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Koi Herpes Virus (KHV or Cyprinid Herpesvirus 3, CyHV-3) is among the most threatening pathogens affecting common carp production as well as the highly valuable ornamental koi carp. To date, no effective commercial vaccine is available for worldwide use. A previous study reported that three intramuscular injections with an ORF25-based DNA vaccine, led to the generation of neutralizing antibodies and conferred significant protection against an intraperitoneal challenge with KHV. In the present study, we set out to optimize an ORF25-based DNA vaccination protocol that required fewer injections and would confer protection upon a challenge that better resembled the natural route of infection. To this end, ORF25 was cloned in pcDNA3 either as a soluble protein or as a full-length transmembrane GFP-fusion protein. We tested our ORF25-based DNA vaccines in multiple vaccination trials using different doses, vaccination routes (i.m. injection and oral gavage) and challenge methods (bath and cohabitation). Furthermore, we analysed local and systemic responses to the i.m. injected DNA vaccine through histological and RT-qPCR analysis. We observed a strong protection when fish received three injections of either of the two DNA vaccines. However, this protection was observed only after bath challenge and not after cohabitation challenge. Furthermore, protection was insufficient when fish received one injection only, or received the plasmid orally. The importance of choosing a challenge model that best reflects the natural route of infection and the possibility to include additional antigens in future DNA vaccination strategies against KHV will be discussed.

This work describes the first confirmed cases of carp oedema virus disease (CEVD) in Slovakia and the Czech Republic and the phylogenetic analysis of Czech and Slovak carp oedema virus (CEV) isolates. Four cases of disease outbreak in the Czech Republic are described, the oldest dating from mid-May 2013 and one case from Slovakia dating from May 2019. In all cases, virus presence was confirmed using nested PCR. PCR products were sequenced and compared with 357-bp nucleotide sequences encoding the CEV P4a protein in GenBank. In four cases of disease outbreak (three common carp breeding facilities and one koi garden pond), CEV detected belonged to genogroup I. In one case (koi garden pond), fish were confirmed as infected with CEV from genogroup II. This work complements data on CEV occurrence in European countries and contributes to a better understanding of the pathways leading to transmission of the virus throughout Europe.

International audience; Koi herpesvirus disease (KHVD) is currently the most serious threat to global carp farming. Prevention is a sensible strategy for tackling this disease and improved genetic resistance of carp strains is a desirable breeding goal. To study the potential for multitrait selection, the objective of the current study was to estimate the genetic correlations between KHVD resistance and production traits in Amur mirror carp. A total of 1500 fingerlings from four factorial crosses of five dams and ten sires were challenged with Koi herpesvirus (KHV). Juvenile growth-related traits were collected on the same individuals before the challenge test. Production traits were measured on siblings of the challenged population at different life stages (yearling to market size). The estimated heritability for resistance to KHVD was 0.43 ± 0.08 on the observed scale and 0.72 ± 0.13 on the underlying liability scale. Most genetic correlations between KHVD resistance and important production traits were insignificant, showing that selection for improved production traits would not increase susceptibility to KHV and vice versa. However, resistance to KHVD was negatively correlated with Fulton's condition factor (FC) after the second overwintering and relative head length (RHL), relative body height (RBH) and relative body width (RBW) from the second growing season to the market size, with a more prolonged body shape of Amur mirror carp (genes from Amur wild scaly carp, Cyprinus rubrofuscus) being associated with higher KHVD resistance. Intermediate favorable genetic correlations between KHVD resistance and log-log residuals of headless carcass yield (0.37 ± 0.14) and fillet yield (0.44 ± 0.13) at market size suggested that selection for improved yields of edible body parts might indirectly lead to a slight improvement in KHVD resistance and vice versa.

