Gene-expression profiling of White spot syndrome virus in vivo

Hendrik Marks1, Oscar Vorst2, Adèle M. M. L. van Houwelingen2, Mariëlle C. W. van Hulten1,{dagger} and Just M. Vlak1

1 Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands
2 Plant Research International, Postbus 16, 6700 AA Wageningen, The Netherlands

Correspondence
Just M. Vlak
just.vlak{at}wur.nl


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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.

{dagger}Present address: CSIRO Livestock Industries, 306 Carmody Road, St Lucia 4067, Brisbane, Australia.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
White spot syndrome virus (WSSV), a member of the virus family Nimaviridae (genus Whispovirus), is a large, enveloped virus that infects a broad range of crustacean species (Wang et al., 1998; Mayo, 2002). In cultured shrimp, WSSV infection can cause a cumulative mortality of up to 100 % within 3–10 days (Lightner, 1996), leading to large economic losses to the shrimp-culture industry. WSSV was first discovered in the Chinese province Fujian in 1992, from where it spread quickly (Cai et al., 1995; Flegel, 1997). Nowadays, the virus has spread to almost all major shrimp-farming areas of the world (Rosenberry, 2002).

Sequencing of three different WSSV isolates (WSSV-TH, WSSV-CN and WSSV-TW) showed that the size of the double-stranded DNA (dsDNA) genome varies from 293 to 307 kb (van Hulten et al., 2001a; Yang et al., 2001; GenBank accession no. AF440570). The completely sequenced isolate WSSV-TH has a genome size of 292 967 bp. The genome encodes 184 putative ORFs, of which only 6 % could be assigned a putative function based on homology with sequences in public databases. Of the remaining ORFs, five major and approximately 40 minor structural virion-protein genes have been identified (van Hulten et al., 2001a; Huang et al., 2002; Tsai et al., 2004). Additionally, three ORFs (ORF3, ORF89 and CN-ORF366) have been suggested to be involved in WSSV latency (Khadijah et al., 2003) and ORF170 was shown to encode an anti-apoptosis protein (Wang et al., 2004). Large regions, each consisting of a variable number of 250 bp-repeat units, were identified dispersed along the viral genome (homologous repeats; hrs). Similar to baculoviruses, these regions may be involved in transcription enhancement and/or DNA replication (Guarino & Summers, 1986; Kool et al., 1993).

WSSV transcriptional analysis performed thus far has focused mainly on WSSV genes that showed homology to known genes, such as the ribonucleotide reductases (Tsai et al., 2000a), the chimeric thymidine kinase–thymidylate kinase (TK-TMK; Tsai et al., 2000b), the DNA polymerase (Chen et al., 2002b), a protein kinase (PK) (ORF2; Liu et al., 2001), the thymidylate synthase (Li et al., 2004b) and the collagen-like ORF (Li et al., 2004a). Also, the major and minor structural protein genes were subjected to transcriptional analysis (Marks et al., 2003; Tsai et al., 2004). Analysis of these genes mostly included RT-PCRs of WSSV-infection time courses and mapping of the 5' and 3' ends of the mRNAs.

To study WSSV gene expression on a genome-wide scale, we constructed a WSSV DNA microarray containing one or more probes for most putative WSSV ORFs. Microarrays have been used successfully to study gene expression of large, dsDNA viruses, such as herpesviruses (Chambers et al., 1999; Stingley et al., 2000; Paulose-Murphy et al., 2001; Ebrahimi et al., 2003), the myovirus bacteriophage T4 (Luke et al., 2002) and the baculoviruses Autographa californica multiple nucleopolyhedrovirus and Bombyx mori nucleopolyhedrovirus (Yamagishi et al., 2003; Iwanaga et al., 2004). By using a WSSV-infection time course in vivo in the shrimp Penaeus monodon, we could show expression of at least 79 % of the WSSV ORFs included on the microarray, indicating that most WSSV computational ORFs are transcriptionally active. For these ORFs, transcription profiles and transcription levels (semi-quantitatively) are analysed.

