Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) 2016/429): infection with Gyrodactylus salaris (GS).

Infection with Gyrodactylus salaris was assessed according to the criteria of the Animal Health Law (AHL), in particular, the criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as laid down in Article 9 and Article 8 for listing animal species related to infection with G. salaris. The assessment was performed following the ad hoc method for data collection and assessment previously developed by AHAW panel and already published. The outcome reported is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with an uncertain outcome. According to the assessment here performed, it is uncertain whether infection with G. salaris can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (33-70% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that Infection with G. salaris does not meet the criteria in Section 1 and 3 (Category A and C; 1-5% and 10-33% probability of fulfilling the criteria, respectively) and it is uncertain whether it meets the criteria in Sections 2, 4 and 5 (Categories B, D and E; 33-80%, 33-66% and 33-80% probability of meeting the criteria, respectively). The animal species to be listed for infection with G. salaris according to Article 8 criteria are provided.

Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) 2016/429): Bacterial kidney disease (BKD).

Bacterial kidney disease (BKD) was assessed according to the criteria of the Animal Health Law (AHL), in particular the criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as laid out in Article 9 and Article 8 for listing animal species related to BKD. The assessment was performed following the ad hoc method on data collection and assessment developed by AHAW Panel and already published. The outcome reported is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with an uncertain outcome. According to this assessment, BKD can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (66-90% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that BKD does not meet the criteria in Sections 1, 2 and 3 (Categories A, B and C; 1-5%, 33-66% and 33-66% probability of meeting the criteria, respectively) but meets the criteria in Sections 4 and 5 (Categories D and E; 66-90% and 66-90% probability of meeting the criteria, respectively). The animal species to be listed for BKD according to Article 8 criteria are provided.

Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU)2016/429): Infection with salmonid alphavirus (SAV).

Infection with salmonid alphavirus (SAV) was assessed according to the criteria of the Animal Health Law (AHL), in particular the criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as laid out in Article 9 and Article 8 for listing animal species related to infection with SAV. The assessment was performed following the ad hoc method on data collection and assessment developed by AHAW Panel and already published. The outcome reported is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with an uncertain outcome. According to the assessment, it was uncertain whether infection with salmonid alphavirus can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (50-80% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that infection with salmonid alphavirus does not meet the criteria in Section 1 (Category A; 5-10% probability of meeting the criteria) and it is uncertain whether it meets the criteria in Sections 2, 3, 4 and 5 (Categories B, C, D and E; 50-90%, probability of meeting the criteria). The animal species to be listed for infection with SAV according to Article 8 criteria are provided.

Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) 2016/429): Spring Viraemia of Carp (SVC).

Spring Viraemia of Carp (SVC) was assessed according to the criteria of the Animal Health Law (AHL), in particular the criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as in Article 9 and Article 8 for listing animal species related to SVC. The assessment was performed following the ad hoc method for data collection and assessment previously developed by the AHAW panel and already published. The outcome reported is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with an uncertain outcome. According to the assessment performed here, it is uncertain whether SVC can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (45-90% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that SVC does not meet the criteria in Section 1 (Category A; 5-33% probability of meeting the criteria) and it is uncertain whether it meets the criteria in Sections 2, 3, 4 and 5 (Categories B, C, D and E; 33-66%, 10-66%, 45-90% and 45-90% probability of meeting the criteria, respectively). The animal species to be listed for SVC according to Article 8 criteria are provided.

Species which may act as vectors or reservoirs of diseases covered by the Animal Health Law: Listed pathogens of molluscs.

Vector or reservoir species of five mollusc diseases listed in the Animal Health Law were identified, based on evidence generated through an extensive literature review, to support a possible updating of Regulation (EU) 2018/1882. Mollusc species on or in which Mikrocytos mackini, Perkinsus marinus, Bonamia exitiosa, Bonamia ostreae and Marteilia refringens were detected, in the field or during experiments, were classified as reservoir species with different levels of certainty depending on the diagnostic tests used. Where experimental evidence indicated transmission of the pathogen from a studied species to another known susceptible species, this studied species was classified as a vector species. Although the quantification of the risk of spread of the pathogens by the vectors or reservoir species was not part of the terms of reference, such risks do exist for the vector species, since transmission from infected vector species to susceptible species was proven. Where evidence for transmission from infected molluscs was not found, these were defined as reservoir. Nonetheless, the risk of the spread of the pathogens from infected reservoir species cannot be excluded. Evidence identifying conditions that may prevent transmission by vectors or reservoir mollusc species during transport was collected from scientific literature. It was concluded that it is very likely to almost certain (90-100%) that M. mackini, P. marinus, B. exitiosa B. ostreae and M. refringens will remain infective at any possible transport condition. Therefore, vector or reservoir species that may have been exposed to these pathogens in an affected area in the wild or at aquaculture establishments or through contaminated water supply can possibly transmit these pathogens. For transmission of M. refringens, the presence of an intermediate host, a copepod, is necessary.

