Publications by authors named "Niels Jørgen Olesen"

25 Publications

  • Page 1 of 1

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

J Virol Methods 2021 Mar 16;292:114132. Epub 2021 Mar 16.

Department of Virus & Microbiological Special Diagnostics, Statens Serum Institut, Artillerivej 5, DK-2300, Copenhagen S, Denmark. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jviromet.2021.114132DOI Listing
March 2021

The (VHSV) Markers of Virulence in Rainbow Trout ().

Front Microbiol 2020 20;11:574231. Epub 2020 Oct 20.

Virologie et Immunologie Moléculaires (VIM), Université Paris-Saclay, Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAE), Université de Versailles Saint-Quentin-en-Yvelines, Jouy-en-Josas, France.

(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 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 phenotype of viral isolates with targeted diagnostic tests and to improve prophylactic methods such as the development of safer live-attenuated vaccines.
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http://dx.doi.org/10.3389/fmicb.2020.574231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606196PMC
October 2020

Emergence and Spread of Genotype 3.

Pathogens 2020 Oct 7;9(10). Epub 2020 Oct 7.

Unit for Fish and Shellfish Diseases, National Institute of Aquatic Resources, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.

(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.
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http://dx.doi.org/10.3390/pathogens9100823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601675PMC
October 2020

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

Dis Aquat Organ 2020 Sep 3;140:219-225. Epub 2020 Sep 3.

Veterinary Institute, Faculty of Veterinary Medicine-Skopje, Ss. Cyril and Methodius University in Skopje, Lazar Pop-Trajkov 5-7, 1000 Skopje, 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.
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http://dx.doi.org/10.3354/dao03507DOI Listing
September 2020

Characterization of ranaviruses isolated from lumpfish L. in the North Atlantic area: proposal for a new ranavirus species (European North Atlantic Ranavirus).

J Gen Virol 2020 02 20;101(2):198-207. Epub 2019 Dec 20.

Technical University of Denmark, National Institute of Aquatic Resources, Aquatic Animal Health Unit, Kgs. Lyngby, Denmark.

The commercial production of lumpfish L. is expanding with the increased demand for their use as cleaner fish, to control sea-lice numbers, at marine Atlantic salmon L. aquaculture sites throughout Northern Europe. A new ranavirus has been isolated from lumpfish at multiple locations in the North Atlantic area. First isolated in 2014 in the Faroe Islands, the virus has subsequently been found in lumpfish from Iceland in 2015 and from Scotland and Ireland in 2016. The Icelandic lumpfish ranavirus has been characterized by immunofluorescent antibody test, optimal growth conditions and transmission electron microscopy. Partial sequences of the major capsid protein gene from 12 isolates showed 99.79-100% nt identity between the lumpfish ranaviruses. Complete genome sequencing from three of the isolates and phylogenetic analysis based on the concatenated 26 iridovirus core genes suggest these lumpfish ranavirus isolates form a distinct clade with ranaviruses from cod L. and turbot L. isolated in Denmark in 1979 and 1999, respectively. These data suggest that these viruses should be grouped together as a new ranavirus species, European North Atlantic Ranavirus, which encompasses ranaviruses isolated from marine fishes in European North Atlantic waters.
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http://dx.doi.org/10.1099/jgv.0.001377DOI Listing
February 2020

The susceptibility of silver crucian carp (Carassius auratus langsdorfii) to infection with koi herpesvirus (KHV).

J Fish Dis 2019 Oct 25;42(10):1333-1340. Epub 2019 Jul 25.

World Organisation for Animal Health Reference Laboratory for KHV, Fisheries Research and Education Agency, National Research Institute of Aquaculture, Mie, Japan.

Koi herpesvirus (KHV) infections cause high mortality in carp (Cyprinus carpio). This study compared the susceptibility of silver crucian carp (Carassius auratus langsdorfii), also called ginbuna, and koi carp to KHV infection. Silver crucian carp and koi carp were challenged with KHV by both intraperitoneal injection and immersion, respectively, and kept in tanks at 22°C. All KHV-exposed koi carp died within 14 days post-infection (dpi), whereas no clinics nor mortality was observed in the KHV-exposed silver crucian carp. KHV DNA was detected in both koi and silver crucian carp shortly after infection. At 7 dpi, the copy numbers of KHV genome were increased in koi carp but decreased in silver crucian carp. Using reverse transcriptase PCR, KHV mRNA was detected in koi carp but not in silver crucian carp. Cell cultivation on common carp brain (CCB) cell samples from koi carp caused KHV-associated cytopathic effects in CCB cells. Therefore, we concluded that KHV replicated in koi carp but not in silver crucian carp and that silver crucian carp is not susceptible to infection with KHV.
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http://dx.doi.org/10.1111/jfd.13054DOI Listing
October 2019

Skin immune response of rainbow trout (Oncorhynchus mykiss) experimentally exposed to the disease Red Mark Syndrome.

