Characterization of a Taura syndrome virus isolate originating from the 2004 Texas epizootic in cultured shrimp

Transcription

1 Arch Virol (2010) 155: DOI /s ORIGINAL ARTICLE Characterization of a Taura syndrome virus isolate originating from the 2004 Texas epizootic in cultured shrimp Arun K. Dhar Dilip K. Lakshman Keenan Amundsen Refugio Robles-Sikisaka Krista N. Kaizer Sribash Roy Kenneth W. Hasson F. C. Thomas Allnutt Received: 8 September 2009 / Accepted: 7 December 2009 / Published online: 5 January 2010 Ó Springer-Verlag 2010 Abstract A comprehensive investigation of the Taura syndrome virus (TSV) isolate that caused epizootics in shrimp farms in Texas in 2004 (Texas isolate) revealed that this virus was more virulent in laboratory bioassays than the TSV reference isolate, Hawaii 1994, causing severe symptom development and rapid mortality. Histopathology A. K. Dhar (&) K. N. Kaizer S. Roy F. C. Thomas Allnutt Advanced BioNutrition Corporation, Columbia, MD 21046, USA ADhar@abn-corp.com; arun_dhar@hotmail.com D. K. Lakshman K. Amundsen USDA-ARS, SASL, Beltsville, MD 20705, USA R. Robles-Sikisaka Hubbs-SeaWorld Research Institute, San Diego, CA 92109, USA K. W. Hasson Texas Veterinary Medical Diagnostic Laboratory, College Station, TX 77841, USA Present Address: A. K. Dhar K. N. Kaizer Viracine Therapeutics Corporation, 7155-H Columbia Gateway Dr., Columbia, MD 21046, USA Present Address: S. Roy Plant Molecular Biology Department, National Botanical Research Institute, Lucknow , UP, India Present Address: F. C. Thomas Allnutt Phycal, LLC, 51 Alpha Park, Highland Heights, OH, USA Present Address: K. W. Hasson Aquadiagnostics, 1103 Rio Bravo Ct, College Station, TX 77845, USA of moribund animals demonstrated epithelial necrosis within the stomach, appendages, general body cuticle and gills, and the surviving animals demonstrated moderate to numerous lymphoid organ spheroids. Purified virions showed icosahedral morphology, with a diameter of 31 nm. Comparative genome analysis showed that the Texas isolate is more closely related to TSV isolates from Thailand and China than to the Hawaii isolate. The predicted tertiary structures of the inhibition of apoptosis protein (IAP) and protease domains of the Texas isolate are very similar to those of the Hawaii isolate. However, the RNA-dependent RNA polymerase (RdRp) of the Texas isolate has significant structural differences from the Hawaii isolate due to point mutation(s) in the RdRp gene. Changes in the RdRp tertiary structure might contribute to the replication fidelity, virulence and ecological adaptability of the Texas isolate. Introduction Taura syndrome (TS) of shrimp is caused by a singlestranded RNA virus, Taura syndrome virus (TSV). TSV is an Office International des Epizooties (OIE) notifiable pathogen and the cause of one of the most economically significant viral diseases affecting shrimp aquaculture [25]. TSV infects a number of commercially important marine penaeid shrimp species. Among American penaeid species, Penaeus (Litopenaeus) vannamei is the principal host of TSV. Other American shrimp species, P. stylirostris, P. schmitti, P. setiferus, P. duorarum, and P. aztecus, as well as the Asian shrimp species P. monodon, P. japonicus, P. chinensis, and Metapenaeus ensis, are also susceptible to TSV, although it may not cause high mortality in all species [21, 26].

