Cyprinid Herpesvirus Research Articles

Akt Inhibitors Prevent CyHV-2 Infection In Vitro

Cyprinid herpesvirus 2 (CyHV-2) is a double-stranded DNA virus that infects goldfish (Carassius auratus) and crucian carp (C. carassius), resulting in substantial mortality rates and significant epidemiological implications. To gain deeper insights into CyHV-2-host interactions and identify potential therapeutic targets, quantitative proteomics analysis was conducted on CyHV-2-infected Ryukin goldfish fin (RyuF-2) cells. Our findings revealed significant alterations in the expression of proteins associated with the PI3K/Akt signaling pathway, which were up-regulated upon viral infection. Building on these observations, we employed LY294002, a specific inhibitor of PI3K, to investigate its impact on viral replication by inhibiting the PI3K/Akt pathway in GiCF cell line derived from the caudal fin of Carassius auratus gibelio (Bloch). Our results demonstrated the inhibition of both CyHV-2 replication and Akt phosphorylation within this pathway. Quercetin, a plant-derived analogue of LY294002, was further investigated for its anti-CyHV-2 effects in vitro as well as its underlying mechanism. The results suggested that quercetin exhibits antiviral properties against CyHV-2 and may exert its effects through mechanisms similar to those observed with LY294002. Given that aquaculture water serves as a vector for aquaculture viral diseases and the release of chemical compounds can lead to pollution of the aquatic environment, our study shifted focus to crude extracts obtained from plants. We confirmed crude quercetin extract derived from Cuminum anisum has antiviral activity against CyHV-2 in vitro. Therefore, based on our identification of the involvement of PI3K/Akt signaling pathway in CyHV-2 replication, along with verification of its antiviral mechanism using LY294002, we propose natural dietary quercetin as a promising candidate for development into a novel anti-CyHV-2 drug.

Cloning and Identification of Common Carp (Cyprinus carpio) PI3KC3 and Its Expression in Response to CyHV-3 Infection.

Phosphoinositide 3-kinases (PI3Ks) are a class of key regulatory factors in eukaryotes that can inhibit viral replication by influencing autophagy. Currently, cyprinid herpesvirus 3 (CyHV-3) poses a serious threat to common carp culture. However, PI3K has not yet been identified in common carp. In this study, full-length PI3KC3 from common carp (CcPI3KC3), consisting of an open reading frame (ORF) of 2664 bp encoding a polypeptide of 887 amino acids, with a predicted molecular mass of 101.19 kDa and a theoretical isoelectric point (pI) of 5.97, was cloned. The amino acid and nucleotide sequences of CcPI3KC3 displayed high similarity to yellow catfish's (Tachysurus fulvidraco) PI3KC3. The tissue expression profile revealed that the mRNA levels of CcPI3KC3 in the liver, spleen, and head kidney were significantly greater than those in the brain, heart, intestines, gills, eyes, testes, and ovaries of common carp. We compared the expression patterns of CcPI3KC3 between "Longke-11" mirror carp (CyHV-3-resistant carp) and German mirror carp (non-resistant to CyHV-3) at different times (0, 48, 96, 144 h, 192, 240, 288 h post-infection (hpi)) after CyHV-3 infection. The results revealed that CcPI3KC3 mRNA expression significantly increased in the early infection stage. In the CyHV-3-resistant mirror carp variety, the relative expression of CcPI3KC3 was significantly greater at 48, 96, and 144 hpi compared with the nonbreeding strain groups after infection (p < 0.001). These results indicate that the full-length CcPI3KC3 sequence was successfully cloned from common carp for the first time, and it might play an important role in the immune system of common carp against CyHV-3 infection. This study provides a theoretical basis for the molecular mechanism of CyHV-3 resistance.

