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More than a decade after its discovery inSouth-East Asia, White Spot Syndrome Virus (WSSV) is still the most important (viral) pathogen in the shrimp culture industry. Despite the shift from culturingPenaeusmonodon towards the presumed less susceptibleLitopenaeusvannamei , the use of specific pathogen free shrimp and the development of more advanced shrimp culturing techniques, WSSV continues to scourge shrimp farms. Therefore there is an urgent need for effective intervention strategies. Vaccination is the generally used method to prevent viral infections in vertebrates. The success of this method depends on the immunological memory generated by the adaptive immune system. Unfortunately, shrimps, as any other arthropod, do not have such an adaptive immune system implying that vaccination would never work. However, some phenomenological observations have been made, indicating that there might be an analogous defense system present in shrimp. With this in mind experiments in this thesis are presented to determine if and how shrimp can be protected against WSSV via vaccination.At the start of this research project several studies were available describing various major structural proteins present in the WSSVvirionincluding the majorvirionenvelope proteins VP28 and VP19 andvirionstructural proteins VP26, VP24 and VP15 (see thesis vanHulten, 2001). In this research a number of these proteins were investigated in more detail as potential vaccine candidates. For one of these, the majornucleocapsidprotein VP15, it was determined that it was probably (one of) the DNA-binding protein(s) of WSSV (Chapter 2). Experiments revealed that VP15 binds non-specifically to double-stranded DNA, but has a strong preference tosupercoiledDNA, suggesting a possible role in the packaging of the WSSV genome. Furthermore, VP15 formshomomultimersbut does not interact with any of the other major WSSV structural proteins and unlike other basic DNA-binding proteins VP15 was notphosphorylated.The

It has been generally accepted that invertebrates such as shrimp do not have an adaptive immune response system comparable to that of vertebrates. However, in the last few years, several studies have suggested the existence of such a response in invertebrates. In one of these studies, the shrimp Penaeus monodon showed increased protection against white spot syndrome virus (WSSV) using a recombinant VP28 envelope protein of WSSV. In an effort to further investigate whether this increased protection is limited to P. monodon or can be extended to other penaeid shrimp, experiments were performed using the Pacific white shrimp Litopenaeus vannamei. As found with P. monodon, a significantly lower cumulative mortality for VP28-fed shrimp was found compared to the controls. These experiments demonstrate that there is potential to use oral application of specific proteins to protect the 2 most important cultured shrimp species, P. monodon and L. vannamei, against WSSV. Most likely, this increased protection is based on a shared and, therefore, general defence mechanism present in all shrimp species. This makes the design of intervention strategies against pathogens based on defined proteins a viable option for shrimp culture

Background: White Spot Syndrome Virus, a member of the virus family Nimaviridae, is a large dsDNA virus infecting shrimp and other crustacean species. Although limited information is available on the mode of transcription, previous data suggest that WSSV gene expression occurs in a coordinated and cascaded fashion. To search in silico for conserved promoter motifs (i) the abundance of all 4 through 8 nucleotide motifs in the upstream sequences of WSSV genes relative to the complete genome was determined, and (ii) a MEME search was performed in the upstream sequences of either early or late WSSV genes, as assigned by microarray analysis. Both methods were validated by alignments of empirically determined 5' ends of various WSSV mRNAs. Results: The collective information shows that the upstream region of early WSSV genes, containing a TATA box and an initiator, is similar to Drosophila RNA polymerase II core promoter sequences, suggesting utilization of the cellular transcription machinery for generating early transcripts. The alignment of the 5' ends of known well-established late genes, including all major structural protein genes, identified a degenerate motif (ATNAC) which could be involved in WSSV late transcription. For these genes, only one contained a functional TATA box. However, almost half of the WSSV late genes, as previously assigned by microarray analysis, did contain a TATA box in their upstream region. Conclusion: The data may suggest the presence of two separate classes of late WSSV genes, one exploiting the cellular RNA polymerase II system for mRNA synthesis and the other generating messengers by a new virus-induced transcription mechanism

