Nervous necrosis virus capsid protein and Protein A dynamically modulate the fish cGAS-mediated IFN signal pathway to facilitate viral evasion.

Nervous necrosis virus (NNV), an aquatic RNA virus belonging to Betanodavirus, infects a variety of marine and freshwater fishes, leading to massive mortality of cultured larvae and juveniles and substantial economic losses. The enzyme cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is widely recognized as a central component in the innate immune response to cytosolic DNA derived from different pathogens. However, little is known about the response of cGAS to aquatic RNA viruses. This study found that Epinephelus coioides cGAS (EccGAS) overexpression inhibited NNV replication, whereas EccGAS silencing promoted NNV replication. The anti-NNV activity of EccGAS was involved in interferon (IFN) signaling activation including tumor necrosis factor receptor-associated factor family member-associated NF-kappa-B activator-binding kinase 1 (TBK1) phosphorylation, interferon regulatory factor 3 (IRF3) nuclear translocation, and the subsequent induction of IFNc and ISGs. Interestingly, NNV employed its capsid protein (CP) or Protein A (ProA) to negatively or positively modulate EccGAS-mediated IFN signaling by simultaneously targeting EccGAS. CP interacted with EccGAS via the arm-P, S-P, and SD structural domains and promoted its polyubiquitination with K48 and K63 linkages in an EcUBE3C (the ubiquitin ligase)-dependent manner, ultimately leading to EccGAS degradation. Conversely, ProA bound to EccGAS and inhibited its ubiquitination and degradation. In regulating EccGAS protein content, CP's inhibitory action was more pronounced than ProA's protective effect, allowing successful NNV replication. These novel findings suggest that NNV CP and ProA dynamically modulate the EccGAS-mediated IFN signaling pathway to facilitate the immune escape of NNV. Our findings shed light on a novel mechanism of virus-host interaction and provide a theoretical basis for the prevention and control of NNV.IMPORTANCEAs a well-known DNA sensor, cGAS is a pivotal component in innate anti-viral immunity to anti-DNA viruses. Although there is growing evidence regarding the function of cGAS in the resistance to RNA viruses, the mechanisms by which cGAS participates in RNA virus-induced immune responses in fish and how aquatic viruses evade cGAS-mediated immune surveillance remain elusive. Here, we investigated the detailed mechanism by which EccGAS positively regulates the anti-NNV response. Furthermore, NNV CP and ProA interacted with EccGAS, regulating its protein levels through ubiquitin-proteasome pathways, to dynamically modulate the EccGAS-mediated IFN signaling pathway and facilitate viral evasion. Notably, NNV CP was identified to promote the ubiquitination of EccGAS via ubiquitin ligase EcUBE3C. These findings unveil a novel strategy for aquatic RNA viruses to evade cGAS-mediated innate immunity, enhancing our understanding of virus-host interactions.

Orange-spotted grouper IFNh response to NNV or MSRV and its potential antiviral activities.

Type I interferon (IFN) plays a crucial role in the antiviral immune response. Nervous necrosis virus (NNV) and Micropterus salmoides rhabdovirus (MSRV) are the most important viruses in cultured larvae and juveniles, causing great economic losses to fish farming. To better understand the antiviral activities and immunoregulatory role of IFN from orange-spotted grouper (Epinephelus coioides), EcIFNh was cloned from NNV infected sample. EcIFNh has an open reading frame (ORF) of 552 bp and encodes a polypeptide of 183 amino acids. Phylogenetic tree analysis showed that EcIFNh was clustered into the IFNh branch. The tissue distribution analysis revealed that EcIFNh was highly expressed in the liver and brain of healthy orange-spotted grouper. The mRNA levels of EcIFNh were significantly upregulated after poly (I:C) stimulation and NNV or MSRV infection. Furthermore, the promoter of EcIFNh was characterized and significantly activated by EcMDA5, EcMAVS, EcSTING, EcIRF3, and EcIRF7 in the luciferase activity assays. We found that EcIFNh overexpression resisted the replication of NNV and MSRV, while EcIFNh silencing facilitated NNV replication in GB cells. In addition, EcIFNh recombinant protein (rEcIFNh) enhanced the immune response by inducing the expression of ISGs in vivo and in vitro, suggesting the potential application of rEcIFNh for anti-NNV and anti-MSRV. Taken together, our research may offer the foundation for virus-IFN system interaction in orange-spotted grouper.

