Search

ABSTRACT White spot syndrome virus (WSSV) and Enterocytozoon hepatopenaei (EHP) represent the most economically destructive pathogens in the current shrimp industry. WSSV causes white spot disease (WSD) responsible for rapid shrimp mortality, while EHP stunts growth and therefore reduces overall productivity. Despite the importance of timely disease detection, current diagnostic methods for WSSV and EHP are typically singleplex, and those offering multiplex detection face issues such as complexity, low field compatibility and/or low sensitivity. Here, we introduce an orthogonal, multiplex CRISPR‐Cas assay for concomitant detection of WSSV and EHP. This method combines recombinase polymerase amplification (RPA) for target DNA enrichment with Cas12a and Cas13a enzymes for fluorescent detection. This assay produces distinct fluorescent colours for different diagnostic outcomes, allowing naked eye visualisation without ambiguity. Further validation reveals that the assay detects as few as 20 and 200 copies of target DNA from EHP and WSSV, respectively, while producing no false positives with DNA from other shrimp pathogens. Moreover, the assay excellently agrees with established PCR methods in evaluation of clinical samples. Requiring only 37°C and less than an hour to complete, multiplex CRISPR‐Cas assay presents a promising tool for onsite diagnostics, offering high accuracy while saving time and resources. [ABSTRACT FROM AUTHOR]

Procambarus clarkii is commonly known as crayfish and has become one of the main species of freshwater aquaculture in China because of its delicious meat and strong adaptability to the environment. The incredible demand promotes the rapid development of the crayfish breeding industry. Viral diseases caused by white spot syndrome virus (WSSV) are widely spread in crustaceans, including P. clarkii. WSSV has become a serious threat to the crayfish breeding industry because of its extremely fast transmission and associated high mortality. Virus infection can directly induce autophagy mechanisms. Autophagosomes can wrap virus particles and transport them to lysosomes for degradation. As a highly conserved cellular defense mechanism, autophagy plays an important role in the regulation of virus infections. However, many viruses have evolved special mechanisms to resist autophagy regulation or use the membrane structure produced by autophagy body formation to complete their own replication. In this study, WSSV in susceptible P. clarkii were explored to determine how autophagy related genes of P. clarkii participate in the regulation of virus infection. To study the role of the autophagy related gene (Atg2) in the innate immunity of P. clarkii, the full-length sequence of the Atg2 gene in P. clarkii (named PcAtg2) was cloned using the total RNA of P. clarkii hepatopancreas as a template with the rapid-amplification of cDNA ends technique (RACE). The bioinformatic analysis showed that the total length of the PcAtg2 gene sequence in P. clarkii was 9 966 bp, including a 582 bp 5' non coding region, 2 817 bp 3' non coding region, and 6 567 bp open reading frame. We speculate it encodes 2 189 amino acids. Multiple sequence alignments showed the PcAtg2 gene had the characteristic sequence of the Atg family, with 65 serine phosphorylation sites, and 48 glycosylation sites. The amino acid sequence of PcAtg2 in P. clarkii had the highest homology with the Homarus americanus Atg2 gene. The distribution of the PcAtg2 gene in the gill, heart, midgut, hepatopancreas, stomach, muscle, hemocyte, epidermis, testis, ovary, abdominal ganglion, and eyestalk of P. clarkii were detected by real-time fluorescence quantitative PCR (RT-qPCR). The results showed that there was no significant difference in the expression of the PcAtg2 gene between male and female individuals. However, there were variations in expression in the different tissues. PcAtg2 was expressed in all tissues of P. clarkii, with the highest expression in the hepatopancreas and the lowest expression in the eyestalk. Under WSSV infection, PcAtg2 was initially up-regulated and then down-regulated in the different tissues, after induced expression. These findings suggest that PcAtg2 is involved in the regulation of autophagy in P. clarkii infected with the WSSV virus, and also plays an important regulatory role in the immune response. RNA interference (RNAi) technology was used to further explore the autophagy related genes PcAtg2 of P. clarkii and their role in WSSV infection. In the WSSV infection experiment with P. clarkii, the copy number of the WSSV virus in the dsPcAtg2 injection group was significantly lower than that in the control group and the dsGFP injection group, indicating that the replication of the WSSV virus was inhibited to some extent during the gene silencing of PcAtg2. The mortality results also showed that silencing PcAtg2 could reduce the mortality of P. clarkii infected with WSSV. In this experiment, after PcAtg2 was silenced, the transmission electron microscope images showed that after 24 and 48 hours of WSSV stress, autophagy vacuoles began to appear in the lysosomes in the hepatopancreas of P. clarkii in the control group, the injected dsPcAtg2 group, and the dsGFP injected group. More autophagosomes appeared and accumulated near the nucleus, indicating that P. clarkii can activate the regulation of cell autophagy under WSSV stress. Among them, more autophagosomes appeared in the hepatopancreas of P. clarkii in the dsPcAtg2 injection group, indicating the PcAtg2 gene promoted the formation of autophagosomes. WSSV virus proliferation can take advantage of autophagy. To avoid using the virus, cells will relatively down regulate the expression of autophagy related genes, and reduce the level of autophagy. In this experiment, by silencing the expression of the PcAtg2 gene, P. clarkii can promote autophagy regulation by up-regulating the expression of other autophagy related genes. In conclusion, the full-length sequences of the autophagy related gene PcAtg2 in P. clarkii were obtained for the first time, allowing us to reveal the effect and mechanism of WSSV infection on autophagy in P. clarkii. The effect of regulating autophagy on WSSV replication was analyzed, and the mechanism of the PcAtg2 gene acting on virus replication by regulating the formation of autophagosome was clarified. The PcAtg2 gene plays an important role in anti-virus immune defense in P. clarkii. We provide a theoretical basis for investigating anti-virus strategies from the perspective of autophagy. Further research on the host defense mechanism regulated by autophagy will provide new antiviral strategies.

Circular, viral copy DNA (cvcDNA) can reveal the existence of endogenous viral elements (EVE) in shrimp genomic DNA. Here we describe isolation and sequencing of cvcDNA from a breeding stock of the whiteleg shrimp Penaeus vannamei. The stock was developed by onward breeding and selection for white spot syndrome virus (WSSV)-free individual survivors of white spot disease outbreaks. The stock exhibits high tolerance to WSSV. A pool of DNA extracted from 10 shrimp from this stock was subjected to cvcDNA isolation and amplification followed by high throughput sequencing. This revealed DNA fragments corresponding to locations covering much of the 300,000 bp WSSV genome. However, high frequency-read-fragments (HFRF) mapped to a surprisingly small region of approximately 1400 bp. We hypothesized that the HFRF reflected their selection due to provision of tolerance to WSSV. Four PCR primer sets were designed to cover the 1365 bp mapped region. One pair (Set 1) targeted the whole 1365 bp mapped region, while another 3 primer sets (Set 2–4), targeted regions within the 1365 bp target. All 4 primer sets were used with DNA samples from each of 36 shrimp from the same breeding stock (including the 10 used for cvcDNA preparation). Individual positive PCR results varied from shrimp to shrimp, ranging from only 1 primer set up to 4 primer sets. Only 1 specimen gave an amplicon of 1365 bp, while others gave single to multiple positive amplicons in both continuous and discontinuous regions. This indicated that the amplicons did not arise from contiguous targets and might vary in insertion length. The results also confirmed that none of the shrimp were infected with WSSV. Sequencing of the PCR amplicons of expected sizes revealed sequence identity to extant WSSV genomes. This data will be used to screen the breeding stock for EVE that provide WSSV tolerance.