White Spot Syndrome Virus (WSSV) is a large enveloped DNA virus that infects shrimp and other crustaceans. The virions are approximately 275 x 120 nm in size and have an ovoid to bacilliform shape and a tail-like appendage at one end. Sequencing revealed that the circular, double stranded (ds) DNA genome of WSSV ranges between 293 and 307 kb in size depending on the WSSV isolate. For a sequenced isolate originating fromThailand(WSSV-TH) 184 putative open reading frames (ORFs) were identified on the genome, most of which are unassigned as they lack homology to known genes in public databases. Based on its unique morphological and genetic features, WSSV has been accommodated in the new virus family Nimaviridae (genus Whispovirus ).WSSV causes serious economic losses in shrimp culture, as 100% cumulative mortalities can be reached within 3-10 days under farming conditions. After its discovery in 1992 in Taiwan WSSV has quickly spread into Southeast-Asia and subsequently to shrimp farming areas all over the world. This thesis aims at obtaining fundamental insights in the genomic structure ("genomics") and transcription regulation ("transcriptomics") of WSSV. This in turn may provide better insight in the molecular basis of WSSV biology and epidemiology, which can be useful in the identification of targets for WSSV intervention strategies.Alignment of the complete genome sequences of the isolates WSSV-TW, WSSV-CN and WSSV-TH, originating from Taiwan, China and Thailand, respectively, revealed that the sequences were very similar (over 99% sequence identity), suggesting that the isolates are variants of the same virus species ( Whispovirus ) and probably evolved recently from a common ancestor (Chapter 2). Two major polymorphic loci were identified, variable region (VR) ORF14/15 and VR ORF23/24, and both appeared to be genomic regions where large deletions occur. Further polymorphisms included loci with variable numbers of tandem repeats (VNTR loci). Next to VR ORF14/15 a

White spot syndrome virus, type species of the genus Whispovirus in the family Nimaviridae, is a large, double-stranded DNA (dsDNA) virus that infects crustaceans. The genome of the completely sequenced isolate WSSV-TH encodes 184 putative open reading frames (ORFs), the functions of which are largely unknown. To study the transcription of these ORFs, a DNA microarray was constructed, containing probes corresponding to nearly all putative WSSV-TH ORFs. Transcripts of 79 % of these ORFs could be detected in the gills of WSSV-infected shrimp (Penaeus monodon). Clustering of the transcription profiles of the individual genes during infection showed two major classes of genes: the first class reached maximal expression at 20 h post-infection (p.i.) (putative early) and the other class at 2 days p.i. (putative late). Nearly all major and minor structural virion-protein genes clustered in the latter group. These data provide evidence that, similar to other large, dsDNA viruses, the WSSV genes at large are expressed in a coordinated and cascaded fashion. Furthermore, the transcriptomes of the WSSV isolates WSSV-TH and TH-96-II, which have differential virulence, were compared at 2 days p.i. The TH-96-II genome encodes 10 ORFs that are not present in WSSV-TH, of which at least seven were expressed in P. monodon as well as in crayfish (Astacus leptodactylus), suggesting a functional but not essential role for these genes during infection. Expression levels of most other ORFs shared by both isolates were similar. Evaluation of transcription profiles by using a genome-wide approach provides a better understanding of WSSV transcription regulation and a new tool to study WSSV gene function

White spot syndrome virus (WSSV) occurs worldwide and causes high mortality and considerable economic damage to the shrimp farming industry. No adequate treatments against this virus are available. It is generally accepted that invertebrates such as shrimp do not have an adaptive immune response system such as that present in vertebrates. As it has been demonstrated that shrimp surviving a WSSV infection have higher survival rates upon subsequent rechallenge, we investigated the potential of oral vaccination of shrimp with subunit vaccines consisting of WSSV virion envelope proteins. Penaeus monodon shrimp were fed food pellets coated with inactivated bacteria overexpressing two WSSV envelope proteins, VP19 and VP28. Vaccination with VP28 showed a significant lower cumulative mortality compared to vaccination with bacteria expressing the empty vectors after challenge via immersion (relative survival, 61%), while vaccination with VP19 provided no protection. To determine the onset and duration of protection, challenges were subsequently performed 3, 7, and 21 days after vaccination. A significantly higher survival was observed both 3 and 7 days postvaccination (relative survival, 64% and 77%, respectively), but the protection was reduced 21 days after the vaccination (relative survival, 29%). This suggests that contrary to current assumptions that invertebrates do not have a true adaptive immune system, a specific immune response and protection can be induced in P. monodon. These experiments open up new ways to benefit the WSSV-hampered shrimp farming industry.

White spot syndrome virus (WSSV) is a member of a new virus family (Nimaviridae) infecting crustaceans. The regulation of transcription of WSSV genes is largely unknown. Transcription of the major WSSV structural virion protein genes, vp28, vp26, vp24, vp19 and vp15, was studied to search for common promoter motifs for coordinate expression. The temporal expression of these genes and both 5' and 3' ends of the mRNA were determined, using infected crayfish gill tissue as a RNA source. RT-PCR showed that all five genes are expressed late in infection compared to the early ribonucleotide reductase large subunit gene. 5' RACE studies revealed a consensus late transcription initiation motif for only two of the five major virion protein genes. This motif was only found in one other upstream region of the putative translational start site of a gene with unknown function (ORF 158). No other conserved sequence motifs could be detected in the sequences surrounding the transcriptional start sites of the five major virion protein genes. All 5' ends were located about 25 nt downstream of an A/T rich sequence, including the consensus TATA-box sequence for vp15. The absence of a consensus motif is distinct from gene regulation of other large dsDNA viruses and suggests a unique regulation of WSSV transcription, in line with its unique taxonomic position.

