Radiographic and clinical outcomes of minimally invasive surgery versus open osteotomies for the correction of hallux valgus.

PURPOSE: Hallux valgus is a common disease which causes pain and dysfunction of the foot. Although numerous methods of procedures have been introduced, a single procedure cannot correct all deformities of hallux valgus. The study aims to evaluate the radiographic and clinical effectiveness of a new minimally invasive surgery (MIS) versus open Chevron-Akin procedures. METHODS: This was a retrospective comparative study. Data were collected from May 2018 to January 2020. A total of 27 patients (31 feet) undergoing MIS for hallux valgus were included in this study. The average age of patients underwent MIS was 59.9 years. The mean follow-up was 25.1 months. Open osteotomies were performed in 30 patients (31 feet) during the same period. The mean age of these patients at the time of surgery was 59.1 years. The mean follow-up was 26.1 months. Pre-operative and post-operative radiographic outcome measures included HVA, IMA, DMAA, the Sgarlato's angle and the length of the first metatarsal, and distance between the dorsal cortex of first and second metatarsal necks. The AOFAS and VAS were used to assess foot function. RESULTS: The preoperative HVA in MIS group and open group were 34.8° and 33.1° respectively. The post-operative HVA were 20.4° and 13.7°. The pre-operative IMA in MIS group and open group were 13.0° and 12.1°. The post-operative IMA were 11.4° and 5.5° respectively. The pre-operative DMAA were 14.8° and 15.1° respectively. The post-operative DMAA were 6.3° and 8.7°. The AOFAS increased from 44.0 to 90.2 in MIS group and from 47.6 to 89.5 in open group. The VAS decreased from 7.3 to 1.3 in MIS group and from 7.1 to 1.2 in open group. CONCLUSION: Although open osteotomies were superior than MIS in HVA and IMA, MIS showed advantages in correcting DMAA. MIS provided equivalent functional outcomes compared to open surgery.

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.

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.

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.

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.

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.

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.

Mandarin fish von Hippel-Lindau protein regulates the NF-κB signaling pathway via interaction with IκB to promote fish ranavirus replication.

The von Hippel-Lindau tumor suppressor protein (VHL), an E3 ubiquitin ligase, functions as a critical regulator of the oxygen-sensing pathway for targeting hypoxia-inducible factors. Recent evidence suggests that mammalian VHL may also be critical to the NF-κB signaling pathway, although the specific molecular mechanisms remain unclear. Herein, the roles of mandarin fish ( Siniperca chuatsi) VHL ( scVHL) in the NF-κB signaling pathway and mandarin fish ranavirus (MRV) replication were explored. The transcription of scVHL was induced by immune stimulation and MRV infection, indicating a potential role in innate immunity. Dual-luciferase reporter gene assays and reverse transcription quantitative PCR (RT-qPCR) results demonstrated that scVHL evoked and positively regulated the NF-κB signaling pathway. Treatment with NF-κB signaling pathway inhibitors indicated that the role of scVHL may be mediated through scIKKα, scIKKß, scIκBα, or scp65. Co-immunoprecipitation (Co-IP) analysis identified scIκBα as a novel target protein of scVHL. Moreover, scVHL targeted scIκBα to catalyze the formation of K63-linked polyubiquitin chains to activate the NF-κB signaling pathway. Following MRV infection, NF-κB signaling remained activated, which, in turn, promoted MRV replication. These findings suggest that scVHL not only positively regulates NF-κB but also significantly enhances MRV replication. This study reveals a novel function of scVHL in NF-κB signaling and viral infection in fish.

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.

The potential role of microfilaments in host cells for infection with infectious spleen and kidney necrosis virus infection.

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.

Entry of tiger frog virus (an Iridovirus) into HepG2 cells via a pH-dependent, atypical, caveola-mediated endocytosis pathway.

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 NH(4)Cl, 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.

Hypoxia triggers the outbreak of infectious spleen and kidney necrosis virus disease through viral hypoxia response elements.

