Genomic Open Reading Frame Research Articles

Abstract PL01-01: Advances in functional cancer genomics

Abstract Recent advances in genomics now make it possible to consider enumerating all of the genetic lesions in specific cancers. While these approaches will yield critical information regarding the identify, number, and types of alterations found in human tumors, a complementary approach to decipher the molecular basis of malignant transformation depends upon the application of genome scale tools to annotate the function of genes involved in cancer initiation and progression. Over the past several years, we have developed genome scale RNAi libraries and open reading frame expression libraries that permit a systematic evaluation of genes involved in cancer initiation and maintenance. Using these libraries, we have now performed screens in a panel of human cancer cell lines to systematically identify cancer vulnerabilities. By combining these functional approaches with information derived from mapping the structural abnormalities present in cancer genomes, we have identified several new oncogenes that contribute to cancer development. The ability to systematically manipulate gene expression at genome scale now provides opportunities to investigate the function of genes involved in cancer initiation and maintenance, which will provide a framework for therapeutic and prevention strategies. Citation Information: Cancer Prev Res 2011;4(10 Suppl):PL01-01.

Abstract SY25-01: Functional genomics, transformation, and resistance

Abstract Recent advances in genomics now make it possible to consider enumerating all of the genetic lesions in specific cancers. While these approaches will yield critical information regarding the identify, number, and types of alterations found in human tumors, a complementary approach to decipher the molecular basis of malignant transformation depends upon the application of genome scale tools to annotate the function of genes involved in cancer initiation and progression. Over the past several years, we have developed genome scale RNAi libraries and open reading frame expression libraries that permit a systematic evaluation of genes involved in cancer initiation and maintenance. Using these libraries, we have now performed screens in a panel of human cancer cell lines to systematically identify cancer vulnerabilities. By combining these functional approaches with information derived from mapping the structural abnormalities present in cancer genomes, we have identified several new oncogenes that contribute to cancer development. In addition, many commonly occurring and well-validated oncogenes and tumor suppressor genes remain refractory to molecularly targeted therapies. An alternative strategy for targeting such cancer drivers is to identify gene products that, when suppressed or inhibited, result in cell death only in the presence of an oncogenic allele. Through the use of systematic RNAi screens, we have identified several genes that act as synthetic lethal partners to known oncogenes. Taken together, these studies suggest that combining forward and reverse genetic approaches with information derived from the cancer genome characterization projects will yield a comprehensive list of cancer vulnerabilities and establish a general approach for the rational identification of oncogenic and co-dependent pathways in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY25-01. doi:10.1158/1538-7445.AM2011-SY25-01

The genome sequence and proteome of bacteriophage ΦCPV1 virulent for Clostridium perfringens

Application of bacteriophages and their lytic enzymes to control Clostridium perfringens is one potential approach to reduce the pathogen on poultry farms and in poultry-processing facilities. Bacteriophages lytic for C. perfringens were isolated from sewage, feces and broiler intestinal contents and ΦCPV1, a virulent bacteriophage, was classified in the family Podoviridae. The purified virus had an icosahedral head and collar of approximately 42 nm and 23 nm in diameter, respectively, with a structurally complex tail of 37 nm lengthwise and a basal plate of 30 nm. The ΦCPV1 double-stranded DNA genome was 16,747 base pairs with a GC composition of 30.5%. Twenty-two open reading frames (ORFs) coding for putative peptides containing 30 or more amino acid residues were identified and analyzed in the genome. Amino acid sequences of the predicted proteins from the ΦCPV1 genome ORFs were compared with those from the NCBI database and potential functions of 12 proteins were predicted by sequence homology. Three putative proteins were similar to hypothetical proteins with unknown functions, whereas seven proteins did not have similarity with any known bacteriophage or bacterial proteins. Identified ORFs formed at least four genomic clusters that accounted for predicted proteins involved with replication of the viral DNA, its folding, production of structural components and lytic properties. One bacteriophage genome encoded lysin was predicted to share homology with N-acetylmuramoyl- l-alanine amidases and a second structural lysin was predicted to be a lysozyme-endopeptidase. These enzymes digest peptidoglycan of the bacterial cell wall and could be considered potential therapeutics to control C. perfringens.

