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White spot syndrome virus (WSSV) is a taxonomically unclassified virus which causes a disease in shrimps worldwide. A 936 bp long open reading frame (ORF) was found on a 7.2 kb HindIII fragment of the DNA genome of WSSV located adjacent to the ribonucleotide reductase small subunit gene. This putative ORF showed homology to prokaryotic and eukaryotic endonucleases, which contain a non-specific endonuclease motif. Alignment with viral and eukaryotic endonuclease ORFs revealed that most catalytically and structurally important amino acid residues were present in the putative WSSV non-specific endonuclease gene. An unrooted parsonimous phylogenetic tree of non-specific endonucleases indicated that the WSSV ORF was located in a well bootstrap supported clade containing only arthopods, including one of WSSV's natural hosts, Penaeus japonicus. A similar conjunction was found for the only other viral homologue, present in Fowlpox virus, which was also found in a well bootstrap-supported clade with its natural host, Gallus gallus. This clustering of virus and host suggests that both WSSV and Fowlpox virus may have acquired their nuclease genes from their respective natural hosts. Because the motif for non-specific nucleases is found in only two viruses, this gene cannot be used to clarify the taxonomic position of WSSV. However, the presence of this type of nuclease rarely found in viruses adds a novel feature to WSSV.

White spot syndrome virus (WSSV) is a virus infecting shrimp and other crustaceans, which is unclassified taxonomically. A 2193 bp long open reading frame, encoding a putative protein kinase (PK), was found on a 8.4 kb EcoRI fragment of WSSV proximal to the gene for the major envelope protein (VP28). The identified PK shows a high degree of homology to other viral and eukaryotic PK genes. Homology in the catalytic domains suggests that this PK is a serine/threonine protein kinase. All of the conserved PK domains are present in the WSSV PK gene product and this allowed the alignment with PK proteins from other large DNA viruses, which encode one or more PK proteins. An unrooted parsonimous phylogenetic tree was constructed and indicated that the PK gene is well conserved in all DNA virus families and hence can be used as a phylogenetic marker. Baculoviruses to date contain only a single PK gene, which is present in a separate well bootstrap-supported branch in the tree. The WSSV PK is not present in the baculovirus clade and therefore is clearly separated phylogenetically from the baculovirus PK genes. Furthermore, the WSSV PK gene does not share a most recent common ancestor with any known PK gene from other viruses. This provides further and independent evidence for the unique position of WSSV in a newly proposed genus named Whispovirus.

Since its first discovery in Taiwan in 1992, White spot syndrome virus (WSSV) has caused major economic damage to shrimp culture. The virus has spread rapidly through Asia and reached the Western Hemisphere in 1995 (Texas), where it continued its devastating effect further into Central- and South-America. In cultured shrimp WSSV infection can reach a cumulative mortality of up to 100% within 3 to 10 days.One of the clinical signs of WSSV is the appearance of white spots in the exoskeleton of infected shrimp, hence its name.WSSV has a remarkably broad host range, it not only infects all known shrimp species, but also many other marine and freshwater crustaceans, including crab and crayfish. Therefore, WSSV can be considered a major threat not only to shrimp, but also to other crustaceans around the world.The WSSV virion is a large enveloped particle of about 275 nm in length and 120 nm in width with an ellipsoid to bacilliform shape and a tail-like extension on one end. The nucleocapsid is rod-shaped with a striated appearance and has a size of about 300 nm x 70 nm. Its virion morphology, nuclear localization and morphogenesis are reminiscent of baculoviruses in insects. Therefore, WSSV was originally thought to be a member of the Baculovirida e.At the onset of the research presented in this thesis, only limited molecular information was available for WSSV, hampering its definitive classification as well as profound studies of the viral infection mechanism. As the first step towards unraveling the molecular biology of WSSV, terminal sequencing was performed on constructed genomic libraries of its genome.This led to the identification of genes for the large (rr 1) and small (rr 2) subunit of ribonucleotide reductase, which were present on a 12.3 kb genomic fragment (Chapter 2). Phylogenetic analyses