Hypoxia frequently occurs in aquatic environments, especially in aquaculture areas. However, research on the relationship between hypoxic aquatic environments with viral diseases outbreak is limited, and its underlying mechanisms remain elusive. Herein, we demonstrated that hypoxia directly triggers the outbreak of infectious spleen and kidney necrosis virus (ISKNV) disease. Hypoxia or activated hypoxia-inducible factor (HIF) pathway could remarkably increase the levels of viral genomic DNA, titers, and gene expression, indicating that ISKNV can response to hypoxia and HIF pathway. To reveal the mechanism of ISKNV respond to HIF pathway, we identified the viral hypoxia response elements (HREs) in ISKNV genome. Fifteen viral HREs were identified, and four related viral genes responded to the HIF pathway, in which the hre-orf077r promoter remarkably responded to the HIF pathway. The level of orf077r mRNA dramatically increased after the infected cells were treated with dimethyloxalylglycine (DMOG) or the infected cells/fish subjected to hypoxic conditions, and overexpressed orf077r could remarkably increase the ISKNV replication. These finding shows that hypoxic aquatic environments induce the expression of viral genes through the viral HREs to promote ISKNV replication, indicating that viral HREs might be important biomarkers for the evaluation of the sensitivity of aquatic animal viral response to hypoxia stress. Furthermore, the frequencies of viral HREs in 43 species aquatic viral genomes from 16 families were predicted and the results indicate that some aquatic animal viruses, such as Picornavirdea, Dicistronviridae, and Herpesviridae, may have a high risk to outbreak when the aquatic environment encounters hypoxic stress.

The Interaction of Mandarin Fish DDX41 with STING Evokes type I Interferon Responses Inhibiting Ranavirus Replication.

DDX41 is an intracellular DNA sensor that evokes type I interferon (IFN-I) production via the adaptor stimulator of interferon gene (STING), triggering innate immune responses against viral infection. However, the regulatory mechanism of the DDX41-STING pathway in teleost fish remains unclear. 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 a high-density cultural mode in mandarin fish, viral diseases have increased and seriously restricted the development of aquaculture, such as ranavirus and rhabdovirus. Herein, the role of mandarin fish DDX41 (scDDX41) and its DEAD and HELIC domains in the antiviral innate immune response were investigated. The level of scDDX41 expression was up-regulated following treatment with poly(dA:dT) or Mandarin fish ranavirus (MRV), suggesting that scDDX41 might be involved in fish innate immunity. The overexpression of scDDX41 significantly increased the expression levels of IFN-I, ISGs, and pro-inflammatory cytokine genes. Co-immunoprecipitation and pull-down assays showed that the DEAD domain of scDDX41 recognized the IFN stimulatory DNA and interacted with STING to activate IFN-I signaling pathway. Interestingly, the HELIC domain of scDDX41 could directly interact with the N-terminal of STING to induce the expression levels of IFN-I and ISGs genes. Furthermore, the scDDX41 could enhance the scSTING-induced IFN-I immune response and significantly inhibit MRV replication. Our work would be beneficial to understand the roles of teleost fish DDX41 in the antiviral innate immune response.

The viral ankyrin repeat protein (ORF124L) from infectious spleen and kidney necrosis virus attenuates nuclear factor-κB activation and interacts with IκB kinase ß.

The ankyrin (ANK) repeat is one of the most common protein-protein interaction motifs, found predominantly in eukaryotes and bacteria, but the functions of the ANK repeat are rarely researched in animal viruses, with the exception of poxviruses. Infectious spleen and kidney necrosis virus (ISKNV) is a typical member of the genus Megalocytivirus in the family Iridoviridae and is a causative agent of epizootics in fish. The genome of ISKNV contains four putative viral ANK (vANK) repeat proteins and their functions remain largely unknown. In the present study, it was found that ORF124L, a vANK repeat protein in ISKNV, encodes a protein of 274 aa with three ANK repeats. Transcription of ORF124L was detected at 12 h post-infection (p.i.) and reached a peak at 40 h p.i. ORF124L was found to localize to both the nucleus and the cytoplasm in mandarin fish fry cells. ISKNV ORF124L interacted with the mandarin fish IκB kinase ß protein (scIKKß), and attenuated tumour necrosis factor alpha (TNF-α)- or phorbol myristate acetate (PMA)-induced activity of a nuclear factor κB (NF-κB)-luciferase reporter but did not interfere with the activity of an activator protein 1 (AP-1)-luciferase reporter. Phosphorylation of IκBα and nuclear translocation of NF-κB were also impaired by ISKNV ORF124L. In summary, ORF124L was identified as a vANK repeat protein and its role in inhibition of TNF-α-induced NF-κB signalling was investigated through interaction with the mandarin fish IKKß. This work may help to improve our understanding of the function of fish iridovirus ANK repeat proteins.

Intra-articular opening osteotomy combined with lateral ligament reconstruction for varus ankle arthritis.

