Suboptimal trophectoderm mitochondrial DNA level is associated with delayed blastocyst development.

PURPOSE: To provide a comprehensive analysis of mtDNA quantity in D5 and D6 blastocysts, as well as a further insight to the origin of delayed blastocyst development. METHODS: A retrospective cohort analysis of 829 D5 and 472 D6 blastocysts from 460 patients who underwent in vitro fertilization (IVF) with next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A). The quantity of trophectoderm mtDNA was extrapolated from the NGS data, followed by the analysis of mean mtDNA levels between D5 and D6 blastocysts of the same ploidy (aneuploid/euploid) and transfer outcomes (positive/negative clinical pregnancy). RESULTS: D5 blastocysts had significantly higher euploidy rate and clinical pregnancy rate when compared with D6 blastocysts. The proportion of blastocysts derived from patients ≧ 40 years old were similar between the D5 and D6 cohorts. When blastocysts with identical ploidy were analyzed, the D5 cohorts all had significantly higher mean mtDNA levels than their D6 counterparts. Similarly, when embryo transfers with identical outcome were analyzed, the D5 cohorts also had significantly higher mean mtDNA levels than the D6 cohorts. Trophectoderm mtDNA level was independent of maternal age and blastocyst morphology grades. CONCLUSIONS: Our data provided further evidence D5 blastocysts contained significantly greater mtDNA quantity than D6 blastocysts, and mtDNA quantity could be a key factor that affects the development rate of blastocysts. Furthermore, one must avoid using an arbitrary threshold when incorporating mtDNA quantity into the embryo selection criteria, as the observed value may have vastly different clinical implication when blastulation rate is also considered.

Evidence for a Novel Antiviral Mechanism of Teleost Fish: Serum-Derived Exosomes Inhibit Virus Replication through Incorporating Mx1 Protein.

Exosomes are associated with cancer progression, pregnancy, cardiovascular diseases, central nervous system-related diseases, immune responses and viral pathogenicity. However, study on the role of exosomes in the immune response of teleost fish, especially antiviral immunity, is limited. Herein, serum-derived exosomes from mandarin fish were used to investigate the antiviral effect on the exosomes of teleost fish. Exosomes isolated from mandarin fish serum by ultra-centrifugation were internalized by mandarin fish fry cells and were able to inhibit Infectious spleen and kidney necrosis virus (ISKNV) infection. To further investigate the underlying mechanisms of exosomes in inhibiting ISKNV infection, the protein composition of serum-derived exosomes was analyzed by mass spectrometry. It was found that myxovirus resistance 1 (Mx1) was incorporated by exosomes. Furthermore, the mandarin fish Mx1 protein was proven to be transferred into the recipient cells though exosomes. Our results showed that the serum-derived exosomes from mandarin fish could inhibit ISKNV replication, which suggested an underlying mechanism of the exosome antivirus in that it incorporates Mx1 protein and delivery into recipient cells. This study provided evidence for the important antiviral role of exosomes in the immune system of teleost fish.

Roles of extracellular matrix components in Tiger frog virus attachment to fathead minnow (Pimephales promelas) cells.

Tiger frog virus (TFV) belongs to the genus Ranavirus (family Iridoviridae) and causes significant harm in cultured frogs, resulting in substantial losses in ecological and economic field in Southern China. Attachment is the first step in viral life cycle, which is dependent on the interactions of virions with extracellular matrix (ECM) components. Studying this process will help in understanding virus infection and controlling viral diseases. In this study, the roles of primary ECM components in TFV attachment were investigated. The results on the kinetics of virus attachment showed TFV successful attachment to the cell surface as a relatively rapid process after TFV was used to inoculate cells for 10 min at 4 °C. Western blot and quantitative PCR analyses results showed that soluble fibronectin, collagen IV, laminin, or hyaluronic acid treatment with TFV caused no significant effect on virus attachment. Soluble heparin, heparan sulfate and chondroitin sulfate A/B could inhibit TFV attachment in a dose-dependent manner. Enzymic digestion by cell surface heparin/heparan sulfate using heparinase I, II, and III could significantly prevent TFV attachment, suggesting that heparan sulfate plays an important role in TFV attachment. Furthermore, the binding assays of heparin-agarose beads and virion showed that TFV virions specifically bound with heparin in a dose-dependent manner. Given that heparin is a structural analogue of heparan sulfate, the above results suggest that heparan sulfate might serve as an attachment factor of TFV infection. Our work would be beneficial to understand the mechanisms of TFV attachment and the interactions of TFV with cellular receptor(s).

