Screening for White Spot Syndrome Virus (WSSV) in pink shrimp (Penaeus paulensis) from Rocha Lagoon, Uruguay.

Penaeus paulensis (pink shrimp) is an important resource for small-scale fisheries in the brackish coastal lagoons of Uruguay. No viral diseases have been detected in shrimp populations in the Uruguayan territory. The presence of viral pathogens, such as White Spot Syndrome Virus (WSSV) and Infectious Hypodermal Haematopoietic Necrosis Virus (IHHNV) in wild shrimp populations has been previously reported in Brazil and Argentina. We investigated the presence of WSSV in wild populations of penaeid shrimp from Rocha Lagoon, Uruguay. We sampled 70 specimens of juvenile P. paulensis and assessed the presence of these viral pathogens using nested PCR and histology. Gill tissue from the 70 samples was divided into 14 pools of 5 individuals for DNA extraction and PCR analysis. We also retested each pooled sample individually. The nested PCR procedure described in the WOAH aquatic animal manual was used. A subset of 20 individual specimens were also processed using standard histological techniques. The results showed that WSSV was not detected in the pooled or individually tested samples. We found no evidence of the presence of the viral genome or gill lesions in the samples analysed. This indicates that the fishery is still likely to be free of WSSV infection. The procedures and information generated can be used as a baseline study for future implementation of surveillance programmes in the country.

Single nucleotide polymorphism associated with disease resistance in Penaeus vannamei.

Despite the considerable number of genetic markers published for Penaeus vannamei, the classification of these markers and their standardization in specific databases is still insufficient. As a consequence, access to these markers is difficult, hampering their application in genetic association studies. In this study, all previously described single nucleotide polymorphisms (SNPs) related to resistance for P. vannamei were revised, and 512 SNPs were identified and classified in detail. We observed that most of the SNPs occurred in the proteins including Toll like receptors 1 and 3, hemocyanin large and small subunits, and anti-lipopolysaccharide factors 1 and 2, allowing to propose to use them as targets in association studies involving resistance in P. vannamei. Additionally, the potential effects of the most frequent non-synonymous coding SNPs in the secondary structure of the main target proteins were evaluated using an in silico approach. These data can serve as the starting point for the development of new genetic and computational tools as well as for the design of new association studies that involve resistance in P. vannamei.

Shedding the Light on Litopenaeus vannamei Differential Muscle and Hepatopancreas Immune Responses in White Spot Syndrome Virus (WSSV) Exposure.

White Spot Syndrome Virus (WSSV) is one of the main threats to farming Litopenaeus vannamei, the most important crustacean commercialized in aquaculture worldwide. Here, we performed RNA-seq analyses in hepatopancreas and muscle from WSSV-negative (healthy) and WSSV-positive (unhealthy) L. vannamei, previously exposed to the virus, to obtain new insights about the molecular basis of resistance to WSSV. We detected 71% of our reads mapped against the recently described L. vannamei genome. This is the first report mapping RNA-seq transcripts from shrimps exposed to WSSV against the species reference genome. Differentially expressed gene (DEG) analyses were performed for four independent comparisons, and 13,338 DEGs were identified. When the redundancies and isoforms were disregarded, we observed 8351 and 6514 DEGs, respectively. Interestingly, after crossing the data, we detected a common set of DEGs for hepatopancreas and healthy shrimps, as well as another one for muscle and unhealthy shrimps. Our findings indicate that genes related to apoptosis, melanization, and the Imd pathway are likely to be involved in response to WSSV, offering knowledge about WSSV defense in shrimps exposed to the virus but not infected. These data present potential to be applied in further genetic studies in penaeids and other farmed shrimp species.

Novel infectious myonecrosis virus (IMNV) genotypes associated with disease outbreaks on Penaeus vannamei shrimp farms in Indonesia.

Infectious myonecrosis virus (IMNV) is one of the most pathogenic viruses that affect Penaeus vannamei shrimp. In 2018, IMNV was reported in grow-out ponds of P. vannamei in Situbondo, Indonesia. Diseased animals displayed clinical signs of infectious myonecrosis (IMN) characterized by white discoloration of skeletal muscle. Histopathology of affected shrimp revealed lesions that are pathognomonic of IMNV infection. The major capsid protein (MCP) gene was amplified and sequenced from representative samples showing IMN pathology. Multiple alignment of predicted amino acid sequences of the MCP gene with known IMNV genotypes in the GenBank database revealed three unique genotypes, SB-A, SB-B and SB-C,in Situbondo samples. The number of amino acid changes in SB-A, SB-B and SB-C compared to known IMNV genotypes ranged from 7-710, including the isolate SB-B, which contains deletion of 622 aa. A phylogenetic analysis using homologous sequences from Brazil and Indonesia showed that these three isolates represent new IMNV genotypes.

