Cyprinid Herpesvirus Research Articles

Investigation of β-Carboline Alkaloid Harmaline Against Cyvirus cyprinidallo3 Infection In Vitro and In Vivo

Cyvirus cyprinidallo3, also known as Cyprinid herpesvirus 3 (CyHV-3), is a common pathogen of koi and common carp (Cyprinus carpio). Infection of CyHV-3 can lead to high mortality in fry under 4 months of age. CyHV-3 can become latent in recovered fish, and latent CyHV-3 can reactivate under stress conditions and spread the virus. Reactivation of CyHV-3 can also lead to mortality and diseases in latently infected fish. No effective drugs are available to prevent CyHV-3 infection or reactivation from latency. There is a need for the discovery of anti-CyHV-3 drugs. Harmine (HAR) and harmaline (HAL) are β-carboline alkaloids found in the medicinal plant Peganum harmala with antiviral activities against many viruses, including HSV. Here, HAL was evaluated against CyHV-3 infection in vitro and in vivo, respectively. Immediately after a one-hour infection exposure of ~1000 FPU/plate or ~500 PFU/plate, cells treated with 5 µM HAL for 2 h can block nearly 50% or 90% plaque formation in vitro. Only around 50% inhibition was observed in cells treated with the common anti-herpesvirus drug acyclovir (ACV) at 10 or 20 µM for 2 h following 1 h post-infection of ~500 PFU/plate. Cells treated with 10 µM HAL for 30 min, 60 min, 2 h, and 6 h can reduce 60%, 65%, 85.5%, and 85% CyHV-3 replication in vitro, respectively. HAL at 20 µM is still effective against CyHV-3 DNA replication and virion production when the treatment started at 3 and 5 days post-infection for 1 or 2 h, respectively. HAL under 50 µM has little toxicity to cells treated for 24 h. Immersion treatment with 10 µM HAL for 3–4 h daily within the first 5 days post-infection can increase the survival of fry by 60%. In addition, IM injection of HAL at 20 µM can reduce the rate of CyHV-3 reactivation induced by heat stress in latently infected koi. This study demonstrated that HAL could potentially be used to prevent CyHV-3 infection or reactivation from latency.

Modulation of Sodium and Ammonia Transporters in the Context of Viral Gill Diseases in Common Carp (Cyprinus carpio).

Osmoregulation and ammonia removal are among key physiological processes that take place in gills and affect fish homeostasis and well-being. These processes can be disrupted by numerous environmental factors, but also by viral infections, especially those leading to severe gill disorders. The mechanisms of how viruses disrupt osmoregulation and ammonia removal in fish have not been extensively studied. We propose further exploration of the molecular and functional basis of viral induced gill disorders by studying the gene expression and enzyme activity of Na+/K+-ATPases (NKA) and ammonia transporters in the gills and kidney of common carp during infection with two viruses: carp edema virus (CEV) and cyprinid herpesvirus 3 (CyHV-3). In the case of NKA, the expression of subunit α of selected NKA was affected by both viruses; however, no discernible trends were observed in the gills and kidneys. The enzyme activity of NKA was significantly reduced in the gills during infection with both CEV and CyHV-3. Moreover, our immunohistochemical studies showed that during infection with CEV and CyHV-3, NKA-rich cells are transferred from the primary lamellae to the more superficial space and to the secondary lamellae of the gills. In the case of ammonia transporters, both CEV and CyHV-3 infection resulted in downregulation of the expression of major transporters gdh1, Rhag, and Rhbg, allowing us to partly explain the ammonia accumulation in blood observed during infection with these viruses. This study highlighted that virus induced gill disorders may lead to disruption of osmoregulation and ammonia removal by dysregulating the expression and activity of NKA and ammonia transporters.

Proteogenomic analysis of Cyprinid herpesvirus 2 using high-resolution mass spectrometry.

