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Introduction Infectious pancreatic necrosis virus belongs to the genus Aquabirnavirus and family Birnaviridae. By VP2 gene similarity, aquatic birnavirus is clustered into seven genogroups. The aim of this study was to genetically analyse IPN viruses occurring on Polish fish farms. Materials and methods Samples from freshwater fish mostly from 2012 to 2013 and from northern Poland were examined for the presence of IPN virus using isolation on cell cultures, real-time RT-PCR and RT-PCR. Fragments of 1,377 and 1,079 bp of the VP2 and VP5 genes, respectively, were sequenced, and the results were assembled into one consensus and analysed by Geneious software. The same VP2 gene region was compared and a phylogenetic tree generated by the neighbour-joining method and MEGA6 software. Results All tested Polish isolates belonged to genogroup 5, like other European Spajurup isolates. Conclusion Our findings prove that there is only one IPN virus genogroup in Poland. Polish isolates show close relationships with each other. There is a close relationship between Polish isolates and isolates from Turkey, Spain and Iran. Isolate 57 is a separate branch related to isolates from the United States and Taiwan. This points to the likelihood of past virus introduction via import of stock from those countries.

Background The mechanism of latency and the ability of the cyprinid herpesvirus 3 (CyHV-3) to establish life-long infections in carp remains poorly understood. To explain the role of miRNAs in this process we applied a range of molecular tools including high-throughput sequencing of RNA libraries constructed from the blood samples of infected fish followed by bioinformatic and functional analyses which show that CyHV-3 profoundly influences the expression of host miRNAs in vivo. Results We demonstrated the changed expression of 27 miRNAs in the clinical phase and 5 in the latent phase of infection. We also identified 23 novel, not previously reported sequences, from which 8 showed altered expressions in control phase, 10 in clinical phase and 5 in latent phase of infection. Conclusions The results of our analysis expand the knowledge of common carp microRNAs engaged during CyHV-3 infection and provide a useful basis for the further study of the mechanism of CyHV-3 induced pathology. Electronic supplementary material The online version of this article (10.1186/s12864-018-5266-9) contains supplementary material, which is available to authorized users.

The red-eared slider (RES) ranavirus (RESRV) was isolated from a free-ranging RES turtle that died with evidence of respiratory disease. The RESRV genome sequence (106,878 bp) was determined, and phylogenetic analysis revealed that it is a common midwife toad virus (CMTV) strain. This study is the first report of CMTV in RES.

Introduction Koi herpesvirus (KHV) has infected farmed common carp in Poland clinically and asymptomatically since 2004. The role of non-carp species as vectors of virus transmission is well known except for in the case of KHV. The aim was to better understand this virus’ infection and transmission pathways in common carp, looking at the potential vector role of fishes kept with them. Material and Methods Eight species were experimentally infected with KHV by immersion in a suspension at 20°C ±1 and transferred to a tank after 45 minutes. Specimens were euthanised at intervals up to 56 days post infection (dpi) and tissue was examined for KHV DNA. Surviving infected fishes were introduced at intervals, each time into a separate tank, to naïve common carp for experimental infection. These were observed daily for symptoms, sacrificed along with controls after three months, and dissected to provide tissue samples. Also fish from 14 species collected from a farm with a history of KHV were sampled from 3 to 22 months after disease was confirmed. Organ sections from single fish were collected in a single tube. Results Viral DNA was detected in tench and roach samples up to 49 dpi, but in three-spined stickleback and stone maroko samples only up to 14 dpi. Transmission of KHV to naïve carp occurred after cohabitation. KHV DNA was detected in three fish species three months after the farm outbreak. Conclusion We confirmed that grass and Prussian carp, tench, roach, and brown bullhead can transfer the virus to naïve common carp.

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.

Aleutian mink disease virus is one of the greatest threats to modern mink farming. The disease reduces fecundity and causes high mortality among kits. The aim of this study was to evaluate the effectiveness of methisoprinol in counteracting the effects of Aleutian disease, both by inhibiting replication of the virus and by mitigating the harmful effects of the disease on the fecundity and weight of infected animals. The study included 300 individuals with confirmed infection, divided according to antibody titres into three experimental groups, which received a 20% methisoprinol solution, and three control groups, which did not receive the immunostimulant. In the mink from the experimental groups, the number of copies of the genetic material of the virus in the spleens and lymph nodes was one order of magnitude lower than in the case of the control groups. Mink receiving the supplement also showed higher fecundity (on average 5.83 in the experimental groups and 4.83 in the control groups), and the weight of their offspring before slaughter was over 200 g higher. Given the lack of effective methods for immunoprophylaxis and treatment, methisoprinol supplementation can be an effective means of counteracting the effects of AMDV on persistently infected farms. Electronic supplementary material The online version of this article (10.1007/s00705-019-04375-x) contains supplementary material, which is available to authorized users.

Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is an aetiological agent of a virulent and lethal disease in common and koi carp. In this study, we examined in vitro the anti-CyHV-3 activity of acyclovir (ACV), nucleoside analogue commonly used against human herpesviruses, as well as acyclovir monophospate (ACV-MP). The cytotoxicity of the ACV and the ACV-MP for two common carp cell lines, CCB (Common carp brain) and KF1 (Koi carp fin 1), was determined by means of MTT and crystal violet assays. In subsequent studies, the concentration of 66.67 μM was applied. The ACV and the ACV-MP (66.67 μM) inhibited a cytopathic effect (CPE) induced by the CyHV-3 virus in the CCB (ACV by 66%, ACV-MP by 58%) and the KF1 (ACV by 25%, ACV-MP by 37%). The viral load measured by the means of TaqMan qPCR was reduced in a range of 67%-93% depending on the analogue, the cell line and the time of incubation. The expression of viral genes (ORF149, ORF3, ORF134 and ORF78) in CCB cells infected with the CyHV-3 was strongly downregulated within the range of 78%-91%. In summary, both the ACV and the ACV-MP can inhibit CyHV-3 replication in vitro.

The aim of this study was the determination of the susceptibility of Polish farmed redfin perch (Perca fluviatilis L.) and rainbow trout (Oncorhynchus mykiss Walbaum) to experimental infection with haematopoietic necrosis virus (EHNV). A bath challenge model was tested at two temperature ranges: 13-15°C and 20-22°C. After 7 d, the first clinical signs and mortality were observed in fish kept at these temperatures. Significantly more mortality cases were reported in the redfin perch population, reaching a maximum of 24% compared with 12% in the rainbow trout group at 20-22°C. EHNV was reisolated from redfin perch and rainbow trout tissue in cell culture and the infection was confirmed by a molecular method and histopathology during the duration of the experiment. This study revealed that fish from Polish farms can be susceptible to EHNV even at lower temperatures.

Invasiveness of alien turtles results from their impact on the functioning of the local ecosystem. It is due to predation on or competing with resident species, but also transfer of new and unknown pathogenic bacteria, viruses, parasites, or fungi. Salmonella is the most often reported microorganism, both in free-living and captive turtles. Zoonotic aspect of Salmonella spp. carriage has led to the definition of RAS (Reptile Associated Salmonellosis) acquired from domestic pet reptiles. Mycobacterium spp., Leptospira spp. and aquatic bacteria are also found in turtles. Additionally, nematode transmissions from invasive turtles to the autochthonic ones have been described. Alien turtles were less affected by parasitic invasion than animals living in a native location, but the infestation of alien parasites in native turtle species was usually more severe. Reports on viral or fungal infections in turtles are scarce. The identified knowledge gaps justify the need for research which will provide basic and systematic data on microbial threats related to alien and invasive turtles present in the natural environment of Poland. It will also give more insight in the scope and the impact of the problem on epidemiology and public health. 1. Introduction. 2. Invasive turtle species. 3. Bacteria. 3.1. Salmonella spp. 3.1.1. Reptile Associated Salmonellosis (RAS). 3.2. Mycobacteria. 3.3. Other bacteria. 4. Parasitofauna of turtles. 4.1. Invasive turtles as a source of helminth invasion of European pond turtle. 4.2. Occurrence and invasiveness of alien parasites in native and alien turtles. 5. Viral infections. 6. Mycotic infection. 7. Conclusions 1. Wstęp. 2. Inwazyjne gatunki żółwi. 3. Bakterie. 3.1. Salmonella spp. 3.1.1. Reptile Associated Salmonellosis (RAS). 3.2. Prątki. 3.3. Inne bakterie. 4. Parazytofauna żółwi. 4.1. Żółwie inwazyjne źródłem zarażenia helmintami żółwia błotnego. 4.2. Występowanie i patogenność obcych pasożytów u natywnych i obcych gatunków żółwi. 5. Infekcje wirusowe. 6. Infekcje grzybicze. 7. Podsumowanie