Two cytokine receptor family B (CRFB) members in orange-spotted grouper Epinephelus coioides, EcCRFB3 and EcCRFB4, negatively regulate interferon immune responses to assist nervous necrosis virus replication.

Receptors of type I interferon (IFNR) play a vital role in the antiviral immune response. However, little is known about the negative regulatory role of the IFNR. Nervous necrosis virus (NNV) is one of the most significant viruses in cultured fish, resulting in great economic losses for the aquaculture industry. In this study, two orange-spotted grouper (Epinephelus coioides) cytokine receptor family B (CRFB) members, EcCRFB3 and EcCRFB4 were cloned and characterized from NNV infected grouper brain (GB) cells. The open reading frame (ORF) of EcCRFB3 consists of 852 bp encoding 283 amino acids, while EcCRFB4 has an ORF of 990 bp encoding 329 amino acids. The mRNA levels of EcCRFB3 or EcCRFB4 were significantly upregulated after NNV infection and the stimulation of poly (I:C) or NNV-encoded Protein A. In addition, EcCRFB3 or EcCRFB4 overexpression facilitated NNV replication, whereas EcCRFB3 or EcCRFB4 silencing resisted NNV replication. Overexpressed EcCRFB3 or EcCRFB4 inhibited the expression of IFN-I-induced ISGs. Taken together, our research provides the first evidence in fish demonstrating the role of IFNRs to regulate the IFN signaling pathway negatively. Our findings enrich the understanding of the functions of IFNRs and reveal a novel escape mechanism of NNV.

Degradation of 2,4-DCP by immobilized laccase on modified biochar carrier.

Rape straw was used as the raw material for the biochar in this study, which was then changed using acid, alkali, and magnetic techniques. The laccase was attached using the adsorptions-crosslinking process, and the three modified biochars served as the carriers. The ideal circumstances for laccase immobilization were explored, and both biochar and immobilized laccase's characteristics were examined. The removal of 2,4-dichlorophenol (2,4-DCP) by immobilized laccase from modified biochar and its degradation products were researched. The main conclusions are as follows: the optimal concentration of glutaraldehyde (GLU) was 4%, and the pH was four, and the enzyme dosage was 1.75 mg/mL for the immobilized laccase of acid-modified biochar (SBC@LAC). The optimal concentration of GLU was 5%; the pH was four, and the enzyme dosage was 2 mg/mL for immobilized laccase from alkali-modified biochar (JBC@LAC). The optimal concentration of GLU was 5%; the pH was four, and the enzyme dosage was 1.75 mg/mL for immobilized laccase from magnetically modified biochar (CBC@LAC). SEM images could show the changes in the surface morphology of biochar caused by three modification methods. The BET results demonstrated that acid and magnetic modification increased the specific surface area of biochar, and alkali modification mainly expanded the pore size of biochar. FT-IR and XRD showed that modification and laccase loading had little effect on the structure of biochar. The stability of immobilized laccase was better than that of free laccase in acid-base, heat, and storage. Among the three modified biochar immobilized laccases, JBC@LAC showed the best acid-base stability and thermal stability, and the relative enzyme activity changed the least when pH and temperature conditions changed. The storage stability of SBC@LAC is the best. After 30 days of storage, the relative enzyme activity is still 83%. The removal rates of 2,4-DCP were 57, 99, and 63%, respectively, by SBC@LAC, JBC@LAC, and CBC@LAC. After five reuses, the removal rates of 2,4-DCP by SBC@LAC, JBC@LAC and CBC@LAC were 26, 42, and 27%, respectively. The intermediate products of 2,4-DCP degradation by immobilized laccase were p-phenol, p-benzoquinone and maleic acid.

On-Orbit Absolute Radiometric Calibration and Validation of ZY3-02 Satellite Multispectral Sensor.

