1 Biotechnological Research and Development Institute, Can Tho University, 3/2 Street Nr 1, 008471 Can Tho City, Vietnam
2 Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands
Correspondence
Just M. Vlak
just.vlak{at}wur.nl
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ABSTRACT |
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These authors contributed equally to this work.
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INTRODUCTION |
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Electron microscopic analysis showed that WSSV consists of a rod-shaped nucleocapsid with a cross-hatched appearance, surrounded by a trilaminar envelope with a unique tail-like appendix at one end (Wongteerasupaya et al., 1995; Durand et al., 1997
; Nadala et al., 1998
). The circular dsDNA genome of WSSV has a size of around 300 kb and is one of the largest animal virus genomes that has been entirely sequenced (van Hulten et al., 2001
; Yang et al., 2001
). Only 6 % of the putative 184 ORFs encoded by the viral genome have homologues in public databases, mainly representing genes encoding enzymes for nucleotide metabolism, DNA replication and protein modification (van Hulten et al., 2001
).
In addition to South-East Asia, WSSV has been reported from the United States in 1995 (Rosenberry, 1996) and from Central and South America since early 1999 (Rosenberry, 2000
). In 2002, WSSV was also detected in France and Iran (Rosenberry, 2002
). The various geographical isolates of WSSV identified so far are very similar in morphology and proteome. Limited differences in RFLP patterns have been reported, suggesting either a high degree of genomic stability or a recent emergence (Nadala & Loh, 1998
; Lo et al., 1999
; Wang et al., 2000a
, b
; Marks et al., 2004
). Preliminary studies indicated that there is also little difference in virulence between various WSSV isolates, although direct comparisons were not made (Wang et al., 1999
; Lan et al., 2002
). After the complete sequencing of three different WSSV isolates originating from Taiwan (WSSV-TW; Wang et al., 1995
), China (WSSV-CN; Yang et al., 2001
) and Thailand (WSSV-TH; van Hulten et al., 2001
), the major variable loci in the WSSV genome were mapped by alignment of these sequences (Marks et al., 2004
). Roughly, the variable loci can be divided into deletions, variable numbers of tandem repeats (VNTRs), single nucleotide indels and single nucleotide polymorphisms (SNPs). The variation within these loci, in particular in the large genomic deletions, suggested a geographical spread from a common ancestor from either side of the Taiwan Strait to Thailand (Marks et al., 2004
), but genetic intermediates were missing to support this hypothesis.
The present study focuses on WSSV isolates from Vietnam (VN), from eight different locations along the central and south coast. The variable loci, as identified by Marks et al. (2004), were subjected to detailed analysis, including sequencing. Using these newly characterized WSSV-VN genotypes, the value of each of the identified loci as a genetic marker for strain identification as well as epidemiological and ecological studies is evaluated. Furthermore, molecular typing was used to analyse the relationship between the eight WSSV isolates from Vietnam and those from Taiwan, China and Thailand. The genetic changes could be correlated with the spread of WSSV radiating out from either side of the Taiwan Strait to Thailand.
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METHODS |
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PCR analysis of WSSV-infected shrimp.
To screen for WSSV, we developed a standardized PCR-based WSSV detection protocol. One microlitre of DNA extract was tested in two similar single-step PCRs with a shrimp 16S rRNA or a WSSV VP26 primer pair (Table 2), using Taq DNA polymerase (Promega). The 16S rRNA primer pair amplifies a shrimp mitochondrial DNA fragment encoding the 16S rRNA and is used as a positive control for the presence of host DNA. The VP26 primer pair amplifies part of the WSSV VP26 ORF (van Hulten et al., 2000b
) and is used to screen for WSSV-positive shrimp. PCR conditions used and sizes of the PCR products are shown in Table 2
.
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Cloning of PCR products.
PCR products were purified from 1 % agarose gels using a DNA extraction kit (MBI Fermentas). These products were subsequently cloned into E. coli DH5 competent cells using the pGEM-T easy vector system I (Promega). Plasmids containing the correct insert, as screened by restriction enzyme analysis and/or by colony PCR, were prepared for sequencing by purification with the High Pure plasmid isolation kit (Roche).
Virus production and purification.
