White Spot Syndrome Virus Copies Research Articles

Identification and functional characterization of two Bcl-2 family proteins in swimming crab, Portunus trituberculatus

Bcl-2 family proteins are regulatory factors of the mitochondrial apoptosis pathway that are vital to immune defenses and many other biological processes. However, the effects of Bcl-2 family proteins on apoptosis and immunity in crustaceans is poorly studied. In the study, two novel genes including Bax gene (PtBax) and Bcl-2 (PtBcl-2) from Portunus trituberculatus were identified and characterized. The deduced amino acid sequences showed that PtBax and PtBcl-2 possess domains of typical Bcl-2-related proteins. Both genes were widely expressed in all tested tissues, with highest expression levels in the hepatopancreas. Fluorescence in situ hybridization revealed that PtBax and PtBcl-2 were mainly expressed in the cytoplasm, where they play an important role in pathogen infection. Both PtBax and PtBcl-2 were significantly upregulated in the hepatopancreas at both mRNA and protein levels following challenge with Vibrio parahaemolyticus or white spot syndrome virus (WSSV). Moreover, double-stranded RNA-mediated silencing of both genes altered apoptosis levels in the hepatopancreas and WSSV copy number in muscles. Interactions between PtBax and PtBcl-2 were analyzed at transcriptional and protein levels. Yeast two-hybrid system analysis showed that PtBcl-2 could interact with PtBax directly. These results suggest that PtBax and PtBcl-2 may participate in pathogen infection by regulating apoptosis.

The Role of microRNA-133 in Hemocyte Proliferation and Innate Immunity of Scylla paramamosain.

MicroRNAs (miRNAs) are important signaling regulators that are involved in regulating the innate immunity of crustacean. However, few studies focus on the role of crustacean miRNAs in the cellular immunity have been reported. In this study, we showed that the expression of miR-133 was significantly up-regulated in the mud crab Scylla paramamosain after infection by white spot syndrome virus (WSSV) or Vibrio parahaemolyticus. The anti-miRNA oligonucleotide AMO-miR-133 was used to knock down miR-133 expression in S. paramamosain. The number of WSSV copies increased significantly in WSSV-infected crabs after miR-133 knockdown. Knockdown of miR-133 also enhanced the mortality rates of WSSV-infected and V. parahaemolyticus-infected mud crabs, and it significantly enhanced the expression of the astakine, which was confirmed by real-time quantitative PCR and western blot analysis. The data also indicate that miR-133 may affect hemocyte proliferation in S. paramamosain by regulating astakine expression. miR-133 Knockdown enhanced the apoptosis or phagocytosis of crab hemocytes, and increased the mortality of mud crabs after WSSV or V. parahaemolyticus infection. These results indicate that miR-133 is involved in the host immune response to WSSV and V. parahaemolyticus infection in mud crabs. Taken together, our research provides new insights for the control of viral or vibrio diseases in S. paramamosain.

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.

WSV056 Inhibits Shrimp Nitric Oxide Synthase Activity by Downregulating Litopenaeus vannamei Sepiapterin Reductase to Promote White Spot Syndrome Virus Replication.

Sepiapterin reductase (Spr) plays an essential role in the biosynthesis of tetrahydrobiopterin (BH4), a key cofactor of multiple enzymes involved in various physiological and immune processes. Suppression of Spr could result in BH4 deficiency-caused diseases in human and murine models. However, information on the biological function of Spr in invertebrates is limited. In this study, two Sprs (CG12116 and Sptr) from Drosophila melanogaster were found to be downregulated in transgenic flies overexpressing white spot syndrome virus (WSSV) immediate-early protein WSV056. CG12116 and Sptr exerted an inhibitory effect on the replication of the Drosophila C virus. A Litopenaeus vannamei Spr (LvSpr) exhibiting similarity of 64.1–67.5% and 57.3–62.2% to that of invertebrate and vertebrate Sprs, respectively, were cloned. L. vannamei challenged with WSSV revealed a significant decrease in LvSpr transcription and Spr activity in hemocytes. In addition, the BH4 co-factored nitric oxide synthase (Nos) activity in shrimp hemocytes was reduced in WSSV-infected and LvSpr knockdown shrimp, suggesting WSSV probably inhibits the LvNos activity through LvSpr downregulation to limit the production of nitric oxide (NO). Knockdown of LvSpr and LvNos caused the reduction in NO level in hemocytes and the increase of viral copy numbers in WSSV-infected shrimp. Supplementation of NO donor DETA/NO or double gene knockdown of WSV056 + LvSpr and WSV056 + LvNos recovered the NO production, whereas the WSSV copy numbers were decreased. Altogether, the findings demonstrated that LvSpr and LvNos could potentially inhibit WSSV. In turn, the virus has evolved to attenuate NO production via LvSpr suppression by WSV056, allowing evasion of host antiviral response to ensure efficient replication.