Abstract IA1-1: Systematic functional genomics and cancer target identification and validation

Abstract Recent advances in genomics now make it possible to consider enumerating all of the genetic lesions in specific cancers. While these approaches will yield critical information regarding the identify, number, and types of alterations found in human tumors, a complementary approach to decipher the molecular basis of malignant transformation depends upon the application of genome scale tools to annotate the function of genes involved in cancer initiation and progression. Over the past several years, we have developed genome scale RNAi libraries and open reading frame expression libraries that permit a systematic evaluation of genes involved in cancer initiation and maintenance. Using these libraries, we have now performed screens in a panel of human cancer cell lines to systematically identify cancer vulnerabilities. By combining these functional approaches with information derived from mapping the structural abnormalities present in cancer genomes, we have identified several new oncogenes that contribute to cancer development. In addition, many commonly occurring and well- validated oncogenes and tumor suppressor genes remain refractory to molecularly targeted therapies. For example, the proto-oncogene KRAS is mutated in a wide array of human cancers, most of which are aggressive and respond poorly to standard therapies. An alternative strategy for targeting KRAS is to identify gene products that, when suppressed or inhibited, result in cell death only in the presence of an oncogenic allele. Through the use of systematic RNAi screens, we have identified two kinases, TBK1 and STK33 that act in a synthetic lethal manner to selectively kill cancer cell lines that depend on mutant KRAS. Taken together, these studies suggest that combining forward and reverse genetic approaches with information derived from the cancer genome characterization projects will yield a comprehensive list of cancer vulnerabilities and establish a general approach for the rational identification of oncogenic and codependent pathways in cancer. Citation Information: Clin Cancer Res 2010;16(14 Suppl):IA1-1.

Automated Yeast Transformation Protocol to Engineer Saccharomyces cerevisiae Strains for Cellulosic Ethanol Production with Open Reading Frames That Express Proteins Binding to Xylose Isomerase Identified Using a Robotic Two-Hybrid Screen

Commercialization of fuel ethanol production from lignocellulosic biomass has focused on engineering the glucose-fermenting industrial yeast Saccharomyces cerevisiae to use pentose sugars. Because S. cerevisiae naturally metabolizes xylulose, one approach involves introducing xylose isomerase (XI), which catalyzes conversion of xylose to xylulose. In this study, an automated two-hybrid interaction protocol was used to find yeast genes encoding proteins that bind XI to identify potential targets for improving xylose utilization by S. cerevisiae. A pDEST32 vector re-engineered for TRP selection and containing the Gal4 binding domain fused with the Piromyces sp. E2 XI open reading frame (ORF) was used as bait with a library of LEU-selectable pOAD vectors containing the Gal4 activation domain in fusion with members of the S. cerevisiae genome ORF collection. Binding of a yeast ORF protein to XI activates two chromosomally located reporter genes in a PJ69–4 yeast strain to give selective growth. Five genes, including ADHI, were identified in the two-hybrid screen, suggesting the proteins encoded by these genes bind to XI. The effect of ADHI overexpression was examined using the pSUMOduoHisADHI vector in an automated protocol to transform eight previously identified yeast strains that showed anaerobic growth on xylose. One transformant consumed all available glucose, xylose, and arabinose during growth on wheat straw hydrolysate.

EPIC-DB: a proteomics database for studying Apicomplexan organisms

BackgroundHigh throughput proteomics experiments are useful for analyzing the protein expression of an organism, identifying the correct gene structure of a genome, or locating possible post-translational modifications within proteins. High throughput methods necessitate publicly accessible and easily queried databases for efficiently and logically storing, displaying, and analyzing the large volume of data.DescriptionEPICDB is a publicly accessible, queryable, relational database that organizes and displays experimental, high throughput proteomics data for Toxoplasma gondii and Cryptosporidium parvum. Along with detailed information on mass spectrometry experiments, the database also provides antibody experimental results and analysis of functional annotations, comparative genomics, and aligned expressed sequence tag (EST) and genomic open reading frame (ORF) sequences. The database contains all available alternative gene datasets for each organism, which comprises a complete theoretical proteome for the respective organism, and all data is referenced to these sequences. The database is structured around clusters of protein sequences, which allows for the evaluation of redundancy, protein prediction discrepancies, and possible splice variants. The database can be expanded to include genomes of other organisms for which proteome-wide experimental data are available.ConclusionEPICDB is a comprehensive database of genome-wide T. gondii and C. parvum proteomics data and incorporates many features that allow for the analysis of the entire proteomes and/or annotation of specific protein sequences. EPICDB is complementary to other -genomics- databases of these organisms by offering complete mass spectrometry analysis on a comprehensive set of all available protein sequences.