2 316 A. K. Dhar et al. The Taura syndrome virion is non-enveloped, icosahedral, nm in diameter, and contains a single, linear, positive-sense, ssrna molecule of *10.2 kb [2, 11]. The TSV genome contains two open reading frames (ORF1 and ORF2) that are separated by an intergenic region (IGR) (Fig. 1a; [22]). ORF1 encodes non-structural proteins that contain the conserved sequence motifs of a helicase, a protease, and an RNA-dependent RNA polymerase (RdRp) (Fig. 1a; [22]), whereas ORF2 encodes the structural proteins. Using SDS-PAGE, three major (VP1, 55 kda; VP2, 40 kda; and VP3, 24 kda), and one minor (VP0, 58 kda) structural proteins were detected in purified TSV preparations [2]. Therefore, the TSV genome organization is similar to that of insect RNA viruses with dicistronic genomes [7, 8] and is classified as a member of the family Dicistroviridae. However, TSV has not been assigned to a specific genus [6]. Since its first report in Ecuador in 1992 and subsequent demonstration of a viral etiology [4, 11, 17, 21], TS has spread progressively throughout North and South America [13]. By the end of 1996, 13 of the 14 shrimp-farming countries in the Western Hemisphere had been affected by TSV. Between 1992 and 1996, the damage caused by TS in the Western Hemisphere was estimated at US$1.2 2 billion [3, 15]. TSV has had a dramatic impact in Asia as well. The disease may have spread from South America to Asia through importation of infected live P. vannamei, which Fig. 1 A schematic diagram of the TSV genome organization (a) and the strategy used to clone the TSV Texas isolate (b). The primers for PCR amplification are indicated above the linear map, and the clone numbers (ABN008, ABN006, ABN003 and ABN002) and the location of unique restriction enzyme sites are indicated below the linear maps. c Agarose gel photographs of RT-PCR products of the TSV Texas isolate. Ma 100-bp ladder (Invitrogen), Mb 1-kb DNA ladder (Invitrogen), Lane 1 *250-bp 5 0 RACE cdna (ABN008), Lane 2 *550-bp cdna (ABN006), Lane 3 *7.2-kb cdna (ABN002), Lane 4 *2.6-kb cdna (ABN003) A B RdRp Helicase Protease Capsid Proteins 5 UTR 3 UTR VP2 VP1 VP3 (A)n 417 ORF ORF F 222R AatII ABN F 722R AatII ApaI ABN F 3000R ApaI ABN F 10205R (A)n ABN002 C Ma Mb Mb 4

3 Properties of 2004 Texas Taura syndrome virus in shrimp 317 had become a major culture species in Asia during the 1990s. The first TS outbreak in Asia was reported in Taiwan during 1998 [35], and subsequently, the disease spread to China, Thailand and Korea. In 2003, the first TS outbreak was confirmed in Thailand [23]. In 2004, mass mortalities due to TS were reported in shrimp farms culturing P. vannamei on the western coast of Korea [9]. In the United States, the state of Texas is the largest producer of shrimp. In 2003, shrimp production in Texas peaked at 4.26 million kilograms [34]. Between 1996 and 2003, there were no significant TS outbreaks in the US, mainly due to heightened biosecurity measures and the practice of stocking specific-pathogen-free (SPF) and TSVresistant P. vannamei. However, in June of 2004, TSV epizootics occurred in the Texas coastal counties of Cameron and Willacy, which are situated in the Rio Grande Valley near the boarder with Mexico (APHIS Report 2004; This epizootic occurred in farms located near shrimp-processing plants that processed frozen commodity and bait shrimp imported from Southeast Asia. The problem was exacerbated as farmers had both overstocked their ponds (AP News 2004 on USMSFP site; news/headlinenews/ txfarmers.htm) and had seeded numerous ponds with a new fast-growing line of P. vannamei that was TSV-susceptible. As a result of these practices and the resurgence of TS, in 2004, shrimp production in Texas dropped to 3.60 million kilograms, resulting in major economic losses for shrimp farmers. The present study was undertaken to determine the biological and genomic properties of a TSV isolate that caused a major epizootic in Texas in To date, only partial capsid sequences of this isolate have been obtained, from which phylogenetic relationships with other TSV isolates have been assessed [36]. However, no biological and genomic properties of this isolate have been determined so far. To gain insights into the virulence of this isolate, bioassays were conducted using the Texas isolate and TSV reference isolate Hawaii 1994 inocula. The genome of the Texas isolate was cloned and sequenced, and phylogenetic analysis was performed. In addition, the predicted tertiary structure of the inhibition of apoptosis protein (IAP), protease, and RNA-dependent RNA polymerase (RdRp) domains of the Texas isolate were determined and compared to the homologous domains of other TSV isolates. Materials and methods Animals Specific-pathogen-free (SPF) P. vannamei were obtained from Shrimp Improvement Systems (SIS; Islamorada, Florida) and the Oceanic Institute (OI; Oahu, Hawaii). The fast growth (FG) line (SIS) or the Kona line (OI) were used for the experiments as indicated. These lines of shrimp are known to be susceptible to TSV. TSV isolates A list of TSV isolates for which the entire genome has been sequenced, along with their geographic location and the year of isolation is provided in Table 1. The 1994 Hawaiian TSV isolate, the first isolate to be characterized and sequenced completely [2, 22], is considered to be the reference isolate. The TSV Texas isolate used in this study was originally collected during a naturally occurring TSV epizootic in Texas in The