Vet Immunol Immunopathol 2019 May 23;211:25-34. Epub 2019 Mar 23.

Unit for Fish and Shellfish Diseases, National Institute of Aquatic Resources, Technical University of Denmark, 2800, Kgs. Lyngby, Denmark.

Red Mark Syndrome (RMS) is a skin disease reported from farmed rainbow trout. Since the turn of the millennium it has been spreading through Europe. RMS is probably a bacterial disease caused by a Midichloria-like organism (MLO). It is non-lethal and causes little obvious changes in appetite or behavior but results in red hyperaemic skin lesions, which may lead to economic losses due to downgrading. Here we transfer RMS to naïve specific pathogen free (SPF) fish by cohabitation with RMS-affected seeder fish. During disease development we characterize local cellular immune responses and regulations of immunologically relevant genes in skin of the cohabitants by immunohistochemistry and qPCR. Skin samples from SPF controls and cohabitants (areas with and without lesions) were taken at 18, 61, 82 and 97 days post-cohabitation. Gene expression results showed that lesions had a Th1-type profile, but with concurrent high expression levels of all three classes of immunoglobulins (IgD, IgM and IgT). The marked local infiltration of IgD + cells in the skin lesions as well as a highly up-regulated expression of the genes encoding sIgD and mIgD indicate that this immunoglobulin class plays an important role in skin immunity in general and in RMS pathology in particular. The co-occurrence of an apparent B cell dominated immune reaction with a Th1-type profile suggests that the local production of antibodies is independent of the classical Th2 pathway.
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http://dx.doi.org/10.1016/j.vetimm.2019.03.008DOI Listing
May 2019

Piscine orthoreovirus subtype 3 (PRV-3) causes heart inflammation in rainbow trout (Oncorhynchus mykiss).

Vet Res 2019 Feb 18;50(1):14. Epub 2019 Feb 18.

National Institute of Aquatic Resources, Technical University of Denmark, Kongens Lyngby, Denmark.

Piscine orthoreovirus (PRV) mediated diseases have emerged throughout salmonid aquaculture. Three PRV subtypes are currently reported as causative agents of or in association with diseases in different salmonid species. PRV-1 causes heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar) and is associated with jaundice syndrome in farmed chinook salmon (Oncorhynchus tshawytscha). PRV-2 causes erythrocytic inclusion body syndrome (EIBS) in coho salmon in Japan. PRV-3 has recently been associated with a disease in rainbow trout (Oncorhynchus mykiss) characterized by anaemia, heart and red muscle pathology; to jaundice syndrome in coho salmon (Oncorhynchus kisutch). In this study, we conducted a 10-week long experimental infection trial in rainbow trout with purified PRV-3 particles to assess the causal relationship between the virus and development of heart inflammation. The monitoring the PRV-3 load in heart and spleen by RT-qPCR shows a progressive increase of viral RNA to a peak, followed by clearance without a measurable change in haematocrit. The development of characteristic cardiac histopathological findings occurred in the late phase of the trial and was associated with increased expression of CD8+, indicating cytotoxic T cell proliferation. The findings indicate that, under these experimental conditions, PRV-3 infection in rainbow trout act similarly to PRV-1 infection in Atlantic salmon with regards to immunological responses and development of heart pathology, but not in the ability to establish a persistent infection.
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http://dx.doi.org/10.1186/s13567-019-0632-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380033PMC
February 2019

Outbreak of viral haemorrhagic septicaemia (VHS) in lumpfish (Cyclopterus lumpus) in Iceland caused by VHS virus genotype IV.

J Fish Dis 2019 Jan 5;42(1):47-62. Epub 2018 Nov 5.

European Union Reference Laboratory for Fish Diseases, National Institute for Aquatic Resources, Technical University of Denmark, Copenhagen, Denmark.

