DETECTION OF WHITE STURGEON IRIDOVIRUS (WSIV) IN STURGEONS (ACTINOPTERYGII: ACIPENSERIFORMES: ACIPENSERIDAE) FROM AQUACULTURE FACILITIES LOCATED IN POLAND, GERMANY, AND ITALY

Background. White sturgeon iridovirus (WSIV) is one of the most serious threats for sturgeon aquaculture as the mortality in juvenile individuals can reach 95%. At the turn of the century WSIV was reported among hatcheryraised white sturgeon, Acipenser transmontanus Richardson, 1836, in North America, Russian sturgeon , Acipenser gueldenstaedtii Brandt et Ratzeburg, 1833, in northern Europe but also pallid sturgeon, Scaphirhynchus albus (Forbes et Richardson, 1905) and shovelnose sturgeon, Scaphirhynchus platorynchus (Rafinesque, 1820) in North and South Dakota. The clinical signs of infection include cessation of feeding and edematous and pale gills. Those pathological changes lead to respiratory problems of the juveniles and deterioration of their overall condition. Rapid detection of pathogen particles under aquaculture conditions is crucial, therefore the principal aim of this study was to establish a molecular method for rapid and effective detection of the WSIV. This is the first study thoroughly comparing WSIV detection methods in sturgeons cultured in Poland, Germany, and Italy. Material and methods. A total of 244 samples were collected from 82 sturgeons representing six species: beluga, Huso huso (Linnaeus, 1758); sterlet, Acipenser ruthenus Linnaeus, 1758; Siberian sturgeon, Acipenser baerii Brandt, 1869; Russian sturgeon, A. gueldenstaedtii; Atlantic sturgeon, Acipenser oxyrinchus Mitchill, 1815; and European sea sturgeon, Acipenser sturio Linnaeus, 1758, as well as five sturgeon hybrids: Siberian × Russian, Adriatic (Acipenser naccarii Bonaparte, 1836) × Siberian, Adriatic × beluga, Siberian × sterlet, and beluga × sterlet. The samples were screened for the WSIV using conventional PCR, real-time PCR, and in situ hybridization (ISH) methods. Results. In total, positive results were obtained in 231 (94.67%) out of 244 analysed samples, except for H. huso where DNA of WSIV was not identified. DNA of WSIV was most frequently detected in the kidney (97.92%), intestine (97.50%), skin (94.97%), and gills (93.29%). The most effective method for detecting DNA of WSIV in sturgeons was real-time PCR (92.62% of), PCR (90.98%), and ISH (49.24%). Conclusion. The host range of the WSIV seems to be wider than expected, however, further studies are necessary since no clinical symptoms were observed in sturgeons and their hybrids.


INTRODUCTION
Fish diseases are a major limiting factor for fish rearing, and viral diseases are responsible for the greatest economic loss (Shchelkunov et al. 2009). The knowledge of sturgeon diseases is insufficient and based primarily on the population of the white sturgeon, Acipenser transmontanus Richardson, 1836, commonly reared in North America (Hua and Wang 2005). A recent phylogenetic study showed that the viruses such as the white sturgeon iridovirus (WSIV), Missouri River sturgeon iridovirus (MRSIV), and shortnose sturgeon virus (SNSV) are only distantly related to Iridoviridae, and are included in a group actually referred to as sturgeon nucleocytoplasmic large DNA viruses (NCLDVs) in the order Megavirales, a new term not formally adopted by DOI: 10.3750/AIEP/02568 the International Committee of the Taxonomy of Viruses (Clouthier et al. 2015(Clouthier et al. , 2018. To date, approximately 10 specific viruses have been found in sturgeons, the majority of which have been detected in fish originating from Canada and the USA. Viral diseases have also been observed in Europe (Italy), where acipenserids from North America have been introduced (Ravetry et al. 2003, Kelley et al. 2005. Among viruses specific for sturgeons are adenoviruses, papovaviruses, iridoviruses, and herpesviruses (Kelley et al. 2005). From an economic point of view, white sturgeon iridovirus (WSIV) and acipenserid herpesvirus-2 (AciHV-2) are of the greatest importance because of the economic loss they may cause. The mortality of WSIV infections in juvenile individuals may reach 95% (Watson et al. 1995, Georgiadis et al. 2001, Kelley et al. 2005. Iridoviruses cause infections mainly in lower invertebrates (Chinchar et al. 2009), but amphibians, insects, and fishes can be their hosts as well. According to the IUCN and CITES data, the viruses constitute a limiting factor also for species threatened with extinction (Mao et al. 1997).