Glycerol monolaurate (GML), one of the medium-chain fatty acid esters, is often used as an emulsifier or preservative. Its biological functions include antibacterial and antiviral activities. In this study, we examined the effects of dietary GML on the resistance of the red claw crayfish to WSSV infection. Crayfish fed with 4 g/kg GML showed higher survival rate and lower WSSV copy numbers than the control after WSSV infection. A RT–qPCR analysis showed that GML supplementation enhanced the expression of immune-related genes, especially JAK and caspase. Our data indicate that GML affects the immune parameters of crayfish, including the total hemocyte counts and phenoloxidase, acid phosphatase, superoxide dismutase, lysozyme, and peroxidase activities. After treatment with GML, the apoptosis of hemocytes increased significantly in both WSSV-infected and uninfected crayfish. In summary, GML reduced the mortality of WSSV-infected crayfish, perhaps by modulating the innate immunity of the crayfish. Our study shows that GML can be used to induce the innate immunity and enhance the immune protection of the red claw crayfish against WSSV infection, either therapeutically or as a preventive measure. • Glycerol monolaurate supplement significantly reduced the mortality caused by WSSV infection. • Glycerol monolaurate significantly reduced the rate of hemocyte apoptosis in crayfish. • The WSSV copy numbers were reduced by glycerol monolaurate supplement. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This study aimed to select high-quality promoters to construct trans- vp28 gene Anabaena sp. PCC7120 and feed Litopenaeus vannamei to assess the effect of L.vannamei against white spot syndrome virus (WSSV). Transgenic algae were created using five plasmids containing PrbcL, Pcpc560, Ptrc, Ptac, and PpsbA. According to the gene expression efficiency and the growth index of transgenic algae, Pcpc560 was determined to be the most efficient promoter. Shrimps were continuously fed trans- vp28 gene Anabaena sp. PCC7120 for one week and then challenged with WSSV. After the challenge, the transgenic algae group (vp28 -7120 group) was continuously immunized [continuous immunization for 0 days (vp28 -7120-0d); continuous immunization for 2 days (vp28 -7120-2d); continuous immunization for 4 days (vp28 -7120-4d)]. After seven days, the daily survival rate of each experimental group was continuously tracked. Following the viral challenge, the hepatopancreas samples were assayed for their levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), thioredoxin peroxidase (TPX), acid phosphatase (ACP), and alkaline phosphatase (AKP) at varying time intervals. In comparison to the positive control group (challenge and no vaccination) and the wild-type group (challenge, fed wild-type Anabaena sp. PCC7120), the vp28 -7120 group (challenge, fed trans- vp28 gene Anabaena sp. PCC7120) exhibited a remarkable increase in survival rates, reaching 50 % (vp28 -7120-0d), 76.67 % (vp28 -7120-2d), and 80 % (vp28 -7120-4d). Furthermore, the vp28 -7120 group consistently displayed significantly higher activities of SOD, CAT, GSH-Px, ACP, and AKP, while exhibiting notably lower TPX activity, when compared to the control group. These results indicate that the Pcpc560 promoter effectively elevated the expression level of the exogenous vp28 gene and spurred the growth of the trans- vp28 gene Anabaena sp. PCC7120. Consequently, trans- vp28 gene Anabaena sp. PCC7120 significantly bolstered the immunity of L.vannamei. Therefore, utilizing the Pcpc560 promoter to develop trans- vp28 gene Anabaena sp. PCC7120 based oral vaccine is highly beneficial for industrial-scale cultivation, advancing its commercialization prospects. [Display omitted] • Construction of Pcpc560-trans- vp28 Anabaena sp. PCC 7120 for the first time. • Promoter Pcpc560 promoted the expression of vp28 and the growth of transgenic algae. [ABSTRACT FROM AUTHOR]

Since the mechanisms by which cellular factors modulate replication of the shrimp viral pathogen white spot syndrome virus (WSSV) are still largely unknown, here we consider the sirtuins, a family of NAD+-dependent protein deacetylases that are known to function as regulatory factors that activate or suppress viral transcription and replication in mammals. In particular, we focus on SIRT1 by isolating and characterizing LvSIRT1 from white shrimp (Litopenaeus vannamei) and investigating its involvement in WSSV infection. DsRNA-mediated gene silencing led to the expression of WSSV genes and the replication of genomic DNAs being significantly decreased in LvSIRT1 -silenced shrimp. The deacetylase activity of LvSIRT1 was significantly induced at the early stage (2 hpi) and the genome replication stage (12 hpi) of WSSV replication, but decreased at the late stage of WSSV replication (24 hpi). Treatment with the SIRT1 activator resveratrol further suggested that LvSIRT1 activation increased the expression of several WSSV genes (IE1 , VP28 and ICP11). Lastly, we used transfection and dual luciferase assays in Sf9 insect cells to show that while the overexpression of LvSIRT1 facilitates the promoter activity of WSSV IE1, this enhancement of WSSV IE1 expression is achieved by a transactivation pathway that is NF-κB-independent. • CDNA encoding SIRT1 was identified and characterized from white shrimp (Litopenaeus vannamei). • LvSIRT1 deacetylase activity was induced at the early stage and genome replication stage of WSSV replication. • LvSIRT1 silencing led to the significant decrease in WSSV genes and WSSV genomic DNAs. • LvSIRT1 activation by SIRT1 activator resveratrol increased the expression of several WSSV genes (IE1 , VP28 and ICP11). • Shrimp SIRT1 activates of the WSSV IE1 promoter independently of the NF-κB binding site. [ABSTRACT FROM AUTHOR]

Nanotechnology a multidisciplinary field involves the design and production of functional systems at the molecular level. In aquaculture, the application of nanotechnology it's still at infant stage and it potent enough to solve many issues related to nutrition, animal production, reproduction, disease diagnosis, prevention and treatment. Worldwide, during the last decade though shrimp culture has been one of the most prevalent practices in marine industry and it has been threatened by viral diseases frequently. Among various shrimp viral pathogens, white spot syndrome virus (WSSV) is exceedingly pathogenic and conscientious for huge economic loss in shrimp culture industry. In this review, the application of nanotechnology in diagnosis and management of WSSV in aquaculture is discussed in detail. [ABSTRACT FROM AUTHOR]

White spot syndrome virus (WSSV) is a notorious virus infecting cultured shrimps affecting the aquaculture industry for more than two decades over the world. WSSV has been isolated from asymptomatic carriers including crabs, artemia, rotifers, polychaetes and insects. Asymptomatic carriers are hosts that do not exhibit infection symptoms but carries infection to other hosts. The potential virulence of asymptomatic carrier of WSSV in multiple hosts were screened in the current study. The symbiotic Octolasmis neptuni isolated from the gill lamellae of moribund mud crab (Scylla olivacea) is suspected to cause WSSV infection. Though there was morphological similarity between healthy and infected animals, the PCR result was positive for WSSV with an amplicon size of 941 base pairs. The amplicon was sequenced and the infection was confirmed. Hypertrophied nuclei, marginalised chromatin and virions of WSSV were observed in the nuclear cells and cytoplasm through transmission electron microscope (TEM). Histopathological study on the pedunculate barnacle showed basophilic inclusion bodies and hypertrophied nuclei. Further series of infectivity experiments were conducted to understand the virulence of the O. neptuni. [ABSTRACT FROM AUTHOR]

White spot syndrome virus has been a threat to the global shrimp industry since it was discovered in Taiwan in 1992. Thus, shrimp‐producing countries have launched regulations to prevent import of WSSV‐infected commodity shrimp from endemic areas. Recently, cooked shrimp that is infected with WSSV tested positive by PCR. However, there is no study to determine the infectivity of WSSV in cooked shrimp that tested positive by PCR. In the present study, WSSV‐infected shrimp were cooked at boiling temperature for different times including 0, 1, 3, 5, 10 and 30 min. Upon exposure to boiling temperature, WSSV‐infected shrimp were fed to SPF shrimp (Litopenaeus vannamei). The result showed experimentally challenged shrimp from 0‐min treatment (positive control) indeed got infected with WSSV. However, experimentally challenged shrimp that were fed tissues boiled at 1, 3, 5, 10 and 30 min were not infected with WSSV. Mortality data showed that only the positive control (0‐min) treatment displayed high mortality, whereas no mortality was observed in any other treatment category. These findings suggest that cooking shrimp at boiling temperature for at least 1 min might prevent any potential spread of WSSV from endemic countries to other geographical areas where WSSV has not yet been reported. [ABSTRACT FROM AUTHOR]

