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Gene regulatory networks (GRNs) are crucial for understanding organismal molecular mechanisms and processes. Construction of GRN in the epithelioma papulosum cyprini (EPC) cells of cyprinid fish by spring viremia of carp virus (SVCV) infection helps understand the immune regulatory mechanisms that enhance the survival capabilities of cyprinid fish. Although many computational methods have been used to infer GRNs, specialized approaches for predicting the GRN of EPC cells following SVCV infection are lacking. In addition, most existing methods focus primarily on gene expression features, neglecting the valuable network structural information in known GRNs. In this study, we propose a novel supervised deep neural network, named MEFFGRN (Matrix Enhancement- and Feature Fusion-based method for Gene Regulatory Network inference), to accurately predict the GRN of EPC cells following SVCV infection. MEFFGRN considers both gene expression data and network structure information of known GRN and introduces a matrix enhancement method to address the sparsity issue of known GRN, extracting richer network structure information. To optimize the benefits of CNN (Convolutional Neural Network) in image processing, gene expression and enhanced GRN data were transformed into histogram images for each gene pair respectively. Subsequently, these histograms were separately fed into CNNs for training to obtain the corresponding gene expression and network structural features. Furthermore, a feature fusion mechanism was introduced to comprehensively integrate the gene expression and network structural features. This integration considers the specificity of each feature and their interactive information, resulting in a more comprehensive and precise feature representation during the fusion process. Experimental results from both real-world and benchmark datasets demonstrate that MEFFGRN achieves competitive performance compared with state-of-the-art computational methods. Furthermore, study findings from SVCV-infected EPC cells suggest that MEFFGRN can predict novel gene regulatory relationships., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Spring viremia of carp virus (SVCV) has a broad fish host spectrum and is responsible for a disease that generally affects juvenile fishes with a mortality rate of up to 90%. In the absence of treatments or vaccines against SVCV, the search for prophylactic or therapeutic solutions is thus relevant, particularly to identify solutions compatible with mass vaccination. In addition to being a threat to aquaculture and ecosystems, SVCV is a unique pathogen to study virus-host interactions in the zebrafish model. Establishing the first reverse genetics system for SVCV and the design of recombinant SVCV (rSVCV) expressing fluorescent or bioluminescent proteins adds a new dimension for the study of these interactions using innovative imaging techniques. The infection by bath immersion of zebrafish larvae with rSVCV expressing mCherry allows us to define the first SVCV replication sites and the host innate immune responses using different transgenic lines of zebrafish. The fins were found as the main initial sites of infection in both zebrafish and carp, its natural host. Hence, new insights into the physiopathology of SVCV infection have been described. We report that neutrophils are recruited at the sites of infection and persist up to the death of the animal leading to an uncontrolled inflammation correlated with the expression of the pro-inflammatory cytokine IL1β. Tissue damage was observed at the site of initial replication, a likely consequence of virus-induced injury or the pro-inflammatory response. Interestingly, SVCV infection by bath immersion triggers a persistent pro-inflammatory response rather than activation of the antiviral IFN signaling pathway as observed following intravenous injection, highlighting the importance of the route of infection on the progression of pathogenicity. Thus, this model of zebrafish larvae infection by rSVCV offers new perspectives to study in detail virus-host interactions and to discover new prophylactic or therapeutic solutions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Souto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In this issue, we established rapid, cost-effective, and simple detection methods including recombines polymerase amplification with lateral flow dipstick (RPA-LFD) and real-time RPA for cyprinid herpesvirus 3(CyHV-3), and evaluated their sensitivity, specificity, and applicability, the real-time RPA method could achieve sensitive diagnosis of CyHV-3 within 1.3 copies per reaction, respectively. The real-time RPA method is 10-fold more sensitive than RPA-LFD method. The exact number of CyHV-3 can be calculated in each sample by real-time RPA. The sera from koi also can be tested in these methods. In addition, no cross-reaction was observed with other related pathogens, including carp oedema virus (CEV), spring viraemia of carp virus (SVCV), cyprinid herpesvirus 1(CyHV-1), cyprinid herpesvirus 2(CyHV-2), type I grass carp reovirus (GCRV-I), type II GCRV (GCRV-II), type III GCRV (GCRV-III), and Aeromonas hydrophila., (© 2024 John Wiley & Sons Ltd.)

Grass carp, one of the major freshwater aquaculture species in China, is susceptible to grass carp reovirus (GCRV). GCRV is a non-enveloped RNA virus and has a double-layered capsid, causing hemorrhagic disease and high mortalities in infected fish. However, the tropism of GCRV infection has not been investigated. In this study, monoclonal antibodies against recombinant VP35 protein were generated in mice and characterized. The antibodies exhibited specific binding to the N terminal region (1-155 aa) of the recombinant VP35 protein expressed in the HEK293 cells, and native VP35 protein in the GCRV-II infected CIK cells. Immunofluorescent staining revealed that viruses aggregated in the cytoplasm of infected cells. In vivo challenge experiments showed that high levels of GCRV-II viruses were present in the gills, intestine, spleen and liver, indicating that they are the major sites for virus infection. Our study showed that the VP35 antibodies generated in this study exhibited high specificity, and are valuable for the development of diagnostic tools for GCRV-II infection., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

BF/C2 is a crucial molecule in the coagulation complement cascade pathway and plays a significant role in the immune response of grass carp through the classical, alternative, and lectin pathways during GCRV infection. In vivo experiments demonstrated that the mRNA expression levels of BF/C2 (A, B) in grass carp positively correlated with GCRV viral replication at various stages of infection. Excessive inflammation leading to death coincided with peak levels of BF/C2 (A, B) mRNA expression and GCRV viral replication. Correspondingly, BF/C2 (A, B) recombinant protein, CIK cells and GCRV co-incubation experiments yielded similar findings. Therefore, 3 h (incubation period) and 9 h (death period) were selected as critical points for this study. Transcriptome sequencing analysis revealed significant differences in the expression of BF/C2A and BF/C2B during different stages of CIK infection with GCRV and compared to the blank control group (PBS). Specifically, the BF/C2A_3 and BF/C2A_9 groups exhibited 2729 and 2228 differentially expressed genes (DEGs), respectively, with 1436 upregulated and 1293 downregulated in the former, and 1324 upregulated and 904 downregulated in the latter. The BF/C2B_3 and BF/C2B_9 groups showed 2303 and 1547 DEGs, respectively, with 1368 upregulated and 935 downregulated in the former, and 818 upregulated and 729 downregulated in the latter. KEGG functional enrichment analysis of these DEGs identified shared pathways between BF/C2A and PBS groups at 3 and 9 h, including the C-type lectin receptor signaling pathway, protein processing in the endoplasmic reticulum, Toll-like receptor signaling pathway, Salmonella infection, apoptosis, tight junction, and adipocytokine signaling pathway. Additionally, the BF/C2B groups at 3 and 9 h shared pathways related to protein processing in the endoplasmic reticulum, glycolysis/gluconeogenesis, and biosynthesis of amino acids. The mRNA levels of these DEGs were validated in cellular models, confirming consistency with the sequencing results. In addition, the mRNA expression levels of these candidate genes (mapk1, il1b, rela, nfkbiab, akt3a, hyou1, hsp90b1, dnajc3a et al.) in the head kidney, kidney, liver and spleen of grass carp immune tissue were significantly different from those of the control group by BF/C2 (A, B) protein injection in vivo. These candidate genes play an important role in the response of BF/C2 (A, B) to GCRV infection and it also further confirmed that BF/C2 (A, B) of grass carp plays an important role in coping with GCRV infection., (© 2024. The Author(s).)

Similar to other RNA viruses, grass carp reovirus, the causative agent of the hemorrhagic disease, replicates in cytoplasmic viral inclusion bodies (VIBs), orchestrated by host proteins and lipids. The host pathways that facilitate the formation and function of GCRV VIBs are poorly understood. This work demonstrates that GCRV manipulates grass carp oxysterol binding protein 1 (named as gcOSBP1) and vesicle-associated membrane protein-associated protein A/B (named as gcVAP-A/B), 3 components of cholesterol transport pathway, to generate VIBs. By siRNA-mediated knockdown, we demonstrate that gcOSBP1 is an essential host factor for GCRV replication. We reveal that the nonstructural proteins NS80 and NS38 of GCRV interact with gcOSBP1, and that the gcOSBP1 is recruited by NS38 and NS80 for promoting the generation of VIBs. gcOSBP1 increases the expression of gcVAP-A/B and promotes the accumulation of intracellular cholesterol. gcOSBP1 also interacts with gcVAP-A/B for forming gcOSBP1-gcVAP-A/B complexes, which contribute to enhance the accumulation of intracellular cholesterol and gcOSBP1-mediated generation of VIBs. Inhibiting cholesterol accumulation by lovastatin can completely abolish the effects of gcOSBP1 and/or gcVAP-A/B in promoting GCRV infection, suggesting that cholesterol accumulation is vital for gcOSBP1- and/or gcVAP-A/B-mediated GCRV replication. Thus, our results, which highlight that gcOSBP1 functions in the replication of GCRV via its interaction with essential viral proteins for forming VIBs and with host gcVAP-A/B, provide key molecular targets for obtaining anti-hemorrhagic disease grass carp via gene editing technology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Li, Zhang, Chen, Le and Chang.)