BACKGROUND: Takakura 3B ankle arthritis is featured as obliteration of ankle space with subchondral bone contact. Among these patients, some have medial distal tibial platform erosion. It is hard to treat this kind of patients. The purpose of this study was to evaluate the therapeutic outcomes of intra-articular opening osteotomy combined with lateral ligament reconstruction for Takakura 3B ankle arthritis with medial distal tibial platform erosion. METHODS: From September 2009 to May 2016, 17 patients with Takakura 3B ankle arthritis were reviewed, including 3 male and 14 female patients. All underwent the operation of intra-articular opening osteotomy combined with lateral ligament reconstruction. All patients were available for analysis. The main outcome measurements included TT angle, AOFAS score, VAS score, SF-36 scale, and AOS scale. RESULTS: All patients were followed for a mean follow-up of 87.2 months (range, 49 to 129 months). The VAS scale improved from 5.5 ± 1.6 to 2.3 ± 1.9. The mean AOFAS score improved from 47.7 ± 15.7 to 75.8 ± 12.0. The SF-36 scale improved from 41.6 ± 14.0 to 67.7 ± 14.6. The AOS improved from 60.9 ± 13.9 to 28.2 ± 17.7. The TT angle improved from 14.3 ± 5.0° to 5.3 ± 4.0°. The TAS and TLS changed from 83.4 ± 2.6° and 77.5 ± 2.3° to 90.7 ± 2.3° and 78.6 ± 2.2°. However, the LTAS was not corrected significantly. CONCLUSION: Intra-articular opening osteotomy combined with lateral ligament reconstruction is an effective method to treat varus ankle arthritis with medial distal tibial platform erosion.

Hallux Valgus Correction Comparing Percutaneous Oblique Osteotomy and Open Chevron Osteotomy at a 2-year Follow-up.

OBJECTIVE: This study aimed to compare the percutaneous oblique osteotomy (POO) and the open chevron osteotomy technique for correction of hallux valgus deformity at a 2-year follow-up. METHODS: This is a retrospective study of consecutive patients undergoing operative correction of hallux valgus using one of two techniques (POO vs open chevron osteotomy) from 2014 to 2018. Forty eight feet (41 patients) that underwent the POO was compared with 64 feet (58 patients) that underwent open chevron osteotomy. The hallux valgus angle (HVA), intermetatarsal angle (IMA) and American Orthopedic Foot & Ankle Society Hallux Metatarsophalangeal-Interphalangeal scores (AOFAS-HMI) were assessed preoperatively and postoperatively at the 1, 2-year follow-up. The Manchester-Oxford Foot Questionnaire (MOXFQ) were assessed preoperatively and postoperatively at the 2-year follow-up. The VAS score was collected preoperatively and on 2 weeks,1 year and 2-year follow-up. RESULTS: Both groups achieved significant correction of the hallux deformity. The HVA in the POO group during the follow-up period were 12.5 ± 2.22 and 17.9 ± 9.31, respectively, and in the open chevron group were 14.1 ± 6.78 and 14.8 ± 7.83, respectively. The IMA in the POO group during the follow-up period were 7.61 ± 1.63 and 6.94 ± 1.53, respectively, and in the open chevron group were 6.89 ± 3.06 and 6.97 ± 2.95, respectively. Postoperative MOXFQ scores in all domains were significantly improved in both groups, however there was no significant difference in the improvement of any domain between POO and open groups at a 2-year follow-up. The AOFAS HMI scores in the POO group during the follow-up period were 86.5 ± 10.7 and 85.2 ± 13.8, respectively, and in the open chevron group were 88.2 ± 10.8 and 79.5 ± 23.7, respectively. The VAS scores in the POO group during the follow-up period were 2.00 ± 0.98, 2.00 ± 0.99 and 1.55 ± 1.11, respectively, and in the open chevron group were 5.51 ± 1.45, 2.56 ± 2.88 and 2.56 ± 2.88 respectively. The 1-year and 2-year follow-up outcomes between POO and open groups showed no significant difference regarding AOFAS HMI scores and VAS scores, however the POO group showed statistically significant improvement of VAS scores in the postoperative 2 weeks (P < 0.001). There was no statistical significance between the POO and open group in terms of complications rates (8.3% vs 12.5%, P = 0.480). CONCLUSION: The POO technique is reliable and shows a comparable outcome to the open chevron osteotomy. However, the POO technique shows significantly less pain in the first 2 weeks after surgery.