A novel viral haemorrhagic septicaemia virus (VHSV) of genotype IV was isolated from wild lumpfish (Cyclopterus lumpus), brought to a land-based farm in Iceland, to serve as broodfish. Two groups of lumpfish juveniles, kept in tanks in the same facility, got infected. The virus isolated was identified as VHSV by ELISA and real-time RT-PCR. Phylogenetic analysis, based on the glycoprotein (G) gene sequences, may indicate a novel subgroup of VHSV genotype IV. In controlled laboratory exposure studies with this new isolate, there was 3% survival in the I.P. injection challenged group while there was 90% survival in the immersion group. VHSV was not re-isolated from fish challenged by immersion. In a cohabitation trial, lumpfish infected I.P. (shedders) were placed in tanks with naïve lumpfish as well as naïve Atlantic salmon (Salmo salar L.). 10% of the lumpfish shedders and 43%-50% of the cohabiting lumpfish survived after 4 weeks. 80%-92% of the Atlantic salmon survived, but no viral RNA was detected by real-time RT-PCR nor VHSV was isolated from Atlantic salmon. This is the first isolation of a notifiable virus in Iceland and the first report of VHSV of genotype IV in European waters.
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http://dx.doi.org/10.1111/jfd.12910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7379627PMC
January 2019

Virulence marker candidates in N-protein of viral haemorrhagic septicaemia virus (VHSV): virulence variability within VHSV Ib clones.

Dis Aquat Organ 2018 Mar;128(1):51-62

Tamaki Laboratory, Research Center for Fish Diseases, National Research Institute of Aquaculture, Fisheries Research Agency, 224-1 Hiruda, Tamaki, Mie 519-0423, Japan.

Four major genotypes of viral haemorrhagic septicaemia virus (VHSV), which have been isolated from many marine and freshwater fish species, are known to differ in virulence. While fast and low-cost genotyping systems based on monoclonal antibodies (MAbs) have been developed for typing of VHSV virulence, there is a need for supplementing the knowledge. In particular, 2 field isolates from viral haemorrhagic septicaemia (VHS) outbreaks in sea-reared rainbow trout Oncorhynchus mykiss in Sweden, SE-SVA-14 and SE-SVA-1033 (both genotype Ib), have yielded contradictory reactions. In the present study, upon cloning by limited dilution, both isolates appeared to be heterogeneous in terms of reactivity with nucleo (N)-protein-specific MAbs as well their gene sequences. Infection trials in rainbow trout further revealed differences in the virulence of these virus clones derived from the same primary isolate. Based on a comparative analysis of the entire genome of the clones tested, we suggest that the differences in virulence are tentatively linked to substitutions of amino acids (aa) in the N-protein region covered by aa 43-46 and aa position 168, or a combination of the two. The fact that such minor naturally occurring genetic differences affect the virulence implies that even low-virulent VHSV isolates in the marine environment should be considered as a potential threat for the trout farming industry. The described MAbs can represent useful tools for initial risk assessment of disease outbreaks in farmed trout by marine VHSV isolates.
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http://dx.doi.org/10.3354/dao03215DOI Listing
March 2018

Piscine orthoreovirus infection in Atlantic salmon (Salmo salar) protects against subsequent challenge with infectious hematopoietic necrosis virus (IHNV).

Vet Res 2018 03 13;49(1):30. Epub 2018 Mar 13.

National Veterinary Institute, Technical University of Denmark, Copenhagen, Denmark.

Infectious hematopoietic necrosis virus (IHNV) is endemic in farmed rainbow trout in continental Europe and in various salmonid fish species at the Pacific coast of North America. IHN has never occurred in European Atlantic salmon (Salmo salar) farms, but is considered as a major threat for the European salmon industry. Another virus, Piscine orthoreovirus (PRV), is widespread in the sea phase of Atlantic salmon, and is identified as the causative agent of heart and skeletal muscle inflammation. The aim of this study was to investigate the interactions between a primary PRV infection and a secondary IHNV infection under experimental conditions. A PRV cohabitation challenge was performed with Atlantic salmon. At peak of PRV viremia the fish were challenged by immersion with an IHNV genogroup E isolate. Clinical signs and morbidity were monitored. Target organs were sampled at selected time points to assess viral loads of both pathogens. Antiviral immune response and presence of histopathological findings were also investigated. Whereas the PRV-negative/IHNV positive group suffered significant decrease in survival caused by IHNV, the PRV infected groups did not suffer any morbidity and showed negligible levels of IHNV infection. Antiviral response genes were induced, as measured in spleen samples, from PRV infected fish prior to IHNV challenge. In conclusion, PRV-infection protects Atlantic salmon against IHNV infection and morbidity, most likely by inducing a protective innate antiviral response.
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http://dx.doi.org/10.1186/s13567-018-0524-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5850924PMC
March 2018

Viral haemorrhagic septicaemia virus (VHSV) remains viable for several days but at low levels in the water flea Moina macrocopa.