Optimization of iron flocculation for enrichment of CyHV-2 in aquaculture water

Cyprinid herpesvirus 2 (CyHV-2) is an important infectious virus endangering the farmed Carassius species industry. The enrichment and identification of CyHV-2 in the water is a vital means of early warning diagnostics, which is helpful to aquatic diseases prevention and control. In this study, we employed and optimized iron flocculation to enrich the concentrations of CyHV-2 in aquaculture water. Different Fe3+ concentrations (0.5, 1.0, and 10.0 mg/L), various membrane filters (mixed cellulose ester, glass fiber, nylon microporous filter membrane), different elution buffers containing reductants (ascorbate, citrate, DL-malate, and their combinations with EDTA or TE buffer) were compared to check the recovery yield of CyHV-2. Viral genome quantitation was determined using quantitative real-time PCR. The results showed that Fe3+ concentration of 1.0 mg/L, the glass fiber and citrate-TE were determined to have high efficiency and stable recovery yield. To evaluate infectious CyHV-2 viral titres, crucian carps (Carassius auratus) were intraperitoneally injected with CyHV-2. The mortality rate was 100 % at 9 days postinoculation (dpi) with the 96.7 % detection of CyHV-2 in the pooled tissues. The histopathological phenotype showed necrotic cell death in the liver, spleen and kidney. The CyHV-2 genome was ranging from 101.1 to 107.5 copies/μl in the deceased fish, and 101.4 to 103.0 copies/μl in the feeding water at various stages from latent infection to onset of disease. The field samples from Huzhou, Zhejiang Province on June 4th and 7th had higher detection rates, 87.5 % and 100 % respectively, corresponding to higher CyHV-2 detected in the aquaculture water. CyHV-2 viral load in cultured waters tends to increase prior to fish death, which provides a new indicator for the prediction of fish diseases. In summary, we have established a unified operational standard for enrichment of CyHV-2 in the aqueous environment by iron flocculation.

Propagation of koi herpesvirus using embryonated chicken eggs: a potential substitute method for fish vaccine production?

Koi herpesvirus disease (KHVD) is caused by a large DNA virus that commonly infects carp, Cyprinus carpio (koi and common carp). KHVD has spread rapidly across the globe and caused high mortality in all ages of common carp and koi. Until now, no effective treatment has been applied to prevent KHV infection impacting the mass production of koi and common carp . An environmentally friendly alternative strategy for controlling fish disease is vaccination. One of the challenges facing conventional viral vaccine production is the requirement for koi or common carp cell cultures, which must be frequently maintained with expensive materials required for virus propagation. This study aims to obtain KHV that has been propagated in embryonated chicken eggs (ECE) to formulate an affordable KHV vaccine for the aquaculture industry. This research consisted of three stages; the first stage was virus inoculation into various parts of eggs (allantoic fluid, chorioallantoic membrane, amniotic fluid, and egg yolk). The second stage was observing viral growth and collecting ECE fluid and membranes. The third stage involved quantitatively determining the viral genomic copy numbers using the quantitative Polymerase Chain Reaction (qPCR) assay. KHV propagation in various parts of ECE resulted in varying viral genomic copy numbers with a high DNA copy number reported in allantoic fluid on the third day after inoculation. Further work is required to monitor virus titer in later passages and optimize methodology for using ECE as the potential alternative to cultured cells as the medium for virus propagation. In the future the system could be developed to produce promising vaccine candidates with more affordable vaccine prices for the Indonesian fish farming industry.

