Penaeus monodon shrimp collected from across the Indo-Pacific region during 1997-2004 were screened for the presence of yellow head-related viruses. Phylogenetic analyses of amplified ORF1b gene segments identified at least six distinct genetic lineages (genotypes). Genotype 1 (YHV) was detected only in shrimp with yellow head disease. Genotype 2 (GAV) was detected in diseased shrimp with the less severe condition described as mid-crop mortality syndrome and in healthy shrimp from Australia, Thailand and Vietnam. Other genotypes occurred commonly in healthy shrimp. Sequence comparisons of structural protein genes (ORF2 and ORF3), intergenic regions (IGRs) and the long 3'-UTR supported the delineation of genotypes and identified both conserved and variant structural features. In putative transcription regulating sequences (TRSs) encompassing the sub-genomic mRNA 5'-termini, a core motif (5'-GUCAAUUACAAC-3') is absolutely conserved. A small (83 nt) open reading frame (ORF4) in the 3'-UTR of GAV is variously truncated in all other genotypes and a TRS-like element preceding ORF4 is invariably corrupted by a A>G/U substitution in the central core motif (5'-UU(G/U)CAAC-3'). The data support previous evidence that ORF4 is a non-functional gene under construction or deconstruction. The 3'-UTRs also contain predicted 3'-terminal hairpin-loop structures that are preserved in all genotypes by compensatory nucleotide substitutions, suggesting a role in polymerase recognition for minus-strand RNA synthesis.

Sequence analysis of the " 20 kb 5h-terminal portion of the ssRNA genome of gill-associated virus (GAV) of Penaeus monodon prawns has previously established that it contains an ORF1a-1b replicase gene equivalent to those of the coronavirus and arterivirus members of the order Nidovirales. Sequence analysis of the remaining " 6n2 kb of the GAV genome downstream of ORF1a-1b to a 3h-poly(A) tail has identified two highly conserved intergenic sequences in which 29/32 nucleotides are conserved. Northern hybridization using probes to the four putative GAV ORFs and either total or poly(A)-selected RNA identified two 3h-coterminal subgenomic (sg) mRNAs of " 6 kb and " 5n5 kb. Primer extension and 5h-RACE analyses showed that the sgmRNAs initiate at the same 5h-AC positions in the central region of the two conserved intergenic sequences. Neither method provided any evidence that the GAV sgmRNAs are fused to genomic 5h-leader RNA sequences as is the case with vertebrate coronaviruses and arteriviruses. Intracellular doublestranded (ds)RNAs equivalent in size to the 26n2 kb genomic RNA and two sgRNAs were also identified by RNase/DNase digestion of total RNA from GAV-infected prawn tissue. The identification of only two sgmRNAs that initiate at the same position in conserved intergenic sequences and the absence of 5h-genomic leader sequences fused to these sgmRNAs confirms that GAV has few genes and suggests that it utilizes a transcription mechanism possibly similar to the vertebrate toroviruses but distinct from coronaviruses and arteriviruses.

