White spot syndrome virus (WSSV), the unique member within the virus family Nimaviridae [10, 17], has been the most devastating aquatic virus infecting shrimp and other crustaceans. It can cause up to 100% cumulative mortality within 3-7 days in infected shrimps and leads to enormous losses to the shrimp farming industry worldwide . Since WSSV was first reported in Fujian province in China in 1991, it has been found in almost all shrimp farming areas all over the world .
The WSSV particle is rod-shaped about 275 nm in length and 120 nm in width with a tail-like appendage at one end . WSSV contains a circular, supercoiled, double-stranded DNA genome, estimated to be about 300 kilobase pairs (kb). Three WSSV isolates have been sequenced so far, WSSV-TW , WSSV-CN and WSSV-TH . Various geographical WSSV isolates share similar genomic and proteomic characteristics. However, there are still several genetic variations along WSSV genome which were widely used for genotyping the WSSV isolates [2, 8, 9, 13]. These variable regions include hot deletion regions in ORF23/24 and ORF14/15 (corresponding to WSSV-TH), and variations in repeat units (RUs) numbers within homologous regions (hrs) . In this study, these variable regions were analyzed in WSSV isolates collected from cultured shrimp in China during 1998-99.
The WSSV-infected shrimp specimens in this study were collected from WSSV outbreak farms in three cities of two provinces along East China Sea and South China Sea: Ning-Bo (NB), Shen-Zhen (SZ) and Guang-Zhou (GZ). The origin of shrimp was documented in Table 1. All samples were stored at -80℃ until further analysis.
Table 1. Sample information of WSSV isolates
Genomic DNA was extracted from the shrimp gill tissue using the standard Phenol/Chloroform DNA extraction protocol. Briefly, 0.1 mg of frozen gill tissue was homogenized using 300 mL of lysis buffer containing 0.4 mol/L NaCl, 2 mmol/L ethy-lenediaminetetraacetic acid (EDTA) and 10 mmol/L Tris-HCL (pH7.5) in a 1.5 mL tube and centrifuged at 6000 ×g for 10 min. Supernatant was collected and supplemented with sodium dodecyl sulphate and Proteinase K to a ﬁnal concentration of 2% and 200 mg/mL, respectively. Samples were incubated at 56 ℃ for 3 h and nucleic acid was separated from the proteinaceous components using phenol-chloroform extraction. Puriﬁed DNA was precipitated with cold ethanol or isopropanol and then washed with 75% cold ethanol. Extracted DNA was stored in TE buffer (10mmol/L Tris-HCl, 1mmol/L EDTA, pH 8.0). The quality and quantity of each sample was determined photometrically using a Biophotometer (Eppendorf). All DNA extractions were stored at -20℃until use.
The shrimp samples were screened for WSSV with a pair of primer designed from WSSV VP26 gene  (Table 2), using Taq polymerase (BioStar). PCR amplification was conducted to pinpoint the 3 variable regions in the WSSV positive samples. Primers VR23/24-3, VR23/24-6 and VR14/15-1F  were used to further characterize variable region ORF23/24 and ORF14/15, respectively. VNTR in ORF94 was analyzed with PCR primers described by Dieu et al . All primers used in this study, PCR conditions and the sizes of the expected PCR products are list in Table 2.
Table 2. Primer sequences and PCR conditions used during PCR analysis
Amplified PCR products were purified using the PCR purification kit (Omega) and bi-directionally sequenced (Invitrogen) using the respective forward and reverse primers described above. The sequence data were analyzed using the DNASTAR 4.2 (DNASTAR Inc.) software package. Alignment of nucleotide sequences was conducted using software ClustalW, version 1.83 software . The number of tandem repeat within ORF94 of each WSSV isolate was determined by using Tandem Repeats Finder (TRF) program . All sequences were compared with three complete WSSV genomes, which are obtained from the NCBI database (http://www.ncbi.nlm.nih.gov/). GenBank accession numbers of the WSSV on the NCBI server are as follows WSSV-TW (AF440570), WSSV-CN (AF332093) and WSSV-TH (AF369029).
PCR analysis of WSSV variable regions
Sequencing and analysis
All shrimps in this study were chosen randomly from samples showing obvious signs of white spots disease. Samples were confirmed as WSSV positive by PCR using primer pair VP26 , and all sequences are identical to the published VP26 gene sequences (data not shown). One shrimp from each place was chosen randomly as the representative geographical isolate. Three categories of variable genetic loci were chosen as the genetic markers to analyze genomic variations among different WSSV isolates.