The diagnostic properties of the one-step real-time reverse-transcription polymerase chain reaction assay for viral haemorrhagic septicaemia virus detection were compared to methods currently in use in the Czech Republic, namely, virus isolation using the cell culture and conventional reverse-transcription polymerase chain reaction followed by the nested polymerase chain reaction. The assays were tested on a panel of 25 archived viral haemorrhagic septicaemia isolates and 8 archived infectious haematopoietic necrosis isolates obtained from monitoring and/or outbreaks of the diseases among farmed salmonids in the Czech Republic. The ability to detect the presence of the virus in the tissues of fish was tested on additional 32 field samples collected from the rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta) and brook trout (Salvelinus fontinalis). The real-time assay showed the highest analytic sensitivity by detecting the presence of viral nucleic acid in samples with 10-7 dilution, whereas the sensitivity of the conventional polymerase chain reaction peaked at 10-5. Diagnostic specificity of both molecular assays was confirmed by absence of cross-reactivity with the infectious haematopoietic necrosis virus isolates. This, along with consistent results in the detection of the virus in the fish tissues, confirms that the one-step real-time reverse-transcription polymerase chain reaction is currently an optimal stand-alone diagnostic method for the detection of the viral haemorrhagic septicaemia virus.

The relationship between koi herpesvirus disease resistance and other production traits inferred from sibling performance in amur mirror carp. Aquaculture Europe 2019

Cyprinids are the most highly produced group of fishes globally, with common carp being one of the most valuable species of the group. Koi herpesvirus (KHV) infections can result in high levels of mortality, causing major economic losses, and is listed as a notifiable disease by the World Organisation for Animal Health. Selective breeding for host resistance has the potential to reduce morbidity and losses due to KHV. Therefore, improving knowledge about host resistance and methods of incorporating genomic data into breeding for resistance may contribute to a decrease in economic losses in carp farming. In the current study, a population of 1,425 carp juveniles, originating from a factorial cross between 40 sires and 20 dams was challenged with KHV. Mortalities and survivors were recorded and sampled for genotyping by sequencing using Restriction Site-Associated DNA sequencing (RADseq). Genome-wideassociation analyses were performed to investigate the genetic architecture of resistance to KHV. A genome-wide significant QTL affecting resistance to KHV was identified on linkage group 44, explaining approximately 7% of the additive genetic variance. Pooled whole genome resequencing of a subset of resistant (n = 60) and susceptible animals (n = 60) was performed to characterize QTL regions, including identification of putative candidate genes and functionalannotation of associated 32 polymorphisms. The TRIM25 gene was identified as a promising positional and functional candidate within the QTL region of LG 44, and a putative premature stop mutation in this gene was discovered.

International audience

International audience; Although spring viremia of carp virus (SVCV) can cause high mortalities in common carp, a commercial vaccine is not available for worldwide use. Here, we report a DNA vaccine based on the expression of the SVCV glycoprotein (G) which, when injected in the muscle even at a single low dose of 0.1 mu g DNA/g of fish, confers up to 100% protection against a subsequent bath challenge with SVCV. Importantly, to best validate vaccine efficacy, we also optimized a reliable bath challenge model closely mimicking a natural infection, based on a prolonged exposure of carp to SVCV at 15 degrees C. Using this optimized bath challenge, we showed a strong age-dependent susceptibility of carp to SVCV, with high susceptibility at young age (3 months) and a full resistance at 9 months. We visualized local expression of the G protein and associated early inflammatory response by immunohistochemistry and described changes in the gene expression of pro inflammatory cytokines, chemokines, and antiviral genes in the muscle of vaccinated fish. Adaptive immune responses were investigated by analyzing neutralizing titers against SVCV in the serum of vaccinated fish and the in vitro proliferation capacity of peripheral SVCV-specific T cells. We show significantly higher serum neutralizing titers and the presence of SVCV-specific T cells in the blood of vaccinated fish, which proliferated upon stimulation with SVCV. Altogether, this is the first study reporting on a protective DNA vaccine against SVCV in carp and the first to provide a detailed characterization of local innate as well as systemic adaptive immune responses elicited upon DNA vaccination that suggest a role not only of B cells but also of T cells in the protection conferred by the SVCV-G DNA vaccine.