The outbreak of white spot disease (SD) caused by White Spot Syndrome Virus (WSSV) has resulted in significant losses in crustacean aquaculture. However, effective measures to control this infectious viral disease are currently lacking. In this study, we screened 15 medicinal plants for their inhibitory activity against WSSV. The results showed that extracts from Paeonia × suffruticosa Andr. (Mu Dan) and Salvia miltiorrhiza Bunge (Dan Shen) exhibited good inhibitory activity against WSSV, with their main component paeonol showing high activity (92.53%, 75 mg/kg). Paeonol demonstrated concentration-dependent reductions in viral load and viral gene transcription levels, leading to enhanced survival rates in WSSV-infected crayfish across various administration routes. Co-incubation experiments demonstrated that paeonol did not affect the infectivity of WSSV particles, but it exhibited significant inhibitory effects on the viral load in crayfish before and after treatment. Mechanistically, paeonol regulated the host's immune function by balancing oxidative stress, reducing inflammatory damage, and normalizing protein levels, thereby inhibiting WSSV replication in various organs of crayfish. Moreover, in a static cohabitation model, paeonol effectively hindered the horizontal transmission of WSSV from infected donor crayfish to healthy individuals. In conclusion, considering the simple structure and ease of synthesis of paeonol, this study provides important theoretical evidence for the development of paeonol as an environmentally friendly and cost-effective drug for controlling WSSV outbreaks in crustacean aquaculture. • Paeonol, a bioactive constituent of P. suffruticosa and S. miltiorrhiza is a potent WSSV inhibitor. • Paeonol inhibits viral replication and improves survival of WSSV-infected crayfish. • Paeonol modulates the host's immune function. • Paeonol effectively suppresses horizontal transmission of WSSV to protect crayfish. [ABSTRACT FROM AUTHOR]

White Spot Disease is one of the most harmful diseases of the red tail shrimp, which can cause devastating economic losses due to the highest mortality up to 100% within a few days. MicroRNAs (miRNAs) are large class of small noncoding RNAs with the ability to post-transcriptionally repress the translation of target mRNAs. MiRNAs are considered to have a significant role in the innate immune response of crustaceans, particularly in relation to antiviral defense mechanisms. Numerous crustacean miRNAs have been verified to be required in host immune defense against viral infection, however, till present, the miRNAs functions of F. penicillatus defense WSSV infection have not been studied yet. Here in this study, for the first time, miRNAs involved in the F. penicillatus immune defense against WSSV infection were identified using high-throughput sequencing platform. A total of 432 miRNAs were obtained including 402 conserved miRNAs and 30 novel predicted miRNAs. Comparative analysis between the WSSV-challenged group and the control group revealed differential expression of 159 microRNAs in response to WSSV infection. Among these, 48 were up-regulated and 111 were down-regulated. Ten candidate MicroRNAs associated with immune activities were randomly selected for qRT-PCR analysis, which confirming the expression profiling observed in the MicroRNA sequencing data. As a result, most differentially expressed miRNAs were down-regulated lead to increase the expression of various target genes that mediated immune reaction defense WSSV infection, including genes related to signal transduction, Complement and coagulation cascade, Phagocytosis, and Apoptosis. Furthermore, the genes expression of the key members in Toll and Imd signaling pathways and apoptotic signaling were mediated by microRNAs to activate host immune responses including apoptosis against WSSV infection. These results will help to understand molecular defense mechanism against WSSV infection in F. penicillatus and to develop an effective WSSV defensive strategy in shrimp farming. • First miRNAs sequencing of red tail shrimp aimed to characterize immune response involved in WSSV infection. • Most differentially expressed miRNAs were down-regulated increase target gene expression for antivirus immune defense. • The genes expression in apoptotic signaling was mediated by microRNAs to active host cell death against WSSV infection. [ABSTRACT FROM AUTHOR]

The outbreak of diseases ordinarily results from the disruption of the balance and harmony between hosts and pathogens. Devoid of adaptive immunity, shrimp rely largely on the innate immune system to protect themselves from pathogenic infection. Two nuclear factor-κB (NF-κB) pathways, the Toll and immune deficiency (IMD) pathways, are generally regarded as the major regulators of the immune response in shrimp, which have been extensively studied over the years. Bacterial infection can be recognized by Toll and IMD pathways, which activate two NF-κB transcription factors, Dorsal and Relish, respectively, to eventually lead to boosting the expression of various antimicrobial peptides (AMPs). In response to white-spot-syndrome-virus (WSSV) infection, these two pathways appear to be subverted and hijacked to favor viral survival. In this review, the recent progress in elucidating microbial recognition, signal transduction, and effector regulation within both shrimp Toll and IMD pathways will be discussed. We will also highlight and discuss the similarities and differences between shrimps and their Drosophila or mammalian counterparts. Understanding the interplay between pathogens and shrimp NF-κB pathways may provide new opportunities for disease-prevention strategies in the future.

White Spot Disease (WSD) caused by the White Spot Syndrome Virus (WSSV) is the most devastating viral disease threatening the shrimp culture industry worldwide, including Madagascar. WDS was first reported on the island in 2012; however, little is known about the circulation of the virus and its genetic diversity. Our study aimed at describing the molecular diversity and the spread of WSSV in the populations of Madagascan crustaceans. Farmed and wild shrimps were collected from various locations in Madagascar from 2012 to 2016 and were tested for WSSV. Amplicons from positive specimens targeting five molecular markers (ORF75, ORF94, ORF125, VR14/15 and VR23/24) were sequenced for genotyping characterizations. Four genotypes were found in Madagascar. The type-I genotype was observed in the south-west of Madagascar in April 2012, causing a disastrous epidemic, then spread to the North-West coast. Type-II strains were detected in October 2012 causing an outbreak in another Penaeus monodon farm. In 2014 and 2015, types II and III were observed in shrimp farms. Finally, in 2016, types II and IV were found in wild species including Fenneropenaeus indicus, Metapenaeus monoceros, Marsupenaeus japonicus and Macrobrachium rosenbergii. Considering the economic importance of the shrimp industry for Madagascar, our study highlights the need to maintain WSSV surveillance to quickly take appropriate countermeasures in case of outbreak and to sustain this industry.

The survival and immune responses of Litopenaeus vannamei were evaluated during white spot syndrome virus (WSSV) or Vibrio parahaemolyticus single and concurrent infections. The mortality, WSSV load, activities of 4 immune enzymes: acid phosphatase (ACP), alkaline phosphatase (AKP), peroxidase (POD) and superoxide dismutase (SOD), and the transcription of Evolutionarily Conserved Signaling Intermediate in Toll pathways of L.vannamei (LvECSIT) were quantified at 0, 3, 6, 12, 24, 48, 72 and 96 h post-infection (pi). The results showed: (i) the cumulative mortality of the co-infection group (WSSV and V. Parahaemolyticus 83%) was significantly lower than the WSSV infection group (97%) (P < 0.05) at 96 hpi; (ii) copies of WSSV in the co-infection group were significantly lower than that of the single infection group from 24 to 96 hpi (P < 0.05); (iii) ACP, AKP,POD and SOD activity in the gills of the co-infection group was higher than that of the WSSV group at12, 48 and 96 hpi (P < 0.05).The expression of LvECSIT mRNA in the co-infection group was significantly higher than in the WSSV infection group from 12 to 72 hpi (P < 0.05).The results indicate that proliferation of WSSV is inhibited by V.parahaemolyticus infection. In addition, infection with WSSV alone causes a significant reduction in some immune responses of shrimp than co-infection with WSSV and V.parahaemolyticus occurs at 26 °C. Third, LvECSIT, an essential member of TLR signaling pathway might play a crucial role in shrimp defense against WSSV – Vibrio co-infection. • Shrimp in co-infection groups suffered lower mortality than WSSV group. • The amount of WSSV in co-infection group was lower than in WSSV group. • ACP and AKP enzyme activity can be used as indicators to co-infection. • The transcription level of LvECSIT was up-regulated in co-infection groups. [ABSTRACT FROM AUTHOR]

Viral diseases are a serious issue for the shrimp aquaculture industry. White spot syndrome virus (WSSV) has been considered one of the most dangerous pathogens infecting cultured shrimp, causing a mortality rate as high as 100% within 7–10 days of viral infection. So far, several protocols have been applied to protect shrimp against virus attacks, but their protection efficiency is very limited. In this study, for the first time, three melanin-containing feedstuffs (F1, F2, and F3) were formulated and fed to cultured shrimp to investigate the ability of melanin to protect shrimp from WSSV. The obtained results showed that F2 had a protection rate of 64% at day 7 and 62% at day 10 after virus challenge. The protection ability of the feedstuff depended on the amount of melanin consumed by shrimp. Moreover, our results also demonstrated that the transcription level of the VP28 gene, which codes for the VP28 protein, a representative for the presence of WSSV, was significantly decreased in shrimp fed F2. Taken together, our study suggests that melanin-containing diets may be applied in aquaculture to protect shrimp against WSSV infection; further, combined protocols with the simultaneous use of melanin-containing diets and other protectants should be studied and applied to increase the protection efficiency. [ABSTRACT FROM AUTHOR]

