Low-temperature immunization attenuates the residual virulence of orf074r gene-deleted infectious spleen and kidney necrosis virus: a candidate immersion vaccine.

IMPORTANCE: Global aquaculture production yielded a record of 122.9 million tons in 2022. However, ~10% of farmed aquatic animal production is lost each year due to various infectious diseases, resulting in substantial economic waste. Therefore, the development of vaccines is important for the prevention and control of aquatic infectious diseases. Gene-deletion live attenuated vaccines are efficacious because they mimic natural pathogen infection and generate a strong antibody response, thus showing good potential for administration via immersion. However, most gene-deletion viruses still have residual virulence, and thus, gene-deletion immersion vaccines for aquatic viruses are rarely developed. In this study, an orf074r deletion strain (Δorf074r) of ISKNV with residual virulence was constructed, and an immunization process was developed to reduce its residual virulence at 22°C, thereby making it a potential immersion vaccine against ISKNV. Our work will aid in the development of an aquatic gene-deletion live-attenuated immersion vaccine.

A Safe and Efficient Double-Gene-Deleted Live Attenuated Immersion Vaccine to Prevent the Disease Caused by the Infectious Spleen and Kidney Necrosis Virus.

Infectious diseases seriously threaten sustainable aquaculture development, resulting in more than $10 billion in economic losses annually. Immersion vaccines are emerging as the key technology for aquatic disease prevention and control. Here, a safe and efficacious candidate immersion vaccine strain (Δorf103r/tk) of infectious spleen and kidney necrosis virus (ISKNV), in which the orf103r and tk genes were knocked out by homologous recombination, is described. Δorf103r/tk was severely attenuated in mandarin fish (Siniperca chuatsi), inducing mild histological lesions, a mortality rate of only 3%, and eliminated within 21 days. A single Δorf103r/tk immersion-administered dose provided long-lasting protection rates over 95% against lethal ISKNV challenge. Δorf103r/tk also robustly stimulated the innate and adaptive immune responses. For example, interferon expression was significantly upregulated, and the production of specific neutralizing antibodies against ISKNV was markedly induced postimmunization. This work provides proof-of-principle evidence for orf103r- and tk-deficient ISKNV for immersion vaccine development to prevent ISKNV disease in aquaculture production. IMPORTANCE Global aquaculture production reached a record of 122.6 million tons in 2020, with a total value of 281.5 billion U.S. dollars (USD). However, approximately 10% of farmed aquatic animal production is lost due to various infectious diseases, resulting in more than 10 billion USD of economic waste every year. Therefore, the development of vaccines to prevent and control aquatic infectious diseases is of great significance. Infectious spleen and kidney necrosis virus (ISKNV) infection occurs in more than 50 species of freshwater and marine fish and has caused great economic losses to the mandarin fish farming industry in China during the past few decades. Thus, it is listed as a certifiable disease by the World Organization for Animal Health (OIE). Herein, a safe and efficient double-gene-deleted live attenuated immersion vaccine against ISKNV was developed, providing an example for the development of aquatic gene-deleted live attenuated immersion vaccine.

Interaction of Teleost Fish TRPV4 with DEAD Box RNA Helicase 1 Regulates Iridovirus Replication.