Rapid antigen detection enzyme-linked immunosorbent assay (ELISA) testing of cell cultures with organ homogenate from fish, collected from farms with a predominance of common carp or in natural aquaculture in the Czech Republic between 1995 and 2008, identified piscine vesiculovirus in 27 of 178 samples. Using reverse transcription semi-nested PCR, targeting a 550 nucleotide region of the glycoprotein (G) gene, piscine vesiculovirus was confirmed in 23 of the 27 organ samples diagnosed by ELISA as infected. PCR products were amplified and sequenced from 18 isolates from common carp Cyprinus carpio (family Cyprinidae), 2 isolates from northern pike Esox lucius (family Esocidae), and 1 isolate each from Siberian sturgeon Acipenser baerii (family Acipenseridae), common barbel Barbus barbus (family Cyprinidae), and koi carp Cyprinus carpio koi (family Cyprinidae). The sequences (based on 401 nucleotides) clustered into 2 genogroups. The majority of isolates (n = 22), including those from sturgeon and pike, grouped with the spring viraemia of carp virus (SVCV) Genogroup I and Subgroup Id. The 22 isolates could be further subdivided into 2 groups: Id1 (n = 20) and Id2 (n = 2). A marker (a non-conservative nucleotide substitution) for the Id1 SVCV group was identified. It was specifically found in all sequences of Id1 isolates when testing SVCV originating from different countries. The remaining isolate from barbel, was classified in the pike fry-like rhabdovirus Genogroup IV. This is the first confirmation of natural SVCV infection in sturgeon and pike, and pike fry-like rhabdovirus infection in barbel. In the case of the pike fry-like rhabdovirus, this is also its first identification in the Czech Republic. According to the presence/absence of evident clinical signs of rhabdoviral disease in the 3 infected hosts, only the sturgeon seemed to be susceptible to the monitored rhabdovirus.

A survey was performed on ornamental fish imported into the EU to detect viral agents belonging to the genus Ranavirus. The objective was to gain knowledge of the potential for these systemic iridoviruses to gain entry into the EU via international trade in ornamental fish. A total of 208 pooled samples, representing 753 individual fish, were tested. The samples included 13 orders and 37 families, originating from different countries and continents. Tissues from fish that died during or just after transport were collected and examined by standard virological techniques in epithelioma papulosum cyprini cells, by transmission electron microscopy and by PCR for the detection of the major capsid protein and DNA polymerase gene sequences of ranaviruses. Virus was isolated from nine fish species but ranavirus was not identified in those samples. The results suggest that ranaviruses are not highly prevalent in ornamental fish imported into the EU.

The present study reports the results of virological survey in fish with an emphasis on the diseases included in the Czech National Legislation. Monitoring of fish both without symptoms and with a manifested disease was performed within this virological surveillance. Between January 1999 and December 2006, viral haemorrhagic septicaemia virus (VHSV), infectious haematopoetic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) were surveyed by the National Reference Laboratory for viral diseases on fish. Among of 3789 sample pools processed, VHSV was detected in seven cases (6× in Oncorhynchus mykiss, 1× in Salmo trutta m. fario), IHNV was detected in seven cases (6× in Oncorhynchus mykiss, and 1× in Esox lucius) and IPNV was diagnosed once in Oncorhynchus mykiss. In four cases, the viral agents were detected in ovarian fluid from broodfish without any clinical symptoms. In the remaining cases the virus was isolated from tissue homogenates. Our results show that ovarian fluid is valuable material for the detection of viral agents during the monitoring of fish diseases.

