CyHV-2 infection triggers mitochondrial-mediated apoptosis in GiCF cells by upregulating the pro-apoptotic gene ccBAX.

Apoptosis is a physiological cell death phenomenon, representing one of the fundamental physiological mechanisms for maintaining homeostasis in living organisms. Previous studies have observed typical apoptotic features in Carassius auratus gibelio caudal fin cell (GiCF) infected with Cyprinid herpesvirus 2 (CyHV-2), and found a significant up-regulation of ccBAX expression in these infected cells. However, the specific apoptotic mechanism involved remains unclear. In this study, we utilized the GiCF cell line to investigate the apoptotic mechanism during CyHV-2 infection. Immunofluorescence staining revealed translocation of ccBAX into mitochondria upon CyHV-2 infection. Flow cytometry analysis demonstrated that overexpression of ccBAX expedited virus-induced apoptosis, characterized by heightened mitochondrial depolarization, increased transcriptional levels of Cytochrome c (Cyto c) in both the cytoplasm and mitochondria, and augmented Caspase 3/7 enzyme activity. Bax inhibitor peptide V5 (BIP-V5), an inhibitor interfering with the function of Bax proteins, inhibited Bax-mediated apoptotic events through the mitochondrial pathway and attenuated apoptosis induced by CyHV-2. In this study, it was identified for the first time that CyHV-2 induces apoptosis via the mitochondrial pathway in GiCF cells, bridging an important gap in our understanding regarding cell death mechanisms induced by herpesvirus infections in fish species. These findings provide a theoretical basis for comprehending viral apoptotic regulation mechanisms and the prevention and control of cellular pathologies caused by CyHV-2 infection.

Mass mortality events associated with cyprinid herpesvirus 2 (CyHV-2) infection in wild Prussian carp Carassius gibelio in the Netherlands, and molecular biology of virus strains.

In 2011 and 2015, four mass mortalities of Prussian carp (Carassius gibelio) were observed in a recreational freshwater lake and open freshwater in the western part of the Netherlands. Cyprinid herpesvirus 2 (CyHV-2) infection was suspected in these cases, based on presumptive gross diagnosis. To elucidate the cause of the mass mortalities diagnostic PCR assays were performed for CyHV-2, based on the helicase gene. Furthermore, the viral isolates were genotyped by sequencing the enlarged marker A and marker B sequences. Diagnostic PCR revealed that three of four samples were positive for CyHV-2, indicating these three mass mortalities were associated with CyHV-2 infection. The marker A sequence from one of the isolates found in this study was identical to those from different locations such as Asia and Middle East, suggesting a link among the isolates. This is the first detailed report on mass mortalities of Prussian carp associated with CyHV-2 infection in natural aquatic environments in the Netherlands. Since 2015, additionally, in total three CyHV-2 associated outbreaks of Dutch Prussian carp were seen in 2016 and 2020. These outbreaks in Prussian carp from lakes and open water suggest that the virus has been spreading in natural freshwaters in the Netherlands.

Immersion immunization with recombinant Saccharomyces cerevisiae displaying ORF25 induced protective immunity against cyprinid herpesvirus 2.

Displaying antigens on yeast surface as an oral vaccine has been widely explored, while its potential as an immersion vaccine has not been evaluated. Here, an immersion vaccine was prepared by displaying ORF25 of Cyprinid herpesvirus 2 (CyHV-2) on the surface of Saccharomyces cerevisiae. Carassius auratus gibelio was immersion immunized by 2 × 107 CFU/mL yeast for 2 h, and reinforce the immunity using the same method 14 days after the first immunization. The results showed that ORF25 specific antibody in immunized crucian carp serum was detected at a high level, and the mRNA expression level of IgM, IgT, IL-1ß, and IFN-1 in vaccinated head-kidney and spleen tissues were higher than the control group, indicating that innate and adaptive immunity were induced. Moreover, the immersion vaccination provided effective protection for fish against CyHV-2, leading to a relative percent survival of 50.2%. Meanwhile, immersion vaccination restrained virus replication and histological damage in CyHV-2 infected crucian carp. Our data suggested that immersion immunization of S. cerevisiae-displayed ORF25 could be served as a candidate vaccine to prevent CyHV-2 infection.