MicroRNAs (miRNAs) are highly conserved endogenous single-stranded non-coding RNA molecules that play a crucial role in regulating gene expression to maintain normal physiological functions in fish. Nevertheless, the specific physiological role of miRNAs in lower vertebrates, particularly in comparison to mammals, remains elusive. Additionally, the mechanisms underlying the control of antiviral responses triggered by viral stimulation in fish are still not fully understood. In this study, we investigated the regulatory impact of miR-1388 on the signaling pathway mediated by IFN regulatory factor 3 ( IRF3 ). Our findings revealed that following stimulation with the viral analog poly(I:C), the expression of miR-1388 was significantly upregulated in primary immune tissues and macrophages. Through a dual luciferase reporter assay, we corroborated a direct targeting relationship between miR-1388 and tumor necrosis factor receptor ( TNFR )-associated factor 3 ( TRAF3 ). Furthermore, our study demonstrated a distinct negative post-transcriptional correlation between miR-1388 and TRAF3 . We observed a significant negative post-transcriptional regulatory association between miR-1388 and the levels of antiviral genes following poly(I:C) stimulation. Utilizing reporter plasmids, we elucidated the role of miR-1388 in the antiviral signaling pathway activated by TRAF3 . By intervening with siRNA- TRAF3 , we validated that miR-1388 regulates the expression of antiviral genes and the production of type I interferons ( IFN-Is ) through its interaction with TRAF3 . Collectively, our experiments highlight the regulatory influence of miR-1388 on the IRF3 -mediated signaling pathway by targeting TRAF3 post poly(I:C) stimulation. These findings provide compelling evidence for enhancing our understanding of the mechanisms through which fish miRNAs participate in immune responses.

Tripartite motif (TRIM) proteins are involved in different cellular functions, including regulating virus infection. In teleosts, two orthologous genes of mammalian TRIM2 are identified. However, the functions and molecular mechanisms of piscine TRIM2 remain unclear. Here, we show that trim2b -knockout zebrafish are more susceptible to spring viremia of carp virus (SVCV) infection than wild-type zebrafish. Transcriptomic analysis demonstrates that NOD-like receptor (NLR), but not RIG-I-like receptor (RLR), signaling pathway is significantly enriched in the trim2b -knockout zebrafish. In vitro , overexpression of Trim2b fails to degrade RLRs and those key proteins involved in the RLR signaling pathway but does for negative regulators NLRP12-like proteins. Zebrafish Trim2b degrades NLRP12-like proteins through its NHL_TRIM2_like and IG_FLMN domains in a ubiquitin-proteasome degradation pathway. SVCV-N and SVCV-G proteins are also degraded by NHL_TRIM2_like domains, and the degradation pathway is an autophagy lysosomal pathway. Moreover, zebrafish Trim2b can interfere with the binding between NLRP12-like protein and SVCV viral RNA and can completely block the negative regulation of NLRP12-like protein on SVCV infection. Taken together, our data demonstrate that the mechanism of action of zebrafish trim2b against SVCV infection is through targeting the degradation of host-negative regulators NLRP12-like receptors and viral SVCV-N/SVCV-G genes.IMPORTANCESpring viremia of carp virus (SVCV) is a lethal freshwater pathogen that causes high mortality in cyprinid fish. In the present study, we identified zebrafish trim2b , NLRP12-L1, and NLRP12-L2 as potential pattern recognition receptors (PRRs) for sensing and binding viral RNA. Zebrafish trim2b functions as a positive regulator; however, NLRP12-L1 and NLRP12-L2 function as negative regulators during SVCV infection. Furthermore, we find that zebrafish trim2b decreases host lethality in two manners. First, zebrafish Trim2b promotes protein degradations of negative regulators NLRP12-L1 and NLRP12-L2 by enhancing K48-linked ubiquitination and decreasing K63-linked ubiquitination. Second, zebrafish trim2b targets viral RNAs for degradation. Therefore, this study reveals a special antiviral mechanism in lower vertebrates., Competing Interests: The authors declare no conflict of interest.

Grass Carp Reovirus (GCRV) and Aeromonas hydrophila (Ah) are the causative agents of haemorrhagic disease in grass carp. This study aimed to investigate the molecular mechanisms and immune responses at the miRNA, mRNA, and protein levels in grass carp kidney cells (CIK) infected by Grass Carp Reovirus (GCRV, NV) and Aeromonas hydrophilus (Bacteria, NB) to gain insight into their pathogenesis. Within 48 h of infection with Grass Carp Reovirus (GCRV), 99 differentially expressed microRNA (DEMs), 2132 differentially expressed genes (DEGs), and 627 differentially expressed proteins (DEPs) were identified by sequencing; a total of 92 DEMs, 3162 DEGs, and 712 DEPs were identified within 48 h of infection with Aeromonas hydrophila . It is worth noting that most of the DEGs in the NV group were primarily involved in cellular processes, while most of the DEGs in the NB group were associated with metabolic pathways based on KEGG enrichment analysis. This study revealed that the mechanism of a grass carp haemorrhage caused by GCRV infection differs from that caused by the Aeromonas hydrophila infection. An important miRNA-mRNA-protein regulatory network was established based on comprehensive transcriptome and proteome analysis. Furthermore, 14 DEGs and 6 DEMs were randomly selected for the verification of RNA/small RNA-seq data by RT-qPCR. Our study not only contributes to the understanding of the pathogenesis of grass carp CIK cells infected with GCRV and Aeromonas hydrophila, but also serves as a significant reference value for other aquatic animal haemorrhagic diseases., Competing Interests: The authors declare no conflict of interest.

The mode and outcome of fish-virus interactions are influenced by many abiotic factors, among which water temperature is especially important in poikilothermic fish. Rare minnow Gobiocypris rarus is a eurythermal small cyprinid fish that is sensitive to infection with genotype II grass carp reovirus (GCRV). HSP70, a conservative and key player in heat shock response, is previously identified as an induced pro-viral factor during GCRV infection in vitro. Here, rare minnow was subjected to heat shock treatment (HST), 1 h treatment at 32 °C followed by reverting to a normal temperature of 24 °C, and subsequently challenged with GCRV-II at a dosage of 1 × LD 50 . The effect of HST on GCRV virulence in vivo was evaluated by calculating virus-associated mortality and viral load in both dead and survival fish. The results revealed that HST enhanced the mortality of rare minnow infected with GCRV; the fact that viral loads in the tissue samples of HST-treated fish were significantly higher than those in samples of the control group at 6, 8 d p.i. reflected a faster infection process due to HST. Quantitative gene expression analysis was further employed to show that the expression levels of Hsp70 in intestine and liver tissues from the HST group declined faster than muscle tissue after HST. HST W/O GCRV challenge upregulated proinflammatory cytokines such as MyD88 and Nf-κB , which was in consistence with the inflammation observed in histopathological analysis. This study shed light on the complexity of the interaction between fish abiotic and biotic stress response, which suggested that HST, an abiotic stress, could enhance the virulence of GCRV in Gobiocypris rarus that involved modulating the gene expression of host heat shock, as well as a pro-inflammatory response.