The outbreak of diseases ordinarily results from the disruption of the balance and harmony between hosts and pathogens. Devoid of adaptive immunity, shrimp rely largely on the innate immune system to protect themselves from pathogenic infection. Two nuclear factor-κB (NF-κB) pathways, the Toll and immune deficiency (IMD) pathways, are generally regarded as the major regulators of the immune response in shrimp, which have been extensively studied over the years. Bacterial infection can be recognized by Toll and IMD pathways, which activate two NF-κB transcription factors, Dorsal and Relish, respectively, to eventually lead to boosting the expression of various antimicrobial peptides (AMPs). In response to white-spot-syndrome-virus (WSSV) infection, these two pathways appear to be subverted and hijacked to favor viral survival. In this review, the recent progress in elucidating microbial recognition, signal transduction, and effector regulation within both shrimp Toll and IMD pathways will be discussed. We will also highlight and discuss the similarities and differences between shrimps and their Drosophila or mammalian counterparts. Understanding the interplay between pathogens and shrimp NF-κB pathways may provide new opportunities for disease-prevention strategies in the future. [ABSTRACT FROM AUTHOR]

Abstract As an important disulfide reductase of the intracellular antioxidant system, Thioredoxin (Trx) plays an important role in maintaining oxidative stress balance and protecting cells from oxidative damage. In recent years, there is increasing evidence that Trx is a key molecule in the pathogenesis of various diseases and a potential therapeutic target for major diseases including lung, colon, cervical, gastric and pancreatic cancer. However, few knowledge is known about the function of Trx in virus infection. In this study, we reported the cloning and functional investigation of a Trx homologue gene, named MjTrx , in shrimp Marsupenaeus japonicus suffered white spot syndrome virus (WSSV) infection. Mj Trx is a 105-amino acid polypeptide with a conservative Cys-Gly-Pro-Cys motif in the catalytic center. Phylogenetic trees analysis showed that Mj Trx has a higher relationship with Trx from other invertebrate and clustered with Trx1 from arthropod. MjTrx transcripts is abundant in the gill and intestine tissues and can be detected in the hemocytes, heart, stomach, and hepatopancreas tissues. The transcription levels of MjTrx in hemocytes, gills and intestine tissues of shrimp were significantly up-regulated after white spot syndrome virus infection. Mj Trx was recombinant expressed in vitro and exhibited obvious disulfide reductase activity. In addition, overexpression Mj Trx in shrimp resulted in the increase of hydrogen peroxide (H 2 O 2) concentration in vivo. All these results strongly suggested that Mj Trx functioned in redox homeostasis regulating and played an important role in shrimp antiviral immunity. Highlights • A Trx homologue gene (Mj Trx)was cloned from Marsupenaeus japonicus and clustered with Trx1 from arthropod. • Mj Trx transcription is widely distributed and abundant in gills and intestine tissues. • Mj Trx was recombinant expressed and exhibited obvious disulfide reductase activity. • Overexpression Mj Trx in shrimp resulted in the increase of hydrogen peroxide (H 2 O 2) concentration in vivo. [ABSTRACT FROM AUTHOR]

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. • Nanotechnology amplifies transgenic algae's efficacy against WSSV. • SHO-LSTM model yields 98.12% accuracy in managing infections. • Transgenic algae boosts shrimp survival to 87% amidst WSSV challenge. • Optimized model enhances ENT-linked neurological disease detection. • PCR and histology confirm enhanced efficacy of algae in WSSV control. [ABSTRACT FROM AUTHOR]

The rapid development of crustacean aquaculture is threatened by white spot syndrome virus (WSSV), which is one of the deadliest pathogens affecting various crustacean species. Based on antiviral plant screening, we found that chlorogenic acid (CGA), a polyphenolic component of Arctium lappa Linn., possessed strong activity against WSSV infection in a red swamp crayfish (Procambarus clarkii) model. The results revealed that CGA dose-dependently inhibited WSSV proliferation in the gill and haemocytes with the inhibition rates (50 mg/kg) were 94.7% and 95.03%, respectively. Through a serial of mechanical experiments, we found that CGA arrested WSSV replication by regulating innate immune defense to reduce viral gene transcription, inducing apoptosis to restrict viral spread, and enhancing anti-inflammatory and antioxidant activities to alleviate oxidative and inflammatory damage caused by WSSV. Significant improvements in survival of WSSV infected crayfish have been made through injection and dietary administration of CGA compared to control group. In a static cohabitation model, pre-incubation of CGA suppressed the horizontal transmission of WSSV between infected donor crayfish and health crayfish. Thus, we have identified CGA as a potent WSSV inhibitor with high potential for further development as prophylaxis and therapy agents against WSSV outbreak in crustacean aquaculture. • Chlorogenic acid, a main bioactive constituent of Arctium lappa Linn. is a potent WSSV inhibitor; • Chlorogenic acid inhibits viral replication and improves survival of WSSV-infected crayfish; • Chlorogenic acid regulates a variety of antiviral immune factors; • Chlorogenic acid effectively suppresses horizontal transmission of WSSV to protect crayfish. [ABSTRACT FROM AUTHOR]

White spot syndrome virus (WSSV) causes severe mortality in farmed shrimps, besides causing infection in a wide range of crustaceans. Among the hosts, mud crabs are reported to be relatively resistant to the virus. To validate this, the present study investigated the susceptibility of two species of mud crabs, Scylla olivacea and S. serrata compared with shrimp, Penaeus vannamei to WSSV through experimental infection. Initially, to find out the pathogenicity of the virus, a quantified, 10-fold serially diluted WSSV inoculum was injected intramuscularly into shrimp and mud crabs. Cumulative mortality of 100% was observed in shrimp injected with 10−1 and 10−2 WSSV dilution at 5 and 6 day post-injection (dpi), respectively. However, injection of the virus inoculum of 10−1 dilution resulted in 100% and 85% cumulative mortality at 7 dpi in S. olivacea and S. serrata, respectively. The LD50 (lethal dose 50% endpoint) value of WSSV in P. vannamei, S. olivacea and S. serrata was determined to be 1.74 × 105, 3.80 × 106 and 8.51 × 106 viral copies per animal, respectively. PCR amplification of DNA extracted from different tissues of the experimental animals at 24, 48 and 72 h post-injection (hpi) confirmed the infection. A SYBR green-based real-time PCR assay revealed comparatively higher viral load in shrimp tissues (4.1 × 103, 2.8 × 106 and 4.3 × 107 copies/ 100 ng DNA in stomach, 1.0 × 103, 2.4 × 106 and 3.5 × 107 copies/ 100 ng DNA in gill and 3.5 × 102 , 1.1 × 104 and 1.1 × 105 copies/ 100 ng DNA in hepatopancreas at 24, 48 and 72 h, respectively). The viral load in S. olivacea was estimated to be 2.9 × 103, 2.2 × 106 and 3.0 × 107 copies/ 100 ng DNA in the stomach, 7.1 × 102, 1.6 × 106 and 2.4 × 107 copies/ 100 ng DNA in gill and 2.0 × 102, 8.6 × 103& 7.8 × 104 copies/ 100 ng DNA and in S. serrata the viral copy numbers detected were 1.1 × 103, 1.7 × 106 and 1.8 × 107 copies/ 100 ng DNA in stomach, 6.6 × 102, 1.3 × 106 and 1.4 × 107 copies/ 100 ng DNA in gill and 5.8 × 101, 7.7 × 102 & 1.1 × 104 copies/ 100 ng DNA at 24, 48 and 72 h, respectively. A higher viral load was detected in the stomach tissue compared to the gill and hepatopancreas of all the experimental animals. Histological examination of the stomach and gill of P. vannamei, S. olivacea and S. serrata at different time points post-infection revealed characteristic WSSV pathology. Hepatopancreatic tubular epithelium showed no viral inclusions, though pathological changes could be noticed in the intertubular connective tissue. The most pronounced changes were found in the stomach epithelium of P. vannamei and S. olivacea, which revealed significant nuclear hypertrophy and large-sized inclusions, compared to S. serrata which showed relatively smaller-sized inclusion bodies. • The study compared the susceptibility of mud crabs, Scylla serrata and S. olivacea to WSSV. • S. serrata was less susceptible to WSSV compared to S. olivacea and P. vannamei. • Molecular and histological evidence suggests different patterns of WSSV infection in three different crustacean hosts. [ABSTRACT FROM AUTHOR]

