Use of brome mosaic virus-like particles in feed, to deliver dsRNA targeting the white spot syndrome virus vp28 gene, reduces Penaeus vannamei mortality.

Numerous strategies have been investigated to combat viral infections in shrimp, specifically targeting the white spot syndrome virus (WSSV) that has caused outbreaks worldwide since the 1990s. One effective treatment involves intramuscular application of dsRNA-mediated interference against the viral capsid protein VP28. However, this approach presents challenges in terms of individual shrimp management, limiting its application on a large scale. To address this, our study aimed to evaluate the efficacy of oral delivery of protected dsRNA using chitosan nanoparticles or virus-like particles (VLPs) synthesized in brome mosaic virus (BMV). These delivery systems were administered before, during, and after WSSV infection to assess their therapeutic potential. Our findings indicate that BMV-derived VLPs demonstrated superior efficiency as nanocontainers for dsRNA delivery. Notably, the treatment involving vp28 dsRNA mixed in the feed and administered simultaneously to shrimp already infected with WSSV exhibited the highest survival rate (48%), while the infected group had a survival rate of zero, suggesting the potential efficacy of this prophylactic approach in commercial shrimp farms.

Virulence and genetic differences among white spot syndrome virus isolates inoculated in Penaeus vannamei.

White spot syndrome virus (WSSV) infects several economically important aquaculture species, and has caused significant losses to the industry. This virus belongs to the Nimaviridae family and has a dsDNA genome ranging between 257 and 309 kb (more than 20 isolate genomes have been 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. Particularly in Penaeus vannamei, differences in isolate virulence have been observed, along with controversy over whether deletions or insertions are associated with virulence gain or loss. The pathogenicity of 3 isolates from 3 localities in Mexico (2 from Sinaloa: 'CIAD' and 'Angostura'; and one from Sonora: 'Sonora') was evaluated in vivo in whiteleg shrimp P. vannamei infection assays. Differences were observed in shrimp mortality rates among the 3 isolates, of which Sonora was the most virulent. Subsequently, the complete genomes of the Sonora and Angostura isolates were sequenced in depth from infected shrimp tissues and assembled in reference to the genome of isolate strain CN01 (KT995472), comprising 289350 and 288995 bp, respectively. Three deletion zones were identified compared to CN01, comprising 15 genes, including 3 envelope proteins (VP41A, VP52A and VP41B), 1 non-structural protein (ICP35) and 11 other encoding proteins whose function is currently unknown. In addition, 5 genes (wsv129, wsv178, wsv204, wsv249 and wsv497) presented differences in their repetitive motifs, which could potentially be involved in the regulation of gene expression, causing virulence variations.

Characterization of putative proteins encoded by variable ORFs in white spot syndrome virus genome.

BACKGROUND: White Spot Syndrome Virus (WSSV) is an enveloped double-stranded DNA virus which causes mortality of several species of shrimp, being considered one of the main pathogens that affects global shrimp farming. This virus presents a complex genome of ~ 300 kb and viral isolates that present genomes with great identity. Despite this conservation, some variable regions in the WSSV genome occur in coding regions, and these putative proteins may have some relationship with viral adaptation and virulence mechanisms. Until now, the functions of these proteins were little studied. In this work, sequences and putative proteins encoded by WSSV variable regions were characterized in silico. RESULTS: The in silico approach enabled determining the variability of some sequences, as well as the identification of some domains resembling the Formin homology 2, RNA recognition motif, Xeroderma pigmentosum group D repair helicase, Hemagglutinin and Ankyrin motif. The information obtained from the sequences and the analysis of secondary and tertiary structure models allow to infer that some of these proteins possibly have functions related to protein modulation/degradation, intracellular transport, recombination and endosome fusion events. CONCLUSIONS: The bioinformatics approaches were efficient in generating three-dimensional models and to identify domains, thereby enabling to propose possible functions for the putative polypeptides produced by the ORFs wsv129, wsv178, wsv249, wsv463a, wsv477, wsv479, wsv492, and wsv497.