) with potential to form an RNA pseudoknot. The structure resides 3 nt downstream of a ribosomal frame-shift 'slippery' sequence (AAAUUUU) and a -1 frame-shift at this site would extend the ORF1 polyprotein by 2616 amino acids (299322 Da). In ORF1b, YHV shares 88.9% amino acid sequence identity with GAV and includes conserved polymerase, metal ion binding, helicase and other domains (Motifs 1 and 3) characteristic of nidoviruses. Compared to GAV, the YHV non-coding region linking the ORF1b and ORF2 genes contains a 263 nt insertion. However, the region contains a conserved core sequence of 46 nucleotides (84.8% identity) that includes a stretch of 20 identical nucleotides surrounding a sub-genomic RNA transcription termination site. The data confirms the taxonomic placement of YHV in the Nidovirales and supports biological and topographical evidence that YHV and GAV may be classified as distinct species. KEY WORDS: Yellow head · Virus · Nidovirus · Polymerase Resale or republication not permitted without written consent of the publisherDis Aquat Org 50: [87][88][89][90][91][92][93] 2002 Morphologically, YHV closely resembles gill-associated virus (GAV) that infects Penaeus monodon in Australia and causes a disease with histological characteristics similar to yellow head disease (Spann et al. 1997). On the basis of genome organization and expression strategy, GAV has recently been characterized as the first invertebrate member of the Nidovirales -a taxonomic order which also includes the coronaviruses, toroviruses and arteriviruses (Cowley et al. 2000a,b, Enjuanes et al. 2000. As for other nidoviruses, the 5'-end of the (+) single-stranded RNA GAV genome expresses a long polyprotein encoded in 2 different reading frames (ORF1a and ORF1b) that are aligned during translation by a -1 ribosomal frameshift (Cowley et al. 2000b). The ORF1b gene encodes sequence motifs for polymerase, metal ion-binding and helicase domains that are also characteristic of nidoviruses. A limited comparison of short regions of the ORF1b gene has indicated that YHV has significant sequence homology with GAV, suggesting the viruses are closely related and should be regarded as geographic topotypes in the yellow head complex (Cowley et al. 1999, Walker et al. 2001.In this paper, we describe the complete nucleotide and deduced amino acid sequences of the YHV ORF1b gene and a long non-coding region immediately downstream of ORF1b that together represent approximately 30% of the genome. Analysis of the sequence indicates that YHV, although clearly distinct from GAV, also has characteristics consistent with classification in the Nidovirales as a member of a new taxon for which the name Okavirus has been proposed (Cowley et al. 2000b, Enjuanes et al. 2000. MATERIALS AND METHODSYHV was obtained from moribund Penaeus monodon showing signs of yellow head disease that were collected from a farm in Chachoengsao province, Thailand, in July 1998. A gill extract from diseased shrimp was passaged once in P. monodon and a stock inoculum was prepared by d...

Gill-associated virus (GAV), a positive-stranded RNA virus of prawns, is the prototype of newly recognized taxa (genus Okavirus, family Roniviridae) within the order Nidovirales. In this study, a putative GAV cysteine proteinase (3C-like proteinase [3CL pro ]), which is predicted to be the key enzyme involved in processing of the GAV replicase polyprotein precursors, pp1a and pp1ab, was characterized. Comparative sequence analysis indicated that, like its coronavirus homologs, 3CLpro has a three-domain organization and is flanked by hydrophobic domains. The putative 3CL pro domain including flanking regions (pp1a residues 2793 to 3143) was fused to the Escherichia coli maltose-binding protein (MBP) and, when expressed in E. coli, was found to possess N-terminal autoprocessing activity that was not dependent on the presence of the 3CL pro C-terminal domain. N-terminal sequence analysis of the processed protein revealed that cleavage occurred at the location 2827 LVTHE2VRTGN2836 . The trans-processing activity of the purified recombinant 3CL pro (pp1a residues 2832 to 3126) was used to identify another cleavage site, 6441 KVNHE2LYHVA 6450 , in the C-terminal pp1ab region. Taken together, the data tentatively identify VxHE2(L,V) as the substrate consensus sequence for the GAV 3CLpro . The study revealed that the GAV and potyvirus 3CL pro s possess similar substrate specificities which correlate with structural similarities in their respective substrate-binding sites, identified in sequence comparisons. Analysis of the proteolytic activities of MBP-3CL pro fusion proteins carrying replacements of putative active-site residues provided evidence that, in contrast to most other 3C/3CL pro s but in common with coronavirus 3CL pro s, the GAV 3CL pro employs a Cys 2968 -His 2879 catalytic dyad. The properties of the GAV 3CL pro define a novel RNA virus proteinase variant that bridges the gap between the distantly related chymotrypsin-like cysteine proteinases of coronaviruses and potyviruses.