An outbreak of koi herpesvirus (KHV) infection associated with high mortality of common and koi carp was recorded in the USA and Israel in 1998. At present, this disease is viewed as one of the most signifi - cant factors that can adversely affect common and koi carp breeds. The disease has spread worldwide including European countries neighbouring with the Czech Republic (CR), i.e. Germany, Poland, and Austria. To monitor the situation in the CR, samples were collected from a total of 138 common and koi carps in seven and eight locations in CR respectively, and were examined between 2005 and 2006. Locations owned by the major produc - ers of common and koi carp were selected with respect of potential occurrence of the KHV virus. No records of increased mortality and morbidity were noticed there. Preferentially carps with non-specific symptoms of disease were sampled, often with isolated skin erosions. To obtain detailed picture about health condition of examined fish the bacteriological and haematological examinations were accomplished. The next part of the examined samples were carp and koi carp for export from professional breeders, imported koi carp and fish from breeds with increased mortality (45 and 21 fish in 2005 and 2006, respectively) The only virological testing was done in this case. The culture and PCR method, according to Gilad et al. (2002) showed negative results for virus KHV in all years. Retrospective investigation by PCR method according to Bercovier et al. (2005) showed positivity in five locations in 2005. In 2006, KHV virus was not detected in any of the selected locations. The aim of our study was to find out the presence/absence of KHV in selected locations and potential correlation of virological, bacteriological and haematological findings. The results of first testing for presence of koi herpesvirus indicates the necessity of regular KHV monitoring in the Czech Republic in the next period.

Monoclonal antibodies (MAbs) against spring viraemia of carp virus (SVCV), a severe disease in cyprinid fish, were prepared. Nine MAbs were characterised using Western blotting (WB) where all reacted with glycoprotein G, except for MAb 2E1, which showed no reactivity in WB. All nine MAbs showed specificity in an immunoperoxidase test. In ELISA assays, their titres ranged between 1:32 000 and 1:128 000. A panel of SVCV isolates from different European regions were set up and examined by sandwich ELISA assay using the MAbs at a concentration of 15 µg/ml. Only MAb 4C12/3C8 showed low sensitivity in most of the isolates at an absorbance of A 450 . Of the other MAbs, even the lowest absorbance value measured exceeded cut-off for evaluation of the whole reaction. No cross-reaction with the infectious haematopoietic necrosis virus (IHNV), viral haemorrhagic septicaemia virus (VHSV) or infectious pancreatic necrosis virus (IPNV) was demonstrated. 2E1 did not show cross-reactivity with PFRV classified in genogroup III-IV and reacted with a Czech SVCV isolate; its identity was confirmed by means of RT PCR assay. The others MAbs reacted positively with PFRV F4 reference strain, isolated from Esox lucius L. (genogroup III).

A method for purification of carp serum immunoglobulin (IgM), intended for the production of monoclonal antibodies, was described in the present study. Hybridomas that produce antibodies against IgM heavy chain were selected by ELISA method and Western blotting. Ascitic fluids were prepared and tested by the above mentioned methods, and their typing followed. Monoclonal antibody with the highest titre of antibodies against carp immunoglobulin was selected for conjugation with horseradish peroxidase. Specificity of conjugated mono- clonal antibody was tested in a panel of various fish species sera. Cross-reactivity was not detected in rainbow trout (Oncorhynchus mykiss) and eleven other fish species. Besides common carp, positive results were also found in goldfish (Carassius auratus) and bighead carp (Aristichthys nobilis), that are members of Cyprinidae family. Among fish other than Cyprinidae, positive results were also detected in sheatfish ( Silurus glanis). The sensitivity in common carp was approximately 10 ng/ml.

The first outbreaks of a disease connected with high mortality of common carp and koi carp caused by koi herpesvirus (KHV) were reported in 1998 in Israel and in the United States. Since then, several cases have been confirmed all over the world. At present, this viral disease is considered to be one of the most risky factors affecting populations of common carp and koi carp. Affected fish become disoriented and swim erratically with high breathing frequency, swollen gills and partially local skin lesions. The virus was isolated from the tissues of fish showing signs of the disease and subsequently cultured on koi fin (KF-1) cells. Electron microscopic examinations revealed morphological signs identical with viruses of the family Herpesviridae. Analysis of virion polypeptides and gene DNA showed the differences between KHV and the well-known herpesvirus of cyprinids, Herpesvirus cyprini (CHV), and Channel catfish virus (CCV). Water temperature is a factor influencing the onset and severity of disease. Fish seem most susceptible at water temperatures of 18-28°C, no morbidities occur at 13°C and 30°C. At present, diagnosis of KHV is mainly based on detection of viral DNA by PCR method.

