White Spot Syndrome Virus Envelope Research Articles

Identification of a FOXO gene and its roles in anti-WSSV infection through regulation of Dicers and Argos in Macrobrachium nipponense

Forkhead box O (FOXO) proteins are a subgroup of the forkhead family of transcription factors that play important roles in the immune response. In this study, we cloned and identified a FOXO gene named MnFOXO from Macrobrachium nipponense. The full-length cDNA of MnFOXO is 2086 bp and contains a 1302 bp open reading frame, which encodes 433 amino acids. MnFOXO consists of five low-complexity regions and a conserved DNA-binding domain (forkhead domain). Evolutionary analyses indicate that MnFOXO proteins cluster with FOXO proteins from crustaceans. Tissue distribution analysis showed that MnFOXO was expressed in all detected tissues, with relatively higher expression levels in the intestine, eyestalks, stomach, and hemocytes than in the hepatopancreas, gills, and heart. The expression levels of MnFOXO in the hepatopancreas and intestine were significantly up-regulated in M. nipponense infected with white spot syndrome virus (WSSV) at 24 and 48 h. Furthermore, knockdown of MnFOXO increased the expression of WSSV envelope protein VP28 during WSSV infection. Further studies showed that knockdown of the MnFOXO gene in M. nipponense inhibited the synthesis of Dicers (MnDicer1, MnDicer2) and Argonautes (MnArgo1, MnArgo2) during WSSV invasion. These findings suggest that MnFOXO positively regulates the expression of Dicers and Argos, and inhibits the expression of VP28. This study provides new evidence for understanding the role of FOXO in antiviral innate immunity in crustaceans.

Insect cells of Spodoptera frugiperda support WSSV gene replication but not progeny virion assembly

The lacking of stable and susceptible cell lines has hampered research on pathogenic mechanism of crustacean white spot syndrome virus (WSSV). To look for the suitable cell line which can sustain WSSV infection, we performed the studies on WSSV infection in the Spodoptera frugiperda (Sf9) insect cells. In consistent with our previous study in vitro in crayfish hematopoietic tissue cells, the WSSV envelope was detached from nucleocapsid around 2 hpi in Sf9 cells, which was accompanied with the cytoplasmic transport of nucleocapsid toward the cell nucleus within 3 hpi. Furthermore, the expression profile of both gene and protein of WSSV was determined in Sf9 cells after viral infection, in which a viral immediate early gene IE1 and an envelope protein VP28 exhibited gradually increased presence from 3 to 24 hpi. Similarly, the significant increase of WSSV genome replication was found at 3–48 hpi in Sf9 cells after infection with WSSV, indicating that Sf9 cells supported WSSV genome replication. Unfortunately, no assembled progeny virion was observed at 24 and 48 hpi in Sf9 cell nuclei as determined by transmission electron microscope, suggesting that WSSV progeny could not be assembled in Sf9 cell line as the viral structural proteins could not be transported into cell nuclei. Collectively, these findings provide a cell model for comparative analysis of WSSV infection mechanism with crustacean cells.

Duox mediated ROS production inhibited WSSV replication in Eriocheir sinensis under short-term nitrite stress

Nitrite stress and white spot syndrome virus (WSSV) infection are major problems threatening the sustainable and healthy development of Eriocheir sinensis. Some studies have found that nitrite stress can lead to the production of reactive oxygen species (ROS), whereas synthetic ROS plays a vital role in the signaling pathway. However, whether nitrite stress influences the infection of crabs by WSSV remains unclear. NADPH oxidases, including NOX1-5 and Duox1-2, are important for ROS production. In the present study, a novel Duox gene (designated as EsDuox) was identified from E. sinensis. The studies found that nitrite stress could increase the expression of EsDuox during WSSV infection and decrease the transcription of the WSSV envelope protein VP28. Moreover, nitrite stress could increase the production of ROS, and the synthesis of ROS relied on EsDuox. These results indicated a potential “nitrite stress–Duox activation–ROS production” pathway that plays a negative role in WSSV infection in E. sinensis. Further studies found that nitrite stress and EsDuox could promote the expression of EsDorsal transcriptional factor and antimicrobial peptides (AMPs) during WSSV infection. Moreover, the synthesis of AMPs was positively regulated by EsDorsal in the process of WSSV infection under nitrite stress. Furthermore, EsDorsal played an inhibitory role in the replication of WSSV under nitrite stress. Our study reveals a new pathway for “nitrite stress–Duox activation–ROS production–Dorsal activation–AMP synthesis” that is involved in the defense against WSSV infection in E. sinensis during short-term nitrite stress.

