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1.  VP08R from Infectious Spleen and Kidney Necrosis Virus Is a Novel Component of the Virus-Mock Basement Membrane 
Journal of Virology  2014;88(10):5491-5501.
ABSTRACT
Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus, family Iridoviridae, brings great harm to fish farming. In infected tissues, ISKNV infection is characterized by a unique phenomenon, in that the infected cells are attached by lymphatic endothelial cells (LECs), which are speculated to wall off the infected cells from host immune attack. A viral membrane protein, VP23R, binds and recruits the host nidogen-1 protein to construct a basement membrane (BM)-like structure, termed virus-mock basement membrane (VMBM), on the surface of infected cells to provide attaching sites for LECs. VMBMs do not contain collagen IV protein, which is essential for maintenance of BM integrity and functions. In this study, we identified the VP08R protein encoded by ISKNV. VP08R was predicted to be a secreted protein with a signal peptide but without a transmembrane domain. However, immunofluorescence assays demonstrated that VP08R is located on the plasma membrane of infected cells and shows an expression profile similar to that of VP23R. Coimmunoprecipitation showed that VP08R interacts with both VP23R and nidogen-1, indicating that VP08R is a component of VMBM and is present on the cell membrane by binding to VP23R. Through formation of intermolecular disulfide bonds, VP08R molecules self-organized into a multimer, which may play a role in the maintenance of VMBM integrity and stability. Moreover, the VP08R multimer was easily degraded when the ISKNV-infected cells were lysed, which may be a mechanism for VMBM disassembly when necessary to free LECs and release the mature virions.
IMPORTANCE Infectious spleen and kidney necrosis virus (ISKNV; genus Megalocytivirus, family Iridovirus) is most harmful to cultured fishes. In tissues, the ISKNV-infected cells are attached by lymphatic endothelial cells (LECs), which are speculated to segregate the host immune system. A viral membrane protein, VP23R, binds and recruits the host nidogen-1 protein to construct virus-mock basement membranes (VMBMs) on the surface of infected cells to provide attaching sites for LECs. Although VMBMs lack the collagen IV network, which is an essential structural part of true BMs, VMBMs still show an intact structure. An ISKNV-encoded VP08R protein can self-assemble into a multimer and bind both VP23R and nidogen-1 to maintain the integrity and stability of VMBMs. On the basis of these facts, we redrew the putative schematic illustration of the VMBM structure. Our study suggests that the virus adopts a strategy to remodel the cellular matrix and may provide an important reference to elucidate BM functions and the mechanisms of lymphangiogenesis.
doi:10.1128/JVI.03776-13
PMCID: PMC4019130  PMID: 24599992
2.  Mandarin Fish Caveolin 1 Interaction with Major Capsid Protein of Infectious Spleen and Kidney Necrosis Virus and Its Role in Early Stages of Infection 
Journal of Virology  2013;87(6):3027-3038.
Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus from the family Iridoviridae. ISKNV is one of the major agents that cause mortality and economic losses to the freshwater fish culture industry in Asian countries, particularly for mandarin fish (Siniperca chuatsi). In the present study, we report that the interaction of mandarin fish caveolin 1 (mCav-1) with the ISKNV major capsid protein (MCP) was detected by using a virus overlay assay and confirmed by pulldown assay and coimmunoprecipitation. This interaction was independent of the classic caveolin 1 scaffolding domain (CSD), which is responsible for interacting with several signaling proteins and receptors. Confocal immunofluorescence microscopy showed that ISKNV MCP colocalized with mCav-1 in the perinuclear region of virus-infected mandarin fish fry (MFF-1) cells, which appeared as soon as 4 h postinfection. Subcellular fractionation analysis showed that ISKNV MCP was associated with caveolae in the early stages of viral infection. RNA interference silencing of mCav-1 did not change virus-cell binding but efficiently inhibited the entry of virions into the cell. Taken together, these results suggested that mCav-1 plays an important role in the early stages of ISKNV infection.
doi:10.1128/JVI.00552-12
PMCID: PMC3592132  PMID: 23283951
3.  Infectious Spleen and Kidney Necrosis Virus (a Fish Iridovirus) Enters Mandarin Fish Fry Cells via Caveola-Dependent Endocytosis 
Journal of Virology  2012;86(5):2621-2631.
Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus from the family Iridoviridae. Megalocytiviruses have been implicated in more than 50 fish species infections and currently threaten the aquaculture industry, causing great economic losses in China, Japan, and Southeast Asia. However, the cellular entry mechanisms of megalocytiviruses remain largely uncharacterized. In this study, the main internalization mechanism of ISKNV was investigated by using mandarin fish fry (MFF-1) cells. The progression of ISKNV infection is slow, and infection is not inhibited when the cells are treated with ammonium chloride (NH4Cl), chloroquine, sucrose, and chlorpromazine, which are inhibitors of clathrin-dependent endocytosis. The depletion of cellular cholesterol by methyl-β-cyclodextrin results in the significant inhibition of ISKNV infection; however, the infection is resumed with cholesterol replenishment. Inhibitors of caveolin-1-involved signaling events, including phorbol 12-myristate 13-acetate (PMA), genistein, and wortmannin, impair ISKNV entry into MFF-1 cells. Moreover, ISKNV entry is dependent on dynamin and the microtubule cytoskeleton. Cofraction analysis of ISKNV and caveolin-1 showed that ISKNV colocates with caveolin-1 during virus infection. These results indicate that ISKNV entry into MFF-1 cells proceeds via classical caveola-mediated endocytosis and is dependent on the microtubules that serve as tracks along which motile cavicles may move via a caveola-caveosome-endoplasmic reticulum (ER) pathway. As a fish iridovirus, ISKNV entry into MFF-1 cells is different from the clathrin-mediated endocytosis of frog virus 3 entry into mammalian cells (BHK-21) at 28°C, which has been recognized as a model for iridoviruses. Thus, our work may help further the understanding of the initial steps of iridovirus infection.
