In-Vivo Comparative Virulence of Different White Spot Syndrome Virus Isolates In Penaeus Vannamei And Whole Genome Comparison Analysis

In-Vivo Comparative Virulence of Different White Spot Syndrome Virus Isolates In Penaeus Vannamei And Whole Genome Comparison Analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In-Vivo Comparative Virulence of Different White Spot Syndrome Virus Isolates In Penaeus Vannamei And Whole Genome Comparison Analysis Álvaro Hernán Hernández-Montiel, Manfred Weidmann, Michael Bekaert, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-824873/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract White Spot Syndrome Virus (WSSV) infects several economically important aquacultural species, causing significant losses to the industry. This virus belongs to the Nimaviridae family, and has a dsDNA genome ranging from 257 to 309 kb (more than 20 isolate genomes fully sequenced and published to date). Multiple routes of infection could be the cause of the high virulence and mortality rates detected in shrimp species. In particular, Penaeus vanname i, differences in isolate virulence have been observed, along with controversy over whether deletions or insertions are associated with gain or loss of virulence. The pathogenicity of three isolates from three locations in Mexico (two from Sinaloa: 'CIAD', and 'Angostura', and one from Sonora: 'Sonora') was evaluated in vivo in white shrimp ( P. vannamei ) infection assays. Differences were observed in the mortality rate of shrimp among the three isolates, with the Sonora isolate being the most virulent. Subsequently, the complete WSSV genomes were sequenced in depth from the tissues of infected shrimp, and assembled in reference to the genome of isolate CN01 (KT995472), identifying genome sizes for Angostura and Sonora of 289,350 bp and 288,995 bp, respectively. Where three deletion zones were identified compared to CN01 comprising 15 genes, including three envelope proteins VP41A, VP52A and VP41B, one non-structural protein ICP35 and 11 others encoding proteins whose function is currently unknown. In addition, five genes (wsv129, wsv178, wsv204, wsv249 and wsv497) show a modified number of repeat motifs. The main implications and possible effects on viral infection of these modifications are discussed. Virology Internal Medicine WSSV Genomics virulence viral isolates envelope proteins deep sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The white spot syndrome virus (WSSV) is a highly contagious pathogen. With a replication cycle of approximately 20 hours at 25 ⁰C, it is capable of causing a 100% mortality rate in white shrimp ( Penaeus vannamei) within a period of 3 to 10 days [ 1 , 2 ], generating massive economic losses in the industry [ 3 ]. The first massive losses due to WSSV occurred in China and Taiwan in the years 1991–1992 especially in Kuruma shrimp ( Marsopeneaus japonicus) , subsequently the massive mortalities due to WSSV spread throughout the rest of Asia and the world [ 4 ]. WSSV appeared in aquaculture farms in Mexico in 1999 [ 5 ] and since then, it has been persistent with regional epidemics [ 6 , 7 ]. Transmission of the virus can be horizontal, through cannibalism or by ingesting water containing WSSV particles, by a vertical transovarial route [ 8 ], with ballast water from ships, birds acting as vectors, and live or frozen imported products [ 9 ]. WSSV belong to the Nimaviridae family, genus Whispovirus (ICTVdb.org) and recently other Nimaviruses were identified in crabs [ 10 ] which they can also infect in aquaculture settings [ 11 ]. WSSV is enveloped, has an elliptical shape with an approximate size of 250–380 nm in length by 80–120 nm in width [ 12 ] and has an filamentous appendage. Its genome is a double-stranded circular DNA [ 13 ]. The largest published genome (CN01 with 309,286 kb) was isolated from China in 1994 ( KT995472.1) [ 14 ]. To date, > 20 WSSV genomes have been published ( https://www.ncbi.nlm.nih.gov/ ), with the latest isolate MG432482 from Mexico [ 7 ](257,675 kb). Two opposite hypotheses have been postulated to explain differences in genome size: 1) the reduction of the genome increases virulence [ 15 ], and 2) the reduction of the genome reduces virulence [ 16 ]. These discrepancies cannot not be fully resolved, because of differences in experimental designs, host species and molecular techniques used. To date a direct cause-effect between genome size and virulence has not been identified [ 17 ]. It has been hypothesized that the pathogenicity depends on the presence of “factors” associated with virulence, which are absent or have lost function in less virulent isolates [ 14 ]. Deletions, mutations in open reading frames (ORFs) are believed to increase virulence rates in their hosts [ 8 ], such as ORFs 75, 94, and 125 involved in receptor recognition which potentially increases tropism. WSSV gene characterization identified some gene functions potentially involved in pathogenicity and pathogen-host interaction [ 18 – 23 ]. However, these analysis in M. japonicus did not include in vivo comparison of virus strains, viral load, and genome analyses altogether. We aimed to compare experimentally ( in vivo ) the virulence of three WSSV isolates from shrimp farms in México isolated during outbreaks in 2008 and 2013, and to analyze specific genomic regions that could be associated with higher virulence by comparing the isolate genomes with each other and with available sequences from public databases. Materials And Methods Origin of the isolates The bioassay was carried with three isolates from different geographical origins and epidemics. One isolate was obtained by Dr. Jorge de la Rosa Vélez † during an outbreak that occurred in Sonora in 2008, called the Sonora isolate. The second isolate was provided by Dr. Leobardo Montoya from the Food and Development Research Center (CIAD) located in Mazatlán Sinaloa, from an outbreak in Sonora in 2013 designated CIAD. The third isolate was provided by MC Joel Lizárraga Valdez from the company Aquapacific S.A. de C.V. obtained from an outbreak in the Angostura, Sinaloa area in 2013. Preparation of WSSV viral inocula Inocula of each isolate were prepared from 25 mg of tissue of four cryopreserved dead shrimps, placed in a 1.5 mL Eppendorf tube, supplemented with 500 µL of TN buffer (Tris/HCl 10mM. pH 7.4, NaCl 3M, passed through 0.2 µm microfilter) and homogenized with a pistil, followed by another 400 µL of TN buffer [ 24 ]. The samples were centrifuged at 1000 ɡ for 5 min, 3000 ɡ for 5 min, 10,000 ɡ for 5 min and 14,000 ɡ for 15 min. The supernatant was recovered with a 3 mL syringe and filtered again with a 0.45 µm microfilter. The filtrate was placed in a new 1.5 mL eppendorf tube and was kept at -4˚C until use, for less than 2 hours. This procedure was repeated every time shrimp were infected. Verification of the viability of isolates White shrimps from the ACUAMOS aquaculture farm (located in San Felipe, Mexicali B.C) were infected with the viral inoculum. Juveniles were maintained at 28 ± 1⁰C, a salinity of 34 ppt, and constant aeration until reaching 15.7 ± 2 g and 14 ± 0.6 cm. For each isolation (n = 5), two replicates were used). 20 µL of inoculum was injected into the fifth abdominal segment of the shrimp. Moribund shrimps were cryopreserved in liquid nitrogen and stored at -80°C. The virus replication was verified by end-point PCR, with a set of primer targeting the structural protein VP664 [ 25 ]. Challenge experiment to compare the isolates virulence White shrimps from the Mahar aquaculture farm (Pichilingue, La Paz, Baja California Sur, Mexico) weighing 4.4 ± 0.9 g and 8.4 ± 0.6 g, were infected with the different viral inocula. A PCR test was done to corroborate animals were wssv-free [ 2 ]. For each isolate challenges of shrimps (n = 7) were performed in triplicate and placed in 40 L tanks, with a temperature of 28 ± 1⁰C, a salinity of 34 ppt, constant aeration, and a biological filter (activated carbon). Three other replicas were used for the non-infected shrimps. DNA extraction to quantify viral load Once bioassays were completed, DNA was extracted from all samples (N = 63).100 mg of tissue was taken from the 5th abdominal segment and DNA was extracted using the Phenol: Chloroform: Isoamyl alcohol [25:24:1] standard protocol [ 26 ]⁠. DNA concentration (260 nm) and quality (radius 260/280) was verified with a Nanodrop LITE (Thermo brand). Additionally, the DNA quality was assessed in a 1% agarose gel electrophoresis in 40 mL of 1X TAE (0.04M Tris-acetate, 1mM EDTA) and visualized by ethidium bromide [10 mg/mL]. Viral load quantification Viral load was determined by real-time PCR (Q-PCR) absolute quantification with standard curve (1:10 dilution factor, with dilutions 10 0 to 10 7 ) according to Durand and Lightner, 2002 (OIE procedure). The 64 bp fragment of vp 664 was inserted on TOPO TA plasmid (Invitrogen, United States) as previously described [ 2 ]. DNA extraction and purification for sequencing of the WSSV genome For deep sequencing of the whole genome of the viruses from the bioassay, the DNA of 4 shrimps tested positive for WSSV by Q-PCR and of two free-wssv were extracted and purified. The extraction was done with the 5 Prime Archive Pure DNA blood kit (# 2900258 5prime GmbH, Germany) with Econo Spin columns (Epoch # 1910 − 250, Epoch Life Science Inc, United States). The column was washed with a Type 1 wash buffer (GE28-9031-70, GElifesciences, United Kingdom). DNA was eluted with 50 µL of water. Preparation of samples for deep sequencing Samples were stabilized with DNAstable Plus® (Biomatrica, UK) following the manufacturer's instructions and were sent for sequencing by regular mail service at the Institute of Aquaculture (University of Stirling in Scotland, UK) with an Illumina MiSeq, V3 75 bp, Paired-End. Deep sequencing data cleaning and mapping The quality of the sequencing raw reads was checked with FastQC [ 27 ]. Quality scores below 30 along with Illumina adapters were removed with Trimmomatic v.0.39 [ 28 ]. The cleaned reads were mapped against the reference genome China CN GCF_000848075.2 (KT995472.1), using the BWA aligner v.0.7.15 [ 29 ] and SAMtools/BCFtools v1.11 ( http://www.htslib.org ). Statistical analysis The survival probability was evaluated by the Kaplan-Meier approach, using the Greenwood formula to estimate the variance, to stabilize the variance, the confidence intervals were calculated by the log (-log) transformation. The comparison between the curves was made with the long-rank test, using the survival package version 