Alternative PCR primers for genotyping of Brazilian WSSV isolates.

White spot syndrome virus (WSSV) is one of the major challenges faced by global shrimp farming in recent decades. The characterization of WSSV genetic variability has been used to determine viral dispersion and is a promising method to determine the association between genotype and virulence. The major variable regions that have been used as markers to differentiate the WSSV genomes include the VNTR loci inside ORF94, ORF75, ORF125, and insertions/deletions interspersing ORF14/15 and ORF23/24. The primers used to amplify these regions were described at least 10 years ago, but some of them do not work efficiently to identify new WSSV variants. The objective of this work was to develop improved PCR primers for WSSV genotyping based on sequence alignments that include new sequences described in recent years. We validated these new primers in a pilot study to verify the genetic variability of the WSSV in Rio Grande do Norte state (northeast Brazil), and efficiency was compared to that of other previously described primers. We confirmed that the primers we developed were more efficient for genotype Brazilian WSSV isolates, enabling us to genotype a larger number of samples. In addition, our results also introduce new data about the genetic characterization of the WSSV isolates that occur in the northeastern region of Brazil.

Molecular variability and genetic structure of white spot syndrome virus strains from northwest Mexico based on the analysis of genomes.

White spot syndrome virus (WSSV) has a ∼300 kb double-stranded DNA genome. It originated in China, spread rapidly through shrimp farms in Asia, and subsequently to America. This study determined complete genome sequences for nine historic WSSV strains isolated from Pacific white shrimp (Litopenaeus vannamei) captured in farm ponds in northwest Mexico (Sinaloa and Nayarit). Genomic DNA was captured by an amplification method using overlapping long-range PCR and sequencing by Ion Torrent-PGM. Complete genome sequences were assembled (length range 255-290 kb) and comparative genome analysis with WSSV strains revealed substantial deletions (3 and 10 kb in two regions) in seven strains, with two strains differing from the rest. Phylogenetic analysis identified that the WSSV strains from the northern area of the state of Sinaloa clustered with strains from China (LC1, LC10, DVI) and Korea (ACF2, ACF4), while those from the southern region of the state of Nayarit (AC1 and JP) differed from both of those and from strains found in Taiwan and Thailand. Our data offer insights into the diversity of the WSSV genome in one country and their divergent origin, suggest that it entered Mexico via multiple routes and that specific genome regions can accommodate substantial deletions without compromising viability.

Specific Molecular Signatures for Type II Crustins in Penaeid Shrimp Uncovered by the Identification of Crustin-Like Antimicrobial Peptides in Litopenaeus vannamei.

Crustins form a large family of antimicrobial peptides (AMPs) in crustaceans composed of four sub-groups (Types I-IV). Type II crustins (Type IIa or "Crustins" and Type IIb or "Crustin-like") possess a typical hydrophobic N-terminal region and are by far the most representative sub-group found in penaeid shrimp. To gain insight into the molecular diversity of Type II crustins in penaeids, we identified and characterized a Type IIb crustin in Litopenaeus vannamei (Crustin-like Lv) and compared Type II crustins at both molecular and transcriptional levels. Although L. vannamei Type II crustins (Crustin Lv and Crustin-like Lv) are encoded by separate genes, they showed a similar tissue distribution (hemocytes and gills) and transcriptional response to the shrimp pathogens Vibrio harveyi and White spot syndrome virus (WSSV). As Crustin Lv, Crustin-like Lv transcripts were found to be present early in development, suggesting a maternal contribution to shrimp progeny. Altogether, our in silico and transcriptional data allowed to conclude that (1) each sub-type displays a specific amino acid signature at the C-terminal end holding both the cysteine-rich region and the whey acidic protein (WAP) domain, and that (2) shrimp Type II crustins evolved from a common ancestral gene that conserved a similar pattern of transcriptional regulation.