Viral diseases are a significant risk to the aquaculture industry. Transient receptor potential vanilloid 4 (TRPV4) has been reported to be involved in regulating viral activity in mammals, but its regulatory effect on viruses in teleost fish remains unknown. Here, the role of the TRPV4-DEAD box RNA helicase 1 (DDX1) axis in viral infection was investigated in mandarin fish (Siniperca chuatsi). Our results showed that TRPV4 activation mediates Ca2+ influx and facilitates infectious spleen and kidney necrosis virus (ISKNV) replication, whereas this promotion was nearly eliminated by an M709D mutation in TRPV4, a channel Ca2+ permeability mutant. The concentration of cellular Ca2+ increased during ISKNV infection, and Ca2+ was critical for viral replication. TRPV4 interacted with DDX1, and the interaction was mediated primarily by the N-terminal domain (NTD) of TRPV4 and the C-terminal domain (CTD) of DDX1. This interaction was attenuated by TRPV4 activation, thereby enhancing ISKNV replication. DDX1 could bind to viral mRNAs and facilitate ISKNV replication, which required the ATPase/helicase activity of DDX1. Furthermore, the TRPV4-DDX1 axis was verified to regulate herpes simplex virus 1 replication in mammalian cells. These results suggested that the TRPV4-DDX1 axis plays an important role in viral replication. Our work provides a novel molecular mechanism for host involvement in viral regulation, which would be of benefit for new insights into the prevention and control of aquaculture diseases. IMPORTANCE In 2020, global aquaculture production reached a record of 122.6 million tons, with a total value of $281.5 billion. Meanwhile, frequent outbreaks of viral diseases have occurred in aquaculture, and about 10% of farmed aquatic animal production has been lost to infectious diseases, resulting in more than $10 billion in economic losses every year. Therefore, an understanding of the potential molecular mechanism of how aquatic organisms respond to and regulate viral replication is of great significance. Our study suggested that TRPV4 enables Ca2+ influx and interactions with DDX1 to collectively promote ISKNV replication, providing novel insights into the roles of the TRPV4-DDX1 axis in regulating the proviral effect of DDX1. This advances our understanding of viral disease outbreaks and would be of benefit for studies on preventing aquatic viral diseases.

Does a rupture of the lateral ankle ligament need to be repaired in supination-adduction type II (OTA/AO 44A2) fractures?

PURPOSE: To evaluate the outcomes of patients with supination-adduction (SAD) type II (OTA/AO 44A2) fractures who had a lateral ankle ligament rupture repaired compared with patients who did not have a lateral ankle ligament repaired using patients who underwent fibula fracture fixation as a control group. METHODS: A retrospective analysis of all 104 patients diagnosed with SAD type II fractures from January 2011 to December 2020 and managed operatively was performed. The patients were divided into three groups: 32 patients with ruptures of the lateral ligaments that were not repaired (group A), 34 patients with ruptures of the lateral ligaments that were repaired (group B), and 38 patients with fibula fracture fixation acting as the control group (group C). The objective outcomes including radiographic findings, the ankle range of motion, the manual ankle stress tests, and complications were gained from the record of the last time in outpatient clinics. The functional outcomes including the identification of functional ankle instability (IdFAI) scores were collected postoperatively at 12-month intervals to assess clinical outcomes. The Manchester Oxford Foot Questionnaire (MOXFQ) and Karlsson scoring scale were also recorded at the last follow-up. RESULTS: The mean follow-up of the objective and subjective functional outcomes was 23.4 (range, 13-42) and 76.9 (range, 25-134) months, respectively. There was no significant difference in the radiographic findings, the ankle range of motion and complications between the three groups. All ankles were found to be stable using the manual ankle stress test in both group A and group B. The IdFAI scores showed a significant difference between group A and group B (1.12 ± 1.3 vs 0.35 ± 0.69; p < 0.001) in the first year of follow-up and no significant difference after the first year. No differences were noted in MOXFQ scores or Karlsson scores among the groups. CONCLUSION: Directly repairing the lateral ligament could minimize the proportion of the first year of postoperative functional ankle instability, although the final stability of the ankle and clinical outcomes were not significantly different in SAD type II fractures. LEVEL OF EVIDENCE: Level III, retrospective comparative case series.

The genome of a vestimentiferan tubeworm (Ridgeia piscesae) provides insights into its adaptation to a deep-sea environment.