doi:10.1128/JVI.06947-11
PMCID: PMC3302275  PMID: 22171272
4.  The impact of pulmonary arterial hypertension-targeted therapy on survival in Chinese patients with idiopathic pulmonary arterial hypertension 
Pulmonary Circulation  2012;2(3):373-378.
The survival rates of Chinese patients with idiopathic pulmonary arterial hypertension (IPAH) and familial pulmonary arterial hypertension (PAH) on conventional therapy at 1 and 3 years were 68.0% and 38.9%, respectively. Our aim was to update recent knowledge on the demographics, clinical course, hemodynamic features, disease management, and survival of adult patients with IPAH. This retrospective and observational study was conducted at the largest tertiary referral center in China. Ninety patients with IPAH who underwent initial evaluation at Fu Wai Hospital from January 2006 through November 2009 were retrospectively enrolled. The primary outcome was death. Statistical analyses used included independent sample t test, nonparametric test, Kaplan-Meier method, and Cox proportional hazards analysis. Of the 90 patients enrolled, the median age was 32 years with female predominance. The median interval from onset of symptoms to diagnosis was 14 months. Patients exhibited severe exercise limitation and hemodynamic abnormalities at diagnosis. Only 10.6% had a positive vasoreactivity test, while calcium channel blockers were given to 22.2% of patients. Fifty-nine patients (65.6%) received PAH-targeted therapies during follow-up. Our survival rates of 84.1%, 73.7%, and 70.6% at 1-, 2-, and 3-years compared favorably with predicted survival based on the National Institutes of Health equation which showed 1-, 2-, and 3-years survival rates of 67.7%, 55.9%, and 47%, respectively. For the patients receiving conventional therapy solely, the 1- and 3-years survival rates were 67.0% and 49.3%, respectively. Younger age, lower body mass index, presence of pericardial effusion, and absence of PAH-targeted therapy were independently associated with mortality. We concluded that patients with IPAH were still diagnosed too late, and while survival rates have improved in the modern treatment era, there is still room for improvement.
doi:10.4103/2045-8932.101655
PMCID: PMC3487306  PMID: 23130106
hemodynamics; pulmonary hypertension; risk factors; survival; targeted therapy
5.  A Novel C-Type Lectin from the Shrimp Litopenaeus vannamei Possesses Anti-White Spot Syndrome Virus Activity▿  
Journal of Virology  2008;83(1):347-356.
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.
doi:10.1128/JVI.00707-08
PMCID: PMC2612311  PMID: 18945787
6.  Effects of azithromycin on Pseudomonas aeruginosa isolates from catheter-associated urinary tract infection 
Pseudomonas aeruginosa is a common pathogenic bacterium in urinary tract infections (UTIs), particularly catheter-associated UTIs. The aim of this study was to investigate the effect of azithromycin (AZM) on P. aeruginosa isolated from UTIs. Isolates were identified by biochemical assays and the Vitek system. Antimicrobial susceptibility was determined using the disk diffusion assay. Biofilm formation and adhesion were assayed using a crystal violet staining method. The swimming motility was assayed on agar plates. The elastase activity and rhamnolipid production were determined by the elastin-Congo red method and orcinol reaction, respectively. A total of 32 bacterial isolates were collected from 159 urinary catheters and eight of them were P. aeruginosa isolates. The results showed that the P. aeruginosa isolates had stronger biofilm formation capability and the biofilms were thicker than those of P. aeruginosa PAO1. AZM inhibited biofilm formation and adhesion on urinary catheters, and also decreased swimming motility and the production of virulence factors. The results of this study indicated that AZM is potentially a good choice for use in the treatment of UTIs.
doi:10.3892/etm.2014.2120
PMCID: PMC4280936  PMID: 25574236
Pseudomonas aeruginosa; azithromycin; urinary tract infection; biofilm; swimming; virulence factor
7.  Characterization of the shrimp neuroparsin (MeNPLP): RNAi silencing resulted in inhibition of vitellogenesis 
FEBS Open Bio  2014;4:976-986.
Highlights
•This is the first comprehensive study of a neuroparsin in a decapod.•Expression of shrimp neuroparsin (MeNPLP) is different after serotonin (5-HT) treatment.•MeNPLP is most likely a secretary product.•MeNPLP is involved in an early stage of vitellogenesis.