3.2-7 for survival analysis. An adjustment of generalized additive models was also carried out, by means of the local regression method, to evaluate the correlation of hours post inoculation (HPI) and viral load (VL) with a confidence interval of 0.95, using the Gam package in R studio version 1.3.959. Genome alignment and phylogenetic analysis The alignment of the isolated genomes from Angostura and Sonora was performed with MAFFT v7,017 [ 30 ] with score matrix of 200PAM/K = 2. GSP open penalty of 1.53 and offset value of 0.123 and automated sequence addressing, together with the sequences KR083866, MG432478, MG432474, AF440570, AF332093, AF369029, KT995470, KY827813, KT995471, JX515788, KT995472, KU216744, MG432479, MG432475, MG432477, MG432482, MG432476, MG432481 and MG432480, extracted from GENBANK and using the genome GCF_000848075.2 corresponding to the sequence CN01_KT995472 isolated from China as a reference since it is the longest WSSV genome Sequence. Sonora and Angostura isolates were also included: ERR5659803, ERR5659804 respectively). The phylogenetic tree was inferred from this alignment with 1,000 bootstrap repeats using RAxML 7.2.8 method, GTR GAMMA nucleotide model, Bootstraping algorithm, genetic distance in annotated as a scale bar. A multiple genome comparison with circular architecture was made with BLAST with the BLAST Ring Image Generator (BRIG) version 0.95 [ 31 ], comparing the all the above listed genomes, using the CN01 isolate as a reference. A multiple genomic alignment to observe genomic conservation and rearrangements was performed with MAUVE version 2.4.0. Repeated motifs (RM) in specific genes were detected with The MEME Suite 5.3.3 (Multiple EM for Motif Elicitation) [ 32 ] on line version https://meme-suite.org/meme/tools/meme . A motif is an approximate sequence pattern that occurs repeatedly in a group of related sequences that usually mediate a common function. Motif function was inferred by comparing motif to motifs databases with Tomtom motif comparison Tool version 5.3.3 [ 33 ] available in the same link. Results Challenge experiment to compare the virulence of WSSV isolates Shrimp infection with the CIAD isolate produced a survival rate of 19% 10 days after challenge, with a first drop in survival at 29 h 40 min post infection (PI) and a final drop at 145 h 50 min PI. On the other hand, the infections carried out with the Angostura and Sonora isolates produced a mortality rate of 100% at 168h 40min PI, with a first drop in survival at 35 HPI and 27 HPI respectively (Fig. 1 A). Comparison of the long-range analysis for the tree isolates (Chisq 0.4, 2 degrees of freedom or d, p 0.81) and the Kaplan-Meier approach indicates a 50% survival for the CIAD isolate at 131 HPI, Angostura at 150 HPI and Sonora at 99 HPI (Fig. 1 A). Viral load quantification of the three treatments Viral loads or viral genomes / mg (VL) were compared between treatments by vp664 qPCR on all infected shrimp (Fig. 1 B). VL ranged from 8.48 x 10^7 (CIAD), 1.26 x 10^8 (Sonora) to 1.91 x 10^8 (Angostura). The variance analysis indicates no correlation between HPI and VL for all isolates (F 0.002459, p 0.001, R² < 0.29). Comparison of WSSV genomes and identification of genes with variations Due to concentration and sample quality problems, it was not possible to sequence the CIAD isolate. However, we obtained a total of 1,717,195 and 1,657,796 reads with a length of 76 bp that were assembled to 155,993 and 105,993 reads for the Angostura and Sonora genomes respectively (KT995472.1). The genome size of the Angostura and Sonora isolates is 289,350 bp and 288,995 bp, respectively (S1), with an overall GC percentage of 41.1%. The Sonora isolate has a slightly shorter genome (355 bp) than the Angostura isolate. The data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI with the accession number PRJEB44096 ( https://www.ebi.ac.uk/ena/browser/view/PRJEB44096 ). Nucleotide alignment with identity % among all genomes ranges from 78.3 % (LG vs GVE05 isolates) to 99.2 % (ACF vs DVI isolates). There is about 99.7% similarity between the Sonora and Angostura isolates (S1, S4). Phylogenetic analysis indicates that Sonora and Angostura isolates are closely related to the Mex2008 isolate and have a 96.4% similarity to isolate CN02 from China. Another clade groups the isolate from Taiwan, AC1 and GVE. The most recent branch contains isolates JP, ACF2, DVI, LG, ACF4, LC1 and LC10. The circular alignment of all genomes with BRIG clearly identified 3 deletion areas with specific genes (shown in red) when compared the CN01 isolate (Figs. 3 A, B) in both Angostura and Sonora genomes (Table 1 ). The genes wsv 463a, wsv 463b, wsv 463c, wsv 463d are absent in both genomes when compared to CN01 (Fig. 3 A and B) and genes wsv 489, wsv 490, wsv 492, wsv 493 and wsv 495 had a < 50% similarity among isolates. The only exception is the Taiwan isolate which has the complete sequences for the mentioned genes, followed by the isolates from China (Mj) and Eg3 containing a deletion of wsv 495 (Fig. 3 A). The genes that code for proteins VP41B, VP52A and VP41A presented deletions or a similarity of less than 50% with respect to CN01, in all the Mexican genomes, with the exception of Mex2008 (Fig. 3 B). Particularly these genes coding are absent in the genome of isolate CN03, while wsv 234 has a low similarity (Fig. 3 A). On the other hand, CN04 presented only the deletions of VP52A, VP41A and wsv 234. Table 1 Deletions identified in the isolates from Angostura and Sonora when compared to CN01 (KT995472). wsv nomenclature corresponds to isolate from China_MJ (or CN) (AF332093) used in this work for annotations, and wssv corresponds to equivalent genes in Taiwan_Pm (AF440570). * Protein name not defined. In parentheses is the name according to Taiwan based studies. No. WSSV gene Protein name Sub-category function Reference 1 wsv 234 * Hypothetical Protein 2 wsv 237 wssv 293 VP41A Envelope protein Huang et al. (2013) 3 wsv 238 wssv 294 VP52A (VP51A) Chang et al . (2008) 4 wsv 242 wssv 298 VP41B Zuo et al. (2011) 5 wsv 244 * Hypothetical Protein 6 wsv 247 * 7 wsv 463 a * 8 wsv 463 b * 9 wsv 463 c * 10 wsv 463 d * 11 wsv 489 * 12 wsv 490 * 13 wsv 492 * 14 wsv 493 wssv 019 ICP35 (VP35) Nonstructural Protein Kang et al . (2013) 15 wsv 495 * Hypothetical Protein An additional multiple MAUVE alignment between Angostura and Sonora genomes in reference to isolate CN01 corroborates the deletion of wsv 247, wsv 244, VP41B, VP52A, VP41A and wsv 234 in both Mexican isolates in reference to the genome of isolate CN01 (Fig. 4 ; S2, orange region in CN01 at 140kb). This analysis showed the deletions of wsv 463a, b, c and d in the 180 kb region of the Angostura genome (S2, red region in Angostura at 180 kb), and deletions corresponding to wsv 489, wsv 490, wsv 492, wsv 493 and wsv 495 in a terminal region close to the 3-prime end of the genome (S2, yellow region in Angostura at 260 kb). A difference of < 30% at nucleotide level was observed in wsv129, wsv178, wsv 204, wsv 249 and wsv 497 genes between the genomes of the Sonora and Angostura isolates (S3). The Sonora isolate wsv 129 has lost several Repeated Motifs (RM) like RM1 and RM2 but gained RM3 (S3, A), wsv 178 has lost five RM1 (S3, B), wsv 204 gene has acquired one RM2 (S3, C), while wsv 249 has lost one RM1 in the Sonora isolate (S3, D, while wsv 497 is similar in both isolates, with 1 more RM2 compared to CN01, (S3, E). RM is a repeated motif, and the number makes reference to the motif number for each case. Discussion Three WSSV isolates from Mexican shrimp farms were characterized by bioassay and through whole genome sequencing. WSSV isolates tested in bioassays generally induce the following clinical signs in infected white shrimp; lethargy, decreased food intake, discoloration of the hepatopancreas, cuticular loosening and red uropods [ 12 ]. Apart from these general clinical signs Shrimp infected with the CIAD isolate in the challenge bioassay showed 19% survival at 10 days post infection similar to the percentage obtained by Sekar, with 100% mortality until day 20 [ 34 ]. In contrast the, isolates from Angostura and Sonora induced 100% mortality between 3- and 10-days post-infection [ 35 , 2 ]. The Angostura isolate produced the highest viral load of the three isolates (Fig. 1 B). The differences between isolates however were in the range of published data reporting the viral load of WSSV in three Penaeus species ( P. vannamei , P. stylirostris and P. monodon ) obtained by real-time PCR in the range of 2 x 10 4 – 2 x 10 9 WSSV copies µg − 1 of total DNA, in infected shrimp [ 25 ]. A review to evaluate the risk associated for the shrimp trade, data on WSSV concentrations obtained from tissues of different species, including P. vannamei , indicated high viral load discharges at the beginning of mortality, with concentrations of 1 x 10 9 – 1 x 10 10 [ 36 ]. The CIAD isolate presented a mortality risk percentage between 95% and 19% with a probability of 50% at 131 h and an overall survival rate of 19%. Isolate Angostura presented a constant 65% mortality risk percentage between 50 and 150 h, for a 50% survival at 150 h (Fig. 1 A). For isolate Sonora, three drops in survival were observed, with risks of death between 64% and 43% from 50 to 150 h, and a probability of survival of 50% at 99 h. This is a possible sign of a faster progressing replication and therefore increased virulence of the isolate Sonora compared to isolate Angostura. Both isolates Angostura and Sonora induced 100% mortality observed at 168 HPI. Therefore, we tentatively classified the isolates as low virulence (CIAD), moderate virulence (Angostura) and high virulence (Sonora) from the mean post-infection lethal time according to [ 37 ]. The genomes of the isolates from Angostura and Sonora, presented a length within the estimated range for Mexican isolates from localities between Sinaloa and Nayarit (257,675 − 290,879 bp) [ 7 ]. To assess genomic differences of the isolates we were able to identify specific sites with differences to isolates from foreign and domestic outbreaks (Fig. 3 A, B). Deletions in the WSSV genome are considered as enhancements of the adaptive response by WSSV as it is reasoned that deleted regions, do not encode proteins vital for WSSV pathogenicity or virulence [ 38 ]. The differences are possibly caused by recombination events mediated by transposons causing insertions or deletions [ 39 ]. An inversion is apparent among the 2008 Sonora and 2013 Angostura isolate as spotted in the MAUVE alignment of motives (Fig S2). Since