Cyprinid herpesvirus 2 (CyHV-2) is the main pathogen responsible for the development of herpesviral hematopoietic necrosis disease (HVHND) in crucian carp (Carassius auratus). The CyHV-2 genome encodes approximately 150 genes that are expressed in a well-defined manner during productive infection. However, CyHV-2 open reading frames (ORFs) are primarily derived from sequence and homology analyses, and most lack protein-level evidence to support their properties. In this study, we used high-resolution mass spectrometry followed by proteogenomic mapping to achieve genome re-annotation of CyHV-2. Based on our results, a total of 1,683 MS/MS spectra could be mapped to the CyHV-2 genome through six-frame translation, with 1,665 corresponding to 117 currently annotated protein-coding ORFs. Three of the remaining 18 peptides were mapped to the N-terminal extension region of known ORFs. However, 12 novel CyHV-2 ORFs, designated nORF1-12, were identified and characterized for the first time based on the remaining 15 peptides that could be mapped to previously unannotated regions of the viral genome. And the sequence differences of the novel phosphorylated nORF1, also referred to as ORF25E, in different CyHV-2 strains indicated that the nORF1 is a prospective molecular marker that can monitor the evolution from the Japan (J) to the China (C) genotype of CyHV-2. These findings further validate existing annotations, expand the genomic landscape of CyHV-2, and provide a rich resource for aquatic virology research.IMPORTANCECyHV-2 is a viral pathogen that poses a significant threat to crucian carp farming. CyHV-2 has a large genome with complex sequence features and diverse coding mechanisms, which complicates accurate genome annotation in the absence of protein-level evidence. Here, we employed various protein extraction and separation methods to increase viral protein coverage and performed an integrated proteogenomic analysis to refine the CyHV-2 genome annotation. A total of 129 viral genes were confidently identified, including 117 currently annotated genes and 12 novel genes. For the first time, we present large-scale evidence of peptide presence and levels in the genome of aquatic viruses and confirm the majority of the predicted proteins in CyHV-2. Our findings enhance the understanding of the CyHV-2 genome structure and provide valuable insights for future studies on CyHV-2 biology.

Molecular characterization and phylogenetic analysis of Cyprinid herpesvirus 3 genotypes in Iran from 2020 to 2023

The aquaculture industry in Iran contributed to about 1% of the world’s aquaculture production in 2020 with a volume of 0.7 million tons. A targeted approach was used to identify positive samples by collecting samples from 342 suspected carp farms showing signs of disease. The carp species was considered as the host for the viruses under investigation and Khuzestan, Mazandaran and Gilan provinces were selected for sampling. A total of 251 farms in Gilan, 68 farms in Mazandaran, and 23 farms in Khuzestan provinces were sampled. Cyprinid herpesvirus 3 (CyHV-3) was characterized by a combination of sequence analysis and duplex PCR. Genetic analyzes and phylogenetic tree construction were performed using MEGA7 software. Of the 342 farms sampled, 85 were infected with koi herpes virus (KHV). Asian 1 and Asian 2 genotypes were identified by sequence analysis of the SphI-5 and TK gene regions. One of the positive samples showed a match in all motif positions within the TK gene, specifically genotype A1, except for positions 814 − 813 where they had the sequence AT, which was a rare exception. Duplex PCR analysis of two variable marker regions between ORF29 and ORF30 (marker I) and ORF133 and its upstream region (marker II) revealed viruses of genotype J (I++ II +), an intermediate genotype (I++ II -), and a new genotype, I++ II -*, identified in viruses from different farms. This new genotype retains the I++ allele of marker I and has a 5-bp deletion in the marker II. The global distribution of CyHV-3 genotypes is not yet fully elucidated. Results indicate the high degree of diversification of CyHV-3 in the West Asian regions, where at least three different genotypes (I++ II +, I++ II -, and I++ II -Δ) currently appear to circulate.