Experimental determination of translational start sites resolves uncertainties in genomic open reading frame predictions – application to Mycobacterium tuberculosis

Correct identification of translational start sites is important for understanding protein function and transcriptional regulation. The annotated translational start sites contained in genome databases are often predicted using bioinformatics and are rarely verified experimentally, and so are not all accurate. Therefore, we devised a simple approach for determining translational start sites using a combination of epitope tagging and frameshift mutagenesis. This assay was used to determine the start sites of three Mycobacterium tuberculosis proteins: LexA, SigC and Rv1955. We were able to show that proteins may begin before or after the predicted site. We also found that a small, non-annotated open reading frame upstream of Rv1955 was expressed as a protein, which we have designated Rv1954A. This approach is readily applicable to any bacterial species for which plasmid transformation can be achieved.

Characterization and phylogeny of two β-cytoskeletal actins fromHemibarbus mylodon(Cyprinidae, Cypriniformes), a threatened fish species in Korea

Complementary DNA and genomic sequences representing two different beta-actins were isolated from a threatened freshwater fish species Hemibarbus mylodon. The beta-actin 1 and 2 encoded an identical number of amino acids (375 aa), and shared 88.8 and 99.7% of identity at coding nucleotide and amino acid levels, respectively. Genomic open reading frame (ORF) sequences of both isoforms contained five translated exons interrupted by four introns with conserved GT/AG exon/intron boundary rule. Semi-quantitative RT-PCR showed that the two isoform mRNAs were ubiquitously detected in all tissues tested, but transcript levels were variable across tissues. Phylogenetic analysis showed that H. mylodon beta-actin 1 and 2 were clustered into two distinct major and minor branches of Cypriniformes, respectively. Comparisons of the 5'-upstream region and 3'-UTR of H. mylodon beta-actin 1 also showed a high degree of homology with those of the major teleost beta-actins and warmblooded vertebrate beta-cytoskeletal actins, suggesting their more recent common origin.

Molecular characterization of astrovirus in stool samples from children in São Paulo, Brazil

The purpose of this study was to characterize astrovirus in faecal samples collected from children with and without diarrhea in São Paulo, Brazil, grouped into two sets: EPM and HU. Detection and genotyping were carried out using reverse transcription nested polymerase chain reaction (RT-PCR) with specific primers directed towards the genome open reading frame 2 (ORF2). Results for EPM set showed that 66/234 (28.2%) were positive: 28/94 (29.7%) from children with acute diarrhea, 14/45 (31.1%) with persistent diarrhea, and 9/55 (16.3%) from control individuals. No data was available for 15/40 (37.5%) of samples. Mixed infections with other viruses were found in 33 samples. In the HU, 18/187 (9.6%) were positive: 12/158 (7.6%) from individuals with acute diarrhea and 6/29 (20.7%) from control children. Four samples were mixed with other viruses. Out of 66 astrovirus positive EPM samples, 18 (27.2%) were characterized as human astrovirus type-1 (HAstV-1), two (3.0%) as HAstV-2, two (3.0%) as HAstV-3, and three (4.5%) as HAstV-8. Among 18 astrovirus positive HU samples, one (5.5%) was characterized as HAstV-1, six (33.3%) as HAstV-2, and one (5.5%) as HAstV-8. Two HAstV-8 genotyped samples were further confirmed by nucleotide sequencing. Our results shows that astroviruses are circulating in a constant manner in the population, with multiple serotypes, in higher frequency than it was described for other Brazilian regions. For the first time in Sao Paulo, Brazil, it was shown that astroviruses play an important role in children gastroenteritis, as described for most locations where they were detected.