Direct sequencing of the white spot syndrome virus from Brazil: Genome assembly and new insights on phylogeny.

White spot syndrome virus (WSSV) has been the cause of great economic losses in world shrimp farming. In this work the genome of a Brazilian WSSV isolate was determined from direct sequencing of total DNA extracted from an infected whiteleg shrimp, and assembled based on a chimera template approach. Comparisons between WSSV-BR and other isolates revealed that the Brazilian virus has a relatively small genome, and is very similar to isolates from Thailand and Mexico. A phylogenetic relationship using different approaches has demonstrated that these isolates share a common evolutionary history. An analysis of conflicting phylogenetic signals also considering genomes of other isolates revealed that the evolutionary history of WSSV may be related to recombination events. We observed that these events can also be traced at some level by analyzing the homologous regions in the WSSV genome. The existence of recombination events introduces a new point of view that must be considered in the evolutionary history of WSSV.

Virulence and genotypes of white spot syndrome virus infecting Pacific white shrimp Litopenaeus vannamei in north-western Mexico.

White spot syndrome virus (WSSV) has caused substantial global economic impact on aquaculture, and it has been determined that strains can vary in virulence. In this study, the effect of viral load was evaluated by infecting Litopenaeus vannamei with 10-fold serial dilution of tissue infected with strain WSSV Mx-H, and the virulence of four WSSV strains from north-western Mexico was assessed along with their variable number of tandem repeat (VNTR) genotypes in ORF75, ORF94 and ORF125. The LD50 of the Mx-H strain was a dilution dose of 10-7.5 ; the mortality titre was 109.2 LD50 per gram. In shrimp injected with 102.5 to 106.5 LD50 , no significant virulence differences were evident. Using mortality data, the four WSSV strains grouped into three virulence levels. The Mx-F strain (intermediate virulence) and the Mx-C strain (high virulence) showed more genetic differences than those observed between the Mx-G (low-virulence) and Mx-H (high-virulence) strains, in ORF94 and ORF125. The application of high-viral-load inocula proved useful in determining the different virulence phenotypes of the WSSV strains from the Eastern Pacific.

A novel viral thymidylate kinase with dual kinase activity.

Nucleotide phosphorylation is a key step in DNA replication and viral infections, since suitable levels of nucleotide triphosphates pool are required for this process. Deoxythymidine monophosphate (dTMP) is produced either by de novo or salvage pathways, which is further phosphorylated to deoxythymidine triphosphate (dTTP). Thymidyne monophosphate kinase (TMK) is the enzyme in the junction of both pathways, which phosphorylates dTMP to yield deoxythymidine diphosphate (dTDP) using adenosine triphosphate (ATP) as a phosphate donor. White spot syndrome virus (WSSV) genome contains an open reading frame (ORF454) that encodes a thymidine kinase and TMK domains in a single polypeptide. We overexpressed the TMK ORF454 domain (TMKwssv) and its specific activity was measured with dTMP and dTDP as phosphate acceptors. We found that TMKwssv can phosphorylate dTMP to yield dTDP and also is able to use dTDP as a substrate to produce dTTP. Kinetic parameters K M and k cat were calculated for dTMP (110 µM, 3.6 s(-1)), dTDP (251 µM, 0.9 s(-1)) and ATP (92 µM, 3.2 s(-1)) substrates, and TMKwssv showed a sequential ordered bi-bi reaction mechanism. The binding constants K d for dTMP (1.9 µM) and dTDP (10 µM) to TMKwssv were determined by Isothermal Titration Calorimetry. The affinity of the nucleotidic analog stavudine monophosphate was in the same order of magnitude (K d 3.6 µM) to the canonical substrate dTMP. These results suggest that nucleotide analogues such as stavudine could be a suitable antiviral strategy for the WSSV-associated disease.

Genotyping WSSV isolates from northwestern Mexican shrimp farms affected by white spot disease outbreaks in 2010-2012.

