Cyprinid herpesvirus 3 (CyHV-3) is the main pathogen of koi herpesvirus disease (KHVD), which has caused serious damage to the ornamental and food-producing carp industry. Effective and rapid on-site detection methods are needed for early diagnosis of CyHV-3. A lateral flow immuno-chromatographic assay (LFIA) using two specific anti-CyHV-3 monoclonal antibodies has been developed and validated for on-site detection of CyHV-3. MAb 3C9 was used to bio-conjugate CyHV-3 antigen with colloidal gold, and MAb 2A8 was used to capture antigen bound colloidal gold on the test line. The control line was lined with goat anti-mouse IgG to capture unbound colloidal gold to validate performance. The test results can be viewed within 10 min after putting the strip into CyHV-3 virus infection fluid. The lowest limit of detection for the LFIA test was found to be 1.5 × 104 copies/μL and it showed no cross-reactivity with other fish viral pathogens. The specificity of the strip was 100% when spleen and kidney tissues of CyHV-3-infected and healthy koi were validated at the field level. The LFIA strip will be an effective device for the early detection of CyHV-3 in the future.

The zebrafish (Danio rerio) represents an increasingly important model organism in virology. We evaluated its utility in the study of economically important viruses from the genus Cyprinivirus (anguillid herpesvirus 1, cyprinid herpesvirus 2 and cyprinid herpesvirus 3 (CyHV-3)). This revealed that zebrafish larvae were not susceptible to these viruses after immersion in contaminated water, but that infections could be established using artificial infection models in vitro (zebrafish cell lines) and in vivo (microinjection of larvae). However, infections were transient, with rapid viral clearance associated with apoptosis-like death of infected cells. Transcriptomic analysis of CyHV-3-infected larvae revealed upregulation of interferon-stimulated genes, in particular those encoding nucleic acid sensors, mediators of programmed cell death and related genes. It was notable that uncharacterized non-coding RNA genes and retrotransposons were also among those most upregulated. CRISPR/Cas9 knockout of the zebrafish gene encoding protein kinase R (PKR) and a related gene encoding a protein kinase containing Z-DNA binding domains (PKZ) had no impact on CyHV-3 clearance in larvae. Our study strongly supports the importance of innate immunity-virus interactions in the adaptation of cypriniviruses to their natural hosts. It also highlights the potential of the CyHV-3-zebrafish model, versus the CyHV-3-carp model, for study of these interactions.

Cyprinid herpesvirus-3 (CyHV-3) is an important pathogen of common carp (Cyprinus carpio, carp) causing significant economic and ecological impacts worldwide. The recent emergence of CyHV-3 in the Upper Midwest region of the United States has raised questions related to the disease ecology and host specificity of CyHV-3 in wild carp populations. To determine the prevalence of CyHV-3 in wild populations of fishes in Minnesota, we surveyed five lakes in 2019 in which the virus was known to have caused mass mortality events in carp from 2017 to 2018. A total of 28 species (n = 756 total fish) of native fishes and 730 carp were screened for the presence of CyHV-3 DNA using specific qPCR. None of the native fish tissues tested positive for CyHV-3 although the prevalence of CyHV-3 in carp was 10%-50% in the five lakes. A single lake (Lake Elysian) with a 50% DNA detection rate and evidence of ongoing transmission and CyHV-3-associated mortality was surveyed again in 2020 from April to September. During this period, none of the tissues from 24 species (n = 607 total fish) tested positive for CyHV-3 though CyHV-3 DNA and mRNA (indicating viral replication) was detected in carp tissues during the sampling period. CyHV-3 DNA was detected most often in brain samples without evidence of replication, potentially indicating that brain tissue is a site for CyHV-3 latency. Paired qPCR and ELISA testing for Lake Elysian in 2019-2020 identified young carp (especially males) to be the primary group impacted by CyHV-3-associated mortality and acute infections, but with no positive detections in juvenile carp. Seroprevalence of carp from Lake Elysian was 57% in 2019, 92% in April of 2020 and 97% in September 2020. These results further corroborate the host specificity of CyHV-3 to carp in mixed wild populations of fishes in Minnesota and provide additional insights into the ecological niche of CyHV-3 in shallow lake populations of carp in North America.