Recently, we made a genomic comparison between the WSSV isolates TH-96-II, containing the largest WSSV genome size identified thus far (around 312 kb), and WSSV-TH, containing the smallest genome size (Marks et al., 2005). The difference in genome size is mainly due to a major genomic polymorphism designated ‘variable region ORF 23/24’, for which WSSV-TH contains a contiguous deletion of ~13·2 kb compared with TH-96-II. The ~13·2 kb fragment encompasses 10 ORFs (Marks et al., 2005). By using our microarray analysis, gene expression of these ORFs of TH-96-II is evaluated in two permissive crustacean hosts for both isolates, P. monodon and crayfish (Astacus leptodactylus). Marks et al. (2005) also demonstrated a higher virulence in P. monodon of WSSV-TH compared with TH-96-II. To correlate this biological feature with differential WSSV gene expression, the complete WSSV transcriptome at 2 days post-infection (p.i.) is compared between the isolates WSSV-TH and TH-96-II in P. monodon. The importance of genome-wide transcription studies using microarrays in understanding the regulation of WSSV gene expression is discussed.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virus infection.
Characteristics of the virus isolates WSSV-TH and TH-96-II were described by van Hulten et al. (2001b) and Marks et al. (2005), respectively. P. monodon (approx. 35 g) or A. leptodactylus (approx. 35 g) was injected intramuscularly with purified WSSV, using a relatively high dose to synchronize infection in the gills as much as possible. At various time points after injection, three animals were selected randomly, frozen in liquid nitrogen and stored at –80 °C.

Poly(A)+ RNA isolation.
Total RNA was isolated from gills as described previously (Marks et al., 2003). For each time point p.i., gills of three animals were pooled. Approximately 70 % of the 184 WSSV genes encode a consensus poly(A) signal (AAUAAA) or another consensus poly(A)-like signal that could be sufficient for polyadenylation (e.g. AUUAAA), within –50 to 300 bp downstream of their translational stop codons (Birnstiel et al., 1985; Sheets et al., 1990; van Hulten et al., 2001a; Yang et al., 2001). Therefore, we used poly(A)-based RNA isolation and Cy3/Cy5-labelling methods. Poly(A)+ RNA was purified by using the PolyATtract mRNA isolation system III (Promega). The yield of poly(A)+ RNA from total RNA was generally between 0·5 and 1·5 %, as quantified by measuring A260 using a spectrophotometer (NanoDrop Technologies).

Construction of WSSV microarrays.
WSSV is known to encode several ORFs with high nucleotide identity, accommodated in so-called gene families (van Hulten et al., 2001a). To minimize the possibility of WSSV transcripts hybridizing with non-specific probes, we decided to use PCR products (~300–1000 bp in size) instead of oligonucleotides (~50 nt in size) as probes on the WSSV microarrays. The viral-array elements include probes for 158 of the 184 putative WSSV-TH ORFs (Table 1). Probes for the 22 putative genes encoded within the WSSV hrs were not included. Probes for WSSV-TH ORF12 and ORF110 were not spotted, as these ORFs are encoded almost completely within the coding regions of ORF13 and ORF109, respectively. For ORF68 and ORF139, we failed to obtain the respective DNA fragments (reason unknown). For all WSSV-TH genes larger than 2100 bp (42 genes), in addition to the 3'-end probe, an extra probe corresponding to the 5' end of the gene was spotted on the microarrays (the 3'-end probes were used for quantification purposes, the 5'-end probes as controls only). For each WSSV hr, one probe was included on the microarrays. Probes to detect the 10 TH-96-II-specific ORFs (CN-ORFs in ‘variable region ORF23/24’; Marks et al., 2005) were also included on the WSSV microarrays. For each WSSV ORF, overlap of the corresponding DNA fragment that was selected for use as a specific probe with (5'/3' untranslated regions of) neighbouring ORFs was avoided. In addition, probes of the following sources were included on the microarrays: (i) 16 cellular shrimp genes, to evaluate normalization between the several chips; (ii) a set of background controls, consisting of four genes of the plant Medicago truncatula and the jellyfish Aequorea victoria green fluorescent protein (GFP) gene, each spotted in quintuplicate; (iii) the complete coding sequence of the firefly luciferase gene and three partial luciferase clones encompassing the 5', middle and 3' parts of the gene, all spotted in quadruplicate (Table 1). As the samples were spiked with luciferase mRNA prior to labelling, this allowed correction of the expression ratios between samples for differences in the preparation of labelled cDNA and for differences during hybridization to the microarrays. The partial luciferase clones were additionally used to monitor the integrity of the labelled sample cDNA. Each of the total of 272 probes was printed in duplicate on each microarray slide, to evaluate the consistency of the signals obtained.


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Table 1. Complete overview of the PCR-amplified DNA fragments spotted on the WSSV microarray, subdivided by WSSV gene probes (according to ORF numbers), shrimp-gene probes and negative-control probes

Luciferase-control probes are not shown.