Study of effects of dietary quercetin (Que) on growth performance and disease resistance mechanism of Litopenaeus vannamei

The purpose of this study was to evaluate effects of dietary quercetin (Que) on growth performance and disease resistance mechanism of Litopenaeus vannamei. Shrimp were randomly divided into 4 groups, with 3 replicants in each group and 70 shrimp in each replicants. The contents of Que. in the four groups were 0, 0.25, 0.5 and 1 g kg−1, respectively. Samples were taken after 56 days, followed by a 3-day white spot syndrome virus (WSSV) challenge experiment. The results showed that Que. significantly improved the growth performance and reduced the content of crude fat in shrimp (P < 0.05). Dietary Que. increased phagocytic activity, bacteriolytic activity and antibacterial activity (P < 0.05), but had no effects on the total hemocytes count. Dietary Que. also improved the expression levels of genes related to immunity of shrimp. In addition, dietary Que. improved the activities of catalase and glutathione peroxidase, total antioxidant capability and the content of glutathione (P < 0.05), but decreased the superoxide dismutase activity (P < 0.05). In the case of intestinal microbiota, Que. reduced microbial diversity and richness. Meanwhile, Que. also increased abundances of functional genes related to the “immune system” and “lipid metabolism”. After WSSV challenge, the cumulative mortality of shrimp decreased with increasing levels of dietary Que., the gene expression of WSSV envelope protein and hemocytes surface receptor protein were down-regulated (P < 0.05), while the gene expression of hemocytes immunity was up-regulated (P < 0.05). In summary, Que. improved the growth performance, enhanced the antioxidant capability and immune response, regulated the composition and structure of intestinal microbiota, and reduced cumulative mortality after WSSV challenge of Litopenaeus vannamei.

Identification of three novel Spätzle genes in Eriocheir sinensis and their roles during white spot syndrome virus infection

Proteins of Spätzle family play an essential role in innate immunity in invertebrates by activating the Toll pathway to induce the expression of antimicrobial peptides. However, little is known about the function of Spätzle in in the immune response of the Chinese mitten crab. In the present study, three novel Spätzle genes (named as EsSpz1, EsSpz2, and EsSpz3) were identified from Eriocheir sinensis. The genome structure of EsSpz1 contains two exons and an intron. Three Spätzle proteins all contain a Pfam Spaetzle domain. In the evolution, EsSpz1-3 cluster with other Spätzle proteins from crustaceans. EsSpz1-3 were widely distributed in multiple immune tissues. The expression levels of EsSpz1-3 in the intestine were remarkably upregulated after white spot syndrome virus (WSSV) challenge. The knockdown of EsSpz1-3 remarkably decreased the expressions of crustins and anti-lipopolysaccharide factors during WSSV infection. Moreover, EsSpz1-3 silencing remarkably increased the expression of WSSV envelope protein VP28. These findings suggest that new-found EsSpz1-3 in E. sinensis could promote the synthesis of antimicrobial peptides and inhibit the expression of VP28 during WSSV infection. Our study indicates that EsSpz1-3 in E. sinensis may participate in the innate immune defenses against WSSV by inducing the expression of antimicrobial peptides. This study provides new knowledge for the function of Spätzle in the antiviral immune defense in crustacean.