Stimulator of interferon genes (STING), as an imperative adaptor protein in innate immune, responds to nucleic acid from invading pathogens to build antiviral responses in host cells. Aberrant activation of STING may trigger tissue damage and autoimmune diseases. Given the decisive role in initiating innate immune response, the activity of STING is intricately governed by several posttranslational modifications, including phosphorylation and ubiquitination. Here, we cloned and characterized a novel RNF122 homolog from common carp (named CcRNF122L). Expression analysis disclosed that the expression of CcRNF122L is up-regulated under spring viremia of carp virus (SVCV) stimulation in vivo and in vitro. Overexpression of CcRNF122L hampers SVCV- or poly(I:C)-mediated the expression of IFN-1 and ISGs in a dose-dependent way. Mechanistically, CcRNF122L interacts with STING and promotes the polyubiquitylation of STING. This polyubiquitylation event inhibits the aggregation of STING and the subsequent recruitment of TBK1 and IRF3 to the signaling complex. Additionally, the deletion of the TM domain abolishes the negative regulatory function of CcRNF122L. Collectively, our discoveries unveil a mechanism that governs the STING function and the precise adjustment of the innate immune response in teleost., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Carp oedema virus (CEV) has distinct molecularly identified genogroups of viral mutations, denoted as I, IIa, and IIb. Failure to propagate CEV in vitro limits studies towards understanding its interactions with host cells. Here, virus isolates belonging to genogroup I collected during natural outbreaks in the Czech Republic were employed for routine CEV cultivation in monolayers of carp-derived primary cells, common carp brain (CCB) cells, and epithelioma papulosum cyprinid (EPC) cells. Induction of cytopathic effects (CPEs) was observed and recorded in affected cells. Cell survival rate was evaluated under serial dilutions of the CEV inoculum. Virus cell entry was quantified and visualized by qPCR and transmission electron microscopy, respectively. Study findings indicate primary gills epithelia likely present the most suitable matrix for CEV growth in vitro. Cells of the head kidney and spleen facilitate virus entry with microscopically confirmed CPEs and the presence of cytoplasmic pleomorphic virus particles. Cells of the trunk kidney and gonads are unlikely to permit virus cell entry and CPEs development. Although CEV cultivation in cell lines was inconclusive, EPC cells were CEV permissible. Monolayers of carp-derived primary cells show promise for CEV cultivation that could enable elaborate study of mechanisms underlying cellular binding and responses., (© 2024 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Introduction: Koi herpesvirus disease (KHVD) is attributed to cyprinid herpesvirus-3 (CyHV-3) and predominantly affects common carp and ornamental koi carp (Cyprinus carpio). This viral infection leads to substantial morbidity and mortality among these fish species. This study aimed to confirm the presence of KHVD in the Kurdistan region of Iraq by employing clinical and optimized molecular assays on fish populations experiencing high mortality among common carp in carp farms., Methodology: The present research was conducted in the Kalar district, situated at the heart of Garmian province in Iraqi Kurdistan. four samples from common carp fish farms were received by our laboratory. These samples specifically displaying clinical signs associated with koi herpesvirus (KHV) infection, were subjected to clinical examinations, and PCR assay in addition to sequence analysis., Results: The results of the current study revealed that the observed clinical signs, particularly gill necrosis, skin lesions, and sunken eyes, closely resembled the clinical signs of KHVD in common carp fish. In addition, PCR, nested PCR, and sequence analysis assay detected appropriate DNA fragments of the CyHV-3 major capsid protein gene confirming the first detection of KHVD in common carp fish in the Kurdistan region of Iraq., Conclusion: In this study, the results confirm the detection of KHVD in the Kurdistan region, Iraq, for the first time. This study revealed that CyHV-3 was responsible for KHVD-related signs and symptoms. Based on these results, it is strongly recommended that comprehensive studies be initiated to investigate the prevalence and distribution of CyHV-3., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Akbar Hasan Al-Jaf et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In aquaculture, viral diseases pose a significant threat and can lead to substantial economic losses. The primary defense against viral invasion is the innate immune system, with interferons (IFNs) playing a crucial role in mediating the immune response. With advancements in molecular biology, the role of non-coding RNA (ncRNA), particularly microRNAs (miRNAs), in gene expression has gained increasing attention. While the function of miRNAs in regulating the host immune response has been extensively studied, research on their immunomodulatory effects in teleost fish, including silver carp ( Hyphthalmichthys molitrix ), is limited. Therefore, this research aimed to investigate the immunomodulatory role of microRNA-30b-5p (miR-30b-5p) in the antiviral immune response of silver carp ( Hypophthalmichthys molitrix ) by targeting cytokine receptor family B5 (CRFB5) via the JAK/STAT signaling pathway. In this study, silver carp were stimulated with polyinosinic-polycytidylic acid (poly (I:C)), resulting in the identification of an up-regulated miRNA (miR-30b-5p). Through a dual luciferase assay, it was demonstrated that CRFB5, a receptor shared by fish type I interferon, is a novel target of miR-30b-5p. Furthermore, it was found that miR-30b-5p can suppress post-transcriptional CRFB5 expression. Importantly, this study revealed for the first time that miR-30b-5p negatively regulates the JAK/STAT signaling pathway, thereby mediating the antiviral immune response in silver carp by targeting CRFB5 and maintaining immune system stability. These findings not only contribute to the understanding of how miRNAs act as negative feedback regulators in teleost fish antiviral immunity but also suggest their potential therapeutic measures to prevent an excessive immune response.

Cyprinid herpesvirus is a causative agent of a destructive disease in common and koi carp (Cyprinus carpio), which leads to substantial global financial losses in aquaculture industries. Among the strains of C. herpesvirus, C. herpesvirus 1 (CyHV-1) and C. herpesvirus 3 (CyHV-3) are known as highly pathogenic to carp fishes in Europe, Asia, and Africa. To date, no effective vaccine has been developed to combat these viruses. This study aimed to develop unique multi-epitope subunit vaccines targeting the CyHV-1 and CyHV-3 using a reverse vaccinology approach. The study began with a comprehensive literature review to identify the most critical proteins, which were then subjected to in silico analyses to predict highly antigenic epitopes. These analyses involved assessing antigenicity, transmembrane topology screening, allergenecity, toxicity, and molecular docking approaches. We constructed two multi-epitope-based vaccines incorporating a suitable adjuvant and appropriate linkers. It revealed that both the vaccines are non-toxic and immunogenic. The tertiary structures of the vaccine proteins were generated, refined, and validated to ensure their suitability. The binding affinity between the vaccine constructs and TLR3 and TLR5 receptors were assessed by molecular docking studies. Molecular dynamics simulations indicated that vaccine construct V1 exhibited greater stability with both TLR3 and TLR5 based on RMSD analysis. Hydrogen bond analysis revealed a stronger binding affinity between the vaccine constructs and TLR5 compared to TLR3. Furthermore, MM-PBSA analysis suggested that both vaccine constructs exhibited a better affinity for TLR5. Considering all aspects, the results suggest that in silico development of CyHV vaccines incorporating multiple epitopes holds promise for management of diseases caused by CyHV-1 and CyHV-3. However, further in vivo trials are highly recommended to validate the efficacies of these vaccines., (© 2024. The Author(s).)

Zalpha (Zα) domains bind to left-handed Z-DNA and Z-RNA. The Zα domain protein family includes cellular (ADAR1, ZBP1 and PKZ) and viral (vaccinia virus E3 and cyprinid herpesvirus 3 (CyHV-3) ORF112) proteins. We studied CyHV-3 ORF112, which contains an intrinsically disordered region and a Zα domain. Genome editing of CyHV-3 indicated that the expression of only the Zα domain of ORF112 was sufficient for normal viral replication in cell culture and virulence in carp. In contrast, its deletion was lethal for the virus. These observations revealed the potential of the CyHV-3 model as a unique platform to compare the exchangeability of Zα domains expressed alone in living cells. Attempts to rescue the ORF112 deletion by a broad spectrum of cellular, viral, and artificial Zα domains showed that only those expressing Z-binding activity, the capacity to induce liquid-liquid phase separation (LLPS), and A-to-Z conversion, could rescue viral replication. For the first time, this study reports the ability of some Zα domains to induce LLPS and supports the biological relevance of dsRNA A-to-Z conversion mediated by Zα domains. This study expands the functional diversity of Zα domains and stimulates new hypotheses concerning the mechanisms of action of proteins containing Zα domains., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The frequent outbreak of grass carp hemorrhagic disease caused by grass carp reovirus (GCRV), especially the mainly prevalent type II GCRV (GCRV-II), has seriously affected the grass carp culture in China. However, its pathogenic mechanism is still far from clear. In this study, the GCRV-II outer capsid protein VP35 was used as bait to capture interacting partners from Ctenopharyngon idellus kidney (CIK) cells, and heat shock protein 90 (Hsp90) was selected and confirmed interacting with VP35 through the C-terminal domain of Hsp90. Knockdown of Hsp90 or inhibition of Hsp90 activity suppressed GCRV-II proliferation, demonstrating that Hsp90 is an essential factor for GCRV-II proliferation. The confocal microscopy and flow cytometry showed that Hsp90 localized at both membrane and cytoplasm of CIK cells. The entry of GCRV-II into CIK cells was efficiently blocked by incubating the cells with Hsp90 antibody or by pretreating the virus with recombinant Hsp90 protein. Whereas overexpression of Hsp90 in CIK cells, grass carp ovary (GCO) cells, or 293T cells promoted GCRV-II entry, indicating that the membrane Hsp90 functions as a receptor of GCRV-II. Furthermore, Hsp90 interacted with clathrin and mediated GCRV-II entry into CIK cells through clathrin endocytosis pathway. In addition, we found that the cytoplasmic Hsp90 acted as a chaperone of VP35 because inhibition of Hsp90 activity enhanced VP35 polyubiquitination and degraded VP35 through the proteasome pathway. Collectively, our data suggest that Hsp90 functions both as a receptor for GCRV-II entry and a chaperone for the maturation of GCRV-II VP35, thus ensuring efficient proliferation of GCRV-II. IMPORTANCE Identification of viral receptors has always been the research hot spot in virus research field as receptor functions at the first stage of viral infection, which can be designed as efficient antiviral drug targets. GCRV-II, the causative agent of the grass carp epidemic hemorrhagic disease, has caused tremendous losses in grass carp culture in China. To date, the receptor of GCRV-II remains unknown. This study focused on identifying cellular receptor interacting with the GCRV-II outer capsid protein VP35, studying the effects of their interaction on GCRV-II proliferation, and revealing the underlying mechanisms. We demonstrated that Hsp90 acts both as a receptor of GCRV-II by interacting with VP35 and as a chaperone for the maturation of VP35, thus ensuring efficient proliferation of GCRV-II. Our data provide important insights into the role of Hsp90 in GCRV-II life cycle, which will help understand the mechanism of reovirus infection.

This experiment was conducted to evaluate the immunomodulatory effect and antiviral activity of Astragalus polysaccharides (APS) in crucian carp and epithelioma papulosum cyprinid (EPC) cells. Two diets containing 0 and 2 g/kg, APS were fed crucian carp for 56 days. The results showed that supplementation with APS significantly upregulated the immune-related indices including the levels of IgM, the activities of LZM, AKP and ACP, and the contents of C3 and C4. At the same time, compared with the CK group, adding APS to the feed significantly upregulated the expression of IL-8, IL-10, IL-1β, IFN-α, IFN-γ, MyD88, TGF-β and TNF-α in the spleen, kidney, liver and intestine of crucian carp. In addition, when the crucian carp were injected with SVCV, the survival rates of fish in the APS group and the control group were 48.87% and 13.76%, respectively. These results indicated that dietary APS could improve the resistance of crucian carp against SVCV infection. APS also significantly decreased viral titer and inhibited apoptosis induced by SVCV in EPC cells. These results indicated that APS could stimulate the immune response of crucian carp and improve the abilities of crucian carp and EPC cells to resist SVCV infection., (Copyright © 2021. Published by Elsevier Inc.)