The virus isolate WSSV-TH used in this study originated from infected P. monodon imported from Thailand in 1996 and was obtained as described before (van Hulten et al., 2000a). The virus WSSV-VN isolate T (Table 1
) originated from a single infected P. monodon. Tissue of a WSSV-VN-T-infected P. monodon was homogenized in 330 mM NaCl. After centrifugation at 1700 g for 10 min, the supernatant was filtered (0·45 µm filter; Schleicher & Schuell) to obtain the virus. Crayfish Orconectes limosus or Astacus leptodactylus were injected intramuscularly with a lethal dose of WSSV (WSSV-TH or WSSV-VN-T), using a 26-gauge needle (Microfine B&D). Virus was isolated and processed according to published procedures (van Hulten et al., 2000a
).
Purification of viral DNA and restriction enzyme analysis.
Viral DNA was isolated from purified virions as described by van Hulten et al. (2000a). WSSV DNA was digested with BamHI (Invitrogen) and fragments were separated by electrophoresis in a 0·6 % agarose gel at 40 V (1·3 V cm1) for 20 h. After separation, the gels were stained with ethidium bromide (0·5 µg ml1 in TAE).
Sequencing and computer analysis.
Plasmid clones were sequenced using universal T7 and/or Sp6 primers, and by primer walking for inserts of >1·5 kb (BaseClear). Sequence data were analysed using the software package DNASTAR 4.2 (DNASTAR Inc.) and the output was edited in GeneDoc, version 2.6.000 (Nicholas et al., 1997). Complete WSSV sequences were obtained from the NCBI database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide) using the accession numbers for WSSV-TW (AF440570), WSSV-CN (AF332093) and WSSV-TH (AF369029). Dot-plot analysis was performed using PIPmaker (http://bio.cse.psu.edu/pipmaker/).
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RESULTS |
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(i) Variable region ORF23/24
Previously, this genomic region was shown to contain deletions of 1·2 and
13·2 kb in WSSV-CN and WSSV-TH, respectively, compared with WSSV-TW (Fig. 2
a; Marks et al., 2004
). Three other unique deletions in this region were reported in Chinese isolates collected in Tong'an and Anhui in south-east China in 2001 (Fig. 2a
; WSSV-CN-A to -C; Lan et al., 2002
).
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We mapped the variable region ORF23/24 in isolates Tv and Kg using a method similar to that used to map the deletion for WSSV-VN isolate K. Cloning and sequencing of the 1·6 kb PCR product obtained with primers VR23/24-south-forward and VR23/24-1-reverse (Table 2
; Fig. 2a
) showed that isolate Tv has a deletion of 11 450 bp relative to the WSSV-TW genome (Fig. 2a
). A PCR with the primer pair VR23/24-south (Table 2
; Fig. 2a
) for isolate Kg resulted in a
2·6 kb PCR product, which, after cloning and sequencing, showed that this isolate contains a deletion of 12 166 bp relative to the WSSV-TW genome (Fig. 2a
). We previously mapped five SNPs and a 1 bp deletion within WSSV-TW coordinates 1644716773 (flanking the deletion) compared with WSSV-CN and WSSV-TH (Marks et al., 2004
). With respect to these genetic differences, isolate Kg is identical to WSSV-CN and WSSV-TH, suggesting that this isolate is more closely related to these isolates than to WSSV-TW.
Dot-plot analysis showed that, except for the hrs (van Hulten et al., 2001), the genomic region in WSSV-TW in which these deletions occur contains the most direct and inverted repeats of the entire WSSV genome (Fig. 2b
). However, for the deletion in the VN-south isolates Tv and Kg as well as in the six VN-central isolates, no direct repeats were identified within 300 bp flanking the putative recombination sites in WSSV-TW that could be involved in recombination (Fig. 2b
; sequence data not shown).