The full-length Metapenaeus ensis neuroparsin (MeNPLP) cDNA was cloned which encodes a shrimp protein homologous to the insect neuroparsin and vertebrate insulin-like growth factor binding protein (IGFBP). MeNPLP cDNA is 1389 bp in length and the longest open reading frame is 303 bp in length. The first 27 aa are predicted to be the signal peptide and aa 28–101 is the mature peptide with an estimated molecular weight of 7.83 kDa and pI of 5. It shows high amino acid sequence similarity (42–68%) to the neuroparsin of insects and N-terminal end of the IGFBP of vertebrates. The cysteine residues in MeNPLP responsible for disulfide bond formation are conserved as in other neuroparsin-like proteins. The expression level of MeNPLP is the highest in the hepatopancreas, followed by the nerve cord, brain, heart, ovary, and muscle. However, it was not expressed in the testis. Using an insect neuroparsin antibody, MeNPLP could only be detected in the hepatopancreatic tubules, suggesting that MeNPLP may be a secretary product. Although MeNPLP expression was stimulated in the ovary, it was inhibited in the hepatopancreas after treatment with neurotransmitter serotonin (5-HT). In vivo gene silencing of MeNPLP could cause a significant decrease of vitellogenin transcript level in the hepatopancreas and ovary. As a result, a corresponding decrease in vitellogenin protein level was observed in the hemolymph and ovary. In conclusion, this study has provided the first evidence that MeNPLP is involved in the initial stage of ovary maturation in shrimp.
doi:10.1016/j.fob.2014.09.005
PMCID: PMC4244560  PMID: 25431753
5-HT, serotonin; CHH, crustacean hyperglycemic hormone; EST, expressed sequence tag; GSI, gonadosomatic index; IGFBP, insulin-like growth factor binding protein; ITP, ion transport peptide; MeNPLP, Metapenaeus ensis neuroparsin; NP, neuroparsin; Shrimp; Neuroparsin; IGF-like binding protein; RNA interference; Vitellogenin
8.  Characterization of Four Novel Caspases from Litopenaeus vannamei (Lvcaspase2-5) and Their Role in WSSV Infection through dsRNA-Mediated Gene Silencing 
PLoS ONE  2013;8(12):e80418.
Apoptosis plays an important role in white spot syndrome virus (WSSV) pathogenesis, and caspases are central players in apoptosis. Here, we cloned four novel caspases (Lvcaspase2-5) from the Pacific white shrimp Litopenaeus vannamei, and investigated their potential roles in WSSV replication using dsRNA-mediated gene silencing. Lvcaspase2-5 have the typical domain structure of caspase family proteins, with the conserved consensus motifs p20 and p10. Lvcaspase2 and Lvcaspase5 were highly expressed in muscle, while Lvcaspase3 was highly expressed in hemocytes and Lvcaspase4 was mainly expressed in intestine. Lvcaspase2-5 could also be upregulated by WSSV infection, and they showed different patterns in various tissues. When overexpressed in Drosophila S2 cells, Lvcaspase2-5 showed different cellular localizations. Using dsRNA-medicated gene silencing, the expression of Lvcaspase2, Lvcaspase3, and Lvcaspase5 were effectively knocked down. In Lvcaspase2-, Lvcaspase3- or Lvcaspase5-silenced L. vannamei, expression of WSSV VP28 gene was significantly enhanced, suggesting protective roles for Lvcaspase2, Lvcaspase3 and Lvcaspase5 in the host defense against WSSV infection.
doi:10.1371/journal.pone.0080418
PMCID: PMC3871164  PMID: 24376496
9.  The shrimp IKK–NF-κB signaling pathway regulates antimicrobial peptide expression and may be subverted by white spot syndrome virus to facilitate viral gene expression 
Cellular and Molecular Immunology  2013;10(5):423-436.
The IκB kinases IKKα and IKKβ and the IKK-related kinases TANK-binding kinase 1 (TBK1) and IKKε are the master regulators of the NF-κB signaling pathway. Although this pathway has been extensively studied in mammals, less attention has been paid in crustaceans, which have significant economic value. Here, we report the cloning and functional studies of two IKK homologs, LvIKKβ and LvIKKε, from Pacific white shrimp, Litopenaeus vannamei. LvIKKβ and LvIKKε mRNAs are widely expressed in different tissues and are responsive to white spot syndrome virus (WSSV) infection. When overexpressed in Drosophila S2 cells, LvIKKβ but not LvIKKε activates the promoters of NF-κB pathway-controlled antimicrobial peptide genes (AMPs), such as the Penaeidins (PENs). In HEK 293T cells, both LvIKKβ and LvIKKε activate an NF-κB reporter. The silencing of LvIKKβ or LvIKKε using double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) decreases the expression of L. vannamei AMPs, including PENs, lysozyme and crustins. Intriguingly, LvIKKβ- or LvIKKε-silenced L. vannamei are resistant to WSSV infection. We hypothesized that successful infection with WSSV requires the activation of the IKK–NF-κB signaling pathway to modulate viral gene expression. We constructed luciferase reporters for 147 WSSV genes. By screening, we found that the WSV051, WSV059, WSV069, WSV083, WSV090, WSV107, WSV244, WSV303, WSV371 and WSV445 promoters can be activated by LvIKKβ or LvIKKε in Drosophila S2 cells. Taken together, our results reveal that LvIKKβ and LvIKKε may participate in the regulation of shrimp AMPs and that WSSV may subvert the L. vannamei IKK–NF-κB signaling pathway to facilitate viral gene expression.
doi:10.1038/cmi.2013.30
PMCID: PMC3759962  PMID: 23954949
antimicrobial peptide; IKK–NF-κB; innate immunity; Litopenaeus vannamei; WSSV
10.  Analysis of Expression, Cellular Localization, and Function of Three Inhibitors of Apoptosis (IAPs) from Litopenaeus vannamei during WSSV Infection and in Regulation of Antimicrobial Peptide Genes (AMPs) 
PLoS ONE  2013;8(8):e72592.
