Genomics and transcriptomics of White spot syndrome virus

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 from Thailand (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 and VR ORF23/24, three of these loci, located in the regions coding for ORF75, ORF94 and ORF125, were identified as useful markers in epidemiological and ecological studies. The highly conserved genomic loci, e.g. the gene encoding the major structural virion protein VP26, are useful for reliable monitoring of WSSV infections in PCR based assays. The observation that the isolate WSSV-TH contains a large deletion in VR ORF23/24 relative to the isolates WSSV-TW and WSSV-CN suggested the evolution and spread of WSSV from a common ancestor, provisionally located near the Southeast coast of China . Further support for this model was obtained by the genomic characterization of eight WSSV isolates collected in 2003 and 2004 along the central- and south-coast of Vietnam (VN) during WSSV outbreaks (Chapter 3). These WSSV-VN isolates contained deletions of intermediate size in VR ORF23/24 relative to WSSV-TW and WSSV-TH. In VR ORF14/15, the WSSV-VN isolates contained deletions of various sizes compared to WSSV-TH. These collective data suggest that the VN isolates and WSSV-TH have a common lineage, which branched off from WSSV-TW and WSSV-CN early on, and that WSSV possibly entered Vietnam by multiple introductions. Further comparisons among the WSSV-VN isolates revealed that the VNTR loci in ORF75 and ORF125, but not in ORF94, are suitable markers to study local and regional spread of WSSV. To study the possible effect of the genetic differences on the fitness and virulence of WSSV, two divergent WSSV isolates (TH-96-II and WSSV-TH) were compared (Chapter 4). TH-96-II was a newly characterized archival WSSV isolate from 1996, which has the largest genome size (~312 kb) of all WSSV isolates identified thus far. As TH-96-II does not contain deletions in either VR ORF14/15 or VR ORF23/24, it may be ancestral to all known WSSV isolates. WSSV-TH contains the smallest genome (~293 kb) identified at present, due to large deletions in VR ORF14/15 and VR ORF23/24. Comparison between TH-96-II and WSSV-TH, when administered to shrimp Penaeus monodon, showed a higher virulence and competitive fitness for the latter. This may suggest that the virus became more virulent over the years during the epidemic while moving south. This enhanced virulence is possibly caused by the continuous contact with susceptible animals, a behavior also seen with some other emerging viruses. Since the more virulent variant (WSSV-TH) has a smaller genome, it may replicate faster to reach a lethal dose. However, it is also possible that the observed differences in virulence are caused by other genetic polymorphisms between the two isolates. As WSSV differs profoundly from other large ds DNA viruses and mainly contains unique genes, the mechanism of gene expression and transcription regulation of this new virus was investigated in the second part of this thesis. To study WSSV gene expression on a genome wide scale, a WSSV DNA microarray was constructed containing probes corresponding to nearly all putative WSSV ORFs (Chapter 5). Using a WSSV infection time course we could show expression of at least 79% of the WSSV ORFs included on the microarray in gill tissue of Penaeus monodon . Clustering of the transcription profiles of the individual genes showed the presence of two major classes of genes, a putative early and a putative late class, suggesting that the WSSV genes at large are expressed in a coordinated and cascaded fashion. Five genes encoding WSSV major virion proteins (VP28, VP26, VP24, VP19 en VP15), which clustered in the late class, were further confirmed to be late by RT-PCR (Chapter 6). Furthermore, the 5' and 3' ends of the mRNA of these late genes were determined for identification of common promoter motifs. To search for common conserved WSSV promoter motifs associated with WSSV early or late gene expression, as determined by the microarrays, two in silico methods were employed (Chapter 7). The abundance of all 4 through 8 nucleotide motifs in the upstream sequences of WSSV genes relative to the complete genome was determined and the upstream sequences of early or late WSSV genes were analyzed for conserved sequences motifs using MEME. Both methods were complemented by alignments of empirically determined 5' ends of various WSSV mRNAs. The collective information shows that the upstream region of WSSV early genes, containing a TATA box and an initiator sequence, is reminiscent to Drosophila RNA polymerase II promoters, suggesting utilization of the cellular transcription machinery for generating early transcripts. The alignment of the 5' ends of known late genes, including the 5' ends determined in chapter 6, identified a degenerate consensus late transcription initiation motif (ATNAC). Of these genes, only one contained a functional TATA box. However, almost half of the WSSV late genes, as assigned by microarrays, did contain a TATA box in their upstream region. This 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. Alignments of the 3' ends of various WSSV mRNAs suggest that there is no difference in polyadenylation between early and late mRNAs. The WSSV polyadenylation characteristics of both classes resemble regular polyadenylation in eukaryotic mRNAs, which is typically located 10 to 25 nt downstream of the sequence AATAAA. In conclusion, the research performed for this thesis has led to a model on the mechanism of WSSV gene expression, and the promoter motifs involved (Chapter 8). The identification of genetic markers has led to more insight in the quick geographical spread of the virus, and the genetic characterization of WSSV isolates may add to the identification of virulence related factors on the WSSV genome. The fundamental insights obtained in the biology and epidemiology of WSSV in this thesis may help in the identification of WSSV genes which can be targets for WSSV intervention strategies.