Natural ingredient paeoniflorin could be a lead compound against white spot syndrome virus infection in Litopenaeus vannamei.

White spot syndrome virus (WSSV) is an important pathogen causing high mortality in the shrimp industry in aquaculture, yet there is no treatment available to date. In order to find a treatment against WSSV infection, this study examined the anti-WSSV activity of eight natural compounds using shrimp larvae as a model. Among the eight compounds, paeoniflorin showed the most obvious anti-WSSV effect, with a maximum protection efficiency of WSSV-infected shrimp >60% at 100μM. Furthermore, pretreatment and post-treatment experiments revealed that paeoniflorin could prevent and treat WSSV infection in shrimp. The antiviral activity of paeoniflorin in aquaculture water decreased rapidly with time, and the results showed that the stable anti-WSSV activity of paeoniflorin could only remain in water for 1day. Thus, the dosing pattern of continuous medication changes was evaluated. Obviously, in the model of continuous change of paeoniflorin, WSSV copy numbers in the virus-treated shrimp group still progressively increased, while the virus content in WSSVpaeoniflorin -treated group continued to decrease. Interestingly, paeoniflorin inhibited horizontal transmission of WSSV to a certain extent. Notably, paeoniflorin significantly increased the expression of antimicrobial peptides of shrimp to resist WSSV. In conclusion, paeoniflorin has the potential to protect shrimp against WSSV.

Field-usable aptamer‑gold nanoparticles-based colorimetric sensor for rapid detection of white spot syndrome virus in shrimp

White spot syndrome virus (WSSV) is one of the most threatening viral pathogens of shrimp worldwide. VP28, the major capsid protein of WSSV, is reported to play an essential role in the interaction with the host cells. Usually, the diagnostic test of WSSV is performed by one-step PCR, which is neither field-usable nor rapid enough. Therefore, the development of early diagnostic tests is imperative for the management of disease and to prevent huge economic losses. In this work, a rapid colorimetric aptasensor with high selectivity and sensitivity was introduced. First, a new ssDNA aptamer for the detection of WSSV was designed from a random pool of aptamer sequences based on the docking score and bioconjugate free energy, and then, evaluated experimentally. The designed aptamer was used to prepare an aptasensor conjugate gold nanoparticles (AuNPs) as a recognition element that increased the resistance of AuNPs to salt-induced aggregation. Therefore, the AuNPs with spherical morphology and average particle size of about 20 nm were synthesized for this reason. The results showed that the aptamer didn't attach to the other pathogens (i.e., IHHN and Vibrio harveyi) and healthy shrimp cells, and remained stable. Interestingly, the gray-blue color of aptasensor was seen only by the presence of WSSV, indicating the aptasensor assay showed good specificity to WSSV. The limit of detection (LOD) of the aptasensor was 104 copies of WSSV that could be detected only utilizing naked eyes. It was concluded that the designed aptamer had excellent potential for the detection of WSSV as a rapid visual colorimetric assay in aquaculture shrimp farmers.