the isolate with the shortest sequence was the most virulent (Sonora), we analyzed the deletions. In our comparative analysis between the genome of the isolates of the present study, with sequences of international and national isolates, a group of deleted genes stands out: wsv 237(VP41A) [ 40 ], wsv 238(VP52A-VP51A) [ 41 ]. wsv 242(VP41B) [ 42 ] and wsv 493(ICP35-VP35) [ 43 ]. The VP41A envelope protein is known to interact with VP26, VP56 and VP28 envelope proteins [ 40 , 44 ] and with the viral envelope protein VP51A (VP52A for AF332093) [ 45 ]. It has been suggested that VP26 is anchored to the envelope with its N-terminal hydrophobic region, as a C-terminal region binds to the nucleocapsid [ 46 ], while VP51A is a type II transmembrane protein, with a transmembrane domain highly hydrophobic at its N-terminus and an exposed C-terminus on the virion surface [ 47 ]. On the other hand, VP41A has only one transmembrane region at its C-terminal end, and this envelope protein is part of a group of 11 proteins that interacts with the chitin-binding protein of P. monodon (PmCBP) [ 16 ] which are known to participate in shrimp immune response [ 48 ]. It also interacts with preitrophin-like protein in L. vannamei (LvPT). LvPT is involved in the formation of the peritrophic membrane [ 49 ] and contains a chitin-binding domain facilitating binding of WSSV to the intestinal epithelium. It has been shown to promote infection, as silencing of LvPT decreased the viral load in pleopods, stomach and intestines in previously WSSV infected shrimp [ 50 ]. Therefore, the absence of the envelope proteins VP41A and VP51A could modify the usual conformation of the protein complex composed of VP26, VP28 and VP56, as well as the interactions of VP26 with VP51A reducing viral recognition by chitin-binding proteins such as PmCBP and LvPT. The observed deletions in Sonora and Angostura isolates could contribute to changes in the WSSV viral envelope and enhance virulence causing high mortality. VP38 acts as a repressor of Penaeus japonicus caspase (Pjcaspase) transcription in M. japonicus , while VP41B activates its expression, regulating the apoptosis mechanism used by shrimp to control viral load [ 42 ]. Elevated Pjcaspasa gene expression of M. japonicus was observed in shrimp survival challenges while siRNA silencing of Pjcaspasa increased post-infection viral load [ 51 ]. So, deletion of VP41B would not allow timely induction of apoptosis of infected cells allowing the virus to replicate without limitation and thus having a major impact on the viral load control mechanism employed by WSSV. The Sonora isolate has noticeable modifications on wsv 129, wsv 178, wsv 204 and wsv 249 as gain or loss of specific sequences equivalent to Repeated Motifs (RM) that strongly suggest a role in the infection process. A high variation from 3 to 20 in the number of repetitive sequences in WSV178 has been related to virulence [ 52 , 53 ]. In contrast a lower mean number of repeated sequences was observed in outbreaks, where WSSV genotypes with 5, 6 and 7 of a 54 bp RM were those that predominated in semi-intensive shrimp crops [ 54 ]. In our study, we identified 4 and 3 repetitions of a 54 bp RM in WSV178 in the Angostura and Sonora genomes respectively. Previous studies identified wsv178 between rr1 and rr2, coding for enzymes that catalyze the formation of deoxyribonucleotide precursors, for the DNA replication process [ 55 – 57 ]. A study conducted in outbreaks which occurred in Sonora during 2005 and between 2010–2013, indicated a higher frequency of genotypes with less than 8 RMs in WSV178. In addition, during mortality events in Tastiota and Atanasia regions in 2008, WSSV genomes were observed which showed between 1 and 4 RM in WSV178. However, the mortality of these events was low at about 1.5% [ 58 ]. In outbreaks on farms in Culiacán Sinaloa in 2000, WSSV genomes with 4, 8, 9 and 12 repetitions in WSV178 were identified. WSSV genomes with 4 repetitions detected on a particular farm in Cruz Blanca, suggested the emergence of WSSV genomes with 4 repetitions in Mexico [ 59 ]. A study analyzing possible variations in WSV178, suggested that VNTRs in the different genotypes are stable during multiple infection cycles in crustaceans [ 60 ]. Therefore, our observation of RM distribution in the Angostura and Sonora isolates in combination with the reported observations on shrimp farms in Mexico could suggest that if the frequency of WSSV genomes with varying and in particular lower number of repetitions in WSV178 reaches statistically significant frequencies in a given population, WSSV outbreaks may develop [ 58 ]. A similarity difference < 30% was observed in wsv 204 between the Sonora and Angostura isolates in comparison to CN01, with Angostura being distinctly different from Sonora and CN01. The gene wsv 204 was identified in isolates CN01 and CN03, but not in CN02 who were respectively shown to be high, low and moderate virulent in bioassays by Li, et al (2016) [ 14 ]. We compared the most variable regions at the protein level from the sequence of isolate CN01 (KT995472.1) [ 61 ], identifying that wsv129, wsv178 and wsv497 could be encoding structure- and packaging-related proteins, and appear to be involved in enhancing WSSV virulence by promoting viral replication. A difference in similarity < 30% in wsv204 was also observed between Sonora and Angostura isolates compared to CN01, with Angostura being clearly different from Sonora and CN01. wsv204 gene was identified in CN01 and CN03 isolates, but not in CN02, which respectively proved to be of high, low and moderate virulence in bioassays performed by Li, et al (2016)[ 14 ]. The function of wsv204, which contains RMs, is currently unknown, but its function could be related to transcription factors, as the RMs were similar to those found in the SOX gene member (wsv129), the EST domain transcription factor (wsv178), the tryptophan cluster factors (wsv129, wsv204 and wsv 249), the homeodomain motif (wsv 249) or the paired box factor motif (wsv497). In eukaryotic organisms, sequences similar to these are found in transcriptional regulatory sites or factors that bind to DNA and control the activity of other genes. They are involved in the developmental process, as modulators or architectural components [ 62 ], in genome accessibility, DNA shape, and interact with many transcriptional partners, in activation of transcriptional programs [ 63 ], in neighbor cell communication, in cellular secretion of abrogation [ 64 ], and in tryptophan regulation. Tryptophan has a vital role in serotonin (5-HT) synthesis that regulates homeostasis, stress and cannibalism among other vital functions in shrimp [ 65 ]. Therefore, it is not entirely clear how wsv204 might be involved in regulatory processes that have an influence on WSSV replication success in host cells. Conclusions The comparison of the virulence of WSSV isolates Angostura and Sonora in in vivo bioassays in Penaeus vannamei resulted in similar survival but differences in time of mortality Sonora being more lethal (99 HPI) than Angostura (150 HPI). Viral load indicated no correlation between HPI, so virulence may be associated with a different factor. In comparison to the Chinese CN01 isolate15 genes are deleted from the genomes of these isolates and they may contribute to changes in the WSSV viral envelope attachment function, however the deletion of proteins associated with chitin binding sites does not appear to be related directly with the increase of virulence since several genomes share this characteristic. The deletion of VP41B may be related to the apoptotic viral load control mechanism employed by WSSV. Specific repeats lost or acquired in wsv 129, wsv 178, wsv 204, wsv 249 and wsv 497 might interact and/or regulate expression of other proteins that may enhance the virulence of the Sonora isolate. Additional molecular epidemiological and transcriptional studies are needed to corroborate these findings. Altogether the study identified several interesting leads on the importance of the presence and absence of nucleocapsids, envelope proteins and proteins involved in immune evasion. Due to the complex interactions and the current state of knowledge on the function and expression of WSSV proteins detailed follow up studies are required to clarify the role of the implicated proteins. Declarations Funding ‘CONACyT-Laboratorios Nacionales Bioseguridad number 264455’ Conflicts of interest/Competing interests ‘Not applicable’ Availability of data and material ‘Yes’ Code availability ‘Yes’ Ethics approval ‘This paper covers the ethical guidelines of the journal’ Consent to participate ‘All authors agree to participate in this publication’ Consent for publication ‘All authors give their authorization to publish this draft’ Acknowledgments We want to thank Dr. Gustavo Ramírez-Paredes. and Dr. Samuel Sánchez-Serrano for contacting the researchers at the University of Stirling with whom we are enormously grateful. We also appreciate the support provided by the Campo Mosqueda shrimp company, which provided the shrimp for the bioassays. 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Aquac Int 25(2):955–968. doi: 10.1007/s10499-016-0098-6 Supplementary Files S1.png S1, Multiple genomic alignment performed with MAFFT. The alignment was done with complete WSSV genome sequences available on public databases (KR083866, MG432478, MG432474, AF440570, AF332093, AF369029, KT995470, KY827813, KT995471, JX515788, KT995472, KU216744, MG432479, MG432475, MG432477, MG432482, MG432476, MG432481, MG432480). ERR5659803 and ERR5659804 were included. Sequence numbers are indicated next to country of origin and isolate. It was performed with Geneious software version 8.1.9 and MAFFT algorithm with score matrix of 200PAM/K=2. GSP open penalty of 1.53 and offset value of 0.123 and automated sequence addressing. Scale bar indicates genetic distance. S2.tif S2, Multiple genomic alignment performed with Mauve using the reference genome CN01 and the Mexican isolates Sonora and Angostura. Homologous regions are coloured by LCB (Locally collinear block). White regions are specific to each virus genome and are not aligned. The multiple alignment shows the conservation of most genomic regions across the different virus genomes. But, one particular region between 260-280 KB coloured in red within the CN01 genome have been shorten for the Sonora isolate and rearranged at a different location around 180 Kb within the genomes of the Angostura isolate. Sequences used for the alignment: ERR5659803, ERR5659804 and KT995472 S3.tif S3, Repeated motifs in Sonora and Angostura. Genes wsv204, wsv249 and wsv497 with lower coverage and similarity from Ring blast analyses comparison of Angostura and Sonora isolates. Graphics were generated with The MEME Suite version 5.3.3. (The p-value describe the probability of each possible letter at each position in the pattern). Motif function was inferred by comparing motif to motif databases with Tomtom motif comparison Tool version 5.3.3 (Gupta et al. 2007). RM, repeated motif. Arrow indicate gain or loss of RM. Specific gene sequences were used taken translated protein ORF from sequences ERR5659803, ERR5659804 and KT995472. S4.tif S4, Matrix of identity percentages. Generated with Geneious software version 8.1.9 and MAFFT alignments presented on S1. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-824873","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":48997153,"identity":"1f8e3317-d11b-4706-9a0b-5d8bfb2b1b3a","order_by":0,"name":"Álvaro Hernán Hernández-Montiel","email":"","orcid":"","institution":"Universidad Autónoma de Baja California: Universidad Autonoma de Baja California","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Álvaro","middleName":"Hernán","lastName":"Hernández-Montiel","suffix":""},{"id":48997154,"identity":"9ebc1d24-3658-4e38-9752-401db3f35703","order_by":1,"name":"Manfred Weidmann","email":"","orcid":"","institution":"Institute of Microbiology and Virology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Manfred","middleName":"","lastName":"Weidmann","suffix":""},{"id":48997155,"identity":"b7529ab0-24ef-4c7b-8298-625819d8a3c6","order_by":2,"name":"Michael Bekaert","email":"","orcid":"","institution":"University of Stirling Institute of Aquaculture","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Bekaert","suffix":""},{"id":48997156,"identity":"dc93dcbb-b8bb-4b1d-a3e3-3fe74f0da4ab","order_by":3,"name":"Kristina Ulrich","email":"","orcid":"","institution":"University of Stirling","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kristina","middleName":"","lastName":"Ulrich","suffix":""},{"id":48997157,"identity":"f1fd6726-78ee-488a-b8d7-367ef7ff4ba5","order_by":4,"name":"Jessica Benkaroun","email":"","orcid":"","institution":"MRC-University of Glasgow Centre for Virus Research","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"","lastName":"Benkaroun","suffix":""},{"id":48997152,"identity":"ea3e359d-20f0-4ed6-ba83-0df0539bc2a4","order_by":5,"name":"Ivone Giffard-Mena","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYBACNgbGhg8VDAyM/SA2EDA2EKGlccYZoMqZDcRqASkCa9lwgFgtfPyLGxsO1NyT3Xwj+dmDHww2shsOcKdJ4HWYxEOglmPFxttupJkb9jCkGW84wLvZAL+Wg+2PP7AlJG67kWAmzcBwOBGoZeMDAlqAtvxLSNw8I/0bUMt/kBagv/Bp4W9sbDjYlpC4QSIHZMsBYmxhBGrpSzCeceZNuWGPQbLxzMME/CLff/xhw4FvCbL97enbHvyosJPtO967DW+IMUgkIPNAxjPjVQ8E/AcIqRgFo2AUjIIRDwDExVXQZeCHkAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-1688-0703","institution":"Universidad Autónoma de Baja California","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ivone","middleName":"","lastName":"Giffard-Mena","suffix":""}],"badges":[],"createdAt":"2021-08-18 13:00:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-824873/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-824873/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":12992425,"identity":"7cb89c2c-7a24-403a-80df-a197340e33ee","added_by":"auto","created_at":"2021-09-01 22:39:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1115051,"visible":true,"origin":"","legend":"In vivo WSSV infection of Penaeus vannamei. A, survival of shrimp from Ecuadorian line exposed to isolates with different virulence. Kaplan-Meier approach, using the Greenwood formula to estimate the variance, to stabilize the variance, the confidence intervals were calculated by the log (-log) transformation (3 replicates for each treatment, n = 21). B, Fitting of generalized additive models, using the local regression method, to evaluate the correlation of HPI and VL with a confidence interval of 0.95.","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-824873/v1/4b78775ca5944aa7b55796fb.png"},{"id":12992603,"identity":"14ff0aaf-1455-450d-a665-607d9a3bb400","added_by":"auto","created_at":"2021-09-01 22:42:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":976873,"visible":true,"origin":"","legend":"Complete genome analysis of available WSSV sequences. The phylogenetic tree was inferred from this alignment with 1,000 bootstrap repeats using RAxML 7.2.8 method, GTR GAMMA I nucleotide model, Bootstraping algorithm, obtaining similarity percentages that are indicated in the branches of the phylogenetic tree. Angostura and Sonora isolates are included (arrow). Sequences used for the alignment: KR083866, MG432478, MG432474, AF440570, AF332093, AF369029, KT995470, KY827813, KT995471, JX515788, KT995472, KU216744, ERR5659803, ERR5659804, MG432479, MG432475, MG432477, MG432482, MG432476, MG432481, MG432480.","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-824873/v1/c0d55a36d95dba1c48825bcf.png"},{"id":12992423,"identity":"4f43f9a5-a469-4995-9ac0-f10cf3087406","added_by":"auto","created_at":"2021-09-01 22:39:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8927141,"visible":true,"origin":"","legend":"BLAST genome comparison of WSSV genomes. A, Blast between the international isolates available to date on the NCBI data base; Taiwan (AF440570), WSSV-MX08 (KU216744), South Korea (JX515788), CN02 (KT995470), China Mj (AF332093), EG3 (KR083866), CN03 (KT995471), CN04 (KY827813), Thailand. B, Blast between Mexican isolates GVE05 (MG432478), ACF2 (MG432475), ACF4 (MG432476), LC1 (MG432481), LC10 (MG432480), DVI (MG432477), AC1 (MG432474), JP (MG432479) and LG (MG432482). The isolate from China (CN01) was used as the reference genome (A and B). In red the genes deleted in Angostura and Sonora genomes. The gradient of identity by samples with respect to the reference ranges between 100% and 50%; with blanks representing percentages less than 50% or deletion. ","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-824873/v1/6a325f8836bc9977f0333517.png"},{"id":12992426,"identity":"b45cd761-f2b8-4afd-ba60-9e3ad98d868d","added_by":"auto","created_at":"2021-09-01 22:39:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7210391,"visible":true,"origin":"","legend":"BLAST genome comparison between the Angostura and Sonora isolates against CN01. Blast analyses indicates depth of coverage of reads in yellow with a scale from 0 to 30x with respect to CN01-CDS, the blue bars indicate a coverage greater than 30x and the red bars the coverage between 0 to 10x. Genes with deletions identified in Angostura and Sonora were are noted in red. Genes with 30% difference are noted in green. BRIG version 0.95 (Alikhan, et al 2011). Sequences used for the alignment: ERR5659803, ERR5659804 and KT995472.","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-824873/v1/502da9b6510171b2d181664c.png"},{"id":18364253,"identity":"64727e11-efed-4841-b3bd-88b05d79aea7","added_by":"auto","created_at":"2022-02-18 14:16:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2903902,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-824873/v1/7f5a1a6c-d0e0-474a-bb1c-c9ccc44e1b2f.pdf"},{"id":12992422,"identity":"65e2f496-e9dd-4062-a779-16aa671ac81e","added_by":"auto","created_at":"2021-09-01 22:39:23","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":510821,"visible":true,"origin":"","legend":"S1, Multiple genomic alignment performed with MAFFT. The alignment was done with complete WSSV genome sequences available on public databases (KR083866, MG432478, MG432474, AF440570, AF332093, AF369029, KT995470, KY827813, KT995471, JX515788, KT995472, KU216744, MG432479, MG432475, MG432477, MG432482, MG432476, MG432481, MG432480). ERR5659803 and ERR5659804 were included. Sequence numbers are indicated next to country of origin and isolate. It was performed with Geneious software version 8.1.9 and MAFFT algorithm with score matrix of 200PAM/K=2. GSP open penalty of 1.53 and offset value of 0.123 and automated sequence addressing. Scale bar indicates genetic distance.","description":"","filename":"S1.png","url":"https://assets-eu.researchsquare.com/files/rs-824873/v1/05a237223551fef24529335b.png"},{"id":12992428,"identity":"d7bc0ece-ee57-4a61-896d-e71df262b9e0","added_by":"auto","created_at":"2021-09-01 22:39:24","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":23605353,"visible":true,"origin":"","legend":"S2, Multiple genomic alignment performed with Mauve using the reference genome CN01 and the Mexican isolates Sonora and Angostura. Homologous regions are coloured by LCB (Locally collinear block). White regions are specific to each virus genome and are not aligned. The multiple alignment shows the conservation of most genomic regions across the different virus genomes. But, one particular region between 260-280 KB coloured in red within the CN01 genome have been shorten for the Sonora isolate and rearranged at a different location around 180 Kb within the genomes of the Angostura isolate. Sequences used for the alignment: ERR5659803, ERR5659804 and KT995472","description":"","filename":"S2.tif","url":"https://assets-eu.researchsquare.com/files/rs-824873/v1/dc744d555b6df94bab620727.tif"},{"id":12992604,"identity":"4e809a66-d714-418d-92cd-dd046a7c58be","added_by":"auto","created_at":"2021-09-01 22:42:23","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2491148,"visible":true,"origin":"","legend":"S3, Repeated motifs in Sonora and Angostura. Genes wsv204, wsv249 and wsv497 with lower coverage and similarity from Ring blast analyses comparison of Angostura and Sonora isolates. Graphics were generated with The MEME Suite version 5.3.3. (The p-value describe the probability of each possible letter at each position in the pattern). Motif function was inferred by comparing motif to motif databases with Tomtom motif comparison Tool version 5.3.3 (Gupta et al. 2007). RM, repeated motif. Arrow indicate gain or loss of RM. Specific gene sequences were used taken translated protein ORF from sequences ERR5659803, ERR5659804 and KT995472.","description":"","filename":"S3.tif","url":"https://assets-eu.researchsquare.com/files/rs-824873/v1/5e177bfa1c4cd57486f6f4ea.tif"},{"id":12992429,"identity":"80930694-2cf1-4d2e-9ceb-19bff69a3503","added_by":"auto","created_at":"2021-09-01 22:39:24","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":14340018,"visible":true,"origin":"","legend":"S4, Matrix of identity percentages. Generated with Geneious software version 8.1.9 and MAFFT alignments presented on