Species which may act as vectors or reservoirs of diseases covered by the Animal Health Law: Listed pathogens of crustaceans.

Vector or reservoir species of three diseases of crustaceans listed in the Animal Health Law were identified based on evidence generated through an extensive literature review, to support a possible updating of Regulation (EU) 2018/1882. Crustacean species on or in which Taura syndrome virus (TSV), Yellow head virus (YHV) or White spot syndrome virus (WSSV) were identified, in the field or during experiments, were classified as reservoir species with different levels of certainty depending on the diagnostic tests used. Where experimental evidence indicated transmission of the pathogen from a studied species to another known susceptible species, the studied species was classified as vector species. Although the quantification of the risk of spread of the pathogens by the vectors or reservoir species was not part of the terms of reference, such risks do exist for the vector species, since transmission from infected vector species to susceptible species was proven. Where evidence for transmission from infected crustaceans was not found, these were defined as reservoirs. Nonetheless, the risk of the spread of the pathogens from infected reservoir species cannot be excluded. Evidence identifying conditions that may prevent transmission by vectors during transport was collected from scientific literature. It was concluded that it is very likely to almost certain (90-100%) that WSSV, TSV and YHV will remain infective at any possible transport condition. Therefore, vector or reservoir species that may have been exposed to these pathogens in an affected area in the wild or aquaculture establishments or by water supply can possibly transmit WSSV, TSV and YHV.

Species which may act as vectors or reservoirs of diseases covered by the Animal Health Law: Listed pathogens of fish.

Vector or reservoir species of five fish diseases listed in the Animal Health Law were identified, based on evidence generated through an extensive literature review (ELR), to support a possible updating of Regulation (EU) 2018/1882. Fish species on or in which highly polymorphic region-deleted infectious salmon anaemia virus (HPR∆ ISAV), Koi herpes virus (KHV), epizootic haematopoietic necrosis virus (EHNV), infectious haematopoietic necrosis virus (IHNV) or viral haemorrhagic septicaemia virus (VHSV) were detected, in the field or during experiments, were classified as reservoir species with different levels of certainty depending on the diagnostic tests used. Where experimental evidence indicated transmission of the pathogen from a studied species to another known susceptible species, the studied species was classified as a vector species. Although the quantification of the risk of spread of the pathogens by the vectors or reservoir species was not part of the terms or reference, such risks do exist for the vector species, since transmission from infected vector species to susceptible species was proven. Where evidence for transmission from infected fish was not found, these were defined as reservoirs. Nonetheless, the risk of the spread of the pathogens from infected reservoir species cannot be excluded. Evidence identifying conditions that may prevent transmission by vectors or reservoir fish species during transport was collected from scientific literature. For VHSV, IHNV or HPR∆ ISAV, it was concluded that under transport conditions at temperatures below 25°C, it is likely (66-90%) they will remain infective. Therefore, vector or reservoir species that may have been exposed to these pathogens in an affected area in the wild, aquaculture establishments or through water supply can possibly transmit VHSV, IHNV or HPR∆ ISAV into a non-affected area when transported at a temperature below 25°C. The conclusion was the same for EHN and KHV; however, they are likely to remain infective under all transport temperatures.

Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) No 2016/429): infectious pancreatic necrosis (IPN).

Infectious pancreatic necrosis (IPN) was assessed according to the criteria of the Animal Health Law (AHL), in particular, the criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as in Article 9, and Article 8 for listing animal species related to IPN. The assessment was performed following a methodology previously published. The outcome reported is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with an uncertain outcome. According to the assessment here performed, it is uncertain whether IPN can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (50-90% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that IPN does not meet the criteria in Section 1 (Category A; 0-1% probability of meeting the criteria) and it is uncertain whether it meets the criteria in Sections 2, 3, 4 and 5 (Categories B, C, D and E; 33-66%, 33-66%, 50-90% and 50-99% probability of meeting the criteria, respectively). The animal species to be listed for IPN according to Article 8 criteria are provided.