Characterization of four spliced isoforms of a transmembrane C-type lectin from Procambarus clarkii and their function in facilitating WSSV infection

C-type lectin (CTL) is an important pattern recognition receptor that play vital functions in the innate immunity. Many soluble CTLs in crustacean participate in the inhibition or promotion of white spot syndrome virus (WSSV) infection. However, whether transmembrane CTLs participate in WSSV infection in crustacean remains unknown. In the present study, four spliced isoforms of a transmembrane CTL (designated as PcTlec) from Procambarus clarkii were identified for the first time. The genome structure of PcTlec contains eight exons, six known introns, and one unknown intron. PcTlec-isoform1 is produced by intron retention, whereas PcTlec-isoform3 and PcTlec-isoform4 are produced by exon skipping. All of them contain the transmembrane domain and characteristic carbohydrate recognition domain (CRD). Four PcTlec isoforms were mainly expressed in the hepatopancreas, stomach, and intestine. After WSSV challenge, the expression levels of PcTlec-isoform1-4 in the intestine were upregulated. The knockdown of the region shared by four PcTlec isoforms evidently decreased the expression of WSSV envelope protein VP28 and the copies of viral particles. A recombinant protein (rPcTlec-CRD) containing the CRD that was shared by four PcTlec isoforms was acquired by procaryotic expression system. The injection of purified rPcTlec-CRD protein evidently increased the VP28 expression and WSSV copies during viral infection. Moreover, rPcTlec-CRD could directly bind to WSSV and interact with VP28 protein. These findings indicate that new-found transmembrane CTL isoforms in P. clarkii may act as viral receptors that facilitate WSSV infection. This study contributes to the recognition and understanding of the functions of transmembrane CTLs in crustacean in the infection of host by WSSV.

Applying Modified VP53A Recombinant Protein as an Anti-White Spot Syndrome Virus Biological Agent in Litopenaeus vannamei Farming.

Shrimp farming is an important economic activity. However, due to the spread of pathogens, shrimp aquaculture is becoming increasingly difficult. Many studies have confirmed that white spot syndrome virus (WSSV) recombinant proteins can inhibit viral infection. Among them, VP53 recombinant protein has been found to reduce mortality upon WSSV challenge. This study was conducted in Kaohsiung, Taiwan and reports the first field feeding trial to demonstrate that WSSV recombinant proteins can improve shrimp survival rates at a farming scale. Prior to the feeding trial, the shrimp were confirmed to be slightly infected with WSSV, Vibrio parahaemolyticus strains causing acute hepatopancreatic necrosis disease (AHPND), non-AHPND V. parahaemolyticus strains, and Enterocytozoon hepatopenaei (EHP), which are common pathogens that shrimp farmers often face. The shrimp were then divided into two groups: a control group (C group) fed with a commercial diet and a protein group (P group) fed with the same commercial feed with VP53 recombinant protein. Our findings indicated that the survival rate and expression of immune genes of the P group were higher than those of the C group. The intestinal microbiota of the two groups were also analysed. Collectively, our results confirmed that the recombinant WSSV envelope protein derivative can be used as an effective anti-virus biological agent in shrimp farms.

The dyslexia-associated KIAA0319-like protein from Marsupenaeus japonicus recognizes white spot syndrome virus and restricts its replication

Dyslexia-associated protein KIAA0319, with immunoglobulin-like polycystic kidney disease (PKD) domains, is a dyslexia susceptibility protein. KIAA0319 participates in neuronal migration and is involved in the interaction between neurons and glial fibers. Recent studies found that KIAA0319 acts as a viral receptor via an interaction between its PKD domains with viruses. KIAA0319 mediates viral internalization via a clathrin-dependent pathway involving interaction with adaptor protein 2 (AP2). In the present study, we identified a cDNA encoding a dyslexia-associated KIAA0319-like protein from Marsupenaeus japonicus (designated as MjKIAA0319L). MjKIAA0319L was upregulated in shrimp challenged by white spot syndrome virus (WSSV). After knockdown of MjKIAA0319L using RNA interference, followed by WSSV infection in shrimp, WSSV replication increased and the shrimp survival rate decreased significantly. Further study found that the extracellular domain of MjKIAA0319L expressed in Escherichia coli bound WSSV particles by interacting with the WSSV envelope protein VP28. Fluorescent immunocytochemical analysis showed that MjKIAA0319L was internalized into the cytoplasm of hemocytes during WSSV infection. The intracellular domain of MjKIAA0319L, comprising a YXXΦ motif, interacted with AP2μ, suggesting that MjKIAA0319L binds to WSSV and induces endocytosis in a clathrin-dependent manner. Therefore, MjKIAA0319L recognizes WSSV via its extracellular domain and induces endocytosis of WSSV through its intracellular domain in shrimp. Thus, the MjKIAA0319L-AP2-clathrin pathway is involved in the phagocytosis of WSSV to restrict viral infection in shrimp.