Vaccination is the best form of protecting fish against viral diseases when the pathogen cannot be contained by biosecurity measures. Vaccines based on live attenuated viruses seem to be most effective for vaccination against challenging pathogens like Cyprinid herpesvirus 3. However, there are still knowledge gaps how these vaccines effectively protect fish from the deadly disease caused by the epitheliotropic CyHV-3, and which aspects of non-direct protection of skin or gill integrity and function are important in the aquatic environment. To elucidate some elements of protection, common carp were vaccinated against CyHV-3 using a double deletion vaccine virus KHV-T ΔDUT/TK in the absence or presence of a mix of common carp beta-defensins 1, 2 and 3 as adjuvants. Vaccination induced marginal clinical signs, low virus load and a minor upregulation of cd4, cd8 and igm gene expression in vaccinated fish, while neutralisation activity of blood serum rose from 14 days post vaccination (dpv). A challenge infection with CyHV-3 induced a severe disease with 80-100% mortality in non-vaccinated carp, while in vaccinated carp, no mortality was recorded and the virus load was >1,000-fold lower in the skin, gill and kidney. Histological analysis showed strongest pathological changes in the skin, with a complete destruction of the epidermis in non-vaccinated carp. In the skin of non-vaccinated fish, T and B cell responses were severely downregulated, inflammation and stress responses were increased upon challenge, whereas vaccinated fish had boosted neutrophil, T and B cell responses. A disruption of skin barrier elements (tight and adherence junction, desmosomes, mucins) led to an uncontrolled increase in skin bacteria load which most likely exacerbated the inflammation and the pathology. Using a live attenuated virus vaccine, we were able to show that increased neutrophil, T and B cell responses provide protection from CyHV-3 infection and lead to preservation of skin integrity, which supports successful protection against additional pathogens in the aquatic environment which foster disease development in non-vaccinated carp.

Cyprinid herpesvirus 3 (CyHV-3) is the etiological agent of koi herpesvirus disease (KHVD) and important pathogen of aquaculture and wild populations of common carp worldwide. Understanding the relative contributions of direct and indirect transmission of CyHV-3 as well as the factors that drive CyHV-3 transmission can clarify the importance of environmental disease vectors and is valuable for informing disease modeling efforts. To study the mechanisms and factors driving CyHV-3 transmission we conducted infection trials that determined the kinetics of KHVD and the contributions of direct and indirect forms of CyHV-3 transmission, as well as the contributions of contact rate, viral load, pathogenicity and contact type. The incubation period of KHVD was 5.88 + 1.75 days and the symptomatic period was 5.31 + 0.87 days. Direct transmission was determined to be the primary mechanism of CyHV-3 transmission (OR = 25.08, 95%CI = 10.73-99.99, p = 4.29 × 10-18) and transmission primarily occurred during the incubation period of KHVD. Direct transmission decreased in the symptomatic period of disease. Transmissibility of CyHV-3 and indirect transmission increased during the symptomatic period of disease, correlating with increased viral loads. Additionally, potential virulence-transmission tradeoffs and disease avoidance behaviors relevant to CyHV-3 transmission were identified.

Cholesterol is essential for building and maintaining cell membranes and is critical for several steps in the replication cycle of viruses, especially for enveloped viruses. In mammalian cells virus infections lead to the accumulation of the oxysterol 25-hydroxycholesterol (25HC), an antiviral factor, which is produced from cholesterol by the cholesterol 25 hydroxylase (CH25H). Antiviral responses based on CH25H are not well studied in fish. Therefore, in the present study putative genes encoding for CH25H were identified and amplified in common carp and rainbow trout cells and an HPLC-MS method was applied for determination of oxysterol concentrations in these cells under virus infection. Our results give some evidence that the activation of CH25H could be a part of the antiviral response against a broad spectrum of viruses infecting fish, in both common carp and rainbow trout cells in vitro. Quantification of oxysterols showed that fibroblastic cells are capable of producing 25HC and its metabolite 7α,25diHC. The oxysterol 25HC showed an antiviral activity by blocking the entry of cyprinid herpesvirus 3 (CyHV-3) into KFC cells, but not spring viremia of carp virus (SVCV) or common carp paramyxovirus (Para) in the same cells, or viral haemorrhagic septicaemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV) into RTG-2 cells. Despite the fact that the CH25H based antiviral response coincides with type I IFN responses, the stimulation of salmonid cells with recombinant type I IFN proteins from rainbow trout could not induce ch25h_b gene expression. This provided further evidence, that the CH25H-response is not type I IFN dependent. Interestingly, the susceptibility of CyHV-3 to 25HC is counteracted by a downregulation of the expression of the ch25h_b gene in carp fibroblasts during CyHV-3 infection. This shows a unique interplay between oxysterol based immune responses and immunomodulatory abilities of certain viruses.