S1.","description":"","filename":"S4.tif","url":"https://assets-eu.researchsquare.com/files/rs-824873/v1/bcce0199f681ae25b72756bb.tif"}],"financialInterests":"","formattedTitle":"\u003cp\u003e\u003cem\u003eIn-Vivo\u003c/em\u003e Comparative Virulence of Different White Spot Syndrome Virus Isolates In \u003cem\u003ePenaeus Vannamei \u003c/em\u003eAnd Whole Genome Comparison Analysis\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe white spot syndrome virus (WSSV) is a highly contagious pathogen. With a replication cycle of approximately 20 hours at 25 ⁰C, it is capable of causing a 100% mortality rate in white shrimp (\u003cem\u003ePenaeus vannamei)\u003c/em\u003e within a period of 3 to 10 days [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], generating massive economic losses in the industry [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The first massive losses due to WSSV occurred in China and Taiwan in the years 1991\u0026ndash;1992 especially in Kuruma shrimp (\u003cem\u003eMarsopeneaus japonicus)\u003c/em\u003e, subsequently the massive mortalities due to WSSV spread throughout the rest of Asia and the world [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWSSV appeared in aquaculture farms in Mexico in 1999 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and since then, it has been persistent with regional epidemics [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Transmission of the virus can be horizontal, through cannibalism or by ingesting water containing WSSV particles, by a vertical transovarial route [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], with ballast water from ships, birds acting as vectors, and live or frozen imported products [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWSSV belong to the \u003cem\u003eNimaviridae\u003c/em\u003e family, genus Whispovirus (ICTVdb.org) and recently other Nimaviruses were identified in crabs [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] which they can also infect in aquaculture settings [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. WSSV is enveloped, has an elliptical shape with an approximate size of 250\u0026ndash;380 nm in length by 80\u0026ndash;120 nm in width [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and has an filamentous appendage. Its genome is a double-stranded circular DNA [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The largest published genome (CN01 with 309,286 kb) was isolated from China in 1994 (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eKT995472.1)\u003c/span\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. To date, \u0026gt;\u0026thinsp;20 WSSV genomes have been published (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003c/span\u003e), with the latest isolate MG432482 from Mexico [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e](257,675 kb).\u003c/p\u003e \u003cp\u003eTwo opposite hypotheses have been postulated to explain differences in genome size: 1) the reduction of the genome increases virulence [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and 2) the reduction of the genome reduces virulence [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These discrepancies cannot not be fully resolved, because of differences in experimental designs, host species and molecular techniques used. To date a direct cause-effect between genome size and virulence has not been identified [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It has been hypothesized that the pathogenicity depends on the presence of \u0026ldquo;factors\u0026rdquo; associated with virulence, which are absent or have lost function in less virulent isolates [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Deletions, mutations in open reading frames (ORFs) are believed to increase virulence rates in their hosts [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], such as ORFs 75, 94, and 125 involved in receptor recognition which potentially increases tropism. WSSV gene characterization identified some gene functions potentially involved in pathogenicity and pathogen-host interaction [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, these analysis in \u003cem\u003eM. japonicus\u003c/em\u003e did not include \u003cem\u003ein vivo\u003c/em\u003e comparison of virus strains, viral load, and genome analyses altogether.\u003c/p\u003e \u003cp\u003eWe aimed to compare experimentally (\u003cem\u003ein vivo\u003c/em\u003e) the virulence of three WSSV isolates from shrimp farms in M\u0026eacute;xico isolated during outbreaks in 2008 and 2013, and to analyze specific genomic regions that could be associated with higher virulence by comparing the isolate genomes with each other and with available sequences from public databases.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOrigin of the isolates\u003c/h2\u003e \u003cp\u003eThe bioassay was carried with three isolates from different geographical origins and epidemics. One isolate was obtained by Dr. Jorge de la Rosa V\u0026eacute;lez\u003csup\u003e\u0026dagger;\u003c/sup\u003e during an outbreak that occurred in Sonora in 2008, called the Sonora isolate. The second isolate was provided by Dr. Leobardo Montoya from the Food and Development Research Center (CIAD) located in Mazatl\u0026aacute;n Sinaloa, from an outbreak in Sonora in 2013 designated CIAD. The third isolate was provided by MC Joel Liz\u0026aacute;rraga Valdez from the company Aquapacific S.A. de C.V. obtained from an outbreak in the Angostura, Sinaloa area in 2013.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of WSSV viral inocula\u003c/h2\u003e \u003cp\u003eInocula of each isolate were prepared from 25 mg of tissue of four cryopreserved dead shrimps, placed in a 1.5 mL Eppendorf tube, supplemented with 500 \u0026micro;L of TN buffer (Tris/HCl 10mM. pH 7.4, NaCl 3M, passed through 0.2 \u0026micro;m microfilter) and homogenized with a pistil, followed by another 400 \u0026micro;L of TN buffer [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The samples were centrifuged at 1000 ɡ for 5 min, 3000 ɡ for 5 min, 10,000 ɡ for 5 min and 14,000 ɡ for 15 min. The supernatant was recovered with a 3 mL syringe and filtered again with a 0.45 \u0026micro;m microfilter. The filtrate was placed in a new 1.5 mL eppendorf tube and was kept at -4˚C until use, for less than 2 hours. This procedure was repeated every time shrimp were infected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eVerification of the viability of isolates\u003c/h2\u003e \u003cp\u003eWhite shrimps from the ACUAMOS aquaculture farm (located in San Felipe, Mexicali B.C) were infected with the viral inoculum. Juveniles were maintained at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;1⁰C, a salinity of 34 ppt, and constant aeration until reaching 15.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g and 14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 cm. For each isolation (n\u0026thinsp;=\u0026thinsp;5), two replicates were used). 20 \u0026micro;L of inoculum was injected into the fifth abdominal segment of the shrimp. Moribund shrimps were cryopreserved in liquid nitrogen and stored at -80\u0026deg;C. The virus replication was verified by end-point PCR, with a set of primer targeting the structural protein VP664 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eChallenge experiment to compare the isolates virulence\u003c/h2\u003e \u003cp\u003eWhite shrimps from the Mahar aquaculture farm (Pichilingue, La Paz, Baja California Sur, Mexico) weighing 4.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 g and 8.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 g, were infected with the different viral inocula. A PCR test was done to corroborate animals were wssv-free [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. For each isolate challenges of shrimps (n\u0026thinsp;=\u0026thinsp;7) were performed in triplicate and placed in 40 L tanks, with a temperature of 28\u0026thinsp;\u0026plusmn;\u0026thinsp;1⁰C, a salinity of 34 ppt, constant aeration, and a biological filter (activated carbon). Three other replicas were used for the non-infected shrimps.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction to quantify viral load\u003c/h2\u003e \u003cp\u003eOnce bioassays were completed, DNA was extracted from all samples (N\u0026thinsp;=\u0026thinsp;63).100 mg of tissue was taken from the 5th abdominal segment and DNA was extracted using the Phenol: Chloroform: Isoamyl alcohol [25:24:1] standard protocol [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]⁠. DNA concentration (260 nm) and quality (radius 260/280) was verified with a Nanodrop LITE (Thermo brand). Additionally, the DNA quality was assessed in a 1% agarose gel electrophoresis in 40 mL of 1X TAE (0.04M Tris-acetate, 1mM EDTA) and visualized by ethidium bromide [10 mg/mL].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eViral load quantification\u003c/h2\u003e \u003cp\u003eViral load was determined by real-time PCR (Q-PCR) absolute quantification with standard curve (1:10 dilution factor, with dilutions 10\u003csup\u003e0\u003c/sup\u003e to 10\u003csup\u003e7\u003c/sup\u003e) according to Durand and Lightner, 2002 (OIE procedure). The 64 bp fragment of \u003cem\u003evp\u003c/em\u003e664 was inserted on TOPO TA plasmid (Invitrogen, United States) as previously described [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction and purification for sequencing of the WSSV genome\u003c/h2\u003e \u003cp\u003eFor deep sequencing of the whole genome of the viruses from the bioassay, the DNA of 4 shrimps tested positive for WSSV by Q-PCR and of two free-wssv were extracted and purified. The extraction was done with the 5 Prime Archive Pure DNA blood kit (# 2900258 5prime GmbH, Germany) with Econo Spin columns (Epoch # 1910\u0026thinsp;\u0026minus;\u0026thinsp;250, Epoch Life Science Inc, United States). The column was washed with a Type 1 wash buffer (GE28-9031-70, GElifesciences, United Kingdom). DNA was eluted with 50 \u0026micro;L of water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of samples for deep sequencing\u003c/h2\u003e \u003cp\u003eSamples were stabilized with DNAstable Plus\u0026reg; (Biomatrica, UK) following the manufacturer's instructions and were sent for sequencing by regular mail service at the Institute of Aquaculture (University of Stirling in Scotland, UK) with an Illumina MiSeq, V3 75 bp, Paired-End.