Antigenic and adjuvant activities of microspheres and ISCOMs (immunity stimulating complexes) with incorporated envelope glycoproteins of bovine herpesvirus-1 (BHV-1) were tested in long-term (31-week) experiments in rabbits. Similar antibody responses were recorded after a single intradermal immunization with BHV-1 glycoproteins incorporated into ISCOM nanoparticles or microspheres prepared from linear co-polymers (50/50) of dl -lactic and glycolic acids. Stronger, longer and markedly biphasic antibody responses were observed in rabbits immunized with BHV-1 M- dl -LA microspheres prepared from branched oligoesters of dl -lactic acid and mannitol. Indirect evidence of the incorporation of the viral envelope proteins into any type of microspheres or ISCOMs was obtained by immunoblotting. A set of precipitation lines was detected in rabbit blood sera collected 7 weeks after the immunization with microsphere- or ISCOM-entrapped glycoproteins. A similar, but more numerous, set was found in the control serum collected from cattle after natural infection by BHV-1.

Under environmental conditions, fish can be exposed to multiple stressors including natural toxins and infectious agents at the same time. This study brings new knowledge on the effects of controlled exposure to multiple stressors in fish. The aim of this study was to test the hypothesis that influence of cyanobacterial biomass and an infection agent represented by the white spot disease can combine to enhance the effects on fish.Common carps were divided into four groups, each with 40 specimens for 20 days: control group, cyanobacterial biomass exposed group, Ichthyophthirius multifiliis-infected fish (Ich) and cyanobacterial biomass-exposed fish + Ichthyophthirius multifiliis-infected fish. During the experiment we evaluated the clinical signs, mortality, selected haematological parameters, immune parameters and toxin accumulation.There was no mortality in control fish and cyanobacterial biomass-exposed fish. One specimen died in Ichthyophthirius multifiliis-infected fish and the combined exposure resulted in the death of 13 specimens. The whole leukocyte counts (WBC) of the control group did not show any significant differences. Cyanobacteria alone caused a significant increase of the WBC on day 13 (p≤0.05) and on day 20 (p≤0.01). Also, I. multifiliis caused a significant elevation of WBC (p≤0.01) on day 20. Co-exposition resulted in WBC increased on day 13 and decrease on day 20, but the changes were not significant. It is evident from the differential leukocyte counts that while the increase of WBC in the group exposed to cyanobacteria was caused by elevation of lymphocytes, the increase in the group infected by I. multifiliis was due to the increase of myeloid cells. It well corresponds with the integral of chemiluminescence in the group infected by I. multifiliis, which is significantly elevated on day 20 in comparison with all other groups.We can confirm additive action of different agents on the immune system of fish. While single agents seemed to stimulate the immune response, the combination of both caused immunosuppression.

Mammalian naïve CD8+ T cells are activated by antigen (signal 1) and CD28 costimulation (signal 2) to undergo several rounds of cell division, but programming for survival, effector function and memory requires a third signal that can be provided by IL-12 and/or type I interferons. Functional studies indicate that the route of antigen presentation and costimulation are conserved from fish to mammals. However, the potential of IL-12 and IFN alpha beta to act as signal-3 cytokines in infections inducing a CTL response has not been examined in fish. We report the cloning of CD8 alpha and CD8 beta homologues, each present in duplicate copies and of two TCR-C alpha isoforms in European common carp. The identification of (cytotoxic) T cell marker sequences and the availability of sequences coding for the signal-3 cytokines in the same fish species, allowed us to investigate by RT-qPCR their kinetics of gene expression during viral and parasitic infection. Our results show that transcription of signal-3 cytokines occurred concomitantly with CD8 alpha beta up-regulation exclusively at 4 days post-primary viral infection. No regulation of IL-12 and IFN alpha beta was observed after parasitic infection. Our data provide evidences for an evolutionary conservation of function for IL-12 and IFN alpha beta to act as third signal during CTL activation. In addition, we suggest that a CD8 alpha 2/beta1 and a p35p40b association could be the preferred combinations for the formation of a functional CD8 co-receptor and an IL-12p70 heterodimer during viral infection. The relevance of our findings to future vaccination strategies in fish is discussed.