Arturo Sánchez-Paz

White spot syndrome virus: an overview on an emergent concern

Viruses are ubiquitous and extremely abundant in the marine environment. One of such marine viruses, the white spot syndrome virus (WSSV), has emerged globally as one of the most prevalent, widespread and lethal for shrimp populations. However, at present there is no treatment available to interfere with the unrestrained occurrence and spread of the disease. The recent progress in molecular biology techniques has made it possible to obtain information on the factors, mechanisms and strategies used by this virus to infect and replicate in susceptible host cells. Yet, further research is still required to fully understand the basic nature of WSSV, its exact life cycle and mode of infection. This information will expand our knowledge and may contribute to developing effective prophylactic or therapeutic measures. This review provides a state-of-the-art overview of the topic, and emphasizes the current progress and future direction for the development of WSSV control strategies.

Invertebrate trypsins: a review

Food protein hydrolysis, a crucial step in digestion, is catalyzed by trypsin enzymes from the digestive apparatus of invertebrates. Trypsin appeared early in evolution and occurs in all phyla and, in the digestive systems of invertebrates, it became the most abundant proteinase. As in vertebrates, invertebrate trypsin is also present in several forms (isoenzymes). Its physiological importance in food protein digestion in several invertebrate species has emerged with compelling evidence; and several other physiological functions, such as regulation of digestive functions, are now settled. Recent advances in the knowledge of invertebrate trypsin synthesis, regulation, genetics, catalytic characteristics; structure, evolution, as well as inhibition, especially in non-Drosophilidae insects and in some crustaceans are reviewed. Most of the existing information is largely based on the use of several tools, including molecular techniques, to answer many still open questions and solve medical, agricultural, and food quality problems.

Differential expression of trypsin mRNA in the white shrimp (Penaeus vannamei) midgut gland under starvation conditions

Regulation of the expression of trypsin encoding genes in juveniles white shrimp (Penaeus vannamei) was studied during starvation for up to 120 h. Since molting is now recognized as a physiologically important process in penaeids, specimens were selected according to molt stage. Starvation led to immediate weight loss and a sharp decrease in hepatosomatic index. Specific DNA probes for trypsin were synthesized and trypsin mRNA concentration was evaluated by dot blot hybridization. Trypsin-encoding RNA levels were strongly influenced by starvation, resulting in an increase after 24 h of starvation, followed by a steep decline to lower level compared to those obtained in fed shrimp. A possible mechanism for regulation of trypsin activity during transcription is proposed. The biological implications of our findings for transcriptional regulation of trypsin mRNA during starvation are discussed.

Gene expression and protein levels of thioredoxin in the gills from the whiteleg shrimp (Litopenaeus vannamei) infected with two different viruses: the WSSV or IHHNV.

The thioredoxin (TRX) system in crustaceans has demonstrated to act as a cell antioxidant being part of the immune response by dealing with the increased production of reactive oxygen species during bacterial or viral infection. Since the number of marine viruses has increased in the last years significantly affecting aquaculture practices of penaeids, and due to the adverse impact on wild and cultured shrimp populations, it is important to elucidate the dynamics of the shrimp response to viral infections. The role of Litopenaeus vannamei thioredoxin (LvTRX) was compared at both, mRNA and protein levels, in response to two viruses, the white spot syndrome virus (WSSV) and the infectious hypodermal and hematopoietic necrosis virus (IHHNV). The results confirmed changes in the TRX gene expression levels of WSSV-infected shrimp, but also demonstrated a more conspicuous response of TRX to WSSV than to IHHNV. While both the dimeric and monomeric forms of LvTRX were detected by Western blot analysis during the WSSV infection, the dimer on its reduced form was only detected through the IHHNV infectious process. These findings indicate that WSSV or IHHNV infected shrimp may induce a differential response of the LvTRX protein.

Development and validation of a quantitative real-time polymerase chain assay for universal detection of the White Spot Syndrome Virus in marine crustaceans

BackgroundThe White Spot Syndrome Virus (WSSV), the sole member of the family Whispoviridae, is the etiological agent that causes severe mortality events in wild and farmed shrimp globally. Given its adverse effects, the WSSV has been included in the list of notifiable diseases of the Office of International Epizootic (OIE) since 1997. To date there are no known therapeutic treatments available against this lethal virus, and a surveillance program in brood-stock and larvae, based on appropriate diagnostic tests, has been strongly recommended. However, some currently used procedures intended for diagnosis of WSSV may be particularly susceptible to generate spurious results harmfully impacting the shrimp farming industry.MethodsIn this study, a sensitive one-step SYBR green-based real-time PCR (qPCR) for the detection and quantitation of WSSV was developed. The method was tested against several WSSV infected crustacean species and on samples that were previously diagnosed as being positive for WSSV from different geographical locations.ResultsA universal primer set for targeting the WSSV VP28 gene was designed. This method demonstrated its specificity and sensitivity for detection of WSSV, with detection limits of 12 copies per sample, comparable with the results obtained by other protocols. Furthermore, the primers designed in the present study were shown to exclusively amplify the targeted WSSV VP28 fragment, and successfully detected the virus in different samples regardless of their geographical origin. In addition, the presence of WSSV in several species of crustaceans, including both naturally and experimentally infected, were successfully detected by this method.ConclusionThe designed qPCR assay here is highly specific and displayed high sensitivity. Furthermore, this assay is universal as it allows the detection of WSSV from different geographic locations and in several crustacean species that may serve as potential vectors. Clearly, in many low-income import-dependent nations, where the growth of shrimp farming industries has been impressive, there is a demand for cost-effective diagnostic tools. This study may become an alternative molecular tool for a less expensive, rapid and efficient detection of WSSV.