The roles of mandarin fish STING in innate immune defense against Infectious spleen and kidney necrosis virus infections.

The mandarin fish Siniperca chuatsi is a cultured freshwater fish species that is popular in China because of its high market value. With the development of high-density cultural mode in mandarin fish, viral diseases such as Infectious spleen and kidney necrosis virus (ISKNV) are becoming increasingly serious. Stimulator of interferon genes (STING) is a central component in the innate immune response to cytosolic DNA and RNA derived from different pathogens. However, the roles of STING in innate immune response of mandarin fish remain unknown. In the present study, S. chuatsi STING (scSTING)-mediated host immune response against ISKNV infection was investigated. ScSTING transcription level increased remarkably in response to ISKNV infection, LPS, PMA, or poly (I:C) stimulation in mandarin fish fry (MFF-1) cells. Immunofluorescence results showed that scSTING localized majorly in the endoplasmic reticulum. scSTING overexpression remarkably increased the expression levels of scIFN-h, scMx, scISG15, scPKR, scViperin, scIL-1ß, scIL-18, and scTNF-α genes. IFN-ß-luciferase report assay results showed that the relative expressions of luciferin were remarkably increased in MFF-1 cells. Site mutation of serine (S) on C-terminus of scSTING showed that both S388 and S396 were important for mediated signaling. Furthermore, scSTING overexpression inhibited ISKNV infection, and knockdown of scSTING promoted ISKNV infection, indicating that scSTING could suppress ISKNV infection in MFF-1 cells. These observations suggested that the scSTING played an important role in innate immune against ISKNV infection. Our work would help elucidate the roles of teleost fish STING in innate immunity.

Tiger frog virus ORF104R interacts with cellular VDAC2 to inhibit cell apoptosis.

Ranaviruses belong to the family Iridoviridae, and have become a serious threat to both farmed and natural populations of fish and amphibians. Previous reports showed that ranaviruses could encode viral Bcl-2 family-like proteins (vBcl-2), which play a critical role in the regulation of cell apoptosis. However, the mechanism of ranaviruses vBcl-2 interactions with host protein in mediating apoptosis remains unknown. Tiger frog virus (TFV) belonging to the genus Ranavirus has been isolated from infected tadpoles of Rana tigrina rugulosa, and it causes a high mortality rate among tiger frog tadpoles cultured in southern China. This study elucidated the molecular mechanism underlying the interaction of TFV ORF104R with the VDAC2 protein to regulate cell apoptosis. TFV ORF104R is highly similar to other ranaviruses vBcl-2 and host Mcl-1 proteins, indicating that TFV ORF104R is a postulate vBcl-2 protein. Transcription and protein expression levels showed that TFV orf104r was a late viral gene. Western blot results suggested that TFV ORF104R was a viral structural protein. Subcellular localization analysis indicated that TFV ORF104R was predominantly colocalized with the mitochondria. Overexpressed TFV ORF104R could suppress the release of cytochrome C and the activities of caspase-9 and caspase-3. These results indicated that TFV ORF104R might play an important role in anti-apoptosis. Furthermore, the interaction between TFV ORF104R and VDAC2 was detected by co-immunoprecipitation in vitro. The above observations suggest that the molecular mechanism of TFV-regulated anti-apoptosis is through the interaction of TFV ORF104R with the VDAC2 protein. Our study provided a mechanistic basis for the ranaviruses vBcl-2-mediated inhibition of apoptosis and improved the understanding on how TFV subverts host defense mechanisms in vivo.

Molecular cloning of Y-Box binding protein-1 from mandarin fish and its roles in stress-response and antiviral immunity.