Inhibitors of apoptosis (IAPs) play important roles in apoptosis and NF-κB activation. In this study, we cloned and characterized three IAPs (LvIAP1-3) from the Pacific white shrimp, Litopenaeusvannamei. LvIAP1-3 proteins shared signature domains and exhibited significant similarities with other IAP family proteins. The tissue distributions of LvIAP1-3 were studied. The expression of LvIAP1-3 was induced in the muscle after white spot syndrome virus (WSSV) infection. LvIAP1 expression in the gill, hemocytes, hepatopancreas, and intestine was responsive to WSSV and Vibrioalginolyticus infections. LvIAP2 expression in the gill, hemocytes, and hepatopancreas was also responsive to WSSV infection. The expression of LvIAP3 in the gill, hemocytes, and intestine was reduced after V. alginolyticus infection. When overexpressed in Drosophila S2 cells, GFP labeled-LvIAP2 was distributed in the cytoplasm and appeared as speck-like aggregates in the nucleus. Both LvIAP1 and LvIAP3 were widely distributed throughout the cytoplasm and nucleus. The expression of LvIAP1, LvIAP2, and LvIAP3 was significantly knocked down by dsRNA-mediated gene silencing. In the gill of LvIAP1- or LvIAP3-silenced shrimp, the expression of WSSV VP28 was significantly higher than that of the dsGFP control group, suggesting that LvIAP1 and LvIAP3 may play protective roles in host defense against WSSV infection. Intriguingly, the LvIAP2-silenced shrimp all died within 48 hours after dsLvIAP2 injection. In the hemocytes of LvIAP2-silenced shrimps, the expression of antimicrobial peptide genes (AMPs), including Penaeidins, lysozyme, crustins, Vibriopenaeicidae-induced cysteine and proline-rich peptides (VICPs), was significantly downregulated, while the expression of anti-lipopolysaccharide factors (ALFs) was upregulated. Moreover, LvIAP2 activated the promoters of the NF-κB pathway-controlled AMPs, such as shrimp Penaeidins and Drosophila drosomycin and attacin A, in Drosophila S2 cells. Taken together, these results reveal that LvIAP1 and LvIAP3 might participate in the host defense against WSSV infection, and LvIAP2 might be involved in the regulation of shrimp AMPs.
doi:10.1371/journal.pone.0072592
PMCID: PMC3743791  PMID: 23967321
11.  Echocardiographic Parameters in Patients with Pulmonary Arterial Hypertension: Correlations with Right Ventricular Ejection Fraction Derived from Cardiac Magnetic Resonance and Hemodynamics 
PLoS ONE  2013;8(8):e71276.
Background
Echocardiography is the most convenient method used to evaluate right ventricular function, and several echocardiographic parameters were studied in previous studies. But the value of these parameters to assess the right ventricular function in patients with pulmonary arterial hypertension (PAH) has not been well defined.
Methods
Patients with PAH were observed prospectively. Right heart catheterization, echocardiography and cardiac magnetic resonance (CMR) were performed within 1 week interval. The correlations between echocardiographic parameters and right ventricular ejection fraction (RVEF) derived from CMR as well as hemodynamics were analyzed.
Results
Thirty patients were enrolled including 24 with idiopathic PAH, 5 with PAH associated with connective tissue diseases and 1 with hereditary PAH. All echocardiographic parameters except right ventricular myocardial performance index (RVMPI) correlated significantly with RVEF (tricuspid annual plane systolic excursion [TAPSE], r = 0.440, P = 0.015; tricuspid annular systolic excursion velocity [S’], r = 0.444, P = 0.016; isovolumic acceleration [IVA], r = 0.600, P = 0.001; right ventricular fraction area change [RVFAC], r = 0.416, P = 0.022; ratio of right ventricular transverse diameter to left ventricular transverse diameter [RVETD/LVETD], r = −0.649, P<0.001; RVMPI, r = −0.027, P = 0.888). After adjusted for mean right atrial pressure, mean pulmonary arterial pressure and pulmonary vascular resistance (PVR), only IVA and RVETD/LVETD could independently predict RVEF. Four echocardiographic parameters displayed significant correlations with PVR (TAPSE, r = −0.615, P<0.001; S’, r = −0.557, P = 0.002; RVFAC, r = −0.454, P = 0.012; RVETD/LVETD, r = 0.543, P = 0.002).
Conclusions
The echocardiographic parameters IVA and RVETD/LVETD can reflect RVEF independently regardless of hemodynamics in patients with PAH. In addition, TAPSE, S’, RVFAC and RVETD/LVETD can also reflect PVR in PAH patients.
doi:10.1371/journal.pone.0071276
PMCID: PMC3743887  PMID: 23967181
12.  Activating Transcription Factor 4 and X Box Binding Protein 1 of Litopenaeus vannamei Transcriptional Regulated White Spot Syndrome Virus Genes Wsv023 and Wsv083 
PLoS ONE  2013;8(4):e62603.
