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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., (© 2024 John Wiley & Sons Ltd.)

Introduction: 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., Material and Methods: 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., Results: 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., Conclusion: 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., Competing Interests: Conflict of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article., (© 2024 Sven Michael Bergmann et al., published by Sciendo.)

Introduction: Carp oedema virus (CEV) is a relatively understudied poxvirus. It exhibits an affinity for gill and skin epithelial cells. Investigations were conducted into selected aspects of CEV biology, with a focus on determining cell and tissue tropism of CEV, acquiring gene sequences and updating CEV tests in fish tissues., Material and Methods: A total of 238 common carp tissue samples from nine aquaculture farms were analysed. The study evaluated the efficacy of intermediate detection of CEV by real-time PCR and in situ hybridisation. The genes encoding protein P4a were sequenced, analysed and aligned in a phylogenetic tree using a molecular evolution model., Results: In situ hybridisation revealed the necessity to validate the Centre for Environment, Fisheries and Aquaculture Science protocols for sampling for CEV detection and to use the tissues for which the virus has the highest tropism, namely the skin and kidneys, rather than solely the gills. The level of genetic variability was determined, and it was shown that CEV mutates systematically. The creation of two distinct phylogenetic clades confirms certain strains' description as Polish isolates., Conclusion: Determining the localisation of CEV genetic material in organs and tissues is pivotal for shaping the World Organisation for Animal Health guidelines. The utility of molecular diagnostics has been demonstrated in the skin and kidney of carp, in addition to the gills, impelling their inclusion in diagnostic protocols. The clusters identified in the phylogenetic tree offer valuable insights for developing the current PCR primers. The prevalence of CEV infection in aquaculture, juxtaposed with its notably lower detection in wild fish, underscores the significance of mandatory molecular diagnostic testing for CEV in carp farming., Competing Interests: Conflict of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article., (© 2024 Natalia Adamkowska et al., published by Sciendo.)

As a widespread epidemic virus, genotype II of the grass carp reovirus poses a significant threat to the grass carp farming industry in China. Different genotype II isolates cause different degrees of virulence, although the underlying pathogenic mechanisms remain largely unknown. In this work, infections of grass carp with the virulent isolate grass carp reovirus (GCRV)-HN1307 and the avirulent isolate GCRV-GD1108 were performed to reveal a possible mutual transcriptional discrepancy. More differentially expressed genes (DEGs) were identified in the HN1307-infected group, which defined a grossly similar gene ontology (GO) pattern and different pathway landscape as the GD1108-infected group. Gene set enrichment analysis revealed that pathways related to innate immunity and metabolism were reciprocally activated and suppressed, respectively, following infection withHN1307, compared with GD1108. The trend analysis further indicated that immune-related pathways were involved in one of the four statistically significant profiles. Network analysis of transcription factor-gene interactions and protein-protein interactions on the immune-related profile suggested that among the core transcriptional factors (TFs) (UBTF, HCFC1, MAZ, MAX, and NRF1) and the hub proteins (Tlr3, Tlr7, Tlr9, Irf3, and Irf7), the latter were highly enriched in the toll-like receptor signaling pathway. Real-time quantitative PCR performed on the selected mRNAs validated the relative expression. This work will provide insights into the distinct transcriptional signatures from avirulent and virulent isolates of GCRV, which may contribute to the development of products for prevention., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Koi herpesvirus (KHV) is the causative agent of a koi herpesvirus disease (KHVD) inducing high mortality rates in common carp and koi (Cyprinus carpio). No widespread effective vaccination strategy has been implemented yet, which is partly due to side effects of the immunized fish. In this study, we present an evaluation of the purification of infectious KHV from host cell protein and DNA, using the steric exclusion chromatography. The method is related to conventional polyethylene glycol (PEG) precipitation implemented in a chromatographic set-up and has been applied for infectious virus particle purification with high recoveries and impurity removal. Here, we achieved a yield of up to 55% of infectious KHV by using 12% PEG (molecular weight of 6 kDa) at pH 7.0. The recoveries were higher when using chromatographic cellulose membranes with 3-5 μm pores in diameter instead of 1 μm. The losses were assumed to originate from dense KHV precipitates retained on the membranes. Additionally, the use of >0.6 M NaCl was shown to inactivate infectious KHV. In summary, we propose a first step towards a purification procedure for infectious KHV with a possible implementation in fish vaccine manufacturing., (© 2023 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Hemorrhagic disease of grass carp, which is induced by grass carp reovirus II (GCRV-II), leads to mass mortality in grass carp culture and causes enormous economic loss. However, there is currently no quantitative analysis method for the detection of GCRV-II, which is greatly restricted the etiological and epidemiological study of the disease. In this study a reverse transcription TaqMan PCR (RT-qPCR) assay was developed for the quantitative detection of GCRV-II. The probe and primers targeted location is the segment 6 (S6) region of the GCRV-II genome which is highly conserved. Standard curves were drawn and criteria were confirmed after the determination of the optimum reaction conditions. The species-specific assay showed that the method is highly specific and has no cross reactions with other pathogens. The assay was sufficiently sensitive to detect as low as 10 copies of virus RNA. Moreover, the method has a very good repeatability for batches and inter-batches sample detection. Then the method was applied to detect the virus in tissue samples from clinically infected grass carp, compared with conventional RT-seminested PCR, the RT-qPCR represents a specific value for detection rate of positive samples. In summary, the RT-qPCR was applied and achieved high sensitivity and specificity for GCRV-II detection., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Lymphocystis disease viruses (LCDVs) are viruses that infect bony fish which has been found in different locations across the globe. Four virus species have been classified by the International Committee on Taxonomy of Viruses (ICTV), despite remarkable discrepancies in genome size. Whole genome sequencing and phylogenetic analysis of LCDVs from wild fish from the North Sea and partial sequences from gilthead sea bream of an aquafarm located in the Aegean Sea in Turkey confirm that the LCDV1 genome at 100 kb is approximately half the size of the genomes of LCDV2-4. Since the fish species, of which LCDV1 was isolated, differ taxonomically at the order level, co-speciation can be excluded as the driver of the adaptation of the genome of this nucleocytoplasmic large DNA virus, but may represent an adaptation to the lifestyle of this demersal fish in the northeast Atlantic.

