Ling Lü

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.

Molecular epidemiology and phylogenetic analysis of a marine fish infectious spleen and kidney necrosis virus-like (ISKNV-like) virus.

SummaryInfectious spleen and kidney necrosis virus-like (ISKNV-like) virus causes a serious systemic disease with high morbidity and mortality of freshwater and marine fishes. Based on the ISKNV putative major capsid protein (MCP), the vascular endothelial growth factor (VEGF), the mRNA capping enzyme (Capping), and the tumor necrosis factor receptor-associated protein (TNFR) genes, primers were designed and used in PCR to determine the host range of ISKNV-like viruses. From the sampling of >1600 marine fishes representing 6 orders, 25 families, and 86 species collected in the South China Sea, 13 cultured fish species (141 fish) and 39 wild fish species (102 fish) were confirmed hosts of ISKNV-like viruses. The average percentage of infection of ISKNV-like viruses was 14.6%. The results from phylogenetic analysis of these genes revealed that ISKNV-like viruses could be placed into two clusters: cluster I was more related to ISKNV; cluster II included OSGIV (orange-spotted grouper iridovirus) and RBIV (rock bream iridovirus), and was quite different from ISKNV. The results of this study can contribute to the prediction and prevention of ISKNV disease outbreaks.

Inhibition of white spot syndrome virus in Litopenaeus vannamei shrimp by sequence-specific siRNA

n Abstractn n RNA interference (RNAi) is a sequence-specific, post-transcriptional process of mRNA degradation. Here, we described specific silencing of five white spot syndrome virus (WSSV) genes in Litopenaeus vannamei in vivo with sequence-specific siRNAs. These genes included DNA polymerase (dnapol), ribonucleotide reductase small subunit (rr2), thymidine kinase and thymidylate kinase (tk-tmk), vp24 and vp28. At 6xa0days post-challenged, the relative survival rates of shrimp injected with siDNApol, siRR2, siTK-TMK, siVP24 and siVP28 (siRNAs for dnapol, rr2, tk-tmk, vp24 and vp28 genes) reached 50%, 50%, 66%, 33% and 33%, respectively. Specific siRNAs of the five WSSV genes could result in suppression of the target genes and a significant reduction in the viral proliferation. In negative controls, sequence-independent siRNA (mutant siRNA) could not inhibit expression of these five genes or viral replication. Consequently, injection of sequence-dependent siRNA could induce anti-WSSV activity in shrimp. These results suggest that siRNA can suppress WSSV efficiently in shrimp, and it may provide a potential approach to the therapy of aquaculture viral disease.n n

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.

Sequence analysis of 12 genome segments of mud crab reovirus (MCRV).

Mud crab reovirus (MCRV) is the causative agent of a serious disease with high mortality in cultured mud crab (Scylla serrata). This study sequenced and analyzed 12 genome segments of MCRV. The 12 genome segments had a total length of 24.464 kb, showing a total G+C content of 41.29% and predicted 15 ORFs. Sequence analysis showed that the majority of MCRV genes shared low homology with the counterpart genes of other reoviruses, e.g., the amino acid identity of RNA-dependent RNA polymerase (RdRp) was lower than 13.0% compared to the RdRp sequences of other reoviruses. Nucleotide and amino acid sequences of RdRp and capping enzyme suggested MCRV as a single group. Further genome-based phylogenetical analysis of conserved termini and reovirus polymerase motif indicates that this MCRV belongs to a new genus of the Reoviridae family, tentatively named as Crabreovirus.

Identification of two novel membrane proteins from the Tiger frog virus (TFV).

The Tiger frog virus (TFV) belongs to the genus Ranavirus in the family Iridoviridae, and its genome was completely sequenced in 2002. In order to better understand the viral structure and functional genes involved in infection and virus-host interactions, two candidate genes, ORF001L and ORF020R, were selected for our study. ORF001L and ORF020R were analyzed by genomic comparison and by using the TMHMM software. Both genes were conserved in the genus Ranavirus, may encode putative membrane proteins, and were determined as late genes by temporal analysis. In order to identify whether these two proteins were structural proteins or not, ORF001L and ORF020R were cloned and expressed in the pET32a (+) vector. Antisera against the two proteins were prepared by immunization of mice with purified proteins. Western blot analysis suggested that both ORF001L and ORF020R were structural proteins. Indirect immunofluorescence assay (IFA) revealed that the subcellular location of the two proteins was confined to the cytoplasm, especially at the viral assembly site (AS). Immunogold electron microscopy (IEM) further localized these two proteins, showing that they were envelope proteins.

VP23R of infectious spleen and kidney necrosis virus mediates formation of virus-mock basement membrane to provide attaching sites for lymphatic endothelial cells.

