Shrimp White Spot Syndrome Virus Research Articles

Functional and genomic characterization of a novel probiotic Lactobacillus johnsonii KD1 against shrimp WSSV infection

White Spot syndrome virus (WSSV) causes rapid shrimp mortality and production loss worldwide. This study demonstrates potential use of Lactobacillus johnsonii KD1 as an anti-WSSV agent for post larva shrimp cultivation and explores some potential mechanisms behind the anti-WSSV properties. Treatment of Penaeus vannamei shrimps with L. johnsonii KD1 prior to oral challenge with WSSV-infected tissues showed a significantly reduced mortality. In addition, WSSV copy numbers were not detected and shrimp immune genes were upregulated. Genomic analysis of L. johnsonii KD1 based on Illumina and Nanopore platforms revealed a 1.87 Mb chromosome and one 15.4 Kb plasmid. Only one antimicrobial resistance gene (ermB) in the chromosome was identified. Phylogenetic analysis comparing L. johnsonii KD1 to other L. johnsonii isolates revealed that L. johnsonii KD1 is closely related to L. johnsonii GHZ10a isolated from wild pigs. Interestingly, L. johnsonii KD1 contains isolate-specific genes such as genes involved in a type I restriction-modification system and CAZymes belonging to the GT8 family. Furthermore, genes coding for probiotic survival and potential antimicrobial/anti-viral metabolites such as a homolog of the bacteriocin helveticin-J were found. Protein–protein docking modelling suggests the helveticin-J homolog may be able to block VP28–PmRab7 interactions and interrupt WSSV infection.

Smart nano generation of transgenic algae expressing white spot syndrome virus in shrimps for inner ear-oral infection treatments using the spotted hyena optimizer (SHO)-Long short-term memory algorithm

Nanotechnology offers a promising avenue to amplify the effectiveness and precision of using transgenic algae in managing WSSV in shrimp by possibly crafting nano-carriers for targeted therapeutic agent delivery or modifying algae cells at a molecular level. Leveraging the capabilities of nano-scale interventions, this study could explore innovative means to manipulate cellular processes, control biological interactions, and enhance treatment efficacy while minimizing undesirable impacts in aquatic environments. The White Spot Syndrome Virus (WSSV) is a double-stranded DNA virus with a tail and rod form that belongs to theNimaviridaefamily. There is no workable way to manage this illness at the moment. This research proposes a new model based on the Long Short-Term Memory (LSTM) and Spotted Hyena Optimizer (SHO) method to control the inner ear-oral infection, utilizing transgenic algae (Chlamydomonas reinhardtii). It is pretty tricky to modify the weight matrix in LSTM. The output will be more accurate if the weight of the neurons is exact. Histological examinations and nested polymerase chain reaction (PCR) testing were performed on the challenged shrimp every 4 h to assess the degree of white spot disease. The SHO-LSTM has shown the highest accuracy and Roc value (98.12% and 0.93, respectively) and the lowest error values (MSE = 0.182 and MAE = 0.48). The hybrid optimized model improves the overall inner ear-oral linked neurological diseases detection ratio. Additionally, with the slightest technical complexity, it effectively controls the forecast factors required to anticipate the ENT. Algal cells were found to be particularly well-suited for inner ear-oral infections, and shrimps fed a transgenic line had the best survival ratio in WSSV infection studies, with 87% of the shrimp surviving. This shows that using this line would effectively stop the spread of WSSV in shrimp populations.

Early detection and identification of white spot syndrome in shrimp using an improved deep convolutional neural network

White Spot Syndrome Virus (WSSV) is a major virus found in shrimp that causes huge economic loss in shrimp farms. A selective diagnostic approach for WSSV is required for the early diagnosis and protection of farms. This work proposes a novel recognition method based on improved Convolutional Neural Network (CNN) namely Dense Inception Convolutional Neural Network (DICNN) for diagnoses of WSSV disease. Initially, the process of data acquisition and data augmentation is carried out. The Inception structure is then used to improve the performance of multi-dimensional feature extraction. As a result, the proposed work has the highest accuracy of 97.22% when compared to other traditional models. The proposed work is targeted to Litopenaeus Vannamei (LV), and Penaeus Monodon (PM) diversities for major threats detection of White Spot Syndrome (WSS). Performance metrics related to accuracy have been compared with other traditional models, which demonstrate that our model will efficiently recognize shrimp WSSV disease.