Cyprinid herpesvirus 2 ORF41 Protein Degrades Pyruvate Dehydrogenase (PDH)-E1β to Promote Viral Replication in Gibel Carp Brain (GiCB) Cells

Cyprinid herpesvirus 2 (CyHV-2) is a major pathogen posing a serious threat to crucian carp farming and has led to major economic losses in China’s aquaculture industry. This research aimed to explore how the CyHV-2-ORF41 protein influences viral replication. Firstly, we found that ORF41 overexpression in Gibel carp brain (GiCB) cells significantly enhanced CyHV-2 replication. Subsequently, GST pull-down and LC-MS/MS analyses were conducted to identify ORF41’s protein interactions. The results showed that ORF41 might interact with pyruvate dehydrogenase (PDH)-E1β, an enzyme connecting glycolysis to the tricarboxylic acid (TCA) cycle. Furthermore, ORF41 expression decreased the PDH-E1β levels, leading to pyruvate and lactic acid accumulation. Molecular docking and dynamics simulations confirmed a stable interaction between ORF41 and PDH-E1β. This research not only deepens our understanding of CyHV-2’s mechanisms of infection but also suggests potential targets for therapeutic strategies in aquaculture.

Occurrence of Infectious Hematopoietic Necrosis, Koi Herpesvirus Disease, and Viral Hemorrhagic Septicemia in North Macedonia Between 2015-2023

Abstract Viral hemorrhagic septicemia (VHS), Infectious hematopoietic necrosis (IHN), and Koi herpesvirus disease (KHVD) are listed diseases by the European Commission that pose significant threats to the global aquaculture industry, resulting in substantial economic losses and impacting fish health and welfare. Due to their rapid spread potential, it is crucial for member states to implement measures preventing their transmission to disease-free areas. In this study, we aimed to assess the presence or absence of these viruses in fish aquaculture facilities in North Macedonia. During 9 years of surveillance from 2015 to 2023, 1,527 samples were tested for VHS and IHN, and 2,760 samples were tested for KHVD from aquaculture sites across North Macedonia using molecular diagnostic techniques. Our results indicated the absence of VHS and KHVD in all tested samples. However, the number of IHN-affected farms increased from two in 2018 to 33 by 2023, persisting across multiple sites. Despite the absence of VHS and KHVD, the ongoing presence and increasing incidence of IHN highlight the need to assess the effectiveness of existing biosecurity measures and disease management practices in the region. Ongoing surveillance and stringent biosecurity measures are essential for controlling IHN and preventing the introduction of other viral pathogens. Strengthening these measures is vital to ensure the long-term sustainability of the aquaculture industry in North Macedonia.

Establishment and Application of a Rapid Detection Method of Cyprinid Herpesvirus 2 (CyHV-2) in Aquacultural Waters by Using a Novel One-Pot RAA-CRISPR/Cas12a Combined With Fe-Iron Flocculation Technology.

Cyprinid herpesvirus 2 (CyHV-2) poses a substantial global threat to goldfish (Carassius auratus) and crucian carp (Carassius carassius). Despite the development of several sensitive molecular diagnostic techniques, there is an ongoing demand for alternative visualisation platforms to streamline the workflow, enhance safety profiles, and improve accessibility for end-users. In this study, we have integrated recombinase-aided amplification (RAA) technology with the CRISPR/Cas12a system to establish a rapid diagnostic system for CyHV-2, termed one-pot RAA-CRISPR/Cas12a. This method enables the results of detection within 60 min. The RAA-CRISPR/Cas12a platform is capable of detecting as few as 10 copies of CyHV-2 per reaction cycle without exhibiting cross-reactivity with other pathogens. The positive detection rate in clinical samples exceeds that of conventional PCR approaches, underscoring its high precision. Furthermore, the method could be used in conjunction with iron flocculation for the concentration and detection of viruses within aquaculture settings. This approach minimises the dissection of aquatic organisms, thereby maximising animal welfare and bolstering detection efficiency. Collectively, our findings validate the RAA-CRISPR/Cas12a method as a robust, specific, confirmatory, user-friendly and promising approach for on-site diagnosis of CyHV-2.