SpSR-B2 functions as a potential pattern recognition receptor involved in antiviral and antibacterial immune responses of mud crab Scylla paramamosain

Although class B scavenger receptors (SR-Bs) in mammals are multifunctional molecules, the functions of SR-Bs in invertebrates remain largely unknown. In this study, we characterized an SR-B homolog, namely SpSR-B2, from Scylla paramamosain. SpSR-B2 shared high similarity with mammalian SR-Bs, and exhibited specific binding activity to ac-LDL, indicating that it may be a new member of SR-B class in invertebrates. SpSR-B2 was upregulated after challenge with white spot syndrome virus (WSSV) or bacteria. Binding assays showed that SpSR-B2 specifically interacted with WSSV envelope protein VP24. Besides, SpSR-B2 could bind to all tested bacterial cells and agglutinate these bacteria. SpSR-B2 also exhibited a strong binding activity to LPS but weak binding activities to other tested polysaccharides. These findings indicated that SpSR-B2 was a potential recognition molecule for viral protein VP24 and bacterial LPS. Knockdown of SpSR-B2 resulted in dramatically decreased expressions of certain antimicrobial peptides (AMPs), and overexpression of SpSR-B2 led to the increased expression of the AMP of SpALF2, suggesting that SpSR-B2 could regulate the expression of AMPs. Taken together, this study revealed that SpSR-B2 functioned as a potential pattern recognition receptor participating in antiviral and antibacterial immunity, and provided new insights into the immune functions of invertebrate SR-Bs.

Anti-lipopolysaccharide factor D from kuruma shrimp exhibits antiviral activity

Anti-lipopolysaccharide factors (ALFs) exhibit a potent antimicrobial activity against a broad range of bacteria, filamentous fungi, and viruses. In previous reports, seven groups of ALFs (groups A–G) were identified in penaeid shrimp. Among them, group D showed negative net charges and weak antimicrobial activity. Whether this group has antiviral function is not clear. In this study, the ALF sequences of penaeid shrimp were analyzed, and eight groups of ALF family (groups A–H) were identified. The four ALFs including MjALF-C2, MjALF-D1, MjALF-D2, and MjALF-E2 from kuruma shrimp Marsupenaeus japonicus were expressed recombinantly in Escherichia coli, and the antiviral activity was screened via injection of purified recombinant ALFs into shrimp following white spot syndrome virus (WSSV) infection. Results showed that the expression of Vp28 (WSSV envelope protein) decreased significantly in the MjALF-D2-injected shrimp only. Therefore, MjALF-D2 was chosen for further study. Expression pattern analysis showed that MjAlf-D2 was upregulated in shrimp challenged by WSSV. The WSSV replication was detected in RNA, genomic DNA, and protein levels using VP28 and Ie1 (immediate-early gene of WSSV) as indicators in MjALF-D2-injected shrimp following WSSV infection. Results showed that WSSV replication was significantly inhibited compared with that in the rTRX- or PBS-injected control groups. After knockdown of MjAlf-D2 in shrimp by RNA interference, the WSSV replication increased significantly in the shrimp. All these results suggested that MjALF-D2 has an antiviral function in shrimp immunity, and the recombinant ALF-D2 has a potential application for viral disease control in shrimp aquaculture.

Identification of a Shrimp E3 Ubiquitin Ligase TRIM50-Like Involved in Restricting White Spot Syndrome Virus Proliferation by Its Mediated Autophagy and Ubiquitination.