BACKGROUND: Vestimentifera (Polychaeta, Siboglinidae) is a taxon of deep-sea worm-like animals living in deep-sea hydrothermal vents, cold seeps, and organic falls. The morphology and lifespan of Ridgeia piscesae, which is the only vestimentiferan tubeworm species found in the hydrothermal vents on the Juan de Fuca Ridge, vary greatly according to endemic environment. Recent analyses have revealed the genomic basis of adaptation in three vent- and seep-dwelling vestimentiferan tubeworms. However, the evolutionary history and mechanism of adaptation in R. piscesae, a unique species in the family Siboglinidae, remain to be investigated. RESULT: We assembled a draft genome of R. piscesae collected at the Cathedral vent of the Juan de Fuca Ridge. Comparative genomic analysis showed that vent-dwelling tubeworms with a higher growth rate had smaller genome sizes than seep-dwelling tubeworms that grew much slower. A strong positive correlation between repeat content and genome size but not intron size and the number of protein-coding genes was identified in these deep-sea tubeworm species. Evolutionary analysis revealed that Ridgeia pachyptila and R. piscesae, the two tubeworm species that are endemic to hydrothermal vents of the eastern Pacific, started to diverge between 28.5 and 35 million years ago. Four genes involved in cell proliferation were found to be subject to positive selection in the genome of R. piscesae. CONCLUSION: Ridgeia pachyptila and R. piscesae started to diverge after the formation of the Gorda/Juan de Fuca/Explorer ridge systems and the East Pacific Rise. The high growth rates of vent-dwelling tubeworms might be derived from their small genome sizes. Cell proliferation is important for regulating the growth rate in R. piscesae.

Development and characterization of a spleen cell line from yellowfin seabream Acanthopagrus latus and its susceptibility to Mandarinfish ranavirus.

Yellowfin seabream (Acanthopagrus latus) is one of the most commercially important marine fish in China. In this study, a new continuous cell line, named ALS cells, was developed from the spleen tissue of A. latus. The cell line was maintained in Dulbecco's modified Eagle medium/Nutrient Mixture F-12 Ham (DMEM/F-12) supplemented with 10% fetal bovine serum (FBS) and successfully cultured up to 50 passages. The cell line was authenticated by amplifying and sequencing mitochondrial cytochrome C oxidase subunit-I (coi-I) gene. The ALS cell line had the maximum growth rate in DMEM/F-12 medium containing 20% FBS at 27°C. Chromosome number analysis showed that the ALS cells have a modal diploid chromosome number of 34. The ALS cell line was transfected with the pEGFP-N1 plasmid, and green fluorescence was observed. The ALS cell line was used for testing Mandarinfish ranavirus (MRV) susceptibility, and the cytopathic effects in the cell line were observed at 4 days post-infection (dpi). Furthermore, the susceptibility of the ALS cell line to MRV and the levels of MRV mRNA and viral loads were found to be significantly increased at 1-7 dpi. This study revealed that the ALS cell line could be useful for molecular, virological, and biotechnological studies on yellowfin seabream.

Comparison of prone-supine versus supine position for the treatment of pilon fractures via modified posteromedial approach combined with anterolateral approach.

BACKGROUND: Most of modified posteromedial approaches require prone position for the treatment of pilon fractures. We describe the technique of modified posteromedial approach under supine position. The goal of the study was to compare the radiographic and clinical outcomes of prone-supine versus supine position for the treatment of pilon fractures via modified posteromedial approach combined with anterolateral approach. METHODS: A total of 50 retrospectively consecutive pilon fractures that underwent open reduction internal fixation via modified posteromedial approach combined with anterolateral approach from 2016 to 2019 were reviewed at least a two-year follow up. The positions of patients were divided into two groups: prone-supine versus supine position (26 vs 24, respectively). The operation time, radiographic outcomes including bone union time and ratio of congruent articular reduction were evaluated. The post-operative function was evaluated using the Manchester Oxford score (MOXFQ) and the visual analogue score (VAS). The motion of ankle joint and complications and were also compared. RESULTS: The mean follow-up was 42.2(24.7-73.0) months in the prone-supine group and 42.7(37.3-56.5) months in the supine group (P = .87). The mean operation time was 141.9 ± 10.1 min in the prone-supine group and 107.5 ± 18.9 min in the supine group (P = .00). There was no significant difference in the bone union time and ratio of congruent articular reduction between the two groups. There was no significant difference in the final MOXFQ score, VAS score, and the mean range of ankle motion between the two groups (P > .05). The total incidence of complications was 11.5% in the prone-supine group and 16.6% in the supine group (P = .66). CONCLUSION: The patient in the prone-supine position versus supine position for pilon fractures via modified posteromedial approach combined with anterolateral approach contributed comparable quality of reduction, bone union time functional outcomes and complications. The supine technique was significantly shorter in terms of operation time.