Evaluation of protective effects of heat-inactivated cyprinid herpesvirus-2 (CyHV-2) vaccine against herpesviral hematopoietic necrosis disease (HVHND) in goldfish (Carassius auratus).

Cyprinid herpesvirus-2 (CyHV-2) is an important virus that causes herpesviral hematopoietic necrosis disease (HVHND) leading to huge economic losses in goldfish (Carassius auratus). However, until now no proper prophylactic measure or treatment is available for CyHV-2 infection in goldfish. Hence, in this experiment, we developed a heat-inactivated CyHV-2 vaccine and evaluated its performance in goldfish. Initially, CyHV-2 was propagated in the fantail goldfish fin (FtGF) cell line and the titer of the viral inoculum was 107.8 TCID50/ml. Subsequently, various temperatures (40 °C, 50 °C, 60 °C, 70 °C, and 80 °C) were evaluated to achieve the complete inactivation of CyHV-2. Only the viral inoculum inactivated at 80 °C for 1 h did not show any cytopathic effect in the FtGF cell line after five blind passages. Hence the heat-inactivated CyHV-2 vaccine developed at 80 °C was further used for immunization trials in goldfish. The experimental goldfish were intraperitoneally immunized with 300 µL of the heat-inactivated CyHV-2 vaccine. Subsequently, the kidney and spleen tissues were sampled at various time points post-vaccination (6th hr, 2nd day, 4th day, 6th day, 10th day, 16th day, and 30th day) to evaluate the expression of immune genes (IL-12, IL-10, IFN-γ, CD8, and CD4). A significant upregulation of immune genes was observed at various time points in the kidney and spleen tissue of the vaccinated goldfish. Furthermore, in order to study the efficacy of the vaccine, the experimental fish were challenged with CyHV-2 (107.8 TCID50/ml) after the 30th day post-vaccination. The survival of the fish in the vaccine group (86.7%) was significantly higher compared to the non-vaccinated group (20%). Moreover, the relative percentage survival of the vaccinated group was 83.34%. In spite of the single dose, the heat-killed vaccine developed in the present study elicited the immune response and offered better protection in goldfish against CyHV-2. However, further large-scale field performance evaluation studies are necessary to develop this vaccine on a commercial scale.

An attenuated strain of cyprinid herpesvirus 2 as a vaccine candidate against herpesviral hematopoietic necrosis disease in gibel carp, Carassius auratus gibelio.

Herpesviral hematopoietic necrosis disease causes by cyprinid herpesvirus 2 (CyHV-2) infection is a high mortality disease that leads to great economic damage to gibel carp, Carassius auratus gibelio aquaculture. In this study, an attenuated strain of CyHV-2 G-RP7 was achieved by subculture on RyuF-2 cells derived from the fin of Ryukin-variety goldfish and GiCF cells derived from fin of gibel carp. As the attenuated vaccine candidate, there are no clinical symptoms of gibel carp that immersion or intraperitoneal injection with G-RP7 strain. The protection rates of G-PR7 to gibel carp by immersion and intraperitoneal injection were 92% and 100%, respectively. In the test for virulence reversion, the candidate was propagated through gibel carp six times by intraperitoneal injection with kidney and spleen homogenate of the inoculated fish. During in vivo passages in gibel carp, no abnormality and mortality of the inoculated fish were observed, and the virus DNA copies maintain a low level from the first passage to the sixth passage. The dynamic of virus DNA in each tissue of G-RP7 vaccination fish increased within 1, 3, and 5 days post-immunization, and subsequently decreased and stabilized within 7 and 14 days. In addition, the increase of anti-virus antibody titer was detected both immersion and injection immunization fish 21 days after vaccination by ELISA. These results demonstrated that G-RP7 can be a promising live attenuated vaccine candidate against the disease.