An overview of vaccine production against shrimp White Spot Syndrome Virus, effects and the possible impact of this technology in Ecuador

Although aquaculture in Ecuador has a high economic and socio-cultural importance, pathogenic microorganisms affect the development and vitality of crustaceans, fish, and mollusks, reducing their production yields. Among these pathogens, White Spot Syndrome Virus (WSSV) is an invertebrate virus that induces high mortality, generating severe economic losses due to its wide geographical distribution and high infection rate finding the most significant devastation worldwide in the shrimp sector. Although several strategies are described to fight against WSSV, this study points to an updated overview of vaccines used against this virus, including types, effects and large-scale production ways. Thus, this research supplies an analysis of possible treatments based on vaccination to combat the WSSV caused-disease that significantly impacts the aquaculture economy and could be helpful to those working in this field. Keywords: Whispovirus, White Spot Syndrome Virus, Shrimp, virus, vaccine, production, Ecuador.

Multiple Nucleocapsid Structural Forms of Shrimp White Spot Syndrome Virus Suggests a Novel Viral Morphogenetic Pathway.

White spot syndrome virus (WSSV) is a very large dsDNA virus. The accepted shape of the WSSV virion has been as ellipsoidal, with a tail-like extension. However, due to the scarcity of reliable references, the pathogenesis and morphogenesis of WSSV are not well understood. Here, we used transmission electron microscopy (TEM) and cryogenic electron microscopy (Cryo-EM) to address some knowledge gaps. We concluded that mature WSSV virions with a stout oval-like shape do not have tail-like extensions. Furthermore, there were two distinct ends in WSSV nucleocapsids: a portal cap and a closed base. A C14 symmetric structure of the WSSV nucleocapsid was also proposed, according to our Cryo-EM map. Immunoelectron microscopy (IEM) revealed that VP664 proteins, the main components of the 14 assembly units, form a ring-like architecture. Moreover, WSSV nucleocapsids were also observed to undergo unique helical dissociation. Based on these new results, we propose a novel morphogenetic pathway of WSSV.

Investigation of the effects of cup plant (Silphium perfoliatum L.) on the growth, immunity, gut microbiota and disease resistance of Penaeus vannamei

To investigate the effects of adding different concentrations of cup plant (Silphium perfoliatum L.) to the feed on the growth performance, hepatopancreas and intestinal microstructure, gene expression, enzyme activity, as well as intestinal microorganisms and resistance to Vibrio parahaemolyticus E1 and White spot syndrome virus (WSSV) infection of the shrimp, cup plant was added to the basal feed at 1%, 3%, 5% and 7% respectively, and fed the shrimp for 6 weeks. It was found that the addition of different concentrations of cup plant could significantly improve the specific growth rate and survival rate of shrimp, reduce the feed conversion rate, and improve the resistance to V. parahaemolyticus E1 and WSSV in shrimp, with the best effect of 5% addition. The tissue sections observations showed that the addition of cup plant significantly improved the hepatopancreas and intestinal tissues of shrimp, especially in alleviating the tissue damage caused by V. parahaemolyticus E1 and WSSV infection, but too high an addition (7%) could also cause side effects on the shrimp intestinal tract. Meantime, the addition of cup plant can also increase the activity of immunodigestive-related enzymes in the hepatopancreas and intestinal tissues of shrimp, and can significantly induce the up-regulation of immune-related genes expression, and it is positively correlated with the amount of addition in a certain range. In addition, it was found that the addition of cup plant has a significant regulating effect on the intestinal flora of shrimp, which can significantly promote the growth of beneficial bacteria such as Haloferula sp., Algoriphagus sp. and Coccinimonas sp., and inhibit pathogenic bacteria Vibrio sp., such as the number of Vibrionaceae_Vibrio and Pseudoalteromonadaceae_Vibrio in the experimental group were significantly reduced, and the lowest level in the 5% addition group. In summary, the study shows that cup plant can promote the growth of shrimp, improve the resistance of shrimp to disease, and is a potential green environmental feed additive that can replace antibiotics.

Identification of a Double-β-Defensin with Multiple Antimicrobial Activities in a Marine Invertebrate.