Evaluation of the relationship between the 14-3-3\u03b5 protein and LvRab11 in the shrimp Litopenaeus vannamei during WSSV infection

The 14-3-3 proteins interact with a wide variety of cellular proteins for many diverse functions in biological processes. In this study, a yeast two-hybrid assay revealed that two 14-3-3ε isoforms (14-3-3ES and 14-3-3EL) interacted with Rab11 in the white shrimp Litopenaeus vannamei (LvRab11). The interaction of 14-3-3ε and LvRab11 was confirmed by a GST pull-down assay. The LvRab11 open reading frame was 645 bp long, encoding a protein of 214 amino acids. Possible complexes of 14-3-3ε isoforms and LvRab11 were elucidated by in silico analysis, in which LvRab11 showed a better binding energy score with 14-3-3EL than with 14-3-3ES. In shrimp challenged with the white spot syndrome virus (WSSV), the mRNA expression levels of LvRab11 and 14-3-3ε were significantly upregulated at 48 h after challenge. To determine whether LvRab11 and binding between 14-3-3ε and LvRab11 are active against WSSV infection, an in vivo neutralization assay and RNA interference were performed. The results of in vivo neutralization showed that LvRab11 and complexes of 14-3-3ε/LvRab11 delayed mortality in shrimp challenged with WSSV. Interestingly, in the RNAi experiments, the silencing effect of LvRab11 in WSSV-infected shrimp resulted in decreased ie-1 mRNA expression and WSSV copy number. Whereas suppression of complex 14-3-3ε/LvRab11 increased WSSV replication. This study has suggested two functions of LvRab11 in shrimp innate immunity; (1) at the early stage of WSSV infection, LvRab11 might play an important role in WSSV infection processes and (2) at the late stage of infection, the 14-3-3ε/LvRab11 interaction acquires functions that are involved in immune response against WSSV invasion.

Effects of dietary quercetin on the innate immune response and resistance to white spot syndrome virus in Procambarusclarkii

In recent years, the use of natural products with immune-stimulating and antimicrobial properties has attracted increasing attention in aquaculture researches. In our study, the effect of diet supplemented with quercetin, a flavonoid commonly found in some types of plants substance on the innate immune response and disease resistance in crayfish (Procambarus clarkii) against white spot syndrome virus (WSSV) is reported. It was found that dietary 40 mg/kg quercetin significantly reduced the mortality of crayfish and WSSV copy number after WSSV challenge. Dietary quercetin increased catalase (CAT), and lysozyme (LZM) activity in crayfish. Dietary quercetin increased the expression of NF-κB, anti-lipopolysaccharide factor (ALF) and toll-like receptor (TLR) genes in crayfish. The apoptosis rate of hemocyte was increased by quercetin supplement in crayfish. Our results suggest that dietary quercetin may affect the innate immunity of crayfish and protect crayfish from WSSV infection.

Identification of four Spätzle genes (MnSpz1, MnSpz2, MnSpz2-isoform, and MnSpz3) and their roles in the innate immunity of Macrobrachium nipponense

Spätzle, an extracellular ligand of the Toll receptor, is involved in the innate immunity of crustaceans. In this study, four Spätzle genes were cloned from Macrobrachium nipponense and designed as MnSpz1, MnSpz2, MnSpz2-isoform, and MnSpz3. The coding region of the four Spätzle genes all contained one intron and two exons, and they were predicted to be produced by gene duplication based on sequence similarities and phylogenetic tree. The predicted MnSpz1, MnSpz2, and MnSpz3 proteins all contained a signal peptide and a Spätzle domain. No signal peptide but a Spätzle domain existed in MnSpz2-isoform because of frameshift mutation caused by 50 bp nucleotide deletion compared with MnSpz2. Quantitative real-time polymerase chain reaction (RT-qPCR) analysis showed that MnSpz1, MnSpz2, and MnSpz3 were expressed in all the detected tissues of M. nipponense, and MnSpz2 was found to be the major isoform in the heart, gills, stomach, and intestine. After stimulation by Vibrio parahaemolyticus, Staphylococcus aureus, or White spot syndrome virus (WSSV), the expression levels of MnSpz1, MnSpz2, and MnSpz3 changed. Given the high similarities among MnSpz1-3, RNA interference (RNAi) using dsRNA of MnSpz1 inhibited the expression of the three Spätzle genes (MnSpz1, MnSpz2 and MnSpz3). Silencing of MnSpz1-3 down-regulated the expression levels of nine antimicrobial peptide (AMP) genes in M. nipponense. After Knockdown of MnSpzs, the number of V. parahaemolyticus, S. aureus and WSSV copies in M. nipponense increased significantly in vivo. Our results suggest that Spätzles are involved in the innate immunity of M. nipponense. The expansion of MnSpz genes through gene duplication is beneficial to enhance the innate immune defense ability of M. nipponense.