Introduction: White spot syndrome virus (WSSV) is one of the most deadly infectious pathogens of the shrimp culture industry. Neither effective vaccines nor efficient treatments are currently available for this disease. Vibrio species are well known dominant bacterial pathogens in the shrimp ponds. As facultative pathogenic bacteria, it is possible that Vibrio spp. along with WSSV to co-infect the shrimp species such as Litopenaeus vannamei. The aim of this study was to investigate the co-administration of gamma-irradiated Vibrio paraheomolyticus as a kind of probiotic and immune-stimulator with gamma-irradiated and inactivated WSSV as an intramuscular vaccine for protection of L. vannamei against WSSV infection. Methods: WSSV was isolated from the infected shrimp samples and multiplied in Astacus leptodactylus crayfish. Titration of WSSV was obtained in post-larvae as 105.4 LD50/ml. The virus was irradiated where D10 value and optimum dose of gamma ray were calculated to be 2.56 and 15 kGy, respectively. The gamma-irradiated WSSV samples, named GI-WSSV were used as a vaccine to immunize L. vannamei shrimps. The freeze-dried V. paraheomolyticus cultures, inactivated by gamma ray (8 kGy) were named GI-V.P and used as a probiotic. Results: Protective dose50 was calculated as 5.61 and 7.94 for the shrimp groups which were vaccinated by GI–WSSV vaccine and GI-WSSV vaccine + GI-V.P, respectively. Calculated RPS values were 73.3%, 86.66% and 26.66% for the GI-WSSV vaccine, GI-WSSV + GI-V.P and isolated probiotic groups, respectively. Significant differences in cumulative mortalities were observed between the vaccination groups and the positive control group (P < 0.05). No significant difference was observed in cumulative mortalities between the two vaccination groups (P > 0.05). Conclusion: GI-WSSV vaccine can induce immune responses in shrimps infected with WSSV and probiotic GI-V.P enhances these responses.

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. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Southern Agriculture is the property of Journal of Southern Agriculture and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Raf is a member in the Ras/Raf/MAPKK/MAPK signaling transduction pathway. To obtain a better understanding of Raf in the interaction between the Chinese shrimp Fenneropenaeus chinensis and white spot syndrome virus (WSSV), the sequence of cDNA of Raf from F. chinensis (FcRaf) was obtained. The FcRaf gene contained a 2421 bp open reading frame (ORF). The FcRaf shared most characteristic of Raf protein, such as the Raf-like Ras-binding domain (RBD), phorbol esters/diacylglycerol binding domain (C1 domain), and catalytic domain of the serine/threonine kinases, Raf (STKc_Raf). The sequence of functional domains of Raf protein was relatively conserved. The FcRaf mRNA was detected in the tissues of gill, muscle, and hepatopancreas from normal F. chinensis. The mRNA abundance level of FcRaf in the gill was the highest, which was 2.7-fold the level in the hepatopancreas. The expression level of FcRaf was significantly (P < 0.05) up-regulated in the tissues of gill, muscle, and hepatopancreas post WSSV-infection, which suggested that FcRaf might be involved in the interaction between F. chinensis and WSSV. Two SNP loci were identified in the ORF, one of which was a C-T mis-sense mutation, where an Ala was replaced by a Val, and induced the predicted protein secondary structure change. Considering the relatively low MAF (0.07), whether this mis-sense mutation was a detrimental mutation needs further investigation. Highlights • A cDNA encoding Raf from Fenneropenaeus chinensis (FcRaf) was cloned and sequenced for the first time. • The sequence of functional domains of FcRaf protein was relatively conserved. • The FcRaf mRNA abundance level in tissues fluctuated post WSSV challenge. • Two SNPs were identified in the FcRaf ORF and one of which was a mis-sense mutation. [ABSTRACT FROM AUTHOR]

Abstract Penaeidins are a unique family of antimicrobial peptides specific to penaeid shrimp and have been reported mainly function as anti-bacterial and anti-fungal. In order to investigate whether penaeidins could also respond to virus or not, we examined the effect of WSSV on MjPen-II (penaeidin in kuruma shrimp, Marsupenaeus japonicus ) expression. In the control group, MjPen-II transcript level can be detected in almost all test tissues but was expressed most strongly in hemocytes. After WSSV infection, MjPen-II transcript level was significantly downregulated in hemocytes. Moreover, the proportion of MjPen-II+ hemocytes was not significantly different between non-infected and WSSV-infected shrimp, but the number of MjPen-II+ highly expressing hemocytes decreased after infection. In addition, MjPen-II was observed in the cytoplasm of granule-containing hemocytes. These results suggest that WSSV suppresses MjPen-II expression in hemocytes. Highlights • MjPen-II transcript level can be detected in various tissues but was expressed most strongly in hemocytes. • MjPen-II was located in the cytoplasm of granule-containing hemocytes in Marsupenaeus japonicus. • MjPen-II transcript level was significantly downregulated after white spot syndrome virus (WSSV) infection. • The proportion of MjPen-II+ highly expressing hemocytes decreased after WSSV infection but not with MjPen-II+ hemocytes. [ABSTRACT FROM AUTHOR]

White spot syndrome virus immediate early (IE) gene wsv249 encodes an E3 ubiquitin ligase that can interact with a shrimp ubiquitin-conjugating enzyme to mediate ubiquitination. In this study, to understand the transcriptional regulation of wsv249 , a serial of 5′-truncated mutations were made on its promoter and the activities of mutated promoters was analyzed. Four 25 bp regions potentially containing either positive or negative regulatory elements were identified. Notably, the deletion of −275/−250, which abolished a cAMP-response element (CRE), greatly reduced the promoter activity by 84.2%. CRE serves as the binding site for proteins belong to the cAMP responsive element-binding proteins (CREBs) family and the activator protein 1 (AP-1) family. Electrophoretic mobility shift assay (EMSA) showed that Lvc-Jun could directly bind to the CRE element in the promoter region of wsv249. In addition, the regulation of shrimp homolog of c-Jun and CREB on wsv249 promoter was further investigated. We found that Lvc-Jun greatly upregulated the activity of wsv249 promoter by ∼12.4 fold, and the CRE at −212/−205 but not the one at −256/−249 was essential for the regulation. In contrast, LvCREB-3 could not activate wsv249 promoter activity. These findings extend our knowledge of the transcriptional regulation of WSSV IE genes. [ABSTRACT FROM AUTHOR]

cDNA of a newly recognized white spot syndrome virus (WSSV)-induced gene (MjVIG1) was characterized from Marsupenaeus japonicus hemocytes; this gene encodes a protein that lack similarity to any known characterized protein. To identify this novel gene, we mainly conducted transcript level analysis, immunostaining and flow cytometry after WSSV infection. MjV1G1 transcript levels were also measured after Yellow head virus (YHV) and Vibrio parahaemolyticus infection tests. In non-infected and WSSV-infected shrimp, MjVIG1 was observed in granule-containing hemocytes. In addition, the MjVIG1 transcript level and ratio of MjVIG1-positive hemocytes both significantly increased, and number of MjVIG1-positive hemocytes slightly increased after WSSV infection. In contrast, MjVIG1 transcript level did not change after YHV and V. parahaemolyticus infection. These results indicated that MjVIG1 might be a WSSV-specific induced gene in M. japonicus hemocytes. [ABSTRACT FROM AUTHOR]