White spot disease (WSD) causes high mortality in cultured shrimp throughout the world. Its etiologic agent is the white spot syndrome virus (WSSV). The genomic repeat regions ORF 75, ORF 94, and ORF 125 have been used to classify WSSV isolates in epidemiological studies using PCR with specific primers and sequencing. The present study investigated the variation in nucleotide sequences from 107, 150, and 143 isolates of WSSV collected from Litopenaeus vannamei shrimp ponds with WSD outbreaks in northwestern Mexico during the period 2010-2012, in the genomic repeat regions ORFs 75, 94, and 125, respectively. The haplotypic nomenclature for each isolate was based on the number of repeat units and the position of single nucleotide polymorphisms on each ORF. We report finding 17, 43, and 66 haplotypes of ORFs 75, 94, and 125, respectively. The study found high haplotypic diversity in WSSV using the complete sequences of ORFs 94 and 125 as independent variables, but low haplotypic diversity for ORF 75. Different haplotypes of WSSV were found from region-to-region and year-to-year, though some individual haplotypes were found in different places and in more than one growing cycle. While these results suggest a high rate of mutation of the viral genome at these loci, or perhaps the introduction of new viral strains into the area, they are useful as a tool for epidemiological surveys. Two haplotypes from some of the ORFs in the same shrimp were encountered, suggesting the possibility of multiple infections.

Identification of highly expressed host microRNAs that respond to white spot syndrome virus infection in the Pacific white shrimp Litopenaeus vannamei (Penaeidae).

MicroRNAs (miRNAs) are known to play an important role in regulating both adaptive and innate immunity. Pacific white shrimp (Litopenaeus vannamei) is the most widely farmed crustacean species in the world. However, little is known about the role miRNAs play in shrimp immunity. To understand the impact of viral infection on miRNA expression in shrimp, we used high-throughput sequencing technology to sequence two small RNA libraries prepared from L. vannamei under normal and white spot syndrome virus (WSSV) challenged conditions. Approximately 19,312,189 and 39,763,551 raw reads corresponding to 17,414,787 and 28,633,379 high-quality mappable reads were obtained from the two libraries, respectively. Twelve conserved miRNAs and one novel miRNA that were highly expressed (>100 RPM) in L. vannamei were identified. Of the identified miRNAs, 8 were differentially expressed in response to the virus infection, of which 1 was upregulated and 7 were downregulated. The prediction of miRNA targets showed that the target genes of the differentially expressed miRNAs were related to immunity, apoptosis, and development functions. Our study provides the first characterization of L. vannamei miRNAs in response to WSSV infection, which will help to reveal the roles of miRNAs in the antiviral mechanisms of shrimp.

A ten-month diseases survey on wild Litopenaeus setiferus (Decapoda: Penaeidae) from Southern Gulf of Mexico.

The development of shrimp aquaculture in Mexican coasts of the Gulf of Mexico began to be explored using the Pacific white shrimp Litopenaeus vannamei in the mid 90's. Many concerns over the risk of disease transmission to the economically important native penaeids, have been the main deterrent for the aquaculture of L. vannamei in the region. Concurrently, more than 10 years of research experience on the aquaculture suitability of the native Litopenaeus setiferus from the Terminos Lagoon, in the Yucatán Peninsula, have been accumulated. The aim of this study was then to determine the seasonal variations of the naturally acquired diseases and the possible detection of exotic pathogens. For this, random subsamples (n-60) of juveniles L. setiferus were collected from monthly captures. In order to detect the widest range of pathogens, including infectious hypodermal and hematopoietic necrosis (IHHNV) and white spot syndrome (WSSV) viruses, both histopathological and molecular methods were employed. Monthly prevalence (%) was calculated for every finding. We were able to detect a total of 16 distinct histological anomalies, most of which the presumptive aetiological agent was readily identified. PCR results for viruses were negative. For some pathogens and symbionts, the prevalence was significantly different between the adult and juvenile populations. Prevalence of diseases tended to be higher in juvenile shrimp than in adults. The results of this study indicated that L. setiferus carry a wide variety of pathogens and symbionts that seem to be endemic to penaeids of the Gulf of Mexico, and those juveniles were more conspicuous to acquire pathogens and symbionts than adults.

