Vol. 106: 1–6, 2013 DISEASES OF AQUATIC ORGANISMS Published September 24 doi: 10.3354/dao02645 Dis Aquat Org Novel, closely related, white spot syndrome virus (WSSV) genotypes from Madagascar, Mozambique and the Kingdom of Saudi Arabia Kathy F. J. Tang1,*, Marc Le Groumellec2, Donald V. Lightner1 1Department of Veterinary Science and Microbiology, University of Arizona, Tucson, Arizona 85721, USA 2AQUALMA, Aquaculture de la Mahajamba, Mahajanga 401, Madagascar ABSTRACT: White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp and has caused significant economic losses in the aquaculture industry around the world. During 2010 to 2012, WSSV caused severe mortalities in cultured penaeid shrimp in Saudi Arabia, Mozambique and Madagascar. To investigate the origins of these WSSV, we performed genotyping analyses at 5 loci: the 3 open reading frames (ORFs) 125, 94 and 75, each containing a variable number of tan- dem repeats (VNTR), and deletions in the 2 variable regions, VR14/15 and VR23/24. We catego- rized the WSSV genotype as {N125, N94, N75, ΔX14/15, ΔX23/24} where N is the number of repeat units in a specific ORF and ΔX is the length (base pair) of deletion within the variable region. We detected 4 WSSV genotypes, which were characterized by a full-length deletion in ORF94/95, a relatively small ORF75 and one specific deletion length in each variable region. There are 2 closely related genotypes in these 3 countries: {6125, del94, 375, Δ595014/15, Δ1097123/24} and {7125, del94, 375, Δ595014/15, Δ1097123/24}, where del is the full-length ORF deletion. In Saudi Arabia, 2 other related types of WSSV were also found: {6125, 794, 375, Δ595014/15, Δ1097123/24} and {8125, 1394, 375, Δ595014/15, Δ1097123/24}. The identical patterns of 3 loci in these 4 types indicate that they have a common lineage, and this suggests that the WSSV epidemics in these 3 countries were from a common source, possibly the environment. KEY WORDS: WSSV genotyping · Variable number of tandem repeats · VNTR analysis · Variable- length deletion · Africa · Saudi Arabia Resale or republication not permitted without written consent of the publisher INTRODUCTION 2001). The viral genome has 181 non-overlapping open reading frames (ORFs). Only about 20% of the Viral diseases have caused significant economic genes are known to encode structural proteins and losses for the shrimp farming industry throughout the those in volved in DNA replication or in modifying world. Among these, white spot syndrome virus other proteins. WSSV was first reported around 1991 (WSSV) caused severe mortalities in many species to 1992 in SE Asia (Huang et al. 1994, Inouye et al. of penaeid shrimp and other aquatic crustaceans 1994). The virus spread rapidly, and by the late 90s, it (Lightner 1996, Lo et al. 1996), and infections can had severely impacted most shrimp farming regions. result in 95 to 100% mortalities in shrimp ponds. Eco- However, shrimp farms in the Indian Ocean re - nomic losses in production and trade approach mained free of WSSV until recently, when the virus US$10 billion (Stentiford et al. 2009, Lightner 2011). was found in Mozambique (OIE 2011) and Madagas- WSSV is a large (70−150 × 275−380 nm), envel- car (OIE 2012a). Before these reports, WSSV was oped, double-stranded DNA virus with a genome detected in shrimp farms in Saudi Arabia and caused size over 300 kb (Van Hulten et al. 2001, Yang et al. >95% mortalities (Tang et al. 2012). *Email: [email protected] © Inter-Research 2013 · www.int-res.com 2 Dis Aquat Org 106: 1–6, 2013 To investigate the origins of WSSV emergence within this Indian Ocean−Red Sea region, we applied variable number of tandem repeats (VNTR) analysis and determined the deletion lengths in specific regions within the viral genome to genotype WSSV isolates collected from this region. The analyses of VNTR and variable-length deletions have been used in several epidemiological studies of WSSV (Wong- teerasupaya et al. 2003, Dieu et al. 2004, Marks et al. 2004, Pradeep et al. 2008, Muller et al. 2010, Hoa et al. 2012). It has been suggested that deletion lengths in the variable regions VR14/15 and VR23/24 are associated with geographic locations (Dieu et al. 2010), and the VNTR analyses in ORFs 125, 94 and 75 are used for epidemiological comparisons among farms or ponds. In this study, we applied the WSSV genotyping analyses within these 5 loci to compare 13 WSSV representative isolates collected in Mada- gascar, Mozambique, and Saudi Arabia. MATERIALS AND METHODS Shrimp samples Samples of Penaeus monodon and P. indicus (taxon- omy according to Holthuis 1980) were collected