Vertical Transmission of Cyprinid Herpesvirus 3 (CyHV-3) in Common Carp Cyprinus carpio: Experimental Insights and Implications

Currently, little is known about vertical transmission of cyprinid herpesvirus 3 (CyHV-3). Here, we assessed potentials for (1) vertical transmission of CyHV-3 and (2) viral infection to fertilized eggs via viral-contaminated environmental water. After CyHV-3 exposure, eggs were incubated, and hatchlings were raised to juvenile stage. To assess occurrence of KHVD during the experiment, samplings were performed at various stages, such as fertilized egg, eyed egg, larval, and juvenile stages. The viral DNA was detected only from eggs at 3 h post fertilization, but not from fish at later stages. No mortality associated with CyHV-3 infection was observed in all the groups during the experimental period, or fish became 91 days post hatch. Additionally, those juveniles were re-exposed to CyHV-3 to investigate whether CyHV-3 infection was established at the egg stage. As a result, statistically significant difference was not observed in the cumulative mortality between the larval fish with or without CyHV-3 infection at the egg stage, suggesting that fish exposed to CyHV-3 at the egg stage did not acquire immunity against the viral infection. All our results suggest that it is improbable that KHVD can be transmitted vertically through eggs of common carp that have survived CyHV-3 infection.

Maternal Immunity and Immune Improvement of Koi Cyprinus rubrofuscus Larvae after DNA Vaccine against Hoi Herpesvirus

Vaccination is an efficient step in preventing disease caused by koi herpesvirus in koi fish (Cyprinus rubrofuscus). This study aimed to investigates the impact of maternal broodstock vaccination with ORF81 DNA vaccine on the transmission of maternal immunity to offspring against KHV. The ORF81 DNA vaccine has been shown to increase RPS, but its effect on maternal immunity in koi offspring after vaccination has not been evaluated. The study examined koi broodstocks vaccinated with the ORF81 DNA vaccine 45 and 60 days before spawning (45V and 60V) at a dose of 12.5 μg/100 g. Immunological parameters were assessed in broodstock, eggs, and larvae. Immune gene expression was also analyzed using the qPCR method. The results showed that lysozyme activity and antibody levels in eggs from vaccinated mothers were higher, though not significantly different. Meanwhile, lysozyme activity and antibody levels in mothers were significantly higher (p&lt;0.05) compared to controls. Immunoglobulin-M gene (IgM) and recombination-activating gene 1 (RAG1), were significantly induced. The relative survival percentage increased significantly in larvae produced from broodstocks with 60V. These findings indicate that broodstocks treated with the ORF81 DNA vaccine can enhance the immune response of their offspring.

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and visual detection of Anguillid herpesvirus 1

China has the largest aquaculture eel production in the world. High-density cultivation pattern often results in an outbreak of epidemic diseases. Since the 1990s, eel “mucus sloughing and hemorrhagic septicemia disease” was often broke out in China, and brought huge economic losses to eel breeders. Anguillid herpesvirus 1 (AngHV) was detected and isolated from the diseased eel, and proved to be the pathogen of the disease. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid, sensitive, and specific detection of AngHV. A set of six primers targeting the ORF51 gene of AngHV was designed, which could effectively detect purified AngHV virions, AngHV-infected cells, or eel tissue samples. The suitable reaction temperature is 63℃, and the reaction time is 40 min. There was no cross-reaction with eel and other fish viruses, including Infectious pancreatic necrosis virus (IPNV), Marine birnavirus (MABV), Rana grylio virus (RGV), Cyprinid herpesvirus 3 (CyHV-3), and Eel iridovirus (EIV). The lower detection limit of the AngHV LAMP assay is 10 copies of AngHV genome DNA, which is at least 100 times more sensitive than conventional PCR in detecting AngHV. The assay could effectively detect AngHV from collected samples with typical clinical symptoms of AngHV infection. It suggested that the LAMP assay could be used in specific detection of AngHV and has great potential for early diagnosis of AngHV infection in the farm.