viral isolate was subsequently amplified in the laboratory by intramuscular injection of FG line P. vannamei with a homogenate prepared from TSV-infected shrimp tissue following a published method [11]. The virus-infected tissue was stored frozen at -80 C until used in the current study. TSV infection was confirmed in test shrimp by histopathology and in situ hybridization using a TSV-specific probe. Experimental challenge Three separate bioassays were conducted for the Texas isolate and one bioassay for the TSV Hawaii isolate utilizing SPF line (Kona) P. vannamei (average weight g) in a 20-l aquarium. For each bioassay, there was one Table 1 The host species, year of isolation, country of origin, and GenBank accession number of TSV isolates for which a full-length genome has been sequenced Source species Date Origin Accession number Reference P. vannamei 1994 Hawaii, USA AF [22] P. vannamei 2001 Belize AY [31] P. vannamei 2004 Texas, USA GQ Present study P. vannamei 2005 China DQ Huang and Lu (unpublished) P. vannamei 2005 Thailand AY [29]

4 318 A. K. Dhar et al. Table 2 List of primers used to clone the TSV Texas isolate genome a Primer location and orientation are with respect to the TSV Hawaii strain (AF277675) Clone Primer ID Nucleotide sequence ( ) Primer location a ABN F CATTCAGACG GCGTTTAGAT 3001 (?) 10205R TTTTTTTTTT AGGAATCCGC CGC (-) ABN F ATGGCCTCTT ATTATCTAAA C 417 (?) 3000R CATCTGCACT ATGAATAGAC 3000 ( ) ABN F GAGACGTCCC GCTCCCCGTC TTATTTC 166 (?) 722R CTTAAGCATT GAAATCAGGC TG 722 (-) ABN RACE adapter GCUGAUGGCG AUGAAUGAAC Not applicable ACUGCGUUUG CUGGCUUUGA UGAAA 222R GTCCACCAAT AGATGTCGAG TCTACGG 222 (-) 253R CAGAAGCAAC TTACGGCGAC TGG 253 (-) tank for the control group and one tank for the TSV Texas isolate, except in bioassay #2, where the control group animals were divided into two tanks (10 in one tank and 11 in the second tank). Overall, there were animals per bioassay for the TSV treatment group and animals per bioassay for the control group (Table 3). Each aquarium contained artificial seawater (25 ppt) at C, which was supplied with continuous aeration through placement of a single airstone and kept covered to prevent both shrimp escape and possible cross-contamination by aerosols. The shrimp were acclimated for 1 2 days prior to the onset of each bioassay. For diagnostic purposes, tissues from the cephalothorax of moribund animals were preserved in Davidson solution, and the tail muscle tissues were frozen at -80 C. Histological analysis was performed using H&E staining, and the severity of TSV infection in the virus-injected shrimp was graded following a published paper [14]. In order to generate virus (Hawaii and Texas isolates) inocula for the bioassay, SPF P. vannamei shrimp were first injected with 0.45-lm-filtered tissue homogenates from shrimp infected with TSV Hawaii and Texas isolates. Tail muscle tissue from only those animals that were showing acute-phase lesions were pooled, minced and fed to SPF Penaeus vannamei shrimp to compare the virulence of the two isolates (Bioassay #1). When the bioassay using Texas isolate was repeated (Bioassay #2), the virus inoculum was derived from the moribund animals of the first bioassay (Bioassay #1) that were showing acute-phase lesions. Similarly for the third bioassay (Bioassay #3), shrimp tissue from moribund animals with acute-phase lesions from Bioassay #2 were used as inoculum. Due to the lack of availability of SPF P. vannamei shrimp, the bioassay for the Hawaii isolate was conducted only one time. TSV per os challenge was performed following an OIE protocol [25]. Briefly, SPF P. vannamei shrimp were fed minced TSV-infected P. vannamei carcasses for three consecutive days (days 0, 1, and 2) at 10% of the biomass per tank. Negative-control shrimp were fed minced healthy SPF P. vannamei carcasses at the same rate. Starting on day 3, shrimp were fed a pelleted ration (SI35 pellet, Ziegler Brothers) ad libitum once per day for the remainder of each study. After siphoning of organic debris, a 50 70% water exchange was conducted on each tank every 3 days during the course of each bioassay. Animals were observed two to four times daily to remove and preserve moribund and dead shrimp. Histopathology and in situ hybridization During each bioassay, all moribund shrimp were preserved for histological analysis with Davidson s AFA fixative following the methods of Bell and Lightner [1]. Upon terminating each bioassay, surviving shrimp were collected, and each abdomen was preserved frozen (-80 C), while the cephalothorax was preserved in Davidson fixative for histological analysis. The resulting paraffin-embedded tissues were sectioned (4 5 lm), mounted on standard glass slides, stained with hematoxylin and eosin (H&E) and examined by light microscopy [20]. Sections containing acute-phase, transition-phase, or presumed chronic-phase TSV lesions were categorized and lesion severity graded according to Hasson et al. [14]. Briefly, acutely infected shrimp were characterized by the presence of multifocal epithelial necrosis, transition-phase animals were those demonstrating both acute-phase lesions (epithelial necrosis) and lymphoid organ spheroids with or without cuticular melanization, and chronic-phase shrimp were those only demonstrating numerous lymphoid organ spheroids. Lesion severity was graded on a scale from 1 to 4, with grade 1 indicating lesion or spheroid presence in \25% of the tissue/organ; grade 2, 25 50%; grade 3, 50 75% and grade 4, [75%. Based on the histological findings, a subset of samples from the TSV-challenged and