Environmental risk characteristics of bacterial antibiotic resistome in Antarctic krill.

Antibiotic resistance genes (ARGs) are ubiquitous in nature, especially in the current era of antibiotic abuse, and their existence is a global concern. In the present study, we discovered that Antarctic krill-related culturable bacteria are resistant to ß-lactam, tetracyclines, aminoglycosides, and sulphamethoxazole/trimethoprim based on the antibiotic efflux mechanism. In addition, the co-occurrence of ARGs with insertion sequence (IS) (tnpA, IS91) and Intl1 on the isolates and the phylogenetic analysis results of the whole-genome revealed low-frequency ARG transfer events, implying the transferability of these ARGs. These findings provide an early warning for the wide assessment of Antarctic microbiota in the spread of ARGs. Our work provides novel insights into understanding ARGs in culturable host-associated microorganisms, and their ecological risks and has important implications for future risk assessments of antibiotic resistance in extreme environments.

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.

Whole genome sequencing and comparative genomic analyses of Planococcus alpniumensis MSAK28401T, a new species isolated from Antarctic krill.

BACKGROUND: Extremophiles have attracted much attention in the last few decades, as they possess different properties by producing certain useful metabolites. However, the secondary metabolism of the extremophiles of Antarctic krill has received little attention. RESULTS: In this study, a new bacterial strain MSAK28401T from Antarctic krill was isolated and identified. The results of analysis on phenotypic, chemotaxonomic, and genomic characteristics showed that the strain MSAK28401T belongs to the genus Planococcus. Cells of this strain were coccoid (0.89-1.05 µm) and aerobic. The majority of the fatty acid content was C15:0 anteiso (37.67 ± 0.90%) followed by C16:1 ω7c alcohol (10.37 ± 1.22%) and C16:0 iso (9.36 ± 0.71%). The calculated average nucleotide identity and DNA-DNA hybridization values between the strain MSAK28401T and type strains P. citreus DSM 20549T and P. rifietoensis M8T were lower than 91 and 70%, respectively. The strain MSAK28401T (=KCTC 43283T and MCCC 1k05448T) represented a new member of the genus Planococcus and was named P. alpniumensis sp. nov. Moreover, genes involved in the degradation of aromatic compounds (e.g., salicylate, gentisate, and quinate) were found in the genome, implying that strain MSAK28401T has an aromatic compound as its potential metabolite. This work will help us understand the genomic characteristics and potential metabolic pathway of Planococcus from Antarctic krill. CONCLUSIONS: This study reported the genomic information and phenotypic characteristics of the new strain P. alpniumensis MSAK28401T isolated from Antarctic krill, and provided the genome information of Planococcus strains for further studying the function roles in aromatic compound metabolism.

Comparing less invasive plate fixation versus screw fixation of displaced intra-articular calcaneus fracture via sinus tarsi approach.