A ten-month diseases survey on wild Litopenaeus setiferus (Decapoda: Penaeidae) from Southern Gulf of Mexico / Diez meses de análisis de las enfermedades en especímenes silvestres de Litopenaeus setiferus en el Sur del Golfo de México

The development of shrimp aquaculture in Mexican coasts of the Gulf of Mexico began to be explored using the Pacific white shrimp Litopenaeus vannamei in the mid 90´s. Many concerns over the risk of disease transmission to the economically important native penaeids, have been the main deterrent for the aquaculture of L. vannamei in the region. Concurrently, more than 10 years of research experience on the aquaculture suitability of the native Litopenaeus setiferus from the Terminos Lagoon, in the Yucatán Peninsula, have been accumulated. The aim of this study was then to determine the seasonal variations of the naturally acquired diseases and the possible detection of exotic pathogens. For this, random subsamples (n~60) of juveniles L. setiferus were collected from monthly captures. In order to detect the widest range of pathogens, including infectious hypodermal and hematopoietic necrosis (IHHNv) and white spot syndrome (WSSv) viruses, both histopathological and molecular methods were employed. Monthly prevalence (%) was calculated for every finding. We were able to detect a total of 16 distinct histological anomalies, most of which the presump- tive aetiological agent was readily identified. PCR results for viruses were negative. For some pathogens and symbionts, the prevalence was significantly different between the adult and juvenile populations. Prevalence of diseases tended to be higher in juvenile shrimp than in adults. The results of this study indicated that L. setiferus carry a wide variety of pathogens and symbionts that seem to be endemic to penaeids of the Gulf of Mexico, and those juveniles were more conspicuous to acquire pathogens and symbionts than adults.

Durante la década de los 90´s se introdujo el camarón blanco del Pacífico Litopenaeus vannamei a los Estados costeros mexicanos del Golfo de México con fines acuícolas, por lo que desde entonces existe preocupación por la posible introducción de enfermedades que puedan afectar a las poblaciones de camarones nativos. La investigación sobre la domesticación de especies nativas para una acuacultura sustentable se ha realizado por más de 10 años, sin embargo, aún existe escasa información sobre las enfermedades que se presentan de manera natural en estas poblaciones y posible trasfaunación. El presente estudio aborda el problema de las enfermedades encontradas en subpoblaciones de jóvenes y adultos de Litopenaeus setiferus del Área natural protegida Laguna de Términos, estado de Campeche, México. Técnicas de histología y biología molecular fueron utilizadas como herramientas de diagnóstico. Se encontró que L. setiferus es portador de patógenos y simbiontes endémicos del Golfo de México, y comparativamente, los jóvenes son más susceptibles en adquirir estas infecciones que los adultos, como probable respuesta al ambiente lacustre que ocupan. No se encontró evidencia de los virus IHHNv y WSSv, aunque en trabajos más recientes en algunos Estados del Norte ya se han detectado en poblaciones silvestres.

Natural occurrence of White spot syndrome virus and Infectious hypodermal and hematopoietic necrosis virus in Neohelice granulata crab.

White spot syndrome virus (WSSV) and Infectious hypodermal and hematopoietic necrosis virus (IHHNV) are two infectious agents associated to economic losses in shrimp aquaculture. As virus spread occurs through vectors and hosts, this study sought to verify the presence of WSSV and IHHNV in Neohelice granulata crab from Lagoa dos Patos estuary in Brazil and nearby shrimp farms. DNA extractions were performed with phenol/chloroform protocol. Molecular diagnosis was carried out by nested PCR for WSSV and one-step PCR for IHHNV. Results showed the presence of WSSV on crabs of both Lagoa dos Patos and farms, while IHHNV was found only on crabs collected in estuary. This is the first study to report IHHNV presence in N. granulata. Moreover, as analyzed crabs had no clinical symptoms or showed in situ mortality, we suggest its use as a bioindicator for virus occurrence in aquatic environments.