Cyprinid herpesvirus 3 (CyHV-3) causes high mortality in carp. Emodin has been shown of the effects of antioxidant, anti-inflammatory and antiviral. In present study, we investigated the preventive effects and mechanism of emodin on CyHV-3 infection. The ornamental koi carp (Cyprinus carpio haematopterus) were intraperitoneally injected with emodin (10 mg/kg, 20 mg/kg, or 40 mg/kg). 72 h later, an intraperitoneal injection of CyHV-3 was administered, and collected the samples one week later to detect the antioxidant parameters, antioxidant genes, inflammatory genes and to perform histopathology assays. The results showed that emodin significantly suppressed CyHV-3 replication (P < 0.05), improved the koi survival rate and slowed the damage caused by CyHV-3. Emodin treatment increased the antioxidant activity and decreased the lipid peroxidation level of the koi. Compared to the CyHV-3 group, emodin treatment resulted in the same antioxidant parameters after CyHV-3 infection. Emodin treatment activated the Nuclear factorery throid 2-related factor 2/Kelch-like ECH-associated protein 1-antioxidatant response element (Nrf2/Keap1-ARE) pathway and upregulated the expression of heme oxygenase 1 (HO-1), superoxide dismutase (SOD), and catalase (CAT) in the hepatopancreas after CyHV-3 infection. Emodin activated the nuclear factor kappa-B (NF-κB) pathway and decreased the expression of interleukin-6 (IL-6), interleukin-8 (IL-8), and tumour necrosis factor-α (TNF-α) in the koi induced by CyHV-3. In conclusion, emodin treatment can suppress CyHV-3 replication and reduce the mortality of koi caused by CyHV-3. Emodin improves antioxidant function, relieves oxidative stress and inflammation cytokines via Nrf2/Keap1-ARE and NF-κB pathways, and protects against the adverse effects induced by CyHV-3.

Common carp (Cyprinus carpio) is a very important fish species for warm-water aquaculture in Croatia. All Croatian carp farms are subjected to a surveillance programme for the presence of koi herpesvirus (KHV), causing a deadly disease called koi herpesvirus disease (KHVD). However, there is no surveillance for other viral pathogens of importance like carp edema virus (CEV), a causative agent of koi sleepy disease (KSD). During regular testing within the KHVD surveillance programme, we tested samples for CEV simultaneously. The screening indicated possible outbreaks of KHVD and KSD. During 2016, KHVD broke out in an isolated area and soon thereafter a KHV eradication programme was successfully performed. However, during 2018 and 2019, two additional mortality events occurred in lakes in the southern part of Croatia during the spring. Samples from both events tested positive for CEV. An epidemiological investigation confirmed the introduction of infected carps from an infected farm to one of the lakes. To prevent the spreading of CEV into open waters, it is of utmost importance to introduce CEV testing before fish movement or to perform regular testing of all carp farms in the country to determine CEV prevalence for the purpose of implementation of control measures.

Cyprinid herpesvirus 3, also known as koi herpesvirus (KHV), is a viral pathogen responsible for mass mortalities of carp worldwide. In this study, we compared the sensitivity and specificity of ELISA and quantitative PCR (qPCR) methods for the diagnosis of KHV in experimentally infected koi Cyprinus carpio over an 11 mo period. Koi were exposed to KHV at 18 ± 1°C (permissive temperatures for KHV disease) in laboratory-controlled conditions. At 21 d post challenge, the temperature in the system was decreased to <15°C (non-permissive temperature for KHV disease), and fish were monitored for the following 11 mo. At different time points throughout the study, samples of blood and gills were collected from exposed and control koi and subjected to qPCR and ELISA. Survival proportions of 53.3 and 98.8% in exposed and control treatments, respectively, were recorded at the end of the challenge. Traditional receiver-operating characteristic analysis was used to compare the sensitivity of the ELISA and blood and gill qPCR during permissive and non-permissive temperatures. ELISA was superior to qPCR of gills and whole-blood samples in detecting previous exposure to KHV. Similar results were obtained in a second experiment exposing koi to KHV and inducing persistent infection at >30°C (non-permissive temperature for KHV disease). Finally, KHV ELISA specificity was confirmed using cyprinid herpesvirus 1-exposed koi through a period of 3 mo. This study demonstrates that the combination of ELISA and gill qPCR should be recommended in the diagnosis of KHV exposure of suspected carrier-state fish.

Cyprinid herpesvirus 3 (CyHV-3) is the etiological agent of koi herpersvirus disease (KHVD), which causes serious economic losses in global common carp and ornamental koi carp production of larvae as well as adult type fish. To control KHVD, vaccines against CyHV-3 utilizing different immunization routes have been developed, among them, oral vaccination is the most desirable method to prevent fish diseases occurring at the early larval stage. Here, we developed an oral subunit vaccine through the Saccharomyces cerevisiae cell surface display of CyHV-3 envelope protein pORF65, then, the recombinant yeast fed to Artemia which served as bio-encapsulation vector by subsequently feeding the common carp (Cyprinus carpio var. Jian) larvae. The fluorescent observation showed that the Artemia and S. cerevisiae could deliver intact antigen to the hindgut of carp larvae suggesting the possibility of the vector for oral immunization. On this basis, after three immunizations at a week interval, the oral vaccine induced high level of specific anti-pORF65 antibody. Meanwhile, a significant difference of immune-related genes expression occurred including cxca, IL-1β, IFN-a1, lysozyme, IgM and CD8α between vaccined group and blank control group. In addition, 30% of relative percent survival of carp larvae after immunization was obtained post the animal infection assay, offered an certain immune protection. Our results indicated that the oral pORF65 subunit vaccine bioencapsulated in Artemia induced the activation of immune response and high level of antibodies, which could be served as an oral vaccine candidate for the prevention of CyHV-3 infection.