Comparison of Immune Responses Induced in Gibel Carp, Carassius auratus gibelio Immersion, and Intraperitoneal Vaccinated With Live Attenuated Cyprinid Herpesvirus 2 (CyHV‐2)

This study investigated the immune responses induced in gibel carp, Carassius auratus gibelio, following immersion and intraperitoneal (i.p.) vaccination with live attenuated Cyprinid herpesvirus 2 (CyHV‐2). Transcriptome sequencing analysis was performed on the kidney tissues of the experimental fish at 3, 7, and 14 days postvaccination. A staggering 948 million readings were assembled into 96,891 genes, with an average length of 1214 bp. We analyzed the KEGG enrichment, differentially expressed genes (DEGs), and DEGs temporal expression patterns between the immersion and i.p. injection groups. Our findings revealed that both i.p. injection and immersion vaccination significantly altered immune‐related genes in gibel carp, suggesting that the prepared attenuated vaccine effectively stimulates the fish’s immune response. Generally, the injection group exhibited higher gene expression levels in the kidney tissue earlier and to a greater extent. In contrast, the immersion group showed delayed immune responses with lower expression levels. The differential expression of 12 immune‐related genes in immunized gibel carp, as detected by real‐time quantitative PCR. Significant differences were observed in the expression of 12 immune‐related genes after vaccination, suggesting variations in the sensitivity of each gene to bodily stimuli and its involvement in immunity. The specific immune mechanisms require further research. These results provide essential data for evaluating vaccines’ immune pathways and efficacy, offering optimism for vaccine development in fish immunology.

Lactobacilli-Derived Postmetabolites Are Broad-Spectrum Inhibitors of Herpes Viruses In Vitro.

Herpes viruses are highly contagious agents affecting all classes of vertebrates, thus causing serious health, social, and economic losses. Within the One Health concept, novel therapeutics are extensively studied for both veterinary and human control and management of the infection, but the optimal strategy has not been invented yet. Lactic acid bacteria are key components of the microbiome that are known to play a protective role against pathogens as one of the proposed mechanisms involves compounds released from their metabolic activity. Previously, we reported the anti-herpes effect of postmetabolites isolated from Lactobacilli, and here, we confirm the inhibitory properties of another nine products against the phylogenetically distant human Herpes simplex virus-1 (HSV-1) and fish Koi Herpes virus (KHV) in cell cultures. Cytotoxicity, cytopathic effect inhibition, virucidal effect, the influence on the adsorption stage of the virus to the cells, as well as the protective effect of the postmetabolites on healthy cells were evaluated. The inhibitory effect was more pronounced against HSV-1 than against KHV at all studied viral cycle stages. Regarding the intracellular replicative steps, samples S7, S8, and S9 (Mix group) isolated from Ligilactobacillus salivarius (vaginal strain) demonstrated the most distinct effect with calculated selective indices (SIs) in the range between 69.4 and 77.8 against HSV-1, and from 62.2 to 68.4 against KHV. Bioactive metabolites from various LAB species significantly inhibit extracellular HSV-1 and, to a lesser extent, KHV virions. The blockage of viral adsorption to the host cells was remarkable, as recorded by a decrease in the viral titer with Δlg ≥ 5 in the Mix group for both herpes viruses. The remaining postmetabolites also significantly inhibited viral adsorption to varying degrees with Δlg ≥ 3. Most metabolites also exerted a protective effect on healthy MDBK and CCB cells to subsequent experimental viral infection. Our results reveal new horizons for the application of LAB and their postbiotic products in the prevention and treatment of herpes diseases.

Development Using Bioluminescence Imaging of a Recombinant Anguillid Herpesvirus 1 Vaccine Candidate Associated with Normal Replication In Vitro but Abortive Infection In Vivo.