This study described the on-orbit vicarious radiometric calibration of Chinese civilian high-resolution stereo mapping satellite ZY3-02 multispectral imager (MUX). The calibration was based on gray-scale permanent artificial targets, and multiple radiometric calibration tarpaulins (tarps) using a reflectance-based approach between July and September 2016 at Baotou calibration site in China was described. The calibration results reveal a good linear relationship between DN and TOA radiances of ZY3-02 MUX. The uncertainty of this radiometric calibration was 4.33%, indicating that radiometric coefficients of ZY3-02 MUX are reliable. A detailed discussion on the validation analysis of the comparison results between the different radiometric calibration coefficients is presented in this paper. To further validate the reliability of the three coefficients, the calibrated ZY3-02 MUX was compared with Landsat-8 Operational Land Imager (OLI). The results also indicate that radiometric characteristics of ZY3-02 MUX imagery are reliable and highly accurate for quantitative applications.

Calibration of the laser pointing bias of the GaoFen-7 satellite based on simulation waveform matching.

The two-beam GaoFen-7 (GF-7) laser altimeter is China's first formal spaceborne laser altimeter system for Earth observations. In this article, a calibration method based on simulation waveform matching is proposed to correct the laser pointing error now that the satellite is in orbit. In the method, the optimal position of the laser footprint is searched using simulated and actual waveforms. Then, the laser pointing is calibrated based on the laser footprint optimal position. In this paper, after calibration of the GF-7 laser pointing, infrared detectors are used to capture laser footprints for accuracy verification. The results show that the GF-7 laser pointing accuracy is greatly improved by the method; the laser pointing accuracy of beam 1 is approximately 5.4 arcsec, and that of beam 2 is approximately 5.7 arcsec. Subsequently, two laser footprints are selected for GF-7 laser calibration in the Helan Mountains, China, and AW3D30 digital surface model (DSM) and GPS/RTK data are used to verify the laser elevation measurement accuracy (EMA). The results show that the EMA of the GF-7 laser is significantly improved after calibration. Over flat terrain, the EMA of the GF-7 laser is improved by 10 times, from 3.74 ± 0.55 m to 0.35 ± 0.50 m, verifying the effectiveness of the proposed method.

Coarse-to-Fine Image Matching-Based Footprint Camera Calibration of the GF-7 Satellite.

The GF-7 satellite is China's first high-resolution stereo mapping satellite that reaches sub-meter resolution, equipped with new-type payloads, such as an area array footprint camera that can achieve synchronization acquisition of laser spots. When the satellite is in space, the variation of camera parameters may occur due to launch vibration and environmental changes, and on-orbit geometric calibration thereby must be made. Coupled with the data from the GF-7 satellite, this paper constructs a geometric imaging model of the area array footprint camera based on the two-dimensional direction angle, and proposes a coarse-to-fine "LPM-SIFT + Phase correlation" matching strategy for the automatic extraction of calibration control points. The single-image calibration experiment shows that the on-orbit geometric calibration model of the footprint camera constructed in this paper is correct and effective. The matching method proposed is used to register the footprint images with the DOM (Digital Orthophoto Map) reference data to obtain dense control points. Compared with the calibration result using a small number of manually collected control points, the root mean square error (RMSE) of the residual of the control points is improved from half a pixel to 1/3, and the RMSE of the same orbit checkpoints in the image space is improved from 1 pixel to 0.7. It can be concluded that using the coarse-to-fine image matching method proposed in this paper to extract control points can significantly improve the on-orbit calibration accuracy of the footprint camera on the GF-7 satellite.

Synthesis of Semiconducting 2H-Phase WTe2 Nanosheets with Large Positive Magnetoresistance.

