Xiaopeng Xu

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.

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.

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.

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.

Shrimp NF-κB binds to the immediate-early gene ie1 promoter of white spot syndrome virus and upregulates its activity

The immediate-early gene ie1 carried by white spot syndrome virus (WSSV) exhibits very strong promoter activity and expresses highly throughout the infection cycle. Here we identified a NF-κB binding motif in the ie1 promoter region. Electrophoretic mobility shift assays indicated that the recombinant Rel homology domain (RHD) of shrimp NF-κB homolog LvRelish bound to the putative NF-κB site in the ie1 promoter. A transactivity assay of the WSSV ie1 promoter in Drosophila Schneider 2 cells demonstrated that LvRelish could increase ie1 promoter activity. These results show that shrimp NF-κB homolog LvRelish transactivates WSSV ie1 gene expression and contributes to its high promoter activity. Further transactivation assays showed that WSSV IE1 protein expression upregulated the promoter activities of WSSV ie1 gene and antimicrobial peptide genes regulated by the NF-κB system. We suggested that WSSV may annex the shrimp NF-κB system, which it uses to enhance the expression of viral immediate-early genes.

Identification, Characterization, and Function Analysis of the Cactus Gene from Litopenaeus vannamei

The nuclear factor-kappa B (NF-κB) pathways play important roles in innate immune responses. IκB is the main cytoplasmic inhibitor of NF-κB. In this study, we identified the LvCactus gene from Litopenaeus vannamei, which is the first cloned IκB homologue in subphylum Crustacea. LvCactus contains six predicted ankyrin repeats, which show similarities to those of Cactus proteins from insects. LvCactus localizes in cytoplasm and interacts with LvDorsal, an L. vannamei homologue to Drosophila melanogaster Dorsal belonging to class II NF-κB family, to prevent its nuclear translocation. Contrary to that of LvDorsal, over-expression of LvCactus down-regulates the activities of shrimp antimicrobial peptides promoters, suggesting LvCactus is an inhibitor of LvDorsal. The promoter of LvCactus was predicted to contain five putative NF-κB binding motifs, among which four were proved to be bound by LvDorsal by chromatin immunoprecipitation assays. Dual-luciferase reporter assays also showed that transcription of LvCactus was promoted by LvDorsal but inhibited by LvCactus itself, indicating a feedback regulatory pathway between LvCactus and LvDorsal. Expression of LvCactus was up-regulated after Lipopolysaccharides, poly (I:C), Vibrio parahaemolyticus, and Staphylococcus aureus injections, suggesting an activation response of LvCactus to bacterial and immune stimulant challenges. Differently, the LvCactus expression levels obviously decreased during white spot syndrome virus (WSSV) infection, indicating the feedback regulatory pathway of LvCactus/LvDorsal could be modified by WSSV.

Proteomic analysis of zebrafish (Danio rerio) infected with infectious spleen and kidney necrosis virus

Iridovirus infections remain a severe problem in aquaculture industries worldwide. Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytovirus in the family Iridoviridae, has caused significant economic losses among freshwater fish in different Asian countries. To investigate the molecular mechanism of iridoviral pathogenesis, we analyzed the differential proteome from the spleen of ISKNV-infected zebrafish through two-dimensional gel electrophoresis (2-DE). Mass spectrometry revealed 35 altered cellular protein spots, including 15 upregulated proteins and 20 downregulated proteins at five days post-infection. The altered host proteins were classified into 13 categories based on their biological processes: cytoskeletal protein, stress response, lipoprotein metabolism, ubiquitin-proteasome pathway, carbohydrate metabolism, signal transduction, proteolysis, ion binding, transport, metabolic process, catabolic process, biosynthesis, and oxidation reduction. Moreover, 14 corresponding genes of the differentially expressed proteins were validated by RT-PCR. Western blot analysis further demonstrated the changes in α-tubulin, β-actin, HSC70, and major capsid protein (MCP) during infection. β-Actin was selected for further study via co-immunoprecipitation analyses, which confirmed that the cellular β-actin interacts with the MCP protein of ISKNV in the infected zebrafish. These findings provide insight into the interactions between iridoviruses (especially ISKNV) and host, as well as the mechanism and pathogenesis of ISKNV infections.

Presence of Tube isoforms in Litopenaeus vannamei suggests various regulatory patterns of signal transduction in invertebrate NF-κB pathway.