Since the end of the1990ies, Cyprinid herpesvirus 3 (also known as koi herpesvirus, KHV) has caused mass mortality events of koi and common carp all over the globe. This induced a high economic impact, since the KHV disease cannot be cured up to now, but only prevented by vaccination. Unfortunately, there is only one commercial vaccine available which is not approved in most countries. Therefore, there is an urgent need for new, safe and available vaccines. In this study, a live attenuated vaccine virus was generated by cell culture passages of virulent KHV, and shown to protect carp or koi after immersion or oral application against wild type challenge. An advantage of boost immunization was demonstrated, especially after oral application. Vaccination induced no or mild clinical signs and protecting antibodies have been measured. Additionally, the vaccine virus allowed differentiation of infected from vaccinated animals (DIVA) by PCR. The attenuation of the newly generated vaccine was tracked down to a partial deletion of open reading frame 150. This was confirmed by the generation of engineered ORF150 deletion mutants of wild-type KHV which exhibited a similar attenuation in vivo., (© 2022. The Author(s).)

Largemouth bass is an important commercially farmed fish in China, but the rapid expansion of its breeding has resulted in increased incidence of diseases caused by bacteria, viruses and parasites. In this study, moribund largemouth bass containing ulcer foci on body surfaces indicated the most likely pathogens were iridovirus and rhabdovirus members and this was confirmed using a combination of immunohistochemistry, cell culture, electron microscopy and conserved gene sequence analysis. We identified that these fish had been co-infected with these viruses. We observed bullet-shaped virions (100−140 nm long and 50−100 nm in diameter) along with hexagonal virions with 140 nm diameters in cell culture inoculated with tissue homogenates. The viruses were plaque purified and a comparison of the highly conserved regions of the genome of these viruses indicated that they are most similar to largemouth bass virus (LMBV) and hybrid snakehead rhabdovirus (HSHRV), respectively. Regression infection experiments indicated fish mortalities for LMBV-FS2021 and HSHRV-MS2021 were 86.7 and 11.1%, respectively. While co-infection resulted in 93.3% mortality that was significantly (p < 0.05) higher than the single infections even though the viral loads differed by >100-fold. Overall, we simultaneously isolated and identified LMBV and a HSHRV-like virus from diseased largemouth bass, and our results can provide novel ideas for the prevention and treatment of combined virus infection especially in largemouth bass.

Carp edema virus (CEV) is the causative agent of koi sleepy disease (KSD), a serious gill disease affecting common carp, Cyprinus carpio, and its ornamental variety, koi. After recent detections of the virus in various countries around the world, KSD has emerged as a new global disease in carp. However, the prevalence of the infection in carp populations in a given geographical region has not been studied thoroughly. The present communication reports an investigation into the presence of CEV in carp and koi populations in Germany. For this purpose, gill samples collected from carp and koi populations suffering from gill diseases or collected for a routine examination of their health status were tested for the presence of CEV by PCR. In total, 651 fish samples from 401 carp or koi cases were examined in 2015 and 2016, additional 118 samples from previous studies were included in the examination. CEV was detected in archive samples from carp dating back to 2007, and in koi samples dating back to 2009. From 2015 to 2016, CEV was detected in 69% of cases from carp populations examined from the main carp-producing areas in Germany, and in 41% of the examined cases from koi populations from all over Germany. Clinical KSD occurred mainly from April to June in carp populations at water temperatures ranging from 8 to 12°C and in koi populations at water temperatures ranging from 18 to 22°C. Most fish from clinically affected carp or koi populations harboured high virus loads of above 10,000 copies of CEV-specific DNA per 250 ng DNA, while gills from fish of other fish species from the ponds, including goldfish, grass carp and European perch were found CEV negative or harboured a low virus load. A phylogenetic analysis revealed the presence of multiple CEV variants from genogroup I in carp and genogroup II in koi populations in Germany. Genetically identical genogroup I isolates were detected in carp from different geographical locations in Germany and in other European carp populations. Some German genogroup II variants were identical to variants previously recorded from koi in Asian and other European countries. The data presented here show that CEV is highly prevalent in German common carp and koi populations and implies the spreading of this virus by intense trading of common carp and koi without necessary risk mitigating measures. As infections with this virus may induce serious disease, CEV diagnostic should be included in health surveillance and disease monitoring programmes., (© 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Largemouth bass ranavirus disease (LMBVD) caused by largemouth bass ranavirus (LMBV) has resulted in severe economic losses in the largemouth bass (Micropterus salmoides) farming industry in China. Early and accurate diagnosis is the key measure for the prevention and control of LMBVD. In this study, a quantitative polymerase chain reaction (qPCR) and a real-time recombinase-aided amplification (real-time RAA) assay were established for the detection of LMBV. The sensitivity and specificity of these two methods, and the efficacy for detection of LMBV from clinical samples were also evaluated. Results showed that the real-time RAA reaction was completed in <30 min at 39℃ with a detection limit of 58.3 copies, while qPCR reaction required 60 min with a detection limit of 5.8 copies. Both methods were specific for LMBV, where no cross-reactions observed with the other tested fish pathogens. Comparing the amplification results of both assays to the results obtained by virus isolation using 53 clinical tissue samples, results showed that the clinical sensitivity of real-time RAA and qPCR were 93.75% and 100% respectively, and the clinical specificity of both were 100%. Our results showed that qPCR is more suitable for quantitative analysis and accurate detection of LMBV in the laboratory, while real-time RAA is more suitable as a point-of-care diagnostic tool for on-site detection and screening of LMBV under farm conditions and in poorly equipped laboratories., (© 2022 John Wiley & Sons Ltd.)