Anguillid herpesvirus 1 (AngHV-1) (recently renamed Cyvirus anguillidallo 1) is the etiologic agent of a lethal disease that affects several eel species. It is thought to be one of the main infectious agents causing a population decline in wild eels and economic loss within the eel aquaculture sector. To date, no vaccines are available against AngHV-1. Recently, we developed a safe and efficacious live attenuated recombinant vaccine against Cyprinid herpesvirus 3 (CyHV-3). This CyHV-3 recombinant vaccine encodes a deletion of ORF57. Orthologues of CyHV-3 ORF57 exist in Cyprinid herpesvirus 2 (CyHV-2, ORF57) and AngHV-1 (ORF35). In the present study, using recombinant strains and bioluminescent in vivo imaging, we investigated the effect of AngHV-1 ORF35 deletion on virus replication in vitro, virulence in vivo, and the potential of an AngHV-1 ORF35-deleted recombinant as a vaccine candidate for the mass vaccination of eels by immersion. With this goal in mind, we produced ORF35-deleted recombinants using two parental strains: a UK strain and a recombinant derived from the former strain by insertion of a Luciferase-GFP reporter cassette into a non-coding intergenic region. Analyses of ORF35-deleted recombinants led to the following observations: (i) AngHV-1 ORF35 is not essential for viral growth in cell culture, and its deletion does not affect the production of extracellular virions despite reducing the size of viral plaque. (ii) In contrast to what has been observed for CyHV-3 ORF57 and CyHV-2 ORF57, in vivo bioluminescent analyses revealed that AngHV-1 ORF35 is an essential virulence factor and that its deletion led to abortive infection in vivo. (iii) Inoculation of the AngHV-1 ORF35-deleted recombinant by immersion induced a protective immune response against a wild-type challenge. This protection was shown to be dose-dependent and to rely on the infectivity of AngHV-1 ORF35-deleted virions. This study suggests that the AngHV-1 ORF35 protein has singular properties compared to its orthologues encoded by CyHV-2 and CyHV-3. It also supports the potential of AngHV-1 ORF35-deleted recombinants for the mass vaccination of eels by immersion.

Genetic Characterization of the Thymidine Kinase of Koi Herpesvirus 3 in Sulaymaniyah Province, Iraq

Koi herpesvirus 3 (KHV-3) is nominated as an emerging, highly devastating, and contagious viral disease with severe economic losses in the carp aquaculture industries worldwide. Carp rearing under intensive culture conditions is constantly vulnerable to infection by various pathogens in the water. Water temperature has a major influence on the onset and severity of the disease. Since common carp have gained importance in the Iraqi fish industry, the investigation and control of associated diseases, including KHV, are warranted. We confirmed the first detection of the KHV from the collected specimens from the suspected cages in the Sulaymaniyah region. Also, an analysis of the thymidine kinase (TK) gene sequence of the KHV showed that the virus strain had a point mutation (proline, P193) instead of threonine, T193. The isolated KHV strains were subdivided into three clades with three distinct genotypes: Asian (Iranian, Indonesian, Japanese, and Chinese), European (UK), and North American (USA and Mexico) genotypes. The virus sequences were more closely related to the Iranian and Indonesian genotypes. This report describes the first isolation and genetic characterization of koi herpesvirus (KHV) in the Sulaymaniyah province, which is important for efficacious management and vaccination programs in the future to control KHV infection in carp cages in Northern Iraq.

Emergence and persistence of Cyvirus cyprinidallo 2 (CyHV-2) in Prussian carp in Serbian lakes

Emergence and persistence of Cyvirus cyprinidallo 2 (CyHV-2) in Prussian carp in Serbian lakes

Breeding evaluations in aquaculture using neural networks

Breeding evaluations in aquaculture using neural networks

Virus susceptibility of a new cell line derived from the muscle of koi (Cyprinus carpio koi).

In this study, a continuous cell line (KM cells) derived from koi (Cyprinus carpio koi) muscle was established and characterized. The KM cells were subcultured for more than 70 passages and showed high viability after long-term cryopreservation. The KM cell line was optimally cultured in medium 199 containing 10% foetal bovine serum at 25°C. A chromosome analysis indicated that the cell line remained diploid, with a mean chromosome count of 100. DNA sequencing and comparative analysis of the 16S rRNA and cytochrome oxidase I gene sequences showed that the KM cell line originated from koi. In transfection experiments using the plasmid pEGFP, KM cells demonstrated a high level of transfection efficiency, suggesting their potential for use in foreign gene expression studies. Inoculation with spring viraemia of carp virus (SVCV) resulted in a substantial cytopathic effect, and the level of production of SVCV in KM cells was higher than that in the epithelioma papulosum cyprinid (EPC) cell line that is normally used to produce the virus. However, no cytopathic effect was observed when these cells were inoculated with koi herpesvirus, carp oedema virus, or grass carp reovirus. These observations suggest that the newly established KM cell line will be a valuable tool for investigating the pathogenesis of infection with spring viraemia of carp virus.

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.

Akt inhibitors prevent CyHV-2 infection in vitro

Akt inhibitors prevent CyHV-2 infection in vitro

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

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

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.