Grass carp reovirus (GCRV) is a highly virulent RNA virus that mainly infects grass carp and causes hemorrhagic disease. The roles of nonstructural proteins NS38 and NS80 of GCRV-873 in the viral replication cycle and viral inclusion bodies have been established. However, the strategies that NS38 and NS80 used to avoid host antiviral immune response are still unknown. In this study, we report the negative regulations of NS38 and NS80 on the RIG-I-like receptors (RLRs) antiviral signaling pathway and the production of IFNs and IFN-stimulated genes. First, both in the case of overexpression and GCRV infection, NS38 and NS80 inhibited the IFN promoter activation induced by RIG-I, MDA5, MAVS, TBK1, IRF3, and IRF7 and mRNA abundance of key antiviral genes involved in the RLR-mediated signaling. Second, both in the case of overexpression and GCRV infection, NS38 interacted with piscine TBK1 and IRF3, but not with piscine RIG-I, MDA5, MAVS, and TNF receptor-associated factor (TRAF) 3. Whereas NS80 interacted with piscine MAVS, TRAF3, and TBK1, but not with piscine RIG-I, MDA5, and IRF3. Finally, both in the case of overexpression and GCRV infection, NS38 inhibited the formation of the TBK1-IRF3 complex, but NS80 inhibited the formation of the TBK1-TRAF3 complex. Most importantly, NS38 and NS80 could hijack piscine TBK1 and IRF3 into the cytoplasmic viral inclusion bodies and inhibit the translocation of IRF3 into the nucleus. Collectively, all of these data demonstrate that GCRV nonstructural proteins can avoid host antiviral immune response by targeting the RLR signaling pathway, which prevents IFN-stimulated gene production and facilitates GCRV replication., (Copyright © 2022 by The American Association of Immunologists, Inc.)

N-ethyl-N-nitrosourea (ENU) selection is a useful technique to generate new mutations that may cause some functional changes in the gene. Through our previous genomic bulked segregant analysis (BSA), one single nucleotide polymorphism (SNP) at the 3' UTR of Toll interacting protein gene (TOLLIP 982T>C ) was identified in grass carp (Ctenopharyngodon idella) subjected to ENU-induced mutagenesis. We found that the overexpression of cid-miR-nov-1043 mimics significantly suppressed the luciferase activity of the TOLLIP 3' UTR, but TOLLIP 982T>C mutation at the target site can decrease the binding affinity between the miRNA cid-miR-nov-1043 and TOLLIP 3' UTR, reducing the inhibition of TOLLIP mRNA transcription in grass carp subjected to ENU-induced mutagenesis. More importantly, we demonstrated that TOLLIP mRNA transcription levels in the gills, liver, kidney and the isolate white cells of the mutant grass carp were significantly (p < 0.01) higher than those in the corresponding tissues from the wild-type grass carp following infection with Grass Carp Reovirus (GCRV) for seven days, while the downstream gene of TOLLIP transforming growth factor β-activated kinase 1 (TAK1) and TAK1-binding protein 1 (TAB1), were higher expressed in wild-type grass carp. As a negative regulator in the pro-inflammatory pathway of NF-κB, TOLLIP inhibits the excessive inflammation in ENU grass carp after GCRV infection. Consistent with the TOLLIP expression, histopathological results demonstrated more severe inflammation in wild-type grass carp, compared to the TOLLIP 982T>C mutant grass carp on the seventh day. Severe inflammation will lead to thoroughly infiltration of chloride and inflammatory cells in the gill filaments. This seriously hindered the exchange of oxygen, which ultimately disrupted blood circulation. Meanwhile, the survival rate of the mutant grass carp was significantly (p < 0.01) higher than that of the wild-type grass carp, indicating that the TOLLIP 982T>C mutants showed strong anti-viral abilities. Our results revealed that an SNP in the TOLLIP 3' UTR may contribute to the suppression of serve inflammation subjected to ENU-induced mutagenesis following GCRV infection, which may be helpful for future resistant breeding development of grass carp., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Gills of fish are involved in respiration, excretion and osmoregulation. Due to numerous interactions between these processes, branchial diseases have serious implications on fish health. Here, "koi sleepy disease" (KSD), caused by carp edema virus (CEV) infection was used to study physiological, immunological and metabolic consequences of a gill disease in fish. A metabolome analysis shows that the moderately hypoxic-tolerant carp can compensate the respiratory compromise related to this infection by various adaptations in their metabolism. Instead, the disease is accompanied by a massive disturbance of the osmotic balance with hyponatremia as low as 71.65 mmol L -1 , and an accumulation of ammonia in circulatory blood causing a hyperammonemia as high as 1123.24 µmol L -1 . At water conditions with increased ambient salt, the hydro-mineral balance and the ammonia excretion were restored. Importantly, both hyponatremia and hyperammonemia in KSD-affected carp can be linked to an immunosuppression leading to a four-fold drop in the number of white blood cells, and significant downregulation of cd4, tcr a2 and igm expression in gills, which can be evaded by increasing the ion concentration in water. This shows that the complex host-pathogen interactions within the gills can have immunosuppressive consequences, which have not previously been addressed in fish. Furthermore, it makes the CEV infection of carp a powerful model for studying interdependent pathological and immunological effects of a branchial disease in fish.

Microalgae are possible sources of antiviral substances, e.g. against cyprinid herpesvirus 3 (CyHV-3). Although this virus leads to high mortalities in aquacultures, there is no treatment available yet. Hence, ethanolic extracts produced with accelerated solvent extraction from six microalgal species (Arthrospira platensis, Chlamydomonas reinhardtii, Chlorella kessleri, Haematococcus pluvialis, Nostoc punctiforme and Scenedesmus obliquus) were examined in this study. An inhibition of the in vitro replication of CyHV-3 could be confirmed for all six species, with the greatest effect for the C. reinhardtii and H. pluvialis crude extracts. At still non-cytotoxic concentrations, viral DNA replication was reduced by over 3 orders of magnitude each compared to the untreated replication controls, while the virus titers were even below the limit of detection (reduction of 4 orders of magnitude). When pre-incubating both cells and virus with C. reinhardtii and H. pluvialis extracts before inoculation, the reduction of viral DNA was even stronger (> 4 orders of magnitude) and no infectious viral particles were detected. Thus, the results of this study indicate that microalgae and cyanobacteria are a promising source of natural bioactive substances against CyHV-3. However, further studies regarding the isolation and identification of the active components of the extracts are needed., (© 2021. The Author(s).)

Complement factor D (Df) is a serine protease well known for activating the alternative pathway (AP) in mammals by promoting the cleavage of complement component 3 (C3), thus becoming involved in innate defense. In teleost fish, however, the functional mechanisms of Df in the AP and against pathogen infection are far from clear. In the present study, we cloned and characterized the Df gene, Ci Df, from grass carp ( Ctenopharyngodon idella ) and analyzed its function in promoting C3 cleavage and expression changes after grass carp reovirus (GCRV) infection. The open reading frame of Ci Df was found to be 753 bp, encoding 250 amino acids with a molecular mass of 27.06 kDa. Ci Df harbors a conserved Tryp&#95;SPc domain, with three conserved residues representing the catalytic triad and three conserved binding sites in the substrate specificity pocket. Pairwise alignment showed that Ci Df shares the highest identity (96%) and similarity (98%) with Df from Anabarilius grahami . Phylogenetic analysis indicated that Ci Df and other fish Dfs formed a distinct evolutionary branch. Similar to most Dfs from other vertebrates, the Ci Df gene structure is characterized by four introns and five exons. The incubation of recombinant Ci Df protein with grass carp serum significantly increased the C3b content, demonstrating the conserved function of Ci Df in the AP in promoting C3 cleavage, similar to Dfs in mammals. Ci Df mRNA expression was widely detected in various tissues and levels were relatively higher in the liver, spleen, and intestine of grass carp. During GCRV infection over a 168-hour period, a high level of Ci Df mRNA expression in the liver, spleen, and intestine was maintained at 144 and 168 h, suggesting AP activity at the late stage of GCRV infection. Collectively, the above results reveal the conserved structure and function of Ci Df and its distinct expression patterns after GCRV infection, which provide a key basis for studying the roles of Df and AP during GCRV infection in the grass carp C. idella .

The crosstalk between the immune system and microbiota drives an amazingly complex mutualistic symbiosis. In mammals, the upper respiratory tract acts as a gateway for pathogen invasion, and the dynamic interaction between microbiota and mucosal immunity on its surface can effectively prevent disease development. However, the relationship between virus-mediated mucosal immune responses and microbes in lower vertebrates remains uncharacterized. In this study, we successfully constructed an infection model by intraperitoneally injecting common carp ( Cyprinus carpio ) with spring viremia of carp virus (SVCV). In addition to the detection of the SVCV in the nose and pharynx of common carp, we also identified obvious histopathological changes following viral infection. Moreover, numerous immune-related genes were significantly upregulated in the nose and pharynx at the peak of SVCV infection, after which the expression levels decreased to levels similar to those of the control group. Transcriptome sequencing results revealed that pathways associated with bacterial infection in the Toll-like receptor pathway and the Nod-like receptor pathway were activated in addition to the virus-related Rig-I-like receptor pathway after SVCV infection, suggesting that viral infection may be followed by opportunistic bacterial infection in these mucosal tissues. Using 16S rRNA gene sequencing, we further identified an upward trend in pathogenic bacteria on the mucosal surface of the nose and pharynx 4 days after SVCV infection, after which these tissues eventually reached new homeostasis. Taken together, our results suggest that the dynamic interaction between mucosal immunity and microbiota promotes the host to a new ecological state., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wu, Meng, Ding, Wu, Han, Zhai, Sun, Yu, Ji and Xu.)