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDeep sequencing data cleaning and mapping\u003c/h2\u003e \u003cp\u003eThe quality of the sequencing raw reads was checked with FastQC [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Quality scores below 30 along with Illumina adapters were removed with Trimmomatic v.0.39 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The cleaned reads were mapped against the reference genome China CN GCF_000848075.2 (KT995472.1), using the BWA aligner v.0.7.15 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and SAMtools/BCFtools v1.11 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.htslib.org\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe survival probability was evaluated by the Kaplan-Meier approach, using the Greenwood formula to estimate the variance, to stabilize the variance, the confidence intervals were calculated by the log (-log) transformation. The comparison between the curves was made with the long-rank test, using the survival package version 3.2-7 for survival analysis. An adjustment of generalized additive models was also carried out, by means of the local regression method, to evaluate the correlation of hours post inoculation (HPI) and viral load (VL) with a confidence interval of 0.95, using the Gam package in R studio version 1.3.959.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGenome alignment and phylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe alignment of the isolated genomes from Angostura and Sonora was performed with MAFFT v7,017 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] with score matrix of 200PAM/K\u0026thinsp;=\u0026thinsp;2. GSP open penalty of 1.53 and offset value of 0.123 and automated sequence addressing, together with the sequences KR083866, MG432478, MG432474, AF440570, AF332093, AF369029, KT995470, KY827813, KT995471, JX515788, KT995472, KU216744, MG432479, MG432475, MG432477, MG432482, MG432476, MG432481 and MG432480, extracted from GENBANK and using the genome GCF_000848075.2 corresponding to the sequence CN01_KT995472 isolated from China as a reference since it is the longest WSSV genome Sequence. Sonora and Angostura isolates were also included: ERR5659803, ERR5659804 respectively). The phylogenetic tree was inferred from this alignment with 1,000 bootstrap repeats using RAxML 7.2.8 method, GTR GAMMA nucleotide model, Bootstraping algorithm, genetic distance in annotated as a scale bar.\u003c/p\u003e \u003cp\u003eA multiple genome comparison with circular architecture was made with BLAST with the BLAST Ring Image Generator (BRIG) version 0.95 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], comparing the all the above listed genomes, using the CN01 isolate as a reference. A multiple genomic alignment to observe genomic conservation and rearrangements was performed with MAUVE version 2.4.0. Repeated motifs (RM) in specific genes were detected with The MEME Suite 5.3.3 (Multiple EM for Motif Elicitation) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] on line version \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/tools/meme\u003c/span\u003e\u003c/span\u003e. A motif is an approximate sequence pattern that occurs repeatedly in a group of related sequences that usually mediate a common function. Motif function was inferred by comparing motif to motifs databases with Tomtom motif comparison Tool version 5.3.3 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] available in the same link.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003eChallenge experiment to compare the virulence of WSSV isolates\u003c/h2\u003e\n \u003cp\u003eShrimp infection with the CIAD isolate produced a survival rate of 19% 10 days after challenge, with a first drop in survival at 29 h 40 min post infection (PI) and a final drop at 145 h 50 min PI. On the other hand, the infections carried out with the Angostura and Sonora isolates produced a mortality rate of 100% at 168h 40min PI, with a first drop in survival at 35 HPI and 27 HPI respectively (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Comparison of the long-range analysis for the tree isolates (Chisq 0.4, 2 degrees of freedom or d, p 0.81) and the Kaplan-Meier approach indicates a 50% survival for the CIAD isolate at 131 HPI, Angostura at 150 HPI and Sonora at 99 HPI (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec16\"\u003e\n \u003ch2\u003eViral load quantification of the three treatments\u003c/h2\u003e\n \u003cp\u003eViral loads or viral genomes / mg (VL) were compared between treatments by vp664 qPCR on all infected shrimp (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). VL ranged from 8.48 x 10^7 (CIAD), 1.26 x 10^8 (Sonora) to 1.91 x 10^8 (Angostura). The variance analysis indicates no correlation between HPI and VL for all isolates (F 0.002459, p 0.001, R\u0026sup2; \u0026lt; 0.29).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec17\"\u003e\n \u003ch2\u003eComparison of WSSV genomes and identification of genes with variations\u003c/h2\u003e\n \u003cp\u003eDue to concentration and sample quality problems, it was not possible to sequence the CIAD isolate. However, we obtained a total of 1,717,195 and 1,657,796 reads with a length of 76 bp that were assembled to 155,993 and 105,993 reads for the Angostura and Sonora genomes respectively (KT995472.1). The genome size of the Angostura and Sonora isolates is 289,350 bp and 288,995 bp, respectively (S1), with an overall GC percentage of 41.1%. The Sonora isolate has a slightly shorter genome (355 bp) than the Angostura isolate. The data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI with the accession number PRJEB44096 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/ena/browser/view/PRJEB44096\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eNucleotide alignment with identity % among all genomes ranges from 78.3 % (LG vs GVE05 isolates) to 99.2 % (ACF vs DVI isolates). There is about 99.7% similarity between the Sonora and Angostura isolates (S1, S4). Phylogenetic analysis indicates that Sonora and Angostura isolates are closely related to the Mex2008 isolate and have a 96.4% similarity to isolate CN02 from China. Another clade groups the isolate from Taiwan, AC1 and GVE. The most recent branch contains isolates JP, ACF2, DVI, LG, ACF4, LC1 and LC10.\u003c/p\u003e\n \u003cp\u003eThe circular alignment of all genomes with BRIG clearly identified 3 deletion areas with specific genes (shown in red) when compared the CN01 isolate (Figs. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, B) in both Angostura and Sonora genomes (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The genes \u003cem\u003ewsv\u003c/em\u003e463a, \u003cem\u003ewsv\u003c/em\u003e463b, \u003cem\u003ewsv\u003c/em\u003e463c, \u003cem\u003ewsv\u003c/em\u003e463d are absent in both genomes when compared to CN01 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and B) and genes \u003cem\u003ewsv\u003c/em\u003e489, \u003cem\u003ewsv\u003c/em\u003e490, \u003cem\u003ewsv\u003c/em\u003e492, \u003cem\u003ewsv\u003c/em\u003e493 and \u003cem\u003ewsv\u003c/em\u003e495 had a\u0026thinsp;\u0026lt;\u0026thinsp;50% similarity among isolates. The only exception is the Taiwan isolate which has the complete sequences for the mentioned genes, followed by the isolates from China (Mj) and Eg3 containing a deletion of \u003cem\u003ewsv\u003c/em\u003e495 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The genes that code for proteins VP41B, VP52A and VP41A presented deletions or a similarity of less than 50% with respect to CN01, in all the Mexican genomes, with the exception of Mex2008 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Particularly these genes coding are absent in the genome of isolate CN03, while \u003cem\u003ewsv\u003c/em\u003e234 has a low similarity (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). On the other hand, CN04 presented only the deletions of VP52A, VP41A and \u003cem\u003ewsv\u003c/em\u003e234.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDeletions identified in the isolates from Angostura and Sonora when compared to CN01 (KT995472). \u003cem\u003ewsv\u003c/em\u003e nomenclature corresponds to isolate from China_MJ (or CN) (AF332093) used in this work for annotations, and \u003cem\u003ewssv\u003c/em\u003e corresponds to equivalent genes in Taiwan_Pm (AF440570). * Protein name not defined. In parentheses is the name according to Taiwan based studies.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eWSSV gene\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtein name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSub-category\u003c/p\u003e\n \u003cp\u003efunction\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewsv\u003c/em\u003e234\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHypothetical Protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewsv\u003c/em\u003e237\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewssv\u003c/em\u003e293\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVP41A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eEnvelope protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuang \u003cem\u003eet al.\u003c/em\u003e (2013)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewsv\u003c/em\u003e238\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewssv\u003c/em\u003e294\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVP52A (VP51A)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChang \u003cem\u003eet al\u003c/em\u003e. (2008)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewsv\u003c/em\u003e242\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewssv\u003c/em\u003e298\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVP41B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZuo et al. (2011)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewsv\u003c/em\u003e244\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"9\"\u003e\n \u003cp\u003eHypothetical Protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewsv\u003c/em\u003e247\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewsv\u003c/em\u003e463 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewsv\u003c/em\u003e463 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewsv\u003c/em\u003e463 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewsv\u003c/em\u003e463 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewsv\u003c/em\u003e489\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewsv\u003c/em\u003e490\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewsv\u003c/em\u003e492\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewsv\u003c/em\u003e493\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewssv\u003c/em\u003e019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eICP35 (VP35)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNonstructural Protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKang \u003cem\u003eet al\u003c/em\u003e. (2013)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ewsv\u003c/em\u003e495\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHypothetical Protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eAn additional multiple MAUVE alignment between Angostura and Sonora genomes in reference to isolate CN01 corroborates the deletion of \u003cem\u003ewsv\u003c/em\u003e247, \u003cem\u003ewsv\u003c/em\u003e244, VP41B, VP52A, VP41A and \u003cem\u003ewsv\u003c/em\u003e234 in both Mexican isolates in reference to the genome of isolate CN01 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e; S2, orange region in CN01 at 140kb). This analysis showed the deletions of \u003cem\u003ewsv\u003c/em\u003e463a, b, c and d in the 180 kb region of the Angostura genome (S2, red region in Angostura at 180 kb), and deletions corresponding to \u003cem\u003ewsv\u003c/em\u003e489, \u003cem\u003ewsv\u003c/em\u003e490, \u003cem\u003ewsv\u003c/em\u003e492, \u003cem\u003ewsv\u003c/em\u003e493 and \u003cem\u003ewsv\u003c/em\u003e495 in a terminal region close to the 3-prime end of the genome (S2, yellow region in Angostura at 260 kb).