNA polymerase (Froussard, 1992 ). cDNAs of variable length were amplified by PCR (Froussard, 1992
) using both the virus-specific primer and UNI-primer and Taq DNA polymerase and buffer (Promega) containing 2·5 mM MgCl2. RTPCR products were either gel purified or processed directly using QIAquick columns and cloned into either pGEM-T vector or EcoRV-cut pBluescript II KS(+).
Analysis of the ORF1a/1b -1 ribosomal frameshift element.
Functional activity of the GAV -1 ribosomal frameshift site at the ORF1a/1b overlap was demonstrated by protein expression in vitro using a T7 TNT coupled transcription/translation system (Promega). A 693 nt sequence encompassing the ORF1a/1b overlap was amplified by PCR from clone P68-15 using primers GAV82 (5' GCAATGGATCCATCCCTGTCGG 3') and GAV83 (5' TTAGCGGATCCATGCAACACATC 3') spanning BamHI-like sites in ORF1a and ORF1b, respectively. Nucleotides modified to generate BamHI sites are underlined. PCR utilized Pwo DNA polymerase (Boehringer Mannheim) and 25 cycles of 95 °C/45 s, 62 °C/45 s, 72 °C/45 s. The 0·7 kb PCR product was purified using a QIAquick column, digested with BamHI, similarly repurified and cloned into a plasmid (pBG) containing the bovine ephemeral fever virus (BEFV) G gene (Walker et al., 1992 ) downstream of a T7 RNA promoter in pBluescript II KS(+). The ORF1a/1b overlap region was cloned into pBG by insertion into a BamHI site, at position G1176C1181 of the BEFV G gene, so that ORF1a and ORF1b were in-frame with the N- and C-terminal ORFs of the G protein, respectively. Clones with the insert in the correct (pBG-F1) and reverse orientations (pBG-R10) were identified and high quality DNA was prepared with a Plasmid Maxi Kit (Qiagen).
Plasmid DNA (0·5 µg) was combined with TNT Rabbit Reticulocyte Lysate reagents (Promega), 0·4 µl [35S]methionine (10001500 Ci/mmol, Amersham) and 5 U T7 RNA polymerase (Promega) in a 10 µl reaction and protein was synthesized by incubating at 30 °C for 2 h. An aliquot (5 µl) of the TNT reaction was added to 20 µl SDSPAGE buffer (125 mM TrisHCl pH 6·8, 2% SDS, 5% 2-mercaptoethanol, 20% glycerol, 0·01% bromophenol blue) and heated at 99 °C for 5 min. Proteins were electrophoresed in an 8% SDSpolyacrylamide gel, fixed, impregnated with Amplify (Amersham) and dried. [35S]Methionine-labelled proteins were detected by fluorography by exposure to Fuji RX film at -70 °C. Radioactivity in proteins was quantified using a Bio-Rad phosphorimaging system.
5'-RACE.
The putative 5' terminus of the GAV genomic RNA was determined by a primer extension anchorPCR method for the random amplification of cDNA ends (5'-RACE) (Dumas et al., 1991 ) using complementary anchorPCR primers (156 and 2668) described previously (Walker et al., 1994
). Briefly, cDNA was synthesized using any of four GAV antisense primers including GAV94 (5' CGTGGTGTGATCATAGTCCTT 3') and GAV95 (5' CTAAGCTCTGGAGGTTGATCAT 3') at different positions toward the N terminus of ORF1a. cDNA was synthesized using either 1 µg lymphoid organ total RNA from GAV-infected prawns or RNA isolated from GAV semi-purified from clarified prawn haemolymph (15000 g for 5 min) by ultracentrifugation at 200000 g, 45 min at 4 °C. The reaction mixture containing RNA, 150 ng primer and 200 U Superscript II RT was incubated at 42 °C for 1 h. RNA was digested with 1·5 U RNase H at 37 °C for 1 h and primer was removed using a Sephadex S400-HR column. Primer 156 (0·2 µg), 5'-phosphorylated using [
-32P]ATP (Bresatec) and T4 polynucleotide kinase (Promega), was ligated to 3' cDNA ends using T4 RNA ligase (NEB) (Walker et al., 1994
). Primer was removed as above and cDNA was amplified by PCR using primer 2668 in combination with GAV-specific primers GAV95 or GAV96 (5' TCTGACGGCAGACAGAACAGT 3'), Taq DNA polymerase and buffer (Promega) containing 2·5 mM MgCl2 and 40 cycles of 95 °C/30 s, 60 °C/30 s and 72 °C/30 s. PCR products were purified using QIAquick columns, cloned into pGEM-T and sequenced.