Pathogenicity of two lineages of infectious hematopoietic necrosis virus (IHNV) to farmed rainbow trout (Oncorhynchus mykiss) in South Korea.

In May 2015, a high mortality event in farmed rainbow trout occurred in Jeollabuk-do province in Korea. Histopathological analysis revealed necrosis in the kidney, liver, branchial arch, and gills of moribund fish, and infectious hematopoietic necrosis virus (IHNV) was detected in the lesions by immunohistochemistry. Cytopathic effects were observed in EPC, FHM, and RTG-2 cell lines after inoculation with kidney and spleen tissues and IHNV was detected by reverse transcription polymerase chain reaction (PCR). The amplified PCR product was sequenced, and phylogenetic analysis placed IHNV in the JRt Nagano group. Both in vivo and in vitro trials were performed to compare the virulence properties between RtWanju15 isolate, which causes 100% mortality in imported fry, and a previous isolate RtWanju09 of the JRt Shizuoka group isolated from eggs of healthy broodfish. In vivo challenge with high dose on specific pathogen free (SPF) rainbow trout fry performed in Denmark with isolates RtWanju09, RtWanju15 and DF04/99 isolates showed a survival rates of 60%, 37.5% and 52.5% (average), respectively without statistical difference. The replication efficiency of the two isolates in the in vitro challenge was similar.

Epitope mapping of the monoclonal antibody IP5B11 used for detection of viral haemorrhagic septicaemia virus facilitated by genome sequencing of carpione novirhabdovirus.

The monoclonal antibody (mAb) IP5B11, which is used worldwide for the diagnosis of viral haemorrhagic septicaemia (VHS) in fish, reacts with all genotypes of VHS virus (VHSV). The mAb exceptionally also reacts with the carpione rhabdovirus (CarRV). Following next generation genome sequencing of CarRV and N protein sequence alignment including five kinds of fish novirhabdoviruses, the epitope recognized by mAb IP5B11 was identified. Dot blot analysis confirmed the epitope of mAb IP5B11 to be associated with the region N219 to N233 of the N protein of VHSV. Phylogenetic analysis identified CarRV as a new member of the fish novirhabdoviruses.

Decreased water temperature enhance Piscine orthoreovirus genotype 3 replication and severe heart pathology in experimentally infected rainbow trout.

Piscine orthoreovirus genotype 3 (PRV-3) was first discovered in Denmark in 2017 in relation to disease outbreaks in rainbow trout (Oncorhynchus mykiss). While the virus appears to be widespread in farmed rainbow trout, disease outbreaks associated with detection of PRV-3 have only occurred in recirculating aquaculture systems, and has predominantly been observed during the winter months. To explore the possible effects of water temperature on PRV-3 infection in rainbow trout, an in vivo cohabitation trial was conducted at 5, 12, and 18°C. For each water temperature, a control tank containing mock-injected shedder fish and a tank with PRV-3 exposed fish were included. Samples were collected from all experimental groups every 2nd week post challenge (WPC) up until trial termination at 12 WPC. PRV-3 RNA load measured in heart tissue of cohabitants peaked at 6 WPC for animals maintained at 12 and 18°C, while it reached its peak at 12 WPC in fish maintained at 5°C. In addition to the time shift, significantly more virus was detected at the peak in fish maintained at 5°C compared to 12 and 18°C. In shedders, fish at 12 and 18°C cleared the infection considerably faster than the fish at 5°C: while shedders at 18 and 12°C had cleared most of the virus at 4 and 6 WPC, respectively, high virus load persisted in the shedders at 5°C until 12 WPC. Furthermore, a significant reduction in the hematocrit levels was observed in the cohabitants at 12°C in correlation with the peak in viremia at 6 WPC; no changes in hematocrit was observed at 18°C, while a non-significant reduction (due to large individual variation) trend was observed at cohabitants held at 5°C. Importantly, isg15 expression was positively correlated with PRV-3 virus load in all PRV-3 exposed groups. Immune gene expression analysis showed a distinct gene profile in PRV-3 exposed fish maintained at 5°C compared to 12 and 18°C. The immune markers mostly differentially expressed in the group at 5°C were important antiviral genes including rigi, ifit5 and rsad2 (viperin). In conclusion, these data show that low water temperature allow for significantly higher PRV-3 replication in rainbow trout, and a tendency for more severe heart pathology development in PRV-3 injected fish. Increased viral replication was mirrored by increased expression of important antiviral genes. Despite no mortality being observed in the experimental trial, the data comply with field observations of clinical disease outbreaks during winter and cold months.