In response to endoplasmic reticulum (ER) stress, the signaling pathway termed unfolded protein response (UPR) is activated. To investigate the role of UPR in Litopenaeus vannamei immunity, the activating transcription factor 4 (designated as LvATF4) which belonged to a branch of the UPR, the [protein kinase RNA (PKR)-like ER kinase, (PERK)]-[eukaryotic initiation factor 2 subunit alpha (eIF2α)] pathway, was identified and characterized. The full-length cDNA of LvATF4 was 1972 bp long, with an open reading frame of 1299 bp long that encoded a 432 amino acid protein. LvATF4 was highly expressed in gills, intestines and stomach. For the white spot syndrome virus (WSSV) challenge, LvATF4 was upregulated in the gills after 3 hpi and increased by 1.9-fold (96 hpi) compared to the mock-treated group. The LvATF4 knock-down by RNA interference resulted in a lower cumulative mortality of L. vannamei under WSSV infection. Reporter gene assays show that LvATF4 could upregulate the expression of the WSSV gene wsv023 based on the activating transcription factor/cyclic adenosine 3′, 5′-monophosphate response element (ATF/CRE). Another transcription factor of L. vannamei, X box binding protein 1 (designated as LvXBP1), has a significant function in [inositol-requiring enzyme-1(IRE1) – (XBP1)] pathway. This transcription factor upregulated the expression of the WSSV gene wsv083 based on the UPR element (UPRE). These results suggest that in L. vannamei UPR signaling pathway transcription factors are important for WSSV and might facilitate WSSV infection.
doi:10.1371/journal.pone.0062603
PMCID: PMC3634759  PMID: 23638122
13.  Liposarcoma of the retropharyngeal space with rapidly worsening dyspnea: A case report and review of the literature 
Oncology Letters  2013;5(6):1939-1942.
Liposarcomas represent a significant proportion of soft-tissue sarcomas. However, their occurrence in the head and neck is infrequent and they are exceedingly rare in the retropharyngeal space. The present study reports the case of a 58-year-old patient with retropharyngeal liposarcoma. Uniquely, the patient presented with rapidly worsening dyspnea. The diagnosis of liposarcoma was established following retropharyngeal tumor excision, although biopsies were performed twice. Adjuvant radiotherapy was refused by the patient. However, during the post-operative follow-up period, no sign of either local tumor recurrence or distant metastasis was observed. Previously reported cases were also reviewed to analyze the diagnosis, treatment and prognosis of this disease.
doi:10.3892/ol.2013.1310
PMCID: PMC3700895  PMID: 23833671
liposarcoma; retropharyngeal space; dyspnea
14.  The potential role of microfilaments in host cells for infection with infectious spleen and kidney necrosis virus infection 
Virology Journal  2013;10:77.
Background
Infectious spleen and kidney necrosis virus (ISKNV) belongs to the genus Megalocytivirus from the family Iridoviridae. Megalocytivirus causes severe economic losses to tropical freshwater and marine culture industry in Asian countries and is devastating to the mandarin fish farm industry in China particularly.
Methods
We investigated the involvement of microfilaments in the early and late stages of ISKNV infection in MFF-1 cells by selectively perturbing their architecture using well-characterized inhibitors of actin dynamics. The effect of disruption of actin cytoskeleton on ISKNV infection was evaluated by indirect immunofluorescence analysis or real-time quantitative PCR.
Results
The depolymerization of the actin filaments with cytochalasin D, cytochalasin B, or latrunculin A reduced ISKNV infection. Furthermore, depolymerization of filamentous actin by inhibitors did not inhibit binding of the virus but affected virus internalization in the early stages of infection. In addition, the depolymerization of actin filaments reduced total ISKNV production in the late stages of ISKNV.
Conclusions
This study demonstrated that ISKNV required an intact actin network during infection. The findings will help us to better understand how iridoviruses exploit the cytoskeleton to facilitate their infection and subsequent disease.
doi:10.1186/1743-422X-10-77
PMCID: PMC3599308  PMID: 23497248
Iridovirus; Infectious spleen and kidney necrosis virus; Microfilaments
15.  Identification and Function of Leucine-Rich Repeat Flightless-I-Interacting Protein 2 (LRRFIP2) in Litopenaeus vannamei 
PLoS ONE  2013;8(2):e57456.
Leucine-rich repeat flightless-I-interacting protein 2 (LRRFIP2) is a myeloid differentiation factor 88-interacting protein with a positive regulatory function in toll-like receptor signaling. In this study, seven LRRFIP2 protein variants (LvLRRFIP2A-G) were identified in Litopenaeus vannamei. All the seven LvLRRFIP2 protein variants encode proteins with a DUF2051 domain. LvLRRFIP2s were upregulated in hemocytes after challenged with lipopolysaccharide, poly I:C, CpG-ODN2006, Vibrio parahaemolyticus, Staphylococcus aureus, and white spot syndrome virus (WSSV). Dual-luciferase reporter assays in Drosophila Schneider 2 cells revealed that LvLRRFIP2 activates the promoters of Drosophila and shrimp AMP genes. The knockdown of LvLRRFIP2 by RNA interference resulted in higher cumulative mortality of L. vannamei upon V. parahaemolyticus but not S. aureus and WSSV infections. The expression of L. vannamei AMP genes were reduced by dsLvLRRFIP2 interference. These results indicate that LvLRRFIP2 has an important function in antibacterials via the regulation of AMP gene expression.
doi:10.1371/journal.pone.0057456
PMCID: PMC3585381  PMID: 23468989
16.  Litopenaeus vannamei Sterile-Alpha and Armadillo Motif Containing Protein (LvSARM) Is Involved in Regulation of Penaeidins and antilipopolysaccharide factors 
PLoS ONE  2013;8(2):e52088.