Grass carp haemorrhagic disease caused by grass carp reovirus II is a serious disease of the aquaculture industry and vaccination is the only effective method of GCRV protection. In this study, Lactococcus lactis was used as oral vaccine delivery to express the GCRV II VP6 protein. We evaluated the protective efficacy of the live vaccine strain to induce mucosal immune protection. After oral administration, the recombinant strains remained in the hindgut for antigen presentation and increased the survival rate 46.7% and the relative percent survival 42.9%, respectively versus control vaccination. Though L. lactis alone can induce the inflammatory response by stimulating the mucosal immune system, the recombinant L. lactis expressing VP6 greatly enhanced nonspecific immune responses via expression of immune related genes of the fish. Furthermore, both systemic and mucosal immunity was elicited following oral immunization with the recombinant strain and this strain also elicited an inflammatory response and cellular immunity to enhance the protective effect. L. lactis can therefore be utilized as a mucosal immune vector to trigger high levels of immune protection in fish at both the systemic and mucosal levels. L. lactis is a promising candidate for oral vaccine delivery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Li, Wang, Wang, Zhang, Shi, Li, Bergmann, Mo, Yin and Wang.)

Tilapia lake virus disease (TiLVD) caused by Tilapia lake virus (TiLV) is a great threat to the global tilapia culture industry. Effective prevention and control strategies have not been developed due to limited basic research of pathogenesis of TiLVD. Cell lines from different fish species have been found to be permissive to TiLV infection. In the current study, we comprehensively analyzed TiLV susceptibilities to 10 permanent growing fish cell lines. We found that the highest viral titers were generated onto TiB cells originated from the tilapia species Oreochromis mossambicus, MSF from the largemouth bass Micropterus salmoides, CAMK from the hybrid snakehead Channa argus × Channa maculata and SS derived from the perch species Siniperca chuatsi. Viral copy numbers from these four cell lines ranged from 4 × 10 7 copies/μL to 4.6 × 10 8 copies/μL. Confocal immunofluorescent microscopy also indicated that all 10 cell lines can support varying degrees of viral infection and replication. TiLV particles can be observed in cells from randomly selected three fish species using electron microscope. This study will assist in research and development of prevention and control of TiLVD., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Tilapia lake virus (TiLV) is a newly emerged pathogen responsible for high mortality and economic losses in the global tilapia industry. Early and accurate diagnosis is an important priority for TiLV disease control. In order to evaluate the methodology in the molecular diagnosis of TiLV, we compared newly developed quantitative real-time PCR (qPCR) and real-time recombinase polymerase amplification (real-time RPA) assays regarding their sensitivities, specificities and detection effect on clinical samples. Real-time RPA amplified the target pathogen in less than 30 min at 39 °C with a detection limit of 620 copies, while qPCR required about 60 min with a detection limit of 62 copies. Both assays were specific for TiLV and there were no cross-reactions observed with other common fish pathogens. The assays were validated using 35 tissue samples from clinically infected and 60 from artificially infected animals. The sensitivities for the real-time RPA and qPCR assays were 93.33 and 100%, respectively, and the specificity was 100% for both. Both methods have their advantages and can play their roles in different situations. The qPCR is more suitable for quantitative analysis and accurate detection of TiLV in a diagnostic laboratory, whereas real-time RPA is more suitable for the diagnosis of clinical diseases and preliminary screening for TiLV infection in poorly equipped laboratories as well as in fish farms., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Gills of fish are involved in respiration, excretion and osmoregulation. Due to numerous interactions between these processes, branchial diseases have serious implications on fish health. Here, "koi sleepy disease" (KSD), caused by carp edema virus (CEV) infection was used to study physiological, immunological and metabolic consequences of a gill disease in fish. A metabolome analysis shows that the moderately hypoxic-tolerant carp can compensate the respiratory compromise related to this infection by various adaptations in their metabolism. Instead, the disease is accompanied by a massive disturbance of the osmotic balance with hyponatremia as low as 71.65 mmol L -1 , and an accumulation of ammonia in circulatory blood causing a hyperammonemia as high as 1123.24 µmol L -1 . At water conditions with increased ambient salt, the hydro-mineral balance and the ammonia excretion were restored. Importantly, both hyponatremia and hyperammonemia in KSD-affected carp can be linked to an immunosuppression leading to a four-fold drop in the number of white blood cells, and significant downregulation of cd4, tcr a2 and igm expression in gills, which can be evaded by increasing the ion concentration in water. This shows that the complex host-pathogen interactions within the gills can have immunosuppressive consequences, which have not previously been addressed in fish. Furthermore, it makes the CEV infection of carp a powerful model for studying interdependent pathological and immunological effects of a branchial disease in fish.