Cyprinid herpesvirus 3 replication is inhibited via decreased ORF3 promoter activity mediated by multiple cellular transcription factors

Cyprinid herpesvirus 3 (CyHV-3) is an enveloped and double-stranded DNA virus that primarily infects common carp and its variants. The transcriptional regulation program of the viral immediate early (IE) genes plays a crucial role in virus replication. However, the transcriptional regulatory mechanism of CyHV-3 IE genes remains largely unclear. In this study, we found that the IE gene ORF3 promoter, which exhibited the highest activity among the eight CyHV-3 IE genes, was inhibited in the host common carp brain (CCB) cells. Deletions of the ORF3 promoter revealed the presence of multiple regulatory elements. Site-directed deletions of multiple transcription factors binding sites (TFBS), including Yin Yang-1(YY1), TATA-binding protein (TBP), sex-determining region Y-box2 (SOX2), and cyclin-dependent kinase 8 (CDK8) binding sites, markedly inhibited the ORF3 promoter activity. Moreover, overexpression of these transcription factors in CCB cells also led to a noticeable reduction in ORF3 promoter activity. Importantly, YY1, TBP, SOX2, and CDK8 played antiviral roles against CyHV-3 infection, as evidenced by decreased viral transcripts (ORF55, ORF81, and ORF92) and genome copies after overexpression of these TFs followed by CyHV-3 infection. Collectively, CyHV-3 replication was markedly inhibited by reducing the promoter activity of ORF3 mediated by multiple cellular TFs. These findings enhance our understanding of the role of IE gene promoters in CyHV-3 and lays a foundation for in-depth studies on viral gene transcriptional regulation mechanisms.

Development of cross monoclonal antibodies for cyprinid herpesvirus 3 and cyprinid herpesvirus 2

Cyprinid herpesvirus 3 (CyHV-3) and Cyprinid herpesvirus 2 (CyHV-2) both belong to Alloherpesviridae family and bring great harm to koi carp, goldfish and crucian carp industry worldwide. In order to prepare a cross monoclonal antibody (MAb) that can simultaneously detect CyHV-3 and CyHV-2, mice were immunized with purified CyHV-3, and their spleens were taken for cell fusion with SP2/0 cells. Hybridoma cells were detected with purified CyHV-3 and CyHV-2. Western blotting was performed on the screened positive cell lines, and then the supernatant of the screened positive cell lines was used to detect common carp brain (CCB) cells infected with CyHV-3 and caudal fin of Carassius auratus gibelio (GiCF) cells infected with CyHV-2. The results showed that two strains of MAbs 3E11 and 4E4 could recognize CyHV-3 and CyHV-2 by dot blot. MAbs 3E11 and 4E4 could detect viruses in CCB cells infected with CyHV-3 and GiCF cells infected with CyHV-2. In this study, two strains of CyHV-3 and CyHV-2 cross MAbs were prepared to detect CyHV-3 and CyHV-2 simultaneously, providing more technical support for the establishment of detection methods for CyHV-3 and CyHV-2.

Rapid cyprinid herpesvirus 3 detection using loop-mediated isothermal amplification (LAMP) combined with gold nanoprobes and SYBR safe

As an etiological agent of a notifiable and emerging disease, Cyprinid herpesvirus 3 (CyHV-3 or koi herpesvirus, KHV) is a highly risky factor that affects koi and common carp yield through increasing the mortality rates. In the current study, a highly rapid and sensitive technique was established to detect CyHV-3 with the help of loop-mediated isothermal amplification (LAMP) method with dual visualizing indicators (SYBR safe and gold nanoparticle probes; AuNP-probes). Six specific primers were used to amplify the thymidine kinase (TK) of CyHV-3 using a LAMP reaction of 45 min at 65 °C along with detecting the products through visual inspection via hybridization at 65 °C for 20 min with a functionalized thiol-AuNP probe. A closed-tube LAMP-SYBR safe assay was also developed under the same condition without AuNP probe. The amplification of KHV-LAMP products were evaluated by the naked eye, through fluorescent emission of LAMP-SYBR safe dye-complex and colorimetric aggregate of LAMP product in the presence of AuNP probe. Furthermore, quantitative measurement was applied to the LAMP-AuNPs assay, using the spectral shift assay. The detection limit was 1.25 fentograms DNA which can compete the latest techniques available. The proposed technique yielded negative outcomes with DNA template from other viruses such as CyHV-1 and CEV. The LAMP-SYBR safe and LAMP-AuNPs assays introduced in this article were very sensitive, fast, and reliable diagnostic tools for the detection of CyHV-3.