Scavenger receptor class B type 2 (SR-B2) is a pattern recognition receptor involved in innate immunity in mammals; however, the immunological function of SR-Bs in fish remains unclear. In this study, the full-length cDNA sequences of SR-B2a and SR-B2b from grass carp (Ctenopharyngodon idellus) were cloned and designated as CiSR-B2a and CiSR-B2b. Multiple alignments and phylogenetic analyses deduced that CiSR-B2a and CiSR-B2b had the highest evolutionary conservation and were closely related to the zebrafish (Danio rerio) homologs, DrSR-B2a and DrSR-B2b, respectively. Both CiSR-B2a and CiSR-B2b were expressed in all the tested tissues, with the highest expression levels found in the hepatopancreas. In Ctenopharyngodon idellus kidney cells (CIK), CiSR-B2a and CiSR-B2b were mainly located in the cytoplasm, and a small amount located on the plasma membrane. After challenge with Grass Carp Reovirus (GCRV), the expression of CiSR-B2a and CiSR-B2b were significantly upregulated in the spleen (about 10.27 and 27.19 times higher than that at 0 day, p < 0.01). With CiSR-B2a or CiSR-B2b overexpressed in CIK, the relative copy number of GCRV in the cells was both significantly increased compared to that in the control group, indicating that CiSR-B2a and CiSR-B2b may be important proteins during the infection processes of GCRV., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Grass carp reovirus (GCRV), the most virulent aquareovirus, causes epidemic hemorrhagic disease and tremendous economic loss in freshwater aquaculture industry. VP56, a putative fibrin inlaying the outer surface of GCRV-II and GCRV-III, is involved in cell attachment. In the present study, we found that VP56 localizes at the early endosome, lysosome, and endoplasmic reticulum, recruits the cytoplasmic viral RNA sensor retinoic acid-inducible gene I (RIG-I) and binds to it. The interaction between VP56 and RIG-I was detected by endogenous coimmunoprecipitation (co-IP), glutathione S -transferase (GST) pulldown, and subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) and was then confirmed by traditional co-IPs and a novel far-red mNeptune-based bimolecular fluorescence complementation system. VP56 binds to the helicase domain of RIG-I. VP56 enhances K48-linked ubiquitination of RIG-I to degrade it by the proteasomal pathway. Thus, VP56 impedes the initial immune function of RIG-I by dual mechanisms (blockade and degradation) and attenuates signaling from RIG-I recognizing viral RNA, subsequently weakening downstream signaling transduction and interferon (IFN) responses. Accordingly, host antiviral effectors are reduced, and cytopathic effects are increased. These findings were corroborated by RNA sequencing (RNA-seq) and VP56 knockdown. Finally, we found that VP56 and the major outer capsid protein VP4 bind together in the cytosol to enhance the degradation of RIG-I and more efficiently facilitate viral replication. Collectively, the results indicated that VP56 allies VP4, recruits, blocks, and degrades RIG-I, thereby attenuating IFNs and antiviral effectors to facilitate viral evasion more effectively. This study reveals a virus attacking target and an escaping strategy from host antiviral immunity for GCRV and will help understand mechanisms of infection of reoviruses. IMPORTANCE Grass carp reovirus (GCRV) fibrin VP56 and major outer capsid protein VP4 inlay and locate on the outer surface of GCRV-II and GCRV-III, which causes tremendous loss in grass carp and black carp industries. Fibrin is involved in cell attachment and plays an important role in reovirus infection. The present study identified the interaction proteins of VP56 and found that VP56 and VP4 bind to the different domains of the viral RNA sensor retinoic acid-inducible gene I (RIG-I) in grass carp to block RIG-I sensing of viral RNA and induce RIG-I degradation by the proteasomal pathway to attenuate signaling transduction, thereby suppressing interferons (IFNs) and antiviral effectors, facilitating viral replication. VP56 and VP4 bind together in the cytosol to more efficiently facilitate viral evasion. This study reveals a virus attacking a target and an escaping strategy from host antiviral immunity for GCRV and will be helpful in understanding the mechanisms of infection of reoviruses.

Cyprinid herpesvirus 2 (CyHV-2), a member of the Alloherpesviridae family belonging to the genus Cyprinivirus , is a fatal contagious aquatic pathogen that affects goldfish ( Carassius auratus ) and crucian carp ( Carassius carassius ). Although crucian carp and goldfish belong to the genus Carassius , it is unclear whether they are susceptible to the same CyHV-2 isolate. In addition, the origin of the crucian carp-derived CyHV-2 virus isolate remains unclear. CyHV-2 SH01 was isolated during herpesviral hematopoietic necrosis disease (HVHN) outbreaks in crucian carp at a local fish farm near Shanghai. CyHV-2 SH01 was confirmed by PCR and Western blot analysis of kidney, spleen, muscle, and blood tissue from the diseased crucian carp. Moreover, histopathological and ultra-pathological analyses revealed pathological changes characteristic of CyHV-2 SH01 infection in the tissues of the diseased crucian carp. In the present study, goldfish and crucian carp were challenged with CyHV-2 SH01 to elucidate viral virulence. We found that CyHV-2 SH01 could cause rapid and fatal disease progression in goldfish and crucian carp 24 h post-injection at 28 °C. Experimental infection of goldfish by injection indicated that the average virus titer in the kidney of the goldfish was 10 3.47 to 10 3.59 copies/mg. In addition, tissues exhibited the most prominent histopathological changes (cellular wrinkling and shrinkage, cytoplasmic vacuolation, fusion of the gill lamellae, and hepatic congestion) in CyHV-2 SH01-infected goldfish and crucian carp. Thus, crucian carp and goldfish showed a high sensitivity, with typical symptoms, to HVHN disease caused by CyHV-2 SH01.

TUFM is a mitochondrial protein and serves as a regulator of antiviral signaling; nevertheless, the character of TUFM in teleosts remains unidentified. In this study, TUFM homologue of black carp (Mylopharyngodon piceus) has been characterized and its role in innate immunity has been explored. Black carp TUFM (bcTUFM) comprises 447 amino acids and shows the high similarity to human TUFM. bcTUFM was about 50 kDa in the Western blot assay and was determined as a cytosolic protein by immunofluorescent microscopy. Knockdown of bcTUFM by shRNA enhanced the antiviral ability of the host cells. The induction fold of interferon promoter transcription in the cells co-expressing bcTUFM and bcMAVS was much lower than that of the cells expressing bcMAVS alone. Our previous study has identified that bcNLRX1 interacted with bcMAVS and functioned as an inhibitor of bcMAVS. The interaction between bcTUFM and bcNLRX1, but not bcTUFM and bcMAVS, was detected through co-immunoprecipitation. The subsequent reporter assay and plaque assay demonstrated that the inhibition of bcMAVS-mediated interferon production and antiviral activity by bcNLRX1 was enhanced by co-expressed bcTUFM. Thus, our data suggests that bcTUFM cooperates with bcNLRX1 to inhibit bcMAVS-mediated antiviral signaling during host antiviral innate immune response against SVCV., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Mammalian Nemo-like kinase (NLK) plays important roles in multiple biological processes including immune response; however, the roles of teleost NLK remain largely unknown. In the present study, the NLK homolog (bcNLK) of black carp (Mylopharyngodon piceus) has been cloned and characterized. The coding region of bcNLK consists of 1427 nucleotides and encodes 476 amino acid, including two low complexity region (LCR) domains at the N-terminus and a serine/threonine protein kinase catalytic (S-TKc) domain in the middle region. The transcription of bcNLK are promoted after spring viremia of carp virus (SVCV) infection and poly (I:C) stimulation in host cells, but not post LPS treatment. bcNLK exhibits weak impact on the transcription of interferon (IFN) promoter in the reporter assay, however, black carp MAVS (bcMAVS)-mediated IFN promoter transcription is remarkably dampened by bcNLK. The interaction between bcNLK and bcMAVS is detected through the co-immunoprecipitation assay. Accordingly, the plaque assay results show that bcMAVS-mediated antiviral ability is impaired by bcNLK. Moreover, knockdown of bcNLK in host cells leads to the enhanced antiviral ability against SVCV. All these data support the conclusion that black carp NLK associates with MAVS and inhibited MAVS-mediated antiviral signaling., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Polyploidy and subsequent diploidization provide genomic opportunities for evolutionary innovations and adaptation. The researches on duplicated gene evolutionary fates in recurrent polyploids have seriously lagged behind that in paleopolyploids with diploidized genomes. Moreover, the antiviral mechanisms of Viperin remain largely unclear in fish. Here, we elaborate the distinct antiviral mechanisms of two viperin homeologs ( Cgviperin-A and Cgviperin-B ) in auto-allo-hexaploid gibel carp ( Carassius gibelio ). First, Cgviperin-A and Cgviperin-B showed differential and biased expression patterns in gibel carp adult tissues. Subsequently, using co-immunoprecipitation (Co-IP) screening analysis, both Cg Viperin-A and Cg Viperin-B were found to interact with crucian carp ( C. auratus ) herpesvirus ( Ca HV) open reading frame 46 right (ORF46R) protein, a negative herpesvirus regulator of host interferon (IFN) production, and to promote the proteasomal degradation of ORF46R via decreasing K63-linked ubiquitination. Additionally, Cg Viperin-B also mediated ORF46R degradation through autophagosome pathway, which was absent in Cg Viperin-A. Moreover, we found that the N-terminal α-helix domain was necessary for the localization of Cg Viperin-A and Cg Viperin-B at the endoplasmic reticulum (ER), and the C-terminal domain of Cg Viperin-A and Cg Viperin-B was indispensable for the interaction with degradation of ORF46R. Therefore, the current findings clarify the divergent antiviral mechanisms of the duplicated viperin homeologs in a recurrent polyploid fish, which will shed light on the evolution of teleost duplicated genes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mou, Li, Lu, Wang, Yu, Li, Tong, Zhang, Wang, Zhang, Wang, Zhou and Gui.)