The aim of the study was to identify the genotype of Polish isolates of salmonid alphaviruses (SAV) and to find the origin of the virus. Samples for virus isolation included the kidneys, spleen, and liver pooled from 10 fish. A typical cytopathic effect was observed after inoculation of samples on cell lines. Total RNA was extracted from cell culture supernatant and submitted to RT-PCR with primers amplifying two informative regions of the genome: a conserved region in the E2 gene and a variable region in the nsP3 gene. The sequences revealed that the strain from Poland belonged to subtype SAV 2, indicating a very strong genetic identity with isolates from Italy and France.

Spring viremia of carp (SVC) is a disease caused by a virus belonging to the genus Vesiculovirus, family Rhabdoviridae. The SVC virus is divided into four genogroups, Ia, Ib, Ic, and Id, due to its geographical distribution. This study aimed to identify the genotype of the SVC virus circulating in Poland. Polish SVC virus isolates were propagated on EPC and FHM cell lines, and genetic material (RNA) was isolated. The virus was detected in test samples by reverse transcription, sequenced and analyzed using MEGA 6.06 software. The phylogenetic tree was constructed by the Neighbor-Joining method. The results of phylogenetic analysis revealed the presence of two genogroups of the SVC virus in Poland. Most of Polish isolates belonged to the genogroup Id, as do isolates AY196200 from the Czech Republic, Z37505 from Belgium and EF593149 from the United States. Only two Polish isolates from Silesian Voivodeship were more closely related to Chinese and US isolates belonging to the genogroup Ia. There were no isolates belonging to the genogroups Ib and Ic. Nucleotide sequence analysis revealed certain point mutations between particular isolates. Knowledge on the genetic variants of the SVC virus circulating in Poland will be useful in epizootic investigations and preventive measures to protect Polish aquaculture from new variants from the neighboring countries

A rhabdovirus was isolated in cell culture inoculated with tissue material from diseased grayling, Thymallus thymallus (L.), originating from a fish farm affected by a mortality episode in Poland. Diagnostics tests showed that the virus was not related to novirhabdoviruses known in Europe, nor to vesiculovirus-like species, except perch rhabdovirus (PRhV) with which it shared moderate serological relations. However, RT-PCR with PRhV probes gave negative results. To identify the virus, a random-priming sequence-independent single primer amplification was adopted. Surprisingly, two of the obtained sequences exhibited a high identity (>99%) with hirame rhabdovirus (HIRRV), a novirhabdovirus usually found in fish in marine Asiatic countries, for instance Japan, China and Korea. The full-length sequence of the phosphoprotein gene (P) demonstrated a higher identity of the present isolate with HIRRV from China compared with the Korean isolate. An identical viral sequence was also found in brown trout, Salmo trutta trutta L., affected by mortalities in a second farm in the same region, after a likely contamination from the grayling farm. To our knowledge, this is the first report of HIRRV in Europe, and in two hosts from fresh water that have not been described before as susceptible species.

Koi herpesvirus disease (KHVD) is an emerging disease that causes mass mortality in koi and common carp,Cyprinus carpioL. Its causative agent is Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV). Although data on the pathogenesis of this deadly virus is relatively abundant in the literature, still little is known about its genomic diversity and about the molecular mechanisms that lead to such a high virulence. In this context, we developed a new strategy for sequencing full-length CyHV-3 genomes directly from infected fish tissues. Total genomic DNA extracted from carp gill tissue was specifically enriched with CyHV-3 sequences through hybridization to a set of nearly 2 million overlapping probes designed to cover the entire genome length, using KHV-J sequence (GenBank accession numberAP008984) as reference. Applied to 7 CyHV-3 specimens from Poland and Indonesia, this targeted genomic enrichment enabled recovery of the full genomes with >99.9% reference coverage. The enrichment rate was directly correlated to the estimated number of viral copies contained in the DNA extracts used for library preparation, which varied between ∼5000 and ∼2×107. The average sequencing depth was >200 for all samples, thus allowing the search for variants with high confidence. Sequence analyses highlighted a significant proportion of intra-specimen sequence heterogeneity, suggesting the presence of mixed infections in all investigated fish. They also showed that inter-specimen genetic diversity at the genome scale was very low (>99.95% of sequence identity). By enabling full genome comparisons directly from infected fish tissues, this new method will be valuable to trace outbreaks rapidly and at a reasonable cost, and in turn to understand the transmission routes of CyHV-3.

