for virus isolation and purification, 1 batch of prawns yielded hemolymph fractions dominated by a previously undescribed non-occluded baculovirus rather than YHV. Injection of test shrimp with a semipurified preparation of this virus gave rapid mortality, and examination with the transmission electron microscope revealed a dual infection where cells containing the new virus dominated, but some cells containing YHV could also be seen. The tissues infected by the 2 viruses were similar. However, in contrast to YHV, the new virus was assembled completely in the nucleus and in the absence of occluding protein (polyhedrin). By normal histology, the most characteristic feature of infection was eosinophilic Cowdry A-type inclusions in hypertrophied nuclei with marginated chromatin, especially in epithelial cells of the stomach. These intranuclear inclusions became lightly basophilic in late stages of infection. In the epithelial cells of the gills, ultrastructural pathology included nuclear hypertrophy and cytoplasmic disintegration leading to large voids at lysed cell sites. By negative staining, completely assembled, enveloped virions were ellipsoid to obovate with a distinctive multifibrillar appendage and they measured 276 x 121 nm (excluding the appendage). Enveloped and unenveloped nucleocapsids were significantly different In size, indicating posslble shortening and thickening of the viral core and nucleocapsid during viral assembly. Isolation and punficat~on of the nucleic acid from the new virus yielded double-stranded DNA of approximately 168 lulo base pairs. This DNA did not cross-hybridize with DNA fragments isolated from YHV-infected shrimp or from monodon baculovirus (MBV). The features placed t h~s virus in the family Baculoviridae, subfamily Nudibaculovirinae as PmNOBII, but for convenience we have named it informally as Systemic Ectodermal and Mesodermal Baculovirus (SEMBV).

) 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...

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.

Corresponding genomic regions of isolates of yellow head virus (YHV) from Thailand and gill-associated virus (GAV) from Australia were compared by RT-PCR and sequence analysis. PCR primers designed from sequences in the GAV ORFlb polyprotein gene amplified the corresponding 577 nucleotide region of the YHV genome. Comparison of the amplified region indicated 85.1 % nucleotide and 95.8% amino acid sequence identity. YHV PCR primers designed to amplify a 135 nucleotide product previously described as a YHV &agnostic probe failed to amplify the correspondmg product from GAV RNA. However, the cognate GAV sequence for this and another recently reported YHV sequence were located in an upstream region of the ORFlb gene. A comparison of these sequences indicated identities of 83.0 and 80.9% at the nucleotide level and 86.7 and 86.5% at the amino acid level, respectively. The data indicate that GAV and YHV are closely related but distinct viruses for whlch differential diagnostic probes can be applied.

No abstract