 
Preparation of probes.
Parts of the DNA fragments used as WSSV-TH probes were obtained by performing PCRs on DNA clones of the WSSV-TH DNA bank constructed for sequencing of the complete viral genome (van Hulten et al., 2001a), using universal primers. The other parts of the WSSV-TH probes and all TH-96-II-specific probes (CN-ORFs) were obtained by performing PCRs on genomic DNA of WSSV-TH and TH-96-II, respectively, using specific primers designed by using PrimeArray (Raddatz et al., 2001). The cellular shrimp genes, negative controls and luciferase controls were obtained by performing PCRs on purified plasmids containing the anticipated fragments using universal primers. The shrimp genes were originally amplified from a cDNA library of uninfected P. monodon that was available in our laboratory by using specific primers (sequences obtained from GenBank) and cloned into the pGEM-T Easy vector (Promega). Sequencing confirmed that these clones contained the anticipated sequences. All PCRs were performed by using the Expand Long Template PCR system (Roche).

Preparation of WSSV microarrays.
PCR products were column-purified by using a High Pure PCR product purification kit (Roche) and diluted to 0·1 µg µl–1 (in a total volume of 100 µl), as measured by A260. All PCR products showed a clear band of the appropriate size and concentration by agarose-gel electrophoresis. A 10 µg aliquot of each PCR product was dried to completion and dissolved in 10 µl 5x SSC (sodium citrate/sodium chloride) buffer. Microarrays were prepared by spotting individual DNA fragments on GAPS amino-silane-coated glass slides (Corning) with a PixSys 7500 arrayer (Cartesian Technologies) equipped with Chipmaker 3 quill pins (Telechem). Spotting volumes were 0·5 nl, resulting in a 120 µm spot diameter at a pitch of 160 µm. After drying overnight, the microarrays were rehydrated with steam, snap-dried (95–100 °C) and UV-cross-linked (150 mJ). The slides were soaked twice in 0·2 % SDS (2 min), twice in MilliQ (Millipore)-treated water (MQ water) (2 min) and placed into boiling MQ water (2 min). After drying (5 min), the slides were rinsed three times in 0·2 % SDS (1 min), once in MQ water (1 min), submerged in boiling MQ water (2 s) and air-dried.

Synthesis of Cy3/Cy5-labelled cDNA.
Purified poly(A)+ RNA preparations were labelled by using a standard protocol for cDNA synthesis: 2·5 µg P. monodon poly(A)+ RNA [spiked with 1 ng luciferase mRNA (Promega); for A. leptodactylus samples, 1 µg poly(A)+ RNA was used] and 2·5 µg (dT)21 primer were heated to 65 °C (3 min) and then placed at 25 °C (10 min) to anneal the primer. RNA was reverse-transcribed by using Superscript II (Invitrogen) according to the protocol of the manufacturer, with the exception that 40 % of the total dTTP was replaced with 5-(3-aminoallyl)-2'dUTP. After precipitation and washing of the cDNA–RNA hybrids, the RNA was hydrolysed with 0·2 M NaOH at 37 °C (10 min). The solution was neutralized with 0·15 M HEPES (pH 6·8) and 0·15 M HCl. After precipitation and washing, the cDNA was resuspended in 5 µl 0·1 M carbonate buffer (pH 9·3). Dyes were bound covalently to the incorporated amino groups by adding 5 µl 5 mM Cy3 or Cy5 reactive dyes (Amersham Biosciences) in DMSO. This mixture was incubated at room temperature in the dark (1 h). Unincorporated dye was removed by performing ethanol precipitation twice, after which the labelled cDNA was dissolved in 5 µl MQ water. Samples under study were labelled with Cy3 and the reference sample was labelled with Cy5. Reference samples used were (i) for the time series: a mixture of poly(A)+ RNA harvested 0, 1 and 2 days after WSSV-TH infection, containing a pool of transcripts representing all of the genes present in the time course; and (ii) for the isolate comparison: a mixture of poly(A)+ RNA harvested 0 and 2 days p.i. from WSSV-TH-infected, as well as from TH-96-II-infected, shrimp and crayfish gill tissue, providing additional Cy5 signals for the TH-96-II-specific ORFs.

Microarray hybridizations.
After prehybridization of the slides for 2 h at 42 °C in hybridization buffer (50 % formamide, 5x Denhardt's reagent, 5x SSC, 0·2 % SDS, 0·1 mg fish DNA ml–1), the slides were washed by dipping in MQ water, followed by dipping in 2-propanol. Slides were dried by centrifugation at 160 g (1 min). Hybridization occurred in a volume of 65 µl, using a covered hybridization frame [Gene Frame 15 mmx15 mm (65 µl); ABgene]. After heating 65 µl hybridization buffer containing both Cy3- and Cy5-labelled cDNA samples to 95 °C for 1 min, it was loaded into the hybridization chamber. The slides were hybridized for 24 h at 42 °C. Following hybridization, the slides were washed in 1x SSC/0·1 % SDS (5 min), 0·1x SSC/0·1 % SDS (5 min) and rinsed briefly in 0·1x SSC. Slides were dried by centrifugation at 160 g (1 min).