Dis Aquat Organ 2017 Dec;127(1):11-18

Tamaki Laboratory, Research Centre for Fish Diseases, National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, 224-1 Hiruda, Tamaki, Mie 519-0423, Japan.

Viral haemorrhagic septicaemia virus (VHSV) Genotype IVb has been isolated from amphipods belonging to the genus Diporeia, but it has yet to be established whether crustacean zooplankton act as vectors of this virus for fish species. Therefore, we evaluated the viability of infectious VHSV in the water flea Moina macrocopa. VHSV was re-isolated from replicate groups of M. macrocopa that had been immersed with 108.0, 107.0, and 105.0 TCID50 ml-1 of VHSV (DK-3592B, Genotype Ia). Furthermore, 40 M. macrocopa that had been immersed with 108.0 TCID50 ml-1 of VHSV for 72 h had VHSV titers of 102.7-104.3 TCID50. Thus, VHSV was clearly taken up by M. macrocopa and remained viable in this crustacean for several days. However, no mortality was observed over a 28 d period in rainbow trout Oncorhynchus mykiss that were fed VHSV-contaminated M. macrocopa for 14 d, and we found that the virus titer significantly decreased after a 4 h incubation with pyloric caecal extracts from rainbow trout, indicating that passage through the gut is likely to result in a significant decrease in viral titer. This may explain why consumption of prey containing low levels of VHSV did not result in clinical VHS.
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http://dx.doi.org/10.3354/dao03185DOI Listing
December 2017

Infection experiments with novel Piscine orthoreovirus from rainbow trout (Oncorhynchus mykiss) in salmonids.

PLoS One 2017 5;12(7):e0180293. Epub 2017 Jul 5.

Norwegian Veterinary Institute, Oslo & Bergen, Norway.

A new disease in farmed rainbow trout (Onchorhyncus mykiss) was described in Norway in 2013. The disease mainly affected the heart and resembled heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar L.). HSMI is associated with Piscine orthoreovirus (PRV), and a search for a similar virus in the diseased rainbow trout led to detection of a sequence with 85% similarity to PRV. This finding called for a targeted effort to assess the risk the new PRV-variant pose on farmed rainbow trout and Atlantic salmon by studying infection and disease pathogenesis, aiming to provide more diagnostic knowledge. Based on the genetic relationship to PRV, the novel virus is referred to as PRV-Oncorhynchus mykiss (PRV-Om) in contrast to PRV-Salmo salar (PRV-Ss). In experimental trials, intraperitoneally injected PRV-Om was shown to replicate in blood in both salmonid species, but more effectively in rainbow trout. In rainbow trout, the virus levels peaked in blood and heart of cohabitants 6 weeks post challenge, along with increased expression of antiviral genes (Mx and viperin) in the spleen, with 80-100% of the cohabitants infected. Heart inflammation was diagnosed in all cohabitants examined 8 weeks post challenge. In contrast, less than 50% of the Atlantic salmon cohabitants were infected between 8 and 16 weeks post challenge and the antiviral response in these fish was very low. From 12 weeks post challenge and onwards, mild focal myocarditis was demonstrated in a few virus-positive salmon. In conclusion, PRV-Om infects both salmonid species, but faster transmission, more notable antiviral response and more prominent heart pathology were observed in rainbow trout.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0180293PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497981PMC
October 2017

Screening for viral hemorrhagic septicemia virus in marine fish along the Norwegian coastal line.

PLoS One 2014 23;9(9):e108529. Epub 2014 Sep 23.

Section of Virology, National Veterinary Institute, Oslo, Norway.

Viral hemorrhagic septicemia virus (VHSV) infects a wide range of marine fish species. To study the occurrence of VHSV in wild marine fish populations in Norwegian coastal waters and fjord systems a total of 1927 fish from 39 different species were sampled through 5 research cruises conducted in 2009 to 2011. In total, VHSV was detected by rRT-PCR in twelve samples originating from Atlantic herring (Clupea harengus), haddock (Melanogrammus aeglefinus), whiting (Merlangius merlangus) and silvery pout (Gadiculus argenteus). All fish tested positive in gills while four herring and one silvery pout also tested positive in internal organs. Successful virus isolation in cell culture was only obtained from one pooled Atlantic herring sample which shows that today's PCR methodology have a much higher sensitivity than cell culture for detection of VHSV. Sequencing revealed that the positive samples belonged to VHSV genotype Ib and phylogenetic analysis shows that the isolate from Atlantic herring and silvery pout are closely related. All positive fish were sampled in the same area in the northern county of Finnmark. This is the first detection of VHSV in Atlantic herring this far north, and to our knowledge the first detection of VHSV in silvery pout. However, low prevalence of VHSV genotype Ib in Atlantic herring and other wild marine fish are well known in other parts of Europe. Earlier there have been a few reports of disease outbreaks in farmed rainbow trout with VHSV of genotype Ib, and our results show that there is a possibility of transfer of VHSV from wild to farmed fish along the Norwegian coast line. The impact of VHSV on wild fish is not well documented.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108529PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172761PMC
January 2016

Evolutionary dynamics and genetic diversity from three genes of Anguillid rhabdovirus.