Mandarin fish (Siniperca chuatsi) is a universally farmed fish species in China and has a large farming scale and economic value. With the high-density cultural mode in mandarin fish, viral diseases, such as infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV), have increased loss, which has seriously restricted the development of aquaculture. Y-Box binding protein 1 (YB-1) is a member of cold shock protein family that regulates multiple cellular processes. The roles of mammalian YB-1 protein in environmental stress and innate immunity have been studied well, but its roles in teleost fishes remain unknown. In the present study, the characteristic of S. chuatsi YB-1 (scYB-1) and its roles in cold stress and virus infection were investigated. The scYB-1 obtained an 1541 bp cDNA that contains a 903 bp open reading frame encoding a protein of 300 amino acids. Tissue distribution results showed that the scYB-1 is a ubiquitously expressed gene found among tissues from mandarin fish. Overexpression of scYB-1 can increase the expression levels of cold shock-responsive genes, such as scHsc70a, scHsc70b, and scp53. Furthermore, the role of scYB-1 in innate immunity was also investigated in mandarin fish fry (MFF-1) cells. The expression level of scYB-1 was significant change in response to poly (I:C), poly (dG:dC), PMA, ISKNV, or SCRV stimulation. The overexpression of scYB-1 can significantly increase the expression levels of NF-κB-responsive genes, including scIL-8, scTNF-α, and scIFN-h. The NF-κB-luciferase report assay results showed that the relative expression of luciferin was significantly increased in the cells overexpressed with scYB-1 compared with those in cells overexpressed with control plasmid. These results indicate that scYB-1 can induce the NF-κB signaling pathway in MFF-1 cells. Overexpressed scYB-1 can downregulate the expression of ISKNV viral major capsid protein (mcp) gene but upregulates the expression of SCRV mcp gene. Moreover, knockdown of scYB-1 using siRNA can upregulate the expression of ISKNV mcp gene but downregulates the expression of SCRV mcp gene. These results indicate that scYB-1 suppresses ISKNV infection while enhancing SCRV infection. The above observations suggest that scYB-1 is involved in cold stress and virus infection. Our study will provide an insight into the roles of teleost fish YB-1 protein in stress response and innate immunity.

Deletion of the Infectious spleen and kidney necrosis virus ORF069L reduces virulence to mandarin fish Siniperca chuatsi.

Mandarin fish (Siniperca chuatsi) is a significant cultured species with high added value in China. With the expansion of farming, diseases of mandarin fish such as Infectious spleen and kidney necrosis virus (ISKNV) diseases are becoming more and more serious. Human endogenous retrovirus subfamily H long terminal repeat associating protein 2 (HHLA2) is a type 1 transmembrane molecule with three extracellular Ig domains (IgV-IgC-IgV) and plays important roles in the T cell proliferation and tumorigenesis. The HHLA2-homologues have not been found in virus. In this study, a viral HHLA2 protein encoded by ISKNV ORF069L was identified and the virulence of the deleted ORF069L reconstruction ISKNV strain (ΔORF069L) was investigated. ISKNV ORF069L gene was predicted to encode a 222-amino acids peptide. The bioinformation analysis revealed that ISKNV ORF069L contained an Ig HHLA2 domain and was homologous to vertebrate B7-CD28 family proteins. The recombinant virus strain of ΔORF069L was constructed by homologous recombination technology. The virus titer and growth curves between ISKNV wild type (WT) and ΔORF069L on cellular level showed no significant differences indicating that the ORF069L did not influence the ISKNV replication. The expression levels of immune-related genes (Mx1, IL-1ß, IL-8, TNF-a and IgM) were increased in fish infected with ΔORF069L, compared to those in fish infected with ISKNV WT. Furthermore, the lethality caused by ΔORF069L declined by 40% compared with ISKNV WT, indicating that ORF069L was a virulence gene of ISKNV. Most importantly, the protection rate was nearly 100% for fish immunized with ΔORF069L strain. Those results suggested that ΔORF069L could be developed as a potential attenuated vaccine against ISKNV. Our work will be beneficial to promote the development of gene deletion attenuated vaccines for ISKNV disease.

Identification and functional analysis of the Mandarin fish (Siniperca chuatsi) hypoxia-inducible factor-1α involved in the immune response.