CD63 is a member of the four-transmembrane-domain protein superfamily and is the first characterized tetraspanin protein. In the present study, we cloned the common carp (Cyprinus Carpio) CD63 (ccCD63) sequence and found that the ccCD63 ORF contained 711 bp and encoded a protein of 236 amino acids. Homology analysis revealed that the complete ccCD63 sequence had 84.08% amino acid similarity to CD63 of Sinocyclocheilus anshuiensis. Subcellular localization analysis revealed that ccCD63 was localized in the cytoplasm. Quantitative real-time PCR (qRT-PCR) analysis indicated that ccCD63 was expressed in the gill, intestine, liver, spleen, brain and kidney, with higher expression in spleen and brain tissues than in the other examined tissues. After koi herpesvirus (KHV) infection, these tissues exhibited various expression levels of ccCD63. The expression level was the lowest in the liver and highest in the brain; the expression level in the brain was 8.7-fold higher than that in the liver. Furthermore, knockdown of ccCD63 promoted KHV infection. Moreover, ccCD63 was correlated with the regulation of RIG-I/MAVS/TRAF3/TBK1/IRF3 and may be involved in the antiviral response through the RIG-I viral recognition signalling pathway in a TRAF3/TBK1-dependent manner. Taken together, our results suggested that ccCD63 upregulated the interaction of KHV with the host immune system and suppressed the dissemination of KHV., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

TRIF is an antiviral adaptor downstream of Toll-like receptors, the roles of teleost TRIF and their regulation remain largely unknown. In this study, a TRIF homologue (bcTRIF) of black carp (Mylopharyngodon piceus) has been cloned, and the transcription of bcTRIF in vivo and ex vivo increased in response to different stimuli. Overexpressed bcTRIF induced the transcription of interferon promoter in the EPC cells and enhanced protection of cells against infection of spring viremia of carp virus (SVCV). The previous study has identified that black carp TRAF2 (bcTRAF2) and TRAF6 (bcTRAF6) functioned positively in RIG-I/MAVS signaling. When co-expressed with bcTRAF2, bcTRIF-induced the transcription of interferon promoter in EPC cells was decreased, and the antiviral activity of bcTRIF was dampened accordingly. On the contrary, co-expressed bcTRAF6 enhanced both bcTRIF-mediated interferon promoter transcription and antiviral activity. The subsequent co-immunoprecipitation identified the interaction between bcTRAF2/6 and bcTRIF. Thus, bcTRIF-mediated antiviral signaling is up-regulated by bcTRAF6 and down-regulated by bcTRAF2., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Anti-disease breeding is becoming the most promising solution to cyprinid herpesvirus-3 (CyHV-3) infection, the major threat to common carp aquaculture. Virus challenging studies suggested that a breeding strain of common carp developed resistance to CyHV-3 infection. This study illustrates the immune mechanisms involved in both sensitivity and anti-virus ability for CyHV3 infection in fish. An integrative analysis of the protein-coding genes and long non-coding RNAs (lncRNAs) using transcriptomic data was performed. Tissues from the head kidney of common carp were extracted at days 0 (the healthy control) and 7 after CyHV-3 infection (the survivors) and used to analyze the transcriptome through both Illumina and PacBio sequencing. Following analysis of the GO terms and KEGG pathways involved, the immune-related terms and pathways were merged. To dig out details on the immune aspect, the DEGs were filtered using the current common carp immune gene library. Immune gene categories and their corresponding genes in different comparison groups were revealed. Also, the immunological Gene Ontology terms for lncRNA modulation were retained. The weighted gene co-expression network analysis was used to reveal the regulation of immune genes by lncRNA. The results demonstrated that the breeding carp strain develops a marked resistance to CyHV-3 infection through a specific innate immune mechanism. The featured biological processes were autophagy, phagocytosis, cytotoxicity, and virus blockage by lectins and MUC3. Moreover, the immune-suppressive signals, such as suppression of IL21R on STAT3, PI3K mediated inhibition of inflammation by dopamine upon infection, as well as the inhibition of NLRC3 on STING during a steady state. Possible susceptible factors for CyHV-3, such as ITGB1, TLR18, and CCL4, were also revealed from the non-breeding strain. The results of this study also suggested that Nramp and PAI regulated by LncRNA could facilitate virus infection and proliferation for infected cells respectively, while T cell leukemia homeobox 3 (TLX3), as well as galectin 3 function by lncRNA, may play a role in the resistance mechanism. Therefore, immune factors that are immunogenetically insensitive or susceptible to CyHV-3 infection have been revealed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jia, Wu, Jiang, Li, Sun, Shi, Li, Ge, Hu, Ye, Tang, Shan, Cheng, Xia and Shi.)

The Krüppel-like factors (KLFs) are a family of transcription factors containing three highly conserved tandem zinc finger structures, and each member participates in multiple physiological and pathological processes. The publication of genome sequences and the application of bioinformatics tools have led to the discovery of numerous gene families in fishes. Here, 24 klf genes were re-annotated in grass carp. Subsequently, the number of klf family members were investigated in some representative vertebrate species. Then, a series of bioinformatics analysis showed that grass carp klfs in the same subfamily had similar genome structure patterns and conserved distribution patterns of motifs, which supported their molecular evolutionary relationships. Furthermore, the mRNA expression profiles showed that 24 grass carp klfs were ubiquitously expressed in 11 different tissues, and some of them displayed tissue-enriched expression patterns. Finally, the expressions of the evolutionarily expanded klf members (klf2a, 2b, 2l, 5a, 5b, 5l, 6a, 6b, 7a, 7b, 11a, 11b, 12a, 12b, 15 and 15l) during GCRV infection were also analyzed. The results suggested that grass carp klf genes with common evolutionary sources may share functional diversity and conservation. In conclusion, this study provides preliminary clues for further researches on grass carp klf members and their underlying transcriptional regulatory mechanisms during GCRV infection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Mortality in wild fish populations represents a challenging issue for public fish health inspectors. When a single fish species is involved, an infective aetiology is frequently suspected, with focus on viral notifiable diseases. However, other viral agents not subjected to regulation and causing mortality in common carp have been reported such as carp edema virus (CEV). In mid-June 2020, a severe common carp mortality was observed in an artificial lake in north-east of Italy. Sleepy fish were noted some days before the beginning of the mortality itself, which lasted several days and involved over 340 adult specimens. During the outbreak, water temperature was around 15°C, water quality was normal, and no adverse meteorological events were reported in the area. Four specimens, which showed severe cutaneous hyperaemia and increased mucus production on skin and gills, were tested by bacteriological methods and virological analysis targeting the main carp pathogens. Molecular analysis performed on gills, kidney and brains from all the fish analysed resulted positive for CEV, which, based on anamnestic information and laboratory findings, was considered the responsible for the mortality event herein described., (© 2021 John Wiley & Sons Ltd.)

Ovarian tumor domain-containing 6B ( OTUD6B ) belongs to the OTU deubiquitylating enzyme family. In this study, we report that zebrafish otud6b is induced upon viral infection, and overexpression of otud6b suppresses cellular antiviral response. Disruption of otud6b in zebrafish increases the survival rate upon spring viremia of carp virus and grass carp reovirus exposure. Further assays indicate that otud6b interacts with irf3 and irf7 and diminishes traf6-mediated K63-linked polyubiquitination of irf3 and irf7. In addition, the OTU domain is required for otud6b to repress IFN-1 activation and K63-linked polyubiquitination of irf3 and irf7. Moreover, otud6b also attenuates tbk1 to bind to irf3 and irf7, resulting in the impairment of irf3 and irf7 phosphorylation. This study provides, to our knowledge, novel insights into otud6b function and sheds new lights on the regulation of irf3 and irf7 by deubiquitination in IFN-1 signaling., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Grass carp ( Ctenopharyngodon idellus ) is an important aquaculture species in China that is affected by serious diseases, especially hemorrhagic disease caused by grass carp reovirus (GCRV). Grass carp have previously shown age-dependent susceptibility to GCRV, however, the mechanism by which this occurs remains poorly understood. Therefore, we performed transcriptome and metabolome sequencing on five-month-old (FMO) and three-year-old (TYO) grass carp to identify the potential mechanism. Viral challenge experiments showed that FMO fish were susceptible, whereas TYO fish were resistant to GCRV. RNA-seq showed that the genes involved in immune response, antigen presentation, and phagocytosis were significantly upregulated in TYO fish before the GCRV infection and at the early stage of infection. Metabolome sequencing showed that most metabolites were upregulated in TYO fish and downregulated in FMO fish after virus infection. Intragroup analysis showed that arachidonic acid metabolism was the most significantly upregulated pathway in TYO fish, whereas choline metabolism in cancer and glycerophospholispid metabolism were significantly downregulated in FMO fish after virus infection. Intergroup comparison revealed that metabolites from carbohydrate, amino acid, glycerophospholipid, and nucleotide metabolism were upregulated in TYO fish when compared with FMO fish. Moreover, the significantly differentially expressed metabolites showed antiviral effects both in vivo and in vitro . Based on these results, we concluded that the immune system and host biosynthesis and metabolism, can explain the age-dependent viral susceptibility in grass carp., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 He, Zhu, Liang, Li, Liao, Yang, Huang, Zhu and Wang.)