from 2 farms on the west coast of Madagascar (labeled as Farms MG-A and MG-B in Fig. 1), 1 farm in Mozam- bique (MZ in Fig. 1), and 4 farms along the Red Sea from central to southern Saudi Arabia (SA-A, -B, -C, Fig. 1. Locations of the white spot syndrome virus (WSSV)- affected shrimp farms in Madagascar (2 farms: MG-A and -D in Fig. 1) between 2010 and 2012 (Table 1). Each MG-B), Mozambique (1 farm: MZ), and Saudi Arabia (4 sample consisted of whole shrimp or pleopods sam- farms: SA-A to SA-D). The WSSV genotypes detected are pled from 1 to 5 individuals. These were preserved in indicated Table 1. Number of repeat units (RU) found within open reading frames (ORFs) 125, 94 and 75, and the length of deletions within variable regions VR14/15 (compared with WSSV TH-96-II; AY753327) and VR23/24 (compared with Taiwan WSSV; AF440570) of WSSV isolated from Madagascar (MG), Mozambique (MZ) and Saudi Arabian (SA) farms (farm sites indicated in Fig. 1). P.: Penaeus; del: full-length ORF deletion Farm site Host species No. of RU Deletion size (bp) Isolate no. Sampling ORF125 ORF94 ORF75 VR14/15 VR23/24 date MG-A Wild P. indicus 6 del 3 5950 10971 12-354A1 Sep 2012 MG-A P. monodon 6 del 3 5950 10971 12-354A2 Sep 2012 MG-B P. monodon 7 del 3 5950 10971 12-171B Apr 2012 MG-B P. monodon 7 del 3 5950 10971 12-213F May 2012 MZ P. monodon 6 del 3 5950 10971 11-312 Sep 2011 SA-A P. indicus 6 7 3 5950 10971 10-143 Apr 2010 SA-A P. indicus 7 del 3 5950 10971 12-380D Oct 2012 SA-A P. indicus 7 del 3 5950 10971 12-404 Dec 2012 SA-B P. indicus 8 13 3 5950 10971 11-065/6 Feb 2011 SA-B P. indicus 8 13 3 5950 10971 11-102/1 Mar 2011 SA-C P. indicus 7 del 3 5950 10971 11-041/20 Jan 2011 SA-C P. indicus 7 del 3 5950 10971 11-208/3 May 2011 SA-D P. indicus 7 del 3 5950 10971 11-394/B Nov 2011 Tang et al.: White spot syndrome virus genotypes 3 95% ethanol and sent to the Aquaculture Pathology Laboratory at the University of Arizona, Tucson USA, for PCR analysis for the presence of WSSV. The pres- ence of the virus was determined by PCR using a World Organisation for Animal Health (OIE) recom- mended method (procedure is described below). DNA extraction, WSSV PCR analysis and genotyping regions VR14/15 and VR23/24 Total DNA was extracted with a Maxwell-16 Cell LEV DNA purification kit (Promega). For WSSV detection, a nested PCR was performed with PuRe- Taq™ Ready-To-Go™ PCR beads (GE Healthcare). Each reaction (final volume: 25 µl) contained 1 µl of extracted DNA (at concentrations of 100 to 300 ng µl−1), 2.5 units of PuReTaq™ DNA polymerase, 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each dNTP, and 0.2 µM WSSV primers (first step primers: 146F1 and 146R1; second step primers: 146F2 and 146R2; OIE 2012b). Amplifications (both first and second step) were carried out as initial denaturation at 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 2 min; Following this, an aliquot of the PCR mixture was analyzed in a 1.2% agarose gel containing ethidium bromide and then Initial Amplification Final photographed with an AlphaImager gel documenta- tion system (Alpha Innotech). For VNTR analysis within the 3 ORF regions (ORFs 125, 94 and 75) and determination of the length of deletions in the 2 variable regions (VR14/15 and VR23/24), PCR was performed using the Ready-To- Go™ PCR beads with specific primers (Table 2). PCR results were visualized as described above. The PCR products were purified with the QIAquick PCR purification kit (Qiagen), and DNA sequencing was performed by the Genomic Analysis and Technology Core facility at University of Arizona by a 3730 DNA Analyzer (Applied Biosystems). From the nucleotide sequence, the number of re- peat units (RU) within the ORFs was determined with the Tandem Repeats Finder program (Benson 1999). The consensus sequence of the RU in each ORF is as follows: ORF125 (69 bp) – AG/TA ACA AGC AGG AAG AAG ACG CGA GGA TCA AGC GTG CAG TCG ACA TGG CTG TTG CAG CCA TCA ACG AAA; ORF94 (54 bp) – CGC AAA AAG CGT GCC GCA CCT CCA CCT GAG GAT GAA GAA GAG GAT GAT TTC TAC; ORF75 (45 bp) – GAA GCA GCT CCC CCA CTT AAA GGT GCA CTT GGA CGT ORF125-flank-FORF125-flank-RORF94-FORF94-R TTG TGC ATG CTT GAT CGA AAT CCA TTG CCC TTC TC CCA TAT ORF73-FORF77-RVR14/15-complete-(F)VR14/15-complete-(R) GAA CGA CGG GTG ATG AAT 94°C for TCT ACT CGA GGA GGT GAC GAC CAG CGC CTC TTC AGVR23/24-south-(F) G ACA TTT CAT ACA GGT GTG TAC 35 cycles: 94°C for 30 s, 55°CVR23/24-south-(R) 3 min 72°C for 7 min CTT TCA CCG CTC CCA AC GGG TTC ACC AGA GAG ACA GG AC GTT TCT CTA GTG CAT GTA for 30 s, 72°C 1 min TTG CTG AGA AG 94°C for AGT TTA GTA min 3 35 cycles: 94°C for 30 s, 55°C Dieu et al.