Use of glycerol monolaurate as a treatment against white spot syndrome virus in crayfish (Procambarus clarkii)

Glycerol monolaurate (GML), a monoester formed from glycerol and lauric acid, is widely used in food, cosmetics, and dietary supplements. GML also has antiviral activity against enveloped viruses. In this study, we conducted a challenge experiment to assess the effects of GML on the survival of crayfish (Procambarus clarkii) infected with white spot syndrome virus (WSSV). Dietary GML treatment reduced the mortality of WSSV-infected crayfish and reduced the WSSV copy number. Quantitative reverse transcriptase polymerase chain reaction analysis showed that GML enhanced the expression of immune-related genes, such as nuclear factor-κB and C-type lectin. GML increased three immune parameters (total hemocyte count, phenoloxidase activity, and superoxide dismutase activity) in crayfish with or without WSSV infection. GML also increased the hemocyte apoptosis rate in crayfish with or without WSSV infection. These data indicated that GML increased hemocyte apoptosis, THC, PO and SOD activity and reduced the mortality in crayfish after oral WSSV challenge, which suggested that GML can be used to keep the crayfish healthy.

A novel antiviral coumarin derivative as a potential agent against WSSV infection in shrimp seedling culture

White spot syndrome virus (WSSV), a double-stranded DNA virus that infects crustaceans, is the most serious viral pathogen affecting shrimp farming worldwide. To reduce the economic losses caused by WSSV, we screened a novel coumarin derivative from a small molecule drug library, N-(4-((4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)sulfonyl)phenyl)acetamide (N2905), to evaluate its anti-WSSV effects in vivo. We determined that compound N2905, up to a concentration of 20 mg/L, significantly decreased the number of WSSV copies in Litopenaeus vannamei post-larvae, with a maximum inhibitory rate of > 90 %, and increased the survival rate of WSSV-infected post-larvae. Pre-treatment and post-treatment assays indicated that N2905 could treat, but not prevent, WSSV infections. When WSSV was preincubated with N2905 for 1−4 h, the incidence of viral infections was significantly reduced and survival time of post-larvae extended to 120 h. A stability study of N2905 provided a reference for its practical use. Considering the antiviral stability of N2905 in culture water within 2 d, continuous N2905 exchange was performed, showing a significant decrease in viral load at 120 h post-infection (hpi) and a 55 % increase in survival of WSSV-infected post-larvae. Overall, our study demonstrated the potential of N2905 as an antiviral agent.

Effect of dietary fucoidan on innate immune response of Procambarus clarkii and disease resistance against white spot syndrome virus

This study has been investigated the effect of dietary fucoidan on the innate immune response of crayfish (Procambarus clarkii) and the disease resistance against white spot syndrome virus (WSSV) infection. Adding fucoidan (30 mg/kg) to the diet can dramatically improved the survival rate of crayfish after WSSV infection and also can reduce the copy number of WSSV. The result of qRT-PCR demonstrated that fucoidan treatment can increase the expression profile of immune-related genes, such as NF-κB and prophenoloxidase. Further evidence indicates that fucoidan also had the influence on three immune parameters, which consist of the total number of hemocyte (THC), phenoloxidase activity (PO) and superoxide dismutase activity (SOD) in crayfish. The hemocytes apoptosis of crayfish post WSSV-infected and uninfected crayfish were both increased significantly after treating with 30 mg/kg fucoidan. Dietary fucoidan delayed and reduced the mortality after oral WSSV challenge, and decreased WSSV copies in 96 h after challenge. Our research demonstrated that fucoidan can be used to the regulation of innate immunity and induce immune protection effect of crayfish from WSSV infection, whether for treatment or prevention.