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-1β, 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.

Genomic and molecular characterization of a cyprinid herpesvirus 2 YC-01 strain isolated from gibel carp

Cyprinid herpesvirus 2 (CyHV-2) is the pathogen of herpesviral hematopoietic necrosis (HVHN), causing the severe economic losses in farmed gibel carp (Carassius gibelio). Further exploration of the genome structure and potential molecular pathogenesis of CyHV-2 through complete genome sequencing, comparative genomics, and molecular characterization is required. Herein, the genome of a CyHV-2 YC-01 strain isolated from diseased gibel carp collected in Yancheng, Jiangsu Province, China was sequenced, then we analyzed the genomic structure, genetic properties, and molecular characterization. First, the complete YC-01 genome comprises 275,367 bp without terminal repeat (TR) regions, with 151 potential open reading frames (ORFs). Second, compared with other representative published strains of the genus Cyvirus, several evident variations are found in YC-01, particularly the orientation and position of ORF25 and ORF25B. ORF107 and ORF156 are considered as potential molecular genetic markers for YC-01. ORF55 (encoding thymidine kinase) might be used to distinguish YC-01 and ST-J1 from other CyHV-2 isolates. Third, phylogenetically, YC-01 clusters with the members of the genus Cyvirus (together with the other six CyHV-2 isolates). Fourth, 43 putative proteins are predicted to be functional and are mainly divided into five categories. Several conserved motifs are found in nucleotide, amino acid, and promoter sequences including cis-acting elements identification of YC-01. Finally, the potential virulence factors and linear B cell epitopes of CyHV-2 are predicted to supply possibilities for designing novel vaccines rationally. Our results provide insights for further understanding genomic structure, genetic evolution, and potential molecular mechanisms of CyHV-2.

Long-term detection of cyprinid herpesvirus 1 genome and tumorous transcriptome in common carp (Cyprinus carpio) infected with the virus

Cyprinid herpesvirus 1 (CyHV-1) disease induced by CyHV-1 (Cyvirus cyprinidallo1) causes mortality in common carp (Cyprinus carpio) and formation of tumors on the skin of infection-tolerant fish. Similar to other viruses in the Orthoherpesviridae family, CyHV-1 appears to establish latent infections. However, information regarding the mechanisms of genetic conservation for a prolonged period and tumorigenesis by CyHV-1 is limited. Here, we aimed to reveal i) potential tissues where the virus establishes persistent infection, and ii) the transcriptome characteristics of tumors formed in common carp infected with CyHV-1. In fish experimentally infected with the virus, the presence of the viral genome was observed in kidney, spleen, and gill tissue at 300 days post injection, a long time after the injection. This result suggests that CyHV-1 may utilize one or more of these tissues for its persistency. In fish naturally infected with CyHV-1 at a fish farm, transcriptome analysis showed substantial quantities of viral transcripts in tumor tissue. Of the 5 most abundant viral transcripts expressed in the tumor tissue, 4 transcripts (ORF81, ORF78, ORF31, and ORF72) are believed to encode virion proteins. This indicates the virus was active in the tumor tissue. Pathways altered in tumor tissue included those related to viral infection or cancer cells, suggesting that viral infection and tumor formation affect the metabolism of the host. This study is the first to report the likely persistent tissues of CyHV-1 and molecular mechanisms involved in tumor formation caused by an oncogenic virus in fish. These findings contribute to the development of quarantine measures for CyHV-1 and provide insights into the oncogenic mechanism of fish viruses.

Development of multi epitope subunit vaccines against emerging carp viruses Cyprinid herpesvirus 1 and 3 using immunoinformatics approach

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.

