Shaoping Weng

A Novel C-Type Lectin from the Shrimp Litopenaeus vannamei Possesses Anti-White Spot Syndrome Virus Activity

ABSTRACT C-type lectins play key roles in pathogen recognition, innate immunity, and cell-cell interactions. Here, we report a new C-type lectin (C-type lectin 1) from the shrimp Litopenaeus vannamei (LvCTL1), which has activity against the white spot syndrome virus (WSSV). LvCTL1 is a 156-residue polypeptide containing a C-type carbohydrate recognition domain with an EPN (Glu99-Pro100-Asn101) motif that has a predicted ligand binding specificity for mannose. Reverse transcription-PCR analysis revealed that LvCTL1 mRNA was specifically expressed in the hepatopancreas of L. vannamei. Recombinant LvCTL1 (rLvCTL1) had hemagglutinating activity and ligand binding specificity for mannose and glucose. rLvCTL1 also had a strong affinity for WSSV and interacted with several envelope proteins of WSSV. Furthermore, we showed that the binding of rLvCTL1 to WSSV could protect shrimps from viral infection and prolong the survival of shrimps against WSSV infection. Our results suggest that LvCTL1 is a mannose-binding C-type lectin that binds to envelope proteins of WSSV to exert its antiviral activity. To our knowledge, this is the first report of a shrimp C-type lectin that has direct anti-WSSV activity.

Molecular cloning, characterization and expression analysis of two novel Tolls (LvToll2 and LvToll3) and three putative Spätzle-like Toll ligands (LvSpz1–3) from Litopenaeus vannamei

Toll-like receptor-mediated NF-κB pathways are essential for inducing immune related-gene expression in the defense against bacterial, fungal and viral infections in insects and mammals. Although a Toll receptor (LvToll1) was cloned in Litopenaeus vannamei, relatively little is known about other types of Toll-like receptors and their endogenous cytokine-like ligand, Spätzle. Here, we report two novel Toll-like receptors (LvToll2 and LvToll3) and three Spätzle-like proteins (LvSpz1-3) from L. vannamei. LvToll2 has 1009 residues with an extracellular domain containing 18 leucine-rich repeats (LRRs) and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain of 139 residues. LvToll3 is 1244 residues long with an extracellular domain containing 23 LRRs and a cytoplasmic TIR domain of 138 residues. The Spätzle-like proteins LvSpz1, LvSpz2 and LvSpz3 are 237, 245 and 275 residues in length, respectively, and all of them have a putative C-terminal cystine-knot domain. In Drosophila Schneider 2 (S2) cells, LvToll1 and LvToll3 were localized to the membrane and cytoplasm, and LvToll2 was confined to the cytoplasm. In Drosophila S2 cells, LvToll2 could significantly activate the promoters of NF-κB-pathway-controlled antimicrobial peptide genes, whereas LvToll1 and LvToll3 had no effect on them. LvSpz1 exerted some degree of inhibition on the promoter activities of Drosophila Attacin A and L. vannamei Penaeidin4. LvSpz3 also inhibited the Drosophila Attacin A promoter, but LvSpz2 could only slightly activate it. LvToll1, LvToll2 and LvToll3 were constitutive expressed in various tissues, while LvSpz1, LvSpz2 and LvSpz3 exhibited tissue-specific expression in the epithelium, eyestalk, intestine, gill and muscle. In the gill, after Vibrio alginolyticus challenge, LvToll1 was upregulated, but LvToll2 and LvToll3 showed no obvious changes. LvSpz1 and LvSpz3 were also strongly induced by V. alginolyticus challenge, but LvSpz2 only showed a slight downregulation. In the gill, after white spot syndrome virus (WSSV) challenge, LvToll1, LvToll2, LvToll3, LvSpz1 and LvSpz3 were upregulated, but LvSpz2 showed no obvious change, except for a slight downregulation at 12h post-injection of WSSV. These findings might be valuable in understanding the innate immune signal pathways of shrimp and enabling future studies on the host-pathogen interactions in V. alginolyticus and WSSV infections.