Biochemical characterization of thymidine monophosphate kinase from white spot syndrome virus: a functional domain from the viral ORF454.

Nucleotide phosphorylation is a key step towards DNA replication and during viral infections the maintenance of the nucleotide triphosphates pool is required. Deoxythymidine triphosphate (dTTP) is the unique nucleotide that is produced either by de novo or salvage pathways. Thymidine monophosphate kinase (TMK) is the enzyme that phosphorylates deoxythymidine monophosphate (dTMP) using adenosine triphosphate (ATP) as a phosphate group donor in presence of Mg2+ yielding deoxythymidine diphosphate (dTDP) and adenosine diphosphate. The TMK region of the WSSV TK-TMK chimeric protein was overexpressed and purified. This recombinant protein had TMK activity, this is that dTMP was phosphorylated to dTDP and we found that the dimeric state of the protein was the functional and a theoretical structural model was built as such. Future work will focus towards a structural characterization as an antiviral target.

Double-stranded RNA against white spot syndrome virus (WSSV) vp28 or vp26 reduced susceptibility of Litopenaeus vannamei to WSSV, and survivors exhibited decreased susceptibility in subsequent re-infections.

The antiviral effect of vp28 or vp26 double-stranded (ds) RNA upon single or consecutive white spot syndrome virus (WSSV) intramuscular challenges with a high infectious dose was evaluated. The vp28 dsRNA showed the highest protection both in single (LT(50)=145h at 10d and 98h at 20d post treatment [dpt]) or consecutive (LT(50)=765h) WSSV challenges compared to vp26 dsRNA (LT(50)=126h at 10 d and 57h at 20dpt vs. consecutive challenge LT(50)=751h). Single WSSV challenges showed that animals treated with vp28 or vp26 dsRNA gradually lost the antiviral effect as virus challenge occurred at 10dpt (cumulative mortality 63% vs. 80%, respectively) or 20dpt (87% vs. 100%, respectively). In contrast, animals treated with vp28 or vp26 dsRNA and consecutively challenged with WSSV showed and extended lower susceptibility to WSSV. All dead animals were WSSV-positive by one-step PCR, whereas all surviving shrimp from single or continuous challenges were WSSV-negative as determined by reverse transcription (RT)-PCR. In conclusion, shrimp treated with a single administration of vp28 or vp26 dsRNA and consecutively challenged with WSSV showed a stronger and longer antiviral response than shrimp exposed once to WSSV at 10 or 20dpt.

White spot syndrome virus Orf514 encodes a bona fide DNA polymerase.

White spot syndrome virus (WSSV) is the causative agent of white spot syndrome, one of the most devastating diseases in shrimp aquaculture. The genome of WSSV includes a gene that encodes a putative family B DNA polymerase (ORF514), which is 16% identical in amino acid sequence to the Herpes virus 1 DNA polymerase. The aim of this work was to demonstrate the activity of the WSSV ORF514-encoded protein as a DNA polymerase and hence a putative antiviral target. A 3.5 kbp fragment encoding the conserved polymerase and exonuclease domains of ORF514 was overexpressed in bacteria. The recombinant protein showed polymerase activity but with very low level of processivity. Molecular modeling of the catalytic protein core encoded in ORF514 revealed a canonical polymerase fold. Amino acid sequence alignments of ORF514 indicate the presence of a putative PIP box, suggesting that the encoded putative DNA polymerase may use a host processivity factor for optimal activity. We postulate that WSSV ORF514 encodes a bona fide DNA polymerase that requires accessory proteins for activity and maybe target for drugs or compounds that inhibit viral DNA replication.