5 Properties of 2004 Texas Taura syndrome virus in shrimp 319 negative control groups were selected for in situ hybridization (ISH) analysis using a TSV-specific probe following standard methods [20]. Virus purification and electron microscopy P. vannamei FG shrimp infected with TSV Texas 2004 were processed for virus purification following a published protocol [2] with minor modifications. Briefly, 20 g of infected tissue was homogenized in 160 ml of ice-cold TN buffer (0.4 M NaCl, 0.01 M Tris HCl, ph 7.4) and the homogenate was centrifuged at 6,000 rpm for 15 min using a SW 28 rotor (Beckman Instruments). The supernatant was collected and then centrifuged at 15,000 rpm for 20 min. The supernatant was extracted using an equal volume of toluene, and the aqueous layer removed and loaded onto a linear sucrose gradient (15 60% v/v) before centrifuging for 90 min at 26,000 rpm using an SW28 rotor. The virus-containing band was diluted with ten volumes of TN buffer and centrifuged at 28,000 rpm for 3 h. The resulting pellet of purified virus was suspended in TN buffer. The purified virus preparation was negatively stained with 2% phosphotungstic acid at ph 7.0 on formvar-coated grids stabilized with evaporated carbon film and observed using a transmission electron microscope (JEOL 1200 EX2, JOEL Ltd., Tokyo, Japan). The sizes of 25 particles were measured. Cloning and sequencing of the TSV Texas isolate In order to clone the genome of the TSV Texas isolate, total RNA was isolated from tail muscle tissue of infected shrimp using TRI Reagent (Molecular Research Laboratory Inc., Cincinnati, OH, USA), following the manufacturer s recommendations. The cloning of the viral genome was done in four segments (ABN008, ABN006, ABN003 and ABN002, Fig. 1b). In order to clone ABN002 and ABN003, cdna was synthesized using 1.0 lg of total RNA and 10 lm ofv 3 -Oligo(dT) 21 primer following the protocol described in the MonsterScript 1 st -Strand cdna Synthesis Kit (Epicentre, Madison, WI, USA). RT- PCR was then performed using MasterAmp TM Extra-Long PCR Premix #4 (MasterAmp TM Extra-Long PCR Kit, EPICENTRE, Madison, WI, USA) and ABN002-and ABN003-specific primers (Table 2). The temperature profile for the amplification included denaturation at 94 C for 1 min followed by 30 cycles of denaturation at 94 C for 30 s, annealing at 60 C for 1 min and extension at 72 C for 8 min (for ABN002) or 3 min (for ABN003) and a final extension at 68 C for 7 min. In order to clone ABN006 and ABN008, tail muscle RNA was processed (treatment with calf intestine alkaline phosphatase and tobacco acid pyrophosphatase and ligation to 5 0 RACE adapter) following the FirstChoice RLM- RACE protocol (FirstChoice Ò RLM-RACE Kit, Ambion, Austin, TX, USA). The cdna was synthesized using the processed RNA, 10 lm primer 760 R (5 0 TTATCCTTTGG CCATCCTTCCCAG3 0 ) (for ABN006) or V 3 -Oligo(dT) 21 primer (for ABN008), following the protocol described in the MonsterScript 1 st -Strand cdna Synthesis Kit (Epicentre, Madison, WI). RT-PCR was then performed using ABN006- and ABN008-specific primers (Table 2) and AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA, USA) following manufacturer s protocol (First- Choice Ò RLM-RACE Kit). The temperature profile for the amplification included denaturation at 94 C for 3 min followed by 30 cycles of denaturation at 94 C for 30 s, annealing at 57 C (for ABN006) or 62 C (for ABN008) for 30 s and extension at 72 C for 30 s and a final extension at 72 C for 7 min. The amplified cdnas representing ABN002, ABN003, ABN006 and ABN008 were electrophoresed in agarose gels, and the amplicons were eluted from the gels using a gel purification kit (Qiagen, Valencia, CA, USA) and cloned into the TOPO XL vector (Invitrogen, Carlsbad, CA, USA). Three clones were sequenced for each of the three constructs ABN002, ABN003, and ABN006, and eight clones were sequenced for ABN008. Sequence analysis Sequence similarities of the five distinct regions (5 0 untranslated region, ORF1, intergeinc region, ORF2 and 3 0 untranslated region) of TSV isolates were determined using the BLAST utility of the National Center for Biotechnology Information (NCBI). Both ORF1 and ORF2 of the Texas isolate were translated with the ExPASy Proteomics tools ( and examined for similarity at the amino acid level using the BLASTP utility of NCBI with the non-redundant protein database. The evolutionary relationships of the Texas isolate with other TSV isolates were determined using the full-length genome sequence of the Hawaii, Belize, China, Thailand, Venezuela and Texas isolates (Table 1). The full-length genome of cricket paralysis virus (CrPV) (GenBank accession number AF218039), also a member of the family Dicistroviridae, was included as an outgroup in the phylogenetic analysis. Phylogenetic analysis was performed using a Bayesian inference approach with the computer program MrBayes [28]. The analyses incorporated a nexus alignment produced by ClustalX [33] and the best-fit model of nucleotide substitution, GTR? I? G, chosen by MrModeltest v.2 [24]. Markov chain Monte Carlo (MCMC) parameters included a run time of generations and sampling every 1,000 generations. Chain convergence was determined when the standard deviation