Characterization of a Novel Infectious Pancreatic Necrosis Virus (IPNV) from Genogroup 6 Identified in Sea Trout (Salmo trutta) from Lake Vänern, Sweden.

In November 2016, infectious pancreatic necrosis virus (IPNV) was isolated from a broodstock female of landlocked sea trout (Salmo trutta) in Lake Vänern in Sweden. VP2 gene sequencing placed the IPNV isolate in genogroup 6, for which pathogenicity is largely unknown. Lake Vänern hosts landlocked sea trout and salmon populations that are endangered, and thus the introduction of new pathogens poses a major threat. In this study we characterized the novel isolate by conducting an infection trial on three salmonid species present in Lake Vänern, whole genome sequencing of the isolate, and prevalence studies in the wild sea trout and salmon in Lake Vänern. During the infection trial, the pathogenicity of the Swedish isolate was compared to that of a pathogenic genogroup 5 isolate. Dead or moribund fish were collected, pooled, and analyzed by cell culture to identify infected individuals. In the trial, the Swedish isolate was detected in fewer sample pools in all three species compared to the genogroup 5 isolate. In addition, the prevalence studies showed a low prevalence (0.2-0.5%) of the virus in the feral salmonids in Lake Vänern. Together the data suggest that the novel Swedish IPNV genogroup 6 isolate is only mildly pathogenic to salmonids.

Viral Haemorrhagic Septicemia Virus (VHSV) Isolated from Atlantic Herring, Clupea harengus, Causes Mortality in Bath Challenge on Juvenile Herring.

Viral hemorrhagic septicaemia virus (VHSV) has been demonstrated to cause high mortalities in a wide range of teleosts, farmed as well as wild. In Europe, VHSV of genotypes Ib, Id, II, and III have been detected in wild fish, including Atlantic herring Clupea harengus, but disease outbreaks have not been observed in Atlantic herring and the effects on wild stocks are not well documented. Here, we have tested two VHSV isolates from herring (genotypes Ib and III, from the western coasts of Norway and Denmark, respectively) in a challenge experiment with herring (mean weight 2.59 g, SD 0.71 g) caught on the west coast of Denmark. The Norwegian genotype Ib isolate (NO-F-CH/2009) showed an accumulated mortality of 47% compared to 6% mortality with the Danish genotype III isolate 4p168 and zero in the unchallenged control group. In both groups, we found positive rt-RT-PCR and positive immunohistochemistry of VHSV from days 6 and 8 onward. With both isolates, the organs mainly affected were the heart and kidney. The results demonstrate the susceptibility of Atlantic herring to VHSV, and both genotypes gave pathological findings in several organs. Genotype III showed a low mortality rate, and the importance of this genotype for herring is therefore not determined. Genotype Ib showed both high prevalence and mortality, and this genotype is therefore likely to have a negative effect on wild Atlantic herring stocks. Further examinations to determine how VHSV can affect wild Atlantic herring stocks are needed.

First confirmation of salmonid alphavirus infection in rainbow trout in Bosnia and Herzegovina and Serbia.

Sleeping disease is a highly infectious viral disease caused by salmonid alphavirus subtype 2 (SAV2 FW), affecting mainly rainbow trout Oncorhynchus mykiss farmed in freshwater. During March to May 2014, disease episodes with clinical signs of sleeping disease in rainbow trout fingerlings occurred almost simultaneously in 2 trout farms located in Bosnia and Herzegovina (BiH) and Serbia. The infection of rainbow trout with SAV2 FW in 2 farms was confirmed by virus isolation and molecular methods. This is the first isolation and molecular characterization of SAV2 FW in BiH and Serbia.

Experimental infection trials with European North Atlantic ranavirus (Iridoviridae) isolated from lumpfish (Cyclopterus lumpus, L.).