Vaccination is the most effective way to control the grass carp haemorrhagic disease (GCHD) with the primary pathogen grass carp reovirus genotype II (GCRV-II). However, due to the large difference in breeding conditions and unclear genetic background of grass carp, the results of the experiment were not reliable, which further hinders the effective prevention and control of GCHD. The rare minnow (Gobiocypris rarus) is highly sensitive to GCRV. Its small size, easy feeding, transparent egg membrane, and annual spawning are in line with the necessary conditions for an experimental aquatic animals culture object. In this study, immunogenicity and protective effects of attenuated and inactivated viruses for grass carp and rare minnow were evaluated in parallel. The expression of immune-related genes increased statistically significant after immunization. With the rise of specific serum antibody titers, the results of rare minnow and grass carp were consistent. In addition, there was no significant residue of adjuvant observed in both fish species injected with an adjuvanted and inactivated virus. Challenge of immunized grass carp and rare minnow with the isolate HuNan1307 resulted in protection rates of 95.8% and 92.6% for attenuated virus, 81.4% and 77.7% for inactivated virus, respectively, as well as the viral load changed consistently. The results indicated that rare minnow can be used as a model for evaluation of experimental vaccines against GCHD., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Koi herpesvirus disease (KHVD) caused by the koi herpesvirus (KHV) is difficult to diagnose in live fish, presenting a challenge to the koi industry. The enzyme-linked immunosorbent assay (ELISA) method cannot be widely used to detect KHV because few commercial anti-KHV antibody exists. Here, we developed an anti-ORF132 polyclonal antibody and confirmed its reactivity via indirect immunofluorescence assay and Western blotting. A double-antibody sandwich ELISA (DAS-ELISA) was established to detect KHV, monoclonal antibody 1B71B4 against ORF92 was used as the capture antibody, and the detection antibody was the polyclonal antibody against the truncated ORF132. The lowest limit was 1.56 ng/ml KHV. Furthermore, the DAS-ELISA reacted with KHV isolates, while no cross-reactions occurred with carp oedema virus, spring viraemia of carp virus, frog virus 3 and grass carp reovirus. Two hundred koi serum samples from Guangdong, China, were used in the DAS-ELISA test, and the positive rate of the koi sera was 13%. The clinical sensitivity and specificity of the DAS-ELISA relative to the traditional PCR method were 66.7% and 97.6%, respectively. Our findings may be useful for diagnosing and preventing KHVD in koi and common carp., (© 2021 John Wiley & Sons Ltd.)

Rainbow trout (Oncorhynchus mykiss) and common carp (Cyprinus carpio) are the two most common species in traditional fish farming in Germany. Their aquaculture is threatened upon others by viruses that can cause a high mortality. Therefore, this work focuses on three viruses-viral haemorrhagic septicaemia virus, infectious hematopoietic necrosis virus and cyprinid herpesvirus 3 (CyHV-3)-that endanger these species. To prevent their spread and contain further outbreaks, it is essential to know how long they can outlast in environmental waters and what affects their infectivity outside the host. Hence, the stability of the target viruses in various water matrices was examined and compared in this work. In general, all three viruses were quite stable within sterile water samples (showing mostly ≤1 log reduction after 96 hr) but were inactivated faster and to a higher extent (up to five log steps within 96 hr) in unsterile environmental water samples. The inactivation of the viruses correlated well with the increasing bacterial load of the samples, suggesting that bacteria had the greatest effect on their stability in the examined samples. In comparison, CyHV-3 seemed to be the most sensitive and maintained its infectivity for the shortest period., (© 2020 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Vaccine immunization is currently the only effective way to prevent and control the grass carp haemorrhagic disease, and the primary pathogen in these infections is grass carp reovirus genotype II (GCRV-II) for which there is no commercial vaccine. In this study, we evaluated the safety of the GCRV-II avirulent strain GD1108 which isolated in the early stage of the laboratory through continuously passed in grass carp. The immunogenicity and protective effects were evaluated after immunization by injection and immersion. The avirulent strain GD1108 could infect and replicate in the fish which did not revert to virulence after continuous passage. No adverse side effects were observed and the vaccine strain did not spread horizontally among fish. Two routes of immunization induced high serum antibody titers of OD 450nm value were 0.79 and 0.76 and neutralization titers of 320 and 320 for the injection and immersion routes of inoculation, respectively. The expression of immune-related genes increased after immunization and the levels were statistically significant. Challenge of immunized fish with a virulent GCRV-II strain resulted in protection rates of 93.88% and 76.00% for the injection and immersion routes, respectively. The avirulent strain GD1108 revealed good safety and immunogenicity via two different inoculation routes. Although the injection route provided the best immune effect, two pathways provided protection against infection with virulent GCRV-II strains in various degrees. These results indicated that the avirulent strain GD1108 can be used for the development and application as live vaccine., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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&#95;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&#95;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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Adamek, Davies, Beck, Jordan, Becker, Mojzesz, Rakus, Rumiac, Collet, Brogden, Way, Bergmann, Zou and Steinhagen.)

Tilapia lake virus (TiLV) is a newly emerging pathogen responsible for high mortality and economic losses in the global tilapia industry. Currently, no antiviral therapy or vaccines are available for the control of this disease. The goal of the present study was to evaluate the immunological effects and protective efficacy of formaldehyde- and β-propiolactone-inactivated vaccines against TiLV in the presence and absence of the Montanide IMS 1312 VG adjuvant in tilapia. We found that β-propiolactone inactivation of viral particles generated a vaccine with a higher protection efficacy against virus challenge than did formaldehyde. The relative percent survivals of vaccinated fish at doses of 10 8 , 10 7 , and 10 6 50% tissue culture infectious dose (TCID 50 )/mL were 42.9%, 28.5%, and 14.3% in the absence of the adjuvant and 85.7%, 64.3%, and 32.1% in its presence, respectively. The vaccine generated specific IgM and neutralizing antibodies against TiLV at 3 weeks following immunization that were significantly increased after a second booster immunization. The steady state mRNA levels of the genes tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interferon γ (IFN-γ), cluster of differentiation 4 (CD4), major histocompatibility complex (MHC)-Ia, and MHC-II were all increased and indicated successful immune stimulation against TiLV. The vaccine also significantly lowered the viral loads and resulted in significant increases in survival, indicating that the vaccine may also inhibit viral proliferation as well as stimulate a protective antibody response. The β-propiolactone-inactivated TiLV vaccine coupled with the adjuvant Montanide IMS 1312 VG and booster immunizations can provide a high level of protection from virus challenge in tilapia.