β-Defensins are a family of cysteine-rich antimicrobial peptides that are generally monodomain. Interestingly, the avian β-defensin 11 (AvBD11) is unique, with two β-defensin motifs with a broad range of antimicrobial activities. However, a double-sized β-defensin has not been identified and functionally characterized in invertebrates. In this study, we cloned and identified a double-β-defensin in shrimp Litopenaeus vannamei (named LvDBD) and explored its potential roles during infection with shrimp pathogens Vibrio parahaemolyticus and white spot syndrome virus (WSSV). LvDBD is an atypical double-sized defensin, which is predicted to possess two motifs related to β-defensin and six disulfide bridges. The RNA interference-mediated knockdown of LvDBD invivo results in phenotypes with increased bacterial loads, rendering the shrimp more susceptible to V. parahaemolyticus infection, which could be rescued by the injection of recombinant LvDBD protein. In vitro, rLvDBD could destroy bacterial membranes and enhance hemocyte phagocytosis, possibly attributable to its affinity to the bacterial wall components LPS and peptidoglycan. In addition, LvDBD could interact with several viral envelope proteins to inhibit WSSV proliferation. Finally, the NF-κB transcription factors (Dorsal and Relish) participated in the regulation of LvDBD expression. Taken together, these results extend the functional understanding of a double-β-defensin to an invertebrate and suggest that LvDBD may be an alternative agent for the prevention and treatment of diseases caused by V. parahaemolyticus and WSSV in shrimp.

An In-silico analysis of the molecular interactions between PmCBP-VP24 and PmCBP-VP28 protein complex to understand the initial initiating events of shrimp WSSV infection

The White Spot Syndrome Virus (WSSV) has been the most pathogenic virus in the shrimp and other crustacean industries across the world in terms of production and financial benefits. Invertebrate vectors, freshwater, and sediments spread it horizontally as well as vertically from diseased brood stocks to post larvae. It is an exceedingly contagious disease that may cause entire mortality within 3–10 days of an outbreak under standard culture conditions. The sequencing and characterization of several WSSV strains has begun to reveal information regarding pathogen biology, pathogenicity, and WSSV must bind to the shrimp digestive system in order to infect it, and failure to do so results in ineffective infection. The peneaus monodon chitin binding protein (PmCBP) as well as the viral envelope proteins VP24 and VP28 are required for viruses to bind to shrimp digestive system. In this study, we have shown the molecular interactions between PmCBP-VP24 and PmCBP-VP28 complex and speculated about first steps of virus ingression in shrimps for the first time.

Validation of qPCR from a crude extract for the rapid detection of white spot syndrome virus.

White spot disease (WSD) has posed a serious threat to the China and the global shrimp aquaculture. In order to diagnose white spot syndrome virus (WSSV) early and prevent the spread and outbreak of WSD, it is necessary to establish a highly sensitive WSSV diagnosis method suitable for shrimp farming sites. In this study, a pre-amplification qPCR assay from the crude extract of samples heated lysis was established, which was further compared with the universal qPCR assay to verify the shrimp samples. The limit of detection (LOD) of pre-amplification qPCR assay and universal qPCR assay was 2.80 copies and 20.57 copies per reaction at 95% CI, respectively. It had good WSSV specificity and did not show cross-detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV), hepatopancreatic parvovirus (HPV), Enterocytozoon hepatopenaei (EHP), acute hepatopancreas necrosis disease (AHPND), necrotizing hepatopancreatitis bacteria (NHPB), and decapod iridescent virus 1 (DIV1). A total of 36 shrimp samples were detected as WSSV DNA positive by pre-amplification qPCR with crude extract from samples heated lysis and universal qPCR with DNA extraction. The diagnostic sensitivity and specificity were 97.22% (85.5 ~ 99.9%, 95% CI) and 100% (81.5 ~ 100%, 95% CI), respectively. The agreement Kappa value was 0.959 (0.879 ~ 1, 95% CI), and the analysis results were basically consistent. Eliminating the tedious steps of extracting DNA and using pre-amplified qPCR to detect WSSV in shrimp, it is a good choice for aquaculture farms.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s10499-022-00920-9.