Mandarin fish (Siniperca chuatsi) is a popular cultured freshwater fish species due to its high market value in China. With increasing density of breeding, mandarin fish is often cultured under low environmental oxygen concentrations (hypoxia). In this study, the relative expression levels of hypoxia response element (HRE)-luciferase reporter and the HIF signaling pathway downstream genes (scldha, scvegf, and scglut-1) were significantly increased by hypoxic stress, thereby indicating that mandarin fish has an HIF signaling pathway. The mandarin fish HIF-1α (scHIF-1α) was also characterized. Multiple sequence alignments showed that scHIF-1α presented similar architectures to other known vertebrates. Subcellular localization analysis showed that scHIF-1α was mainly located in the nucleus of the mandarin fish fry-1 (MFF-1) cells. The role of scHIF-1α in the regulation of the HIF signaling pathway was confirmed. Overexpression of scHIF-1α could induce the HIF signaling pathway, whereas knockdown of scHIF-1α inhibited the activity of the HIF-1 signaling pathway. Tissue distribution analysis showed that schif-1α was significantly highly expressed in the blood, heart, and liver, which indicated that the main function of scHIF-1α was closely related to the circulatory system. Furthermore, scHIF-1α expression was significantly induced by poly I:C, poly dG:dC or PMA, thereby indicating that scHIF-1α was involved in the immune response. HIF-1α plays an important role in pathogen infections in mammals, but its role in fish is rarely investigated. Overexpression of scHIF-1α could inhibit MRV and SCRV infections, whereas knockdown of scHIF-1α could promote such infections. Those results suggested that scHIF-1α played an important role in fish virus infection. Our study will help understand the hypoxia associated with the outbreaks of aquatic viral disease.

Litopenaeus vannamei activating transcription factor 6 alpha gene involvement in ER-stress response and white spot symptom virus infection.

A previous study found that inositol-requiring enzyme-1-X-box binding protein 1 (IRE1-XBP1) pathway and the protein kinase RNA (PKR)-like ER kinase-eIF2α (PERK-eIF2α) pathway of shrimp play roles in the unfolded protein response (UPR). And they also be proved that was involved in white spot symptom virus (WSSV) infection. Yet the functions of the third branch in shrimp UPR are still unclear. In this study, we showed that upon UPR activation, activating transcription factor 6 alpha (LvATF6α) of Litopenaeus vannamei was cleaved and transferred from the cytoplasm to the nucleus in 293T cells, indicating that the ATF6 pathway in shrimp is also a branch of UPR. Furthermore, LvATF6α could reduce the apoptosis rate of Drosophila Schneider 2 (S2) cells treated with actinomycin, and knock-down expression of LvATF6α increased the apoptosis rate of shrimp hemocytes. In vivo testing revealed that the short from LvATF6α (LvATF6α-s) was obviously increased after UPR activation or WSSV infection, indicating that the ATF6 pathway was activated in L. vannamei gills under such circumstances. Moreover, knock-down expression of LvATF6α could reduce the cumulative mortality and WSSV copy number in WSSV-infected shrimp. Further study revealed that WSSV may profit from shrimp ATF6 pathway activation in two aspects. First, LvATF6α-s significantly upregulated the expression of the WSSV genes (wsv023, wsv045, wsv083, wsv129, wsv222, wsv249, and wsv343). Second, LvATF6α-s inhibited apoptosis by negatively regulating the apoptosis signal-regulating kinase 1 - (c-Jun N-terminal kinase) pathway. All of these evidences suggested that the ATF6 pathway is a member of the L. vannamei UPR, and it is also engaged in WSSV infection.

Functional characterization of a reactive oxygen species modulator 1 gene in Litopenaeus vannamei.

Reactive oxygen species (ROS) imparts a dual effect on multicellular organisms, wherein high levels are usually harmful, and low levels could facilitate in combating pathogenic microorganisms; therefore, the regulation of ROS production is critical. Previous studies have suggested that ROS contributes to resistance to the white spot syndrome virus (WSSV) or Vibrio alginolyticus in Litopenaeus vannamei. However, the regulation of ROS metabolism in L. vannamei remains elusive. In the present study, we proved that the overexpression of L. vannamei reactive oxygen species modulator 1 (LvROMO1) increases ROS production in Drosophila Schneider 2 (S2) cells. Real-time RT-PCR analysis indicated that LvROMO1 is induced by WSSV or V. alginolyticus infection and ß-glucan or microcystin (MC-LR) injection. Further investigation showed that LvROMO1 responding to MC-LR, thereby inducing hemocytes to undergo apoptosis, and ultimately resulting in hepatopancreatic damage. And LvROMO1 downregulation induced an increase in the cumulative mortality of WSSV-infected shrimp by reducing ROS production and suppressing the expression of antimicrobial peptides genes. The findings of present study suggest that LvROMO1 plays an important role in ROS production in L. vannamei and is involved in innate immunity.

Interaction of infectious spleen and kidney necrosis virus ORF119L with PINCH leads to dominant-negative inhibition of integrin-linked kinase and cardiovascular defects in zebrafish.