DNA sequencing and analyses.
Plasmid DNA was prepared using the RPM (BIO 101) or PerfectPREP (5 Prime3 Prime) kits and sequenced using either the Thermo-Sequenase (Amersham) or Big Dye (ABI) dye-terminator systems. Sequencing utilized T7, SP6, universal pUC forward and reverse primers or GAV-specific oligonucleotides. Sequences were determined using an ABI Model 377 automated sequencer at the Australian Genome Reference Facility. SeqEd 1.0.3 and MacVector 6.0.1 were used for sequence alignments, ORF determinations and sequence translations. Database sequence similarity searches were done with the BLAST 2.0 program (Altschul et al., 1997 ). Multiple sequence alignments were undertaken using Clustal W 1.7 (Thompson et al., 1994
). Phylogenetic trees were constructed using the maximum parsimony (PROTPARS) and neighbour-joining (NEIGHBOR) methods in Phylip 3.5 (Felsenstein, 1993
). The COMPARE and GAP programs in the Genetics Computer Group (GCG) package were used for ORF1a and ORF1b sequence comparisons. RNA folding structures were predicted using MFOLD and PLOTFOLD (Zuker, 1989
).
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Results |
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ORF1a sequence
The ORF1a polypeptide has a deduced molecular mass of 460273 Da, a pI of 6·63, 43 potential N-linked glycosylation sites and four significant hydrophobic regions comprising putative multiple membrane-spanning domains (Fig. 2). The two C-terminal hydrophobic regions (aa 25752722 and 31763434) are also evident in nidovirus ORF1a polyproteins (Boursnell et al., 1987
; Lee et al., 1991
; Herold et al., 1993
). Comparisons of the predicted ORF1a polyprotein of GAV with those of bird (avian infectious bronchitis virus, IBV) and mammalian (mouse hepatitis virus, MHV; human coronavirus, HCV; transmissible gastroenteritis virus, TGEV) coronaviruses using the GAP program identified levels of amino acid sequence similarity (40·441·9%) and identity (15·717·2%) that were significantly lower than those evident among the coronavirus proteins (50·762·4% and 27·342·3%, respectively).
Of the putative functional motifs, including the papain-like Cys protease, chymotrypsin-like Ser protease (CSP), 3C-like Cys protease (3CLP) and growth factor/receptor-like domain, identified in nidovirus ORF1a polyproteins (Gorbalenya et al., 1989 ; Boursnell et al., 1987
; Lee et al., 1991
; Herold et al., 1993
; Eleouet et al., 1995
; den Boon et al., 1991
), only a 3CLP homologue was readily identified in GAV. As in coronaviruses, the GAV 3CLP domain is encompassed by two highly hydrophobic regions located in similar positions in ORF1a (Fig. 2
). Although the GAV 3CLP possesses a Cys catalytic nucleophile as in coronaviruses, alignment of upstream Thr and downstream His and Gly residues, important to formation of the substrate-binding pocket, suggests a structural similarity to arterivirus CSPs (Fig. 4A
) (Godeny et al., 1993
; Snijder & Spaan, 1995
; Snijder et al., 1996
). This was also supported by BLAST searches, in which the most significant alignments occurred with the related NIa Cys protease of tobacco etch virus (TEV) and other plant potyviruses (Carrington & Dougherty, 1987
; Snijder et al., 1996
). The GAV ORF1ab polyprotein contains numerous Q(G/S/A) and E(G/S/A) dipeptides representing potential coronavirus 3CLP and arterivirus CSP cleavage sites, respectively. However, further studies are required to determine which of these or related sites are utilized.