Argemone mexicana called as Mexican prickly poppy is a species of poppy found in Mexico and now widely naturalized in many parts of the world with broad range of bioactivities including anthelmintic, cures lepsory, skin-diseases, inflammations and bilious fevers. Plant parts of A. mexicana were serially extracted with hexane, ethyl acetate, methanol and performed antiviral and immunostimulant screening against WSSV and Vibrio harveyi respectively. The control groups succumbed to death 100% within three days, whereas the mortality was significantly (P < 0.5) reduced to 17.43 and 7.11 in the ethyl acetate extracts of stem and root treated shrimp group respectively. The same trend was reflected in the immunostimulant screening also. Different diets were prepared by the concentrations of 100 (AD-1), 200 (AD-2), 300 (AD-3) and 400 (AD-4) mg kg −1 using A. mexicana stem and root ethyl acetate extracts and fed to Pacific white leg shrimp Litopenaeus vannamei weighed about 9.0 ± 0.5 g for 30 days. The control groups fed with the normal diets devoid of A. mexicana extracts. The antiviral screening results revealed that, the ethyl acetate extract of the stem and root were effectively suppressed the WSSV and it reflected in the lowest cumulative mortality of treated shrimps. After termination of feeding trials, group of shrimps from control and each experimental group were challenged with virulent WSSV by intramuscular (IM) injection and studied cumulative mortality, molecular diagnosis by quantitative real time PCR ( q RT-PCR), biochemical, haematological and immunological parameters. Control group succumbed to 100% death within four days, whereas the survival was significantly (P < 0.001) increased to 30, 45, 75 and 79% in AD1, AD-2, AD-4 and AD-5 diets fed shrimp groups respectively. q RT PCR results with positive correlation analysis revealed that, the WSSV copies were gradually decreased when increasing the A. mexicana extracts in the diets. The highest concentrations (300 and 400 mg g −1 ) of A. mexicana extracts in the diets helped to reduce the protein level significantly (P < 0.05) after WSSV challenge. The diets AD-3 and AD-4 also helped to decrease the coagulation time of maximum 64–67% from control groups and maintained the normal level of total haemocyte, oxyhaemocyanin level after WSSV challenge. The proPO level was significantly increased (Column: F = 35.93; P ≤ 0.001 and Row: F = 37.14; P ≤ 0.001) in the AD1-AD-4 diet fed groups from the control diet fed groups. The lowest intra-agar lysozyme activity of 1.63 mm found in control diet fed group and the activity were significantly (P < 0.05) increased to 4.86, 7.89, 9.12 and 10.45 mm of zone of inhibition respectively in AD1 to AD4 diet fed groups. [ABSTRACT FROM AUTHOR]

Shrimp is one of the primary aquaculture species worldwide. The white spot syndrome virus (WSSV) is a highly lethal, stress-dependent virus that has led to enormous economic losses in shrimp aquaculture. WSSV can trigger a persistent infection in shrimp (latency), which can be reactivated to become an acute infection following various stimuli. However, the molecular mechanisms underlying the WSSV latency establishment and the induction of reactivation are poorly understood. A WSSV latency model using low multiplicity infection via intramuscular injection was developed in this study. This research examined the effects of multiple host factors and stimuli on WSSV latency. These effects generated latency reactivation in this model. The outcomes revealed that low multiplicity infection via intramuscular injection could be applied to establish WSSV latency in shrimp. Besides, the expression levels of host genes IRF, Dorsal and Relish were found to be substantially correlated with viral loads during WSSV latent infection in vivo. The knockdown of IRF resulted in elevated viral loads and rendered the reactivation of WSSV latency to acute infection that caused mass mortality among shrimp. Furthermore, several bacterial infection and low salinity stress could induce WSSV transitions from latent to acute infection. Noteworthily, the expression levels of Dorsal and Relish, which are considered to promote the replication of WSSV, are upregulated following bacterial infection and low salinity stress. These results indicated that a delicate balance between host and WSSV interaction regulates the latency establishment. This balance could be broken by several stimuli, such as bacterial infection and low salinity stress, which will provide insights into developing management strategies for WSSV prevention and control. • A stable WSSV latent infection model was established in shrimp through artificial infection. • The delicate balance between the host-WSSV interaction is responsible for regulating the establishment of WSSV latency. • Bacterial infection and low salinity stress can induce the transition of WSSV from a latent to an acute infection. [ABSTRACT FROM AUTHOR]

The article offers information on diagnosis of shrimp diseases using polymerase chain reaction and DNA chromatography technique. Topics discussed include loss faced by shrip farmers through bacterial, fungal diseases and microspordium, common viral diseases such as infectious hypodermal, white spot disease (WSD) and haematopoietic necrosis and other methods including strand tag hybridization (STH) chromatographic printed array strip.

The peritrophic membrane plays an important role in the defense system of the arthropod gut. The digestive tract is considered one of the major tissues targeted by white spot syndrome virus (WSSV) in shrimp. In this study, the nucleotide sequence encoding peritrophin-like protein of Litopenaeus vannamei (LvPT) was amplified from a yeast two-hybrid library of L. vannamei. The epitope peptide of LvPT was predicted with the GenScript OptimumAntigen™ design tool. An anti-LvPT polyclonal antibody was produced and shown to specifically bind a band at ~27 kDa, identified as LvPT. The LvPT protein was expressed and its concentration determined. LvPT dsRNA (4 μg per shrimp) was used to inhibit LvPT expression in shrimp, and a WSSV challenge experiment was then performed with reverse gavage. The pleopods, stomachs, and guts were collected from the shrimp at 0, 24, 48, and 72 h post-infection (hpi). Viral load quantification showed that the levels of WSSV were significantly lower in the pleopods, stomachs, and guts of shrimp after LvPT dsRNA interference than in those of the controls at 48 and 72 hpi. Our results imply that LvPT plays an important role during WSSV infection of the digestive tract. [ABSTRACT FROM AUTHOR]

To improve the efficacy of WSSV protection, multimeric (tetrameric) recombinant VP28 (4XrVP28) was produced and tested in comparison with those of monomeric VP28 (1XrVP28). In vitro binding of either 1XrVP28 or 4XrVP28 to shrimp hemocyte surface was evident as early as 10 min after protein inoculation. Similar results were obtained in vivo when shrimp were injected with recombinant proteins that the proteins bound to the hemocyte surface could be detected since 5 min after injection. Comparison of the WSSV protection efficiencies of 1XrVP28 or 4XrVP28 were performed by injection the purified 1XrVP28 or 4XrVP28 (22.5 μg/shrimp) and WSSV inoculum (1000 copies/shrimp) into shrimp. At 10 dpi, while shrimp injected with WSSV inoculum reached 100% mortality, shrimp injected with 1XrVP28 + WSSV or 4XrVP28 + WSSV showed relative percent survival (RPS) of 67% and 81%, respectively. PCR quantification revealed high number of WSSV in the moribund shrimp of WSSV- and 1XrVP28+WSSV-injected group. In contrast, lower number of WSSV copies were found in the survivors both from 1XrVP28+WSSV- or 4XrVP28+WSSV- injected groups. Histopathological analysis demonstrated the WSSV infected lesions found in the moribund from WSSV-infected group and 1XrVP28+WSSV-injected group, but less or none in the survivors. ELISA demonstrated that 4XrVP28 exhibited higher affinity binding to rPmRab7, a WSSV binding protein essential for WSSV entry to the cell than 1XrVP28. Taken together, the protection against WSSV in shrimp could be improved by application of multimeric rVP28. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA) is a class of small noncoding RNA, which is involved in the post-transcriptional regulation in all metazoan eukaryotes. MiRNAs might play an important role in the host response to virus infection. However, miRNAs in the aquatic crustacean species were not extensively investigated. To obtain a better understanding of the response of Chinese shrimp Fenneropenaeus chinensis to white spot syndrome virus (WSSV) infection, the sequence and expression profile of miRNAs in the hepatopancreas of WSSV-infected F . chinensis were obtained by the high-throughput Illumina HiSeq 2500 deep sequencing technique. A total number of 129 known miRNAs and 44 putative novel miRNAs were identified from the deep sequencing data. The peak size of miRNAs was 22 nt (37.0%). 25 miRNAs were significantly ( P < 0.05) differentially expressed post WSSV infection. Six of the differentially expressed miRNAs were randomly selected for further verification by the real-time RT-PCR technique. The results showed that there was a consistency between the deep sequencing and real-time RT-PCR assay. The target genes of differentially expressed miRNAs were predicted. Each miRNA had 4 target genes on average. The results suggested that some specific miRNAs might be involved in the response of F . chinensis to WSSV infection, and further provided basic information for the investigation of specific miRNAs in F . chinensis . [ABSTRACT FROM AUTHOR]