The toll-like receptor (TLR)/NF-κB signaling pathways play critical roles in the innate immune system. The intracellular signal transduction of most TLR pathways in invertebrate cells is triggered by formation of a heterotrimeric complex composed of MyD88, Tube and Pelle. In this study, we identified a Litopenaeus vannamei Pelle (LvPelle) and an isoform of L. vannamei Tube (LvTube) designated as LvTube-1. The interactions among LvPelle, LvTube/LvTube-1 and LvMyD88/LvMyD88-1 were elucidated and their functions during pathogen infections were investigated. Knockdowns of LvPelle and LvTube/LvTube-1 using RNAi strategy led to higher mortalities of shrimps during Vibrio parahemolyticus infection, and could reduce the genome copy number of white spot syndrome virus (WSSV) in the infected muscle tissue but did not affect the mortality caused by WSSV infection. The effects of LvPelle and LvTube/LvTube-1 on promoters containing NF-κB binding motifs were analyzed by dual-luciferase reporter assays and the results demonstrated that LvTube-1 could activate the NF-κB activity to significantly higher level than LvTube did. Moreover, tissue distributions of LvTube and LvTube-1 mRNAs and their expression profiles during pathogen and immune stimulant challenges were different, indicating that they could play different roles in immune responses. This is the first report of Tube isoforms in invertebrates. Together with our previous study on LvMyD88 isoforms, our results suggest that various isoforms of adaptor components may be involved in various regulatory patterns of signal transduction in invertebrate TLR/NF-κB pathway and this could be a strategy adopted by invertebrates to modulate immune responses.

Litopenaeus vannamei NF-κB is required for WSSV replication

Many viruses can hijack the host cell NF-κB as part of their life cycle, diverting NF-κB immune regulatory functions to favor their replications. There were several reports on the functions of Litopenaeus vannamei NF-κB (LvNF-κB) in White spot syndrome virus (WSSV) replication in vitro. Here, we studied the relationship between LvNF-κB family protein Dorsal (LvDorsal) and Relish (LvRelish) with WSSV replication in vivo. The expressions of LvDorsal and LvRelish were significantly upregulated by WSSV challenge. Virus loads and expression of viral envelope protein VP28 in LvDorsal or LvRelish silencing shrimps were significantly lower than the control shrimps injected with EGFP-dsRNA or PBS after challenge with 1×10(5) copies WSSV/shrimp. In addition to the LvDorsal activation of WSV069 (ie1) and WSV303 promoter that we have reported, LvRelish can also activate WSV069 (ie1) and WSV303 promoter by dual luciferase reporter assays through screening 40 WSSV gene promoters that have putative multiple NF-κB binding sites. The promoter activity of the WSV069 (ie1) by LvDorsal activation was significantly higher than that by LvRelish activation. WSSV replication in LvDorsal, LvRelish or WSV303 silencing shrimps were significantly inhibited. These results indicate that the L. vannamei NF-κB family proteins LvDorsal and LvRelish expressions are significantly activated by WSSV challenge and WSSV replication partially relied on the activations of LvDorsal and LvRelish in vivo.

Administration of recombinant IFN1 protects zebrafish (Danio rerio) from ISKNV infection.

In the present study, we used the infectious spleen and kidney necrosis virus (ISKNV) and zebrafish model system to investigate the inhibitory effect of recombinant zebrafish interferon 1 (zfrIFN1) on acute viral infection and the impact of time of zfrIFN1 administration on its protective efficacy. In vivo experiments showed that administration of recombinant zfrINF1 up-regulated expression of several IFN-stimulated genes within 24 h of injection, and expression levels of these genes dropped to normal levels similar to those in control fish within three days. However, the transcriptions of two viral genes, the major capsid protein and virus protein 48 genes, were significantly inhibited for at least three days, indicating a longer duration of the zfrIFN1-mediated innate immune effect. To evaluate the protective efficacy of zfrIFN1 against ISKNV infection, we compared the relative percentage survival (RPS) of ISKNV-infected zebrafish by intraperitoneally (IP) injecting the fish with zfrIFN1 at different time points before or after infection. IP injection with 1 microg zfrIFN1/g fish body weight at 24, 6 or 0 h before virus infection or 6 h after virus infection significantly improved fish survival. However, IP injection with an equal dose of zfrIFN1 24 h post-infection did not provide significant protection to the fish. Our results suggest that zfrIFN1 is potent in inhibiting ISKNV acute infection and initiating the innate immune response in zebrafish, but its efficiency depends on the time of administration. This study shows the protective effects of interferon against a DNA-virus in fish for the first time and provides information about the efficacy of fish interferon that will prove useful in possible therapeutic applications.