IntroductionHirame rhabdovirus (HIRRV) is one of the four recognized species within the Novirhabdovirus genus, represented by the type species Infectious Haematopoietic Necrosis (IHNV). HIRRV was first isolated during an outbreak on cultured flounder (Paralichthys olivaceus) and ayu (Plecoglossus altivelis) in Japan (1). It was also found on other marine fish in Asia, such as stone flounder (Kareius bicoloratus) in China (2). Furthermore, it was shown to be pathogenic for a range of salmonids species, including rainbow trout, experimentally challenged in freshwater. The major clinical signs of HIRRV infection were congestion of the gonads, focal hemorrhages of the skeletal muscle and fins and ascitic fluid collection (3).We report the first description of HIRRV in Europe, isolated from grayling and brown trout in a farm in Poland.Materials & methodsThirty adult graylings (Thymallus thymallus) with clinical signs and thirty asymptomatic adult brown trouts (Salmo trutta m. fario) from the same farm in Poland were tested for the presence of novirhabdoviruses by cell culture. Pools of kidney and spleen from a maximum of 10 fish were homogenized. For virus propagation, epithelioma papulosum cyprini (EPC), fathead minnow (FHM), rainbow trout gonad (RTG) and bluegill fry (BF-2) cell lines were inoculated and incubated at 15°C. Cell cultures were collected for virus identification when cytopathic effect (CPE) appeared, usually 4 to 7 d later.Starting from RNA extracted from cell culture supernatant, a random-priming sequence-independent single primer amplification (SISPA) was adopted to search for viral sequences (4). PCR products were cloned and sequenced according to the Sanger method.Transmission studies were carried out on rainbow trout (Oncorhynchus mykiss) fry and grayling fry. Virus was propagated in EPC cells and a harvested at maximum CPE, about 4-5 days post inoculation (dpi). Experimental fish were kept in 10 l aquaria supplied with freshwater, the temperature was maintained at 10 or 12°C.ResultsAfter inoculation on various cell lines, the homogenates from graylings induced a strong cytopathic effect (CPE) after 72 hours, suggesting the presence of a virus.The virus isolated in cell culture induced mortalities on experimentally infected graylings, reaching 10-25% after 21 days. In moribund graylings, light petechiae and congestions in rump muscles and also in internal organs were observed.Although some antigenic similarities with perch rhabdovirus (PRhV) were observed, RT-PCR with several sets of generic primers amplifying all fish vesiculo-like viruses, gave consistently negative results. Therefore, we used a sequence independent single primer amplification (SISPA) strategy to obtain and identify viral genomic fragments with similarities to other viruses in GenBank. Surprisingly, of the 60 clones sequenced, two of them showed high sequence similarities (> 99%) with either the L gene or the N gene of HIRRV, a viral species that had been reported only in Japan, China and Korea till now. By amplifying specifically the P gene, we observed that the virus exhibited a higher identity with the Chinese strain compared to the Korean, suggesting that the virus was imported from China, maybe in frozen food.A specific qPCR was developed and used to demonstrate that the same virus was also present in cell culture inoculated with brown trouts extracts from the same farm.Discussion & conclusionsThis study identified for the first time the presence of HIRRV in grayling and brown trouts in a farm in fresh water in Europe. The European isolate was highly similar to two other Asiatic strains, from Korean and China. Meanwhile, the sequence of the P gene revealed a stronger similarity with the Chinese strain, which would be consistent with the hypothesis of the introduction of the virus via frozen food imported from China and used in the farm. This finding raises concerns about the spread of this virus out of Asiatic countries and its potential emergence in freshwater conditions.Any symptom was visible on the graylings and brown trouts from the affected farm, suggesting a latent infection. However, the virus, once produced in cell culture, provoked mortalities during an infectious challenge on graylings and rainbow trouts. The conditions of virulence should be further investigated to estimate the epizootic risks in Europe on grayling and other freshwater fish. It must be mentioned that at the same period of viral isolation, a massive mortality occurred on wild grayling in a river in the same region. Although no samples could be analyzed at that time, the possibility of an HIRRV outbreak is hypothesized. We now have the specific diagnostic tools for routine surveillance and investigationof any other mortality event.