\u003c/p\u003e\n \u003cp\u003eA difference of \u0026lt;\u0026thinsp;30% at nucleotide level was observed in wsv129, wsv178, \u003cem\u003ewsv\u003c/em\u003e204, \u003cem\u003ewsv\u003c/em\u003e249 and \u003cem\u003ewsv\u003c/em\u003e497 genes between the genomes of the Sonora and Angostura isolates (S3). The Sonora isolate \u003cem\u003ewsv\u003c/em\u003e129 has lost several Repeated Motifs (RM) like RM1 and RM2 but gained RM3 (S3, A), \u003cem\u003ewsv\u003c/em\u003e178 has lost five RM1 (S3, B), \u003cem\u003ewsv\u003c/em\u003e204 gene has acquired one RM2 (S3, C), while \u003cem\u003ewsv\u003c/em\u003e249 has lost one RM1 in the Sonora isolate (S3, D, while \u003cem\u003ewsv\u003c/em\u003e497 is similar in both isolates, with 1 more RM2 compared to CN01, (S3, E). RM is a repeated motif, and the number makes reference to the motif number for each case.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThree WSSV isolates from Mexican shrimp farms were characterized by bioassay and through whole genome sequencing. WSSV isolates tested in bioassays generally induce the following clinical signs in infected white shrimp; lethargy, decreased food intake, discoloration of the hepatopancreas, cuticular loosening and red uropods [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Apart from these general clinical signs Shrimp infected with the CIAD isolate in the challenge bioassay showed 19% survival at 10 days post infection similar to the percentage obtained by Sekar, with 100% mortality until day 20 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In contrast the, isolates from Angostura and Sonora induced 100% mortality between 3- and 10-days post-infection [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Angostura isolate produced the highest viral load of the three isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The differences between isolates however were in the range of published data reporting the viral load of WSSV in three Penaeus species (\u003cem\u003eP. vannamei\u003c/em\u003e, \u003cem\u003eP. stylirostris\u003c/em\u003e and \u003cem\u003eP. monodon\u003c/em\u003e) obtained by real-time PCR in the range of 2 x 10\u003csup\u003e4\u003c/sup\u003e \u0026ndash; 2 x 10\u003csup\u003e9\u003c/sup\u003e WSSV copies \u0026micro;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of total DNA, in infected shrimp [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A review to evaluate the risk associated for the shrimp trade, data on WSSV concentrations obtained from tissues of different species, including \u003cem\u003eP. vannamei\u003c/em\u003e, indicated high viral load discharges at the beginning of mortality, with concentrations of 1 x 10\u003csup\u003e9\u003c/sup\u003e \u0026ndash; 1 x 10\u003csup\u003e10\u003c/sup\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe CIAD isolate presented a mortality risk percentage between 95% and 19% with a probability of 50% at 131 h and an overall survival rate of 19%. Isolate Angostura presented a constant 65% mortality risk percentage between 50 and 150 h, for a 50% survival at 150 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). For isolate Sonora, three drops in survival were observed, with risks of death between 64% and 43% from 50 to 150 h, and a probability of survival of 50% at 99 h. This is a possible sign of a faster progressing replication and therefore increased virulence of the isolate Sonora compared to isolate Angostura. Both isolates Angostura and Sonora induced 100% mortality observed at 168 HPI. Therefore, we tentatively classified the isolates as low virulence (CIAD), moderate virulence (Angostura) and high virulence (Sonora) from the mean post-infection lethal time according to [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe genomes of the isolates from Angostura and Sonora, presented a length within the estimated range for Mexican isolates from localities between Sinaloa and Nayarit (257,675\u0026thinsp;\u0026minus;\u0026thinsp;290,879 bp) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. To assess genomic differences of the isolates we were able to identify specific sites with differences to isolates from foreign and domestic outbreaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Deletions in the WSSV genome are considered as enhancements of the adaptive response by WSSV as it is reasoned that deleted regions, do not encode proteins vital for WSSV pathogenicity or virulence [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The differences are possibly caused by recombination events mediated by transposons causing insertions or deletions [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. An inversion is apparent among the 2008 Sonora and 2013 Angostura isolate as spotted in the MAUVE alignment of motives (Fig S2).\u003c/p\u003e \u003cp\u003eSince the isolate with the shortest sequence was the most virulent (Sonora), we analyzed the deletions. In our comparative analysis between the genome of the isolates of the present study, with sequences of international and national isolates, a group of deleted genes stands out: \u003cem\u003ewsv\u003c/em\u003e237(VP41A) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], \u003cem\u003ewsv\u003c/em\u003e238(VP52A-VP51A) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. \u003cem\u003ewsv\u003c/em\u003e242(VP41B) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and \u003cem\u003ewsv\u003c/em\u003e493(ICP35-VP35) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe VP41A envelope protein is known to interact with VP26, VP56 and VP28 envelope proteins [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and with the viral envelope protein VP51A (VP52A for AF332093) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. It has been suggested that VP26 is anchored to the envelope with its N-terminal hydrophobic region, as a C-terminal region binds to the nucleocapsid [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], while VP51A is a type II transmembrane protein, with a transmembrane domain highly hydrophobic at its N-terminus and an exposed C-terminus on the virion surface [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. On the other hand, VP41A has only one transmembrane region at its C-terminal end, and this envelope protein is part of a group of 11 proteins that interacts with the chitin-binding protein of \u003cem\u003eP. monodon\u003c/em\u003e (PmCBP) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] which are known to participate in shrimp immune response [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. It also interacts with preitrophin-like protein in \u003cem\u003eL. vannamei\u003c/em\u003e (LvPT). LvPT is involved in the formation of the peritrophic membrane [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and contains a chitin-binding domain facilitating binding of WSSV to the intestinal epithelium. It has been shown to promote infection, as silencing of LvPT decreased the viral load in pleopods, stomach and intestines in previously WSSV infected shrimp [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, the absence of the envelope proteins VP41A and VP51A could modify the usual conformation of the protein complex composed of VP26, VP28 and VP56, as well as the interactions of VP26 with VP51A reducing viral recognition by chitin-binding proteins such as PmCBP and LvPT. The observed deletions in Sonora and Angostura isolates could contribute to changes in the WSSV viral envelope and enhance virulence causing high mortality.\u003c/p\u003e \u003cp\u003eVP38 acts as a repressor of \u003cem\u003ePenaeus japonicus\u003c/em\u003e caspase (Pjcaspase) transcription in \u003cem\u003eM. japonicus\u003c/em\u003e, while VP41B activates its expression, regulating the apoptosis mechanism used by shrimp to control viral load [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Elevated Pjcaspasa gene expression of \u003cem\u003eM. japonicus\u003c/em\u003e was observed in shrimp survival challenges while siRNA silencing of Pjcaspasa increased post-infection viral load [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. So, deletion of VP41B would not allow timely induction of apoptosis of infected cells allowing the virus to replicate without limitation and thus having a major impact on the viral load control mechanism employed by WSSV.