Most tripartite motif (TRIM) family proteins are critical components of the autophagy machinery and play important roles in host defense against viral pathogens in mammals. However, the roles of TRIM proteins in autophagy and viral infection have not been studied in lower invertebrates, especially crustaceans. In this study, we first identified a TRIM50-like gene from Penaeus monodon (designated PmTRIM50-like), which, after a white spot syndrome virus (WSSV) challenge, was significantly upregulated at the mRNA and protein levels in the intestine and hemocytes. Knockdown of PmTRIM50-like led to an increase in the WSSV quantity in shrimp, while its overexpression led to a decrease compared with the controls. Autophagy can be induced by WSSV or rapamycin challenge and has been shown to play a positive role in restricting WSSV replication in P. monodon. The mRNA and protein expression levels of PmTRIM50-like significantly increased with the enhancement of rapamycin-induced autophagy. The autophagy activity induced by WSSV or rapamycin challenge could be inhibited by silencing PmTRIM50-like in shrimp. Further studies showed that rapamycin failed to induce autophagy or inhibit WSSV replication after knockdown of PmTRIM50-like. Moreover, pull-down and in vitro ubiquitination assays demonstrated that PmTRIM50-like could interact with WSSV envelope proteins and target them for ubiquitination in vitro. Collectively, this study demonstrated that PmTRIM50-like is required for autophagy and is involved in restricting the proliferation of WSSV through its ubiquitination. This is the first study to report the role of a TRIM family protein in virus infection and host autophagy in crustaceans.

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

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

Identification and functional characterization of a novel C-type lectin from the kuruma shrimp, Marsupenaeus japonicus

C-type lectins (CTLs) are immune molecules that are crucial to the invertebrate innate immune system with the primary function of recognizing invading pathogens. In the present study, a novel CTL was cloned from Marsupenaeus japonicus (MjCTL), and its tissue distribution and expression patterns over time in response to white spot syndrome virus (WSSV) and Vibrio parahaemolyticus were further investigated. The open reading frame (ORF) of MjCTL was 513 bp and encoded a polypeptide of 170 amino acids, which contained a signal peptide and a carbohydrate recognition domain (CRD) that are typical for CTLs. MjCTL was primarily expressed in the hepatopancreas and weakly expressed in hemocytes, gill, stomach, intestine, heart, muscle and eyestalk. The expression level of MjCTL in the hepatopancreas was dramatically increased at 48 h post-injection with WSSV at a dosage of 1 × 105 virions. Glutathione-S-transferase (GST) pull-down assays showed that MjCTL could directly bind to several WSSV envelope proteins, including VP19, VP24, VP26 and VP28. Moreover, MjCTL displayed antibacterial activity against V. parahaemolyticus. Our results indicated that MjCTL exhibited multiple functions in innate immune response against pathogens.

Synthesis and evaluation of polyamine carbon quantum dots (CQDs) in Litopenaeus vannamei as a therapeutic agent against WSSV

White spot syndrome virus (WSSV) is the causative agent of white spot syndrome (WSS), a disease that has led to severe mortality rates in cultured shrimp all over the world. The WSSV is a large, ellipsoid, enveloped double-stranded DNA virus with a wide host range among crustaceans. Currently, the main antiviral method is to block the receptor of the host cell membrane using recombinant viral proteins or virus antiserum. In addition to interference with the ligand-receptor binding, disrupting the structure of the virus envelope may also be a means to combat the viral infection. Carbon quantum dots (CQDs) are carbonaceous nanoparticles that have many advantageous characteristics, including small size, low cytotoxicity, cheap, and ease of production and modification. Polyamine-modified CQDs (polyamine CQDs) with strong antibacterial ability have been identified, previously. In this study, polyamine CQDs are shown to attach to the WSSV envelope and inhibit the virus infection, with a dose-dependent effect. The results also show that polyamine CQDs can upregulate several immune genes in shrimp and reduce the mortality upon WSSV infection. This is first study to identify that polyamine CQDs could against the virus. These results, indeed, provide a direction to develop effective antiviral strategies or therapeutic methods using polyamine CQDs in aquaculture.

A shrimp glycosylase protein, PmENGase, interacts with WSSV envelope protein VP41B and is involved in WSSV pathogenesis

Viral glycoproteins are expressed by many viruses, and during infection they usually play very important roles, such as receptor attachment or membrane fusion. The mature virion of the white spot syndrome virus (WSSV) is unusual in that it contains no glycosylated proteins, and there are currently no reports of any glycosylation mechanisms in the pathogenesis of this virus. In this study, we cloned a glycosylase, mannosyl-glycoprotein endo-β-N-acetylglucosaminidase (ENGase, EC 3.2.1.96), from Penaeus monodon and found that it was significantly up-regulated in WSSV-infected shrimp. A yeast two-hybrid assay showed that PmENGase interacted with both structural and non-structural proteins, and GST-pull down and co-immunoprecipitation (Co-IP) assays confirmed its interaction with the envelope protein VP41B. In the WSSV challenge tests, the cumulative mortality and viral copy number were significantly decreased in the PmEngase-silenced shrimp, from which we conclude that shrimp glycosylase interacts with WSSV in a way that benefits the virus. Lastly, we speculate that the deglycosylation activity of PmENGase might account for the absence of glycosylated proteins in the WSSV virion.