6 320 A. K. Dhar et al. of the split frequencies dropped below Clade support was estimated by Bayesian posterior probabilities. Fiftypercent majority rule consensus trees were visualized using Fig Tree v [27]. Structural analysis In order to compare and differentiate the non-structural proteins of six TSV isolates at the tertiary structure level, the SWISS-MODEL server was used in First Approach mode to generate theoretical models of the RdRp and IAP protein domains from each virus. The models were built from the Protein Data Bank (PDB) structures, accession numbers 1XR7 (RNA-dependent RNA polymerase of human rhinovirus serotype 16) and 1E31 (human apoptosis inhibitor protein, survivin) ( home/home.do). To determine the tertiary structure of the protease domain, the amino acid sequence of the Texas isolate was aligned with the protease domain of PDB entry 2A4O, a hepatitis A virus 3C protease, using ClustalW ( The alignment mode of the SWISS-MODEL server ( was then used to model the C-terminal portion of the protease domains. Theoretical structures were refined by manually adjusting the sequence alignments to improve the superimposition of the model structures on the PDB reference structures in the DeepView-Swiss PDB Viewer software 3.9 ( Root mean square (RMS) values for each hypothetical structure compared with the reference PDB structure, Ramachandran plots, and number of unfavorable contacts were reviewed to determine the quality of the theoretical models. Results and discussion Bioassay, histopathology, and electron microscopy Three separate bioassays were conducted using the inoculum of the TSV Texas isolate. The mortality in challenged P. vannamei started 4 days post-challenge and peaked by day 6. The cumulative percent mortalities with the Texas isolate ranged from 84 to 100% and were 0 30% in the negative control groups (Fig. 2). The loss of three negative-control shrimp during Bioassay 1 is attributed to molting and cannibalism (Fig. 2). We performed one bioassay using the Hawaii isolate following the bioassay protocol described above. The mortality recorded for the Hawaii isolate was 71%, similar to what has been reported in earlier studies [2, 11, 30]. A recent study by Srisuvan and colleagues compared the pathogenicity of TSV isolates from Belize, Thailand, Venezuela, and the USA by oral challenge [30]. The Belize isolate was the most virulent (100% mortality), followed by the Thailand (95%), Venezuela (89%) and Hawaii (79%) isolates. Therefore, the mortality numbers for the Texas isolate are very similar to those reported by Srisuvan and colleagues (2006) for their Belize, Thailand, and Venezuela isolate-infected groups, and much higher than for the Hawaii isolate. This suggests that the Texas isolate, like the Belize, Thailand, and Venezuela isolates, is more virulent than the reference strain, Hawaii The principal clinical signs of TS observed in our study included opacity of the abdominal musculature, anorexia, lethargy and, in some cases, partial molting. Reddening of the uropods/antennae and darkening of the body due to chromatophore expansion was also observed among the Fig. 2 Cumulative mortality of Kona shrimp (P. Vannamei) infected with the TSV Texas isolate compared to healthy shrimp. The solid line represents data from TSV-infected shrimp, while the dotted line indicates the healthy shrimp control. Data presented are the average of three separate bioassays (see Table 3). Error bars represent the 95% confidence intervals of the three data sets Cumulative Survival Us04Pv1 (Texas TSV) Control Days post-infection

7 Properties of 2004 Texas Taura syndrome virus in shrimp 321 moribund shrimp (Fig. 3b). Some moribund and surviving animals displayed multifocal, variably sized and shaped, dark brown to black melanized lesions on the cephalothorax and tail regions, typical of transition-phase TSV lesions (Fig. 3a). Histological analysis of three to seven negative control shrimp collected upon termination of each bioassay demonstrated that they were free of TSV as well as any other known shrimp viruses. In contrast, pathodiagnostic acuteor transition-phase TSV lesions were detected histologically in 4 of 8 samples (grade 2 4 severity) that were collected moribund or recently dead during Bioassay 1, 12 of 16 shrimp (grade 1 2 severity) from Bioassay 2 and 1 of 2 shrimp (grade 4 severity) from Bioassay 3 (Table 3). Epithelial necrosis characteristic of TSV infection was observed within the stomach, appendages, general body cuticle, and gills (Fig. 3c). Three of the eight treatment shrimp collected during Bioassay 1 demonstrated moderate to numerous lymphoid organ spheroids, suggestive of a chronic-phase TSV infection (Table 3). ISH analysis utilizing a TSV-specific probe was conducted on two to seven representative treatment samples from each of the three bioassays (Fig. 3d). TSV-positive signals were obtained for four of four samples from Bioassay 1, five of seven samples from Bioassay 2 and two of two samples from Bioassay 3 (Table 3), thus corroborating the histology findings. The failure of one treatment shrimp sample from Bioassays 2 and 3 to produce a probe-positive signal was likely due to a prolonged fixation in Davidson s fixative resulting in acid hydrolysis of the TSV genome and false negative ISH results, as described by Hasson et al. [12]. Transmission electron microscopy of the purified virus preparation of the Texas isolate showed that the virions had icosahedral morphology, with an average size of 31 nm. The morphology and size of the virions were very similar to the virions of the Hawaii isolate [2, 11]. Cloning and sequence analysis