The white spot syndrome virus hijacks the expression of the Penaeus vannamei Toll signaling pathway to evade host immunity and facilitate its replication.

The white spot syndrome virus (WSSV), the most lethal pathogen of shrimp, is a dsDNA virus with approximately a 300,000 base pairs and contains approximately 180-500 predicted open reading frames (ORFs), of which only 6% show homology to any known protein from other viruses or organisms. Although most of its ORFs encode enzymes for nucleotide metabolism, DNA replication, and protein modification, the WSSV uses some of its encoded proteins successfully to take control of the metabolism of the host and avoid immune responses. The contribution of the shrimp innate immune response to prevent viral invasions is recognized but yet not fully understood. Thus, the role of several components of Toll pathway of the shrimp Penaeus vannamei against WSSV has been previously described, and the consequential effects occurring through the cascade remain unknown. In the current study the effects of WSSV over various components of the shrimp Toll pathway were studied. The gene expression of Spätzle, Toll, Tube, Cactus and Dorsal was altered after 6-12 h post inoculation. The expression of LvToll3, LvCactus, LvDorsal, decreased ~4.4-, ~3.7- and ~7.3-fold at 48, 24 and 48 hpi, respectively. Furthermore, a remarkable reduction (~18-fold) in the expression of the gene encoding LvCactus in WSSV infected specimens was observed at 6 hpi. This may be a sophisticated strategy exploited by WSSV to evade the Toll-mediated immune action, and to promote its replication, thereby contributing to viral fitness.

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.

Potential immunomodulatory and protective effects of the Arthrospira-based dietary supplement on shrimp intestinal immune defenses.

Herein, we evaluated the immunomodulatory and the antiviral protective properties of a cyanobacteria-enriched diet on the immune responses of the Pacific white shrimp Litopenaeus vannamei challenged with the White spot syndrome virus (WSSV). Shrimp were fed with an Arthrospira platensis supplemented feed during 20 days, and its effects were examined by evaluating well-known standardized shrimp immune parameters (total hemocyte counts, total protein concentration, phenoloxidase activity, and serum agglutination titer). Additionally, we assessed the expression of crucial genes involved in both hemolymph- and gut-based immunities related to the shrimp capacity to circumvent viral and microbial infections. Dietary supplementation improved shrimp survival rates after challenge with a median lethal dose of WSSV. From all immune parameters tested, only the serum agglutination titer was higher in treated animals. On the other hand, the expression of some representative marker genes from different immune response pathways was only modulated in the midgut and not in the circulating hemocytes, suggesting that this feed supplementation can be used as an attractive strategy to enhance immunity in shrimp gut. Altogether, our results evidence the immunomodulatory properties of A. platensis supplemented feed in shrimp humoral and intestinal defenses and highlight the potential use of cyanobacteria-based immunostimulants in shrimp farming for protection against infectious diseases.

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.

Effects of a co-culture of marine algae and shrimp (Litopenaeus vannamei) on the growth, survival and immune response of shrimp infected with Vibrio parahaemolyticus and white spot virus (WSSV).

In aquaculture, fighting infectious diseases is a necessity. This study measured the immuno-stimulating effect of live macroalgae consumption on Litopenaeus vannamei against Vibrio parahaemolyticus and WSSV infection in two independent bioassays. Shrimps and macroalgae were cultivated in a co-culture with two species of macroalgae separately (Gracilaria vermiculophylla and Dictyota dichotoma), and later, shrimp were infected with V. parahaemolyticus. In another bioassay, shrimp and macroalgae (G. vermiculophylla, D. dichotoma and Ulva lactuca) were grown and subsequently infected with WSSV. For both bioassays, survival after 120 h was determined, the total hemocyte count (TCH) was measured and the activity of superoxide dismutase (SOD) and catalase (CAT) in tissue were measured. The results indicate that the use of macroalgae in co-culture with L. vannamei provides a nutritional benefit that achieves higher growth than the control organisms, as well as improvements of the ammonium concentration and immune response after infection with V. parahaemolyticus and WSSV. A better immune response was obtained in organisms cultured with macroalgae in both bioassays at a ratio of 1.6-1.9 for organisms infected with bacteria and 1.4 to 1.6 times for organisms infected with the virus. In turn, the enzymatic activity of SOD and CAT were higher in the treated organisms relative to the controls in both experiments.