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Visual scanning of the GAV ORF1b coding sequence and alignments using the GAP and COMPARE programs identified homologues of the conserved polymerase, metal ion binding (MIB), helicase, and the Motif 1 and Motif 3 (C-terminal) domains of nidoviruses (de Vries et al., 1997 ; Snijder & Spaan, 1995
; Lai & Cavanagh, 1997
). Although the motifs are ordered similarly, the regions linking the polymeraseMIB and helicaseMotif 1 domains are considerably longer than in corona- or toroviruses (Fig. 5
) (Boursnell et al., 1987
; Bredenbeek et al., 1990
; Snijder et al., 1990a
). Sequence relationships of the GAV domains to those of coronaviruses (IBV, MHV-A59, HCV-229E, TGEV) and ETV-Berne were determined using Clustal W multiple alignments (Fig. 4B
). Similar comparisons of the ORF1b domains of corona- and torovirus with those of arteriviruses are described elsewhere for the arteriviruses equine arteritis virus (EAV) (den Boon et al., 1991
), lactate dehydrogenase elevating virus (LDV) (Godeny et al., 1993
) and porcine reproductive and respiratory syndrome virus (PRRSV) (Meulenberg et al., 1993
). The GAV polymerase core contains homologies in the eight domains described for (+)-RNA viruses and all absolutely conserved amino acids in Supergroup I viruses are preserved (Koonin, 1991
). The most striking feature is the presence of the SDD, rather than GDD, motif in the polymerase core which is unique to nidoviruses.
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The ORF1b helicase domain contains the characteristic purine NTP-binding motifs A (GppGtGKT/S) and B (DE) believed to be associated with dsRNA duplex unwinding during RNA replication (Hodgman, 1988 ; Gorbalenya & Koonin, 1989
) (Fig. 4B
). At the terminal position of motif A, GAV possesses a Thr residue characteristic of toro- and arteriviruses rather than a Ser residue conserved among coronaviruses (Lee et al., 1991
; Snijder et al., 1990a
; den Boon et al., 1991
; Godeny et al., 1993
).
In addition to the three domains with predicted functions, ORF1b contains a region (aa 20862199) homologous to the Motif 1 domain (Fig. 4B) identified in corona- and toroviruses, but not arteriviruses (de Vries et al., 1997
). Sequence identity, however, is restricted primarily to conserved Cys and His residues in the C-terminal half of Motif 1. The near C terminus of GAV ORF1b (aa 25282600) also displays limited homology to the C-terminal region of Motif 3 of corona- and toroviruses (Snijder et al., 1990a
; den Boon et al., 1991
; Godeny et al., 1993
; de Vries et al., 1997
)
The phylogenetic relationship of GAV to nidoviruses was determined for the polymerase and helicase domains described in Fig. 4(B). Previous analyses of coronavirus and torovirus polymerases indicate they are distinct from other (+)-RNA virus polymerases (Koonin, 1991
). Phylogenetic comparisons based on Clustal W alignments and the neighbour-joining or maximum parsimony methods generated similar phenograms which placed the GAV polymerase on a branch distinct from the corona-, toro- and arteriviruses, which clustered into the expected lineages (Godeny et al., 1993
) (Fig. 6A
). Indeed, GAV was almost as distantly related to nidoviruses as the potyvirus TEV included as a representative of Supergroup I virus polymerases (Koonin, 1991
). Phylogenetic comparisons of the helicase clustered corona-, toro- and arteriviruses, although less tightly, into the same general relationships (Fig. 6B
). In this case, however, GAV was placed on a branch marginally more closely related to coronaviruses than was the torovirus ETV-Berne.
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Function analysis of the -1 ribosomal frameshift site
Functional activity of the GAV -1 ribosomal frameshift site was examined in vitro by expression in a coupled transcription/translation system. A 693 nt sequence encompassing the ORF1a/1b overlap was inserted into the BEFV G protein gene (Walker et al., 1992 ) such that ORF1a and ORF1b were in-frame with the N- and C-terminal portions of the G ORF, respectively (Fig. 8 A
). SDSPAGE identified three major polypeptides (52·5, 41 and 37·5 kDa) expressed from the BEFV G-gene plasmid (Fig. 8B
, lane 3). The size of these proteins is consistent with their translation from the first three in-frame AUG codons downstream of the G protein initiation codon. The reason for the translational bypass of the N-terminal AUG is not known. Consistent with this pattern of initiation, the plasmid with the GAV frameshift site in the reverse orientation translated only a truncated N-terminal portion (30·5 kDa) of the G protein (Fig. 8B
, lane 2) whilst that with the in-frame insertion translated three major (46, 33·5 and 29·5 kDa) and three minor larger polypeptides (81, 69·5 and 66 kDa) (Fig. 8B
, lane 1). The sizes of the major polypeptides suggest they terminated at the GAV ORF1a stop codon. The sizes of the larger proteins indicate they were translated by read-through of the ORF1a/1b frameshift site, linking the N- and C-terminal ORFs of the BEFV G protein. Radioactivity in the terminated and extended polypeptides was quantified using a phosphorimager and normalized for the number of Met residues. This indicated that translational read-through at the GAV ORF1a/1b frameshift site occurred at 2325% efficiency.