(ii) Variable region ORF14/15
The variable region ORF14/15 is centred in a region of 842 bp in WSSV-CN, of which 257 bp of its 5' end is only present in WSSV-TH, while the remaining 585 bp of its 3' end is only present in WSSV-TW (Fig. 3a; Marks et al., 2004
). This locus was thought to be a variable region prone to recombination (Marks et al., 2004
). However, a partly characterized isolate recently studied by our laboratory contains at least all unique sequences present in this locus, suggesting that WSSV-TW, WSSV-CN and WSSV-TH are derived from a common ancestor by deletions of various sizes (Fig. 3a
). Because WSSV-TW, WSSV-CN and WSSV-TH each contain unique sequences, these isolates seem to be distinct and probably evolved separately. Using the same strategy as used for the variable region ORF23/24, this locus was mapped for all VN isolates using primer set VR14/15-screen (Table 2
; Fig. 3a
). WSSV-TH DNA, taken as a positive control for the PCR, showed the expected fragment of 1254 bp, whereas the VN isolates showed fragments of different sizes ranging from
500 to
700 bp (Fig. 3b
). Cloning and sequencing of these fragments revealed that all VN isolates had deletions relative to WSSV-TH (Fig. 3a
). The flanking sequences of the deletions present in the
500 to
700 bp fragments were identical to the sequences of WSSV-TH. The VN isolates K, T, L, Tv and Kg had the same deletion of 714 bp, VN isolates X and S had a deletion of 634 bp, while VN isolate A had the smallest deletion, of 563 bp, compared with WSSV-TH (Fig. 3a
).
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(iv) Genetic variation in VNTR loci
Three non-hr unidirectional tandem repeats, in the region encoding ORF75, ORF94 and ORF125, have been shown to be variable in the number of repeat units (RUs) between the WSSV isolates identified so far (Table 3; Wongteerasupaya et al., 2003
; Marks et al., 2004
). The repeats are positioned in the middle of the ORFs, which have non-repeated 5' and 3' ends. For both ORF75 and ORF94, around 50 % of the coding region consists of repeats, while for ORF125 around 20 % of the coding region consists of repeats. Differences in the number of RUs do not cause frameshifts for the respective ORFs, since the length of these RUs is always a multimer of 3 bp. The protein encoded by ORF75 has been shown to be present in WSSV virions (Huang et al., 2002
). ORF94 may have a similar function to ORF75, as the repeat units of the two ORFs share a common motif at the protein level consisting of four basic amino acids (arginine or lysine) followed by two alanines, two or three prolines and a stretch of acidic amino acids (aspartate or glutamate).
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The number of RUs present in ORF75 of the WSSV-VN-central isolates is summarized in Table 3, together with the exact order of appearance of the 45 and 102 bp RUs. The number of RUs identified for each isolate corresponded to the respective sizes of their PCR fragments shown in Fig. 4
. VN isolates K, T, L and X are identical at this point. VN isolate A has an extra RU of 45 bp, which is, based on the SNPs, located after the second repeat unit (sequence data not shown). VN isolate S has a larger number of RUs and, based on the SNPs, more closely resembles the genotype of WSSV-CN (sequence data not shown).
ORF94.
In all WSSV isolates characterized so far, ORF94 has tandem RUs of 54 bp with an SNP at position 48 (either guanine or thymine) when comparing the RUs mutually within one isolate (Table 3). The number of RUs was highly variable between the various isolates for which this locus has been characterized: WSSV-TW, WSSV-CN, WSSV-TH and 55 other isolates originating from Thailand. The number of RUs varied from six to 20 repeat units (van Hulten et al., 2000a
; Wongteerasupaya et al., 2003
; Marks et al., 2004
).
The WSSV-VN-central isolates contained between seven and 17 RUs (Table 3), corresponding to the respective sizes of their PCR fragments (Fig. 4
). The identity of the nucleotide at position 48 of each of the VN isolates is shown in Table 3
. Isolates X and S are identical, while the other isolates, although some have the same number of RUs, all have a unique pattern of nucleotides at position 48. VN isolates K, T and L had a thymine deletion at position 143149 (WSSV-TH coordinates), located in the 3' end flanking the repeat. As this is outside the coding region, it will not cause a frameshift in ORF94.
ORF125.
ORF125 contains tandem RUs of 69 bp, of which the first two as well as the last can be recognized by their specific SNPs when comparing the RUs mutually within one isolate (Table 3). The other RUs (the third to the penultimate) contain SNPs at positions 8, 18, 25, 66 and 69 (Marks et al., 2004
). The WSSV-VN-central isolates contained between five and seven RUs (Table 3
), corresponding to the respective sizes of their PCR fragments (Fig. 4
). VN isolates X and S, as well as VN isolates A and L, are identical in this locus (Table 3
). The genotype of the VN isolates A and L is identical to the genotype of WSSV-TH (Table 3
).