The Toll-like receptor (TLR)-mediated NF-κB pathway is tightly controlled because overactivation may result in severe damage to the host, such as in the case of chronic inflammatory diseases and cancer. In mammals, sterile-alpha and armadillo motif-containing protein (SARM) plays an important role in negatively regulating this pathway. While Caenorhabditis elegans SARM is crucial for an efficient immune response against bacterial and fungal infections, it is still unknown whether Drosophila SARM participates in immune responses. Here, Litopenaeus vannamei SARM (LvSARM) was cloned and functionally characterized. LvSARM shared signature domains with and exhibited significant similarities to mammalian SARM. Real-time quantitative PCR analysis indicated that the expression of LvSARM was responsive to Vibrio alginolyticus and white spot syndrome virus (WSSV) infections in the hemocyte, gill, hepatopancreas and intestine. In Drosophila S2 cells, LvSARM was widely distributed in the cytoplasm and could significantly inhibit the promoters of the NF-κB pathway-controlled antimicrobial peptide genes (AMPs). Silencing of LvSARM using dsRNA-mediated RNA interference increased the expression levels of Penaeidins and antilipopolysaccharide factors, which are L.vannamei AMPs, and increased the mortality rate after V. alginolyticus infection. Taken together, our results reveal that LvSARM may be a novel component of the shrimp Toll pathway that negatively regulates shrimp AMPs, particularly Penaeidins and antilipopolysaccharide factors.
doi:10.1371/journal.pone.0052088
PMCID: PMC3566147  PMID: 23405063
17.  Identification and Function of Myeloid Differentiation Factor 88 (MyD88) in Litopenaeus vannamei 
PLoS ONE  2012;7(10):e47038.
Myeloid differentiation factor 88 (MyD88) is a universal and essential signaling protein in Toll-like receptor/interleukin-1 receptor-induced activation of nuclear factor-kappa B. In this study, two MyD88 protein variants (LvMyD88 and LvMyD88-1) were identified in Litopenaeus vannamei. The LvMyD88 cDNA is 1,848 bp in length and contains an open reading frame (ORF) of 1,428 bp, whereas the LvMyD88-1 cDNA is 1,719 bp in length and has an ORF of 1,299 bp. Both variants encode proteins with death and Toll/interleukin-1 receptor domains and share 91% sequence identity. In healthy L. vannamei, the LvMyD88 genes were highly expressed in hemocytes but at a low level in the hepatopancreas. The LvMyD88s expression was induced in hemocytes after challenge with lipopolysaccharide, CpG-ODN2006, Vibrio parahaemolyticus, Staphyloccocus aureus, and white spot syndrome virus, but not by poly I∶C. Overexpression of LvMyD88 and LvMyD88-1 in Drosophila Schneider 2 cells led to activation of antimicrobial peptide genes and wsv069 (ie1), wsv303, and wsv371. These results suggested that LvMyD88 may play a role in antibacterial and antiviral response in L. vannamei. To our knowledge, this is the first report on MyD88 in shrimp and a variant of MyD88 gene in invertebrates.
doi:10.1371/journal.pone.0047038
PMCID: PMC3470552  PMID: 23071706
18.  Comparison of 18F-FDG uptake by right ventricular myocardium in idiopathic pulmonary arterial hypertension and pulmonary arterial hypertension associated with congenital heart disease 
Pulmonary Circulation  2012;2(3):365-372.
This study measured glucose uptake in the right ventricle (RV) of patients with pulmonary hypertension and investigated the relationship to hemodynamics and survival. Myocardial 18F-fluorodeoxy-glucose (FDG) uptake was measured using single-photon positron emission tomography (SPECT) in 24 patients with idiopathic pulmonary arterial hypertension (IPAH) and 43 patients with congenital heart disease (CHD). In both IPAH and CHD-PAH, RV FDG uptake (RV/LV ratio) was associated with pulmonary vascular resistance (PVR). A second SPECT scan was performed in nine patients after 6 months treatment with sildenafil. PVR decreased from 1683±426 to 1207±383 dyn s-1 cm-5 (P < 0.05) and cardiac index improved from 2.2±0.2 to 2.8±0.5 L/min/m2 (P < 0.01). RV/LV FDG uptake decreased from 1.28±0.32 before treatment to 0.99±0.23 (P < 0.05). Survival in the IPAH group with a baseline RV/LV FDG uptake greater than the median value of 1.20 was significantly lower than that of the group with RV/LV FDG uptake below 1.20 (log-rank test, P < 0.05). In contrast, baseline RV/LV FDG was of little informative value in CHD. FDG uptake by the RV reflects the severity of PVR in PAH. Increased RV FDG uptake is a marker of poor prognosis in IPAH and is reduced in patients receiving effective therapy. It could prove useful in the early clinical assessment of novel therapies for PAH.
doi:10.4103/2045-8932.101651
PMCID: PMC3487305  PMID: 23130105
pulmonary arterial hypertension; right ventricle; FDG uptake; PET imaging
19.  The Viral TRAF Protein (ORF111L) from Infectious Spleen and Kidney Necrosis Virus Interacts with TRADD and Induces Caspase 8-mediated Apoptosis 
PLoS ONE  2012;7(5):e37001.
Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus of the Iridoviridae family. It causes a serious and potentially pandemic disease in wild and cultured fishes. ISKNV infection induces evident apoptosis in mandarin fish (Siniperca chuatsi) and zebrafish (Danio renio). However, the mechanism is still unknown. After a genome-wide bioinformatics analysis of ISKNV-encoded proteins, the ISKNV open reading frame 111L (ORF111L) shows a high similarity to the tumour necrosis factor receptor-associated factor (TRAF) encoded by fish, mice and mammals, which is essential for apoptotic signal transduction. Moreover, ORF111L was verified to directly interact with the zebrafish TNF receptor type 1 associated death domain protein (TRADD). A recombinant plasmid containing the DNA sequence of ORF111L was constructed and microinjected into zebrafish embryos at the 1–2 cell stage to investigate its biological function in vivo. ORF111L overexpression in the embryos resulted in increased apoptosis. ORF111L-induced apoptosis was clearly associated with significant caspase 8 upregulation and activation. The knockdown of zebrafish caspase 8 expression effectively blocked the apoptosis induced by ORF111L overexpression. Significantly, ORF111L overexpression resulted in much stronger effect on caspase 8 and caspase 3 upregulation compared to zebrafish TRAF2. This is the first report of a viral protein similar to TRAF that interacts with TRADD and induces caspase 8-mediated apoptosis, which may provide novel insights into the pathogenesis of ISKNV infection.
doi:10.1371/journal.pone.0037001
PMCID: PMC3352826  PMID: 22615868
20.  Bisulfite Sequencing Reveals That Aspergillus flavus Holds a Hollow in DNA Methylation 
PLoS ONE  2012;7(1):e30349.
Aspergillus flavus first gained scientific attention for its production of aflatoxin. The underlying regulation of aflatoxin biosynthesis has been serving as a theoretical model for biosynthesis of other microbial secondary metabolites. Nevertheless, for several decades, the DNA methylation status, one of the important epigenomic modifications involved in gene regulation, in A. flavus remains to be controversial. Here, we applied bisulfite sequencing in conjunction with a biological replicate strategy to investigate the DNA methylation profiling of A. flavus genome. Both the bisulfite sequencing data and the methylome comparisons with other fungi confirm that the DNA methylation level of this fungus is negligible. Further investigation into the DNA methyltransferase of Aspergillus uncovers its close relationship with RID-like enzymes as well as its divergence with the methyltransferase of species with validated DNA methylation. The lack of repeat contents of the A. flavus' genome and the high RIP-index of the small amount of remanent repeat potentially support our speculation that DNA methylation may be absent in A. flavus or that it may possess de novo DNA methylation which occurs very transiently during the obscure sexual stage of this fungal species. This work contributes to our understanding on the DNA methylation status of A. flavus, as well as reinforces our views on the DNA methylation in fungal species. In addition, our strategy of applying bisulfite sequencing to DNA methylation detection in species with low DNA methylation may serve as a reference for later scientific investigations in other hypomethylated species.
doi:10.1371/journal.pone.0030349
PMCID: PMC3262820  PMID: 22276181
21.  Entry of Tiger Frog Virus (an Iridovirus) into HepG2 Cells via a pH-Dependent, Atypical, Caveola-Mediated Endocytosis Pathway▿ 
Journal of Virology  2011;85(13):6416-6426.
Tiger frog virus (TFV), in the genus Ranavirus of the family Iridoviridae, causes high mortality of cultured tiger frog tadpoles in China. To explore the cellular entry mechanism of TFV, HepG2 cells were treated with drugs that inhibit the main endocytic pathways. We observed that TFV entry was inhibited by NH4Cl, chloroquine, and bafilomycin, which can all elevate the pH of acidic organelles. In contrast, TFV entry was not influenced by chlorpromazine or overexpression of a dominant-negative form of Esp15, which inhibit the assembly of clathrin-coated pits. These results suggested that TFV entry was not associated with clathrin-mediated endocytosis, but was related to the pH of acidic organelles. Subsequently, we found that endocytosis of TFV was dependent on membrane cholesterol and was inhibited by the caveolin-1 scaffolding domain peptide. Dynamin and actin were also required for TFV entry. In addition, TFV virions colocalized with the cholera toxin subunit B, indicating that TFV enters as caveola-internalized cargo into the Golgi complex. Taken together, our results demonstrated that TFV entry occurs by caveola-mediated endocytosis with a pH-dependent step. This atypical caveola-mediated endocytosis is different from the clathrin-mediated endocytosis of frog virus 3 (FV3) by BHK cells, which has been recognized as a model for iridoviruses. Thus, our work may help further the understanding of the initial steps of iridovirus infection in lower vertebrates.
doi:10.1128/JVI.01500-10
PMCID: PMC3126490  PMID: 21543502
22.  The Shrimp NF-κB Pathway Is Activated by White Spot Syndrome Virus (WSSV) 449 to Facilitate the Expression of WSSV069 (ie1), WSSV303 and WSSV371 
PLoS ONE  2011;6(9):e24773.
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.
doi:10.1371/journal.pone.0024773
PMCID: PMC3171479  PMID: 21931849
23.  Global Landscape of Structural Proteins of Infectious Spleen and Kidney Necrosis Virus ▿  
Journal of Virology  2011;85(6):2869-2877.
Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus in the family Iridoviridae, causes severe damage to mandarin fish cultures in China. Little is known about the proteins of ISKNV virions. In this study, a total of 38 ISKNV virion-associated proteins were identified by four different workflows with systematic and comprehensive proteomic approaches. Among the 38 identified proteins, 21 proteins were identified by the gel-based workflows (one-dimensional [1-D] and two-dimensional [2-D] gel electrophoresis). Fifteen proteins were identified by 1-D gel electrophoresis, and 16 proteins were identified by 2-D gel electrophoresis, with 10 proteins identified by both methods. Another 17 proteins were identified only by liquid chromatography (LC)-based workflows (LC-matrix-assisted laser desorption ionization [MALDI] and linear trap quadrupole [LTQ]-Orbitrap). Among these 17 LC-identified proteins, 5 proteins were identified uniquely by the LC-MALDI workflow, whereas another 6 proteins were identified only by the LTQ-Orbitrap workflow. These results underscore the importance of incorporation of multiple approaches in identification of viral proteins. Based on viral genomic sequence, genes encoding these 38 viral proteins were cloned and expressed in vitro. Antibodies were produced against these 38 proteins to confirm the ISKNV structural proteins by Western blotting. Of the newly identified proteins, ORF 056L and ORF 118L were identified and confirmed as two novel viral envelope proteins by Western blotting and immunoelectron microscopy (IEM). The ISKNV proteome reported here is currently the only characterized megalocytivirus proteome. The systematic and comprehensive identification of ISKNV structural proteins and their localizations in this study will facilitate future studies of the ISKNV assembly process and infection mechanism.
doi:10.1128/JVI.01444-10
PMCID: PMC3067969  PMID: 21209107
24.  Infectious Spleen and Kidney Necrosis Virus ORF48R Functions as a New Viral Vascular Endothelial Growth Factor▿  
Journal of Virology  2008;82(9):4371-4383.
Infectious spleen and kidney necrosis virus (ISKNV) causes a pandemic and serious disease in fish. Infection by ISKNV causes epidermal lesions, in which petechial hemorrhages and abdominal edema are prominent features. ISKNV ORF48R contains a domain similar to that of the platelet-derived growth factor and vascular endothelial growth factor (VEGF) families of proteins. ISKNV ORF48R showed higher similarity to the VEGFs encoded by Megalocytivirus and Parapoxvirus than to those encoded in fish and mammals. We used zebrafish as a model and constructed a recombinant plasmid containing the DNA sequence of ISKNV ORF48R to study ISKNV infection. The plasmid was microinjected into zebrafish embryos at the one-cell stage. Overexpression of the ISKNV ORF48R gene results in pericardial edema and dilation at the tail region of zebrafish embryos, suggesting that ISKNV ORF48R induces vascular permeability. ISKNV ORF48R is also able to stimulate a striking expression of flk1 in the zebrafish dorsal aorta and the axial vein. Furthermore, ISKNV ORF48R, while cooperating with zebrafish VEGF121, can stimulate more striking expression of flk1 than can either ISKNV ORF48R or zebrafish VEGF121 alone. However, decreased expression of FLK-1 by gene knockdown results in the disappearance of pericardial edema and dilation at the tail region of zebrafish embryos induced by overexpression of ISKNV ORF48R in the early stages of embryonic development.
doi:10.1128/JVI.02027-07
PMCID: PMC2293046  PMID: 18305039
25.  The shrimp IKK–NF-κB signaling pathway regulates antimicrobial peptide expression and may be subverted by white spot syndrome virus to facilitate viral gene expression 
Cellular & Molecular Immunology  2013;10(5):423-436.
The IκB kinases IKKα and IKKβ and the IKK-related kinases TANK-binding kinase 1 (TBK1) and IKKε are the master regulators of the NF-κB signaling pathway. Although this pathway has been extensively studied in mammals, less attention has been paid in crustaceans, which have significant economic value. Here, we report the cloning and functional studies of two IKK homologs, LvIKKβ and LvIKKε, from Pacific white shrimp, Litopenaeus vannamei. LvIKKβ and LvIKKε mRNAs are widely expressed in different tissues and are responsive to white spot syndrome virus (WSSV) infection. When overexpressed in Drosophila S2 cells, LvIKKβ but not LvIKKε activates the promoters of NF-κB pathway-controlled antimicrobial peptide genes (AMPs), such as the Penaeidins (PENs). In HEK 293T cells, both LvIKKβ and LvIKKε activate an NF-κB reporter. The silencing of LvIKKβ or LvIKKε using double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) decreases the expression of L. vannamei AMPs, including PENs, lysozyme and crustins. Intriguingly, LvIKKβ- or LvIKKε-silenced L. vannamei are resistant to WSSV infection. We hypothesized that successful infection with WSSV requires the activation of the IKK–NF-κB signaling pathway to modulate viral gene expression. We constructed luciferase reporters for 147 WSSV genes. By screening, we found that the WSV051, WSV059, WSV069, WSV083, WSV090, WSV107, WSV244, WSV303, WSV371 and WSV445 promoters can be activated by LvIKKβ or LvIKKε in Drosophila S2 cells. Taken together, our results reveal that LvIKKβ and LvIKKε may participate in the regulation of shrimp AMPs and that WSSV may subvert the L. vannamei IKK–NF-κB signaling pathway to facilitate viral gene expression.
doi:10.1038/cmi.2013.30
PMCID: PMC3759962  PMID: 23954949
antimicrobial peptide; IKK–NF-κB; innate immunity; Litopenaeus vannamei; WSSV

Results 1-25 (25)