Characteristics and expression profiles of MHC class â molecules in Carassius auratus.

Major histocompatibility complex class Ⅰ (MHC Ⅰ) molecules play a vital role in adaptive immune systems in vertebrates by presenting antigens to effector T cells. Understanding the expression profiling of MHC Ⅰ molecules in fish is essential for improving our knowledge of the relationship between microbial infection and adaptive immunity. In this study, we conducted a comprehensive analysis of MHC Ⅰ gene characteristics in Carassius auratus, an important freshwater aquaculture fish in China that is susceptible to Cyprinid herpesvirus 2 (CyHV-2) infection. We identified approximately 20 MHC Ⅰ genes discussed, including U, Z, and L lineage genes. However, only U and Z lineage proteins were identified in the kidney of Carassius auratus using high pH reversed-phase chromatography and mass spectrometry. The L lineage proteins were either not expressed or present at an extremely low level in the kidneys of Carassius auratus. We also used targeted proteomics to analyze changes in protein MHC Ⅰ molecules abundance in healthy and CyHV-2-infected Carassius auratus. We observed that five MHC Ⅰ molecules were upregulated, and Caau-UFA was downregulated in the diseased group. This study is the first to reveal the expression of MHC Ⅰ molecules at a large scale in Cyprinids, which enhances our understanding of fish adaptive immune systems.

Proteomic and Phosphoproteomic Analyses Reveal Gibel Carp Responses to Cyprinid Herpesvirus 2 Infection.

Cyprinid herpesvirus 2 (CyHV-2) is a typical linear double-stranded DNA virus, which can induce severe herpesviral hematopoietic necrosis disease (HVHND) in gibel carp. However, the CyHV-2 infection mechanisms still remain unresolved till now. Here, we combined the isobaric tag for relative absolute quantitation (iTRAQ)-labeled quantitative proteomic and phosphoproteomic analysis enriched by Ti4+-immobilized titanium ion affinity chromatography (IMAC) to uncover the host responses to CyHV-2 infection in the kidneys of symptomatic and diseased gibel carp. We totally identified 192 differential expression proteins and 951 high-confident phosphopeptides involved in 657 proteins. After being infected with CyHV-2, the proteins involved in energy generation and ion balance were significantly downregulated in the host, and the phosphorylated proteins induced by viral infection mainly participated in the regulation for RNA processing, translation, cytoskeleton organization, immunoreaction, etc. Furthermore, 11 phosphorylated CyHV-2 viral proteins were found in the diseased group by the host proteome. The virus-host protein-protein interactions were investigated, in which the potential host kinases casein kinase II (CK-II) and cyclin-dependent kinase (CDK) that interacted with viral ORF88 or ORF89 were identified and can serve as candidate targets for disease treatment in the future. Overall, our study provides a comprehensive understanding of CyHV-2-induced perturbations at the protein and phosphorylation levels in gibel carp, forming a base for the treatment of HVHND.

Immune gene expression and protective effects in goldfish (Carassius auratus L.) immunized with formalin-inactivated cyprinid herpesvirus-2 (CyHV-2) vaccine.

The goldfish hematopoietic necrosis viral disease (GHNVD) has led to worldwide economic losses in goldfish aquaculture. The present study has focused on the development of an inactivated vaccine for the cyprinid herpesvirus (CyHV-2) and to check the immunogenicity of the vaccine in the host. The fantail goldfish fin (FtGF) cell line was used in the propagation of the CyHV-2 and the viral titer obtained were of 107.8 TCID50/ml. Followed by the virus was inactivated using 0.1% formalin for 2 days. Various concentrations of formalin-inactivated CyHV-2 (1%, 0.7%, 0.5%, 0.3% and 0.1%) were studied in the FtGF cell line. Morphological changes were observed in the FtGF cell line in all other concentrations of formalin except 0.1% formalin-inactivated CyHV-2 vaccine. The goldfishes were intraperitoneally injected with 300 µl of vaccine and various immune gene responses were studied for a period of 30 days. The gene expression of the adaptive markers CD8, CD4, IFN-ϒ, the cytokines (IL-10, IL-12) was studied in kidney and spleen tissues. Formalin-inactivated CyHV-2 vaccine showed a significant up-regulation of the genes CD8 and IFN-ϒ by the 6th hr post-vaccination onwards. The experimental fish were challenged intraperitoneally with CyHV-2 virus of concentration 107.8 TCID50/ml after 30 days of post-vaccination. A significant difference in cumulative mortality rate was observed for the vaccinated fishes from the unvaccinated fishes. The relative percent survival for formalin immunized fish was 74.03%. Our results have proven that the formalin-inactivated vaccines were efficient and it resulted in triggering the immune gene expression in goldfish. The development and further enhanced studies for this vaccine will lead to a promising low-cost commercial vaccine for CyHV-2 viral infection.