Analysis of Litopenaeus vannamei Transcriptome Using the Next-Generation DNA Sequencing Technique

Background Pacific white shrimp (Litopenaeus vannamei), the major species of farmed shrimps in the world, has been attracting extensive studies, which require more and more genome background knowledge. The now available transcriptome data of L. vannamei are insufficient for research requirements, and have not been adequately assembled and annotated. Methodology/Principal Findings This is the first study that used a next-generation high-throughput DNA sequencing technique, the Solexa/Illumina GA II method, to analyze the transcriptome from whole bodies of L. vannamei larvae. More than 2.4 Gb of raw data were generated, and 109,169 unigenes with a mean length of 396 bp were assembled using the SOAP denovo software. 73,505 unigenes (>200 bp) with good quality sequences were selected and subjected to annotation analysis, among which 37.80% can be matched in NCBI Nr database, 37.3% matched in Swissprot, and 44.1% matched in TrEMBL. Using BLAST and BLAST2Go softwares, 11,153 unigenes were classified into 25 Clusters of Orthologous Groups of proteins (COG) categories, 8171 unigenes were assigned into 51 Gene ontology (GO) functional groups, and 18,154 unigenes were divided into 220 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. To primarily verify part of the results of assembly and annotations, 12 assembled unigenes that are homologous to many embryo development-related genes were chosen and subjected to RT-PCR for electrophoresis and Sanger sequencing analyses, and to real-time PCR for expression profile analyses during embryo development. Conclusions/Significance The L. vannamei transcriptome analyzed using the next-generation sequencing technique enriches the information of L. vannamei genes, which will facilitate our understanding of the genome background of crustaceans, and promote the studies on L. vannamei.

The shrimp NF-κB pathway is activated by white spot syndrome virus (WSSV) 449 to facilitate the expression of WSSV069 (ie1), WSSV303 and WSSV371.

The Toll-like receptor (TLR)-mediated NF-κB pathway is essential for defending against viruses in insects and mammals. Viruses also develop strategies to utilize this pathway to benefit their infection and replication in mammal hosts. In invertebrates, the TLR-mediated NF-κB pathway has only been well-studied in insects and has been demonstrated to be important in antiviral responses. However, there are few reports of interactions between viruses and the TLR-mediated NF-κB pathway in invertebrate hosts. Here, we studied Litopenaeus vannamei Pelle, which is the central regulator of the Toll pathway, and proposed that a similar TLR/MyD88/Tube/Pelle/TRAF6/NF-κB cascade may exist in shrimp for immune gene regulation. After performing genome-wild analysis of white spot syndrome virus (WSSV) encoded proteins, we found that WSSV449 shows 15.7-19.4% identity to Tube, which is an important component of the insect Toll pathway. We further found that WSSV449 activated promoters of Toll pathway-controlled antimicrobial peptide genes, indicating WSSV449 has a similar function to host Tube in activating the NF-κB pathway. We suspected that WSSV449 activated the Toll-mediated NF-κB pathway for regulating viral gene expression. To test this hypothesis, we analyzed the promoters of viral genes and found 40 promoters that possess NF-κB binding sites. A promoter screen showed that the promoter activities of WSSV069 (ie1), WSSV303 and WSSV371 can be highly induced by the shrimp NF-κB family protein LvDorsal. WSSV449 also induced these three viral promoter activities by activating the NF-κB pathway. To our knowledge, this is the first report of a virus that encodes a protein similar to the Toll pathway component Tube to upregulate gene expression in the invertebrate host.

Litopenaeus vannamei tumor necrosis factor receptor-associated factor 6 (TRAF6) responds to Vibrio alginolyticus and white spot syndrome virus (WSSV) infection and activates antimicrobial peptide genes

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a key signaling adaptor protein not only for the TNFR superfamily but also for the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. To investigate TRAF6 function in invertebrate innate immune responses, Litopenaeus vannamei TRAF6 (LvTRAF6) was identified and characterized. The full-length cDNA of LvTRAF6 is 2823bp long, with an open reading frame (ORF) encoding a putative protein of 594 amino acids, including a RING-type Zinc finger, two TRAF-type Zinc fingers, a coiled-coil region, and a meprin and TRAF homology (MATH) domain. The overall amino acid sequence identity between LvTRAF6 and other known TRAF6s is 22.2-33.3%. Dual luciferase reporter assays in Drosophila S2 cells revealed that LvTRAF6 could activate the promoters of antimicrobial peptide genes (AMPs), including Drosophila Attacin A and Drosomycin, and shrimp Penaeidins. Real-time quantitative PCR (qPCR) indicated that LvTRAF6 was constitutively expressed in various tissues of L. vannamei. After Vibrio alginolyticus and white spot syndrome virus (WSSV) challenge, LvTRAF6 was down-regulated, though with different expression patterns in the intestine compared to other tissues. After WSSV challenge, LvTRAF6 was up-regulated 2.7- and 2.3-fold over the control at 3h in gills and hepatopancreas, respectively. These results indicated that LvTRAF6 may play a crucial role in antibacterial and antiviral responses via regulation of AMP gene expression.