(v) Fragment encoding part of DNA polymerase
To classify the WSSV-VN isolates further, a PCR was performed on a conserved genomic fragment encoding part of WSSV DNA polymerase using primer set Polymerase (Table 2). Within this genomic fragment, a single nucleotide deletion occurs in WSSV-CN (WSSV-TH coordinate 36030) compared with WSSV-TW and WSSV-TH, causing a frameshift in the polymerase gene (Chen et al., 2002
; Marks et al., 2004
). The WSSV-VN isolates gave a PCR fragment of a similar size to the positive control WSSV-TH. Cloning and sequencing of the eight PCR fragments from the central and south VN isolates failed to detect an adenine deletion as is present in WSSV-CN. The PCR fragments showed 100 % nucleotide identity to the respective fragments of WSSV-TW and WSSV-TH.
Restriction enzyme analysis of VN isolate T
RFLP analysis between WSSV-TH and WSSV-VN-T is shown in Fig. 5. The BamHI restriction pattern of WSSV-TH exactly matches the expected pattern based on the complete nucleotide sequence (van Hulten et al., 2001
), except for the three smallest fragments, which are not visible due to their estimated size of <1 kb. Two clear polymorphisms (shifts) are visible between WSSV-TH and VN isolate T, indicated in Fig. 5
by A and B. Shift A, in which a fragment of
27·5 kb for VN isolate T shifts to
24·5 kb for WSSV-TH, can be explained by the observed sequence diversity in variable region ORF 14/15 and variable region ORF 23/24, which are both located on this large fragment. The approximately 3 kb discrepancy is the sum of the observed differences in PCR mapping of both variable regions of approximately 0·7 kb and 3·7 kb, respectively (Figs 2a and 3a
). Shift B, in which a corresponding fragment has a size of
11·2 kb for WSSV-TH and of
11·8 kb for VN isolate T, can be explained by the sequence variation of the repeat in ORF94 (Table 3
). The difference of 11 RUs of each 54 bp results in a shift of 594 bp. The differences in the repeats in ORF75 and ORF125 are not clearly visible. ORF75 is located on a large fragment (
20 kb) for which the 350 bp difference in size will only show a minor shift, whereas the difference in the repeats of ORF125 between WSSV-TH and VN isolate T is marginal (138 bp).
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DISCUSSION |
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Based on both variable region ORF23/24 and variable region ORF14/15, we propose a model to explain the genotypic changes of WSSV during its geographical spread from either side of the Taiwan Strait towards the west to Thailand between 1992 and 1995 (Fig. 1b). In this model, the two loci evolved independently and both deletions in the variable regions showed a progressive increase in length during the spread of WSSV. The WSSV common ancestor (Fig. 1b
) contains a genotype similar to WSSV-TW in the variable region ORF23/24 (Fig. 2a
) and a genotype similar to the putative common ancestor in variable region ORF14/15 (Fig. 3a
). WSSV-TW evolved from this common ancestor by a deletion in variable region ORF14/15, while WSSV-CN evolved by a deletion of
1·2 kb in variable region ORF23/24 (Fig. 1b
; An-1) followed by a deletion in variable region ORF14/15. Based on the observation that the genotypes of the VN isolates seem to have evolved from a genotype similar to WSSV-TH in variable region ORF14/15 by separate unique deletions of different sizes, the VN isolates and WSSV-TH probably have a common lineage, which branched off at an early stage from WSSV-TW and WSSV-CN. However, the extra sequences in the variable region ORF23/24 present in the VN isolates compared with WSSV-TH exclude the possibility that the WSSV-VN isolates are derived from WSSV-TH. Therefore, WSSV-TH and the WSSV-VN isolates probably have a common ancestor, An-3 (Fig. 1b
), which could contain the genotype of WSSV-TH in variable region ORF14/15, but the
8·5 kb deletion similar to the VN-central isolates in variable region ORF23/24. Within the three different WSSV-VN genotypes in variable region ORF14/15, each contains unique sequences and thus probably evolved separately. Therefore, WSSV entered Vietnam by multiple introductions from the common ancestor An-3, from where it spread further within Vietnam (VN isolate Kg; Fig. 1b
). WSSV isolates collected further along the coast of South-East Asia [i.e. isolates from North Vietnam, China (Hainan) and Cambodia] should be genotyped to confirm and further detail this model.
