VIRION COMPOSITION AND GENOMICS OF WHITE SPOT SYNDROME VIRUS OF SHRIMP, 2001

PhD Thesis by M.C.W. van Hulten

Wageningen University, the Netherlands

ISBN: 90-5808-516-3, 115 pp.

Summary:

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 Baculoviridae.

At the onset of the research presented in this thesis, only limited molecular information was available to WSSV, hampering its definitive classification as well as profound studies of the viral infection mechanism. As the first step towards unravelling 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 (rr1) and small (rr2) 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 (pk) 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/theonine 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 Baculoviridae.

As a further step to analyze WSSV in more detail, its major virion proteins were analysed. In general, structural proteins are well conserved within virus families and therefore represent good phylogenetic markers. Furthermore, knowledge on these proteins 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 analysed 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 studies (Chapter 7). An in vivo neutralization assay was performed in P. monodon, 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 rr1,rr2, pk and pol genes, which were also present in other large dsDNA virus families (Chapter 9). Based on the 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.  

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