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
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ABSTRACT |
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Present address: CSIRO Livestock Industries, 306 Carmody Road, St Lucia 4067, Brisbane, Australia.
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INTRODUCTION |
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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 kinasethymidylate 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.
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METHODS |
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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 (
3001000 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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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 µl1 (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 (95100 °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 cDNARNA 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 ml1), 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 %.
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RESULTS |
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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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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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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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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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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
).
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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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Received 14 January 2005;
accepted 4 April 2005.
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