of thetsv Texas isolate The genome of the TSV Texas isolate was cloned in four segments (222 bp, 557 bp, 2.6 kb and 7.2 kb) and sequenced (Fig. 1a, b). An agarose gel photograph showing the amplification of four different segments of the Texas isolate is presented in Fig. 1c. The amplified cdna fragments were cloned and sequenced, and the consensus sequence was used for sequence analysis. The genome of Fig. 3 a A juvenile P. vannamei (Kona) showing clinical signs of TSV Texas isolate infection. Tail fan showing reddening of the uropod and multifocal necrotic lesions. b Multifocal melanized lesions on the cuticle are indicative of transition- or chronic-phase infection. I Infected, H healthy animals. c Sequential tissue sections showing a focal acute-phase infection by the TSV Texas isolate in the ventral cephalothorax of a per os-exposed P. vannamei juvenile by routine H&E histology and d by in situ hybridization. In panel c, infected cuticular epithelial cells (arrow) are characterized by pyknotic nuclei, cytoplasmic eosinophilia and detachment from the surrounding cell matrix. In panel d, TSV-infected cells display a probe-positive signal characterized by cytoplasmic blue-black coloration (arrow), while normal cells are golden orange due to counterstaining with Bismarck brown

8 322 A. K. Dhar et al. Table 3 A summary of mortality, histopathology and in situ hybridization (ISH) results with shrimp (Penaeus vannamei, Kona stock) infected with the TSV Texas isolate Bioassay Treatments/# animals a Cumulative mortality (%) Histology by H&E Infection phase # animals examined In situ hybridization (# animals examined) 1 Control/N = Negative 7 Negative (2) Us04Pv1/N = Acute 3 (G2, G3, G4) c Positive (2) Transition 1 (G3) Positive (1) Chronic 3 Positive (1) Unresolved b 1 2 Control/N = 21 5 Negative 5 Us04Pv1/N = Acute 10 (six G1, four G2) Positive (2) Transition 2 (G1) Positive (1) Unresolved 4 Positive (2), Negative (2) 3 Control/N = 13 0 Negative 3 Us04Pv1/N = Acute 1 (G4) Positive (1) Unresolved 1 Positive (1) From each bioassay, representative moribund animals were taken for histopathology by H&E. Based on the histopathology results, a subset of samples was taken for ISH analysis a In each bioassay, there was one tank for the control group and one tank for the TSV Texas isolate, except in bioassay #2, where the controlgroup animals were kept in two tanks (10 in one tank and 11 in the second tank) b Unresolved = TSV infection not definitive, might be due to over-fixation of sample or that the sample was not infected with TSV c Lesion severity as described by Hasson et al. [14]. Grade 1 indicates lesion or spheroid presence in\25% of the tissue/organ, grade 2, 25 50%; grade 3, 50 75%; and grade 4, [75% the Texas isolate was found to be 10,205 nucleotides long, and the sequence has been deposited in the GenBank database under the accession number GQ In order to understand both structural and pathological variations among geographically diverse TSV isolates, a comparison of genome sequences of the six isolates was carried out. Unlike the and 3 0 -UTRs, the nucleotide lengths of ORF1s, IGR and ORF2 were strictly maintained among the isolates, with the exception of the isolate from Venezuela, which had a two-base shorter IGR sequence. The 5 0 -UTR of the Texas (416 nts), Hawaii (416 nts), China (413 nts) and Thailand (416 nts) isolates showed 98% similarities among themselves. For the Belize (153 nts) and Venezuela (344) isolates, only partial 5 0 -UTR sequences have been deposited in the database. Within the sequenced region of 5 0 -UTR, the Belize and Venezuela isolates showed 98 and 96.5% similarity with the corresponding region of Texas isolate. Sequence identities of TSV isolates with respect to the Texas isolate ORF1 were 98% with the China and Thailand isolates, 97% with Hawaii and Belize isolates and 92% with the Venezuela isolate. Sequence identities with respect to Texas isolate IGR sequence were 98% with the Hawaii, China, Belize and Thailand isolates and 95% with the Venezuela isolate. The nucleotide identities with respect to the Texas isolate ORF2 were 97% with the Hawaii, China, and Thailand isolates, 96% with the Belize isolate and 93% with the Venezuela isolate. Trends of comparable homologies were also observed at the amino acid level. For example, similarities above 96% were observed for both ORFs and among all of the TSV isolates except Venezuela, which showed 93 and 94% similarity for ORF1 and ORF2, respectively. Recently, the VP1 capsid protein sequence of a Texas isolate has been published [36]. The VP1 sequence of the Texas isolate presented in this study showed seven nucleotide differences (4 G s to A s, 1 G to C, 1 A to T, 1 A to C) resulting 4 aa changes (1 E to G, 1 H to Q, 1 N to K, 1 G to E) compared to the sequence published by Weirtheim et al. (2009). Considering the high substitution rate within VP1 of TSV ( substitutions/site/year, [36]), this is perhaps not surprising. However, it suggests that the Texas isolate sequence described in the present study may be a different strain than the one presented by Weirtheim et al. [36]. Within the 3 0 -UTR region, the Texas (223 nts), Hawaii (223 nts), China (223 nts) and Thailand (223 nts) isolates showed 98% similarities among themselves. For the Belize (203 nts) and Venezuela (188 nts) isolates, only partial 3 0 -UTR sequences are available, and these two isolates showed 96 and 97% similarities to the corresponding region of Texas isolate.