Identification and Molecular Characterization of a Pellino Protein in Kuruma Prawn (Marsupenaeus Japonicus) in Response to White Spot Syndrome Virus and Vibrio Parahaemolyticus Infection.

Kuruma prawn, Marsupenaeus japonicus, has the third largest annual yield among shrimp species with vital economic significance in China. White spot syndrome virus (WSSV) is a great threat to the global shrimp farming industry and results in high mortality. Pellino, a highly conserved E3 ubiquitin ligase, has been found to be an important modulator of the Toll-like receptor (TLR) signaling pathways that participate in the innate immune response and ubiquitination. In the present study, the Pellino gene from Marsupenaeus japonicus was identified. A qRT-PCR assay showed the presence of MjPellino in all the tested tissues and revealed that the transcript level of this gene was significantly upregulated in both the gills and hemocytes after challenge with WSSV and Vibrio parahaemolyticus. The function of MjPellino was further verified at the protein level. The results of the three-dimensional modeling and protein–protein docking analyses and a GST pull-down assay revealed that the MjPellino protein was able to bind to the WSSV envelope protein VP26. In addition, the knockdown of MjPellino in vivo significantly decreased the expression of MjAMPs. These results suggest that MjPellino might play an important role in the immune response of kuruma prawn.

RPS27, a sORF-Encoded Polypeptide, Functions Antivirally by Activating the NF-κB Pathway and Interacting With Viral Envelope Proteins in Shrimp.

A small open reading frame (smORF) or short open reading frame (sORF) encodes a polypeptide of <100 amino acids in eukaryotes (50 amino acids in prokaryotes). Studies have shown that several sORF-encoded peptides (SEPs) have important physiological functions in different organisms. Many ribosomal proteins belonging to SEPs play important roles in several cellular processes, such as DNA damage repair and apoptosis. Several studies have implicated SEPs in response to infection and innate immunity, but the mechanisms have been unclear for most of them. In this study, we identified a sORF-encoded ribosomal protein S27 (RPS27) in Marsupenaeus japonicus. The expression of MjRPS27 was significantly upregulated in shrimp infected with white spot syndrome virus (WSSV). After knockdown of MjRPS27 by RNA interference, WSSV replication increased significantly. Conversely, after MjRPS27 overexpression, WSSV replication decreased in shrimp and the survival rate of the shrimp increased significantly. These results suggested that MjRPS27 inhibited viral replication. Further study showed that, after MjRPS27 knockdown, the mRNA expression level of MjDorsal, MjRelish, and antimicrobial peptides (AMPs) decreased, and the nuclear translocation of MjDorsal and MjRelish into the nucleus also decreased. These findings indicated that MjRPS27 might activate the NF-κB pathway and regulate the expression of AMPs in shrimp after WSSV challenge, thereby inhibiting viral replication. We also found that MjRPS27 interacted with WSSV's envelope proteins, including VP19, VP24, and VP28, suggesting that MjRPS27 may inhibit WSSV proliferation by preventing virion assembly in shrimp. This study was the first to elucidate the function of the ribosomal protein MjRPS27 in the antiviral immunity of shrimp.

Identification of two novel C-type lectins involved in immune defense against white spot syndrome virus and Vibrio parahaemolyticus from Marsupenaeus japonicus