Development and characterization of monoclonal antibodies specific for cyprinid herpesvirus 2.

Infections of Cyprinid herpesvirus 2 in goldfish and farmed crucian carp (Carassius auratus gibelio) are still an urgent problem worldwide. Detection and prevention are necessary for the control of haematopoietic necrosis disease caused by CyHV-2. Although many sensitive molecular diagnostic methods have been developed, effective immunodiagnosis and neutralization approaches based on monoclonal antibodies (MAbs) against CyHV-2 are still important to CyHV-2 study. In this experiment, purified CyHV-2 was used as antigens to produce monoclonal antibodies (Mabs). Six Mabs bound to different proteins were selected by Dot-blot screening and Western-blot analysis, and no one had cross-reactivity with closely related koi herpesvirus. Among them, Mabs 2E1-B10, 1F5-A3 and 4C4-A7 belonged to IgG1 isotype, while other three Mabs 3G9-B11, 3B4-G5 and 4F4-B7 belonged to IgM isotype. These six Mabs all could specifically detect CyHV-2 in CyHV-2 infected caudal fin of Carassius auratus gibelio (GiCF) cells by immunofluorescence assays. Then, the neutralization ability was tested in vitro, and the result showed that all six Mabs can attenuate CPE by CyHV-2 in vitro among which 2E1-B10 had the best neutralization ability. The virus proteins recognized by these six Mabs were identified by mass spectrometry identification, and the result showed they probably were ORF88, ORF55R, ORF115 and ORF151R. This study is the first to prepare Mabs by purifying CyHV-2, which will provide a practical basis for the in-depth study of CyHV-2 virus.

Expression and regulation of ccBAX by miR-124 in the caudal fin cell of C. auratus gibelio upon cyprinid herpesvirus 2 infection.

Bcl2 family proteins play a critical role in cell death or survival. BAX, the death-promoting protein of bcl2 family, mediated mitochondrial pathway inducing cells' apoptosis in mammal. MiRNAs have been implicated as negative regulators down-regulating genes' expression after post-transcriptional level. At present, little is known about the regulatory mechanism of miRNA on the Bcl2 family proteins during CyHV-2 infection in silver crucian carp (Carassius auratus gibelio). In this study, the ccBAX (silver crucian carp BAX) gene was cloned and expressed, and polyclonal antibodies were raised in mouse against the purified ccBAX-GST fusion protein. The structure analysis indicated that ccBAX protein included four conserve domains (BH1, BH2, BH3 and transmembrane domains) and the expression of ccBAX protein occurred throughout the cells. Furthermore, two miRNAs (miR-124 and miRNA-29b) were identified to negatively regulate ccBAX gene expression in GiCF cell. miR-124 was found to suppress the expression of WT-ccBAX (wild type), but not the MT-ccBAX (mutant). Overall, the results demonstrated that the expression of the ccBAX gene was significantly down-regulated by miR-124 in silver crucian carp (Carassius auratus gibelio) during CyHV-2 infection.

Detection of cyprinid herpesvirus 2 by loop-mediated isothermal amplification in combination with a lateral flow dipstick.