Spring viremia of carp virus (SVCV) causes a systemic hemorrhagic disease that poses a significant risk to wild and cultured fish and is listed as notifiable by the World Organization for Animal Health. Validated molecular diagnostic tools for SVCV are required to accurately describe and analyze the ecology of the virus. Here, the diagnostic specificity (DSp) and sensitivity (DSe) (i.e. accuracy) of three SVCV diagnostic tests - 2 reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays Q1G and Q2N and virus isolation by cell culture (VI) - were evaluated using 2-class latent class models run in maximum likelihood (ML) and Bayesian frameworks. Virus-free or experimentally-infected koi were sorted into three populations with low, moderate or high prevalence levels of SVCV (n = 269 fish in total). Koi kidney tissues were tested using Q2N and Q1G and for the VI assay, pools of kidney, spleen and gill tissues were used. All samples were blinded and analyzed in one laboratory. The ML and Bayesian approaches successfully estimated the diagnostic accuracy of the 3 tests with the exception of 1 ML model. The estimates were consistent across the two frameworks. The DSe estimates were higher for Q1G (>98 %) and Q2N (>96 %) compared to VI (>60 %). The DSp of all three tests varied by 12-15 % (79-91 % for Q1G, 79-94 % for Q2N and 81-97 % for VI) across same-fish samples revealing the potential range in test performance for one sample. The 3 fish populations had distinct SVCV prevalence levels estimated at 0-3 % (low), 70-73 % (moderate) and 95-96 % (high). The Bayesian covariance models revealed minor DSe dependence between Q1G and Q2N. The results suggested that SVCV diagnostic tests Q2N and Q1G are suitable for use as diagnostic assays and are fit for presumptive diagnosis, surveillance, and certification of populations or individuals as SVCV free., (Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)

The hemorrhagic disease of grass carp (HDGC) caused by grass carp reovirus (GCRV) still poses a great threat to the grass carp industry. Isolation and identification of the GCRV genotype I (GCRV-I) has been rarely reported in the past decade. In this study, a new GCRV was isolated from diseased fish with severe symptoms of enteritis and mild hemorrhages on the body surface. The isolate was further identified by cell culture, transmission electron, indirect immunofluorescence, and SDS-PAGE electrophoretic pattern analysis of genomic RNA. The results were consistent with the new isolate as a GCRV-I member and tentatively named GCRV-GZ1208. Both grass carp and rare minnow infected by the GCRV-GZ1208 have no obvious hemorrhagic symptoms, and the final mortality rate was ≤10%, indicating that it may be a low virulent isolate. GZ1208 possessed highest genomic homology to 873/GCHV (GCRV-I) and golden shiner reovirus (GSRV). Additionally, it was found a 90.7-98.3% nucleotide identity, a 96.4-100% amino acid identity, and <50% identity with GCRV-II and III genotypes. Interestingly, the sequences of some segments of GZ1208 were similar to GCRV-8733/GCHV, whereas the remaining segments were more closely related to GSRV, suggesting that a recombination event had occurred. Bootscan analysis of the complete genomic sequence confirmed this hypothesis, and recombination events between 873/GCHV and other GSRV-like viruses were also accompanied by gene mutations.

A new grass carp reovirus (GCRV), healthy grass carp reovirus (HGCRV), was isolated from grass carp in 2019. Its complete genome sequence was determined and contained 11 dsRNAs with a total size of 23,688 bp and 57.2 mol% G+C content, encoding 12 proteins. All segments had conserved 5' and 3' termini. Sequence comparisons showed that HGCRV was closely related to GCRV-873 (GCRV-I; 69.57-96.71% protein sequence identity) but shared only 22.65-45.85% and 23.37-43.39% identities with GCRV-HZ08 and Hubei grass carp disease reovirus (HGDRV), respectively. RNA-dependent RNA-polymerase (RdRp) protein-based phylogenetic analysis showed that HGCRV clustered with Aquareovirus -C ( AqRV -C) prior to joining a branch common with other aquareoviruses. Further analysis using VP6 amino acid sequences from Chinese GCRV strains showed that HGCRV was in the same evolutionary cluster as GCRV-I. Thus, HGCRV could be a new GCRV isolate of GCRV-I but is distantly related to other known GCRVs. Grass carp infected with HGCRV did not exhibit signs of hemorrhage. Interestingly, the isolate induced a typical cytopathic effect in fish cell lines, such as infected cell shrank, apoptosis, and plague-like syncytia. Further analysis showed that HGCRV could proliferate in grass carp liver (L28824), gibel carp brain (GiCB), and other fish cell lines, reaching a titer of up to 7.5 × 10 4 copies/μL.

Mre11A is considered as a cytosolic DNA receptor in mammals. However, it is rarely known about Mre11A in other vertebrates. Recently, a mammalian ortholog of Mre11A has been identified in grass carp (Ctenopharyngodon idellus) in our lab. Phylogenetic-tree analysis provided evidence for a close genetic relationship between C.idellus Mre11A and Carassius auratus Mre11A. The tissue expression profile of CiMre11A was detected, with a relatively higher level of expression in kidney, intestines, liver and spleen than that in other tissues after grass carp reovirus (GCRV) infection. Similarly, CiMre11A was also up-regulated in CIK cells after treatment with GCRV. Q-PCR and dual-luciferase assays indicated that the transcription levels of IFN1 and ISG15 were inhibited by CiMre11A knockdown, but were gradually augmented after CIK cells were transfected with increasing amounts of CiMre11A. Subcellular localization assays showed that a part of CiMre11A was translocated from the nucleus to the cytoplasm. Co-immunoprecipitation and co-localization assays demonstrated that CiMre11A interacts with CiSTING in response to GCRV infection. In CIK cells, the expressions of both IFN1 and ISG15 were acutely up-regulated by CiMre11A overexpression, as well as by co-overexpression of CiMre11A and CiSTING. CiMre11A and CiSTING induced the phosphorylation and cytoplasmic-to-nuclear translocation of IRF7 in CIK cells. The multiplication of GCRV in CIK cells was inhibited by the overexpression of CiMre11A and CiSTING., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cholesterol is essential for building and maintaining cell membranes and is critical for several steps in the replication cycle of viruses, especially for enveloped viruses. In mammalian cells virus infections lead to the accumulation of the oxysterol 25-hydroxycholesterol (25HC), an antiviral factor, which is produced from cholesterol by the cholesterol 25 hydroxylase (CH25H). Antiviral responses based on CH25H are not well studied in fish. Therefore, in the present study putative genes encoding for CH25H were identified and amplified in common carp and rainbow trout cells and an HPLC-MS method was applied for determination of oxysterol concentrations in these cells under virus infection. Our results give some evidence that the activation of CH25H could be a part of the antiviral response against a broad spectrum of viruses infecting fish, in both common carp and rainbow trout cells in vitro . Quantification of oxysterols showed that fibroblastic cells are capable of producing 25HC and its metabolite 7α,25diHC. The oxysterol 25HC showed an antiviral activity by blocking the entry of cyprinid herpesvirus 3 (CyHV-3) into KFC cells, but not spring viremia of carp virus (SVCV) or common carp paramyxovirus (Para) in the same cells, or viral haemorrhagic septicaemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV) into RTG-2 cells. Despite the fact that the CH25H based antiviral response coincides with type I IFN responses, the stimulation of salmonid cells with recombinant type I IFN proteins from rainbow trout could not induce ch25h&#95;b gene expression. This provided further evidence, that the CH25H-response is not type I IFN dependent. Interestingly, the susceptibility of CyHV-3 to 25HC is counteracted by a downregulation of the expression of the ch25h&#95;b gene in carp fibroblasts during CyHV-3 infection. This shows a unique interplay between oxysterol based immune responses and immunomodulatory abilities of certain viruses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Adamek, Davies, Beck, Jordan, Becker, Mojzesz, Rakus, Rumiac, Collet, Brogden, Way, Bergmann, Zou and Steinhagen.)