Grass carp reovirus (GCRV) leads to severe hemorrhagic disease in grass carp ( Ctenopharyngodon idella ) and causes economic losses in grass carp aquaculture. Recent epidemiological investigations showed that GCRV genotype II is the dominant subtype in China. Therefore, it is very important to develop a novel vaccine for preventing diseases caused by GCRV genotype II. In this study, we employed a bac-to-bac expression system to generate GCRV-II-based virus-like particles (VLPs). Previous studies have shown that the structural proteins VP3, VP4, and VP38 encoded by the segments S3, S6, and S10 of type II GCRV are immunogenic. Hence, the GCRV-VLPs were produced by co-infection of sf9 cells with recombinant baculoviruses PFBH-VP3, PFBH-VP4, and PFBH-VP38. The expressions of VP3, VP4, and VP38 proteins in GCRV-VLPs were tested by IFA and Western blot analysis. By electron microscopic observations of ultrathin sections, purified VLPs showed that the expressed proteins are similar in shape to GCRV genotype II with a size range from 40 nm to 60 nm. The immunogenicity of GCRV-VLPs was evaluated by the injection immunization of grass carp. The analysis of serum-specific IgM antibody showed that grass carp immunized with GCRV-VLPs produced GCRV-specific antibodies. Furthermore, injection with GCRV-VLPs increased the expressions of immune-related genes (IgM, IFN, TLR3, TLR7) in the spleen and kidney. In addition, grass carp immunized with a GCRV-VLPs-based vaccine showed a relative percent survival rate (RPS) of 83.33% after challenge. The data in this study showed that GCRV-VLPs demonstrated an excellent immunogenicity and represent a promising approach for vaccine development against GCRV genotype II infection.

Introduction: White sturgeon iridovirus (WSIV) disease is caused by a virus of the eponymous family and is mostly triggered by stressful environmental conditions, i.e . high rearing density, excessive handling, or temporary loss of water. The aim of this study was to develop the most effective diagnostic method for quick and efficient confirmation or exclusion of the presence of WSIV., Material and Methods: A total of 42 samples (spleen, gills, intestine, skin, kidney, and brain) were collected from eight sturgeon ( Acipenser gueldenstaedtii and A. oxyrinchus ) aged ≤5+ farmed or caught between 2010 and 2014 in open waters (Dąbie Lake and Szczecin Lagoon). They were tested for WSIV presence using conventional PCR, qPCR, and in situ hybridisation (ISH)., Results: In gross examination, all fish appeared to be healthy. Neither species showed clinical signs typical of WSIV infection. In the majority of cases, fragments of iridoviral DNA were found using molecular methods in the kidneys, and also in the liver, gills, and skin. The detection rate using ISH was 47.37% and most commonly the brain and kidney tissues were positive. The most efficient of the methods used was real-time PCR, with 100% effectiveness in detection of WSIV DNA., Conclusion: The study demonstrates the capabilities for WSIV diagnosis available to sturgeon farmers and water administrators, indicating useful methods of adequate sensitivity as well as organs to sample in order to achieve the highest probability of viral detection., Competing Interests: Conflict of Interest Conflict of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article., (© 2020 P. Hofsoe-Oppermann et al. published by Sciendo.)

Over the last years, there has been an enormous increase in the knowledge on koi herpesvirus (KHV), koi herpesvirus disease (KHVD), pathogenesis and virus variants. Different KHV lineages have clearly been identified, possible genomic changes during replication in different cell cultures at different temperatures but also in several hosts have been identified, a persistent stage of infection has been specified and it has been shown that infection with KHV is not host specific at all, but KHVD is. Additionally, it has been shown that it is possible to combat KHVD by immunization with inactivated and attenuated live vaccines using different delivery systems but also to benefit from alternative treatments with e.g. exopolysaccharids obtained from Arthrospira platensis., (© 2020 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Introduction: The aim of the study was to determine the transmission potential of carp edema virus (CEV) and koi herpesvirus (KHV) introduced to Europe by the invasive round goby ( Neogobius melanostomus )., Material and Methods: A total of 70 round goby specimens were collected from the Szczecin Lagoon, Poland, and locations in Germany in the third and fourth quarters of 2018. The fish were analysed to detect KHV and CEV by PCR., Results: Six fish specimens were positive for the presence of KHV, while none of the gobies examined showed the presence of CEV., Conclusion: The CEV genome was detected in the goby specimens from Germany and from Poland. Considering the high pace of the spread of the round goby and its effectiveness in acquisition of new ecological niches, it should be kept out during refilling of carp ponds. Further studies should focus on experimental cohabitation of CEV-infected round gobies and specific-pathogen-free (SPF) carp to investigate the potential for active virus transfer., Competing Interests: Conflict of Interest Conflict of Interests Statements: The authors declare that there is no conflict of interests regarding the publication of this article., (© 2020 Y. Jin et al., published by Sciendo.)