We developed a convenient technique to detect Herpesviral haematopoietic necrosis attributed to cyprinid herpes virus 2 (CyHV-2), a serious disease of Crucian carp and goldfish related to high mortality. In the present study, we employed a lateral flow dipstick (LAMP-LFD) to present a loop-mediated isothermal amplification assay. The specificity was ascertained via other six viruses, and the sensitivity was compared using PCR method, which are the reaction conditions changes for the method improved. The results revealed that CyHV-2 performance was observable at 64 °C in a separated tube within 60 min, when the samples hybridized using an FITC-labeled probe. As the LAMP-LFD method's specificity was high, with its sensitivity identical to that of traditional PCR, the overall DNA collected revealed the lowest detection limit of 0.18 pg/µl from goldfish diseased by CyHV-2. In summary, the development of LAMP-LFD's method does not require expensive instruments, and it can be regarded as a fast, simple, and reliable method for CyHV-2 detection.

Transcriptomic and proteomic analyses reveal new insights into the regulation of immune pathways during cyprinid herpesvirus 2 infection in vitro.

Carassius auratus gibelio is susceptible to the herpesviral hematopoietic necrosis (HVHN) disease caused by cyprinid herpesvirus 2 (CyHV-2) infection during the breeding process. Nevertheless, the report on biological response of CyHV-2 with C. auratus gibelio was limited, especially in vitro. In this study, host gene expression profiling was mostly analyzed in caudal fin cells of Carassius auratus gibelio (GiCF) underlying CyHV-2 infection. Transcriptomics and proteomics were employed to study the differential expression gene and revealed the host genes involved in pathway during the CyHV-2 infection. Transcriptome analysis revealed that compared with the control group, there were 11 335 and 19 421 differentially expressed unigenes at 48 h and at 96 h, respectively. Furthermore, proteome analysis showed that there were a total of 9008 proteins, among which 169 proteins were differential expression in the 48 h group and 502 proteins in the 96 h group. Notably, 10 and 158 differentially co-expressed genes at mRNA and protein levels (cDEGs) were reliably quantified at 48 h and 96 h, respectively. Interestingly, significantly different expressed genes both in the transcriptome and the proteome were identified, including GNG7, Hsp90a, THBS1 and RRM2. The result suggested that PI3k-AKT pathway was activated, but the p53 signaling pathway was suppressed. The above result will lay the foundation for understanding the mechanisms of host defense virus invasion during CyHV-2 infection.

Reactivation of cyprinid herpesvirus 2 (CyHV-2) in asymptomatic surviving goldfish Carassius auratus (L.) under immunosuppression.

Cyprinid herpesvirus 2 (CyHV-2) is a highly contagious pathogen of goldfish (Carassius auratus) and Prussian carp (Carassius auratus gibelio) causing herpesviral hematopoietic necrosis. Our previous study revealed that CyHV-2 can persistently infect the kidney and spleen of goldfish that recovered from a primary infection. In this study, we tried to identify the cells persistently infected with the virus in surviving fish and investigated virus reactivation in the survivors injected with immunosuppressants, namely dexamethasone (Dex) and cyclosporine A (CsA). Virus DNA was detected from the monocytes that were isolated from the trunk kidney of the asymptomatic survivors, suggesting that monocytes/macrophages are major cells that may be persistently infected with CyHV-2. A significant increase of virus DNA levels was detected in the group injected with Dex at 10 and 21 days post-injection (dpi). In the fish group injected with CsA, the virus DNA level was the same as that in the control group at 10 dpi but increased in some organs at 21 dpi. Compared with Dex-injected fish at 10 dpi, the group injected with both Dex and CsA showed a greater increase in virus DNA levels. The gene expression of phagocytosis-associated genes, major histocompatibility complex (MHC) class II and p47phox, and anti-virus antibody levels increased in the CsA group due to virus reactivation in the infected cells but not in the Dex and Dex & CsA groups, indicating that Dex effectively suppressed monocyte/macrophage function and antibody production. In addition, recombinant interferon γ (IFNγ) supplementation in the kidney leukocyte culture that was isolated from survivors showed a reduction of virus DNA. CsA may inhibit T-helper 1 (Th1) cells and consequently IFNγ production, causing a synergetic effect with Dex on virus reactivation. The results suggest that the activity of monocytes/macrophages stimulated by IFNγ can relate to virus latency and reactivation in asymptomatic virus carriers.