The mechanism(s) by which the changes or (gradual) deletions in both variable regions occur is unclear. For WSSV-TH, it was suggested that the deletions in variable region ORF23/24 might have occurred by homologous recombination, as a direct repeat is present at both ends of the deletion in WSSV-TW (Marks et al., 2004). However, no direct repeats that could be involved in recombination were identified for the deletion in the VN-south isolates Tv and Kg or in the six VN-central isolates (Fig. 2b
). Maybe the deletions in the variable region ORF23/24 can be explained by genomic pressure on the virus to discard redundant sequences, as Fig. 2(b)
shows that WSSV-TW contains a lot of duplicated sequences and ORFs (especially genes of WSSV gene family 4; van Hulten et al., 2001
) in this region. It is also possible that the host species or an intermediate host has an effect on the size of the deletion, as WSSV-CN-A (Metapenaeus ensis), -B (P. japonicus) and -C (P. vannamei, P. monodon, P. chinensis) were isolated from different host species (Fig. 2a
; Lan et al., 2002
). However, within one host species, WSSV isolates can show different sizes of deletion, as WSSV-TW, WSSV-TH, WSSV-CN-C and the VN isolates were all obtained from P. monodon and WSSV-CN and WSSV-CN-B were both isolated from P. japonicus. To date, there seems to be no difference in host range between the characterized WSSV isolates (Wang et al., 1998
, 1999
; Chen et al., 2000
; Lan et al., 2002
; Hameed et al., 2003
).
Based on the genetic make-up in the two variable regions and the thymine deletion shared by isolates K, T and L in the 3' flanking region of the repeat located in ORF94, three groups of VN-central isolates can be distinguished [(K, T, L), (X, S) and (A); Fig. 1b]. Within these groups, each of the non-hr unidirectional tandem repeats located in ORF75, ORF94 and ORF125 seem to have their own, independent genesis in terms of insertion or deletion of repeat units (Table 3
). Possibly, insertions or deletions of repeat units are generated during homologous recombination or replication slippage, as is proposed for repeats such as the baculovirus homologous repeats (hrs) (Garcia-Maruniak et al., 1996
) and the herpesvirus direct repeats (DRs) (Umene, 1991
).
Compared to the other two non-hr unidirectional tandem repeats (ORF94, ORF125), the repeats in ORF75 seem to be rather conserved within and between the three groups of VN-central isolates. The additional repeat unit in VN isolate A could be explained by a single insertion event. The large number of repeat units present in ORF75 for WSSV-VN-S is surprising, especially because the VN isolates X and S, whose geographical origins are very close (10 km) and which may even originate from postlarvae from the same supplier, are completely identical in all other loci screened for. Analysis of more WSSV isolates at this locus from different infected shrimp from the same pond may provide clarification of whether this is the common genotype of WSSV isolates derived from pond S or whether it is an irregularity. Also, for the repeats in ORF125, the genotypic differences in VN isolates can be explained by a one-step deletion or insertion of a single repeat unit (Table 3
). Analysis of the genotypes present within the WSSV-VN group K, T, L suggests that this locus has a higher mutation frequency than ORF75.
The largest genomic variation among the VN-central isolates was observed for the non-hr unidirectional tandem repeats located in ORF94. The number of repeat units within ORF94, as well as the SNP located at position 48, already appeared highly variable for WSSV isolates within Thailand (Wongteerasupaya et al., 2003). Between the isolates characterized within Vietnam, a wide range of genotypic variation was also found for this locus, without any obvious correlation with its geographical location. It is interesting to note that the repeats of ORF94 are highly variable in number, whereas the repeat in ORF75 seems to be more stable, although the two repeat regions share structural properties on the protein level. In conclusion, the repeats of ORF75 and ORF125, each having its own mutation dynamics different from both more stable variable regions, seem suitable to study WSSV spread on a more local or regional scale.
This paper shows the potential to use genetic markers to study WSSV epidemiology and ecology. However, more information about the mode of spread of WSSV is necessary in order to understand further the relationship between the VN isolates. Often, WSSV infection in a pond can be traced back to the broodstock supplier or the postlarvae producers. Therefore, on a regional scale, most likely the virus spreads in myriad ways during the turnover of shrimp. However, on a global scale, this study provides support for the contention that WSSV originated from either side of the Taiwan Strait and evolved concurrently with its geographical spread over time in South-East Asia.
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ACKNOWLEDGEMENTS |
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Received 4 June 2004;
accepted 25 August 2004.