Tungsten ditelluride (WTe2) is provoking immense interest because of its unique electronic properties, but studies about its semiconducting hexagonal (2H) phase are quite rare. Herein, we report the synthesis of semiconducting 2H WTe2 nanosheets with large positive magnetoresistance, for the first time, by a simple lithium-intercalation-assisted exfoliation strategy. Systematic characterizations including high-resolution transmission electron microscopy, X-ray diffraction, and Raman and X-ray photoelectron spectroscopies provide clear evidence to distinguish the structure of 2H WTe2 nanosheets from the orthorhombic (Td) phase bulk counterpart. The corresponding electronic phase transition from metal to semiconductor is also confirmed by density of states calculation, optical absorption, and electrical transport property measurements. Besides, the 2H WTe2 nanosheets exhibit large positive magnetoresistance with values of up to 29.5% (10 K) and 16.2% (300 K) at 9 T. Overall, these findings open up a promising avenue into the exploration of WTe2-based materials in the semiconductor field.

Laser Spot Center Location Method for Chinese Spaceborne GF-7 Footprint Camera.

The Gaofen-7 (GF-7) satellite is equipped with two area array sensor footprint cameras to capture the laser altimeter spot. In order to establish a direct correspondence between the laser data and the stereo image data, a new method is proposed to fit the center of the spot using the brightness difference between the spot image and the footprint image. First, the geometric registration between the spot image and the footprint image is completed based on feature matching or template matching. Then, the brightness values between the two images are extracted from the corresponding image position to form a measurement, and the least squares adjustment method is used to calculate the parameters of the brightness conversion model between the spot image and the footprint image. Finally, according to the registration relationship, the center of the identified spots is respectively positioned in the footprint images, so that the laser spots are accurately identified in the along-track stereo footprint images. The experimental results show that the spot error of this method is less than 0.7 pixel, which has higher reliability and stability, and can be used for a GF-7 satellite footprint camera.

Overview of Earth Observation Satellite Platform Microvibration Detection Methods.

Satellite platform microvibration is a common phenomenon in earth observation satellite orbits that directly affects the imaging quality and accuracy of surveying and mapping. With the continuous improvement in the spatial resolution, the influence of satellite platform microvibration on image geometric accuracy is becoming increasingly significant. High-precision microvibration detection and compensation are key technologies for eliminating image distortion and location deviation caused by satellite platform microvibration. In this paper, the microvibration detection methods of different satellite platforms are summarized, and the verification and analysis are performed on the data downloaded from the Resource-3 satellite (ZY-3) platform, to provide technical support for subsequent refined processing of satellite attitude.

Enhanced Superoxide Generation on Defective Surfaces for Selective Photooxidation.

Photocatalytic selective oxidation reactions hold great promise for the design of high-value-added organic intermediates, but many of these reactions suffer from low conversion efficiency and selectivity due to uncontrollable oxidation processes. In view of using photogenerated reactive oxygen species as the key oxidant in a selective oxidation reaction, we propose that a highly selective oxidation reaction can be achieved by modulating the corresponding photocatalytic molecular oxygen (O2) activation processes. Using cubic indium sulfide (ß-In2S3) nanosheets as a model system, we show that the charge carriers involved in O2 activation can be optimized with the introduction of surface S vacancies. Benefiting from the enhanced charge separation and transfer processes, the In2S3 nanosheets with S vacancies could simultaneously activate O2 into superoxide radicals via electron transfer under visible-light irradiation to display outstanding activity for the selective oxidation of alcohols to aldehydes with high conversion and selectivity. This study offers a new strategy to optimize photocatalytic selective oxidation reactions.

An affinity peptide exerts antiviral activity by strongly binding nervous necrosis virus to block viral entry.