ABSTRACT Putative open reading frames (ORFs) encoding laminin-like proteins are found in all members of the genus Megalocytivirus, family Iridoviridae. This is the first study that identified the VP23R protein encoded by ORF23R of the infectious spleen and kidney necrosis virus (ISKNV), a member of these genes of megalocytiviruses. The VP23R mRNA covering the ISKNV genomic coordinates 19547 to 22273 was transcribed ahead of the major capsid protein. Immunofluorescence analysis demonstrated that VP23R was expressed on the plasma membrane of the ISKNV-infected cells and could not be a viral envelope protein. Residues 292 to 576 of VP23R are homologous to the laminin γ1III2-6 fragment, which covers the nidogen-binding site. An immunoprecipitation assay showed that VP23R could interact with nidogen-1, and immunohistochemistry showed that nidogen-1 was localized on the outer membrane of the infected cells. Electron microscopy showed that a virus-mock basement membrane (VMBM) was formed on the surface of the infected cells and a layer of endothelial cells (ECs) was attached to the VMBM. The VMBM contained VP23R and nidogen-1 but not collagen IV. The attached ECs were identified as lymphatic endothelial cells (LECs), which have unique feature of overlapping intercellular junctions and can be stained by immunohistochemistry using an antibody against a specific lymphatic marker, Prox-1. Such infection signs have never been described in viruses. Elucidating the functions of LECs attached to the surface of the infected cells may be useful for studies on the pathogenic mechanisms of megalocytiviruses and may also be important for studies on lymphangiogenesis and basement membrane functions.

VP15R from infectious spleen and kidney necrosis virus is a non-muscle myosin-II-binding protein.

VP15R protein, encoded by the 15th open reading frame of infectious spleen and kidney necrosis virus (ISKNV), was identified. VP15R is a 263-residue protein that is first transcribed within 12xa0h post-infection. The VP15R mRNA is transcribed beginning at ISKNV genomic coordinate 12111, extending 167xa0bp upstream of the initiation codon. No signal peptides, transmembrane fragments, or nuclear localization signal sequences were predicted in the VP15R sequence. The 102–202 sequence of VP15R is homologous to the 1153–1253 sequence of the filamin C protein of Danio rerio (zebrafish), with an identity of 29%. Immunofluorescence and VP15R-GFP fusion protein subcellular localization assays showed that VP15R is localized in the cytoplasm. Pull-down and MALDI-TOF–TOF/MS assays demonstrated that VP15R can bind to the non-muscle myosin II (NM II) protein. Co-immunoprecipitation assays confirmed that VP15R can bind to the heavy chains of the NM II protein of mandarin fish, mice, and humans.

Effective Polyethyleneimine-Mediated Gene Transfer into Zebrafish Cells

Polyethyleneimine (PEI) has been broadly studied as a leading nonviral gene delivery carrier because of its relatively high transfection efficiency in a wide range of cell types. Here, we report gene transfer in zebrafish cells (ZF4) using PEI as a gene carrier and lipofectamine as a control. Formations of PEI-DNA complexes were characterized by a series of measurements. The particle size of PEI-DNA complexes decreased from 274 to 132 nm, the surface charge gradually increased from -26 to 29 mV, and the cytotoxicity for zebrafish cells was observed with increasing proportion of PEI. Gel retardation assay showed that DNA was completely bound by PEI with a negative-to-positive charge ratio of 4. It was observed by transmission electron microscopy that the morphology of PEI-DNA complexes was spherical with smooth surfaces. Flow cytometry revealed that the optimum transfection efficiency (27%) mediated by PEI was obtained at an negative-to-positive charge ratio of 8, which was higher than that with lipofectamine. Luciferase activity assay confirmed the increase in reporter gene expression probably due to a more efficient formation of complex between DNA and PEI than DNA and lipofectamine. In conclusion, our study demonstrates that PEI may be applied as an effective gene carrier to mediate gene transfer into zebrafish cells.

Identification of the VP92R gene from infectious spleen and kidney necrosis virus

Megalocytiviruses, which belong to the family Iridoviridae, are among the most harmful viruses to cultured fishes. Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus. The genome of ISKNV was sequenced and annotated by He et al. (Virology 291:126–139, 2001) in 2001 and re-annotated by Eaton et al. (Virol. J. 4:11, 2007) in 2007. The ORF092R of ISKNV was identified in the annotations of He et al. but was not reported by Eaton et al. In this study, by using Northern-blot and RACE assays, we identified the ORF092R transcript, indicating that ORF092R was indeed transcribed in the ISKNV-infected cells. Immunofluorescence and Western-blot showed that VP92R protein was expressed in the ISKNV-infected cells, and the molecular mass of VP92R is consistent with the theoretical one. Sequence comparison demonstrated that the VP92R orthologous protein is present in large yellow croaker iridovirus (LYCIV), but not in rock bream iridovirus (RBIV) or orange-spotted grouper iridovirus (OSGIV). Therefore, VP92R may have specific functions during ISKNV pathogenesis and could be a subject for studying the differences between megalocytiviruses.