Experimental evidence of metabolic disturbance in the white shrimp Penaeus vannamei induced by the Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV).

The Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) is a single-stranded DNA virus that infects several penaeid shrimp species, provoking economic losses in farmed shrimp populations estimated at several million of dollars. Furthermore, IHHNV has historically been considered an important threat for wild shrimp populations, but its real measurable impact remains unknown. Currently no treatments are available against IHHNV, and research to develop potential antiviral strategies depends on a detailed understanding of the viral life cycle. However, the exact pathophysiological events underlying the development of metabolic changes in IHHNV-infected shrimp are still unknown. Thus, the biochemical changes caused by the IHHNV infection in plasma and hepatopancreas of the economically important shrimp species Penaeus vannamei were evaluated. Glucose, lactate, total protein, glycogen, triacylglycerides, cholesterol, and total lipids were measured in healthy and IHHNV-infected shrimp. Significant changes were observed in energy substrates (glucose, lactate, triacylglycerides and cholesterol), in plasma and hepatopancreas. These changes may indicate a temporal sequestration of the host-cell metabolic pathways by the virus to maximize its replication and propagation.

Invertebrates Mitochondrial Function and Energetic Challenges

Oliviert Martinez-Cruz1, Arturo Sanchez-Paz2, Fernando Garcia-Carreno3, Laura Jimenez-Gutierrez1, Ma. de los Angeles Navarrete del Toro3 and Adriana Muhlia-Almazan1 1Molecular Biology Laboratory, Centro de Investigacion en Alimentacion y Desarrollo (CIAD), Hermosillo, Sonora, 2Laboratorio de Sanidad Acuicola, Centro de Investigaciones Biologicas del Noroeste (CIBNOR), Hermosillo, Sonora, 3Biochemistry Laboratory, Centro de Investigaciones Biologicas del Noroeste (CIBNOR), La Paz, Mexico

Genome dynamics in three different geographical isolates of white spot syndrome virus (WSSV).

White spot syndrome virus (WSSV), the sole member of the monotypic family Nimaviridae, is considered an extremely lethal shrimp pathogen. Despite its impact, some essential biological characteristics related to WSSV genome dynamics, such as the synonymous codon usage pattern and selection pressure in genes, remain to be elucidated. The results show that compositional limitations and mutational pressure determine the codon usage bias and base composition in WSSV. Furthermore, different forces of selective pressure are acting across various regions of the WSSV genome. Finally, this study points out the possible occurrence of two major recombination events.

An improved validated SYBR green-based real-time quantitative PCR assay for the detection of the Penaeus stylirostris densovirus in penaeid shrimp

The Penaeus stylirostris densovirus (PstDV) (also known as infectious hypodermal and hematopoietic necrosis virus, IHHNV), one of the major shrimp pathogens, has a worldwide distribution in farmed and wild shrimp populations. Outbreaks of IHHNV have been associated with substantial economic losses which are accompanied by a negative social impact. Current diagnostic PCR tests may result in false-positive results as several parts of PstDV genome may be endogenized in the nuclear genome of the shrimp P. stylirostris. A one-step qPCR SYBR-Green based quantitative real-time polymerase chain reaction (qPCR) assay to detect different isolates of the IHHNV in shrimp samples was developed. The detection limit of the assay was 81 viral copies of targeted DNA per reaction. The specificity of the assay was evaluated by melting curve analysis, which showed that the IHHNV product generated a single melt peak at 81.4±0.044°C. The assay was more sensitive than conventional PCR. The standardized PCR was shown to be highly sensible, specific, robust, and reproducible, which makes it an economical and powerful tool for both diagnostic applications and general research of IHHNV.

Prevalence of the infectious hypodermal and hematopoietic necrosis virus in shrimp (Penaeus vannamei) broodstock in northwestern Mexico

The Penaeus stylirostris densovirus (PstDNV or IHHNV) is the smallest of the known shrimp viruses. It causes severe mortalities in juveniles and sub-adults of the blue shrimp Penaeus stylirostris, while specimens of the white shrimp Penaeus vannamei infected by this virus exhibit reduced growth rates and negative effects on the feed-conversion rate (FCR). To date, no descriptive epidemiological surveys on the prevalence of this virus in shrimp broodstock have been performed. In this study, the prevalence of IHHNV in broodstock of the white shrimp P. vannamei from hatcheries on the northwest of Mexico region was estimated. Prevalence vary across different regions from high (63%) to low (6%) in shrimp broodstock. Several factors, as transport of pathogens by human activities, or the absence or implementation of ineffective biosecurity measures, may explain the observed differences. To the best of our knowledge, the present study is the first to examine the prevalence of IHHNV on broodstock.