Efficacy of double-stranded RNA against white spot syndrome virus (WSSV) non-structural (orf89, wsv191) and structural (vp28, vp26) genes in the Pacific white shrimp Litopenaeus vannamei

White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. RNA interference (RNAi) is a promising tool against viral infections. Previous works with RNAi showed different antiviral efficacies depending on the silenced gene. This work evaluated the antiviral efficacy of double-stranded (ds) RNA against two non-structural (orf89, wsv191) WSSV genes compared to structural (vp26, vp28) genes to inhibit an experimental WSSV infection. Gene orf89 encodes a putative regulatory protein and gene white spot virus (wsv)191 encodes a nonspecific nuclease; whereas genes vp26 and vp28 encode envelope proteins, respectively. Molecules of dsRNA against each of the WSSV genes were intramuscularly injected (4 μg per shrimp) into a group of shrimp 48 h before a WSSV challenge. The highest antiviral activity occurred with dsRNA against orf89, vp28 and vp26 (cumulative mortalities 10%, 10% and 21%, respectively). In contrast, the least effective treatment was wsv191 dsRNA (cumulative mortality 83%). All dead animals were WSSV-positive by one-step PCR, whereas reverse-transcription PCR of all surviving shrimp confirmed inhibition of virus replication. This study showed that dsRNA against WSSV genes orf89, vp28 and vp26 were highly effective to inhibit virus replication and suggest an essential role in WSSV infection. Non-structural WSSV genes such as orf89 can be used as novel targets to design therapeutic RNAi molecules against WSSV infection

Revisión de patogénesis y estrategias moleculares contra el virus del síndrome de la mancha blanca en camarones peneidos

White spot syndrome virus (WSSV) causes high mortality to farmed shrimp and serious economic losses. Its unique sequence and genome structure has placed WSSV in its own new family Nimaviridae. Recently, high performance molecular techniques have made it possible to identify and characterize several WSSV structural proteins. These include �shotgun� sequencing and isobaric tags for relative and absolute quantification (iTRAQ). Such techniques have made it possible to characterize 14 new WSSV proteins. Location and characterization of structural proteins can help to understand WSSV morphogenesis and pathogenesis. Both processes are essential to understand the mechanism of infection and to develop novel control methods. At present no effective treatments exist to fight WSSV in the field. WSSV structural proteins such as VP28 and VP19 have been evaluated to reduce the impact of WSSV. These molecules are essential early in the infection. Neutralization assays using specific antibodies against WSSV structural proteins have shown an increased survival of treated shrimp. Recently, RNA interference (RNAi) constructs directed against structural proteins have been used as a new tool to reduce/inhibit WSSV replication. A better comprehension of the host-pathogen interaction would allow the development of new methods to control WSSV. The use of high throughput techniques to determine the location and function of structural proteins will contribute to develop new strategies against infection. Intervention strategies aimed to block virus entry into the host cells may be a valuable output from these studies.El virus del síndrome de mancha blanca (WSSV) provoca graves mortandades en granjas de cultivo de camarones peneidos y serias pérdidas económicas. La secuencia y estructura genética excepcionales de WSSV lo colocan en su propia nueva familia, Nimaviridae. Recientemente, novedosas técnicas moleculares de alto rendimiento han permitido identificar y caracterizar varias proteínas estructurales de WSSV. Estas incluyen la secuenciación por �shotgun� y marcadores isobáricos para cuantificación absoluta y relativa (iTRAQ). Dichas técnicas han permitido caracterizar 14 nuevas proteínas de WSSV. La caracterización y localización de proteínas estructurales pueden ayudar a conocer la morfogénesis y patogénesis de WSSV. Ambos procesos son esenciales para entender el mecanismo de infección y para desarrollar nuevos métodos de control. Hasta ahora no existen tratamientos efectivos para combatir este virus en campo. Proteínas estructurales de WSSV como VP28 y VP19 han sido evaluadas para reducir el impacto de WSSV. Estas moléculas son esenciales en las etapas tempranas de infección. Bioensayos de neutralización usando anticuerpos específicos contra proteínas estructurales de WSSV han aumentado la supervivencia de camarones tratados. Recientemente, construcciones de RNA de interferencia (RNAi) dirigidos contra proteínas estructurales han sido usadas como una nueva herramienta para reducir/inhibir la replicación de WSSV. Una mejor comprensión de las interacciones hospedero-patógeno permitirá desarrollar nuevos métodos para controlar este virus. La localización y función de proteínas estructurales usando métodos de alto rendimiento contribuirá a implementar nuevas estrategias contra la infección. Métodos de intervención para bloquear la entrada del virus a la célula podrían ser valiosos productos de este tipo de investigaciones