Genomic comparison using data mining techniques based on a possibilistic fuzzy sets model

Current copiousness of genomic information stored in biological databases [Mar Albà, M., Lee, M., Pearl, D., Shepherd, F.M.G., Martin, A.J., Orengo, N., Kellam, C.A., 2001. P. VIDA: a virus database system for the organisation of virus genome open reading frames. Nuleic Acids Res. 133–136] makes ultimately feasible the proposal for an application of knowledge management aimed to discover general rules in subcellular phenomena. The goal of this work is primarily to discover relationships between genes by microarray analysis. The tools exploited come from clustering techniques and are mainly based on Knowledge Discovery in Databases (KDD) concepts [Fayyad, U., Piatetsky-Shapiro, G., Smyth, P., 1996. From data mining to knowledge discovery in databases. AI Magazine 17(3), 37–54]. Starting from a data set, each element can be represented by a characteristic matrix, which sums up all data attributes. In this case data mining is oriented to perform a Pattern Recognition of related sequences, hidden in databases [Hand, D.J., Nicholas, A., 2005. Heard finding groups in gene expression data. J. Biomed. Biotechnol. 215–225]. Following a bottom up approach, the next refinement is to compare retrieved data to gather similar features, by dedicated clustering algorithms [Kaufman, L., Rousseeuw, P.J., 1990. Finding groups in data. An Introduction to Cluster Analysis. John Wiley & Sons, New York; Forman, G., Zhang, B., 2000. Distributed Data clustering can be efficient and exact HP. Laboratories Palo Alto HPL-2000, p. 158], driven by fuzzy logic, allowing us to perceive by intuition a common denominator for various genomic families and to anticipate likely future developments.

Differential expression of a putative riboflavin-aldehyde-forming enzyme (raf) gene during development and post-harvest storage and in different tissue of the sporophore in Agaricus bisporus

Cloning and characterisation of a putative riboflavin-aldehyde-forming enzyme gene (raf) from the cultivated mushroom Agaricus bisporus and its expression during morphogenesis are described. Three cDNA clones were isolated following differential screening of cDNA libraries from rapidly expanding sporophores and post-harvest stored sporophores. The cDNA sequence and predicted translation analysis revealed an open reading frame (ORF) of 348 nucleotides encoding a polypeptide of 115 amino acids, with three introns (56-66 bases) interrupting the genomic ORF. Blast X searches of the databases with the gene sequence showed homology (40% identity and 56% similarity) to the riboflavin-aldehyde-forming enzyme gene from Schizophyllum commune. In A.bisporus, the raf gene sequence upstream of the ORF contained a large CT-rich putative regulatory element (-64 to -24 bases) found in highly expressed genes in various mushrooms, and a 6-base motif present in the 3' end of the genomic sequence, but not in the corresponding 3' non-coding part of the cDNA, was identified. The raf gene transcripts increased abundantly in rapidly developing sporophores as well in post-harvest stored sporophores. Differential expression of the raf gene transcripts in different tissues of the sporophore was also observed, with higher levels in the stipe compared with the cap and gills. The temporal and spatial expression patterns observed suggest transcriptional regulation of the raf gene during A. bisporus morphogenesis.

Novel gene and gene model detection using a whole genome open reading frame analysis in proteomics

High-throughput mass spectroscopy data combined with a six-frame translation of the human genome can be used to identify novel protein encoding genes, as demonstrated with a search for plasma proteins.

Genetic Analysis of Feline Caliciviruses Associated with a Hemorrhagic-Like Disease

Feline calicivirus (FCV) is 1 of the most common causes of upper respiratory tract disease in cats. Other disease syndromes associated with FCV infection have been reported. Recently, calicivirus infection associated with a hemorrhagic-like disease leading to significant mortality in cats has been reported. The clinical signs are similar to those observed with the calicivirus of rabbit hemorrhagic disease. This study characterized 2 FCV isolates associated with hemorrhagic-like disease. Nucleotide sequencing of the complete genome has been done for these 2 isolates as well as for 4 additional isolates representing other disease syndromes. Previously reported sequence data for the entire genome of classical FCV (6 isolates) and a portion of the capsid gene for hemorrhagic-like FCV (3 isolates), isolated in different regions of United States were used in the genetic analysis. Sequence data were used to determine relationships among the isolates and any correlation with phenotype. Nucleotide sequence comparisons of the entire genome and individual open reading frames revealed high homology among all isolates. Data suggest that the virulence may have genetic determinants on the basis of phylogenetic clustering of the isolates associated with hemorrhagic-like disease.