using the RR1 and RR2 proteins indicated that WSSV belongs to the eukaryotic branch of an unrooted parsimonious tree and further showed that WSSV and baculoviruses do not share a recent common ancestor.Subsequently two protein kinase (p k) genes were located on the WSSV genome, showing low homology to other viral and eukaryotic pk genes (Chapter 3). The presence of conserved domains, suggested that these PKs are serine/threonine protein kinases. A considerable number of large DNA viruses contains one or more pk genes and these were used to construct an unrooted parsimonious phylogenetic tree. This tree indicated that the two WSSV pk genes originated most likely by gene duplication. Furthermore, the tree provided strong evidence that WSSV takes a unique position among large DNA virus families and was clearly separated from the Baculovirida e.As a further step to analyze WSSV in more detail, its major virion proteins were analyzed. In general, structural proteins are well conserved within virus families and therefore represent good phylogenetic markers. Furthermore, knowledge on these proteins117 can lead to better insight in the viral infection mechanism. Five major proteins of 28 kDa (VP28), 26 kDa (VP26), 24 kDa (VP24), 19 kDa (VP19), and 15 kDa (VP15) in size were identified (Chapter 4, 5 and 6). VP26, VP24 and VP15 were found associated with the nucleocapsid, while VP28 and VP19 were found associated with the viral envelope. Partial amino acid sequencing was performed on these proteins to identify their respective genes in the WSSV genome.The first structural genes to be identified on the WSSV genome were those coding for VP28 and VP26, which are most abundant in the virion (Chapter 4). The correct identification of these genes was confirmed by heterologous expression in the baculovirus insect cell expression system and detection by Western analysis using a polyclonal antiserum against total WSSV virions. Subsequently, VP24 was characterized (Chapter 5) and computer-assisted analysis revealed a striking amino acid and nucleotide similarity between VP24, VP26 and VP28 and their genes, respectively. This strongly suggests that these genes have evolved by gene duplication and subsequently diverged into proteins with different functions within the virion, i.e. envelope and nucleocapsid. All three proteins contained a putative transmembrane domain at their N-terminus and multiple putative N- and O-glycosylation sites. The putative transmembrane sequence in VP28 may anchor this protein in the viral envelope. The hydrophobic sequences may also be involved in the interaction of the structural proteins to form homo- or heteromultimers. In Chapter 6 the identification of the structural proteins VP19 and VP15 is described.The VP19 polypeptide contained two putative transmembrane domains, which may anchor this protein in the WSSV envelope. Also this protein contained multiple putative glycosylation sites. N-terminal sequencing on VP15 showed that this protein was expressed from the second translational start codon within its gene and that the first methionine was cleaved off. As VP15 is a very basic protein and resembles histone proteins, it is tempting to assume that this protein functions as a DNA binding protein within the viral nucleocapsid.None of the identified structural proteins showed homology to viral proteins in other viruses, which further supports the proposition that WSSV has a unique taxonomical position.As the theoretical sizes determined of the various structural proteins, as derived from their genes, were smaller than the apparent sizes on SDS-PAGE, it was suspected that some of these proteins were glycosylated (Chapter 6). All five identified proteins were expressed in insect cells using baculovirus vectors, resulting in expression products of similar sizes as in the WSSV virion. The glycosylation status of the proteins was analyzed and this indicated that none of the five major structural proteins was glycosylated. This is a very unusual feature of WSSV, as enveloped viruses of vertebrates and invertebrates contain glycoproteins in their viral envelopes, which often play important roles in the interaction between virus and host, such as attachment to receptors and fusion with cell membranes.To study the mode of entry and systemic infection of WSSV in the black tiger shrimp, Penaeus monodon, the role of the major envelope protein VP28 in the systemic infection in shrimp was studied (Chapter 7). An in vivo neutralization assay was performed in P. monodo n, using a specific polyclonal antibody generated against VP28. The VP28 antiserum was able to neutralize WSSV infection of P. monodon