Revisión de patogénesis y estrategias moleculares contra el virus del síndrome de la mancha blanca en camarones peneidos

El virus del síndrome de mancha blanca (WSSV) provoca graves mortandades en granjas de cultivo de camarones peneidos y serias pérdidas económicas. La secuencia y estructura genética excepcionales de WSSV lo colocan en su propia nueva familia, Nimaviridae. Recientemente, novedosas técnicas moleculares de alto rendimiento han permitido identificar y caracterizar varias proteínas estructurales de WSSV. Estas incluyen la secuenciación por "shotgun" y marcadores isobáricos para cuantificación absoluta y relativa (iTRAQ). Dichas técnicas han permitido caracterizar 14 nuevas proteínas de WSSV. La caracterización y localización de proteínas estructurales pueden ayudar a conocer la morfogénesis y patogénesis de WSSV. Ambos procesos son esenciales para entender el mecanismo de infección y para desarrollar nuevos métodos de control. Hasta ahora no existen tratamientos efectivos para combatir este virus en campo. Proteínas estructurales de WSSV como VP28 y VP19 han sido evaluadas para reducir el impacto de WSSV. Estas moléculas son esenciales en las etapas tempranas de infección. Bioensayos de neutralización usando anticuerpos específicos contra proteínas estructurales de WSSV han aumentado la supervivencia de camarones tratados. Recientemente, construcciones de RNA de interferencia (RNAi) dirigidos contra proteínas estructurales han sido usadas como una nueva herramienta para reducir/inhibir la replicación de WSSV. Una mejor comprensión de las interacciones hospedero-patógeno permitirá desarrollar nuevos métodos para controlar este virus. La localización y función de proteínas estructurales usando métodos de alto rendimiento contribuirá a implementar nuevas estrategias contra la infección. Métodos de intervención para bloquear la entrada del virus a la célula podrían ser valiosos productos de este tipo de investigaciones. Palabras clave: WSSV, proteínas estructurales, secuenciación "shotgun", iTRAQ, mecanismo de infección, métodos de contro

A review on the morphology, molecular characterization, morphogenesis and pathogenesis of white spot syndrome virus (WSSV).

Since it first appeared in 1992, white spot syndrome virus (WSSV) has become the most threatening infectious agent in shrimp aquaculture. Within a decade, this pathogen has spread to all the main shrimp farming areas and has caused enormous economic losses amounting to more than seven billion US dollars. At present, biosecurity methods used to exclude pathogens in shrimp farms include disinfecting ponds and water, preventing the entrance of animals that may carry infectious agents and stocking ponds with specific pathogen-free post-larvae. The combination of these practices increases biosecurity in shrimp farming facilities and may contribute to reduce the risk of a WSSV outbreak. Although several control methods have shown some efficacy against WSSV under experimental conditions, no therapeutic products or strategies are available to effectively control WSSV in the field. Furthermore, differences in virulence and clinical outcome of WSSV infections have been reported. The sequencing and characterization of different strains of WSSV has begun to determine aspects of its biology, virulence and pathogenesis. Knowledge on these aspects is critical for developing effective control methods. The aim of this review is to present an update of the knowledge generated so far on different aspects of WSSV organization, morphogenesis, pathology and pathogenesis

In vivo titration of white spot syndrome virus (WSSV) in specific pathogen-free Litopenaeus vannamei by intramuscular and oral routes