Proteomic profiling of the white shrimp Litopenaeus vannamei (Boone, 1931) hemocytes infected with white spot syndrome virus reveals the induction of allergy-related proteins.

To elucidate the proteomic responses of shrimp hemocytes to white spot syndrome virus (WSSV) infection at the proteome level, a quantitative shotgun proteomic analysis was performed to detect differentially synthesized proteins in infected hemocytes of white shrimp (Litopenaeus vannamei). We identified 1528 proteins associated to 203 gene ontology (GO) categories. The most representative GO categories were regulation of cellular processes, organic substance metabolic processes and nitrogen compound metabolic processes. Most of the 83 detected up-regulated proteins are involved in DNA regulation and organization and cell signaling. In contrast, most of the 40 down-regulated proteins were related to immune defense processes, protein folding, and development. Differentially induced proteins were further analyzed at the transcript level by RT-qPCR to validate the results. This work provides new insights into the alterations of L. vannamei hemocytes at the protein level at 12 h post-infection with WSSV. Interestingly, several of the up-regulated proteins are allergy-related proteins in humans. Based on our results, we suggest a deeper analysis of the effects of this interaction on the regulation of allergy related-proteins as their up-regulation during WSSV could represent a threat to human health.

Silver nanoparticles enhance survival of white spot syndrome virus infected Penaeus vannamei shrimps by activation of its immunological system.

The global aquaculture has shown an impressive growth in the last decades contributing with a major part of total food fish supply. However, it also helps in the spread of diseases that in turn, causes great economic losses. The White Spot Syndrome Virus (WSSV) is one of the major viral pathogen for the shrimp aquaculture industry. Several attempts to eliminate the virus in the shrimp have been addressed without achieving a long-term effectiveness. In this work, we determine the capacity of the commercial non-toxic PVP-coated silver nanoparticles to promote the response of the immune system of WSSV-infected shrimps with or without an excess of iron ions. Our results showed that a single dose of metallic silver in the nanomolar range (111 nmol/shrimp), which is equivalent to 12 ng/mL of silver nanoparticles, produces 20% survival of treated infected shrimps. The same concentration administered in healthy shrimps do not show histological evidence of damage. The observed survival rate could be associated with the increase of almost 2-fold of LGBP expression levels compared with non-treated infected shrimps. LGBP is a key gene of shrimp immunological response and its up-regulation is most probably induced by the recognition of silver nanoparticles coating by specific pathogen-associated molecular pattern recognition proteins (PAMPs) of shrimp. Increased LGBP expression levels was observed even with a 10-fold lower dose of silver nanoparticles (1.2 ng/shrimp, 0.011 nmol of metallic silver/shrimp). The increase in LGBP expression levels was also observed even in the presence of iron ion excess, a condition that favors virus proliferation. Those results showed that a single dose of a slight amount of silver nanoparticles were capable to enhance the response of shrimp immune system without toxic effects in healthy shrimps. This response could be enhanced by administration of other doses and might represent an important alternative for the treatment of a disease that has still no cure, white spot syndrome virus.

PirVP genes causing AHPND identified in a new Vibrio species (Vibrio punensis) within the commensal Orientalis clade.

Acute hepatopancreatic necrosis disease (AHPND) has extended rapidly, causing alarming shrimp mortalities. Initially, the only known causative agent was Vibrio parahaemolyticus carrying a plasmid coding for the mortal toxins PirVP. Recently, it has been found that the plasmid and hence the disease, could be transferred among members of the Harveyi clade. The current study performs a genomic characterization of an isolate capable of developing AHPND in shrimp. Mortality studies and molecular and histopathological analyses showed the infection capacity of the strain. Multilocus sequence analysis placed the bacteria as a member of the Orientalis clade, well known for containing commensal and even probiotic bacteria used in the shrimp industry. Further whole genome comparative analyses, including Vibrio species from the Orientalis clade, and phylogenomic metrics (TETRA, ANI and DDH) showed that the isolate belongs to a previously unidentified species, now named Vibrio punensis sp. nov. strain BA55. Our findings show that the gene transfer capacity of Vibrio species goes beyond the clade classification, demonstrating a new pathogenic capacity to a previously known commensal clade. The presence of these genes in a different Vibrio clade may contribute to the knowledge of the Vibrio pathogenesis and has major implications for the spread of emerging diseases.