European North Atlantic ranavirus (ENARV, Iridoviridae), is a ranavirus species recently isolated from lumpfish (Cyclopterus lumpus, L.), which are used as cleaner fish in Atlantic salmon (Salmo salar) farming in Northern Europe. This study aimed to investigate (1) the virulence of ENARV isolates from Ireland, Iceland and the Faroe Islands to lumpfish; (2) horizontal transmission between lumpfish; and (3) virulence to Atlantic salmon parr. Lumpfish were challenged in a cohabitation model using intraperitoneally (IP) injected shedders, and naïve cohabitants. IP challenge with isolates from Iceland (1.9 × 107 TCID50  ml-1 ) and the Faroe Islands (5.9 × 107 TCID50  ml-1 ) reduced survival in lumpfish, associated with consistent pathological changes. IP challenge with the Irish strain (8.6 × 105 TCID50  ml-1 ) did not significantly reduce survival in lumpfish, but the lower challenge titre complicated interpretation. Horizontal transmission occurred in all strains tested, but no clinical impact was demonstrated in cohabitants. Salmon parr were challenged by IP injection with the Irish isolate, no virulence or virus replication were demonstrated. A ranavirus qPCR assay, previously validated for fish ranaviruses, was first used to detect ENARV in tissues of both in lumpfish and Atlantic salmon. This study provides the first data on the assessment of virulence of ENARV isolates to lumpfish and salmon, guidelines for the diagnosis of ENARV infection, and poses a basis for further investigations into virulence markers.

Modifications of the nucleoprotein of viral haemorrhagic septicaemia virus showed gain of virulence in intraperitoneally infected rainbow trout.

Viral haemorrhagic septicaemia virus (VHSV) is the cause of an important listed disease in European rainbow trout (Oncorhynchus mykiss) aquaculture and can be present in a wide range of fish species, including marine fish, which can act as viral reservoir. Recent studies revealed putative genetic virulence markers of VHSV to rainbow trout highlighting the roles of the nucleoprotein, phosphoprotein and non-virion protein. Using reverse genetics, we produced recombinant viruses by introducing parts of or the entire nucleoprotein from a high-virulent isolate VHSV into a low-virulent backbone. Furthermore, we also made recombinant viruses by introducing residue modifications in the nucleoprotein that seem to play a role in virulence. Rainbow trout challenged with these recombinant viruses (rVHSVs) by intraperitoneal injection (IP) developed clinical signs and showed lower survival when compared to the parental rVHSV whereas fish challenged by immersion did not show clinical signs except for the high-virulent control. The mutations did not influence the viral growth in cell culture. The recombinant viruses and parental recombinant were unable to replicate and show cytopathic effect in EPC cells whereas the high-virulent control was well adapted in all the fish cell lines tested. We showed evidence that corroborates with the hypothesis that the nucleoprotein has virulence motifs associated with VHSV virulence in rainbow trout.

Technical challenges in the development of reverse genetics for a viral haemorrhagic septicaemia virus (VHSV) genotype Ib isolate: Alternative cell lines and general troubleshooting.

Several reverse genetics systems for viral haemorrhagic septicaemia virus (VHSV) have been developed over the last decade. These systems have been based on genotype Ia, IVa and IVb isolates and have used the fish cell line EPC, which is less susceptible to some VHSV isolates belonging to genotype I and genotypes II and III. While developing a reverse genetics system in our laboratories for VHSV genotype Ib, we realized that the isolate in interest (SE SVA 1033 9C) did not grow in EPC cells and it was necessary to adapt the reverse genetics protocols to the BF-2 fish cell line. This cell line is very sensitive to high temperatures and is therefore not compatible with the original protocols based on the use of recombinant vaccinia virus (vTF7-3) as a provider of the T7 RNA polymerase (T7-RNAP) to the system, which includes incubation periods at 37 °C. Transfection efficiency was assessed in BF-2 cells using a reporter plasmid and it showed to be highest when using Lipofectamine™ 3000 compared to other transfection reagents. A luciferase assay was performed to determine the optimal activity of T7-RNAP in BF-2 cells with different amounts of vTF7-3. We successfully recovered recombinant VHSV (rVHSV) in BF-2 cells by reducing the incubation time at 37 °C after transfection to both 3 and 6 h. Another strategy we attempted successfully was to transfect mammalian BHK-21 cells, which are routinely used to propagate vTF7-3, and after the 37 °C incubation period, a BF-2 cell suspension was added hypothesizing that the virions formed in the transfected mammalian cells would infect the subsequently added fish cells at 15 °C incubation over the following days. We have successfully recovered rVHSV from both BHK-21 with a BF-2 cells suspension as well as a new protocol for VHSV reverse genetics in BF-2 cells has been established.

The Viral Hemorrhagic Septicemia Virus (VHSV) Markers of Virulence in Rainbow Trout (Oncorhynchus mykiss).