Poly-ß-hydroxybutyrate (PHB) in bioflocs alters intestinal microbial community structure, immune-related gene expression and early Cyprinid herpesvirus 2 replication in gibel carp (Carassius auratus gibelio).

The aquaculture system based on biofloc technology (BFT) showed positive effects on prevention of Cyprinid herpesvirus 2 (CyHV-2) infection in gibel carp (Carassius auratus gibelio), which is detrimental to health and causes seriously economic losses to aquaculture. However, the enhancement mechanism of BFT regarding immunity and disease resistance of cultured species is scarce. Poly-ß-hydroxybutyrate (PHB) has been proved as one of bioactive compounds in bioflocs. In this study, two groups (4% PHB supplementation diets and control with basal diets) with 30-day feeding were set to study the effect of PHB supplementation on immune-related gene expression by qRT-PCR, time-course CyHV-2 replication in vivo by qPCR and intestinal microbiota by illumine high-throughput sequencing. PHB supplementation significantly up-regulated transcriptional levels of eight immune-related genes, decreased cumulative mortality of gibel carp and early CyHV-2 replication in spleen in vivo (P < 0.05). Additionally, PHB changed the microbial structure but not diversity, and significantly increased beneficial bacteria such as Bacillus sp. KEGG pathway analysis by PICRUSt demonstrated that oral administration of PHB up-regulated abundances of genes responsible for seven pathways and down-regulated genes in eleven pathways. Histological structures of foregut, mindgut and hindgut were also affected. Our findings suggested that profitable effects of PHB on immunity and disease resistance might be gut microbiota-related, and regulated through pathways of enzymes secretion, replication and repair, and host immune system. This study will provide new insights into understanding the enhancing mechanism of BFT on immunity and disease resistance of cultured animals, and developing prebiotics/probiotics-based immunotherapies to improve animal health and disease resistance.

Transcriptome analysis provides insights into the antiviral response in the spleen of gibel carp (Carassius auratus gibelio) after poly I: C treatment.

Gibel carp (Carassius auratus gibelio) is an important commercial fish that has become one of the most cultured fishes in the region of Yangtze River in China. However, the fish faces increasing hazard due to cyprinid herpesvirus 2 (CyHV-2) infection, which has caused great economic losses. In this study, healthy gibel carp were intraperitoneally injected with different doses of poly I:C at 24 h before CyHV-2 challenge. Results showed that the mortality decreased and peak death time appeared later in the fish injected with poly I:C at a dose of 10 µg/g body weight. To explore what gene plays an important role after poly I:C treatment, the transcriptome analysis of the gibel carp spleen was further performed. Compared with the PBS group, 1286 differentially expressed genes (DEGs) were obtained in the poly I:C-treated fish, including 1006 up-regulated and 280 down-regulated DEGs. GO analysis revealed that the most enriched DEGs responded to "biological regulation", "regulation of cellular process" and "regulation of biological process". Meanwhile, KEGG enrichment analysis showed that the DEGs were mainly mapped on the immune pathways like "TNF signal pathway", "p53 signal pathway" and "JAK-STAT signal pathway", suggesting that these signal pathways may be responsible for the delayed peak of CyHV-2 infection in gibel carp after poly I:C treatment. Taken together, this study provides insights into the immune protection effect of poly I:C against CyHV-2 infection, as well as providing useful information for antiviral defense in gibel carp.

ß-glucan and anisodamine can enhance the immersion immune efficacy of inactivated cyprinid herpesvirus 2 vaccine in Carassius auratus gibelio.