Death-associated protein 1 (DAP1) is a small proline-rich cytoplasmic protein that functions both in the apoptosis and autophage process of mammalian and in the clinical cancer of human. However, little knowledge is known about the homologue gene of DAP1 and its roles in the physiological process of invertebrates. In this paper, we report a novel function of DAP1 in the antivirus immunity of shrimp. A homologue gene of DAP1 was cloned from Marsupenaeus japonicus and named as Mjdap -1. The full-length of Mjdap -1 was 1761 bp with a 309 bp open reading frame that encoded 102 amino acids. Reverse transcription-PCR results showed that Mjdap- 1 was expressed in all tested tissues, including hemocytes, gills, intestines, stomach, heart, hepatopancreas, testes, and ovaries. In shrimp, Mjdap- 1 transcripts were up-regulated by white spot syndrome virus (WSSV) infection; Mjdap- 1 knockdown decreased the virus copy in vivo and the mortality of M. japonicus to WSSV challenge. Conversely, injecting the purified recombinant Mj DAP1 protein promoted the amplification of virus in shrimp. Flow cytometric assay showed, the virus infection-induced apoptosis of hemocytes was enhanced by Mj DAP1 protein injection and inhibited in Mj DAP1 knockdown shrimp. Furthermore, the expression of apoptosis-inducing factor (AIF) was regulated by Mjdap- 1, but the caspase transcripts were not affected. Our results suggested that Mj DAP1 facilitated the amplification of virus in shrimp, which may be attributed to the promotion of hemocyte apoptosis in an AIF-dependent manner. These results provided a new insight into the function of this protein that may be used for virus disease control. [ABSTRACT FROM AUTHOR]

In the present study a cDNA encoding a phosphopyruvate hydratase (enolase) was cloned from the muscle of the Chinese shrimp ( Fenneropenaeus chinensis ) and named as FcEnolase . The cDNA of FcEnolase encoded a protein of 434 amino acid residues with a molecular mass 47.22 kDa. The residues 342–355 constituted the signature motif “LLLKVNQIGSVTES”. A SNP locus (C96T) in the ORF at 96 bp was identified. The results showed that the FcEnolase was a conserved gene. In the normal F . chinensis , the mRNA level in the muscle was much higher ( P < 0.05) than the mRNA level in the gill and hepatopancreas. To verify the mRNA level of FcEnolase in the F . chinensis post WSSV infection, a real-time RT-PCR was performed. In the WSSV-infected F . chinensis , the FcEnolase mRNA level was significantly ( P < 0.05) up-regulated in the muscle at 12 and 24 h post challenge (hpc) to approximately 2.7-fold and 2.7-fold the mRNA level in the controls, respectively. The FcEnolase mRNA level in the gill was significantly ( P < 0.05) down-regulated at 6 hpc to approximately 0.3-fold the mRNA level in the control, followed by a significant ( P < 0.05) up-regulation at 12 hpc to approximately 2.8-fold the mRNA level in the control. There was no obvious change of FcEnolase mRNA level in the hepatopancreas during the infection process. The expression profile coincided with the fact that WSSV primarily infects the tissues of muscle and gill, but hardly infects hepatopancreas. To verify the protein level of FcEnolase post WSSV infection, a Western blot was performed. The FcEnolase protein level in the muscle at 24 hpc significantly ( P < 0.05) increased to approximately 2.1-fold the level in the control. These results showed the characterization of FcEnolase and suggested that the FcEnolase might be involved in the response of F . chinensis to WSSV infection. [ABSTRACT FROM AUTHOR]

Two monoclonal antibodies (MAbs) specific to different epitopes on ICP11, a non-structural protein of white spot syndrome virus (WSSV), were employed to develop a highly sensitive immunochromatographic strip test (ICP11 strip test). One MAb (WI-11) was conjugated to colloidal gold to bind to ICP11 and another MAb (WI-16) was used to capture colloidal gold MAb-protein complexes at the test line (T) on the nitrocellulose strip. A downstream control line (C) of goat anti-mouse immunoglobulin G (GAM) antibody was used to capture the free colloidal gold conjugated MAb to validate the test performance. Homogenate of shrimp sample in application buffer could be applied directly to the application well and the test result was obtained within 15 min. The sensitivity of the kit is 40 times more sensitive than the detection limit of previously reported strip test using MAbs specific to VP28, and approximately 400 times more sensitive than that of Shrimple®, but approximately 50 times less sensitive than that of one-step PCR. For experimentally WSSV-infected shrimps, the ICP11 strip could detect WSSV as early as 12–18 h post infection. Due to its high specificity, simplicity and no requirement on sophisticated equipment or specialized skills, the strip test could be adopted to surveillance of early WSSV infection at the pond site. [ABSTRACT FROM AUTHOR]

National Aquatic Animal Disease Surveillance Program (NAADSP) has started since 2007 to conduct nationwide surveillance for the prevalence of major crustacean pathogens in China, including white spot syndrome virus (WSSV). Standard diagnostic procedures and reliable detection capability are critical for ensuring accurate and comparable surveillance results, hence competent authority of China launched annual proficiency test (PT) synchronously to screen competent aquatic clinical diagnostic laboratories to undertake NAADSP. Here we present the results of PT for molecular detection of WSSV in 2021. Six tissue samples containing different WSSV concentrations were provided as blind samples to a total of 170 laboratories nationwide. All the participating laboratories were asked to perform qualitative or quantitative analyses by using standard molecular assays or commercial detection kits. In the qualitative assessments, 86.8% (125/144) of the participants achieved satisfied results by using conventional nested-PCR. Comparing to the strong/medium positive samples, the identification of negative samples and weak-positive samples have been shown to be more challenging. A relatively higher error rates were reported in results of sample S3 and S4. In the quantitative assessments, cycle threshold (Ct) values of real-time PCR submitted by 47 participants had been analyzed by using robust statistical method. The overall satisfied performance was achieved by 74.5% (35/47) of the participants. Acceptable between-laboratories z -score (ZB) and within-laboratory z -score (ZW) was achieved by 93.3% and 91.1%, respectively, in the analysis of the pair of identical samples S6 and S8. WSSV-PT 2021 supplied valuable reference information for selecting competent laboratories to participate in the NAADSP. Furthermore, the national PT scheme in the study provided an effective tool to access the performance of laboratories in molecular tests, and also gathered practical experience for organizing PTs on other emerging crustacean pathogens in the future. • A total of 170 laboratories of national wide participated in the ring of WSSV-PT 2021 in China. • In the qualitative assessments, 86.8% (125/144) of the participants correctly reported all samples in the panel. • In the quantitative assessments, all but 12 (25.5%) of the quantitative, participating laboratoriesshowed well performance. • Acceptable ZB and ZW-scores were achieved by 89.4% and 85.1% of participants in the analysis of paired samples. [ABSTRACT FROM AUTHOR]

VP28 is the most abundant membrane protein of WSSV, and the recombinant protein VP28 (VP26 or VP24) was constructed for the immune protection experiment in this study. Crayfish were immunized by intramuscular injection of recombinant protein V28 (VP26 or VP24) at a dose of 2 μg/g. The survival rate of crayfish immunized by VP28 showed a higher value than by VP26 or VP24 after WSSV challenge. Compared with the WSSV-positive control group, the VP28-immunized group could inhibit the replication of WSSV in crayfish, increasing the survival rate of crayfish to 66.67% after WSSV infection. The results of gene expression showed that VP28 treatment could enhance the expression of immune genes, mainly JAK and STAT genes. VP28 treatment also enhanced total hemocyte counts and enzyme activities including PO, SOD, and CAT in crayfish. VP28 treatment reduced the apoptosis of hemocytes in crayfish, as well as after WSSV infection. In conclusion, VP28 treatment can enhance the innate immunity of crayfish and has a significant effect on resistance to WSSV, and can be used as a preventive tool. • The survival rate of crayfish immunized by VP28 showed a higher value than by VP26 or VP24 after WSSV challenge. • VP28 treatment also enhanced total hemocyte counts and enzyme activities including PO, SOD, and CAT in crayfish. • VP28 treatment reduced the apoptosis of hemocytes in crayfish, as well as after WSSV infection. [ABSTRACT FROM AUTHOR]

White Spot disease is a devastating disease of shrimps caused by White Spot Syndrome Virus in multifarious shrimp species. At present there is no absolute medication to suppress the disease hence, there is an urgent need for development of drug against the virus. Molecular interaction between viral envelope protein VP28 and shrimp receptor protein especially chitins play a pivotal role in ingression of WSSV. In the present study, we have tried to shed light on structural aspects of lectin protein in Marsupenaeus japonicus (MjsvCL). A structural insight to the CTLD-domain of MjsvCL has facilitated the understanding of the binding mechanism between the two proteins that is responsible for entry of WSSV into shrimps. Further, incorporation of molecular dynamics simulation and MMPBSA studies revealed the affinity of binding and certain hotspot residues, which are critical for association of both the proteins. For the first time we have proposed that these amino acids are quintessential for formation of VP28-MjsvCL complex and play crucial role in entry of WSSV into shrimps. Targeting the interaction between VP28 and CTLD of MjsvCL may possibly serve as a potential drug target. The current study provides information for better understanding the interaction between VP28 and MjsvCL that could be a plausible site for future inhibitors against WSSV in shrimps.Communicated by Ramaswamy H. Sarma.

