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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).

The heterogeneity of calf histones is limited. The histones are divided into five major electrophoretic groups, several of which are further subdivided to make a total of twelve species of histone molecule. The heterogeneity described is probably not due to impurity (though this is hard to assess), to failure of extraction, to degradation during extraction, or to multiple polymerization through disulfide

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Proteolytic processing of the dengue virus polyprotein is mediated by host cell proteases and the virusencoded NS2B-NS3 two-component protease. The NS3 protease represents an attractive target for the development of antiviral inhibitors. The three-dimensional structure of the NS3 protease domain has been determined, but the structural determinants necessary for activation of the enzyme by the NS2B cofactor have been characterized only to a limited extent. To test a possible functional role of the recently proposed ⌽x 3 ⌽ motif in NS3 protease activation, we targeted six residues within the NS2B cofactor by site-specific mutagenesis. Residues Trp62, Ser71, Leu75, Ile77, Thr78, and Ile79 in NS2B were replaced with alanine, and in addition, an L75A/I79A double mutant was generated. The effects of these mutations on the activity of the NS2B(H)-NS3pro protease were analyzed in vitro by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of autoproteolytic cleavage at the NS2B/NS3 site and by assay of the enzyme with the fluorogenic peptide substrate GRR-AMC. Compared to the wild type, the L75A, I77A, and I79A mutants demonstrated inefficient autoproteolysis, whereas in the W62A and the L75A/I79A mutants self-cleavage appeared to be almost completely abolished. With exception of the S71A mutant, which had a k cat /K m value for the GRR-AMC peptide similar to that of the wild type, all other mutants exhibited drastically reduced k cat values. These results indicate a pivotal function of conserved residues Trp62, Leu75, and Ile79 in the NS2B cofactor in the structural activation of the dengue virus NS3 serine protease.Infection by dengue viruses is now widely recognized as a major public health concern, with more than 1 million cases of dengue hemorrhagic fever per year and case fatality rates ranging from 1 to 10% (23). There are four serotypes of dengue virus, which cause dengue hemorrhagic fever and dengue shock syndrome (21,22). Dengue viruses, members of the Flaviviridae family, are small, enveloped, positive-stranded RNA viruses which are transmitted by Aedes mosquitoes (7). At present, neither a commercial vaccine nor a causative treatment is available for the prevention or cure of acute dengue virus diseases.The genomic RNA of dengue virus serotype 2 contains 10,723 nucleotides and encodes a large polyprotein precursor of 3,391 amino acid residues which consists of three structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (26). The polyprotein is co-and posttranslationally processed by proteases of the host cell and the virus-encoded two-component protease NS2B-NS3 to generate individual viral proteins (11,18). Optimal activity of the NS3 serine protease (flavivirin, EC 3.4.21.91) is an essential requirement for maturation of the virus, and inhibition of this enzyme offers the prospect of an effective antiviral chemotherapy for severe cases of dengue hemorrhagic fever and dengue shock syndrome (for review see references 6, 39, and 41 and...

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) 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.