As one of the most important fish in freshwater aquaculture, gibel carp (Carassius auratus gibelio) is easily susceptible to Cyprinid herpesvirus 2 (CyHV-2). Immersion vaccination has attracted many researchers due to its simple operation in preventing infectious diseases. However, the unavoidable disadvantage is that the immersion vaccine must be used with adjuvants to get a better performance. In this study, gibel carps were vaccinated by a 60 min bath in a ß-propiolactone-inactivated Cyprinid herpesvirus 2, mixed with DTT, ß-glucan, anisodamine and scopolamine, respectively. After immunization, the fishs were challenged by CyHV-2 in 2 weeks. By analyzing pathological section, we found that ß-glucan, anisodamine and scopolamine groups protected the gibel carp compared to the control group, which was consistent with the trend of survival rate. Specifically, ß-glucan group in serum appeared best on lysozyme, TSOD and complement C3. Real time quantitative RT-PCR results demonstrated that in both spleen and head kidney tissues, mRNA expressions of typical Th1 immune response cytokines IL-2 and IFN-γ2 in ß-glucan group and anisodamine group were significantly higher than other groups and the level of immunoglobulins related to systemic immunity (IgM) and mucosal immunity (IgZ) were also enhanced in the immune period. DTT group slightly affected immune gene and serum enzyme activity, while did not show an adjuvant effect on survival rate. In addition, four adjuvant groups could obviously inhibit CyHV-2 replication. This study explored and proved the good efficiency of ß-glucan or anisodamine as immersion immune adjuvant and also provided reference for improving the efficiency of immersion immunity.

Identification, expression profiles and antiviral activities of a type I IFN from gibel carp Carassius auratus gibelio.

Type I interferons, as a class of multipotent cytokines, play a key role in host antiviral immune responses. In this study, a type I IFN coding gene of gibel carp, Carassius auratus gibelio, CagIFNa was cloned and sequenced. The full-length cDNA sequence of CagIFNa consists of 724 nucleotides that encode a predicted protein of 183 amino acids. CagIFNa has two highly conserved cysteine residues in the deduced protein, which is mostly conserved in the fish group I type I IFNs. CagIFNa was identified as a member of the IFNa subgroup of group I type I IFNs by phylogenetic analysis. CagIFNa transcripts were detected in all investigated tissues with higher levels in the liver, intestine, spleen and head kidney of gibel carp. Following injection with Cyprinid herpesvirus 2 (CyHV-2), CagIFNa gene expression was significantly inhibited in the spleen but delayed and then increased in head kidneys. Similarly, while CagIFNa expression was rapidly induced in gibel carp brain (GiCB) cells by poly I:C stimulation and its high induction level was delayed following CyHV-2 infection. CagIFNa overexpression in GiCB cells drastically reduced virus CPE and titer. Furthermore, several genes associated with type I IFN signaling pathway including IRF3, IRF7, IRF9, STAT1, Mx1 and PKR were induced in GiCB cells overexpressing CagIFNa upon CyHV-2 infection. These results show that CagIFNa plays a role in antiviral immune system in gibel carp.

Biofloc technology (BFT): An alternative aquaculture system for prevention of Cyprinid herpesvirus 2 infection in gibel carp (Carassius auratus gibelio).