Visual LAMP Method for Detection of White Spot Syndrome Virus in Shrimp and Crayfish

White spot syndrome virus (WSSV) has caused large economic losses to the aquaculture industry, and thus far there are no efficient therapeutic treatments available against this lethal virus. Therefore, rapid detection of white spot syndrome virus (WSSV) in shrimp is crucial. This study was conducted to establish a rapid visual detection method for WSSV in shrimp and on the basis of loop-mediated isothermal amplification (LAMP). The color change of the amplification products was observed using a fluorescence amplification curve (with the SYTO-9 fluorescent dye) and LAMP visualization (with calcein), and the method`s specificity, sensitivity, and reproducibility were analyzed. It showed that this method can specifically detect WSSV, with a detection sensitivity of 1 fg/μL and has good reproducibility and reliability. This method was used to analyze cultured shrimp and crayfish samples; among 329 suspected clinical samples, the positive diagnostic rate of WSSV was 27.96%. The infection activity of WSSV was the highest at temperatures of 20–26°C. The LAMP-based method for rapid visual detection of WSSV does not need complex experimental instruments, and an equipment with a stable heat source is sufficient for the reaction. It is easy to use and can be used at shrimp breeding sites.

Circulating Phylotypes of White Spot Syndrome Virus in Bangladesh and Their Virulence.

White Spot Syndrome Virus (WSSV) has emerged as one of the most prevalent and lethal viruses globally and infects both shrimps and crabs in the aquatic environment. This study aimed to investigate the occurrence of WSSV in different ghers of Bangladesh and the virulence of the circulating phylotypes. We collected 360 shrimp (Penaeus monodon) and 120 crab (Scylla sp.) samples from the south-east (Cox’s Bazar) and south-west (Satkhira) coastal regions of Bangladesh. The VP28 gene-specific PCR assays and sequencing revealed statistically significant (p < 0.05, Kruskal–Wallis test) differences in the prevalence of WSSV in shrimps and crabs between the study areas (Cox’s Bazar and Satkhira) and over the study periods (2017–2019). The mean Log load of WSSV varied from 8.40 (Cox’s Bazar) to 10.48 (Satkhira) per gram of tissue. The mean values for salinity, dissolved oxygen, temperature and pH were 14.71 ± 0.76 ppt, 3.7 ± 0.1 ppm, 34.11 ± 0.38 °C and 8.23 ± 0.38, respectively, in the WSSV-positive ghers. The VP28 gene-based phylogenetic analysis showed an amino-acid substitution (E→G) at the 167th position in the isolates from Cox’s Bazar (referred to as phylotype BD2) compared to the globally circulating one (BD1). Shrimp PL artificially challenged with BD1 and BD2 phylotypes with filtrates of tissue containing 0.423 × 109 copies of WSSV per mL resulted in a median LT50 value of 73 h and 75 h, respectively. The in vivo trial showed higher mean Log WSSV copies (6.47 ± 2.07 per mg tissue) in BD1-challenged shrimp PL compared to BD2 (4.75 ± 0.35 per mg tissue). Crabs infected with BD1 and BD2 showed 100% mortality within 48 h and 62 h of challenge, respectively, with mean Log WSSV copies of 12.06 ± 0.48 and 9.95 ± 0.37 per gram tissue, respectively. Moreover, shrimp antimicrobial peptides (AMPs), penaeidin and lysozyme expression were lower in the BD1-challenged group compared to BD2 challenged shrimps. These results collectively demonstrated that relative virulence properties of WSSV based on mortality rate, viral load and expression of host immune genes in artificially infected shrimp PL could be affected by single aa substitution in VP28.

A Glutathione Peroxidase Gene from Litopenaeus vannamei Is Involved in Oxidative Stress Responses and Pathogen Infection Resistance.

In shrimp, several glutathione peroxidase (GPX) genes have been cloned and functionally studied. Increasing evidence suggests the genes’ involvement in white spot syndrome virus (WSSV)- or Vibrio alginolyticus-infection resistance. In the present study, a novel GXP gene (LvGPX3) was cloned in Litopenaeus vannamei. Promoter of LvGPX3 was activated by NF-E2-related factor 2. Further study showed that LvGPX3 expression was evidently accelerated by oxidative stress or WSSV or V. alginolyticus infection. Consistently, downregulated expression of LvGPX3 increased the cumulative mortality of WSSV- or V. alginolyticus-infected shrimp. Similar results occurred in shrimp suffering from oxidative stress. Moreover, LvGPX3 was important for enhancing Antimicrobial peptide (AMP) gene expression in S2 cells with lipopolysaccharide treatment. Further, knockdown of LvGPX3 expression significantly suppressed expression of AMPs, such as Penaeidins 2a, Penaeidins 3a and anti-lipopolysaccharide factor 1 in shrimp. AMPs have been proven to be engaged in shrimp WSSV- or V. alginolyticus-infection resistance; it was inferred that LvGPX3 might enhance shrimp immune response under immune challenges, such as increasing expression of AMPs. The regulation mechanism remains to be further studied.