\u003c/p\u003e \u003cp\u003eThe Sonora isolate has noticeable modifications on \u003cem\u003ewsv\u003c/em\u003e129, \u003cem\u003ewsv\u003c/em\u003e178, \u003cem\u003ewsv\u003c/em\u003e204 and \u003cem\u003ewsv\u003c/em\u003e249 as gain or loss of specific sequences equivalent to Repeated Motifs (RM) that strongly suggest a role in the infection process. A high variation from 3 to 20 in the number of repetitive sequences in WSV178 has been related to virulence [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In contrast a lower mean number of repeated sequences was observed in outbreaks, where WSSV genotypes with 5, 6 and 7 of a 54 bp RM were those that predominated in semi-intensive shrimp crops [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In our study, we identified 4 and 3 repetitions of a 54 bp RM in WSV178 in the Angostura and Sonora genomes respectively. Previous studies identified wsv178 between rr1 and rr2, coding for enzymes that catalyze the formation of deoxyribonucleotide precursors, for the DNA replication process [\u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. A study conducted in outbreaks which occurred in Sonora during 2005 and between 2010\u0026ndash;2013, indicated a higher frequency of genotypes with less than 8 RMs in WSV178. In addition, during mortality events in Tastiota and Atanasia regions in 2008, WSSV genomes were observed which showed between 1 and 4 RM in WSV178. However, the mortality of these events was low at about 1.5% [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. In outbreaks on farms in Culiac\u0026aacute;n Sinaloa in 2000, WSSV genomes with 4, 8, 9 and 12 repetitions in WSV178 were identified. WSSV genomes with 4 repetitions detected on a particular farm in Cruz Blanca, suggested the emergence of WSSV genomes with 4 repetitions in Mexico [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. A study analyzing possible variations in WSV178, suggested that VNTRs in the different genotypes are stable during multiple infection cycles in crustaceans [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Therefore, our observation of RM distribution in the Angostura and Sonora isolates in combination with the reported observations on shrimp farms in Mexico could suggest that if the frequency of WSSV genomes with varying and in particular lower number of repetitions in WSV178 reaches statistically significant frequencies in a given population, WSSV outbreaks may develop [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA similarity difference\u0026thinsp;\u0026lt;\u0026thinsp;30% was observed in \u003cem\u003ewsv\u003c/em\u003e204 between the Sonora and Angostura isolates in comparison to CN01, with Angostura being distinctly different from Sonora and CN01. The gene \u003cem\u003ewsv\u003c/em\u003e204 was identified in isolates CN01 and CN03, but not in CN02 who were respectively shown to be high, low and moderate virulent in bioassays by Li, et al (2016) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe compared the most variable regions at the protein level from the sequence of isolate CN01 (KT995472.1) [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], identifying that wsv129, wsv178 and wsv497 could be encoding structure- and packaging-related proteins, and appear to be involved in enhancing WSSV virulence by promoting viral replication.\u003c/p\u003e \u003cp\u003eA difference in similarity\u0026thinsp;\u0026lt;\u0026thinsp;30% in wsv204 was also observed between Sonora and Angostura isolates compared to CN01, with Angostura being clearly different from Sonora and CN01. wsv204 gene was identified in CN01 and CN03 isolates, but not in CN02, which respectively proved to be of high, low and moderate virulence in bioassays performed by Li, et al (2016)[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The function of wsv204, which contains RMs, is currently unknown, but its function could be related to transcription factors, as the RMs were similar to those found in the SOX gene member (wsv129), the EST domain transcription factor (wsv178), the tryptophan cluster factors (wsv129, wsv204 and wsv 249), the homeodomain motif (wsv 249) or the paired box factor motif (wsv497).\u003c/p\u003e \u003cp\u003eIn eukaryotic organisms, sequences similar to these are found in transcriptional regulatory sites or factors that bind to DNA and control the activity of other genes. They are involved in the developmental process, as modulators or architectural components [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], in genome accessibility, DNA shape, and interact with many transcriptional partners, in activation of transcriptional programs [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], in neighbor cell communication, in cellular secretion of abrogation [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], and in tryptophan regulation. Tryptophan has a vital role in serotonin (5-HT) synthesis that regulates homeostasis, stress and cannibalism among other vital functions in shrimp [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Therefore, it is not entirely clear how wsv204 might be involved in regulatory processes that have an influence on WSSV replication success in host cells.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe comparison of the virulence of WSSV isolates Angostura and Sonora in \u003cem\u003ein vivo\u003c/em\u003e bioassays in \u003cem\u003ePenaeus vannamei\u003c/em\u003e resulted in similar survival but differences in time of mortality Sonora being more lethal (99 HPI) than Angostura (150 HPI). Viral load indicated no correlation between HPI, so virulence may be associated with a different factor. In comparison to the Chinese CN01 isolate15 genes are deleted from the genomes of these isolates and they may contribute to changes in the WSSV viral envelope attachment function, however the deletion of proteins associated with chitin binding sites does not appear to be related directly with the increase of virulence since several genomes share this characteristic. The deletion of VP41B may be related to the apoptotic viral load control mechanism employed by WSSV. Specific repeats lost or acquired in \u003cem\u003ewsv\u003c/em\u003e129, \u003cem\u003ewsv\u003c/em\u003e178, \u003cem\u003ewsv\u003c/em\u003e204, \u003cem\u003ewsv\u003c/em\u003e249 and \u003cem\u003ewsv\u003c/em\u003e497 might interact and/or regulate expression of other proteins that may enhance the virulence of the Sonora isolate. Additional molecular epidemiological and transcriptional studies are needed to corroborate these findings. Altogether the study identified several interesting leads on the importance of the presence and absence of nucleocapsids, envelope proteins and proteins involved in immune evasion. Due to the complex interactions and the current state of knowledge on the function and expression of WSSV proteins detailed follow up studies are required to clarify the role of the implicated proteins.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding \u0026lsquo;CONACyT-Laboratorios Nacionales Bioseguridad number 264455\u0026rsquo;\u003c/p\u003e\n\u003cp\u003eConflicts of interest/Competing interests \u0026lsquo;Not applicable\u0026rsquo;\u003c/p\u003e\n\u003cp\u003eAvailability of data and material \u0026lsquo;Yes\u0026rsquo;\u003c/p\u003e\n\u003cp\u003eCode availability \u0026lsquo;Yes\u0026rsquo;\u003c/p\u003e\n\u003cp\u003eEthics approval \u0026lsquo;This paper covers the ethical guidelines of the journal\u0026rsquo;\u003c/p\u003e\n\u003cp\u003eConsent to participate \u0026lsquo;All authors agree to participate in this publication\u0026rsquo;\u003c/p\u003e\n\u003cp\u003eConsent for publication \u0026lsquo;All authors give their authorization to publish this draft\u0026rsquo;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe want to thank Dr. Gustavo Ram\u0026iacute;rez-Paredes. and Dr. Samuel S\u0026aacute;nchez-Serrano for contacting the researchers at the University of Stirling with whom we are enormously grateful. We also appreciate the support provided by the Campo Mosqueda shrimp company, which provided the shrimp for the bioassays. This project was financed by the project CONACyT-Laboratorios Nacionales Bioseguridad number 264455 granted to IGM.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDV. 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Aquac Int 25(2):955\u0026ndash;968. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10499-016-0098-6\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"WSSV Genomics, virulence, viral isolates, envelope proteins, deep sequencing","lastPublishedDoi":"10.21203/rs.3.rs-824873/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-824873/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWhite Spot Syndrome Virus (WSSV) infects several economically important aquacultural species, causing significant losses to the industry. This virus belongs to the \u003cem\u003eNimaviridae\u003c/em\u003e family, and has a dsDNA genome ranging from 257 to 309 kb (more than 20 isolate genomes fully sequenced and published to date). Multiple routes of infection could be the cause of the high virulence and mortality rates detected in shrimp species. In particular, \u003cem\u003ePenaeus vanname\u003c/em\u003ei, differences in isolate virulence have been observed, along with controversy over whether deletions or insertions are associated with gain or loss of virulence.\u003c/p\u003e\u003cp\u003eThe pathogenicity of three isolates from three locations in Mexico (two from Sinaloa: 'CIAD', and 'Angostura', and one from Sonora: 'Sonora') was evaluated \u003cem\u003ein vivo\u003c/em\u003e in white shrimp (\u003cem\u003eP. vannamei\u003c/em\u003e) infection assays. Differences were observed in the mortality rate of shrimp among the three isolates, with the Sonora isolate being the most virulent. Subsequently, the complete WSSV genomes were sequenced in depth from the tissues of infected shrimp, and assembled in reference to the genome of isolate CN01 (KT995472), identifying genome sizes for Angostura and Sonora of 289,350 bp and 288,995 bp, respectively.\t\u0026nbsp;\u003c/p\u003e\u003cp\u003eWhere three deletion zones were identified compared to CN01 comprising 15 genes, including three envelope proteins VP41A, VP52A and VP41B, one non-structural protein ICP35 and 11 others encoding proteins whose function is currently unknown. In addition, five genes (wsv129, wsv178, wsv204, wsv249 and wsv497) show a modified number of repeat motifs. The main implications and possible effects on viral infection of these modifications are discussed.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"In-Vivo Comparative Virulence of Different White Spot Syndrome Virus Isolates In Penaeus Vannamei And Whole Genome Comparison Analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-09-01 22:39:21","doi":"10.21203/rs.3.rs-824873/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b9425604-28d1-4fa5-b9fb-b05138373b6e","owner":[],"postedDate":"September 1st, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":6889899,"name":"Virology"},{"id":6889900,"name":"Internal Medicine"}],"tags":[],"updatedAt":"2022-02-18T14:16:24+00:00","versionOfRecord":[],"versionCreatedAt":"2021-09-01 22:39:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-824873","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-824873","identity":"rs-824873","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}