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.

Silencing of HIF-1 in WSSV-infected white shrimp: Effect on viral load and antioxidant enzymes.

Hypoxia inducible factor-1 (HIF-1) is a transcriptional factor that induces genes involved in glucose metabolism. HIF-1 is formed by a regulatory α-subunit (HIF-1α) and a constitutive ß-subunit (HIF-1ß). The white spot syndrome virus (WSSV) induces a shift in glucose metabolism and oxidative stress. HIF-1α is associated with the induction of metabolic changes in tissues of WSSV-infected shrimp. However, the contributions of HIF-1 to viral load and antioxidant responses in WSSV-infected shrimp have been not examined. In this study, the effect of HIF-1 silencing on viral load and the expression and activity of antioxidant enzymes (superoxide dismutase-SOD, glutathione S-transferase-GST, and catalase) along with oxidative damage (lipid peroxidation and protein carbonyl) in tissues of white shrimp infected with the WSSV were studied. The viral load increased in hepatopancreas and muscle after WSSV infection, and the accumulative mortality was of 100% at 72 h post-infection. The expression and activity of SOD, catalase, and GST decreased in each tissue evaluated after WSSV infection. Protein carbonyl concentrations increased in each tissue after WSSV infection, while lipid peroxidation increased in hepatopancreas, but not in muscle. Silencing of HIF-1α decreased the WSSV viral load in hepatopancreas and muscle of infected shrimp along with shrimp mortality. Silencing of HIF-1α ameliorated the antioxidant response in a tissue-specific manner, which translated to a decrease in oxidative damage. These results suggest that HIF-1 is essential for restoring the antioxidant response, which counters the oxidative injury associated with WSSV infection.

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.

Quantitative risk assessment of WSSV transmission through partial harvesting and transport practices for shrimp aquaculture in Mexico.

This quantitative risk assessment provided an analytical framework to estimate white spot syndrome virus (WSSV) transmission risks in the following different scenarios: (1) partial harvest from rearing ponds and (2) post-harvest transportation, assuming that the introduction of contaminated water with viral particles into shrimp culture ponds is the main source of viral transmission risk. Probabilities of infecting shrimp with waterborne WSSV were obtained by approaching the functional form that best fits (likelihood ratio test) published data on the dose-response relationship for WSSV orally inoculated through water into shrimp. Expert opinion defined the ranges for the following uncertain factors: (1) the concentrations of WSSV in the water spilled from the vehicles transporting the infected shrimp, (2) the total volume of these spills, and (3) the dilution into culture ponds. Multiple scenarios were analysed, starting with a viral load (VL) of 1×102mL-1 in the contaminated water spilled that reached the culture pond, whose probability of infection of an individual shrimp (Pi) was negligible (1.7×10-7). Increasing the VL to 1×104.5mL-1 and 1×107mL-1 yielded results into very low (Pi=5.3×10-5) and high risk (Pi=1.6×10-2) categories, respectively. Furthermore, different pond stocking density (SD) scenarios (20 and 30 post-larvae [PL]/m2) were evaluated, and the probability of infection of at least one out of the total number of shrimp exposed (PN) was derived; for the scenarios with a low VL (1×102mL-1), the PN remained at a negligible risk level (PN, 2.4×10-7 to 1.8×10-6). For most of the scenarios with the moderate VL (1×104.5mL-1), the PN scaled up to a low risk category (PN, 1.1×10-4 to 5.6×10-4), whereas for the scenarios with a high VL (1×107mL-1), the risk levels were high (PN, 2.3×10-2 to 3.5×10-2) or very high (PN, 1.1×10-1 to 1.6×10-1) depending on the volume of contaminated water spilled in the culture pond (VCWSCP, 4 or 20L). In the sensitivity analysis, for a SD of 30 PL/m2, it was shown that starting with a VL of 1×105mL-1 and a VCWSCP of 12L, the PN was moderate (1.05×10-3). This was the threshold for greater risks, given the increase in either the VCWSCP or VL. These findings supported recommendations to prevent WSSV spread through more controlled transportation and partial harvesting practices.