Sturgeons are hosts to a group of specific nucleocytoplasmic large DNA viruses (NCLDV) initially classified in the family Iridoviridae based on the virion morphology (Pallandre et al. 2018). The first identified virus of this group was the white sturgeon iridovirus (WSIV). The WSIV was first isolated in the 1990s from white sturgeon (A. transmontanus) cell lines and then characterized molecularly (Hedrick et al. 1990(Hedrick et al. , 1992. With the size of 120-200 nm, the virus replicates in the cytoplasm, and its genetic material is double-stranded genomic DNA. Among the primary problems facilitating the emergence of serious outbreaks of the disease are stressors such as high stock density, sudden changes in the environmental conditions (e.g., changing water temperature or oxygen content), or inadequately selected feed. Unfortunately, horizontal transmission is not the only route that can lead to infection. There is a possibility of transmitting the virus from adult individuals to offspring, i.e., vertical transmission. This transmission route is limited by the use of iodine used as a disinfectant to remove viruses from the egg sheath (LaPatra et al. 1996). The presently reported study was intended to elucidate whether after such treatment the WSIV will be sufficiently degraded to prevent its further transmission. The first significant sign of infection is the cessation of feeding by the fish, which leads to the deterioration of their overall condition. It is hypothesized that this is due to the infection of the sensory epithelium in the olfactory organ (Watson et al. 1998). Other clinical signs are edematous and pale gills, which lead to problems in oxygen uptake. A characteristic but less common sign of infection is reddening along the ventral plates which may subsequently turn into bloody petechiae (LaPatra et al. 1994).
Since this initial characterization, several viruses, related, although distinct from the WSIV, have been detected in North America and Europe, infecting various farmed sturgeon species (Kurobe et al. 2011, Clouthier et al. 2013, Ciulli et al. 2016, Bigarré et al. 2017.
The presence of iridovirus, observed to date in the white sturgeon, has also been confirmed in other species. In 1993, WSIV arrived in Northern Europe and was identified as the cause of mass kills of the Russian sturgeon, Acipenser gueldenstaedtii Brandt et Ratzeburg, 1833 (see Adkinson et al. 1998). To date, the occurrence of a virus similar to the WSIV was detected in the shovelnose sturgeon, Scaphirhynchus platorynchus (Rafinesque, 1820), Russian sturgeon, A. gueldenstaedtii), and Siberian sturgeon, Acipenser baerii Brandt, 1869 (see Ciulli et al. 2016, Chesneau 2018, pallid sturgeon, Scaphirhynchus albus (Forbes et Richardson, 1905), and sturgeons originating from the Missouri River (Clouthier et al. 2013, MacConnell et al. unpublished * ). From the mid-19th century, a rapid reduction in the population of the majority of Acipenseridae species occurred. It was caused by the developing industry, the spread of hydrotechnical installations in rivers, water pollution, intensive fishing (especially in the periods of spawning migrations), overfishing, as well as regulation of rivers associated with the deepening of riverbeds. The broadly understood anthropogenic impact and other adverse factors caused the destruction of spawning sites and an overall deterioration in the conditions of natural reproduction, resulting in the dramatic reduction and sometimes extinction of sturgeon populations (Bemis and Findeis 1994, Boreman 1997, Birstein et al. 1998). Currently, 26 species of the order Acipenseriformes are threatened with extinction. This was one of the reasons for including all sturgeon species in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) in 1998 (Williot et al. 2001, Pikitch et al. 2005, Rosenthal et al. 2006.
Acipenseriformes play a significant role in world aquaculture production due to the increasing demand for their meat and caviar, but also due to the on-going protection and restitution programs (Jennings and Zigler 2000, Chebanov and Billard 2001, Williot et al. 2001, Geßner et al. 2010, Bronzi et al. 2011, Bronzi and Rosenthal 2014. Currently, 35 countries in the world specialize in sturgeon rearing, and the main European producers are Italy, France, Germany, Poland, and Spain (Bronzi et al. 2011, Anonymous unpublished ** ). With the increasing production of this species, there is no information regarding the current spread of the WSIV across Europe, particularly in aquaculture facilities where fish do not show signs of the disease. The overall epidemiological situation is poorly understood, with different iridoviruses infecting distinct specific species of sturgeon in various regions worldwide. In particular, no full-length iridovirus genome is available, except that of FV3 (Clouthier et al. 2013, Bigarré et al. 2017.
Therefore, in light of the risk of virus introduction into rearing centres and subsequently into open waters through reintroduction material, an attempt was made to determine the prevalence of WSIV in selected sturgeon farms located in Poland, Germany, and Italy.

MATERIAL AND METHODS Fish sampling.