UNLABELLED: Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus, Iridoviridae family, causing a severe systemic disease with high mortality in mandarin fish (Siniperca chuatsi) in China and Southeast Asia. At present, the pathogenesis of ISKNV infection is still not fully understood. Based on a genome-wide bioinformatics analysis of ISKNV-encoded proteins, we found that ISKNV open reading frame 119L (ORF119L) is predicted to encode a three-ankyrin-repeat (3ANK)-domain-containing protein, which shows high similarity to the dominant negative form of integrin-linked kinase (ILK); i.e., viral ORF119L lacks the ILK kinase domain. Thus, we speculated that viral ORF119L might affect the host ILK complex. Here, we demonstrated that viral ORF119L directly interacts with particularly interesting Cys-His-rich protein (PINCH) and affects the host ILK-PINCH interaction in vitro in fathead minnow (FHM) cells. In vivo ORF119L overexpression in zebrafish (Danio rerio) embryos resulted in myocardial dysfunctions with disintegration of the sarcomeric Z disk. Importantly, ORF119L overexpression in zebrafish highly resembles the phenotype of endogenous ILK inhibition, either by overexpressing a dominant negative form of ILK or by injecting an ILK antisense morpholino oligonucleotide. Intriguingly, ISKNV-infected mandarin fish develop disorganized sarcomeric Z disks in cardiomyocytes. Furthermore, phosphorylation of AKT, a downstream effector of ILK, was remarkably decreased in ORF119L-overexpressing zebrafish embryos. With these results, we show that ISKNV ORF119L acts as a domain-negative inhibitor of the host ILK, providing a novel mechanism for the megalocytivirus pathogenesis. IMPORTANCE: Our work is the first to show the role of a dominant negative inhibitor of the host ILK from ISKNV (an iridovirus). Mechanistically, the viral ORF119L directly binds to the host PINCH, attenuates the host PINCH-ILK interaction, and thus impairs ILK signaling. Intriguingly, ORF119L-overexpressing zebrafish embryos and ISKNV-infected mandarin fish develop similar disordered sarcomeric Z disks in cardiomyocytes. These findings provide a novel mechanism for megalocytivirus pathogenesis.

Identification and differential expression analysis of MicroRNAs encoded by Tiger Frog Virus in cross-species infection in vitro.

BACKGROUND: Tiger frog virus (TFV), dsDNA virus of the genus Ranavirus and family Iridoviridae, causes a high mortality of tiger frog tadpoles cultured in Southern China. MicroRNAs (miRNAs) have been identified in many viruses especially DNA viruses such as Singapore Grouper Iridoviruses (SGIV). MicroRNAs play important roles in regulating gene expression for virus subsistence in host. Considering that TFV infects cells of different species under laboratory conditions, we aim to identify the specific and essential miRNAs expressed in ZF4 and HepG2 cells. METHODS: We identified and predicted novel viral miRNAs in TFV-infected ZF4 and HepG2 cells by deep sequencing and software prediction. Then, we verified and described the expression patterns of TFV-encoded miRNAs by using qRT-PCR and Northern blot. RESULTS: Deep sequencing predicted 24 novel TFV-encoded miRNAs, and qRT-PCR verified 19 and 23 miRNAs in TFV-infected ZF4 (Group Z) and HepG2 (Group H) cells, respectively. Northern blot was performed to validate eight and five TFV-encoded miRNAs in Groups H and Z, respectively. We compared the expression of TFV-encoded miRNAs from two groups and defined TFV-miR-11 as the essential viral miRNA and TFV-miR-13 and TFV-miR-14 as the specific miRNAs that contribute to HepG2 cell infection. CONCLUSIONS: We identified novel viral miRNAs and compared their expression in two host cells. The results of this study provide novel insights into the role of viral miRNAs in cross-species infection in vitro.

Identification and characterization of a mitochondrial unfolded protein response transcription factor ATFS-1 in Litopenaeus vannamei.