DNA microarray analysis.
Slides were scanned for fluorescence emission with a ScanArray ExpressHT (Perkin Elmer) at 75 % laser power and a resolution of 10 µm, using an attenuation of 65 % (Cy3) or 60 % (Cy5). The resulting Cy3 and Cy5 images were stored as TIFF files and processed individually. For each array element, the integrated OD was determined within a defined circle, using AIS software (Imaging Research). Mean background values, calculated from the hybridization signals of the M. truncatula and A. victoria probes, were subtracted to correct for non-specific fluorescence. Next, Cy3 or Cy5 signals not reaching 0·5x background value were set to this cut-off (0·5x background value) for the respective dye. Elements for which neither the Cy3 and Cy5 signal reached 0·5x background value were discarded from further analysis.

Normalization of the two samples in each hybridization was done with the mean hybridization signal of the full-length luciferase probes. Finally, the Cy3/Cy5 ratio (Rij) was calculated for each element. The reference sample used on each slide was the same within the time series or within the isolate comparison, allowing direct comparison of the different hybridization experiments. Expression ratios for the on-array duplicates (Rij1, Rij2) were calculated separately and the mean of both values was used for further analysis. Improper duplicates [|2log(Rij1/Rij2)|>1] were filtered out. Microsoft Excel was used for organizing data and for statistical analyses. The Cy3 background threshold for WSSV gene expression was set at 1·5x the expression value obtained initially from the Cy3 background-control probes. WSSV genes with a lower Cy3 expression value (below the background threshold) were considered to be not detected.

Analysis of expression data.
For WSSV genes, mean normalized ratios obtained for each gene were converted into percentages of the maximal expression of each gene over the time series. The WSSV probes with a signal above the background threshold at 0 h p.i. and showing no increase in ratio later in WSSV infection were discarded from the analysis (five probes; Table 1). The remaining WSSV probes showed a signal below the background threshold at 0 and 8 h p.i. and the percentages at these time points were set at 0 %. Next, cluster and correlation analysis of WSSV transcription profiles was performed by using GeneMaths software (Applied Maths). For individual shrimp genes, the 2log expression ratios were normalized by subtracting the mean of the 2log ratios for the respective genes over the time course. To enable comparison with WSSV genes, these normalized ratios were converted to percentages, setting the mean expression of the 16 shrimp genes (Table 1) over the time course at 100 %.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To evaluate WSSV gene expression during infection in shrimp, a viral microarray was designed that contained nearly all of the putative WSSV ORFs, based on the published sequences of WSSV-TH, WSSV-CN and TH-96-II (van Hulten et al., 2001a; Yang et al., 2001; Marks et al., 2005). The WSSV genome contains nine hrs, each consisting of 250 bp repeats with a nucleotide identity of between 74 and 91 %. For each hr, a probe representing the respective hr was included on the microarray. In addition to the viral sequences, various other probes were included on the microarray as controls. Table 1 shows a complete overview of the probes present on the microarray.

Poly(A)+ RNA was isolated from WSSV-TH-infected P. monodon gill tissue at 0, 8, 20, 32 and 48 h p.i. The gills are one of the primary target tissues of WSSV infection (Lo et al., 2004). The poly(A)+ RNA was Cy3-labelled and mixed with a Cy5-labelled reference sample to normalize for differences in probes on the microarray slides. In the experiments performed for this time series, a single, standard reference sample was used for each hybridization, allowing direct comparison of the various hybridization experiments. Each of the time-course Cy3/Cy5 mixtures was hybridized to an individual microarray. Fig. 1(a) shows images of parts of the microarrays that were constructed after the scanning.



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Fig. 1. (a) Pseudocolour microarray images (Cy3 and Cy5 are coloured green and red, respectively) of one of the on-array duplicate sets of probes. An equal mix of red and green results in a yellow pseudocolour; other ratios result in intermediate colours. Probes for WSSV and shrimp genes, as well as background and luciferase controls, are distributed randomly over the array. For probes representing WSSV genes (examples are boxed) that are red at 0 and 8 h post-WSSV infection, a yellowish colour can be observed at 20, 32 and 48 h p.i., indicating WSSV gene expression. White probes (saturation) indicate a very high Cy3/Cy5 signal. (b) Scatter plot of the 2log ratios of the on-array duplicates (A and B) present for each probe on the microarrays. Each point in the graph represents a probe. The trend lines of the scatterings shown in the graphs were almost equal to y=x with a regression coefficient of R2>0·93, indicating that the duplicate sets of results are very similar. (c) Expression level (%) of the 16 shrimp genes shown in Table 1 (mean expression over the time course is set to 100 %; SD is indicated for each time point).