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.

Immunoinformatics Design of Multi-Epitope Peptide-Based Vaccine Against Cyprinid Herpesvirus-3 (CyHV-3) Targeting Thymidine Kinase Proteins

The common carp Cyprinus carpio is a freshwater teleost and is among the most economically significant fishes in aquaculture throughout the world. Taxonomically, C. carpio are a complex of species including subspecies Cyprinus carpio carpio. C. carpio are now threatened by Cyprinid Herpesvirus-3 (CyHV-3), the causative agent of Koi Herpesvirus Disease (KHVD), which causes severe morbidity and mortality in ornamental koi and common carp and can infect or be transmitted by other species. Despite these devastating circumstances, effective vaccinations or other medications for the control of KHVD are not readily available. For this reason, the aim of the current study was to formulate a multi-epitope vaccine against Cyprinid Herpesvirus-3 (CyHV-3) using an immunoinformatics approach. To assess the immunodominant T- and B-cell epitopes, the CyHV-3 proteomes were employed. Following a thorough evaluation, we constructed a strategy for vaccination employing four possible epitopes selected from among each of the three relevant epitope groups: cytotoxic T-lymphocyte, helper T-lymphocyte and linear B-lymphocyte. Important qualities used in the evaluation of the resultant vaccine are that it will be highly soluble, antigenic, immunogenic and non-allergenic. Among acceptable physicochemical qualities, the anticipated structure of the vaccine bears a close resemblance to that of the original protein. Additional considerations include a robust and sustained predicted binding between the vaccine and the Toll-Like Receptor (TLR9). Simulations of molecular dynamics confirm the likelihood of a strong binding stability and structural tightness. Moreover, the computer-generated immunological simulation revealed that the vaccine, when administered to fish, should induce immune responses comparable to those in real life. Finally, codon optimization based on Escherichia coli K12 produced favorable indications of GC content and acceptably high CAI value, as applicable to the cloning vector pET28+ (a). Overall, these results show that the proposed peptide vaccine is a promising option for CyHV-3 prophylaxis.

Autophagy mediated degradation of MITA/TBK1/IRF3 by a hnRNP family member attenuates interferon production in fish

HnRNP A/B belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) family and plays an important role in regulating viral protein translation and genome replication. Here, we found that overexpression of hnRNP A/B promoted spring viremia of carp virus (SVCV) and cyprinid herpesvirus 3 (CyHV3) replication. Further, hnRNP A/B was shown to act as a negative regulator of type I interferon (IFN) response. Mechanistically, hnRNP A/B interacted with MITA, TBK1 and IRF3 to initiate their degradation. In addition, hnRNP A/B bound to the kinase domain of TBK1, the C terminal domain of MITA and IAD domain of IRF3, and the RRM1 domain of hnRNP A/B bound to TBK1, RRM2 domain bound to IRF3 and MITA. Our study provides novel insights into the functions of hnRNP A/B in regulating host antiviral response.

The Molecular Characterization of the Cyprinid herpesvirus 3 (CyHV-3) ORF24 Protein and its effect on the expression of immune genes (in vitro)

Cyprinid herpesvirus 3 (CyHV-3), known as koi herpesvirus (KHV), is highly contagious and lethal. In this study, we aimed to characterize the ORF24-encoding protein of CyHV-3, investigate its sub-cellular localization, and determine its impact on the expression of immune factors through in vitro experiments. The results showed that the CyHV-3 ORF24 protein comprises 579 amino acids. Interestingly, multiple comparisons with homologous proteins from three carp herpesvirus origins showed no significant similarity. The fluorescence localization experiment showed that the green fluorescence signal, representing the protein pEGFP-ORF24, was primarily diffused in the cytoplasm. Notably, the overexpression of ORF24 effectively suppressed the expression of immune factors in both CCO (Channel catfish ovary) and FHM (Fathead minnow muscle cell line) cells. Bioinformatic analysis indicated that the CyHV-3 ORF24 gene exhibited significant differences from the corresponding genes in the other two carp herpesviruses. This suggests its unique functional role in the evolutionary context. Moreover, our findings demonstrated that overexpression of CyHV-3 ORF24 can effectively inhibit the expression of immune factors, underscoring its crucial role as a viral immune escape factor. These results provide further insights into the immune function of the CyHV-3 ORF24 protein and offer a theoretical foundation for developing new vaccines against CyHV-3 virus infections.