Viral hemorrhagic septicemia virus (VHSV) is a highly contagious virus leading to high mortality in a large panel of freshwater and marine fish species. VHSV isolates originating from marine fish show low pathogenicity in rainbow trout. The analysis of several nearly complete genome sequences from marine and freshwater isolates displaying varying levels of virulence in rainbow trout suggested that only a limited number of amino acid residues might be involved in regulating the level of virulence. Based on a recent analysis of 55 VHSV strains, which were entirely sequenced and phenotyped in vivo in rainbow trout, several amino acid changes putatively involved in virulence were identified. In the present study, these amino acid changes were introduced, alone or in combination, in a highly-virulent VHSV 23-75 genome backbone by reverse genetics. A total of 35 recombinant VHSV variants were recovered and characterized for virulence in trout by bath immersion. Results confirmed the important role of the NV protein (R116S) and highlighted a major contribution of the nucleoprotein N (K46G and A241E) in regulating virulence. Single amino acid changes in these two proteins drastically affect virus pathogenicity in rainbow trout. This is particularly intriguing for the N variant (K46G) which is unable to establish an active infection in the fins of infected trout, the main portal of entry of VHSV in this species, allowing further spread in its host. In addition, salmonid cell lines were selected to assess the kinetics of replication and cytopathic effect of recombinant VHSV and discriminate virulent and avirulent variants. In conclusion, three major virulence markers were identified in the NV and N proteins. These markers explain almost all phenotypes (92.7%) observed in trout for the 55 VHSV strains analyzed in the present study and herein used for the backward validation of virulence markers. The identification of VHSV specific virulence markers in this species is of importance both to predict the in vivo phenotype of viral isolates with targeted diagnostic tests and to improve prophylactic methods such as the development of safer live-attenuated vaccines.

Emergence and Spread of Piscine orthoreovirus Genotype 3.

Piscine orthoreovirus (PRV) is a relevant pathogen for salmonid aquaculture worldwide. In 2015, a new genotype of PRV (genotype 3, PRV-3) was discovered in Norway, and in 2017 PRV-3 was detected for first time in Denmark in association with complex disease cases in rainbow trout in recirculating aquaculture systems (RAS). To explore the epidemiology of PRV-3 in Denmark, a surveillance study was conducted in 2017 to 2019. Fifty-three farms, including both flow through and RAS, were screened for PRV-3. Of the farms examined, PRV-3 was detected in thirty-eight (71.7%), with the highest prevalence in grow-out farms. Notably, in Denmark disease outbreaks were only observed in RAS. Additionally, wild Atlantic salmon and brown trout populations were included in the screening, and PRV-3 was not detected in the three years where samples were obtained (2016, 2018, and 2019). Historical samples in the form of archived material at the Danish National Reference Laboratory for Fish Diseases were also tested for the presence of PRV-3, allowing us to establish that the virus has been present in Denmark at least since 1995. Sequence analyses of segment S1 and M2, as well as full genome analyses of selected isolates, did not reveal clear association between genetic makeup in these two segments and virulence in the form of disease outbreaks in the field.

First detection of infectious haematopoietic necrosis virus in farmed rainbow trout in North Macedonia.

Infectious haematopoietic necrosis virus (IHNV) is the causative agent of infectious haematopoietic necrosis (IHN), a disease of salmonids responsible for great economic losses. The disease occurs in most parts of the world where rainbow trout is reared but has not been previously reported in North Macedonia. In this study, 150 pooled samples in total, each consisting of organ mix of 10 freshly killed rainbow trout Oncorhynchus mykiss, were collected from 50 trout farms by the Food and Veterinary Agency of North Macedonia as part of the annual surveillance plan for IHN and viral haemorrhagic septicaemia (VHS) control. Screening of samples was done by cell culture and real-time RT-PCR (qRT-PCR). All 150 tested samples were VHS virus (VHSV) qRT-PCR negative. Two samples from different trout farms were IHNV qRT-PCR positive. On cell culture, 1 IHNV qRT-PCR positive sample caused cytopathic effect after 2 passages on EPC cells. The virus, isolated from an asymptomatic rainbow trout fry, was identified by qRT-PCR and designated as MAKIHNV1. The phylogenetic reconstruction indicates that the isolated virus belongs to the European E genogroup, more specifically within the E-1 clade, and is similar to the German, Italian and Iranian isolates. This study has revealed for the first time the presence of IHNV in rainbow trout in North Macedonia. However, it is not possible to make interpretations about the source of infection from the phylogenetic analysis, and the origin of MAKIHNV1 remains unclear.