Variable region ORF23/24, a hot deletion region, is within ORF23 and ORF24 coding frames of WSSV-TH genome. Th-96-II and WSSV-TW isolates contain the longest fragment in this region (Fig. 1). Other WSSV isolates identified from various geographical locations all contain deletions with varying sizes [2, 6, 8, 13, 14]. In this study, with the primer pair VR23/24-southF and VR23/24-1R  WSSV-98NB2 and WSSV98SZ3 isolates gave an amplicon of 1980 bp, and WSSV99GZ of 3756 bp. These results indicated that WSSV98NB2 and WSSV98SZ3 contain a deletion of 11093 bp and the WSSV99GZ of 9316 bp, compared with WSSV-TW (Fig. 1). Surprisingly, with the primer pair VR23/24-3 , a 546 bp PCR was obtained from WSSV98SZ3. Sequence of this PCR product revealed that the WSSV98SZ3 sample contains a second isolate with a deletion of 1168 bp in ORF23/24 and has a similar genomic structure to WSSV-CN, which was isolated in 1996 in Fujian province, China (Fig. 1). With the extended flanking primer pair VR23/24-6 designed in this study (Table 2), a 1910 bp fragment was amplified from WSSV98NB1 and WSSV98NB2, a 1669 bp fragment from WSSV98SZ1, WSSV98SZ2, and WSSV98SZ4 isolates, indicating that WSSV98NB1 and WSSV 98NB2 contain a deletion of 5657 bp, and WSSV 98SZ1, WSSV98SZ2, and WSSV98SZ4 contain a deletion of 5898 bp compared with WSSV-TW (Fig. 2). The WSSV98NB2 were coinfected with two isolates, one with a deletion of 11093 and another with 5657 bp in ORF23/24 region. All the flanking sequences of the deletion showed 100% identity to the counterpart of WSSV-TW and WSSV-CN.
Figure 1. Schematic representation of the variable region ORF23/24. The WSSV isolates in this study are shown in italics. Nucleotide positions are denoted according to the respective WSSV isolates in GenBank database. Nucleotide coordinates of the Chinese WSSV isolates are assigned according to the annotation for the WSSV-CN isolate. The length of the flanking nucleotide sequence is indicated in the boxes. WSSV strains marked by asterisks indicate the coinfection with multiple genotypes in one individual shrimp. Arrows indicate the annealing location of each primer.
Figure 2. Schematic representation of the variable region ORF14/15. The WSSV isolates in this study are shown in italics. Nucleotide positions are denoted according to the respective WSSV isolates in GenBank database. Nucleotide coordinates of the Chinese WSSV isolates assigned according to the annotation for the WSSV-TW isolate. The length of the flanking nucleotide sequence is indicated in the boxes. The bidirectional arrow indicates insertion of 4481 bp of TH-96-II in variable region ORF14/15. Short arrows indicate annealing location of each primer.
Compared with other published WSSV isolates, WSSV98NB1, WSSV98SZ1, WSSV98SZ2, and WSSV98SZ4 contain deletions of similar sizes with WSSV-CN-A and WSSV-CN-B which contain deletions of 5717 and 5926 bp, respectively, and WSSV99GZ has a deletion fragment as WSSV-CN-C . The larger deletion of 11093 bp in WSSV98SZ3 and WSSV98NB2 is similar to that of WSSV-VN (Tv) which contains a deletion of 11450 bp (Fig. 2) .
Variable region ORF14/15 locates within the WSSV-TH segment coding for ORF14 and ORF15, and has been confirmed to be prone to recombination [2, 8, 13]. Up to now, it was found that the TH-96-II isolate contains the largest fragment in this region . Using the same mapping strategy as used for variable region ORF23/24, we pinpointed the variation within ORF14/15 among different WSSV isolates. Primer pair VR14/15-screen  successfully produced a 1819 bp amplicon in the WSSV98SZ2 isolate, and a 1431 bp amplicons in WSSV98SZ2, WSSV98SZ4, and WSSV99GZ isolates, but not in other samples. Sequencing result showed that WSSV98NB2 contains a 4749 bp deletion compared to TH-96-II, and a 381 bp insertion compared to WSSV-CN. The WSSV-98SZ2, WSSV98SZ4 and WSSV99GZ isolates have identical structures relative to WSSV-TW, which contain a 5140 bp deletion compared to TH-96-II. Subsequently, by extending the forward primer, VR14/15-1F/VR14/15-screenR produced a 1095 bp amplicon in WSSV98SZ1, WSSV98SZ3 and WSSV-98NB1, indicating that these isolates contain a 5719 bp deletion compared to TH-96-II, and part of 585 bp in the corresponding region of WSSV-TW (Fig. 2).
The ORF94 (corresponding to WSSV-TH) locates between genes encoding the large (RR1) and small (RR2) subunits of ribonucleotide reductase. In this ORF, various WSSV isolates show different numbers of 54 bp of Repeat Unit (RU), furthermore, the position 48 of each RU shows a single nucleotide polymorphism (SNP) (guanine or thymine) [2, 5, 11, 12, 19]. After PCR amplification with the flanking primer pair ORF94 F/R  of this region and sequences analysis, 4 different repeat types ranging from 6 to 14 RUs were obtained among the analyzed WSSV isolates, with the 6 RUs as predominant type (Table 3). Consistently, three categories of tandem repeat sequences were obtained from WSSV98NB2: 14, 9 and 6 RUs, further confirmed that WSSV98NB2 was coinfected with two or three WSSV isolates. Nucleotide polymorphism analysis at position 48 of each RU indicated that thymine is more common than guanine.
Table 3. Variable number of tandem repeats within ORF94 in analyzed WSSV isolates from shrimp samples