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Discussion |
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ORF1a of GAV contains a 3C-like Cys protease motif likely to be involved in processing the large (759 kDa) ORF1ab polyprotein into functional units. Interestingly, BLAST searches gave the closest match with the NIa proteases of TEV and other plant potyviruses that have a QG/S cleavage specificity similar to coronavirus 3CLPs (Carrington & Dougherty, 1987 ). Although the GAV 3CLP contains a Cys as its catalytic nucleophile, alignment of other residues important to substrate binding and cleavage suggests a structural similarity to arterivirus chymotrypsin-like Ser proteases (Snijder et al., 1996
). However, the GAV 3CLP is distantly related and cleavage sites in the ORF1ab polyprotein will need to be identified to determine whether its site specificity resembles that of the equivalent coronavirus or arterivirus proteases. Its unique sequence, however, may provide definitive evidence of their common ancestry.
The ORF1b coding region of GAV contains functional motifs associated with RNA synthesis, including a polymerase, a metal ion binding domain and a helicase. It also contains distantly related homologues of the Motif 1 and 3 domains that have undefined functions and are present in toro- and coronaviruses but not arteriviruses (Lai & Cavanagh, 1997 ; Snijder & Spaan, 1995
; de Vries et al., 1997
). Although ordered similarly, the spatial distribution of the ORF1b domains is distinct from corona- and toroviruses and no significant similarity exists in the intervening sequences. Pair-wise comparisons of the ORF1b sequence using the GAP and COMPARE programs indicate that coronaviruses and the torovirus ETV-Berne are more closely related to each other than to GAV. Moreover, phylogenetic analyses using the polymerase and helicase domains suggest that corona-, toro- and arteriviruses have diverged from GAV to similar extents. Nevertheless, its ancestral relationship to nidoviruses is clearly established by the presence of the SDD polymerase motif unique to these (+)-ssRNA viruses (Koonin, 1991
; Snijder & Spaan, 1995
). Considering that GAV replicates in penaeid shrimp, the low levels of homology in the conserved functional components of the replicase polyproteins of the vertebrate nidoviruses are not unexpected. Indeed, as marine invertebrates were abundant prior to the evolution of higher plants and animals, GAV may be a relict of an ancient progenitor nidovirus.
A prerequisite for translation of nidovirus ORF1b coding regions is the presence of a -1 ribosomal frameshift site at the ORF1a/1b overlap (Spaan et al., 1988 ; Lai, 1990
; Lai & Cavanagh, 1997
; de Vries et al., 1997
). In GAV, the ORF1a/1b overlap contains a recognized slippery heptanucleotide, AAAUUUU, and a stable but complex RNA pseudoknot 3 nt downstream. Although unlike the G/UUUAAAC heptanucleotides and H-type pseudoknots characteristic of the -1 frameshift elements of vertebrate nidoviruses (Brierley et al., 1989
; Pleij & Bosch, 1989
; ten Dam et al., 1990
; Snijder et al., 1990a
; Bredenbeek et al., 1990
; Lee et al., 1991
), the GAV frameshift site promotes translational read-through of the ORF1a/1b overlap at an efficiency (2325%) comparable to these viruses (Brierley et al., 1989
, 1992
). Interestingly, a frameshift site similar to that of GAV, utilizing an AAAUUUU sequence and complex pseudoknot formed by base-pairing of a downstream sequence with a bulge region of a long stemloop, facilitates a translational frameshift at the gag/pol junction of the retrovirus Rous sarcoma virus (Jacks et al., 1988
; Brierley et al., 1989
). Although diverse heptanucleotides and accompanying RNA folding structures can promote -1 ribosomal frameshifting, those used by vertebrate nidoviruses and GAV are among the most efficient yet identified (Brierley et al., 1992
). It is apparent that the requirement for efficient translation of the GAV ORF1ab replicase polyprotein is fulfilled by a frameshift site quite distinct from those of vertebrate nidoviruses.