Cyprinid herpesvirus 2 (CyHV-2), which infects silver crucian carp including goldfish (Carassius auratus auratus) and Crucian carp (Carassius auratus gibelio) with high mortality, is an emerging viral pathogen worldwide. Previous studies showed that berberine (BBR), a bioactive plant-derived alkaloid, demonstrated potential antiviral actions against many different viruses. Here, we assessed the effect of berberine hydrochloride (BBH) on the replication of CyHV-2 in vitro and in vivo. Cytotoxicity assay indicated that 5-25 μg/mL BBH was non-toxic to the RyuF-2 cells. In viral inhibition assays, real time PCR was employed to titrate the genomic copy number of progeny virus, real time RT-PCR was applied to monitor the transcriptional levels of viral genes, and Western blot analysis was performed to detect the synthetic levels of viral proteins. The results demonstrated that BBH systematically impedes the viral gene transcription and suppressed the replication of CyHV-2 in RyuF-2 cells. In animal challenge test, BBH was confirmed to protect Crucian carps from CyHV-2 infection in a dose-dependent manner, which was supported by suppressed viral replication levels, reduced viral pathogenesis and higher survival rates. Furthermore, pharmacokinetics data of BBH in Crucian carp revealed its rapid absorption (T max of 1.5 h), suitable plasma half-life (t 1/2 z/h of 7-12 h depending on oral dosage), and dose-dependent drug exposure properties following oral administration (revealed by AUC 0-t values). These findings shed light on repurposing BBH to treat CyHV-2 infections in silver crucian carp., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Protein phosphatase-1 (PP1) has an important role in many cell functions, such as cell differentiation, development, immune response and tumorigenesis. However, the specific role of PP1 in the antiviral response in fish remains to be elucidated. In this study, the PPP1R3G homolog was identified in the grass carp ( Ctenopharyngodon idella ) and its role in defence against the GCRV infection was investigated. Phylogenetic analysis demonstrated that CiPPP1R3G clustered with homologues from other teleosts. Temporal expression analysis in vivo revealed that the expression level of CiPPP1R3G was significantly up-regulated in response to GCRV infection in grass carps, especially in the intestine and head-kidney. Cellular distribution analysis revealed that CiPPP1R3G was located in the nucleus and cytoplasm. Overexpression of CiPPP1R3G significantly negatively regulated the expression of CiIRF3, thus inhibiting its activation. In summary, we systematically analyzed the PPP1R3G gene in grass carp and illustrated its function as a negative regulator in the anti-GCRV immune responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hu, Wang, Feng, Zhou, Lin and Cao.)

Cyprinid herpesvirus-3 (CyHV-3, syn. koi herpesvirus) is an important pathogen worldwide and a common cause of mass mortality events of wild common carp (Cyprinus carpio) in North America, however, reference strains and genomes obtained from wild carp are not available. Additionally, it is unclear if fishes in North America are susceptible to CyHV-3 infection due to incomplete susceptibility testing. Here we present the first North American type strain and whole-genome sequence of CyHV-3 isolated from wild carp collected from a lake with a history and recent incidence of carp mortality. Additionally, the strain was used in an in-vivo infection model to test the susceptibility of a common native minnow (Pimephales promelas) and goldfish (Carrasius auratus) which is invasive in North America. Detection of CyHV-3 DNA was confirmed in the tissues of a single fathead minnow but the same tissues were negative for CyHV-3 mRNA and samples from exposed fathead minnows were negative on cell culture. There was no detection of CyHV-3 DNA or mRNA in goldfish throughout the experiment. CyHV-3 DNA in carp tissues was reproducibly accompanied by the detection of CyHV-3 mRNA and isolation on cell culture. Additionally, environmental CyHV-3 DNA was detected on all tank filters during the study. These findings suggest that fathead minnows and goldfish are not susceptible to CyHV-3 infection and that detection of CyHV-3 DNA alone in host susceptibility trials should be interpreted with caution.

Phenylpropanoids, common natural compounds, possess many different biological activities such as antioxidant, anti-inflammatory and antiviral. Spring viraemia of carp virus (SVCV) can cause a high mortality in common carp (Cyprinus carpio). However, there are currently no licenced drugs that effectively cure this disease. In this study, we designed and synthesized a phenylpropanoid derivative 4-(4-methoxyphenyl)-3,4-dihydro-2H-chromeno[4,3-d]pyrimidine-2,5(1 H)-dione (E2), and explored the antiviral effect against SVCV in vitro and in vivo. Up to 25 mg/L of E2 significantly inhibited the expression levels of SVCV protein genes in the epithelioma papulosum cyprini (EPC) cell line by a maximum inhibitory rate of >90%. As expected, E2 remarkably declined the apoptotic of SVCV-infected cells and suppressed potential enhancement of the mitochondrial membrane potential (ΔΨm), these data implied that E2 could protect mitochondria from structural damage in response to SVCV. Meanwhile, E2 was added to EPC cells under four different conditions: time-of-addition, time-of-removal, pre-treatment of viruses and pre-treatment of cells indicated that E2 may block the post-entry transport process of the virus. Additionally, the up-regulation of six interferon (IFN)-related genes also demonstrated that E2 indirectly activated IFNs for the clearance of SVCV in common carp. Drug cure effect showed that treatment with E2 at 0.5 d post infection (dpi) is more effective than at 0, 1 or 2 dpi. Most importantly, intraperitoneal therapy of E2 markedly improved common carp survival rate and reduced virus copies in body. Therefore, the E2 has potential to be developed into a novel anti-SVCV agent., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Grass carp reovirus (GCRV) causes devastating viral haemorrhagic disease in farmed grass carp (Ctenopharyngon idellus). As novel molecular probes, aptamers have been widely applied in rapid diagnosis and efficient therapies against virus or diseases. In this study, three single-stranded DNA (ssDNA) aptamers were selected against GCRV-infected CIK cells via SELEX (systematic evolution of ligands by exponential enrichment technology). Secondary structures predicted by MFOLD indicated that aptamers formed stem-loop structures, and GVI-11 had the lowest ΔG value of -30.84 KJ/mol. Three aptamers could specifically recognize GCRV-infected CIK cells, with calculated dissociation constants (Kd) of 220.86, 176.63 and 278.66 nM for aptamers GVI-1, GVI-7 and GVI-11, respectively, which indicated that they could serve as specific delivery system for antiviral therapies. The targets of aptamers GVI-1, GVI-7 and GVI-11 on the surface of GCRV-infected cells could be membrane proteins, which were trypsin-sensitive. Furthermore, FAM-labelled aptamer GVI-7 could be applied to detect GCRV infection in vivo. It is the first time to generate and characterize aptamers against GCRV-infected cells. These aptamers have great potentials in development of rapid diagnosis technology and antiviral agents against GCRV infection in aquaculture., (© 2020 John Wiley & Sons Ltd.)

Selenium, as an essential trace element, interferes through selenoproteins in many physiological processes of plants and mammals. Its antiviral activity has recently attracted much attention because selenium improves the antiviral capacity of animal cells against a few viruses relevant to human diseases. In this study, the red elemental selenium was purified from the fermentative culture of Herbaspirillum camelliae WT00C and then used to culture epithelioma papulosum cyprinid (EPC) cells or feed crucian carp and zebrafish. Finally, its antiviral effects were investigated at the cell level and living fishes after spring viraemia of carp virus infection. At the cell level, 5, 10 and 20 μg ml -1 red elemental selenium significantly induced the expression of interferon (IFN) and ISG15 genes in EPC cells. The viral TCID 50 (50% tissue culture infective dose) values in the EPC cells incubated with 5, 10 and 20 μg ml -1 red elemental selenium were significantly less than those of the control. More expression of IFN and ISG15 genes and less TCID 50 values indicate that red elemental selenium indeed improves the antiviral capability of EPC cells. In the crucian carp fed with the food containing 5 and 10 μg g -1 red elemental selenium, IFN expressions showed 13- and 39-fold increases at the 16th day of post-injection, and its expression was dependent on selenium concentrations. Meanwhile, no fish death occurred in all the experimental groups. In the zebrafish fed with the red worm containing 5 μg g -1 red elemental selenium, IFN and Mx expressions and survival rate were significantly higher than those of the control. The results of this study show that red elemental selenium indeed improves the antiviral activity of fish. The antiviral effects of selenium mainly come from its immune regulation through its incorporation into selenoproteins. The optimum level of selenium contributes to improving fish immunity, whereas excess selenium causes excessive immune and inflammatory responses., (© 2020 Fisheries Society of the British Isles.)

Gut microbiota could facilitate host to defense diseases, but fish-microbiota interactions during viral infection and the underlying mechanism are poorly understood. We examined interactions and responses of gut microbiota to grass carp reovirus (GCRV) infection in Ctenopharyngodon idellus, which is the most important aquaculture fish worldwide. We found that GCRV infection group with serious haemorrhagic symptoms (G7s) showed considerably different gut microbiota, especially with an abnormally high abundance of gram-negative anaerobic Cetobacterium somerae. It also showed the lowest (p < 0.05) alpha-diversity but with much higher ecological process of homogenizing dispersal (28.8%), confirming a dysbiosis of the gut microbiota after viral infection. Interestingly, signaling pathways of NOD-like receptors (NLRs), toll-like receptors (TLRs), and lipopolysaccharide (LPS) stimulation genes were significantly (q-value < 0.01) enriched in G7s, which also significantly (p < 0.01) correlated with the core gut microbial genera of Cetobacterium and Acinetobacter. The results suggested that an expansion of C. somerae initiated by GCRV could aggravate host inflammatory reactions through the LPS-related NLRs and TLRs pathways. This study advances our understanding of the interplay between fish immunity and gut microbiota challenged by viruses; it also sheds new insights for ecological defense of fish diseases with the help of gut microbiota., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)