J Gen Virol 2014 Nov 31;95(Pt 11):2390-2401. Epub 2014 Jul 31.

European University of Brittany, France.

Wild freshwater eel populations have dramatically declined in recent past decades in Europe and America, partially through the impact of several factors including the wide spread of infectious diseases. The anguillid rhabdoviruses eel virus European X (EVEX) and eel virus American (EVA) potentially play a role in this decline, even if their real contribution is still unclear. In this study, we investigate the evolutionary dynamics and genetic diversity of anguiillid rhabdoviruses by analysing sequences from the glycoprotein, nucleoprotein and phosphoprotein (P) genes of 57 viral strains collected from seven countries over 40 years using maximum-likelihood and Bayesian approaches. Phylogenetic trees from the three genes are congruent and allow two monophyletic groups, European and American, to be clearly distinguished. Results of nucleotide substitution rates per site per year indicate that the P gene is expected to evolve most rapidly. The nucleotide diversity observed is low (2-3 %) for the three genes, with a significantly higher variability within the P gene, which encodes multiple proteins from a single genomic RNA sequence, particularly a small C protein. This putative C protein is a potential molecular marker suitable for characterization of distinct genotypes within anguillid rhabdoviruses. This study provides, to our knowledge, the first molecular characterization of EVA, brings new insights to the evolutionary dynamics of two genotypes of Anguillid rhabdovirus, and is a baseline for further investigations on the tracking of its spread.
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http://dx.doi.org/10.1099/vir.0.069443-0DOI Listing
November 2014

Spatio-temporal risk factors for viral haemorrhagic septicaemia (VHS) in Danish aquaculture.

Dis Aquat Organ 2014 May;109(2):87-97

National Veterinary Institute, Technical University of Denmark, 8200 Århus N, Denmark.

Viral haemorrhagic septicaemia (VHS) is an economically very important fish disease in the northern hemisphere. When the VHS virus was first isolated in Denmark 50 yr ago, more than 80% of the 800 Danish fish farms were considered to be infected, but vigilant surveillance and eradication programmes led to a drastic reduction in prevalence, and finally, to complete eradication of VHS. Denmark thus obtained official status as an approved VHS-free member state within the European Union in November 2013. Data on outbreaks within the country have been collected since 1970, and here we combined these data with the geographical coordinates of fish farms to identify clusters of high disease prevalence and other risk factors. Our analyses revealed a statistically significant cluster in the southwestern part of the country, which persisted throughout the study period. Being situated within such a cluster was a significant risk factor for VHS. For freshwater rainbow trout farms situated inland, the number of upstream farms was a determining risk factor for VHS, as was distance to the nearest VHS-infected farm and year. Whether the farm used fresh or marine water in production did not have any influence on the risk of VHS, when accounting for whether the farm was situated inside a cluster of high risk. This information can be used when implementing risk-based surveillance programmes.
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http://dx.doi.org/10.3354/dao02706DOI Listing
May 2014

Susceptibility of various Japanese freshwater fish species to an isolate of viral haemorrhagic septicaemia virus (VHSV) genotype IVb.

Dis Aquat Organ 2013 Nov;107(1):1-8

Tamaki Laboratory, Aquatic Animal Health Division, National Research Institute of Aquaculture, Fisheries Research Agency, 224-1 Hiruta, Tamaki, Mie 519-0423, Japan.