 
Microarray controls
Interpretation of microarray experiments is highly dependent on the quality of the data obtained, as well as on the normalization between the several microarrays used. Therefore, the most important controls are summarized.

Reproducibility.
After processing of the data, the Cy3/Cy5 ratio for each gene was obtained in duplicate for the individual time points, due to the on-array duplicates. Fig. 1(b) shows that the ratios of the duplicates were very similar. The mean of the duplicate values was used for further analysis. Additionally, the hybridizations for the five time points were repeated with independently isolated RNA preparations. The data obtained in the duplicate experiment were consistent with those of the experiment described.

Normalization.
To evaluate the normalization procedure [we used the same amount of poly(A)+ RNA for each time point], the transcription profiles of 16 cellular shrimp genes present on the microarray were evaluated (Fig. 1c). Although limited information is available for shrimp transcription, we chose to spot probes on our WSSV microarray for shrimp genes that were likely to be expressed constitutively before and during virus infection. Except for the time point at 0 h p.i., the difference in expression of the 16 selected shrimp genes is within 20 % of the mean expression (set at 100 %) between the several time points. The expression of some of the individual cellular genes shown in Fig. 1(c) might be influenced by WSSV infection, but the transcriptional profile of the 16 shrimp genes combined confirmed the robustness of the normalization procedure used for analysis of the time course.

Expression of WSSV genes
Detectable WSSV genes.
Pilot experiments revealed that the probes of M. truncatula and A. victoria, used as background controls, showed a minimal signal compared with the other probes on the WSSV microarray when labelled cDNA of WSSV-infected shrimp was hybridized to the microarrays. Except for some false-positive signals (see Table 1), the Cy3 signal of every WSSV-specific probe was below the background threshold at the time points 0 and 8 h p.i. of the WSSV-infection time course, indicating that we could not detect any WSSV transcripts at these time points. At 20, 32 and 48 h p.i., we detected 125 of the 158 WSSV ORFs (79 %) for which probes were present on the microarray (Table 1). The ORFs corresponding to the WSSV probes that did not reach the background threshold at any of the time points (21 %) were excluded from further analysis. Four probes representing hrs gave a specific signal, indicating transcriptional activity within these hrs (Table 1). These hrs were included in our further analysis.

Transcription profiling.
The Cy3/Cy5 ratios of the 125 WSSV genes that we could detect specifically were normalized to the maximal expression of each gene (100 %). The constructed transcription profiles are presented in Table 2. To examine the relationship between the WSSV genes, these relative expression data were analysed by hierarchical clustering (Euclidean distance). Fig. 2(a) shows that the WSSV genes clustered into two major groups, boxed yellow and blue. The mean transcription patterns of these clusters are shown in Fig. 2(b and c), respectively.


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Table 2. (Relative) expression levels of the WSSV ORFs on the microarray, sorted by intensities of the probes

 


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Fig. 2. (a) Hierarchical clustering (Euclidean distance-clustering algorithm) of relative WSSV gene-expression profiles in P. monodon gill tissue. Columns indicate separate time points and every row displays the expression profile of a single ORF. The dendrogram at the left clusters the ORFs based on the relatedness of their gene-expression patterns. (b, c) Mean expression profiles (% of maximal expression; SD is indicated for each time point) of the ORFs of (b) cluster ‘E’ and (c) cluster ‘L’ indicated in (a).

 
After the genes of the ‘yellow’ cluster reached (almost) a maximal amount of mRNA in the shrimp gill tissue at 20 h p.i., the total amount of transcripts stayed at this level or declined slightly, depending on the gene (Fig. 2b). Most WSSV putative-early genes, such as the ribonucleotide reductases (rr1 and rr2), the chimeric tk-tmk and both pk genes, which are considered to be expressed before viral DNA replication, were present within this group (Fig. 2a). Therefore, we designated the genes in this cluster ‘E’ (‘putative-early type’). For the ‘blue’ cluster, almost all genes showed a maximal amount of mRNA in the gill tissue at 48 h p.i. The genes of the ‘blue’ cluster include all major structural virion-protein genes (VPs), as well as most minor virion-protein genes (vps; Fig. 2a). Structural virion-protein genes are supposed to be expressed after virus replication and are therefore considered late genes. Genes of the blue cluster were designated ‘L’ (‘putative-late type’).