A mitochondrial specific stress response termed mitochondrial unfolded protein response (UPR(mt)) is activated in responding to disturbance of protein homeostasis in mitochondria. The activating transcription factor associated with stress-1 (designated as ATFS-1) is the key regulator of UPR(mt). To investigating the roles of ATFS-1 (LvATFS-1) in Litopenaeus vannamei mitochondrial stress remission and immunity, it's full length cDNA was cloned. The open reading frame of LvATFS-1 was 1, 557 bp in length, deducing to a 268 amino acids protein. LvATFS-1 was highly expressed in muscle, hemocytes and eyestalk. Subcellular location assays showed that N-terminal of LvATFS-1 contained a mitochondrial targeting sequence, which could directed the fused EGFP located to mitochondria. And the C-terminal of LvATFS-1, which had a nuclear localization signal, expressed in nucleus. The in vitro experiments verified that LvATFS-1 could reduced the level of intracellular reactive oxygen species (ROS). And results of real-time RT-PCR indicated that LvATFS-1 might scavenge excess ROS via ROS-eliminating genes regulation. Reporter gene assays showed that LvATFS-1 could upregulated the expression of the antimicrobial peptide genes in Drosophila Schneider 2 cells. Results of real-time RT-PCR showed that Vibrio alginolyticus or white spot syndrome virus (WSSV) infection induced the expression of LvATFS-1. And knocked-down LvATFS-1 by RNAi resulted in a higher cumulative mortality of L. vannamei upon V. alginolyticus or WSSV infection. These results suggested that LvATFS-1 not only rolled in mitochondrial specific stress responding, but also important for L. vannamei immunologic defence.

Down-regulation apoptosis signal-regulating kinase 1 gene reduced the Litopenaeus vannamei hemocyte apoptosis in WSSV infection.

Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, is crucial in various cellular responses. In the present study, we identified and characterized an ASK1 homolog from Litopenaeus vannamei (LvASK1). The full-length cDNA of LvASK1 was 5400 bp long, with an open reading frame encoding a putative 1420 amino acid protein. LvASK1 was highly expressed in muscle, hemocyte, eyestalk and heart. Real-time RT-PCR analysis showed that the expression of the LvASK1 was upregulated during the white spot syndrome virus (WSSV) challenge. The knocked-down expression of LvASK1 by RNA interference significantly reduced the apoptotic ratio of the hemocytes collected from WSSV-infected L. vannamei. Furthermore, the down-regulation of LvASK1 also decreased the cumulative mortality of WSSV-infected L. vannamei. These results suggested that down-regulation of LvASK1 decreased the apoptotic rate of hemocytes in WSSV-infected shrimp, and that it could contribute to the reduction of cumulative mortality in WSSV-infected L. vannamei.

Flightless-I (FliI) is a potential negative regulator of the Toll pathway in Litopenaeus vannamei.

Flightless-I (FliI) is a protein negatively modulates the Toll-like receptor (TLR) pathway through interacting with Myeloid differentiation factor 88 (MyD88). To investigate the function of FliI in innate immune responses in invertebrates, Litopenaeus vannamei FliI (LvFliI) was identified and characterized. The full-length cDNA of LvFliI is 4, 304 bp long, with an open reading frame (ORF) encoding a putative protein of 1292 amino acids, including 12 leucine-rich repeat (LRR) domains at the N-terminus and 6 gelsolin homology (GEL) domains at the C-terminus. The LvFliI protein was located in the cytoplasm and LvFliI mRNA was constitutively expressed in healthy L. vannamei, with the highest expression level in the muscle. LvFliI could be up-regulated in hemocytes after lipopolysaccharide (LPS), poly I:C, CpG-ODN2006, Vibrio parahaemolyticus, Staphylococcus aureus, and white spot syndrome virus (WSSV) challenges, suggesting a stimulation response of LvFliI to bacterial and immune stimulant challenges. Upon LPS stimulation, overexpression of LvFliI in Drosophila Schneider 2 cells led to downregulation of Drosophila and shrimp antimicrobial peptide (AMP) genes. Knockdown of LvFliI by RNA interference (RNAi) resulted in an increase of the expression of three shrimp AMP genes (PEN2, crustin, and Lyz1). However, the mortality rates of LvFliI-knockdown shrimp in response to V. parahaemolyticus, S. aureus or WSSV infections were not significantly different from those of the control group. Taken together, all the results suggested that LvFliI may play a negative role in TLR signaling response in L. vannamei.

Identification of mud crab reovirus VP12 and its interaction with the voltage-dependent anion-selective channel protein of mud crab Scylla paramamosain.

