Tripartite motif 2b (trim2b) restricts spring viremia of carp virus by degrading viral proteins and negative regulators NLRP12-like receptors.

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.

A fish herpesvirus highlights functional diversities among Zα domains related to phase separation induction and A-to-Z conversion.

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.

Grass Carp Reovirus Nonstructural Proteins Avoid Host Antiviral Immune Response by Targeting the RLR Signaling Pathway.

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.

Potential to grow carp oedema virus (genogroup I) in monolayers of carp-derived primary cells with further implication in cell analysis.

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.

Development of real-time recombinase polymerase amplification (RPA) and RPA combined with lateral flow dipstick (LFD) assays for the rapid and sensitive detection of cyprinid herpesvirus 3.

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.

Comparative Analysis of mRNA, microRNA of Transcriptome, and Proteomics on CIK Cells Responses to GCRV and Aeromonas hydrophila.

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.

Poly (I:C)-Induced microRNA-30b-5p Negatively Regulates the JAK/STAT Signaling Pathway to Mediate the Antiviral Immune Response in Silver Carp (Hypophthalmichthys molitrix) via Targeting CRFB5.

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.

Hsp90 Regulates GCRV-II Proliferation by Interacting with VP35 as Its Receptor and Chaperone.

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.

Zebrafish otud6b Negatively Regulates Antiviral Responses by Suppressing K63-Linked Ubiquitination of irf3 and irf7.

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.

An SNP at the target site of cid-miR-nov-1043 in the TOLLIP 3' UTR decreases mortality rate in grass carp subjected to ENU-induced mutagenesis following grass carp reovirus infection.

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 (TOLLIP982T>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 TOLLIP982T>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 ß-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 TOLLIP982T>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 TOLLIP982T>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.

Zebrafish NIK Mediates IFN Induction by Regulating Activation of IRF3 and NF-κB.

Type I IFN mediates the innate immune system to provide defense against viral infections. NF-κB-inducing kinase (NIK) potentiates the basal activation of endogenous STING, which facilitates the recruitment of TBK1 with the ectopically expressed IRF3 to induce IFN production. Moreover, NIK phosphorylates IKKα and confers its ability to phosphorylate p100 (also known as NF-κB2) in mammals. Our study demonstrated that NIK plays a critical role in IFN production in teleost fish. It was found that NIK interacts with IKKα in the cytoplasm and that IKKα phosphorylates the NIK at the residue Thr432, which is different from the mammals. Overexpression of NIK caused the activation of IRF3 and NF-κB, which in turn led to the production of IFN and IFN-stimulated genes (ISGs). Furthermore, the ectopic expression of NIK was observed to be associated with a reduced replication of the fish virus, whereas silencing of endogenous NIK had an opposite effect in vitro. Furthermore, NIK knockdown significantly reduced the expression of IFN and key ISGs in zebrafish larvae after spring viremia of carp virus infection. Additionally, the replication of spring viremia of carp virus was enhanced in NIK knockdown zebrafish larvae, leading to a lower survival rate. In summary, our findings revealed a previously undescribed function of NIK in activating IFN and ISGs as a host antiviral response. These findings may facilitate the establishment of antiviral therapy to combat fish viruses.

Host-microbiota interactions and responses to grass carp reovirus infection in Ctenopharyngodon idellus.

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.

Generation and characterization of aptamers against grass carp reovirus infection for the development of rapid detection assay.

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.

Carp edema virus-related mortality in wild adult common carp (cyprinus carpio) in Italy.

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.

Functional Identification of Complement Factor D and Analysis of Its Expression during GCRV Infection in Grass Carp (Ctenopharyngodon idella).

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, CiDf, 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 CiDf was found to be 753 bp, encoding 250 amino acids with a molecular mass of 27.06 kDa. CiDf harbors a conserved Tryp_SPc domain, with three conserved residues representing the catalytic triad and three conserved binding sites in the substrate specificity pocket. Pairwise alignment showed that CiDf shares the highest identity (96%) and similarity (98%) with Df from Anabarilius grahami. Phylogenetic analysis indicated that CiDf and other fish Dfs formed a distinct evolutionary branch. Similar to most Dfs from other vertebrates, the CiDf gene structure is characterized by four introns and five exons. The incubation of recombinant CiDf protein with grass carp serum significantly increased the C3b content, demonstrating the conserved function of CiDf in the AP in promoting C3 cleavage, similar to Dfs in mammals. CiDf 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 CiDf 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 CiDf 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.

Red elemental selenium (Se0 ) improves the immunoactivities of EPC cells, crucian carp and zebrafish against spring viraemia of carp virus.

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 TCID50 (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 TCID50 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.

Identification of coronavirus sequences in carp cDNA from Wuhan, China.