Shrimp protected from a virus by feed containing yeast with a surface-displayed viral binding protein

Recombinant Pichia pastoris biomass surface-expressing the viral binding protein PmRab7 (YSD-PmRab7) was prepared by fed-batch, aerobic fermentation with methanol induction for 48 h. By cell based ELISA assay, immunofluorescence and flow cytometry, 45% of the YSD-PmRab7 cells were positive for PmRab7. Freeze dried YSD-PmRab7 cells were added to formulated shrimp feed pellets at 0.25 g and 0.5 g per g feed and fed to 2 shrimp groups for 7 days prior to challenge with white spot syndrome virus (WSSV). Controls consisted of 1 shrimp group fed normal pellets and one fed pellets containing P. pastoris carrying an empty gene cassette. At 10 days post challenge, survival in the two control groups was 6.7 ± 6.6%, while it was 26.7 ± 6.6% in the 0.25 g YSD-PmRab7 group and significantly higher (p < 0.05) in the 0.5 g YSD-PmRab7 group at 46.7 ± 10.1%. Nested PCR assays and histopathological analysis revealed significantly lower WSSV replication levels in the 0.5 g YSD-PmRab7 group. The results indicated potential for development of YSD-PmRab7 cells as an oral prophylactic against WSSV in shrimp.

An invertebrate gene encoding a Mab21-containing protein involves in antiviral response through regulating the STING pathway

The cGAS-STING pathway plays essential roles in detecting cytosolic dsDNA and initiating antiviral and antibacterial responses in vertebrates. However, knowledge about its function in antiviral response of invertebrates is very limited. In the present study, a gene encoding a Mab21-containing protein, a cGAS homologue, was identified from a decapod crustacean Litopenaeus vannamei and designated as LvMab21cp. LvMab21cp was mainly distributed in intestine and hepatopancreas, showing similar expression profile with other genes in the cGAS-STING pathway, such as LvSTING and LvIRF. The expression levels of LvMab21cp, LvSTING and LvIRF were up-regulated in intestine and hepatopancreas of shrimp after white spot syndrome virus (WSSV) infection. Knockdown of LvMab21cp by dsRNA-mediated RNA interference could decrease the expression levels of its putative downstream genes, including LvSTING, LvIRF, LvVago4 and LvVago5, and enhance the in vivo propagation of WSSV in shrimp. Overexpression of LvMab21cp and LvSTING in HEK 293T cells activated the expression of mammalian IFNs upon simulation with interferon stimulatory DNA (ISD). These data suggest that LvMab21cp was a cGAS homologue, a member of the shrimp cGAS-STING pathway, and play an important role during WSSV infection. To our knowledge, this is the first report to show the role of the cGAS-STING pathway in the antiviral response of invertebrates, which will provide new insights into the innate immunity of invertebrates.

Development of a VP28-pseudotyped baculovirus expression system for efficient gene transfer in penaeid shrimps

Both efficient gene transfer and expression tools and suitable reporter genes are lacking for penaeid shrimps and their isolated cells. In this study a VP28-pseudotyped insect baculovirus expression system: Bacmid-GUS/VP28, carrying a reporter gene of GUS (β-glucuronidase) and an exogenous envelope protein of VP28 from shrimp white spot syndrome virus (WSSV), has been successfully developed for the first time and well used for efficient gene transfer and expression in adult shrimps with an extremely high infection efficiency of up to 100%, but in a tissue-specific manner because it could only infect the adult tissues of gill, heart and intestine, but not the Oka organ and muscle under the tested infection doses. The aforementioned VP28-pseudotyped recombinant baculovirus of Bacmid-GUS/VP28 could also infect the primarily cultured hemolymph cells of shrimp (Metapenaeus ensiss) and produced strong GUS signals with a relatively low efficiency of 1.21%, but failed in the primary embryonic cells of shrimp (Penaeus japonicus), showing an obvious cell-specificity. A possible explanation for the above-mentioned low infection efficiency is the occurrence of mitosis-arrest in the infected shrimp cells. In contrast, no obvious GUS signals could be detected in any of the uninfected and wild type recombinant baculovirus (Bacmid-GUS)-infected tissues and cells of adult shrimps, confirming the suitability of GUS as a reporter gene and the great contribution of VP28 to the tissue- and cell- tropism of pseudotyped recombinant baculovirus (Bacmid-GUS/VP28) in penaeid shrimps. In a word, the successful development of VP28-pseudotyped recombinant baculovirus system will provide us a useful tool for efficient gene transfer and expression in adult shrimps as well as an assay system for the examination of the contribution of different WSSV envelope proteins to the viral tissue tropism.