Genotype IVb of viral haemorrhagic septicaemia virus (VHSV) was isolated for the first time in the Great Lakes basin in 2003, where it spread and caused mass mortalities in several wild fish species throughout the basin. In order to prevent further spreading of the disease and to assess risks of new genotypes invading new watersheds, basic microbiological information such as pathogenicity studies are essential. In this study, experimental infections were conducted on 7 indigenous freshwater fish species from Japan by immersion with a VHSV genotype IVb isolate. In Expt 1, cumulative mortalities in bluegill Lepomis macrochirus used as positive controls, Japanese fluvial sculpin Cottus pollux, and iwana Salvelinus leucomaenis pluvius were 50, 80 and 0%, respectively. In Expt 2, cumulative mortalities of 100, 100 and 10% were observed in Japanese fluvial sculpin C. pollux, Japanese rice fish Oryzias latipes and yoshinobori Rhinogobius sp., respectively. No mortality was observed in honmoroko Gnathopogon caerulescens, akaza Liobagrus reini or Japanese striped loach Cobitis biwae. VHSV was detected by RT-PCR from samples of kidney, spleen, and brain from all dead fish, and virus re-isolation by cell culture was successful from all dead fish. We detected the virus in the brain from a few surviving bluegill 50 d post exposure by both cell culture and RT-PCR. These results revealed that VHSV IVb could become a serious threat to wild freshwater fish species in Japan, and that some surviving fish might become healthy carriers of the virus.
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http://dx.doi.org/10.3354/dao02667DOI Listing
November 2013

Typing of viral hemorrhagic septicemia virus by monoclonal antibodies.

J Gen Virol 2012 Dec 12;93(Pt 12):2546-2557. Epub 2012 Sep 12.

National Veterinary Institute, Technical University of Denmark, Hangøvej 2, DK-8200 Århus N, Denmark.

Seven mAbs with specific reaction patterns against each of the four genotypes and eight subtypes of viral hemorrhagic septicemia virus (VHSV) were produced, aiming to establish an immunoassay for typing VHSV isolates according to their genotype. Among the mAbs, VHS-1.24 reacted with all genotypes except genotype Ie, whilst mAb VHS-9.23 reacted with all genotypes except genotype III. mAb VHS-3.80 reacted with genotypes Ib, Ic, Id and II. mAb VHS-7.57 reacted with genotypes II and IVa, and mAb VHS-5.18 with genotype Ib only. Interestingly, mAb VHS-3.75 reacted with all of the genotype III isolates except a rainbow trout-pathogenic isolate from the west coast of Norway, and reacted in addition with the IVb isolate, CA-NB00-01, from the east coast of the USA. Finally, mAb VHS-1.88 reacted with all genotype IVb isolates from the Great Lakes, but not with CA-NB00-01. In conclusion, we can distinguish between all four genotypes and between five of eight subtypes of VHSV by testing isolates in immunoassay using a panel of nine mAbs. By Western blotting and transfection of cell cultures, it was shown that mAb VHS-1.24 recognized an epitope on the viral phosphoprotein (P), whilst all others recognized antigenic determinants on the nucleoprotein (N). From amino acid alignments of the various genotypes and subtypes of VHSV isolates, it was possible to determine the epitope specificity of mAb VHS-1.24 to be aa 32-34 in the P-protein; the specificities of mAbs VHS-3.80, VHS-7.57 and VHS-3.75 were found to be aa 43 and 45-48, aa 117 and 121, and aa 103, 118 and 121 of the N-protein, respectively.
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http://dx.doi.org/10.1099/vir.0.043091-0DOI Listing
December 2012

First isolation and genotyping of viruses from recent outbreaks of viral haemorrhagic septicaemia (VHS) in Slovenia.

Dis Aquat Organ 2010 Oct;92(1):21-9

Virology Unit, Institute of Microbiology and Parasitology, Veterinary Faculty, University of Ljubljana, Gerbiceva 60, 1115 Ljubljana, Slovenia.

In November and December 2007, the virus causing viral haemorrhagic septicaemia (VHS) was detected in rainbow trout Oncorhynchus mykiss from 2 fish farms in Slovenia. During 2008 and 2009 the infection spread only among rainbow trout farms and 4 new outbreaks were confirmed. High mortality and clinical signs of VHS were observed among the diseased fish. VHSV was confirmed by virus isolation, immunoperoxidase test, reverse transcriptase polymerase chain reaction (RT-PCR) and phylogenetic analysis. Based on 1 complete (1524 nucleotides [nt]) and 9 partial (600 nt) glycoprotein gene nucleotide sequences, 9 VHSV isolates from the 6 VHS outbreaks were genetically closely related (99 to 100% identity), and were classified into the Subgroup I-a of Genotype I, most closely related to the German isolates Dstg21-07, Dstg36-06, and Dstg54-1-07 (99 to 100% identity). Phylogenetic analysis and epidemiological investigations confirmed that the VHS virus had been (re)introduced with imported live fish, and that subsequent outbreaks were linked to the initial infection. Our study shows that direct nucleotide sequencing of RT-PCR products, amplified from the tissue of VHSV-infected fish, represents a reliable tool for fast routine genotyping in diagnostic laboratories. This is the first report of a natural epidemic associated with VHSV infection in Slovenia since the eradication of the disease in 1977.
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http://dx.doi.org/10.3354/dao02251DOI Listing
October 2010

Comparative study of ranavirus isolates from cod (Gadus morhua) and turbot (Psetta maxima) with reference to other ranaviruses.