Nervous necrosis virus (NNV) causes viral nervous necrosis (VNN), a disease that leads to almost 100% mortality among larvae and juvenile fish, severely affecting the aquaculture industry. VNN vaccines based on inactivated viruses or virus-like particles (VLPs) are unsuitable for fish fry with immature adaptive immune systems. Here, we applied an anti-NNV strategy based on affinity peptides (AFPs). Three phage display peptide libraries were screened against RBS, the VLP of orange-spotted grouper nervous necrosis virus (OGNNV). From the positive clones, a dodecapeptide with the highest binding capacity (BC) to RBS was selected. This AFP agglutinated or disrupted virion particles, inhibiting RBS entry into sea bass (SB) cells. To enhance BC and solubility, we amended the AFP sequence as "LHWDFQSWVPLL" and named as 12C. One to three copies of 12C in tandem were prokaryotically expressed with a maltose binding protein (MBP) linked by a flexible peptide. Of the recombinant proteins expressed, MBP-triple-12C (MBP-T12C) exhibited the highest BC, efficiently blocked RBS entry, and strongly inhibited OGNNV infection at viral entry. Moreover, MBP-T12C bound the VLPs of all NNV serotypes, displaying broad-spectrum anti-NNV ability, and recognized only OGNNV and mud crab virus, demonstrating binding specificity. Therefore, these anti-NNV AFPs specifically bound NNV, aggregating or disrupting the viral particles, to reduce the contact probability between the virus and cell surface, subsequently inhibiting viral infection. Our results not only provided a candidate of anti-NNV AFP, but a framework for the development of antiviral AFP.

Promoted water splitting by efficient electron transfer between Au nanoparticles and hematite nanoplates: a theoretical and experimental study.

An ideal interface model combining a hematite nanoplate-based photoanode with Au nanoparticles (NPs) is proposed for elucidating the specific role of Au NPs in photoelectrochemical performances. The theoretical and experimental results reveal that Au/Fe2O3 nanoplates can lead to an enhanced localized electric field at the metal-semiconductor interface upon the formation of surface plasmon resonance and hot electrons, which can be injected into the conduction band of the semiconductor, thus improving the efficiency of the generation and separation of electron-hole pairs. As expected, the Au/Fe2O3 nanoplate-based photoelectrode possessed a higher carrier density and a photocurrent of 1.7 mA cm-2 and 3.8 mA cm-2 at 1.23 V and 1.5 V vs. RHE, which are nearly 5 times and 30 times larger than that of the Au/Fe2O3 nanocrystals and pristine Fe2O3 nanoplate-based photoelectrodes, respectively.

Fluorometric determination of the activity of alkaline phosphatase and its inhibitors based on ascorbic acid-induced aggregation of carbon dots.

The authors describe a fluorometric method for determination of the activity of alkaline phosphatase (ALP) and its inhibitors. Nitrogen and boron co-doped carbon dots (C-dots) with excitation/emission peaks at 490/540 nm act as the fluorescent probe. The C-dots were prepared by hydrothermal carbonization starting from 3-aminophenylboronic acid as the sole precursor. On the basis of the boronic acid-triggered specific reaction with cis-diols, the boronic acid modified C-dots can bind to ascorbic acid that is generated by ALP-catalyzed hydrolysis of ascorbic acid 2-phosphate. This results in particle aggregation and quenching of fluorescence. If the ALP inhibitor Na3VO4 is introduced into the system, the activity of ALP is reduced and the fluorescence of C-dots recovers. This fluorometric method allows for the determination of ALP activity in the range from 0.2 to 6.0 mU mL-1 with a detection limit of 0.16 mU mL-1. The IC50 value for the inhibitor Na3VO4 is 3.6 µM. The method is convenient and cost-effective. It does not require complicated operations and in our perception widens the scope of applications of C-dots in bioanalytical sciences. Graphical abstract Schematic presentation of the nitrogen and boron co-doped carbon dot-based fluorometric method for determination of alkaline phosphatase (ALP) activity.

Spectrophotometric determination of the activity of alkaline phosphatase and detection of its inhibitors by exploiting the pyrophosphate-accelerated oxidase-like activity of nanoceria.