Occurrence of herpesvirus in fish.

Herpesviruses are common agents in animals of the aquatic environment. They infect many species of fish but only lead to disease in one or two species. Nevertheless, infected fish without clinical symptoms can actively transfer infectious agents to disease-susceptible species. The aim of the study was to identify and prove the natural presence of different herpesviruses. Koi, Nile tilapia, grass carp, goldfish and crucian carp were infected with a herpesvirus isolate 99% identical to goldfish herpesvirus (GHV) or cyprinid herpesvirus 2 (CyHV-2) obtained from crucian carp. Before and after infection, samples were collected non-lethally at different time points from all five fish species to identify and evaluate the replication of viruses naturally infecting the fish as well as the CyHV-2 experimentally infecting them. Gill swabs and separated leukocytes were subjected to PCR and the results compared. These samples yielded DNA of koi herpesvirus (KHV, also referred to as CyHV-3), GHV and a new herpesvirus. While Asian-lineage CyHV-3 DNA was detected in samples from crucian carp and goldfish, CyHV-2 DNA was found in samples from koi and tilapia. A new, hitherto unknown herpesvirus was identified in samples from grass carp, and was confirmed by nested PCR and sequence analysis. The survival rates were 5% for grass carp, 30% for tilapia, 55% for crucian carp, 70% for koi and 100% for goldfish at 20 days post infection. Evolutionary analyses were conducted and five clusters were visible: CyHV-1 (carp pox virus), CyHV-2 with sequences from koi and tilapia, CyHV-3 with sequences from crucian carp and goldfish, probable CyHV-4 from sichel and a newly discovered herpesvirus - CyHV-5 - from grass carp. The results obtained with the molecular tools as well as from the animal experiment demonstrated the pluripotency of aquatic herpesviruses to infect different fish species with and without visible clinical signs or mortality.

Transcriptomic Responses to Koi Herpesvirus in Isolated Blood Leukocytes from Infected Common Carp.

Koi herpesvirus (KHV, CyHV-3) causes severe economic losses in carp farms. Its eradication is challenging due to the establishment of latency in blood leukocytes and other tissues. To understand the molecular mechanisms leading to KHV infection in leukocytes, common carp were bath-exposed to KHV at 17 °C. After confirming the presence of viral transcripts in blood leukocytes at ten days post infection, RNA-Seq was performed on peripheral blood leukocytes on the Illumina NovaSeq. KHV infection triggered a robust immune response mediated by pattern recognition receptors, mainly toll-like receptors (tlr2, tlr5, tlr7, and tlr13), urokinase plasminogen activator surface receptor-like, galectin proteins, and lipid mediators such as leukotriene B4 receptor 1. Enriched pathways showed increased mitochondria oxidative phosphorylation and the activation of signalling pathways such as mitogen-activated protein kinases (MAPKs) and vascular endothelial growth factor (VEGF). KHV-infected leukocytes showed low production of reactive oxygen species (ROS) and glutathione metabolism, high iron export and phagocytosis activity, and low autophagy. Macrophage polarization was deduced from the up-regulation of genes such as arginase non-hepatic 1-like, macrophage mannose receptor-1, crem, il-10, and il-13 receptors, while markers for cytotoxic T cells were observed to be down-regulated. Further work is required to characterise these leukocyte subsets and the molecular events leading to KHV latency in blood leukocytes.