Protection of Caridea Against White Spot Syndrome Virus

White spot syndrome virus (WSSV) belongs to a new virus family, Nimaviridae, genus Whispovirus and contains a large circular double-stranded DNA genome of 292,967 bp. WSSV virions are ellipsoid to bacilliform, enveloped particles with a distinctive tail-like appendage at one end. They can be found throughout the body of infected shrimp. The virions contain one nucleocapsid with a typical striated appearance and 5 major and at least 13 minor proteins. WSSV, which was first discovered in Southeast Asia around 1992, is currently the most serious viral pathogen of shrimp worldwide. It causes up to 100% mortality within 7 to 10 days in commercial shrimp farms, resulting in large economic losses amounting to billions of US dollars across different countries to the shrimp farming industry. In a natural situation, shrimp become infected through both oral and water-borne routes, and the gills are thought to be a major point of viral entry. Considering the global economic and sociological importance of shrimp farming and its continued high growth, the development of novel control measures becomes necessary against the outbreak of WSSV. A number of strategies have been used to control WSSV, each with some limitations. Conventional control strategies such as improvement of environmental conditions, stocking of pathogen-free post-larvae, and augmentation of disease resistance by oral immune-stimulants or probiotics are currently employed to control WSSV infection. Use of recombinant viral proteins as vaccines that induce a specific immune response and protection has been demonstrated to control WSSV. Other studies have shown successful vaccination of shrimp with DNA vaccines that have prolonged effects. The RNA interference (RNAi) mediated silencing of targeted viral mRNAs holds tremendous potential for controlling shrimp diseases. The silencing of viruses using RNAi has been experimentally demonstrated for WSSV in shrimp by injecting or feeding synthetic siRNA, long double-stranded RNA (dsRNA), and short/long-hairpin RNA (shRNA/lhRNA) prepared by in vitro transcription or expressed in bacteria. In addition to targeting viral proteins, protection of WSSV has also been achieved by dsRNA targeted against shrimp PmRab7, a protein important for viral entry into the host cells. Antisense constructs offered strong protection in WSSV challenged shrimp, P. monodon, with a corresponding decrease in viral load. Antisense constructs expressing VP24 and VP28 offered the best protection with a consistent reduction in WSSV copy number in both cell culture and in experimental shrimp. The advantage of using antisense constructs is their lack of toxicity and immunogenicity and their high specificity towards the desired target. The usage of edible pellet feed coated with dsRNA against WSSV has shown promising results. Overall, the present investigation clearly demonstrates that it is possible to induce strong protection in shrimp against WSSV infection using host promoter-driven antisense constructs in controlled laboratory-scale experiments. However, it is important to develop a simple and efficient delivery system for extending this study to the field level.

Detection of shrimp hemocyte iridescent virus by recombinase polymerase amplification assay

Shrimp hemocyte iridescent virus (SHIV), which was first identified in white leg shrimp (Litopenaeus vannamei) in China in 2014, can cause extensive shrimp mortality and major economic losses in the shrimp farming industry in China. In this study, a novel real-time isothermal recombinase polymerase amplification (RPA) assay was developed using a TwistAmp exo kit for SHIV detection. First, five primers and a probe were designed for the major capsid protein gene (GenBank: KY681039.1) according to the TwistDx manual; next, the optimal primers were selected by a comparison experiment. The primers and probe were specific for SHIV and did not react with shrimp white spot syndrome virus (WSSV), shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp enterocytozoon hepatopenaei (EHP), and macrobrachium rosenbergii nodavirus (MrNV) samples, as well as pathogens of acute hepatopancreatic necrosis disease (AHPND). The RPA assay reached a detection limit of 11 copies per reaction according to probit regression analysis. In addition, RPA assay detected the positive plasmid samples at concentration of 1000 copies/μL within 16.04 ± 0.72 min at a single low operation temperature (39 °C). The results proved that the proposed RPA method was an accurate, sensitive, affordable, and rapid detection tool that can be suitably applied for the diagnosis of SHIV in field conditions and in resource-poor settings.