We investigated differential gene expression in Tilapia infected with the Tilapia Lake virus (TiLV).We used high-throughput sequencing to identify mRNAs and miRNAs involved in TiLV infection progression We identified 25,359 differentially expressed genes that included 863 new genes. We identified 1770, 4142 and 4947 differently expressed genes comparing non-infected controls with 24 and 120 h infections and between the infected groups, respectively. These genes were enriched to 291 GO terms and 62 KEGG pathways and included immune system progress and virion genes. High-throughput miRNA sequencing identified 316 conserved miRNAs, 525 known miRNAs and 592 novel miRNAs. Furthermore, 138, 198 and 153 differently expressed miRNAs were found between the 3 groups listed above, respectively. Target prediction revealed numerous genes including erythropoietin isoform X2, double-stranded RNA-specific adenosine deaminase isoform X1, bone morphogenetic protein 4 and tapasin-related protein that are involved in immune responsiveness. Moreover, these target genes overlapped with differentially expressed mRNAs obtained from RNA-seq. These target genes were significantly enriched to GO terms and KEGG pathways including immune system progress, virion and Wnt signaling pathways. Expression patterns of differentially expressed mRNA and miRNAs were validated in 20 mRNA and 19 miRNAs by qRT-PCR. We also were able to construct a miRNA-mRNA target network that can further understand the molecular mechanisms on the pathogenesis of TiLV and guide future research in developing effective agents and strategies to combat TiLV infections in Tilapia., (Copyright © 2020. Published by Elsevier Ltd.)

Koi herpesvirus (KHV) also named Cyprinid Herpesvirus 3 (CyHV-3) is one of the most threatening pathogens affecting common carp production as well as the valued ornamental koi carp. The current commercial vaccines available are costly and potentially cause severe stress caused by live virus. KHV ORF149 gene has been proved encoding one of the main immunogenic proteins for KHV. In this study, we coupled a plasmid expression vector for ORF149 to single walled carbon nanotubes (SWCNTs) for an anti-KHV vaccine. The vaccine conferred an 81.9% protection against intraperitoneal challenge with KHV. Importantly, SWCNTs as a promising vehicle can enhanced the protective effects 33.9% over that of the naked DNA vaccine at the same dose. The protection was longer and serum antibody production, enzyme activities and immune-related gene expression were all induced in fish vaccinated with the nanotube-DNA vaccine compared with the DNA alone. Thereby, this study demonstrates that the ORF149 DNA vaccine loaded onto SWCNTs as a novel vaccine might provide an effective method of coping with KHV disease using intra-muscular vaccination., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Grass carp hemorrhagic disease caused by grass carp reovirus (GCRV) is the most important disease for grass carp aquaculture. Its typical clinical symptom is haemorrhaging, although the mechanism was remained unclear. In this study, we investigated the differences in blood parameters and histopathological features between grass carp infected with a virulent and avirulent isolates of genotype II GCRV. Infection with the virulent isolate resulted in increases in 8 routine blood and 2 serum biochemical parameters (P < 0.05); while 9 routine blood and 5 biochemical parameters were significantly decreased (P < 0.05) compared with fish infected with the avirulent isolate. The majority of these alterations were related to hemorrhage, inflammatory reactions and organic damage. The histopathologic changes were primarily vasodilation and hyperaemia in multiple organs, lymphocyte and macrophage infiltration as well as severe vacuolar degeneration in spleen, kidney and liver. The histopathology changes in fish infected with the avirulent isolate were minimal. These results indicated that the pathogenicity of GCRV was primarily reflected in destruction of the blood circulatory system and parenchymatous organs. This study lays the foundation for further research on the pathogenesis of bleeding caused by GCRV infection and the use of blood parameters and histopathology as tools for disease diagnosis., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In a search for alternative, environmentally friendly and effective disinfecting agents, a commercially available protease-Neutrase ® -was tested in this work for inactivation of koi herpesvirus (KHV) and of viral haemorrhagic septicaemia virus (VHSV). For comparison, the stability of these viral pathogens in similar configurations at various pH values and concentrations of peracetic acid or quicklime, typically used for disinfection, was tested. Therefore, virus suspensions were incubated with various concentrations of different agents for 24 hr and the titre of the remaining infectious particles was determined by virus titration. Furthermore, the treatment of both viruses, with the agents at concentrations that were previously appointed as effective, was also examined in the presence of solid material (quartz sand). All procedures investigated in this study, including the protease treatment, were able to reduce the titre of KHV and VHSV below the detection limit of the titration. Although further studies are necessary, this is the first report of the application of a protease for the inactivation of the selected fish pathogens, demonstrating the great potential of the latter for disinfection., (© 2019 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

A brain cell line (CAMB) derived from hybrid snakehead (Channa argus (♂) × Channa maculata (♀)) was established by trypsin and collagenase combined digestion. The culturing conditions and cell biological characteristics were systematically studied. For growth of the cells, M199 medium containing 10% fetal bovine serum was used and at 27 °C incubated. Based on morphological analysis, CAMB cells were confirmed to be epithelial. The cell line has been subcultured more than 80 times since its initial primary culture. Chromosome analysis revealed that CAMB cells had an abnormal chromosome number 2n = 64, whereas the chromosome number in the hybrid snakehead was 45. The suitability of CAMB for tilapia lake virus (TiLV) was demonstrated. A CPE was observed after infection with TiLV-2017A. The highest TiLV titer was observed after 12 days post infection (dpi) and reached 107.2 TCID50/mL. The virus replication was confirmed by electron microscopic observations. Additionally, immunofluorescence assay confirmed the presence of TiLV-2017A after infection of CAMB. Therefore, CAMB cells can be a useful tool for the investigation of the pathogenesis of the TiLV induced disease in tilapia., (Published by Elsevier Ltd.)