Genotyping of white spot syndrome virus (WSSV) geographical isolates from Brazil and comparison to other isolates from the Americas.

White spot syndrome virus (WSSV) is a viral pathogen that has caused significant economic losses in shrimp farming. Variable-number tandem repeats (VNTRs) (open reading frame [ORF] 94, 125 and 75), a large deletion (ORF 23/24) and a transposase were proposed as molecular markers for genotyping. WSSV-infected shrimp Litopenaeus vannamei were collected in 2 Brazilian regions (Santa Catarina and Bahia) from 2005 to 2008. DNA was extracted and PCR of the variable regions was performed, followed by sequencing. All Santa Catarina samples showed the same number of repeats for the minisatellites analyzed. Bahia samples showed a different pattern for the regions, indicating that there are at least 2 different WSSV genotypes in Brazil. Both Brazilian isolates have an 11453 bp deletion in ORF 23/24 when compared with WSSV-TW (Taiwan), which has the full sequence for this locus. The Brazilian WSSV isolates were compared with WSSV isolates from other countries in the Americas (USA, Panama, Honduras, Mexico and Nicaragua); the repeat number patterns for the 3 VNTR regions analyzed were different between the Brazilian isolates and the other western-hemisphere isolates. This may be due to mutations in WSSV after its introduction into the different countries. Our results also show that WSSV found in Bahia and Santa Catarina very likely originated from different sources of contamination.

Nucleotide sequence variations of the major structural proteins (VP15, VP19, VP26 and VP28) of white spot syndrome virus (WSSV), a pathogen of cultured Litopenaeus vannamei in Mexico.

White spot syndrome virus (WSSV) was first reported in farmed Litopenaeus vannamei stocks in Sinaloa and Sonora, Mexico during 1999 and continues to cause severe shrimp losses. WSSV genes encoding nucleocapsid (VP26 and VP15) and envelope proteins (VP19 and VP28) of a Mexican isolate were cloned in the pMosBlue vector. The nucleotide sequences of these genes were compared with WSSV isolates in GenBank. VP15 is highly conserved, and VP26 showed 99% homology to a Chinese isolate. The VP28 fragment demonstrated 100% homology to the majority of the isolates analysed (UniProt accession no. Q91CB7), differing from two Indian WSSV and one Chinese WSSV isolates by two non-conserved and one conserved replacements, respectively. Because of their highly conserved nature, these three structural proteins are good candidates for the development of antibody-based WSSV diagnostic tools or for the production of recombinant protein vaccines to stimulate the quasi-immune response of shrimp. In contrast, VP19 of the Mexican isolate was distinguishable from almost all isolates tested, including an American strain of WSSV (US98/South Carolina, GenBank accession no. AAP14086). Although homology was found with isolates from Taiwan (GenBank accession no. AAL89341) and India (GenBank accession no. AAW67477), VP19 may have application as a genetic marker.

Temperature modifies gene expression in subcuticular epithelial cells of white spot syndrome virus-infected Litopenaeus vannamei.

Subtractive suppressive hybridization was used to identify differentially expressed genes in subcuticular tissues from white spot syndrome virus(WSSV)-infected shrimp kept at different temperatures. Subtractive libraries I and II contained genes expressed at 26 and 33 degrees C, respectively. Three hundred and seventy-nine insert positive clones were selected to confirm differential expression by dot-blot hybridization. Twenty-two clones from library I and eight from library II were sequenced. All sequences from Library I corresponded to white spot syndrome virus genes. From library II, five clones were homologous with previously reported expressed sequence tags of Litopenaeus vannamei, two had similarity with beta-actin and one transcript represented an unknown gene. Over-expression of VP15 in shrimp at 26 degrees C was further confirmed by real-time polymerase chain reaction (PCR), whereas beta-actin expression was similar in animals kept at both temperatures. Together, our results show that hyperthermia reduces the expression of WSSV genes on shrimp subcuticular epithelial cells.