WSSV gene-expression levels
Semi-quantitative levels of gene expression were evaluated by comparing the normalized, absolute values of the Cy3 signals at time points of maximal expression (100 %) of each particular gene (Fig. 3; Table 2). These absolute gene-expression levels should be interpreted semi-quantitatively, as the DNA fragments spotted on the microarray are different in length and G+C content (resulting in different hybridization efficiencies) and various amounts of DNA might have been spotted for different genes and on different microarray slides. Also, not all WSSV transcripts present in the time-course samples might be labelled with equal efficiency, especially because it is not known whether all mRNAs of WSSV are polyadenylated. Except for vp24, all major structural-protein genes show a very high expression level (Table 2). Most genes with a relatively high expression level (semi-quantitative) cluster in the putative late-type group and encode a consensus poly(A) signal (Table 2). Fig. 3 shows that putative early- and late-type genes, as well as genes of different classes of gene-expression level, are distributed randomly over the genome. The presence of a TATA box in the promoter region of a gene is not correlated positively with its temporal expression class or expression level (Table 2).



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Fig. 3. Semi-quantitative expression levels (arbitrary units) of WSSV genes. Bars representing genes of cluster ‘E’ are coloured grey; bars representing genes of cluster ‘L’ are coloured black.

 
5'/3' probes
For detection of the 42 largest WSSV genes, two probes per gene were spotted on the microarray, corresponding to the 5' and 3' ends. Of the 42 5'-end probes, 28 (67 %) did not give a signal above the background threshold during the time course, whereas, for the 3'-end probes, only eight (19 %) could not be detected. This is probably caused by the reverse-transcriptase reaction, which proceeds from the poly(A) tail at the 3' end of the transcripts. As most 5'-end probes represent parts of the genes that are at least 1·5–2 kb upstream of the poly(A) tail, these 5' ends of the mRNA were probably not reverse-transcribed as efficiently. For the ORFs of which both 5' and 3' ends were detectable, the profile over the time course was very similar, suggesting that both were indeed detecting the same messenger. These data confirm the current annotation of ORFs on the WSSV genome based on the computational analyses by van Hulten et al. (2001a) and Yang et al. (2001), which are very similar (Marks et al., 2004).

Comparison of the transcriptomes of WSSV-TH and TH-96-II
TH-96-II contains a large genomic fragment of ~13·2 kb in a locus known as ‘variable region ORF23/24’, which is absent in WSSV-TH (Marks et al., 2005). Probes for the 10 genes encoded by the ~13·2 kb fragment of TH-96-II were included on our WSSV microarray (Table 1; CN-ORFs). During the complete time course of the previous section, for which WSSV-TH was used, a signal below the background threshold was obtained for these 10 probes, excluding the possibility of (non-specific) cross-hybridization of WSSV-TH genes to these probes. Poly(A)+ RNA was isolated from WSSV-TH- and from TH-96-II-infected P. monodon gill tissue at 2 days post-WSSV infection, labelled and hybridized to different microarrays, as described in the previous section.

As the data obtained from the on-array duplicates were very similar (data not shown), the mean was used for further analysis. The scatter plot in Fig. 4 shows a comparison between the mean ratios obtained from infected tissue of WSSV-TH (one microarray slide) and TH-96-II (other microarray slide). The majority of the probes are between the dotted lines, indicating an expression difference of <2x between WSSV-TH and TH-96-II. Genes showing a twofold expression difference or more are marked.



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Fig. 4. Comparison of the transcriptomes of WSSV-TH (x axis) and TH-96-II (y axis) at 2 days post-infection (d.p.i.) in shrimp and crayfish. Scatter plots of the expression levels obtained for WSSV-TH and TH-96-II. The dotted lines indicate a twofold difference in expression between the isolates. The labelled probes (letters or numbers in case of CN-ORFs) are >2x differentially expressed between WSSV-TH and TH-96-II and represent probes for: 1, CN-ORF479; 2, CN-ORF482; 3, CN-ORF483; 4, CN-ORF492; 5, CN-ORF493; 6, CN-ORF495; 7, CN-ORF500; a, ORF12/13; b, ORF14; c, 3' probe ORF24; d, 5' probe ORF24; e, ORF23; f, ORF77; g, ORF85; h, ORF44; i, ORF118; j, ORF127; k, ORF131; l, ORF145; m, ORF170; n, ORF178.

 
For the TH-96-II-infected P. monodon tissue, we could detect seven of the 10 genes encoded by ‘variable region ORF23/24’ (Table 1). CN-ORF486, CN-ORF489 and CN-ORF497 expression [ORF assignment by Yang et al. (2001)] could not be detected. CN-ORF482, CN-ORF493 (VP35) and CN-ORF495 were expressed highly, indicated by ++ in Table 2. As the CN-ORFs are assigned the background-threshold Cy3 value for WSSV-TH, because these genes are absent from this genome, most of the CN-ORFs (marked l–r) appear to be expressed highly by TH-96-II compared with WSSV-TH in the graphs of Fig. 4. ORF12/13 (as these two genes overlap completely, they cannot be distinguished by using our microarray), ORF14, ORF23, ORF24 (both 5' and 3' probes) and ORF85 were present at around 2x excess in the TH-96-II-infected P. monodon gill tissue, whilst the only gene that showed a relative expression of >2x in WSSV-TH-infected tissue was ORF77 (Fig. 4).