Arch Virol 2010 Aug 15;155(8):1261-71. Epub 2010 Jun 15.

National Veterinary Institute, Technical University of Denmark, Arhus, Denmark.

Two iridovirus isolates recovered from cod (Gadus morhua) and turbot (Psetta maxima) in Denmark were examined in parallel with a panel of other ranaviruses including frog virus 3 (FV3), the reference strain for the genus Ranavirus. The isolates were assessed according to their reactivity in immunofluoresent antibody tests (IFAT) using both homologous and heterologous antisera and their amplification in PCR using primers targeting five genomic regions. The corresponding PCR fragments were sequenced, and the sequences obtained were used in phylogenetic analysis. In addition, the pathogenicity to rainbow trout under experimental challenge conditions was investigated. The viruses were serologically and genetically closely related to highly pathogenic ranaviruses such as European catfish iridovirus (ECV), European sheatfish iridovirus (ESV) and epizootic haematopoietic necrosis virus (EHNV). The challenge trials indicate that rainbow trout fry cultured at 15 degrees C are not target species for the virus isolates in the present panel. We suggest that the two isolates belong in the genus Ranavirus and propose the name Ranavirus maxima (Rmax) for the turbot isolate.
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http://dx.doi.org/10.1007/s00705-010-0715-zDOI Listing
August 2010

Viral haemorrhagic septicaemia (VHS) outbreaks in Finnish rainbow trout farms.

Dis Aquat Organ 2006 Oct;72(3):201-11

Finnish Food Safety Authority Evira, Department of Animal Diseases and Food Safety Research, Virology Unit, Mustialankatu 3, 00790 Helsinki, Finland.

In Finland, viral haemorrhagic septicaemia virus (VHSV) was diagnosed for the first time in 2000 from 4 rainbow trout farms in brackish water. Since then the infection has spread and, by the end of 2004, VHSV had been isolated from 24 farms in 3 separate locations: 2 in the Baltic Sea and 1 in the Gulf of Finland. The pathogenicity of 3 of these isolates from 2 separate locations was analysed in infection experiments with rainbow trout fry. The cumulative mortalities induced by waterborne and intraperitoneal challenge were approximately 40 and 90 %, respectively. Pair-wise comparisons of the G and NV gene regions of Finnish VHSV isolates collected between 2000 and 2004 revealed that all isolates were closely related, with 99.3 to 100% nucleotide identity, which suggests the same origin of infection. Phylogenetic analysis revealed that they were closely related to the old freshwater isolates from rainbow trout in Denmark and to one old marine isolate from cod in the Baltic Sea, and that they were located close to the presumed ancestral source. As the Finnish isolates induce lower mortality than freshwater VHSV isolates in infection experiments, they could represent an intermediate stage of marine isolates evolving towards pathogenicity in rainbow trout.
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http://dx.doi.org/10.3354/dao072201DOI Listing
October 2006

Prevalence of viral haemorrhagic septicaemia virus in Danish marine fishes and its occurrence in new host species.

Dis Aquat Organ 2005 Sep;66(2):145-51

Danish Institute for Food and Veterinary Research, Department of Poultry, Fish and Fur Animals, Hangøvej 2, 8200 Arhus N, Denmark.

In order to analyse the occurrence of viral haemorrhagic septicaemia virus (VHSV) in the marine waters around Denmark, staff from the Danish Institute for Food and Veterinary Research participated in 5 research cruises during 1998 to 2002 as a follow-up to 4 research cruises performed in 1996 to 1997. In total, 16,655 fish were examined virologically as 3569 samples. Forty fish species and 3 invertebrate species were represented. VHSV was isolated from 133 samples representing 8 species: herring Clupea harengus, sprat Sprattus sprattus, dab Limanda limanda, flounder Platichthys flesus, plaice Pleuronectes platessa, cod Gadus morhua, sand eel Ammodytes sp. and sand goby Pomatochistus minutus. Calculations showed that VHSV was more prevalent in the Baltic Sea in an area between Zealand and the island of Bornholm and the waters surrounding Bornholm than in the Kattegat, Skagerrak and along the North Sea coast of Denmark. This is the first report on the isolation of VHSV from dab, flounder and plaice and the first publication on VHSV from sand eel from Europe and sand goby.
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http://dx.doi.org/10.3354/dao066145DOI Listing
September 2005

Experimental infection of rainbow trout Oncorhynchus mykiss with viral haemorrhagic septicaemia virus isolates from European marine and farmed fishes.