The SNP polymorphisms associated with WSSV-resistance of prophenoloxidase in red swamp crayfish (Procambarus clarkii) and its immune response against white spot syndrome virus (WSSV)

Prophenoloxidase (proPO), a critical enzyme in innate immune, has been extensively studied in various crustacean species including red swamp crayfish Procambarus clarkii. However, little is known about the genetic structure of proPO from P. clarkii and its role in antivirals. In this study, the expression pattern of proPO upon white spot syndrome virus (WSSV) infection was investigated, and the proPO genomic DNA sequence was obtained from P. clarkii for the first time and analyzed. The results indicated that the mRNA level of proPO could be significantly upregulated in haemocytes, hepatopancreas, gill and midgut. The proPO genomic DNA sequence is 9085 bp in length, which includes five exons interrupted by four introns, an 82 bp 5′-flanking region, and a 1671 bp 3′-untranslated region. Two TATA boxes as well as three repeat sequences were predicted in the intron region of proPO. Subsequently, the single nucleotide polymorphisms (SNPs) of proPO were identified and genotyped in the crayfish population and the association between SNPs and anti-WSSV traits was investigated. Three SNPs in intron regions were identified, and the SNP 7081 T > C showed a significant association (P < 0.05) with anti-WSSV traits. In addition, the role of purified recombinant protein of proPO in anti-WSSV infection was explored. The results showed that recombinant proPO significantly reduced the amount of WSSV copy number in hepatopancreas and gill compared to the control group injected with bovine serum albumin (BSA). It was found that recombinant proPO could enhance the expression of proPO in hepatopancreas, peroxinectin in midgut, and the C-type lectin (ctl) in gill and midgut. Intramuscularly injection of juvenile crayfish with recombinant proPO could significantly decrease 65% mortality of WSSV-infected crayfish at 10 days as compared to the control group with BSA. The findings of this study suggested that the proPO is an important molecule in the antiviral immune system of crustacean and could provide theoretical for molecular marker-assisted WSSV resistance breeding of P. clarkii.

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.

Rab9 defense against white spot syndrome virus by participation in autophagy in Marsupenaeus japonicas

White spot syndrome virus (WSSV) is the main pathogen of shrimp and has led to considerable economic losses to the shrimp industry around the world. However, so far there are still no effective strategies to address this problem. In this paper, the tissue distribution of Rab9 as well as its defense mechanism against WSSV in Japanese shrimp (Marsupenaeus japonicas) was investigated. The results revealed that Rab9 had a higher expression in hemocyte and gill while expression was lower in heart, muscle, intestine, liver, indicating Rab9 was involved in the innate immune process. The results showed that the Rab9 expression increased when shrimp was challenged with WSSV compared with that of control, while the silence of Rab9 led to the increase of WSSV copies. In order to explore the antiviral mechanism of Rab9, it was demonstrated that the expression level of Rab9 changed during autophagy process, which indicated that Rab9 is participated in the autophagy procedure of shrimp. The fact that autophagy decreased after Rab9 silenced, may also suggest that Rab9 protein could affect autophagy. In short, the results showed Rab9 played a key role in antivirus through regulating autophagy. The results not only enlarge the limited views about molecular mechanism of Rab in invertebrate, but also help to enrich the immunological content in marine invertebrate.