White spot syndrome virus (WSSV) is a devastating pathogen in shrimp aquaculture. Standardized challenge procedures using a known amount of infectious virus would assist in evaluating strategies to reduce its impact. In this study, the shrimp infectious dose 50% endpoint (SID50 ml–1) of a Thai isolate of WSSV was determined by intramuscular inoculation (i.m.) in 60 and 135 dold specific pathogen-free (SPF) Litopenaeus vannamei using indirect immunofluorescence (IIF) and 1-step polymerase chain reaction (PCR). Also, the lethal dose 50% endpoint (LD50 ml–1) was determined from the proportion of dead shrimp. The median virus infection titers in 60 and 135 d old juveniles were 106.8 and 106.5 SID50 ml–1, respectively. These titers were not significantly different (p ≥ 0.05). The titration of the WSSV stock by oral intubation in 80 d old juveniles resulted in approximately 10-fold reduction in virus titer compared to i.m. inoculation. This lower titer is probably the result of physical and chemical barriers in the digestive tract of shrimp that hinder WSSV infectivity. The titers determined by infection were identical to the titers determined by mortality in all experiments using both i.m. and oral routes at 120 h post inoculation (hpi), indicating that every infected shrimp died. The determination of WSSV titers for dilutions administered by i.m. and oral routes constitutes the first step towards the standardization of challenge procedures to evaluate strategies to reduce WSSV infection

Pathogenesis of a Thai strain of white spot syndrome virus (WSSV) in juvenile, specific pathogen-free Litopenaeus vannamei

White spot syndrome virus (WSSV) causes disease and mortality in cultured and wild shrimp. A standardized WSSV oral inoculation procedure was used in specific pathogen-free (SPF) Litopenaeus vannamei (also called Penaeus vannamei) to determine the primary sites of replication (portal of entry), to analyze the viral spread and to propose the cause of death. Shrimp were inoculated orally with a low (101.5 shrimp infectious dose 50% endpoint [SID50]) or a high (104 SID50) dose. Per dose, 6 shrimp were collected at 0, 6, 12, 18, 24, 36, 48 and 60 h post inoculation (hpi). WSSVinfected cells were located in tissues by immunohistochemistry and in hemolymph by indirect immunofluorescence. Cell-free hemolymph was examined for WSSV DNA using 1-step PCR. Tissues and cell-free hemolymph were first positive at 18 hpi (low dose) or at 12 hpi (high dose). With the 2 doses, primary replication was found in cells of the foregut and gills. The antennal gland was an additional primary replication site at the high dose. WSSV-infected cells were found in the hemolymph starting from 36 hpi. At 60 hpi, the percentage of WSSV-infected cells was 36 for the epithelial cells of the foregut and 27 for the epithelial cells of the integument; the number of WSSV-infected cells per mm2 was 98 for the gills, 26 for the antennal gland, 78 for the hematopoietic tissue and 49 for the lymphoid organ. Areas of necrosis were observed in infected tissues starting from 48 hpi (low dose) or 36 hpi (high dose). Since the foregut, gills, antennal gland and integument are essential for the maintenance of shrimp homeostasis, it is likely that WSSV infection leads to death due to their dysfunctio

Standardized white spot syndrome virus (WSSV) inoculation procedures for intramuscular or oral routes

In the past, strategies to control white spot syndrome virus (WSSV) were mostly tested by infectivity trials in vivo using immersion or per os inoculation of undefined WSSV infectious doses, which complicated comparisons between experiments. In this study, the reproducibility of 3 defined doses (10, 30 and 90 shrimp infectious doses 50% endpoint [SID50]) of WSSV was determined in 3 experiments using intramuscular (i.m.) or oral inoculation in specific pathogen-free (SPF) Litopenaeus vannamei. Reproducibility was determined by the time of onset of disease, cumulative mortality, and median lethal time (LT50). By i.m. route, the 3 doses induced disease between 24 and 36 h post inoculation (hpi). Cumulative mortality was 100% at 84 hpi with doses of 30 and 90 SID50 and 108 hpi with a dose of 10 SID50. The LT50 of the doses 10, 30 and 90 SID50 were 52, 51 and 49 hpi and were not significantly different (p > 0.05). Shrimp orally inoculated with 10, 30 or 90 SID50 developed disease between 24 and 36 hpi. Cumulative mortality was 100% at 108 hpi with doses of 30 and 90 SID50 and 120 hpi with a dose of 10 SID50. The LT50 of 10, 30 and 90 SID50 were 65, 57 and 50 hpi; these were significantly different from each other (p < 0.05). A dose of 30 SID50 was selected as the standard for further WSSV challenges by i.m. or oral routes. These standardized inoculation procedures may be applied to other crustacea and WSSV strains in order to achieve comparable results among experiments

Screening for potential probiotic bacteria to reduce prevalence of WSSV and IHHNV in whiteleg shrimp (Litopenaeus vannamei) under experimental conditions