C-type lectins (CTLs) are vital molecules in crustacean innate immunity with the capacity to recognize and eliminate invaders, such as viruses and bacteria. Here, two novel CTLs were identified from the kuruma shrimp Marsupenaeus japonicus, and their molecular characteristics and immune function were investigated. Sequence analysis revealed that the two CTLs possessed the typical CTL structure and function features. Tissue distribution analysis showed that the two CTLs were most abundantly expressed in the hepatopancreas and weakly expressed in other examined tissues. The transcription of the two CTLs significantly increased in the hepatopancreas of shrimp challenged with both white spot syndrome virus (WSSV) and Vibrio parahaemolyticus, and MjCTL4 was found to be more sensitive to the two pathogens than MjCTL3, being induced at relatively faster and higher increments. GST pull-down assays showed that the two CTLs could directly interact with several WSSV envelope proteins (VP19, VP24, VP26 and VP28). Moreover, the two CTLs displayed obvious binding and antibacterial ability to V. parahaemolyticus, and MjCTL3 exhibited stronger anti-V. parahaemolyticus activity than MjCTL4. These results suggest that the two novel CTLs might function as pattern recognition receptors (PRRs) and antibacterial molecules in M. japonicus innate immunity, and the two CTLs may be alternative agents for the prevention and treatment of diseases caused by WSSV and V. parahaemolyticus.

A novel RING finger protein CqRNF152-like with self-ubiquitination activity inhibits white spot syndrome virus infection in a crustacean Cherax quadricarinatus

Really Interesting New Gene (RING) finger proteins are highly conserved molecules that participate in a variety of biological processes such as regulation of development, apoptosis and antiviral immunity in vertebrates. However, the functions of RING finger proteins are still poorly understood in crustaceans. Previously, we found that the transcript of a homolog of RING finger protein 152 (CqRNF152-like) was up-regulated in a differentially expressed transcriptome library of the haematopietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus upon white spot syndrome virus (WSSV) infection, which is one of the most devastating viral diseases for crustaceans like shrimp and crayfish. The full-length cDNA sequence of CqRNF152-like was then identified with 975 bp, including an ORF of 685 bp that encoded a 195 amino acids protein, a 5′- UTR of 180 bp, and a 3′-UTR with a poly (A) tail of 207 bp. The conserved domain prediction showed that CqRNF152-like contained a conserved RING-finger domain. Gene expression analysis showed that CqRNF152-like was distributed in all tissues examined and the transcript is significantly up-regulated after WSSV challenge both in vivo in Hpt tissue and in vitro in cultured Hpt cells. Furthermore, the transcripts of both an immediate early gene ie1 and a late envelope protein gene vp28 of WSSV were clearly increased in the Hpt tissues, hemocytes and cultured Hpt cells after gene silencing of CqRNF152-like, which were further proved to be significantly decreased after overloading of recombinant CqRNF152-like protein in Hpt cell cultures. Meanwhile, CqRNF152-like was found to bind with WSSV envelope protein VP28 by proteins pull-down assay. Similar to most of RNF proteins, CqRNF152-like protein sequence contained a conserved RING-finger domain and showed self-ubiquitination activity in a RING finger domain dependent manner. Taken together, CqRNF152-like is likely to function as an antiviral molecular against WSSV infection through interaction with the envelope protein VP28 in a crustacean C. quadricarinatus. This is the first report that a RING finger protein with directly antiviral functions via interaction with viral protein and self-ubiquitination activity in crustacean, which sheds new light on the molecular mechanism of WSSV infection and the control of white spot disease.

MD-2 Homologue Recognizes the White Spot Syndrome Virus Lipid Component and Induces Antiviral Molecule Expression in Shrimp.

The myeloid differentiation factor 2 (MD-2)-related lipid-recognition (ML) domain is found in multiple proteins, including MD-2, MD-1, Niemann-Pick disease type C2, and mite major allergen proteins. The significance of ML proteins in antibacterial signal transduction and in lipid metabolism has been well studied. However, their function in host-virus interaction remains poorly understood. In the current study, we found that the ML protein family is involved in resistance against white spot syndrome virus in kuruma shrimp, Marsupenaeus japonicus One member, which showed a high similarity to mammalian MD-2/MD-1 and was designated as ML1, participated in the antiviral response by recognizing cholesta-3,5-diene (CD), a lipid component of the white spot syndrome virus envelope. After recognizing CD, ML1 induced the translocation of Rel family NF-κB transcription factor Dorsal into the nucleus, resulting in the expression of Vago, an IFN-like antiviral cytokine in arthropods. Overall, this study revealed the significance of an MD-2 homologue as an immune recognition protein for virus lipids. The identification and characterization of CD-ML1-Dorsal-Vago signaling provided new insights into invertebrate antiviral immunity.