Shark (Scyliorhinus torazame) metallothionein: cDNA cloning, genomic sequence, and expression analysis

Novel metallothionein (MT) complementary DNA and genomic sequences were isolated from a cartilaginous shark species, Scyliorhinus torazame. The full-length open reading frame (ORF) of shark MT cDNA encoded 68 amino acids with a high cysteine content (29%). The genomic ORF sequence (932 bp) of shark MT isolated by polymerase chain reaction (PCR) comprised 3 exons with 2 interventing introns. Shark MT sequence shared many conserved features with other vertebrate MTs: overall amino acid identities of shark MT ranged from 47% to 57% with fish MTs, and 41% to 62% with mammalian MTs. However, in addition to these conserved characteristics, shark MT sequence exhibited some unique characteristics. It contained 4 extra amino acids (Lys-Ala-Gly-Arg) at the end of the beta-domain, which have not been reported in any other vertebrate MTs. The last amino acid residue at the C-terminus was Ser, which also has not been reported in fish and mammalian MTs. The MT messenger RNA levels in shark liver and kidney, assessed by semiquantitative reverse transcriptase PCR and RNA blot hybridization, were significantly affected by experimental exposures to heavy metals (cadmium, copper, and zinc). Generally, the transcriptional activation of shark MT gene was dependent on the dose (0-10 mg/kg body weight for injection and 0-20 microM for immersion) and duration (1-10 days); zinc was a more potent inducer than copper and cadmium.

Developing an epitope-driven tuberculosis (TB) vaccine

Epitope-driven vaccines are created from selected sub-sequences of proteins, or epitopes, derived by scanning the protein sequences of pathogens for patterns of amino acids that permit binding to human MHC molecules. We developed a prototype tuberculosis (TB) vaccine that contains epitopes derived by (1) EpiMer mapping of previously published secreted proteins derived from Mycobacterium tuberculosis (Mtb), and (2) EpiMatrix mapping of selected Mtb genome open reading frames (ORFs). Each of the epitopes contains at least three distinct class II MHC binding motif matches. These Mtb epitope selections were validated by measuring T cell responses from peripheral blood mononuclear cells (PBMC) obtained from healthy, asymptomatic tuberculin skin test-positive donors. Twenty-four validated Mtb epitopes were selected for inclusion in a DNA plasmid vector. We immunized HLA-DR B*0101 transgenic mice with this vaccine prototype augmented by co-administration of rIL-15. Following administration of three immunizations at 14-day intervals in conjunction with rIL-15, epitope-specific T cell responses were observed to eight of the 24 epitopes contained in the DNA construct, one week following the last injection. The systematic application of bioinformatics tools to whole genomes, in combination with in vitro methods for screening and confirming epitopes, may lead to the development of novel vaccines for infectious diseases like TB.

An X-to-autosome retrogene is required for spermatogenesis in mice.

We identified the gene carrying the juvenile spermatogonial depletion mutation (jsd), a recessive spermatogenic defect mapped to mouse chromosome 1 (refs. 1,2). We localized jsd to a 272-kb region and resequenced this area to identify the underlying mutation: a frameshift that severely truncates the predicted protein product of a 2.3-kb genomic open reading frame. This gene, Utp14b, evidently arose through reverse transcription of an mRNA from an X-linked gene and integration of the resulting cDNA into an intron of an autosomal gene, whose promoter and 5' untranslated exons are shared with Utp14b. To our knowledge, Utp14b is the first protein-coding retrogene to be linked to a recessive mammalian phenotype. The X-linked progenitor of Utp14b is the mammalian ortholog of yeast Utp14, which encodes a protein required for processing of pre-rRNA and hence for ribosome assembly. Our findings substantiate the hypothesis that mammalian spermatogenesis is supported by autosomal retrogenes that evolved from X-linked housekeeping genes to compensate for silencing of the X chromosome during male meiosis. We find that Utp14b-like retrogenes arose independently and were conserved during evolution in at least four mammalian lineages. This recurrence implies a strong selective pressure, perhaps to enable ribosome assembly in male meiotic cells.