Gibel carp (Carassius auratus gibelio), a major aquaculture species in China, has emerged in a seriously epizootic disease caused by Cyprinid herpesvirus 2 (CyHV-2). There are no effective methods to prevent or control this serious disease. Biofloc technology (BFT) can improve water quality, reduce pathogens introduction, enhance cultured species immunity and disease resistance. In this study, a 30-day experiment was conducted to investigate the effect of BFT on innate immune response and disease resistance of gibel carp against CyHV-2 infection. Gibel carp was cultured at different total suspended solid (TSS) concentrations of 10, 300, 600, 800 and 1000 mg L-1, which were named as groups BF0, BF300, BF600, BF800 and BF1000. Results showed that fish in groups BF600/800 had significantly higher weight gain (WG) and specific growth rate (SGR) than them in control group (BF0). The transcriptional levels of seven immune-related genes in BF300/600/800 groups, including myeloid-specific- peroxidase (MPO), keratin 8 (KRT 8), dual specificity phosphatase 1 (DUSP 1), interleukin-11 (IL-11), intelectin (ITLN), purine nucleoside phosphorylase 5α (PNP 5α) and c-type lysozyme (c-lys), were up-regulated significantly compared to BF0 group. Furthermore, cumulative mortality of gibel carp in BF600 group after being challenged with CyHV-2 reduced significantly. In vivo viral replication in kidney demonstrated that CyHV-2 load at 168 h post injection in BF600 group was significantly higher than that in BF0 group. In conclusion, BFT could improve growth, immune response and disease resistance of gibel carp, and the effect was related with TSS concentration. The optimal TSS concentration of 600-800 mg L-1 was recommended in the present study.

Identification, structural characterization, and expression analysis of toll-like receptors 2 and 3 from gibel carp (Carassius auratus gibelio).

Toll-like receptors (TLRs) are important components of innate immunity. TLRs recognize pathogen-associated molecular patterns (PAMPs) and initiate downstream signaling pathways in response. In present study, we report the identification of two TLRs from gibel carp (Carassius auratus gibelio), TLR2 and TLR3 (designated CagTLR2 and CagTLR3, respectively). We report on the genomic structures and mRNA expression patterns of CagTLR2 and CagTLR3. Five exons and four introns were identified from the genomic DNA sequence of CagTLR3 (4749 bp in total length); this genomic organization is similar to that of TLR3 in zebrafish and human. However, only one intron was identified from the CagTLR2 genomic locus (3166 bp in total length); this unique genomic organization of CagTLR2 is different from that of TLR2 in fish and humans. The cDNAs of CagTLR2 and CagTLR3 encoded 791 and 904 amino acid residues, respectively. CagTLR2 and CagTLR3 contained two distinct structural/functional motifs of the TLR family: a leucine-rich repeat (LRR) domain in the extracellular portion and a toll/interleukin-1 receptor (TIR) domain in the intracellular portion. The positions of critical amino acid residues involed in PAMP recognition and signaling pathway transduction in mammalian TLRs were conserved in CagTLR2 and CagTLR3. Phylogenetic analysis revealed a closer clustering of CagTLR2 and CagTLR3 with TLRs from freshwater fish than with marine fish species. In healthy gibel carp, transcripts of these genes were detected in all examined tissues, and high expression levels of CagTLR2 and CagTLR3 were observed in liver and brain, respectively. Following injection with CyHV-2, expression levels of CagTLR2 and CagTLR3 were significantly upregulated in the spleens of gibel carp after three days, and CagTLR3 transcript levels were rapidly increased in head kidney after 12 h. These results suggest that CagTLR2 and CagTLR3 are functionally involved in the induction of antiviral immune response.

Rapid visual detection of cyprinid herpesvirus 2 by recombinase polymerase amplification combined with a lateral flow dipstick.

Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus 2 (CyHV-2), causes significant losses in crucian carp (Carassius carassius) aquaculture. Rapid and convenient DNA assay detection of CyHV-2 is useful for field diagnosis. Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that can amplify DNA within 30 min at ~37°C by simulating in vivo DNA recombination. Herein, a rapid and convenient detection assay based on RPA with a lateral flow dipstick (LFD) was developed for detecting CyHV-2. The highly conserved ORF72 of CyHV-2 was targeted by specific and sensitive primers and probes. The optimized assay takes only 15 min at 38°C using a water bath, with analysis of products by 2% agarose gel electrophoresis within 30 min. A simple lateral flow strip based on the unique probe in reaction buffer was developed for visualization. The entire RPA-LFD assay takes 50 min less than the routine PCR method, is 100 times more sensitive and displays no cross-reaction with other aquatic viruses. The combined isothermal RPA and lateral flow assay (RPA-LFD) provides a simple, rapid, reliable method that could improve field diagnosis of CyHV-2 when resources are limited.