9 Properties of 2004 Texas Taura syndrome virus in shrimp 323 Phylogenetic relationships of the Texas isolate with other TSV isolates The Bayesian phylogenetic inferences based on the whole genome sequence revealed that the Texas isolate groups with TSV isolates from Thailand and China with a high posterior probability (Fig. 4). TSV epizootics in Texas first occurred in shrimp farms in Cameron County, located near the shrimp-processing plants that imported shrimp from Southeast Asia. Subsequently, it spread to farms located in nearby counties. This may explain the close relationship of the Texas isolate to isolates from Thailand and China. A detailed epidemiological study to examine the potential role of these shrimp-processing plants in spreading TSV and to elucidate the origin of the Texas isolate remains to be performed. The phylogenetic analysis using full-length genomic sequences described in the present study agrees with a phylogenetic study recently published by Wertheim et al. [36] using TSV VP1 capsid sequences. In that study, Wertheim and colleagues assessed the evolutionary relationships among 83 TSV isolates originating from 16 countries [36]. Although the partial sequence (1,303 nts in length) shed light on the phylogenetic relationships among TSV isolates worldwide, some of the basal structure of the phylogenetic tree remained unresolved (see Fig. 1 in [36]). To help elucidate these relationships, these authors suggested using full-length TSV genome sequences. In the present study, although we used full-length genome sequences, some of the relationships were only partially resolved, as indicated by the lower posterior probability of the branch connecting Venezuela with the rest of the Fig. 4 Evolutionary relationships of the Texas isolate and other TSV geographic isolates as depicted by unrooted 50% majority consensus trees generated by Bayesian inference of the full-genome sequences. Numbers above the branches represent Bayesian posterior probabilities. The scale bar represents number of substitutions per site isolates (Fig. 4). Our finding tends to support to the hypothesis of rapid diversification of TSV isolates, as suggested by Wertheim et al. [36]. However, in order to further clarify the population structure of TSV isolates, a larger number of full-length genome sequences of TSV isolates needs to be incorporated into the analysis. The position of the Belize isolate in the phylogenetic tree suggests that this isolate probably derived from the Hawaiian TSV isolate (posterior probability = 0.89, Fig. 4) and may represent an intermediate between the American and the Asian TSV strains. Overall, the close and highly supported relationships observed among TSV isolates suggest a recent evolutionary process among geographically distinct TSV isolates, and the close association of the Texas isolate with the Asian isolates reflects a TSV population structure that has been shaped mainly by human-mediated transport in aquaculture activities. Protein structural analysis Information on the tertiary structure of viral proteins is important for understanding how they interact with cell receptors and other cellular proteins during viral pathogenesis. Although the primary structures (nucleotide and the predicted amino acid sequences) have been determined for a number of TSV isolates, no information is available on the tertiary structures of TSV-encoded proteins. Using computational modeling, we predicted the tertiary structures of three TSV-encoded proteins, IAP, protease and RdRp because crystal structures of homologous proteins are available in the PDB. Other than these three proteins, there are no other proteins that show a high degree of similarity with TSV-encoded proteins whose crystal structures are available in the database. The crystal structure of the capsid protein of cricket paralysis virus (CrPV), the type member of the family Dicistroviridae, to which TSV belongs, has been published [32]. However, the amino acid sequence of the capsid protein of TSV Texas isolate does not align well with the CrPV structural capsid protein (CrPV GenBank accession number AF218039) and has only 27.3% identity in a 176-residue overlap; score 151.0; gap frequency 3.4% ( pl?prot). As a result, even a theoretical model of TSV capsid protein based on the crystal structure of CrPV is not reliable. The SWISS-MODEL server was used to generate theoretical three-dimensional protein models of the IAP, protease, and RdRp domains of the Texas, Hawaii, China, Thailand, Venezuela, and Belize isolates. The methods used were modeled after those used previously to elucidate the structure of a novel RdRp from Homalodisca coagulata virus-1 [16]. The predicted IAP domains corresponding to the Texas isolate domain from A (182) to Q (213) were