Automated Identification of Putative Methyltransferases from Genomic Open Reading Frames

We have analyzed existing methodologies and created novel methodologies for the automatic assignment of S-adenosylmethionine (AdoMet)-dependent methyltransferase functionality to genomic open reading frames based on predicted protein sequences. A large class of the AdoMet-dependent methyltransferases shares a common binding motif for the AdoMet cofactor in the form of a seven-strand twisted beta-sheet; this structural similarity is mirrored in a degenerate sequence similarity that we refer to as methyltransferase signature motifs. These motifs are the basis of our assignments. We find that simple pattern matching based on the motif sequence is of limited utility and that a new method of "sensitized matrices for scoring methyltransferases" (SM2) produced with modified versions of the MEME and MAST tools gives greatly improved results for the Saccharomyces cerevisiae yeast genome. From our analysis, we conclude that this class of methyltransferases makes up approximately 0.6-1.6% of the genes in the yeast, human, mouse, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, and Escherichia coli genomes. We provide lists of unidentified genes that we consider to have a high probability of being methyltransferases for future biochemical analyses.

Visualization of aligned genomic open reading frame data*

AbstractStudents can better appreciate the value of genomic data if they are asked to use the data themselves. However, in general the enormous volume of data involved makes detailed examination difficult. Here we present a web site that allows students to study one particular aspect of sequenced genomes. They are able to align the open reading frames (ORFs) of any available genome that is of reasonable size. The ORFs may be aligned using either the start codon or the stop codon as the starting points. Results will readily show the presence of common ribosome binding sites as well as reveal interesting order within the ORFs that is nonexistent outside of them. Students will be able to ask various questions involving comparisons of genomes and see the results presented in both a tabular and graphic format. An example problem is presented under “Results.”

The Identification of Pats1, a Novel Gene Locus Required for Cytokinesis inDictyostelium discoideum

Here, we describe the identification and characterization of the cytokinesis-deficient mutant cell line 17HG5, which was generated in a restriction enzyme-mediated integration mutagenesis screen designed to isolate genes required for cytokinesis in Dictyostelium discoideum. Phenotypic characterization of the 17HG5 cell line revealed no apparent defects in the global functionality of the actomyosin cytoskeleton except for the observed cytokinesis defect when grown in suspension culture. Plasmid rescue was used to identify the disrupted gene locus (pats1; protein associated with the transduction of signal 1). that caused the cytokinesis defect. Disruption of the pats1 locus was recreated through homologous recombination in several independent cell lines, each recapitulating the cytokinesis-defective phenotype and thereby confirming that this gene locus is important for proper cytokinesis. Sequence data obtained by analysis of the genomic region flanking the inserted restriction enzyme-mediated integration plasmid revealed an 8892-bp genomic open reading frame encoding a 2964-amino-acid protein. The putative pats1 protein contains 3 regulatory domains (RI-phosphatase, RII-GTP-binding, R-III protein kinase), 13 leucine-rich repeats, and 8 WD-40 repeats. These regulatory domains coupled with the protein-protein interacting domains suggest that pats1 is involved in signal transduction during cytokinesis in Dictyostelium.

Characterization of a novel envelope protein (VP281) of shrimp white spot syndrome virus by mass spectrometry.

The primary structure of a novel envelope protein from shrimp white spot syndrome virus (WSSV) was characterized using a combination of SDS-PAGE and mass spectrometry. The resulting amino acid sequence matched an open reading frame (ORF), ORF1050, of the WSSV genome ORF database. ORF1050 contained 843 nt, encoding 281 aa, and was termed the vp281 gene. Computer-assisted analysis showed that both the vp281 gene and its product shared no significant homology with other known viruses. However, they shared striking identity/similarity with another WSSV structural protein, VP292, at both the nucleotide and amino acid sequence level, suggesting that vp281 and vp292 might have evolved by gene duplication from a common ancestral gene. WSSV VP281 cDNA was cloned into a pET32a(+) expression vector containing a T7 RNA polymerase promoter to produce (His)(6)-tagged fusion proteins in Escherichia coli strain BL21. Specific mouse antibodies were raised using the purified fusion protein (His)(6)-VP281. Western blot analysis showed that the mouse anti-(His)(6)-VP281 antibodies bound specifically to VP281 of WSSV, without cross-reactivity with VP292. The transmission electron microscope immunogold-labelling method was used to localize VP281 in the WSSV virion as an envelope protein. The cell attachment 'Arg-Gly-Asp' motif in VP281 indicated that this protein might play an important role in mediating WSSV infectivity.