Yellow head virus (YHV) is a major agent of disease in farmed penaeid shrimp. YHV virions purified from infected shrimp contain three major structural proteins of molecular mass 116 kDa (gp116), 64 kDa (gp64) and 20 kDa (p20). Two different staining methods indicated that the gp116 and gp64 proteins are glycosylated. Here we report the complete nucleotide sequence of ORF3, which encodes a polypeptide of 1666 amino acids with a calculated molecular mass of 185 713 Da (pI=6?68). Hydropathy analysis of the deduced ORF3 protein sequence identified six potential transmembrane helices and three ectodomains containing multiple sites for potential N-linked and O-linked glycosylation. N-terminal sequence analysis of mature gp116 and gp64 proteins indicated that each was derived from ORF3 by proteolytic cleavage of the polyprotein between residues Ala 228 and Thr 229 , and Ala 1127 and Leu 1128 , located at the C-terminal side of transmembrane helices 3 and 5, respectively. Comparison with the deduced ORF3 protein sequence of Australian gill-associated virus (GAV) indicated 83 % amino acid identity in gp64 and 71 % identity in gp116, which featured two significant sequence deletions near the N terminus. Database searches revealed no significant homology with other proteins. Recombinant gp64 expressed in E. coli with and without the C-terminal transmembrane region was shown to react with antibody raised against native gp64 purified from virions.

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The ORF2 gene of Gill-associated virus (GAV) of Penaeus monodon prawns resides 93 nucleotides downstream of the ORF1a-ORF1b gene and encodes a 144-amino-acid hydrophilic polypeptide (15,998 Da; pI, 9.75) containing 20 basic (14%) and 13 acidic (9%) residues and 19 prolines (13%). Antiserum to a synthetic ORF2 peptide or an Escherichia coli-expressed glutathione S-transferase-ORF2 fusion protein detected a 20-kDa protein in infected lymphoid organ and gill tissues in Western blots. The GAV ORF2 fusion protein antiserum also cross-reacted with the p20 nucleoprotein in virions of the closely related Yellow head virus. By immuno-gold electron microscopy, it was observed that the ORF2 peptide antibody localized to tubular GAV nucleocapsids, often at the ends or at lateral cross sections. As GAV appears to contain only two structural protein genes (ORF2 and ORF3), these data indicate that GAV differs from vertebrate nidoviruses in that the gene encoding the nucleocapsid protein is located upstream of the gene encoding the virion glycoproteins. Gill-associated virus (GAV) ofPenaeus monodon prawns is a type species of the genus Okavirus in the Roniviridae of the order Nidovirales (5, 7, 22). Chronic GAV infection, in which replication is restricted to the foci of hypertrophied cells in the lymphoid organ (LO), is ubiquitous in wild and farmed P. monodon prawns on the east coast of Australia (8,33,39). Acute-phase infection, in which GAV spreads to a wide range of tissues, has been linked to farm disease outbreaks since at least 1996 (33,34,35). The tubular helical nucleocapsids and rod-shaped, enveloped virions of GAV are morphologically identical to those of Yellow head virus (YHV), which has caused mass deaths in P. monodon prawns cultured in Asia, and both viruses cause similar cytopathologies (2,3,20,34,35,36). Sequence similarity levels in the ORF1b gene and the ORF3 glycoprotein gene indicate that GAV and YHV are closely related geographic topotypes (6,16,28).The complete sequencing of the 26,235-nucleotide (nt) RNA genome of GAV has identified five genes ordered 5Ј-ORF1a/ORF1b-ORF2-ORF3-ORF4-(A) n -3Ј (5, 7). YHV also has a long (Ͼ22-kb) (plus-strand) single-stranded RNA genome (24, 36, 43), but sequences have been reported only for the region from the ORF1b gene to the 3Ј poly(A) tail (16,28,29). In GAV, ORF1a contains a 3C-like proteinase with VxHE2(L,V) cleavage site specificity (44); ORF1b contains SDD-type polymerase, zinc finger, and helicase motifs; and the polyprotein pp1ab is C terminally extended from pp1a by Ϫ1 ribosomal frameshifting at an AAAUUUU slippage site preceding an RNA pseudoknot (7). GAV transcribes two 3Ј-coterminal subgenomic mRNAs (sgmRNAs) with 5Ј AC termini that map to sites within conserved intergenic sequences upstream of ORF2 and ORF3 (9). Unlike coronaviruses (27) and arteriviruses (37), but as identified in the shorter three of the four sgmRNAs of the Berne equine torovirus (31, 38), the two GAV sgmRNAs do not contain 5Ј genomic leader sequences.Purified YHV virions contain three s...