10 324 A. K. Dhar et al. Fig. 5 Structural analysis of the predicted protein domains of the c TSV Texas isolate virus compared to the structures predicted for TSV isolates from Belize, China, Hawaii, Thailand and Venezuela. Panel a compares the predicted IAP domains, panel b compares the C-terminal region of the protease domains, and panel c compares the predicted RdRp domains. The Ramachandran plots and the corresponding tables of RMS values are given below the tertiary structures A (a) 1e31 (b) Texas 2004 TSV used to build theoretical models of the IAP domains from each of the 4 isolates. PDB structure 1E31, apoptosis inhibitor survivin, was used as the template for homology modeling to generate the theoretical structures. The threedimensional structure of 1E31, along with the theoretical structure of the IAP domain of the Texas isolate, was predicted using this approach (Fig. 5a). The theoretical structures of the IAP domains of Hawaii, China, Thailand, Venezuela, and Belize isolates did not differ from that of the Texas isolate and are not shown (128 backbone atoms compared with RMS values of 0.01 Å). The RMS values for alpha carbon and all backbone atoms in IAP were\1.0. The lower the RMS value, the higher the correlation between the database model and our predicted model. In a recent paper, Jonassen and colleagues suggested an RMS value of \6.0 Å as a cutoff value to compare protein structures [18]. Since the RMS values of TSV IAP is\1.0, the proposed tertiary structure is quite reliable. The full-length protease domain of the Texas isolate did not share enough sequence identity to any PDB entry to predict the tertiary structure by the First Approach mode of the SWISS-MODEL server. Only the C-terminal portion of the protease domain (P1503 to M1565 of the Texas isolate) shared a high degree of sequence similarity to PDB entry 2A4O, a hepatitis A virus 3C protease (Fig. 5b). Therefore, the 2A4O structure was used to build the theoretical model of the C-terminal end of protease of each of the six isolates. This C-terminal region has been shown for other 3C proteases to contain part of the catalytic domain and residues involved in substrate binding. The C-terminal domain of the six viral isolates studied here did not differ from each other, so the representative Texas isolate structure is shown along with the corresponding domains of 2A4O (Fig. 5b). Like the RMS values of IAP, the RMS values for alpha carbon and all backbone atoms in the protease domain are \1.0, and the proposed tertiary structure were considered quite reliable. The theoretical tertiary structures of the P1605 to L2037 (Texas isolate) portion of the RdRp domain from each of the six TSV isolates were built from PDB accession number 1XR7. The RdRp sequences formed the basic fingers-palm-thumb domain structure that is commonly observed for polymerases [5]. Interestingly, the Hawaii isolate had a very large RMS value (15.96 Å for all backbone atoms) when compared to 1XR7, while the Texas, China, Belize, Thailand, and Venezuela isolates had B Carbon alpha Backbone All Atoms Num Atoms RMS Num Atoms RMS Texas Belize Chinese Hawaii Thailand Venezuela (a) 2A4O RMS values of 1.08 Å or less (Fig. 5c). Most of the higher RMS value of the Hawaii isolate could be traced to the finger region of its RdRp. An amino acid sequence (b) Texas 2004 TSV Carbon Alpha Backbone Num Atoms RMS Num Atoms RMS Texas Belize Chinese Hawaii Thailand Venezuela

11 Properties of 2004 Texas Taura syndrome virus in shrimp 325 C 1XR7:A Texas Belize China Hawaii Thailand Venezuela Carbon alpha < 1.0 RMS Backbone < 3.0 RMS Backbone All Atoms Num Atoms RMS Num Atoms RMS Num Atoms RMS Texas Belize China Hawaii Thailand Venezuela Fig. 5 continued Thumb Palm Fingers Num Atoms RMS Num Atoms RMS Num Atoms RMS Texas Belize China Hawaii Thailand Venezuela comparison of the RdRp domain of the six isolates shows four amino acids of the Hawaii isolate that differ from the consensus (L1622, N1673, K1769, S1994). Each of these four amino acids was mutated separately in silico to its respective consensus amino acid, and then theoretical model structures were generated using the SWISS- MODEL server. The Hawaii RdRp structure had an RMS value of Å when comparing all backbone atoms (1,612 atoms) to the reference structure. The mutant structure S1944N has an RMS value of 15.95, K1769E has an RMS value of 15.96, N1673D has an RMS value of 15.96, and L1622R has an RMS value of It is interesting that the L1622R mutant structure of the Hawaii isolate had a comparable RMS value to those of the Texas, China, Belize, Thailand, and Venezuela isolates. To summarize, a comprehensive investigation of the TSV Texas isolate that caused epizootics in shrimp farms in Texas in 2004 was carried out. The isolate was found to be highly virulent in laboratory bioassays, causing severe symptom development and rapid mortality. Genomic and phylogenetic analyses showed that the Texas isolate is most closely related to the TSV isolates from Thailand and China. Using computational modeling, we predicted and compared the tertiary structures of the three domains of ORF1 (IAP, protease and RdRp) of the six completely sequenced TSV isolates, including the Texas isolate. Although the proposed tertiary structures of IAP and protease domains of TSV isolates did not show any difference, the data from our 3-D modeling showed that the RdRp of TSV reference strain Hawaii 1994 has significant structural differences from the corresponding domains of five other TSV isolates. It is not known whether changes in the tertiary structure due to point mutations in the RdRp gene contribute to the replication fidelity in the Texas isolate and thus enable the virus to overcome host immune responses by reaching a lethal viral load soon after entering into the cells. If point mutations indeed contribute to the virulence of the Texas isolate, these mutations could serve as markers for virulence. In bovine viral diarrhea disease virus (BVDV), an RNA virus belonging to the family Flaviviridae, it has been shown that mutations in the palm region of the RdRp result in reduction in viral RNA synthesis and

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