Within 2010-2014 a total of 244 samples were collected from 82 sturgeons that aged between 0+ and 20+ and originated from fish farms located in Poland, Germany, and Italy (Table 1). The material consisted of pieces of the spleen, gills, intestine, skin, kidney, brain samples, as well as gill swabs. The following sturgeon species were examined beluga, Huso huso (Linnaeus, 1758); sterlet, Acipenser ruthenus Linnaeus, 1758; Siberian sturgeon, A. baerii; Russian sturgeon, A. gueldenstaedtii; Atlantic sturgeon, Acipenser oxyrinchus Mitchill, 1815; and European sturgeon, Acipenser sturio Linnaeus, 1758, as well as from five hybrids: None of the fish included showed clinical symptoms typical of WSIV infection. Samples were kept at 4°C until arrival in the laboratory and then stored at -20°C or preserved in 70% isopropanol alcohol until analysed. Data concerning the names and locations of the rearing centres cannot be disclosed due to the confidentiality of such information. WSIV detection. Samples were used for DNA extraction by using the DNeasy Blood and Tissue Kit (Qiagen) according to the procedures outlined in the protocol. A polymerase chain reaction (PCR) assay was applied for detection of DNA amplicon from the WSIV genome that encodes the putative major capsid protein (MCP) gene using WS 229F/WS 245R primers and cycling conditions described by Kwak et al. (2006). Based on thermal gradient PCR, a primer annealing temperature of 55°C was established. The 25 μL PCR mixtures contained REDTaq® ReadyMix™ PCR Reaction Mix (Sigma-Aldrich, Germany), 0.2 mM of each primer pair and 1 μL of DNA template. In total, the PCR-based tests for the detection of WSIV were conducted in 6 species and 5 interspecific hybrids. The PCR products were resolved by gel electrophoresis and verified by comparison with a molecular size standard (GeneRuler 100 bp Plus DNA ladder, Thermo Fisher Scientific). Bidirectional sequencing of the obtained PCR products was ordered from Genomed, Warsaw, Poland. DNA sequence assembly and similarity analyses were completed using Geneious 8.0, BLAST and MEGA5 software (Altschul et al. 1990, Tamura et al. 2007, Kearse et al. 2012). Detection of WSIV by real-time PCR. Primers and probe for detection 75 bp of WSIV genome were designed based on MCP consensus sequence obtained during the previous step. Real-time PCR was performed using 1 μL of DNA template, 5 μL of RT 2x HS PCR MasterMix (A&A Biotechnology), 0.2 μL of 0.2 mM PHofFor (5′-CSTCAACCRCGTGCCCRA-3′), 0.2 μL of 0.2 mM PHofRev (5′-TCCCYGATGCGGACAAGT-3′), 0.13 μL of 0.13 mM TaqMan probe (5′-FAM-CTTACTGGA YGCACCTATCTTCTCT-BHQ-3′) and 3.47 μL of H 2 O DEPC. The real-time PCR reaction was carried out in a Mastercycler ep realplex 2 (Eppendorf). The thermal profile consisted of polymerase activation at 95°C for 1 min followed by 40 cycles of denaturation at 95°C at 15 s and annealing/elongation at 61°C for 1 min. In each run, positive control and negative control were included. In situ hybridization (ISH). In-situ means of a molecular probe and virus detection was conducted using a kit of reagents from Roche by PCR (Eppendorf Mastercycler) in accordance to a certified methodology developed in the Institute of Infectology in Riems (Germany). Pieces of tissue were formalin fixed and paraffin embedded according to standard protocols. Organ specimens 5-7 μm were placed on Superfrost® microscope slides (Microm International) for 18 h at 62°C, dewaxed by 2 × Rotihistol (Roth) for 10 min, followed by 2 × 100% ethanol for 10 min, and air dried. Prepared slides were framed by PapPen (Merck), treated with proteinase K (100 μg proteinase K · mL -1 ) in TE buffer (50 mM Tris, 10 mM EDTA) for 20 min at 37°C and fixed again by 95% ethanol followed by 100% ethanol for 1 min, respectively. After air drying sections were framed by Pap Pen again and, for equilibration, covered by hybridization mixture (ISH-M) containing 4 × standard saline citrate (SSC), 50% formamide, 1 × Denhardt's reagent, 250 μg yeast tRNA · mL -1 and 10% dextran sulphate and incubated for 1 h at 42°C in a humid chamber. DIG-labelled probes prepared with the primer pair (Kwak et al. 2006) were added to the sections and covered by an ordinary coverslip, placed on the in-situ plate of a thermal cycler (Eppendorf Master Gradient), and heated to 95°C for 5 min for denaturation of DNAs. Then slides were cooled on ice for 2 min and incubated overnight at 42°C in a humid chamber for hybridization. The next day, coverslips were removed by washing in 2 × SSC twice for 10 min. For removal of unspecific bound probes, slides were incubated in 0.4 × SSC at 42°C for 10 min. Sections were counterstained with Bismarck-Brown Yellow (BBY) for sharpening and contrasting a possible positive signal which becomes visible as violet-black foci in infected cells. Analysis of in situ hybridization results was conducted using a fluorescence microscope (Nikon Eclipse E-600), and after all arithmetic data were calculated using MS Excel spreadsheet.