Sequencing of 5'-RACE clones suggests that the 5' end of the GAV genome initiates at a 5' AC sequence 68 nt upstream of the putative ORF1a start codon. The short length of the putative 5' untranslated sequence contrasts with the relatively long sequences present in the torovirus ETV-Berne (>700 nt) (Snijder et al., 1991 ) and in the coronaviruses IBV, HCV and MHV (209528 nt) (Boursnell et al., 1987
; Shieh et al., 1987
; Herold et al., 1993
; Bonilla et al., 1994
). For coronaviruses, the 5' end of their long intracellular mRNA 1 is equivalent to that of the genomic (+)-ssRNA packaged in virions. Although our data suggest that this is also the case with GAV, we have identified subgenomic (sg) mRNAs that initiate at common 5' AC positions in highly conserved regions of intergenic sequences downstream of ORF1ab (J. A. Cowley and others, unpublished). Thus, we cannot as yet discount the possibility that the RNA 5' end identified by 5'-RACE is a product of transcription initiation at a similar internal sequence upstream of ORF1a. Further characterization of the 5' termini of the putative genomic and sgmRNAs will be required to determine whether GAV utilizes a transcription mechanism like that suggested for the torovirus ETV-Berne (Snijder et al., 1990b
) or a leader-fusion mechanism like that used by coronaviruses (Lai et al., 1984
; Spaan et al., 1988
; Lai, 1990
) and arteriviruses (de Vries et al., 1990
).
We have recently shown that regions of ORF1b coding sequences of GAV from Australia and YHV from Thailand share 8185% amino acid and 8796% nucleotide identity, suggesting that they represent geographical topotypes (Cowley et al., 1999 ). The viruses are morphologically indistinguishable. Their tubular helical nucleocapsids (1618 nm diameter) vary considerably in length (166435 nm) and their rod-shaped virions (3550x150200 nm) possess ~11 nm surface projections and are quite flexible, often forming pleomorphic bent or doughnut-shaped structures. Although longer, there is a conspicuous resemblance to the rod forms of the highly pleomorphic torovirions (Weiss et al., 1983
) but not to the spherical coronavirions (Lai & Cavanagh, 1997
). However, in common with these viruses, GAV and YHV virions mature by nucleocapsid budding primarily at endoplasmic reticulum and Golgi membranes, often leading to the formation of virion arrays in cytoplasmic vesicles (Boonyaratpalin et al., 1993
; Chantanachookin et al., 1993
; Spann et al., 1995
, 1997
).
Based on its rod-shaped morphology and data indicating an ssRNA genome, YHV was suggested to be a rhabdovirus or coronavirus (Wongteerasupaya et al., 1995 ). Evidence that YHV contains four major structural proteins (170, 135, 67 and 22 kDa) and that its ~22 kb genomic ssRNA may have (-) polarity led to its provisional classification as a rhabdovirus (Nadala et al., 1997
; Loh et al., 1997
). More recent strand-specific hybridization analyses, however, suggest that YHV possesses a (+)-ssRNA genome (Tang & Lightner, 1999
). The molecular data presented here for the Australian yellow head-like virus GAV unequivocally support a relationship, albeit distant, to the (+)-ssRNA nidoviruses. This evidence includes: (i) cloning of viral RNA from an extremely large (~22 kb) intracellular dsRNA likely to represent a genomic replicative intermediate; (ii) an ORF1a polyprotein containing a 3C-like Cys protease; (iii) an ORF1b coding sequence with replicase functions including an SDD polymerase, a metal ion binding domain, a helicase domain and other motifs conserved in corona- and toroviruses; and (iv) an efficient -1 ribosomal frameshift site at the ORF1a/1b overlap that will facilitate translation of a 759 kDa ORF1ab polyprotein. As such, the penaeid shrimp virus GAV and the closely related YHV (Cowley et al., 1999
) appear to be new members of the Nidovirales and the first to be isolated from an invertebrate. Additional data on the nature and organization of the structural genes and on the replication strategy employed by these viruses will be needed to resolve the issue of their taxonomic classification within the Order. As GAV and YHV replicate primarily in the prawn lymphoid (or Oka) organ, and as a defining clinical feature of YHV infection is a yellow (or ochre; meaning pale brownish-yellow colour) discoloration of the prawn cephalothorax, we suggest the name Okavirus to describe these viruses.
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Acknowledgments |
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Footnotes |
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References |
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Received 21 October 1999;
accepted 3 February 2000.