PURPOSE: The purpose of this study was to compare the post-operative radiographic and clinical outcomes of less invasive plate fixation versus screw fixation of displaced intra-articular calcaneus fractures (DIACFs) via sinus tarsi approach. METHODS: A total of 165 consecutive DIACFs that underwent open reduction internal fixation via sinus tarsi approach from 2013 to 2018 were reviewed at least a two year follow-up. The methods of fixation were divided into two groups: less invasive plate fixation versus screw fixation of calcaneus fracture (59 vs 106, respectively). The radiographic outcomes including pre- and post-operative Bohler's and Gissane's angles were evaluated. The post-operative function was evaluated using the American Orthopaedic Foot and Ankle Society (AOFAS) hindfoot score, the Olerud and Molander Scale and the Visual Analogue Scale (VAS). The complications, the rates of implant removal and cost were also compared. RESULTS: The average follow-up was 44.2 months in the plate groups and 47.9 months in the screw groups (P > 0.05). There was no significant difference in the Bohler's angle and Gissane's angle between the plate group and screw group during the pre-operation and the last follow-up. There was no significant difference in the final AOFAS score, Olerud and Molander score and VAS score between the two groups (P > 0.05). The total incidence of complications was 6.7% in the plate group and 6.6% in the screw group (P > 0.05). The rates of implant removal and total cost during the hospitalization in the plate group were significantly higher compared with screws group (P < 0.05). CONCLUSION: The less invasive plate fixation versus screw fixation of DIACFs via sinus tarsi approach contributed comparable quality of reduction, complications and post-operative functional outcomes. The less invasive plate technique was significantly higher in terms of implant costs and the rate of implant removal.

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.

Identification of association between rs1057317 polymorphism in TLR4 3'-untranslated region and the susceptibility to osteoporosis.

It has been proved that the expression of TLR4 is associated with a reduced risk of osteoporosis (OP). One single-nucleotide polymorphism located within the 3'-untranslated region (3'-UTR) of TLR4 may "generate" binding site of miR-34a and thereby associated with risk of OP. Bioinformatics analysis and luciferase reporter assay were used to specify the effect of polymorphisms on the interaction between miR-34a and TLR4 gene. Western blot analysis and real-time polymerase chain reaction were used to study the expressions of miR-34a, TLR4 in different groups or cells transfected with miR-34a mimics or inhibitor. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to estimate the effect of miR-34a on the apoptosis of osteoblast. TLR4 was identified as a target of miR-34a, with negative regulatory relationship predicted. The expression levels of miR-34a was comparable with each other between CC, CA, and AA groups, and the expression levels of TLR4 was evidently lower in CC compared with GG and GC groups. Also, TLR4 level in culture osteoblast (genotyped as CC) treated with miR-34a mimics was substantially downregulated compared with scramble control, while those cells (genotyped as CC) treated with miR-34a inhibitors showed increased expression of TLR4. Additionally, the apoptosis of osteoblast genotyped as CC was decreased following transfection with miR-34a mimics, while evidently promoted subsequent to transfect with miR-34a inhibitor. The regulatory association between rs1057317 polymorphism in TLR4 3'-UTR led to an inhibitory effect on the expression of TLR4 by miR-34a, which may explain the observed association between the polymorphism and the susceptibility to OP.

TRAF6 neddylation drives inflammatory arthritis by increasing NF-κB activation.

Neddylation is a process similar to ubiquitination, and is critical in various inflammatory diseases; however, its importance in the pathogenesis of inflammatory arthritis is not well understood. Here, we investigated the role of neddylation in collagen-induced arthritis (CIA) and its clinical relevance. We showed that neddylation-related genes, including NEDD8 and CULLIN-1, were significantly upregulated in inflamed arthritic synovia. Functionally, neddylation activation was crucial for synovitis of CIA, as the inhibition of neddylation by MLN4924 significantly suppressed synovial cell proliferation and inflammatory responses. Mechanistically, neddylation mediated inflammatory arthritis by regulating NF-κB activation in fibroblast-like synovial cells (FLSs). Furthermore, TNF receptor-associated factor 6 (TRAF6) neddylation at Lys124 was essential for IL-17A-induced NF-κB activation. Replacing the Lys-124 residue with Arg (K124R) resulted in significantly impaired conjugation of NEDD8 to TRAF6, as well as markedly attenuated IL-17A-induced NF-κB activity. Therefore, the pathogenic role of neddylation in CIA as well as its mechanism of action demonstrated here provides a new insight into understanding the role of post-transcriptional modifications in the arthritis inflammatory response.

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.