Severe acute respiratory syndrome (SARS)-like coronavirus sequences were identified in two separate complementary DNA (cDNA) pools. The first pool was from a Carassius auratus (crusian carp) cell line and the second was from Ctenopharyngodon idella (grass carp) head kidney tissue. BLAST analysis suggests that these sequences belong to SARS-like coronaviruses, and that they are not evolutionarily conserved in other species. Investigation of the submitting laboratories revealed that two laboratories from the Institute of Hydrobiology at the Chinese Academy of Sciences in Wuhan, China performed the research and submitted the cDNA libraries to GenBank. This institution is very close in proximity to the Wuhan South China Seafood Wholesale Market where SARS-CoV-2 first amplified in the human population. It is possible that these sequences are an artifact of the bioinformatics pipeline that was used. It is also possible that SARS-like coronaviruses are a common environmental pathogen in the region that may be in aquatic habitats.

Comparison of the blood parameters and histopathology between grass carp infected with a virulent and avirulent isolates of genotype II grass carp reovirus.

Grass carp hemorrhagic disease caused by grass carp reovirus (GCRV) is the most important disease for grass carp aquaculture. Its typical clinical symptom is haemorrhaging, although the mechanism was remained unclear. In this study, we investigated the differences in blood parameters and histopathological features between grass carp infected with a virulent and avirulent isolates of genotype II GCRV. Infection with the virulent isolate resulted in increases in 8 routine blood and 2 serum biochemical parameters (P < 0.05); while 9 routine blood and 5 biochemical parameters were significantly decreased (P < 0.05) compared with fish infected with the avirulent isolate. The majority of these alterations were related to hemorrhage, inflammatory reactions and organic damage. The histopathologic changes were primarily vasodilation and hyperaemia in multiple organs, lymphocyte and macrophage infiltration as well as severe vacuolar degeneration in spleen, kidney and liver. The histopathology changes in fish infected with the avirulent isolate were minimal. These results indicated that the pathogenicity of GCRV was primarily reflected in destruction of the blood circulatory system and parenchymatous organs. This study lays the foundation for further research on the pathogenesis of bleeding caused by GCRV infection and the use of blood parameters and histopathology as tools for disease diagnosis.

Ginsenoside Rg3 inhibits grass carp reovirus replication in grass carp ovarian epithelial cells.

Ginseng exhibits multiple medicinal properties, including the improvement of immune function and enhancing disease resistance. In this study, we investigated the inhibitory effects of ginsenoside Rg3 on grass carp reovirus (GCRV) infection of grass carp ovarian (CO) epithelial cells, in order to provide a baseline framework for future high-efficacy antiviral drug screening investigations. Ginsenoside Rg3 was added to GCRV-infected CO cells, and cells were cultured at 27 °C before cell proliferation was measured by MTT assays. Label-free real-time cellular analysis (RTCA) after 72 h of experimentation demonstrated that 100 µg/mL ginsenoside Rg3 treatment had the highest inhibitory effect on GCRV (among 1,10,100 µg/mL treatments). We then measured the capacity for cellular antioxidant ability. Cells treated with 1,10,100 µg/mL ginsenoside Rg3 exhibited increases in Total Antioxidant Capacity activity relative to controls, respectively. Furthermore, Antioxidant assay and reverse transcript quantitative polymerase chain reaction (RT-qPCR) showed that ginsenoside Rg3 were efficient to restrain the replication of GCRV in CO cells. Expression analysis of immune-related genes via RT-qPCR showed that treatment with ginsenoside Rg3 promoted expression of IRF-3 and IRF-7 increases, respectively. Moreover, expression of IFN-1 was induced, which then inhibition the expression of tumor necrosis factor-alpha (TNF-α). In conclusion, we demonstrated that ginsenoside Rg3 promotes CO cell proliferation, inhibits GCRV activity, promotes CO cell immune activities, and thereby enhances the resistance of CO to GCRV infection.

Development of a real-time reverse transcription recombinase polymerase amplification assay for rapid detection of spring viremia of carp virus.

Spring viremia of carp virus (SVCV) is a significant pathogenic agent that can cause large-scale outbreaks of spring viremia of carp (SVC) in many types of fish and bring huge economic losses to the aquaculture industry. A simple and convenient detection method is imperative for SVCV diagnosis. In this study, the real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed and validated. Primers and probe targeting the conserved region of M gene were designed and applied to the real-time RT-RPA assay that performed at 39 °C for 20 min. The specificity analysis showed that no cross-reaction with other pathogenic viruses of fish was found, indicating appropriate specificity of the assay. In vitro transcribed RNA standards were used to estimate the sensitivity of the assay and the detection limit was 102copies/reaction. To further evaluate the assay, 65 clinical samples were tested using both real-time RT-RPA assay and real-time RT-PCR method. The same detection results were observed, suggesting the potential application of real-time RT-RPA assay in clinical sample detection. This is the first report on RPA assay for SVCV detection and this new developed assay would be useful in both laboratory and in the field for diagnosis of SVCV.