A truncated version of the white spot syndrome virus (WSSV) 27.5 kDa envelope protein was expressed as a histidine tag fusion protein in Escherichia coli. The bacterial expression system allowed the production of up to 10 mg of purified recombinant protein per liter of bacterial culture. Antiserum from a rabbit immunized with the recombinant protein recognized the 27.5 kDa viral envelope protein of WSSV isolated from different geographic regions. The antiserum did not recognize any of the other known WSSV structural proteins. A sensitive immunodot assay for WSSV was developed using the specific rabbit polyclonal antiserum

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

White spot syndrome virus (WSSV) is a large DNA virus infecting shrimp and other crustaceans. The virus particles contain at least five major virion proteins, of which three (VP26, VP24, and VP15) are present in the rod-shaped nucleocapsid and two (VP28 and VP19) reside in the envelope. The mode of entry and systemic infection of WSSV in the black tiger shrimp, Penaeus monodon, and the role of these proteins in these processes are not known. A specific polyclonal antibody was generated against the major envelope protein VP28 using a baculovirus expression vector system. 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 WSSV infection in shrimp

White spot syndrome virus (WSSV) is at present a major scourge to worldwide shrimp cultivation. We have determined the entire sequence of the double-stranded, circular DNA genome of WSSV, which contains 292,967 nucleotides encompassing 184 major open reading frames (ORFs). Only 6 f the WSSV ORFs have putative homologues in databases, mainly representing genes encoding enzymes for nucleotide metabolism, DNA replication, and protein modification. The remaining ORFs are mostly unassigned, except for five, which encode structural virion proteins. Unique features of WSSV are the presence of a very 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. The collective information on WSSV and the phylogenetic analysis on the viral DNA polymerase suggest that WSSV differs profoundly from all presently known viruses and that it is a representative of a new virus family.

White Spot Syndrome Virus (WSSV) is an invertebrate virus, causing considerable mortality in shrimp. Two structural proteins of WSSV were identified. WSSV virions are enveloped nucleocapsids with a bacilliform morphology with an approximate size of 275 x 120 nm, and a tail-like extension at one end. The double-stranded viral DNA has an approximate size 290 kb. WSSV virions, isolated from infected shrimps, contained four major proteins: 28 kDa (VP28), 26 kDa (VP26), 24 kDa (VP24), and 19 kDa (VP19) in size, respectively. VP26 and VP24 were found associated with nucleocapsids; the others were associated with the envelope. N-terminal amino acid sequences of nucleocapsid protein VP26 and the envelope protein VP28 were obtained by protein sequencing and used to identify the respective genes (vp26 and vp28) in the WSSV genome. To confirm that the open reading frames of WSSV vp26 (612) and vp28 (612) are coding for the putative major virion proteins, they were expressed in insect cells using baculovirus vectors and analyzed by Western analysis. A polyclonal antiserum against total WSSV virions confirmed the virion origin of VP26 and VP28. Both proteins contained a putative transmembrane domain at their N terminus and many putative N- and O-glycosylation sites. These major viral proteins showed no homology to baculovirus structural proteins, suggesting, together with the lack of DNA sequence homology to other viruses, that WSSV may be a representative of a new virus family, Whispoviridae.

The causative agent of white spot syndrome (WSS) is a large double-stranded DNA virus, WSSV, which is probably a representative of a new genus, provisionally called Whispovirus. From previously constructed WSSV genomic libraries of a Taiwan WSSV isolate, clones with open reading frames (ORFs) that encode proteins with significant homology to the class I ribonucleotide reductase large (RR1) and small (RR2) subunits were identified. WSSV rr1 and rr2 potentially encode 848 and 413 amino acids, respectively. RNA was isolated from WSSV-infected shrimp at different times after infection and Northern blot analysis with rr1- and rr2-specific riboprobes found major transcripts of 2.8 and 1.4 kb, respectively. 5′ RACE showed that the major rr1 transcript started at a position of −84 (C) relative to the ATG translational start, while transcription of the rr2 gene started at nucleotide residue −68 (T). A consensus motif containing the transcriptional start sites for rr1 and rr2 was observed (TCAc/tTC). Northern blotting and RT-PCR showed that the transcription of rr1 and rr2 started 4–6 h after infection and continued for at least 60 h. The rr1 and rr2 genes thus appear to be WSSV "early genes."