The rare minnow, Gobiocypris rarus, is small experimental fish proven to be sensitive to Grass Carp Reovirus (GCRV) infection. In present study we established a new cell (GrE) from eggs of G. rarus. GrE cells grew well at 28 °C in M199 medium containing 10% fetal bovine serum, and has been subcultured for over 70 passages. Chromosome analysis indicated that 40% of the cells were diploid 2n = 66 while the chromosome number of the fish is 2n = 50. Viral replication in GrE cells was confirmed by transmission electron microscopy, immunofluorescence assays and virus titration experiments. GrE cells and Cyenopharyngodon idellus kidney cells were infected with two GCRV genotypes while the virus copies of GCRV II in GrE peaked at 2.25 × 10 5 on 12th dpi. In vivo challenge experiments using GCRV I and II isolates at generations 1 and 20 indicated that GCRV II reproduce similar symptoms and histopathological changes of the disease in the rare minnow. These results indicated that GrE is permissive for GCRV genotype II propagation and can be used for pathogenesis studies and vaccine development of the predominant genotype of GCRV., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Viruses are able to evolve in vitro by mutations after serial passages in cell cultures, which can lead to either a loss, or an increase, of virulence. Cyprinid herpesvirus 3 (CyHV-3), a 295-kb double-stranded DNA virus, is the etiological agent of the koi herpesvirus disease (KHVD). To assess the influence of serial passages, an isolate of CyHV-3 (KHV-T) was passaged 99 times onto common carp brain (CCB) cells, and virus virulence was evaluated during passages through the experimental infections of common carp. After 78 CCB passages, the isolate was much less virulent than the original form. A comparative genomic analysis of these three forms of KHV-T (P0, P78 and P99) revealed a limited number of variations. The largest one was a deletion of 1363 bp in the predicted ORF150, which was detected in P78, but not in P99. This unexpected finding was confirmed by conventional PCR and digital PCR. The results presented here primarily suggest that, CyHV-3 evolves, at least in vitro, through an assemblage of haplotypes that alternatively become dominant or under-represented.

Recently, substantial mortality of farmed and wild tilapia caused by tilapia lake virus (TiLV) infection has been observed worldwide. However, sensitive and reliable diagnostic method is limited. A reverse transcription-loopmediated isothermal amplification (RT-LAMP) assay has been applied for the detection of TiLV nucleotide sequence. Six primers targeting two locations on the target gene based on a highly conserved sequence in the segment 1 (S1) region of the TiLV genome have been designed. The optimized RT-LAMP reaction was maintained at the isothermal condition of 63°C for 45 min. And the amplifications could be verified by turbidity or a colour change with the addition of SYBR Green I. Subsequently, RT-LAMP products could be observed by a ladder pattern following gel electrophoresis. The species-specific assay showed that the method was sensitive enough to detect as low as 1.6 copies of viral particle, and the assay was highly specific because no cross-reactivity was observed with other pathogens, and had a diagnostic sensitivity and specificity of 100% when TiLV-positive samples and non-target virus were tested. In summary, all the results demonstrate that this RT-LAMP is a rapid, effective and sensitive method for TiLV detection in tilapia aquaculture., (© 2019 John Wiley & Sons Ltd.)

Carp from breeding strains with different genetic background present diverse levels of resistance to viral pathogens. Carp strains of Asian origin, currently being treated as Cyprinus rubrofuscus L., especially Amur wild carp (AS), were proven to be more resistant to koi herpesvirus disease (KHVD; caused by cyprinid herpesvirus 3, CyHV-3) than strains originating from Europe and belonging to Cyprinus carpio L., like the Prerov scale carp (PS) or koi carp from a breed in the Czech Republic. We hypothesised that it can be associated with a higher magnitude of type I interferon (IFN) response as a first line of innate defence mechanisms against viral infections. To evaluate this hypothesis, four strains of common carp (AS, Rop, PS and koi) were challenged using two viral infection models: Rhabdovirus SVCV (spring viremia of carp virus) and alloherpesvirus CyHV-3. The infection with SVCV induced a low mortality rates and the most resistant was the Rop strain (no mortalities), whereas the PS strain was the most susceptible (survival rate of 78%). During CyHV-3 infection, Rop and AS strains performed better (survival rates of 78% and 53%, respectively) than PS and koi strains (survival rates of 35% and 10%, respectively). The evaluation of virus loads and virus replication showed significant differences between the carp strains, which correlated with the mortality rate. The evaluation of type I IFN responses showed that there were fundamental differences between the virus infection models. While responses to the SVCV were high, the CyHV-3 generally induced low responses. Furthermore, the results demonstrated that the magnitude of type I IFN responses did not correlate with a higher resistance in infected carp. In the case of a CyHV-3 infection, reduced type I IFN responses could be related to the potential ability of the virus to interfere with cellular sensing of foreign nucleic acids. Taken together, the results broaden our understanding of how common carp from different genetic strains interact with various viral pathogens., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Koi herpesvirus (KHV, Cyprinidherpesvirus 3) causes a fatal disease of koi and common carp. To obtain safe and efficacious live vaccines, we generated deletion mutants of KHV lacking the nonessential genes encoding two enzymes of nucleotide metabolism, thymidine kinase (TK, ORF55) and deoxyuridine-triphosphatase (DUT, ORF123). Since single-deletion mutants based on a KHV isolate from Israel (KHV-I) only exhibited partial attenuation (Fuchs W, Fichtner D, Bergmann SM, Mettenleiter TC. Arch Virol 2011;156 : 1059-1063), a corresponding double mutant was generated and tested in vivo, and shown to be almost avirulent but still protective. To overcome the low in vitro virus titres of KHV-I (≤10 5  p.f.u. ml -1 ), single and double TK and DUT deletions were also introduced into a cell culture-adapted KHV strain from Taiwan (KHV-T). The deletions did not affect in vitro virus replication, and all KHV-T mutants exhibited wild-type-like plaque sizes and titres exceeding 10 7  p.f.u. ml -1 , as a prerequisite for economic vaccine production. Compared to wild-type and revertant viruses, the single-deletion mutants of KHV-T were significantly attenuated in vivo, and immersion of juvenile carp in water containing high doses of the double mutant caused almost no fatalities. Nevertheless, the deletion mutants induced similar levels of KHV-specific serum antibodies to the parental wild-type virus, and conferred solid protection against disease after challenge with wild-type KHV. For the convenient differentiation of DNA samples prepared from gill swabs of carp infected with wild-type and TK-deleted KHV we developed a triplex real-time PCR. Thus, KHV-TΔDUT/TK might be suitable as a genetic DIVA vaccine in the field.