Abstract This study evaluated the effect of Pediococcus pentosaceus and Staphylococcus hemolyticus as probiotics in whiteleg shrimp Litopenaeus vannamei naturally infected with WSSV and IHHNV. All bacteria were isolated from the gut of wild brown shrimp (Farfantepenaeus californiensis). Presumptive lactic acid bacteria were characterized for hemolytic and enzymatic activity, hydrophobicity, growth, and molecular identification. Two mixtures of four isolates were tested and their effect measured on the hemocyte number, survival, and prevalence of WSSV and IHHNV. Each mixture was applied at two different concentrations in a 15-day bioassay with shrimp naturally infected with WSSV and IHHNV as determined by single and/or nested PCR. In the treated animals total hemocyte count and survival were similar to control group. All shrimp fed with bacterial mixtures showed a decrease in the prevalence of WSSV but not IHHNV. The results obtained in this preliminary study revealed a protective effect of the two bacterial mixtures against WSSV latent infections

Screening for potential probiotic bacteria to reduce prevalence of WSSV and IHHNV in whiteleg shrimp (Litopenaeus vannamei) under experimental conditions

This study evaluated the effect of Pediococcus pentosaceus and Staphylococcus hemolyticus as probiotics in whiteleg shrimp Litopenaeus vannamei naturally infected with WSSV and IHHNV. All bacteria were isolated from the gut of wild brown shrimp (Farfantepenaeus californiensis). Presumptive lactic acid bacteria were characterized for hemolytic and enzymatic activity, hydrophobicity, growth, and molecular identification. Two mixtures of four isolates were tested and their effect measured on the hemocyte number, survival, and prevalence of WSSV and IHHNV. Each mixture was applied at two different concentrations in a 15-day bioassay with shrimp naturally infected with WSSV and IHHNV as determined by single and/or nested PCR. In the treated animals total hemocyte count and survival were similar to control group. All shrimp fed with bacterial mixtures showed a decrease in the prevalence of WSSV but not IHHNV. The results obtained in this preliminary study revealed a protective effect of the two bacterial mixtures against WSSV latent infections

Effect of high water temperature (33 °C) on the clinical and virological outcome of experimental infections with white spot syndrome virus (WSSV) in specific pathogen-free (SPF) Litopenaeus vannamei

White spot syndrome virus (WSSV) is the most lethal pathogen of cultured shrimp. Previous studies done with undefined WSSV titers showed that high water temperature (32–33 °C) reduced/delayed mortality of WSSV-infected shrimp. This study evaluated the effect of high water temperature on the clinical and virological outcome of a WSSV infection under standardized conditions. Groups of specific pathogen-free Litopenaeus vannamei were challenged either by intramuscular or oral routes with a low (30 SID50) or a high (10,000 SID50) virus titer. Shrimp were kept (i) continuously at 27 °C, (ii) 30 °C or (iii) 33 °C; (iv) maintained at 33 °C before challenge and 27 °C afterwards, or (v) kept at 27 °C before challenge and 33 °C afterwards. Shrimp were maintained at the respective temperatures for 120 h before challenge and 120–144 h post challenge (hpc). Gross signs and mortality were monitored every 12 h until the end of the experiment. Dead and surviving shrimp were screened for WSSV infection (VP28-positive cells) by indirect immunofluorescence (IIF). Shrimp kept continuously at 27 °C or 30 °C, or switched to 27 °C post challenge developed gross signs within 24 hpc, first mortalities at 36–60 hpc and 100% cumulative mortality between 60 and 144 hpc depending on the virus titer. All dead shrimp were WSSV-positive. In contrast, shrimp kept at 33 °C continuously or after WSSV challenge showed no signs of disease and low mortalities (0–30%) regardless of the virus titer. Dead and surviving shrimp were WSSV-negative. Further, early virus replication was studied in two groups of shrimp: one maintained at 27 °C before and after challenge and one switched from 27 °C to 33 °C after challenge with 10,000 SID50. Immunohistochemistry (IHC) analysis showed that WSSV-positive cells were first displayed at 12 hpc in shrimp kept at 27 °C and by 24 hpc the infection became systemic. In contrast, shrimp kept at 33 °C did not display WSSV-positive cells at 12 or 24 hpc. This work confirms previous reports that high water temperature prevents the onset of disease and significantly reduces mortality of WSSV-inoculated shrimp regardless of the route of inoculation or virus titer used. This strategy may have practical applications to control WSSV in tropical shrimp farming countries