White spot syndrome virus (WSSV) is an important viral pathogen that infects farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus infection to shrimp farming has become increasingly severe. Viral and bacterial cross or superimposed infections may induce higher shrimp mortality. We used a feeding method to infect Litopenaeus vannamei with WSSV and then injected a low dose of V. parahaemolyticus (WSSV+Vp), or we first infected L. vannamei with a low-dose injection of V. parahaemolyticus and then fed the shrimp WSSV to achieve viral infection (Vp+WSSV). The eff ect of V. parahaemolyticus and WSSV co-infection on survival of L. vannamei was evaluated by comparing cumulative mortality rates between experimental and control groups. We also spread L. vannamei hemolymph on thiosulfate citrate bile salt sucrose agar plates to determine the number of Vibrio, and the WSSV copy number in L. vannamei gills was determined using an absolute quantitative polymerase chain reaction (PCR) method. LvMyD88 and Lvakt gene expression levels were detected in gills of L. vannamei by real-time PCR to determine the cause of the diff erent mortality rates. Our results show that (1) the cumulative mortality rate of L. vannamei in the WSSV+Vp group reached 100% on day 10 after WSSV infection, whereas the cumulative mortality rate of L. vannamei in the Vp+WSSV group and the WSSV-alone control group approached 100% on days 11 and 13 of infection; (2) the number of Vibrio in the L. vannamei group infected with V. parahaemolyticus alone declined gradually, whereas the other groups showed significant increases in the numbers of Vibrio ( P<0.05); (3) the WSSV copy numbers in the gills of the WSSV+Vp, Vp+WSSV, and the WSSV-alone groups increased from 10 to 10 /mg tissue 72, 96, and 144 h after infection, respectively. These results suggest that V. parahaemolyticus infection accelerated proliferation of WSSV in L. vannamei and vice versa. The combined accelerated proliferation of both V. parahaemolyticus and WSSV led to massive death of L. vannamei. [ABSTRACT FROM AUTHOR]

Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp ( Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future. [ABSTRACT FROM AUTHOR]

Infectious hypodermal and haematopoietic necrosis virus ( IHHNV) and white spot syndrome virus ( WSSV) are two widespread shrimp viruses. The interference of IHHNV on WSSV was the first reported case of viral interference that involved crustacean viruses and has been subsequently confirmed. However, the mechanisms underlying the induction of WSSV resistance through IHHNV infection are practically unknown. In this study, the interference mechanisms between IHHNV and WSSV were studied using a competitive ELISA. The binding of WSSV and IHHNV to cellular membrane of Litopenaeus vannamei was examined. The results suggested that there existed a mutual competition between IHHNV and WSSV for binding to receptors present on cellular membrane of L. vannamei and that the inhibitory effects of WSSV towards IHHNV were more distinct than those of IHHNV towards WSSV. [ABSTRACT FROM AUTHOR]

ADP ribosylation factors (Arf), as highly conserved small guanosine triphosphate (GTP)-binding proteins, participates in intracellular trafficking and organelle structure. In this study, a full-length cDNA of Arf1 (designated EcArf1) was cloned from Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EcArf1 was 1428 bp, which contains an open reading frame (ORF) of 549 bp, encoding a 182 amino-acid polypeptide with the predicted molecular weight of 20.69 kDa and estimated isoelectric point was 7.24. Sequence analysis revealed that the conserved Arf protein family signatures were identified in EcArf1. The deduced amino acid sequence of EcArf1 shared high identity (95%–98%) with that of other species and clustered together with Arf1 of other shrimp in the NJ phylogenetic tree, indicating that EcArf1 should be a member of the Arf1 family. Quantitative real-time RT-qPCR analysis indicated that EcArf1 was expressed in hemocytes, hepatopancreas, gills, muscle, ovary, intestine, stomach and heart, and the most abundant level was in hemocytes and gills, which were also the two main target tissues of pathogen infection and environmental stress. After Vibrio parahaemolyticus challenge, EcArf1 transcripts level significantly increased in hemocytes and hepatopancreas at 3 h and 6 h, respectively. The expression of EcArf1 in hemocytes and hepatopancreas significantly up-regulated at 12 h and 6 h respectively, and down-regulated at 72 h and 48 h, respectively. EcArf1 expression in hepatopancreas and gills both significantly increased at 6 h and decreased at 24 h under ammonia-N stress. The results suggested that EcArf1 might be involved in immune responses to pathogens ( V. parahaemolyticus and WSSV) challenge and ammonia-N stress in E. carinicauda . [ABSTRACT FROM AUTHOR]

Mitogen-activated kinase kinase (MAPKK) is an important gene involved in the host-virus interaction process. To obtain a better understanding of MAPKK in the interaction process between the Chinese shrimp Fenneropenaeus chinensis and white spot syndrome virus (WSSV), we cloned the sequence of an MAPKK cDNA from F . chinensis ( FcMAPKK ) and investigated the effect of FcMAPKK on WSSV infection. The results showed that the FcMAPKK gene contained a 1227 bp open reading frame (ORF), which encoded a highly conserved protein with a serine/threonine protein kinase catalytic (S_TKc) domain. The deduced amino acid sequence of FcMAPKK shared identities between 11.9 and 92.6% with MAPKKs from vertebrate, invertebrate, plant and fungus species. The FcMAPKK was expressed in all the examined tissues in the normal F . chinensis . FcMAPKK expression level was highest in the hepatopancreas where it was approximately 2.6-fold the expression level in the gill, and lowest in the muscle where it was approximately 0.3-fold the expression level in the hepatopancreas. The FcMAPKK expression levels in the muscle, gill, and hepatopancreas were all changed post WSSV challenge. The FcMAPKK expression was significantly ( P < 0.01) up-regulated in the muscle of F . chinensis at 48 h post WSSV infection. The WSSV began to replicate quickly in the normal F . chinensis at 48 h post infection, while the WSSV replication in the U0126-treated F . chinensis could be significantly ( P < 0.05) inhibited. The results suggested that FcMAPKK might be involved in the WSSV infection process, and hijacking of FcMAPKK might be required for WSSV replication in F . chinensis . [ABSTRACT FROM AUTHOR]

White spot syndrome virus (WSSV) is one of the most prevalent and lethal viruses for shrimp, and oral administration of VP28 is a promising approach to protect shrimp against WSSV. Although seven heterologous expression systems have been successful since 2004, no one has been commercialized in shrimp industry. This paper presented the idea of 'both drug and feed from the same feedstock' to develop oral vaccine against WSSV. Filamentous, heterocystous cyanobacterium, Anabaena sp. PCC 7120, was used to express major envelope protein, VP28, for feeding post larvae of shrimp. The expression of vp28 gene in transgenic mutant was analyzed by Western blotting and real-time quantitative polymerase chain reaction (RT-qPCR). For vaccination, Litopenaeus vannamei was orally administrated by feeding the wild type and mutants of Anabaena cells for 10 days, and was challenged with WSSV during 10 days post vaccination. The post larvae were fed with the mutant harboring vp28 (10 μg shrimp). The post larvae were divided five groups and their survivals were as follows: (1) negative control (neither vaccination nor challenge), 100 %; (2) positive control (challenge and no vaccination), 0 %; (3) feeding the wild type, 15.5 %; (4) feeding blank, 20.6 %; and (5) feeding the mutant harboring vp28, 68.0 %. Moreover, the post larvae fed the mutant harboring vp28 grew better than no feed. Also, the photosynthetic features indicated the transgenic mutant was easier to cultivate. These data show that transgenic mutant harboring vp28 was favorable not only to use both for drug and feed but also to cultivate in scale. [ABSTRACT FROM AUTHOR]