Infections with koi herpesvirus (KHV) in carp are still a severe problem worldwide. Detection and elimination of infected fish are necessary for control of the Koi herpesvirus disease (KHVD). Serum is an excellent specimen for KHV testing because of high survivability of KHV in serum and ease of collection, storage, and handling. The direct detection of fish viruses based on the sandwich ELISA has emerged as a practical and reliable means of diagnosis. Thus, it is important to create monoclonal antibodies (MAbs) against purified KHV. By using hybridoma-monoclonal antibody technology, two hybridoma cell lines secreting MAbs against the KHV were established. By Western blot and IFAT analysis, the secreted MAbs from cell line IB7IB4 and cell line 7C72F7 recognized proteins of KHV. The result demonstrated that the MAbs were highly specific and sensitive to the KHV, and can be used for monitoring the virus quantification of carp, for example, the direct KHV diagnosis by sandwich enzyme-linked immunosorbent assay(ELISA). An antigen sandwich ELISA applying the biotin-avidin system was established using the biotinylated MAb IB7IB4 and 7C72F7 to detect virus in koi sera. These MAbs did not react with any of the tested other viruses by ELISA except KHV. The detection limit of the test was 3.923ng/ml KHV. Thus, this antigen sandwich ELISA is suitable for recognition of KHV., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cyprinid herpesvirus 3 (CyHV-3) or koi herpesvirus is a global pathogen causing mass mortality in koi and common carp, against which improved vaccines are urgently needed. In this study we investigated the role of four nonessential, but immunogenic envelope glycoproteins encoded by members of the ORF25 gene family (ORF25, ORF65, ORF148 and ORF149) during CyHV-3 replication. Single deletion of ORF65 did not affect in vitro replication, and deletion of ORF148 even slightly enhanced virus growth on common carp brain (CCB) cells. Deletions of ORF25 or ORF149 led to reduced plaque sizes and virus titers, which was due to delayed entry into host cells. An ORF148/ORF149 double deletion mutant exhibited wild-type like growth indicating opposing functions of the two proteins. Electron microscopy of CCB cells infected with either mutant did not indicate any effects on virion formation and maturation in nucleus or cytoplasm, nor on release of enveloped particles. The ORF148, ORF149 and double deletion mutants were also tested in animal experiments using juvenile carp, and proved to be insufficiently attenuated for use as live virus vaccines. However, surviving fish were protected against challenge with wild-type CyHV-3, demonstrating that these antibody inducing proteins are dispensable for an efficient immune response in vivo., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The production of piscine viruses, in particular of koi herpesvirus (KHV, CyHV-3) and infectious salmon anaemia virus (ISAV), is still challenging due to the limited susceptibility of available cell lines to these viruses. A number of cell lines from different fish species were compared to standard diagnostic cell lines for KHV and ISAV regarding their capability to exhibit a cytopathic effect (CPE) and to accumulate virus. Two cell lines, so far undescribed, appeared to be useful for diagnostic purposes. Fr994, a cell line derived from ovaries of rainbow trout (Oncorhynchus mykiss), produced constantly high ISA virus (ISAV) titres and developed a pronounced CPE even at high cell passage numbers, while standard cell lines are reported to gradually loose these properties upon propagation. Another cell line isolated from the head kidney of common carp (Cyprinus carpio), KoK, showed a KHV induced CPE earlier than the standard cell line used for diagnostics. A third cell line, named Fin-4, established from the fin epithelium of rainbow trout did not promote efficient replication of tested viruses, but showed antigen sampling properties and might be useful as an in vitro model for virus uptake or phagocytosis., (© 2018 John Wiley & Sons Ltd.)

Currently, serological assays for grass carp reovirus genotype II (GCRV-II) diagnosis are not available. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against GCRV-II was developed. The structural protein VP38 of GCRV-II was used as the coating antigen. Monoclonal antibodies (mAb) against IgM of grass carp labelled with HRP were used as a secondary antibody. The antigen concentration and serum dilution were optimized using chess board titration. Furthermore, the specificity of indirect ELISA assay was confirmed by cross check with sera positive for other grass carp pathogens. In comparison with results obtained from indirect immunofluorescence assay (IFA) and Western blot by testing of 60 serum samples to evaluate the sensitivity and specificity of the ELISA, agreement between 90% and 96.7% was reached, respectively. A serological survey was performed using the assay with grass carp field serum samples. The seropositive rate of the 242 serum samples was 69.8%. In conclusion, the developed indirect ELISA is a very specific and sensitive test that will be useful for large-scale serological surveys to detect indirectly GCRV II infections as well as to monitor the changes of antibody level after immunization., (© 2018 John Wiley & Sons Ltd.)