RESULTS
In total, positive results were obtained in 231 (94.67%) out of 244 analysed samples. Only for beluga (H. huso), no positive results were obtained. Fragments of WSIV DNA were in the majority of cases found in the kidney and the intestine, representing 97.92% and 97.50% of all tests, respectively. Other two types of samples in which WSIV was commonly detected were skin (94.97%) and gill (93.29%). As regards gill swabs, positive results were obtained in 80% of the samples. Sequencing and subsequent processing steps yielded in 79 assembled sequences identified in 34 sturgeon individuals. Sequence comparisons revealed 23 single nucleotide substitutions, i.e., 15 transitions and 8 transversions. Employing realtime PCR to detect WSIV yielded positive results in 226 (92.62%) out of 244 analysed isolates (Table 2). It was found that the threshold level was not exceeded (negative results) by a total of 17 samples, representing 6.97% of all tested DNA isolates. Detailed information on the number of positive and negative results in different organs is given in Table 3.
WSIV detection based on in situ hybridization (ISH) was conducted in samples obtained from 13 individuals belonging to four species, i.e., A. ruthenus, A. baerii, A. gueldenstaedtii, A. oxyrinchus, and two hybrids: A. baerii × A. gueldenstaedtii and bester. Microscopic analyses of the specimens revealed a detection rate of 49.24%. Infected tissues were characterized by an altered structure apparent already in the histological analysis. The most noticeable changes occurred in the skin. A clear cytopathic effect (CPE) in the form of morphological changes was visible as the so-called 'balloon-like' cells (Fig.  1A, 1B). Within these structures, hybridization of the probe with WSIV DNA fragments occurred. The presence of the pathogen was also indicated by the specimen colour changed due to a reaction at the site of probe hybridization with the defined fragment of the viral genome in the tissue (Fig. 1C).

DISCUSSION
Viral infections in sturgeons and their interspecific hybrids pose a threat not only due to complicated diagnostics but also especially due to the cessation of systematic monitoring in rearing centres. This situation favours uncontrolled carrying of viral diseases between rearing centres as constitutes a threat to open waters. Introduction of stocking material under the sturgeon restitution programs is not always preceded by mandatory diagnostic tests. As shown in this study, a high risk for farmers is posed not only by A. oxyrinchus or A. sturio, but also by sturgeon hybrids such as A. naccarii × A. baerii or bester. In all the above-mentioned cases, a 100% level of WSIV infection was detected. In light of the fact that only samples collected from beluga and the beluga × A. naccarii hybrid were 100% free from the virus. Therefore, it can be presumed that there is a correlation between the resistance to WSIV and the genotype of beluga. WSIV probably penetrates the fish through the gill lamellae, as evidenced by the 'balloon-like' cells observed in the samples after in situ hybridization. The route of penetration of the virus in sturgeons is limited by the presence of scutes Sterlet, Acipenser ruthenus 6 Liver Siberian sturgeon, Acipenser baerii 14 Russian sturgeon, Acipenser gueldenstaedtii Atlantic sturgeon, Acipenser oxyrinchus Table continues on next page.
that hinder the direct penetration through the skin. This means, however, that juvenile individuals are particularly vulnerable, and their anatomical features, including skin thickness, may not be a sufficient barrier to protect them against the penetration of the virus directly from the water. Because of the high stock density in the rearing centres, and particularly the absence of natural immune mechanisms before the end of the third month of life, it can be presumed that infection originating from other fishesthe carriers (including spawning individuals)-may be permanent. As indicated by Drennan et al. (2006), vertical transmission of WSIV is possible from adult sturgeons of the A. transmontanus species to their offspring, which indicates that wild individuals are carriers of this virus. Therefore, it is not only diagnostics that poses a problem, but also the feasibility of obtaining virus-free offspring.
Adriatic sturgeon × beluga, To this end, iodophor preparations of limited efficiency were used. The study demonstrated that the offspring of not all wild sturgeons of the A. transmontanus species showed WSIV-positive results after a bath in iodophor preparations. For the practice of restitution of sturgeons and stocking initiatives, this means that there is currently no method that would guarantee to obtain virus-free material. Russian researchers (Bauer et al. 2002) indicate that no viruses have been found in the health status monitoring of sturgeons. The authors state, however, that WSIV detection in Russia is only a matter of time, increasing the frequency of virological monitoring and implementation of more effective detection methods.
According to the results of observations associated with programs of restitution and stocking of various sturgeon species into natural waters, this issue poses a threat, as currently there is no information about the possible WSIV transmission by other species. Moreover, the possible scenarios for the evolution of the WSIV genome in its adoption of new niches, i.e., replication capabilities in other species than sturgeons, are not known. This constitutes a threat to the local ichthyocoenoses and species that have not had previous contact with viruses of the family Iridoviridae.