Hsp70 gene polymorphisms in farmed marine shrimp Litopenaeus vannamei populations exposed to white spot disease and infectious myonecrosis.

Single nucleotide polymorphisms (SNPs) are the best genetic markers for associative studies of the immune system in invertebrates. In the marine shrimp Litopenaeus vannamei, SNPs linked to disease resistance have been reported for some genes, such as hemocyanin, anti-lipopolysaccharide factor, and heat-shock protein 70 (Hsp70). In the present study, polymorphisms in the Hsp70 gene were investigated among three commercial L. vannamei populations bred in Northeast and South Brazil. The first population withstood a strong white spot disease outbreak; the second population suffered extended exposure to infectious myonecrosis; the third population was a high health population, which was experimentally infected with white spot syndrome virus (WSSV) in the present study. All five previously known SNPs (C661A, T712C, C782T, C892T, and C1090T) were detected in the coding region of Hsp70, by Sanger sequencing of 119 shrimp. Significant differences in genetic and genotype frequencies among populations were observed for C661A, C892T, and C1090T. In the population submitted to WSSV challenge, no frequency differences were found between dead and surviving shrimp groups. These results indicate that the Hsp70 polymorphisms described here cannot be associated with WSSV tolerance. However, significant frequency differences were observed for the population exposed to infectious myonecrosis virus. This is the first time that L. vannamei Hsp70 gene polymorphisms were studied in correlation with these important shrimp viruses.

Antiviral Activity of Ctn[15-34], A Cathelicidin-Derived Eicosapeptide, Against Infectious Myonecrosis Virus in Litopenaeus vannamei Primary Hemocyte Cultures.

The shrimp farming has been converted into a mature aquaculture industry dealing with over millions of metric tonnes of processed commodities. Nevertheless, the global shrimp productions are constantly threatened by disease outbreaks, mainly triggered by rapidly disseminating viruses. Infectious myonecrosis virus (IMNV) is one of these epizootic agents affecting shrimp production in Brazil, of which no treatment exists. Herein, the antiviral activity against IMNV of an eicosapeptide, named Ctn[15-34], derived from a member of the cathelicidin family of antimicrobial peptides, was demonstrated. Cultures of hemocytes from Litopenaeus vannamei were established that support IMNV replication and infectivity titration. The cytotoxic effect of IMNV in culture and the in vitro anti-IMNV activity of Ctn[15-34] were assessed using a high-sensitive fluorescent-based method in combination with quantitative PCR. The Ctn[15-34] (<12.5 µM) neutralized the toxic effects of IMNV at loads sufficient to kill 50% of shrimp hemocytes. This study reported for the first time the replication of IMNV in vitro and the employment of a straightforward methodology to assess cell viability and viral/antiviral activities. In addition, it provided the basis for the development of the anti-infective multi-effector Ctn[15-34] eicosapeptide and analogs as components of antiviral formulations against shrimp viral diseases.

Selection and validation of candidate reference genes for quantitative real-time PCR studies in the shrimp Penaeus vannamei under viral infection.

The decapod Penstyldensovirus 1 (PstDV-1) represents one of the most serious threats for penaeid shrimp farming. Studies aimed at defining relevant molecular effects of this virus over its host are imperative in the attempt to increase our understanding of its pathogenesis. Unfortunately, few studies have focused on the definition of the expression profile of reference genes in shrimp challenged with a pathogen. As a result, there are no studies on the selection of reference genes for the normalization of target gene expression changes yielding reliable data of the effects following PstDV-1 infection in shrimp. Therefore, the aim of the present study was to evaluate and validate the appropriateness of four candidate reference genes (ef1-α, gapdh, rpl8 and ß-tubulin) for their use as reference genes to normalize qPCR data in gene expression studies of PstDV-1-shrimp interactions. By analyzing the expression profile of those genes, gapdh was validated as a suitable reference gene to normalize expression data gathered from a PstDV1-challenge, while ef1-α, ß-tubulin, and rpl8 were identified as unstably expressed during the infectious process. The suitability of gapdh as a common reference gene in studies of host gene response to viral infections is underlined.