Development of a mandarin fish Siniperca chuatsi fry cell line suitable for the study of infectious spleen and kidney necrosis virus (ISKNV).

Infectious spleen and kidney necrosis virus (ISKNV) is a typical species of the genus Megalocytivirus in the family Iridoviridae. However, until recently, no suitable piscine cell line is stably susceptible to ISKNV. Here, a continuous cell culture derived from the mandarin fish fry (MFF-1) was developed and has been subcultured over 60 passages during a period of 18 months. MFF-1 consists predominantly of epithelial-like cells and grows well in DMEM supplemented with 10% fetal bovine serum. MFF-1 could produce high titers of ISKNV by continuous viral passages which were further confirmed by indirect immunofluorescence assay and RT-PCR analysis. Flow cytometry analyse showed that approximately 80.3% cells could be infected by ISKNV at 3 days post-infection. Abundant ISKNV particles were observed in the cytoplasm of the ISKNV infected MFF-1 cells by transmission electron microscopy. Mandarin fish injected with the filtrate from the infected cell suspension developed clinical signs and died, which is in accordance with the infectious spleen and kidney necrosis virus disease (ISKNVD). In addition, apoptosis was observed in the MFF-1 cells upon ISKNV infection by FITC-annexin V staining. ISKNV was purified and the viral protein profiles were also determined in this research. To our knowledge, MFF-1 is the first cell line originated from mandarin fish and it can be an efficient tool for the study of ISKNV.

An immune deficiency homolog from the white shrimp, Litopenaeus vannamei, activates antimicrobial peptide genes

Invertebrates rely on innate immunity as the first line defense against microbes. In Drosophila, the inducible antimicrobial peptides (AMPs) regulated by the Toll and immune deficiency (Imd) pathways are important effectors in innate immunity. Here we report an immune deficiency homolog (LvIMD) from the white shrimp, Litopenaeus vannamei. The full-length cDNA of LvIMD is 758 bp with an open reading frame of 483 bp that encodes a putative protein of 160 amino acids including a death domain at the C-terminus. LvIMD death domain shows similarity to that of Drosophila IMD and human receptor interacting protein 1 (RIP1) of the tumor necrosis factor receptor (TNFR) pathway, with 27.9% and 26.4% identity, respectively. Phylogenetic analysis shows that LvIMD clusters with a predicted protein from the starlet sea anemone (Nematostella vectensis) independent to insect IMDs and vertebrates RIP1s. LvIMD mRNA is expressed in most tissues and is induced in hepatopancreas and hemocytes after immune challenge. Luciferase reporter assays confirm that LvIMD is able to induce the expression of AMP genes, including Drosophila Attacin A and shrimp Penaeidin 4 in S2 cells. To our knowledge, this is the first report that LvIMD participates in innate signaling to activate the expression of AMP genes in shrimp.

Identification and functional characterization of Dicer2 and five single VWC domain proteins of Litopenaeus vannamei