A similar comparison between WSSV-TH and TH-96-II in the crayfish A. leptodactylus (Fig. 4) could detect the same seven of the 10 genes encoded by ‘variable region ORF23/24’ in TH-96-II-infected tissue as were detected for P. monodon. Also, ORF12/13, ORF14 and ORF24 (5' and 3' probes) were present >2x in excess in the TH-96-II-infected A. leptodactylus gill tissue (Fig. 4). Furthermore, ORF127, ORF145 and ORF170 were expressed >2x in excess in TH-96-II-infected tissue, whilst ORF44, ORF118, ORF131 and ORF178 showed a relative expression of >2x in WSSV-TH-infected tissue (Fig. 4).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The WSSV genome contains 184 putative WSSV ORFs based on computational analysis (van Hulten et al., 2001a; Yang et al., 2001). Yang et al. (2001) confirmed transcription of around 50 of these ORFs (28 %) by using RT-PCR on a cDNA cocktail, whilst Tsai et al. (2004) detected 39 minor virion-protein genes by using a small, single dye (Cy3)-based microarray. By using our WSSV microarray analysis (Cy3/Cy5), we could detect transcription of 79 % of all putative WSSV ORFs (excluding those encoded within hrs; Table 1), indicating that most WSSV predicted ORFs are transcriptionally active. However, as we could not detect strand-specific mRNAs due to the fact that dsDNA fragments were used as probes on the microarray, it cannot be excluded that some of the signals obtained originate from non-annotated ORFs encoded by the opposite strand. Except for ORF6 (vp800), ORF71 (dUTPase) and ORF149 (TATA box-binding protein), all currently annotated genes were detected. We could also show expression of almost all genes detected previously (Yang et al., 2001; Tsai et al., 2004). Most baculovirus hrs, which have a structure similar to that of WSSV hrs, are non-coding regions (Possee & Rohrmann, 1997). Our data suggest transcriptional activity within WSSV hr5, hr6, hr7 and hr8. Further research should elucidate the nature of the transcripts that are detected within these WSSV hrs.

Around 20 % of the putative WSSV ORFs did not reach the background threshold (Table 1). It is possible that these genes are not expressed in gills, but it is more likely that it is caused by the detection limits of our microarray experiment. About half of the ORFs that were not detected encode a consensus poly(A) signal, indicating that their transcripts are probably polyadenylated. Therefore, it is not likely that we could not detect these genes because of our poly(A)-based detection methods. More plausible explanations are: (i) a (very) low expression of the gene; or (ii) inferior hybridization properties of the probe spotted on the microarray. Other detection methods, such as quantitative RT-PCRs, can be used to obtain more information about the expression of these genes.

Our data provide evidence that WSSV gene transcription is regulated in a cascaded fashion. At least two major classes of gene products were distinguished, designated putative-early (E) and putative-late (L) (Fig. 2a). The transcription profiles (Fig. 2b and c) follow the classical expression patterns of these gene types, shown for other large, invertebrate, dsDNA viruses, such as baculoviruses, in cell culture (Friesen, 1997; Lu & Miller, 1997). In the case of baculoviruses, transcription of early genes declines when late-gene transcription becomes very high, as the downregulation of early transcription is due directly or indirectly to late-gene expression or viral DNA replication. The fact that the ‘putative-early’ cluster of WSSV does not show this decline (Fig. 2b) is probably caused by asynchronous infection of the gill tissue. Cells infected in a second or third round of infection in the gills will express early genes at a later stage after injection. The presence of most putative-early genes (genes involved in nucleotide metabolism, DNA replication and protein modification) in the ‘putative-early’ class and most putative-late genes (virion-protein genes) in the other class (‘putative late’) supports the accommodation of transcripts into two classes. The arrangement of WSSV genes into different kinetic classes of gene expression is also supported by the analysis of individual genes using RT-PCR (Tsai et al., 2000a, b, 2004; Liu et al., 2001; Chen et al., 2002b; Marks et al., 2003; Li et al., 2004a, b). To obtain further support, future microarray studies could include the testing of different inhibitors, such as cycloheximide and phosphonoacetic acid, to distinguish between gene expression before and after protein synthesis and virus replication, respectively. Such experiments could also shed some light on the classification of the WSSV DNA polymerase and thymidylate synthase as ‘late’ genes by our microarray analysis (Fig. 2a), which is inconsistent with previous reports (Chen et al., 2002b; Li et al., 2004b). A synchronized infection would enable a more precise time schedule of gene expression, but awaits the availability of a suitable shrimp cell-culture system.