ISOLATION OF WHITE SPOT SYNDROME VIRUS (WSSV) IN EGYPTIAN SHRIMP USING CONVENTIONAL PCR AND REAL TIME PCR (QPCR) TECHNIQUES

Shrimp aquaculture industry threated by high mortality rates and severe economic losses as a result of white spot syndrome virus (WSSV) infection. Early-screening and diagnosis of WSSV are great strategies to decrease the economic losses of the disease on shrimp aquaculture. Therefore, this study was carried out to detect of white WSSV infected shrimp under using two molecular based methods, conventional PCR and qPCR. A total number of 90 samples of red (Aristeus antennatus) and gray (Penaeus latisclcatus) shrimp were collected from Kafr El-Sheikh and Alexandria governorates. External examination of shrimps collected from Kafr El-Sheikh Governorate revealed typical WSSV clinical signs (including loose and easily detached cuticle with appearance of small white spots (3 mm in diameter) and /or larger patches in the external surface of carapace and cephalothorax. The internal examination showed yellowish white, fragile and swollen hepatopancreas and swollen or shrunken lymphoid tissue. Red shrimp showed slightly obvious white spots without any internal lesions. PCR results confirmed the clinical investigation and postmortem (PM) examination and revealed presence of WSSV partial sequences with a size of 190 bp in shrimp samples from Kafr El-Sheikh Governorate. In contrast, samples collected from Alexandria (Borg El-Arab) gave negative results. The result of qPCR confirmed that obtained by conventional PCR and showed that all positive results of WSSV by conventional PCR gave cycle threshold (Ct) values ranged from 34.81 to 40.06. Our results concluded that, WSSV Diseases of shrimp attack shrimp markets of Kafr El-Sheikh Governorate. The conventional PCR and qPCR based methods for isolation and identification of shrimp WSSV, provided accurate results.Key words: WSSV; PCR; qPCR; shrimp

Design of epitope based vaccine against shrimp white spot syndrome virus (WSSV) by targeting the envelope proteins: An immunoinformatic approach

Design of epitope based vaccine against shrimp white spot syndrome virus (WSSV) by targeting the envelope proteins: An immunoinformatic approach

The Improvement and Application of Lentivirus-Mediated Gene Transfer and Expression System in Penaeid Shrimp Cells.

This study first reported the improvement and application of lentivirus-mediated gene transfer and expression system in shrimp cells. After modified by the inclusion of two envelope proteins (VP19 and VP28) of shrimp white spot syndrome virus (WSSV) into the envelope of the packaged lentivirus, and insertion of a truncated promoter of immediate-early gene 1 (Pie1-504) of shrimp WSSV virus into the lentiviral reporter plasmid, the second-generation lentiviral expression system (pLVX-PEF1α-IRES-mCherry, psPAX2, and PMD2.G) was found to behave better in the mitosis-arrested shrimp cells than the similarly modified retrovirus expression system did. Results from the insect sf9 cells indicated that the inclusion of VP19 and VP28 into the envelope of packaged lentiviruses could significantly improve the tropism or infectivity of the modified lentiviruses to insect cells in a cumulative way. Notably, the VP28 contributed about 86% of the total increase of the tropism. In the shrimp primary lymphoid cells infected by modified lentivirus IV with both VP19 and VP28 included, the infection efficiency was up to 11% (non-confocal) and 19% (confocal) and no background fluorescent signal was observed. However, background fluorescent signal was observed in the shrimp primary Oka organ cells although only under a confocal microscope. In the lentivirus IV-infected Oka organ cells, the actual infection efficiencies were calculated up to 8% (non-confocal) and 19% (confocal), significantly higher than those of commercial intact lentivirus I of 0 (non-confocal) and 3% (confocal). The insertion of WSSV promoter (Pie1-504) had interrupted the effective expression of reporter plasmid encoding lentiviral construct of pLVX-PEF1α-Pie1-504-IRES-mCherry in the HEK293T cells, but markedly increased its efficiencies up to 14% (non-confocal) and 26% (confocal) in the Oka organ cells. This improved lentivirus expression system will provide us a useful tool for efficient gene transfer and expression in shrimp cells.