Dicer (Dcr) is the key protein of the RNA interference (RNAi) pathway. To investigate the role of the RNAi pathway in shrimp anti-viral immunity, Litopenaeus vannamei Dcr2 (designated as LvDcr2) was identified and characterized. The full-length cDNA of LvDcr2 was 5513bp long, with an open reading frame encoding a putative protein of 1502 amino acids. In addition, five proteins homologous to the single von Willebrand factor type C (VWC) domain protein (SVC) were also identified in L. vannamei and named LvSVC1-5. These LvSVCs were between 102 and 190 amino acids in length and all contained a motif similar to Drosophila melanogaster SVC proteins (DmSVCs). By co-immunoprecipitation assays and pull-down assays, we demonstrated that LvDcr2, L. vannamei Argonaute 2 (LvAgo2), and L. vannamei transactivating response RNA-binding protein isoform 1 (LvTRBP1) interacted with each other. A luciferase reporter assay indicated that the promoters of LvSVC1, LvSVC4, LvSVC5, and DmSVC Vago (DmVago) were activated by LvDcr2 as well as by Drosophila Dcr2 (DmDcr2). Real-time RT-PCR showed that LvDcr2 and LvSVCs were up-regulated in immune responses against Poly(C-G) or WSSV challenge. These results suggested that LvDcr2 formed complexes with LvAgo2 and LvTRBP1 to act as the cores of shrimp small interfering RNA (siRNA)-induced silencing complex (siRISC)/siRISC-loading complex (siRLC), role in shrimp siRNA pathway. Furthermore, these results also suggested that LvDcr2 may engage in non-specific activation of anti-viral immunity.

A zebrafish (Danio rerio) model of infectious spleen and kidney necrosis virus (ISKNV) infection

Zebrafish is a model animal for studies of genetics, development, toxicology, oncology, and immunology. In this study, infectious spleen and kidney necrosis virus (ISKNV) was used to establish an infection in zebrafish, and the experimental conditions were established and characterized. Mortality of adult zebrafish infected with ISKNV by intraperitoneal (i.p.) injection exceeded 60%. ISKNV can be passed stably in zebrafish for over ten passages. The ailing zebrafish displayed petechial hemorrhaging and scale protrusion. Histological analysis of moribund fish revealed necrosis of tissue and enlarged cells in kidney and spleen. The real-time RT-PCR analysis of mRNA level confirmed that ISKNV was replicated in zebrafish. Immunohistochemistry and immunofluorescence analyses further confirmed the presence of ISKNV-infected cells in almost all organs of the infected fish. Electron microscope analyses showed that the ISKNV particle was present in the infected tissues. The establishment of zebrafish infection model of ISKNV can offer a valuable tool for studying the interactions between ISKNV and its host.

Molecular cloning, characterization and expression analysis of the tumor necrosis factor (TNF) superfamily gene, TNF receptor superfamily gene and lipopolysaccharide-induced TNF-α factor (LITAF) gene from Litopenaeus vannamei

In vertebrates, the tumor necrosis factor (TNF)-receptor (TNFR) system participates in diverse physiological and pathological events, such as inflammation and protective immune responses to microbial infections. There are few reports about the role of the invertebrate TNF-TNFR system in immune responses. Here, we isolated and characterized the TNF superfamily (LvTNFSF) gene, TNFR superfamily (LvTNFRSF) gene and lipopolysaccharide-induced TNF-α factor (LvLITAF) gene from Litopenaeus vannamei. LvTNFSF consists of 472 amino acids with a conserved C-terminal TNF domain and has 89.8% identity with the Marsupenaeus japonicus TNF superfamily gene. LvTNFRSF consists of 296 amino acids with a conserved TNFR domain and has 18.0% identity with Chlamys farreri TNFR, 14.6% identity with Drosophila melanogaster Wengen and 14.6% identity with Homo sapiens TNFR1. LvLITAF consists of 124 amino acids with the LITAF domain and shows 62.6% identity with D. melanogaster LITAF and 32.3% identity with H. sapiens LITAF. The promoter region of LvTNFSF was cloned and used to construct a luciferase reporter. In Drosophila S2 cells, the promoter of LvTNFSF can be activated by LvLITAF, L. vannamei NF-κB family proteins (LvRelish and LvDorsal) and LvSTAT. Unlike its mammalian counterparts, LvTNFRSF could not activate the NF-κB pathway in Drosophila S2 cells. Using real-time quantitative PCR, we obtained expression profiles of LvTNFSF, LvTNFRSF and LvLITAF in the gill, intestine and hepatopancreas of L. vannamei after challenge with Gram-negative Vibrio alginolyticus, Gram-positive Staphylococcus aureus, the fungus Candida albicans and white spot syndrome virus (WSSV). Taken together, our results reveal that LvTNFSF, LvTNFRSF and LvLITAF may be involved in shrimp immune responses to pathogenic infections.