Fish samples initially collected by local veterinarians on the common and koi carp farms in Poland between 2013 and 2015 as part of a KHV surveillance programme, when the water temperature was between 16 and 26 °C, and were also tested for CEV by qPCR. A partial 478 nucleotide fragment of the 4a gene was subsequently generated from 17 qPCR-positive common carp Cyprinus carpio samples from 36 farm sites tested during the period. Sequence alignments and analysis revealed the presence of CEV in Poland both in common carp as well as in koi carp farms, and phylogenetic analysis assigned the Polish CEV sequences into three distinct genogroups. A lineage which includes the original sequences obtained from koi carp in Japan (genogroup II) included sequences from both koi carp and common carp, and the second lineage (genogroup I) contained sequences from common carp only. A third lineage (genogroup III) which was more closely related to the genogroup II also consisted of sequences from common carp only. The latter represents a lineage of CEV not previously described in the literature.

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Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is the etiological agent of a virulent and lethal disease in common and koi carp. This study aimed to determine the genetic basis underlying the common carp immune response to the CyHV-3 virus. Two common carp lines (R3 and K) were infected with CyHV-3 by immersion. The R3 line presented a 20% higher survival rate compared to the K line and significantly lower viral loads as measured at day 3 post infection (p.i.). Microarray analysis using a common carp slides containing a number of 10,822 60-mer probes, revealed that 581 genes in line K (330 up-regulated, 251 down-regulated) and 107 genes in line R3 (77 up-regulated, 30 down-regulated), showed at least a 2-fold difference in expression at day 3 p.i. compared to day 0. Genes which showed at least a 4-fold difference in expression in both lines were selected as potential markers of a CyHV-3 infection in common carp. Additionally, 76 genes showed at least 2-fold differentially expression between K and R3 lines at day 3 p.i. Significantly higher expression of several immune-related genes including number of those which are involve in pathogen recognition, complement activation, MHC class I-restricted antigen presentation and development of adaptive mucosal immunity was noted in more resistant R3 line. Further real-time PCR based analysis provided evidence for higher activation of CD8(+) T cells in R3 line. This study uncovered wide array of immune-related genes involved into antiviral response of common carp toward CyHV-3. It is also demonstrated that the outcome of this severe disease in large extent could be controlled by genetic factors of the host.

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22Highlights 24 1. Differences in mortality rates during SVCV and CyHV-3 infections were recorded in carp 25 strains. 26 2. The higher resistance of the Rop strain was related to lower virus load and replication. 27 3. The magnitude of type I IFN response was not positively correlated with survival. 28 4. CyHV-3 has an ability to limit IFN response induced by sensing viral DNA by cells. 29 30 Abstract 34 Carp from breeding strains with different genetic background present diverse levels of 35 resistance to viral pathogens. Carp strains of Asian origin, currently being treated as Cyprinus 36 rubrofuscus L., especially Amur wild carp (AS), were proven to be more resistant to koi 37 herpesvirus disease (KHVD; caused by cyprinid herpesvirus 3, CyHV-3) than strains originating 38 from Europe and belonging to Cyprinus carpio L., like the Prerov scale carp (PS) or koi carp 39 from a breed in the Czech Republic. We hypothesised that it can be associated with a higher 40 magnitude of type I interferon (IFN) response as a first line of innate defence mechanisms against 41 viral infections. To evaluate this hypothesis, four strains of common carp (AS, Rop, PS and koi) 42were challenged using two viral infection models: Rhabdovirus SVCV (spring viremia of carp 43 virus) and alloherpesvirus CyHV-3. Infection experiments confirmed significant differences in 44 mortality between the analysed carp strains. The infection with SVCV induced a low mortality 45 rate and the most resistant was the Rop strain (no mortalities), whereas the PS strain was the most 46 susceptible (survival rate of 78%). During CyHV-3 infection, Rop and AS strains performed 47 better (survival rates of 78% and 53%, respectively) than PS and koi strains (survival rates of 48 35% and 10%, respectively). The evaluation of virus loads and virus replication showed 49 significant differences between the carp strains, which correlated with the mortality rate. The 50 evaluation of type I IFN responses showed that there were fundamental differences between the 51 virus infection models. While responses to the SVCV were high, the CyHV-3 generally induced 52 low responses. Furthermore, the results demonstrated that the magnitude of type I IFN responses 53 did not correlate with a higher resistance in infected carp. In the case of a CyHV-3 infection, 54 reduced type I IFN responses could be related to the potential ability of the virus to interfere with 55 cellular sensing of foreign nucleic acids. Taken together, the results broaden our understanding of 56 how common carp from different genetic lines interact with various viral pathogens. 57 monitoring the development of the infection in four tissues and subsequently measuring type I 113 IFN responses. Further in vitro studies were performed to better characterise anti-IFN actions of 114 CyHV-3. 115 116 117 For the infection experiments, the carp were transported to the National Veterinary 131Research Institute in Pulawy, Poland at a mean weight of 10.3 ± 8.2 g and placed at 18 °C in a 132 flow through system two week...

During a PCR‐based CEV survey in Poland in 2015–2017, the virus was detected in many farms both in clinical and asymptomatic cases and in common as well as in koi carp (Cyprinus carpio). In order to evaluate the potential carrier role of fish species that share the same habitats with carp, an experimental trial was performed. Investigations carried out on specimens of bleak (Alburnus alburnus), crucian carp (Carassius carassius), European perch (Perca fluviatilis), Prussian carp (Carassius gibelio), roach (Rutilus rutilus) and tench (Tinca tinca) cohabited with CEV‐infected carp yielded positive results. These species of fish were experimentally cohabited with CEV‐infected common carp at a temperature of 16°C ± 1. Material from the brain, gills, spleen, kidneys, intestine and skin was investigated for the presence of CEV DNA. Similar investigations were performed with uninfected fish designated controls. Samples were tested for CEV by qPCR.

The infection of common carp and its ornamental variety, koi, with the carp edema virus (CEV) is often associated with the occurrence of a clinical disease called 'koi sleepy disease'. The disease may lead to high mortality in both koi and common carp populations. To prevent further spread of the infection and the disease, a reliable detection method for this virus is required. However, the high genetic variability of the CEV p4a gene used for PCR-based diagnostics could be a serious obstacle for successful and reliable detection of virus infection in field samples. By analysing 39 field samples from different geographical origins obtained from koi and farmed carp and from all 3 genogroups of CEV, using several recently available PCR protocols, we investigated which of the protocols would allow the detection of CEV from all known genogroups present in samples from Central European carp or koi populations. The comparison of 5 different PCR protocols showed that the PCR assays (both end-point and quantitative) developed in the Centre for Environment, Fisheries and Aquaculture Science exhibited the highest analytical inclusivity and diagnostic sensitivity. Currently, this makes them the most suitable protocols for detecting viruses from all known CEV genogroups.