The oxidase-like activity of nanoceria is low. This limits its practical applications. It is demonstrated here that pyrophosphate ion (PPi) can improve the oxidase-like activity of nanoceria. Specifically, nanoceria catalyzes the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to give a blue product (oxTMB) with an absorption peak at 645 nm in the presence of PPi. If, however, alkaline phosphatase (ALP) is present, it will hydrolyze PPi, and this results in a decreased oxidase-like activity of nanoceria. Hence, less blue oxTMB willl be formed. On the other hand, if the ALP inhibitor Na3VO4 is added to the system, the oxidase-like activity of nanoceria is gradually restored. On the basis of the above results, a spectrophotometric method was developed for determination of the activity of ALP. It works in the 0.5 to 10 mU.mL-1 activity range and has a 0.32 mU.mL-1 detection limit. Na3VO4 causes a 50% ALP inhibition if present in 71 µM concentration. The assay was successfully applied to the determination of ALP in spiked human serum and gave good recoveries. Graphical abstract Schematic presentation of pyrophosphate (PPi)-induced acceleration of the oxidase-like activity of nanoceria (CeO2) for determination of alkaline phosphatase enzyme (ALP) activity and its inhibitor NaVO3.

Iron-Incorporated α-Ni(OH)2 Hierarchical Nanosheet Arrays for Electrocatalytic Urea Oxidation.

An iron-incorporated α-Ni(OH)2 nanosheet array catalyst characterized by hierarchical surface nanobelts and robust urea oxidation performance are reported. The unique single-crystalline belt-on-sheet hierarchical nanostructure was identified and it endows more reactive edges for the Ni2+ -to-Ni3+ pre-oxidation process to boost the generation of active high-valence species for the urea oxidation reaction (UOR). Benefitting from the optimal Fe concentration, the UOR activity was further optimized owing to the favorable reaction kinetics. With the synergistic benefits of the increased surface areas, improved charge transfer behavior, favorable reaction kinetics and excellent structural stability, the iron-incorporated α-Ni(OH)2 hierarchical nanosheet array catalyst displays significantly improved UOR performance with both high activity and outstanding operational stability. This work could guide the design of advanced UOR catalysts for wastewater treatment and clean energy production in the future.

Protein A from orange-spotted grouper nervous necrosis virus triggers type I interferon production in fish cell.

Family Nodaviridae consists of two genera: Alphanodavirus and Betanodavirus, and the latter is classified into four genotypes, including red-spotted grouper nervous necrosis virus, tiger puffer nervous necrosis virus, striped jack nervous necrosis virus, and barfin flounder nervous necrosis virus. Type I interferons (IFNs) play a central role in the innate immune system and antiviral responses, and the interactions between IFN and NNV have been investigated in this study. We have found that the RNA-dependent RNA polymerase (RdRp) from orange-spotted nervous necrosis virus (OGNNV), named protein A, was capable of activating IFN promoter in fathead minnow (FHM) cells. Transient expression of protein A was found to induce IFN expression and secretion, endowing FHM cells with anti-tiger frog virus ability. Protein A from SJNNV can also induce IFN expression in FHM cells but that from Flock House virus (FHV), a well-studied representative species of genus Alphanodavirus, cannot. RdRp activity and mitochondrial localization were shown to be required for protein A to induce IFN expression by means of activating IRF3 but not NFκB. Furthermore, DsRNA synthesized in vitro transcription and poly I:C activated IFN promoter activity when transfected into FHM cells, and dsRNA were also detected in NNV-infected cells. We postulated that dsRNA, a PAMP, was produced by protein A, leading to activation of innate immune response. These results suggest that protein As from NNV are the agonists of innate immune response. This is the first work to demonstrate the interaction between NNV protein A and innate immune system, and may help to understand pathogenesis of NNV.

Spatio-Temporal Super-Resolution Reconstruction of Remote-Sensing Images Based on Adaptive Multi-Scale Detail Enhancement.

There are many problems in existing reconstruction-based super-resolution algorithms, such as the lack of texture-feature representation and of high-frequency details. Multi-scale detail enhancement can produce more texture information and high-frequency information. Therefore, super-resolution reconstruction of remote-sensing images based on adaptive multi-scale detail enhancement (AMDE-SR) is proposed in this paper. First, the information entropy of each remote-sensing image is calculated, and the image with the maximum entropy value is regarded as the reference image. Subsequently, spatio-temporal remote-sensing images are processed using phase normalization, which is to reduce the time phase difference of image data and enhance the complementarity of information. The multi-scale image information is then decomposed using the L0 gradient minimization model, and the non-redundant information is processed by difference calculation and expanding non-redundant layers and the redundant layer by the iterative back-projection (IBP) technique. The different-scale non-redundant information is adaptive-weighted and fused using cross-entropy. Finally, a nonlinear texture-detail-enhancement function is built to improve the scope of small details, and the peak signal-to-noise ratio (PSNR) is used as an iterative constraint. Ultimately, high-resolution remote-sensing images with abundant texture information are obtained by iterative optimization. Real results show an average gain in entropy of up to 0.42 dB for an up-scaling of 2 and a significant promotion gain in enhancement measure evaluation for an up-scaling of 2. The experimental results show that the performance of the AMED-SR method is better than existing super-resolution reconstruction methods in terms of visual and accuracy improvements.