Dis Aquat Organ 2004 Mar;58(2-3):99-110

Danish Institute for Food and Veterinary Research, Department of Poultry, Fish and Fur Animals, Hangøvej 2, 8200 Arhus N, Denmark.

The susceptibility of rainbow trout Oncorhynchus mykiss to infection with various isolates of viral haemorrhagic septicaemia virus (VHSV) was examined. A total of 8 experiments with rainbow trout ranging from 0.6 to 6.2 g was conducted for 139 isolates originating from wild marine fishes in European waters (115 isolates), farmed turbot from Scotland and Ireland (2 isolates), and farmed rainbow trout (22 isolates). The isolates were tested by immersion and/or intraperitoneal injection either as pooled or single isolates. The isolates from wild marine fishes did not cause mortality by immersion while some of the isolates caused mortality when injected. All VHSV isolates from farmed rainbow trout caused significant mortality by immersion. Currently, pathogenicity trials are the only way to differentiate VHSV isolates from wild marine fishes and farmed rainbow trout. The 2 farmed turbot isolates did not cause mortality by immersion, supporting the view that they originated from the marine environment.
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http://dx.doi.org/10.3354/dao058099DOI Listing
March 2004

Age- and weight-dependent susceptibility of rainbow trout Oncorhynchus mykiss to isolates of infectious haematopoietic necrosis virus (IHNV) of varying virulence.

Dis Aquat Organ 2003 Aug;55(3):205-10

Federal Research Centre for Virus Diseases of Animals, Boddenblick 5a, 17493 Greifswald-Insel Riems, Germany.

The virulence of 5 European and 1 North American isolate of infectious haematopoietic necrosis virus (IHNV) was compared by infecting female sibling rainbow trout ('Isle of Man' strain) of different weights and ages (2, 20 and 50 g). The fish were exposed to 10(4) TCID50 IHNV per ml of water by immersion, and the mortality was recorded for 28 d. Two new IHNV isolates from Germany were included in the investigation. One was isolated from European eels kept at 23 degrees C (+/- 2 degrees C) and the other was not detectable by immunofluorescence with commercially available monoclonal antibodies recognising the viral G protein. The results showed that IHNV isolates of high or low virulence persisted in rainbow trout of all ages/weights for 28 d, with the exception of fish over 15 g in the eel IHNV (DF [diagnostic fish] 13/98)-infected groups from which the virus could not be reisolated on Day 28. The smallest fish were most susceptible to an infection with any of the IHNV isolates. The lowest cumulative mortality (18%) was observed in fingerlings infected with the North American isolate HAG (obtained from Hagerman Valley), and the highest mortality (100%) in DF 04/99 infected fish. The DF 04/99 and O-13/95 viruses caused mortality in fish independent of their weight or age. The isolates FR-32/87 and I-4008 were virulent in fish up to a weight of 20 g and caused no mortality in larger fish. In the IHNV HAG- and DF 13/98 (eel)-infected rainbow trout, no signs of disease were observed in fish weighing between 15 and 50 g. An age/weight related susceptibility of rainbow trout was demonstrated under the defined conditions for all IHNV isolates tested.
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http://dx.doi.org/10.3354/dao055205DOI Listing
August 2003

A novel fish rhabdovirus from sweden is closely related to the Finnish rhabdovirus 903/87.

Virus Genes 2002 Oct;25(2):127-38

Institute of Parasitology, Department of Biology, Abo Akademi University, Turku, Finland.

A novel rhabdovirus, preliminary designated as the Sea trout rhabdovirus 28/97 (STRV 28/97), was isolated from sea trout (Salmo trutta trutta) in Sweden in 1996. The fish showed central nervous symptoms, and at the autopsy petechial bleedings in the mesenteric fat were visible. STRV 28/97 was shown to be serologically related to the vesiculotype rhabdovirus 903/87 isolated from brown trout (Salmo trutta lacustris) in Finland [1,3]. The sequences for the nucleocapsid protein, phosphoprotein, matrix protein, glycoprotein and beginning of the polymerase protein of STRV 28/97 were determined. At the amino acid level the genes were over 97% similar to virus 903/87. The nucleocapsid proteins, glycoproteins and beginning of the polymerase protein of STRV 28/97 and virus 903/87 were clustered with the vesiculoviruses and the phosphoproteins close to the vesiculoviruses in protein parsimony analysis. The matrix proteins formed a distinct clade in protein parsimony analysis.
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http://dx.doi.org/10.1023/a:1020153616947DOI Listing
October 2002