In the case of large, dsDNA viruses, such as herpesviruses (Ebrahimi et al., 2003) and baculoviruses (Lu & Miller, 1997), late genes are often transcribed abundantly. Our data show similar results for WSSV late genes, as most of the highly expressed genes are of the late type. Furthermore, all WSSV major structural-protein genes show a very high expression level, except for vp24 (Table 2). RT-PCRs performed for these genes (Marks et al., 2003) confirm these results, showing high expression levels late in infection and a lower expression level of vp24. Also, ORF75 and ORF94, two ORFs containing regions of tandem repeats that are highly polymorphic between several WSSV isolates (Marks et al., 2004; Dieu et al., 2004), show high expression levels (Table 2). The protein encoded by ORF75 is probably located in the virion (vp357; Huang et al., 2002). No functional data are available for ORF94. Of the three ‘putative-early’ genes with a very high expression level, our findings obtained for the chimeric tk-tmk gene (ORF171) confirmed the results of Tsai et al. (2000b) concerning temporal expression class (early) and expression level (very high). No data are available on the function of the other two genes, ORF115 and ORF146.

A comparison of transcriptomes was made between the WSSV isolates WSSV-TH and TH-96-II, both in P. monodon and in A. leptodactylus. The main difference between these isolates is the presence of two large genomic fragments in TH-96-II, ~5·3 kb at a locus known as ‘variable region ORF14/15’ and ~13·2 kb at ‘variable region ORF23/24’, that are both absent in WSSV-TH (Marks et al., 2005). The genes encoded by the additional fragments are dispensable for infection and replication in these species, as both are permissive hosts for WSSV-TH (Marks et al., 2004). Our experiments show that most genes encoded by the ~13·2 kb fragment in TH-96-II are transcriptionally active in these two crustacean species, some even to a relatively high level. Therefore, it is likely that the gene products, although they are not essential, do have a functional role in both species.

Although the expression level of most ORFs shared by both isolates was similar between WSSV-TH and TH-96-II (Fig. 4), some ORFs were expressed differentially (>2x difference). The expression of ORF12/13, ORF14, ORF23 and ORF24 was higher for TH-96-II- than for WSSV-TH-infected gill tissue, both in P. monodon and in A. leptodactylus (except for ORF23, which was only higher in P. monodon). These genes are located at the junction sites of ‘variable region ORF14/15’ and ‘variable region ORF23/24’, but are completely present in both isolates. As the TH-96-II 5' upstream regions of ORF14 and ORF24 are absent in WSSV-TH, they could contain important promoter elements involved in expression of these genes. ORF14 is not essential for virus infection and replication, at least in P. monodon, as a WSSV isolate lacking ORF14 (WSSV-TW; GenBank accession no. AF440570) has been isolated from this species. The significance of the decreased expression of this gene in WSSV-TH-infected tissue remains unclear. As the region encoding ORF24 and the complete coding regions of ORF12/13 and ORF23, including the putative promoter regions, are present in all WSSV isolates characterized thus far (Marks et al., 2004), these ORFs probably have an essential function during virus infection. Recent data suggest a higher virulence of WSSV-TH compared with TH-96-II in P. monodon (Marks et al., 2005). Although the difference in virulence could be explained by a replication advantage of WSSV-TH, which has a smaller-sized genome, the ORFs expressed differentially between WSSV-TH and TH-96-II in P. monodon could also play a role. In this respect, ORF12/13, ORF23 and ORF24, but also ORF85, which are all expressed at a lower level in WSSV-TH-infected P. monodon tissue, and ORF77, the only gene present >2x in excess in WSSV-TH-infected tissue (Fig. 4), are of interest.

Evaluation of in vivo transcription profiles by using microarrays provides a first step in understanding WSSV transcription regulation and gene function on a genome-wide scale. Besides providing insights into the basic biology of the virus, the microarray can also be used to test the effect of drugs on virus replication and gene expression and, consequently, produce information that can aid in the development of effective treatments against WSSV.


   ACKNOWLEDGEMENTS
 
We thank Professor Dr Rob Goldbach, Dr Douwe Zuidema, Dr Marcel Westenberg, Jeroen Witteveldt and Angela Vermeesch for stimulating discussions and their help in the course of these experiments. Roel Staps is acknowledged for his assistance during the hybridizations and scanning of the microarrays. We thank Dr Erik Limpens and Joop Arts for providing us with the M. truncatula probes and the shrimp probes, respectively. This work was supported by Intervet International BV, Boxmeer, the Netherlands.


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DISCUSSION
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Received 14 January 2005; accepted 4 April 2005.



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