Betanodavirus-like particles enter host cells via clathrin-mediated endocytosis in a cholesterol-, pH- and cytoskeleton-dependent manner.

Betanodavirus, also referred to nervous necrosis virus (NNV), is the causative agent of the fatal disease, viral nervous necrosis and has brought significant economic losses in marine and freshwater cultured fish, especially larvae and juveniles. Here, we used an established invasion model with virus-like particle (VLP)-cells, mimicking orange-spotted grouper nervous necrosis virus (OGNNV), to investigate the crucial events of virus entry. VLP were observed in the perinuclear regions of Asian sea bass (SB) cells within 1.5 h after attachment. VLP uptake was strongly inhibited when cells were pretreated with biochemical inhibitors (chlorpromazine and dynasore) blocking clathrin-mediated endocytosis (CME) or transfected with siRNA against clathrin heavy and light chains. Inhibitors against key regulators of caveolae/raft-dependent endocytosis and macropinocytosis had no effect on VLP uptake. In contrast, disruption of cellular cholesterol by methyl-ß-cyclodextrin or reduction of cholesterol fluidity by Cholera toxin B subunit significantly decreased VLP entry. Furthermore, VLP entry is dependent on low pH and cytoskeleton, demonstrated by inhibitor (chloroquine, ammonia chloride, cytochalasin D, wiskostatin, and nocodazole) perturbation. Therefore, OGNNV VLP enter SB cells via CME depending on dynamin-2, cholesterol and its fluidity, low pH, and cytoskeleton. In addition, ten more cell lines were screened for VLP entry and VLP can only enter NNV-sensitive cells, GB and SSN-1, via CME, indicating that CME is the common endocytosis pathway for VLP. These results may provide the data for NNV entry without the influence of the viral genome, an ideal model for exploring the behaviour of betanodavirus in cells, and valuable references to vaccine development.

The core ubiquitin system of mandarin fish, Siniperca chuatsi, can be utilized by infectious spleen and kidney necrosis virus.

The process of ubiquitination regulates various cellular processes. The ubiquitin-proteasome system (UPS) in fish, which is important for the generation of innate and adaptive immune responses to pathogens, is the target of aquatic viruses to achieve immune evasion. We cloned and characterized three genes, namely, a ubiquitin-activating enzyme (ScE1), a ubiquitin-conjugating enzyme (ScE2), and a HECT-type ubiquitin ligase (ScE3) of mandarin fish Siniperca chuatsi. The genes were expressed in all tissues and the highest levels were observed in the blood. In infectious spleen and kidney necrosis virus (ISKNV)-infected mandarin fish fry cells, the expression levels of the three genes in vitro were almost identical, and upregulated during the early stage and downregulated at the late stage. In the blood of ISKNV-infected mandarin fish, their expressions in vivo were downregulated equally although peaking at different timepoints, indicating the suppression of UPS by viral infection. Furthermore, these recombinant proteins were determined to function well in ubiquitination assays in vitro. Moreover, ScE1 and ScE2 can be utilized by four RING-type viral E3s (vE3s) that are encoded by ISKNV. The in vitro activity of vE3 was stronger than that of ScE3, suggesting that the fish UPS may be hijacked by ISKNV via E3 activity competition and expression modulation. The present study investigated the function of mandarin fish UPS as well as its response to iridovirus infection, providing insights to better understand the virus-host interactions and the mechanism of ISKNV in evading host immune responses.