Cloning and characterization of chst11 from Procambarus clarkii involved in the host immune response of white spot syndrome virus and Aeromonas hydrophila

Carbohydrate sulfotransferases 11 (chst11) is one of the enzymes that synthesize chondroitin sulfate (CS), which has extensive immune functions in vitro and plays a critical role in mediating the infection of host by pathogenic microorganisms. However, whether it has immune functions in crayfish is still poorly understood. In our previous study of transcriptome, chst11 was differentially expressed in susceptible individuals and resistant individuals of Procambarus clarkii after white spot syndrome virus (WSSV) injection. Thus, in this study, the sequence of chst11 was obtained from P. clarkii for the first time and analyzed, and the expression pattern of chst11 was investigated. Besides, the purified recombinant protein of chst11 effect in protection in WSSV infection was explored. The full length of chst11 was 1536 bp with an 831-bp open reading frame (ORF), which encoding 276 amino acids residues with a calculated molecular mass of 33.1 kDa. The chst11 contains a Sulfotransfer_2 domain, one N-glycosylation site and three O-glycosylation sites. Phylogenetic analysis results showed that chst11 had the highest similarity to Penaeus vannamei (79.93%). The expression pattern of chst11 in different tissues indicated that chst11 was expressed highest in gut, gill and hypodermis, lowest in testicular duct, periesophageal nerve and hemocytes. The chst11 had different expression patterns in different tissues when the crayfish was challenged by WSSV, Aeromonas hydrophila and CpG ODN. Recombinant chst11 protein significantly reduced the amount of WSSV copy number in hepatopancreas at 6 h and 12 h post injection compared to the control group injected with bovine serum albumin (BSA). It was found that chst11 protein enhanced the expression of peroxinectin, proPO in hepatopancreas and midgut and the C-type lectin (ctl) in hemocytes and hepatopancreas. Intramuscularly injection of juvenile crayfish with chst11 protein decreased 60% mortality compared to the control group with BSA. This study is the first report on the antiviral function of chst11 in the immune system of crustacean.

MicroRNA-589-5p modulates the expression of hemocyanin as part of the anti-WSSV immune response in Litopenaeus vannamei

The respiratory glycoprotein, hemocyanin (HMC) has multiple immune-related functions, including antiviral activity. In this study, in silico methods were used to predict seven miRNAs targeting Litopenaeus vannamei HMC (LvHMC), out of which miR-589-5p was selected for further investigation because of its role in immune response. Transcript levels of miR-589-5p were ubiquitously distributed in all shrimp tissues examined, and significantly induced in hemocytes and hepatopancreas upon challenge with white-spot syndrome virus (WSSV) as well as by marine bacterial pathogens, which suggest that miR-589-5p is involved in shrimp immune response to pathogens. Morever, using Drosophila S2 cells stably overexpressing EGFP-LvHMC, flow cytometry and dual luciferase reporter assays, miR-589-5p was shown to significantly inhibit the in vitro expression of LvHMC. In addition, in vivo knockdown of miR-589-5p using antagomir-589-5p resulted in significant down-regulation in LvHMC expression, while overexpression of miR-589-5p using agomir-589-5p decreased the level of LvHMC expression in shrimp hemocytes and hepatopancreas. Further, the increased expression of miR-589-5p resulted in high shrimp mortality following WSSV challenge, coupled with an increase in the number of WSSV copies in hemocytes and hepatopancreas. These results suggest that miR-589-5p is involved in shrimp immune response to WSSV by negatively regulating the expression of LvHMC.

Apoptosis of hemocytes is associated with the infection process of white spot syndrome virus in Litopenaeus vannamei