in a concentration-dependent manner upon intramuscular injection. This result suggests that VP28 is located on the surface of the virus particle and is likely to play a key role in the initial steps of the systemic infection of shrimp.To analyze the genome structure and composition, the entire sequence of the double-stranded, circular DNA genome of WSSV was determined (Chapter 8). On the 292,967 nucleotide genome 184 open reading frames (ORFs) of 50 amino acids or larger were identified. Only 6% of the WSSV ORFs had putative homologues in databases, mainly representing genes encoding enzymes for nucleotide metabolism, DNA replication and protein modification. The remaining ORFs were mostly unassigned except for the five encoding the structural proteins. Unique features of the WSSV genome are the presence of an extremely long ORF of 18,234 nucleotides with unknown function, a collagen-like ORF, and nine regions, dispersed along the genome, each containing a variable number of 250-bp tandem repeats. When this WSSV genome sequence was compared to that of a second isolate from a different geographic location, the isolates were found to be remarkably similar (over 99% homology) (Chapter 9). The major difference was a 12 kbp deletion in the WSSV isolate, described here, which is apparently dispensable for virus infectivity.To complete the taxonomic research on WSSV, its DNA polymerase gene was used in a phylogenetic study (Chapter 8), confirming the results of the phylogeny performed on PK.To obtain a consensus tree, combined gene phylogeny analysis was performed using the rr 1, rr 2, pk and pol genes, which were also present in other large dsDNA virus families (Chapter 9). Based on this consensus tree no relationship was revealed for WSSV with any of the established families of large DNA viruses. The collective information on WSSV and the phylogenetic analysis suggest that WSSV differs profoundly from all presently known viruses and is a representative of a new virus family, with the proposed name 'Nimaviridae' (nima = thread).The present knowledge on the WSSV genome and its major structural proteins, has created a good starting point for further studies on the replication strategy and infection mechanism of the virus, and last but not least, will open the way for the design of novel strategies to control this devastating pathogen.

White spot syndrome virus (WSSV) is an invertebrate virus causing considerable mortality in penaeid shrimp. The oval-to-bacilliform shaped virions, isolated from infected Penaeus monodon, contain four major proteins: VP28, VP26, VP24 and VP19 (28, 26, 24 and 19 kDa, respectively). VP26 and VP24 are associated with the nucleocapsid and the remaining two with the envelope. Forty-one N-terminal amino acids of VP24 were determined biochemically allowing the identification of its gene (vp24) in the WSSV genome. Computer-assisted analysis revealed a striking similarity between WSSV VP24, VP26 and VP28 at the amino acid and nucleotide sequence level. This strongly suggests that these structural protein genes may have evolved by gene duplication and subsequently diverged into proteins with different functions in the WSSV virion, i.e. envelope and nucleocapsid. None of these three structural WSSV proteins showed homology to proteins of other viruses including baculoviruses, underscoring the distinct taxonomic position of WSSV among invertebrate viruses.

White spot syndrome is a worldwide disease of penaeid shrimp. The disease agent is a bacilliform, enveloped virus, white spot syndrome virus (WSSV), with a double-stranded DNA genome that probably contains well over 200 kb. Analysis of a 12?3 kb segment of WSSV DNA revealed eight open reading frames (ORFs), including the genes for the large (RR1) and small (RR2) subunits of ribonucleotide reductase. The rr1 and rr2 genes were separated by 5760 bp, containing several putative ORFs and two domains with multiple sequence repeats. The first domain contained six direct repeats of 54 bp and is part of a coding region. The second domain had one partial and two complete direct repeats of 253 bp at an intergenic location. This repeat, located immediately upstream of rr1, has homologues at several other locations on the WSSV genome. Phylogenetic analysis of RR1 and RR2 indicated that WSSV belongs to the eukaryotic branch of an unrooted parsimonious tree and, further, seems to suggest that WSSV and baculoviruses probably do not share an immediate common ancestor. The present analysis of WSSV favours the view that this virus is either a member of a new genus (Whispovirus) within the Baculoviridae or a member of an entirely new virus family.