Mud crab reovirus (MCRV) is the causative agent of a severe disease in cultured mud crab (Scylla paramamosain), which has caused huge economic losses in China. MCRV is a double-stranded RNA virus with 12 genomic segments. In this paper, SDS-PAGE, mass spectrometry and Western blot analyses revealed that the VP12 protein encoded by S12 gene is a structural protein of MCRV. Immune electron microscopy assay indicated that MCRV VP12 is a component of MCRV outer shell capsid. Yeast two hybrid cDNA library of mud crab was constructed and mud crab voltage-dependent anion-selective channel (mcVDAC) was obtained by MCRV VP12 screening. The full length of mcVDAC was 1180 bp with an open reading frame (ORF) of 849 bp encoding a 282 amino acid protein. The mcVDAC had a constitutive expression pattern in different tissues of mud crab. The interaction between MCRV VP12 and mcVDAC was determined by co-immunoprecipitation assay. The results of this study have provided an insight on the mechanisms of MCRV infection and the interactions between the virus and mud crab.

Mandarin fish caveolin 1 interaction with major capsid protein of infectious spleen and kidney necrosis virus and its role in early stages of infection.

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.

Identification and functional characterization of heat shock transcription factor1 in Litopenaeus vannamei.

Heat shock transcription factors belong to the heat shock factor (HSF) protein family, which are involved in heat shock protein (HSP) gene regulation. They are critical for cell survival upon exposure to harmful conditions. In this study, we identified and characterized a HSF1 (LvHSF1) gene in Litopenaeus vannamei, with a full-length cDNA of 2841 bp and an open reading frame encoding a putative protein of 632 amino acids. Through multiple sequence alignment and phylogenetic analysis, it was revealed that LvHSF1 was closed to insect HSF family, which contained a highly conserved DNA-binding domain, oligomerization domains with HR-A/B, and a nuclear localization signal. Tissues distribution showed that LvHSF1 was widely expressed in all tissues tested. And it was upregulated in hemocytes and gills after Vibrio alginolyticus or Staphylococcus aureus infection. Dual-luciferase reporter assays indicated that LvHSF1 activated the promoters of L. vannamei HSP70 (LvHSP70) and L. vannamei Cactus (LvCactus), while inhibited the expressions of Drosophila antimicrobial peptide (AMP) Atta, Mtk, and L. vannamei AMP PEN4 through NF-κB signal transduction pathway modification. Knocked-down expression of LvHSF1 by dsRNA resulted in downregulations of LvHSP70 and LvCactus, as well as cumulative mortality decreasing under V. alginolyticus or S. aureus infection in L. vannamei. Taken together, our data strongly suggest that LvHSF1 is involved in LvHSP70 regulation, therefore plays a great role in stress resistance. And it also takes part in LvCactus/LvDorsal feedback regulatory pathway modification of L. vannamei, which is in favor of V. alginolyticus or S. aureus infection.

An activating transcription factor of Litopenaeus vannamei involved in WSSV genes Wsv059 and Wsv166 regulation.

Members of activating transcription factor/cyclic adenosine 3', 5'-monophosphate response element binding protein (ATF/CREB) family are induced by various stress signals and function as effector molecules. Consequently, cellular changes occur in response to discrete sets of instructions. In this work, we found an ATF transcription factor in Litopenaeus vannamei designated as LvATFß. The full-length cDNA of LvATFß was 1388 bp long with an open reading frame of 939 bp that encoded a putative 313 amino acid protein. The protein contained a basic region-leucine zipper (bZip) domain that was a common feature among ATF/CREB transcription factors. LvATFß was highly expressed in intestines, gills, and heart. LvATFß expression was dramatically upregulated by white spot syndrome virus (WSSV) infection. Pull-down assay revealed that LvATFß had strong affinity to promoters of WSSV genes, namely, wsv059 and wsv166. Dual-luciferase reporter assay showed that LvATFß could upregulate the expression of wsv059 and wsv166. Knocked down LvATFß resulted in decreased expression of wsv059 and wsv166 in WSSV-challenged L. vannamei. Knocked down expression of wsv059 and wsv166 by RNA interference inhibited the replication and reduce the mortality of L. vannamei during WSSV challenge inoculation. The copy numbers of WSSV in wsv059 and wsv166 knocked down group were significant lower than in the control. These results suggested that LvATFß may be involved in WSSV replication by regulating the expression of wsv059 and wsv166.

Infectious spleen and kidney necrosis virus (a fish iridovirus) enters Mandarin fish fry cells via caveola-dependent endocytosis.

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 (NH(4)Cl), chloroquine, sucrose, and chlorpromazine, which are inhibitors of clathrin-dependent endocytosis. The depletion of cellular cholesterol by methyl-ß-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.