Previous studies have demonstrated that white spot syndrome virus (WSSV) could induce hemocytes apoptosis in shrimps, however the inter-relationship between apoptotic process and the WSSV infection status is still currently underexplored. In the present work, the apoptosis and the viral proliferation in hemocytes of Litopenaeus vannamei were simultaneously investigated post WSSV infection by two-color immunofluorescence flow cytometry and real-time quantitative PCR. The apoptotic hemocytes of WSSV-infected shrimp was significantly increased at 12 h post infection (hpi), whereas underwent a slight decline at 24 hpi subsequently. Since 24 hpi, the apoptotic rate of hemocytes in the WSSV-infected shrimp exhibited a rapid and significant increase, and reached the peak level at 48 hpi with the ratio of 18.1 ± 2.0%. Meanwhile, the percentage of WSSV-infected hemocytes and WSSV copies in hemocytes significantly increased at 24 hpi and maintained at a high level afterwards. With the rapid increase of hemocytes apoptosis, hemocyte density in hemolymph decreased dramatically to less than 20% of the mean value of control. Co-localization assay showed that the apoptotic WSSV-infected hemocytes occupied the dominant proportion of total apoptotic hemocytes, which reached the peak at 48 hpi with 12.6 ± 1.5%. The expression profiles of seven pro-apoptotic genes and two apoptosis-inhibiting genes showed significant differential responses at different stages of WSSV infection, reflecting the interplay between the virus and the host immune response. Our results demonstrated that the apoptotic response of shrimp hemocytes could be significantly influenced by the WSSV infection process, which might provide an insight into deeper relationships between viral infection and apoptosis.

SpBAG1 promotes the WSSV infection by inhibiting apoptosis in mud crab (Scylla paramamosain)

Bcl-2 associated athanogene-1 (BAG1) is involved in various signalling pathways including apoptosis, cell proliferation, gene transcriptional regulation and signal transduction in animals. However the functions of BAG1 during the antiviral response of mud crab Scylla paramamosain is still unclear. In this study, the mud crab BAG1 (SpBAG1) was characterized to consist of 1761 nucleotides, containing an opening frame of 630bp encoding 209 amino acids with an ubiquitin domain and a BAG1 domain. SpBAG1 was found to be significantly up-regulated at 6 h–24 h, but down-regulated from 48 h–72 h in the hemocytes of mud crab after challenge with white spot syndrome virus (WSSV). RNAi knock-down of SpBAG1 significantly reduced the copies of WSSV and increased the apoptotic rate in mud crabs. The finding from this study suggested that SpBAG1 could promote the WSSV infection by inhibiting apoptosis in mud crab. Therefore, to the best of our knowledge, this is the first study demonstrating the role of SpBAG1 as a novel apoptosis inhibitor to promote virus infection in mud crab.

Molecular characterization of carboxypeptidase B-like (CPB) in Scylla paramamosain and its role in white spot syndrome virus and Vibrio alginolyticus infection

Carboxypeptidase plays an important physiological role in the tissues and organs of animals. In this study, we cloned an entire 2316 bp carboxypeptidase B-like (CPB) sequence with a 1302 bp open reading frame encoding a 434 amino acid peptide from Scylla paramamosain. The CPB gene was expressed highly in hepatopancreas and decreased in crab hemocytes after challenges with white spot syndrome virus (WSSV) or Vibrio alginolyticus. After CPB gene knockdown using double-stranded RNA (CPB-dsRNA), the expression of JAK, STAT, C-type lectin, crustin antimicrobial peptide, Toll-like receptors, prophenoloxidase, and myosin II essential light chain-like protein were down-regulated in hemocytes at 24 h post dsRNA treatment. CPB knockdown decreases total hemocyte count in crabs indicated that CPB may negatively regulate crab hemocyte proliferation in crabs. CPB showed an inhibitory effect on hemocyte apoptosis in crabs infected with WSSV or V. alginolyticus. The phagocytosis rate of WSSV by hemocytes was increased after CPB-dsRNA treatment. After WSSV challenge, the mortality and WSSV copy number were both decreased but the rate of hemocyte apoptosis was increased in CPB-dsRNA-treated crabs. The results indicate that the antiviral activity of the crabs was enhanced when CPB was knocked down, indicating WSSV may take advantage of CPB to benefit its replication. In contrast, the absence of CPB in crabs increased mortality following the V. alginolyticus challenge. The phagocytosis rate of V. alginolyticus by hemocytes was increased after CPB-dsRNA treatment. It was revealed that CPB may play a positive role in the immune response to V. alginolyticus through increasing the phagocytosis rate of V. alginolyticus. This research further adds to our understanding of the CPB and identifies its potential role in the innate immunity of crabs.