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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.

Infectious salmon anaemia virus (ISAV) binds circulating Atlantic salmon erythrocytes, but the relevance of this interaction for the course of infection and development of disease remains unclear. We here characterise ISAV-erythrocyte interactions in experimentally infected Atlantic salmon and show that ISAV-binding to erythrocytes is common and precedes the development of disease. Viral RNA and infective particles were enriched in the cellular fraction of blood. While erythrocyte-associated ISAV remained infectious, erythrocytes dose-dependently limited the infection of cultured cells. Surprisingly, immunostaining of blood smears revealed expression of ISAV proteins in a small fraction of erythrocytes in one of the examined trials, confirming that ISAV can be internalised in this cell type and engage the cellular machinery in transcription and translation. However, viral protein expression in erythrocytes was rare and not required for development of disease and mortality. Furthermore, active transcription of ISAV mRNA was higher in tissues than in blood, supporting the assumption that ISAV replication predominantly takes place in endothelial cells. In conclusion, Atlantic salmon erythrocytes bind ISAV and sequester infective virus particles during infection, but do not appear to significantly contribute to ISAV replication. We discuss the implications of our findings for infection dynamics and pathogenesis of infectious salmon anaemia.

International audience; 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.

In recent years, the use of cleaner fish for biological control of sea lice has increased considerably. Along with this, a number of infectious diseases have emerged. The aim of this study was to investigate the susceptibility of lumpfish (Cyclopterus lumpus) to Betanodavirus since it was detected in asymptomatic wild wrasses in Norway and Sweden. Three betanodaviruses were used to challenge lumpfish: one RGNNV genotype and two BFNNV genotypes. Fish were injected and monitored for 4 weeks. Brain samples from clinically affected specimens, from weekly randomly selected fish and survivors were subjected to molecular testing, viral isolation, histopathology and immunohistochemistry. Reduced survival was observed but was attributed to tail-biting behaviour, since no nervous signs were observed throughout the study. Betanodavirus RNA was detected in all samples, additionally suggesting an active replication of the virus in the brain. Viral isolation confirmed molecular biology results and revealed a high viral titre in BFNNV-infected groups associated with typical lesions in brains and eyes of survivor fish. We concluded that lumpfish are susceptible to Betanodavirus, as proven by the high viral titre and brain lesions detected, but further studies are necessary to understand if Betanodavirus can cause clinical disease in this species.

Piscine orthoreovirus (PRV) infects farmed and wild salmon and trout species in North America, South America, Europe, and East Asia. PRV groups into three distinct genotypes (PRV-1, PRV-2, and PRV-3) that can vary in distribution, host specificity, and/or disease potential. Detection of the virus is currently restricted to genotype specific assays such that surveillance programs require the use of three assays to ensure universal detection of PRV. Consequently, herein, we developed, optimized, and validated a real-time reverse transcription quantitative PCR assay (RT-qPCR) that can detect all known PRV genotypes with high sensitivity and specificity. Targeting a conserved region at the 5′ terminus of the M2 segment, the pan-PRV assay reliably detected all PRV genotypes with as few as five copies of RNA. The assay exclusively amplifies PRV and does not cross-react with other salmonid viruses or salmonid host genomes and can be performed as either a one- or two-step RT-qPCR. The assay is highly reproducible and robust, showing 100% agreement in test results from an inter-laboratory comparison between two laboratories in two countries. Overall, as the assay provides a single test to achieve highly sensitive pan-specific PRV detection, it is suitable for research, diagnostic, and surveillance purposes.

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.

International audience; 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.

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.

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.

Pedigree reconstruction via DNA markers and subsequent quantitative genetic analyses in production stands of trees is an alternative to traditional forest tree breeding. The method requires variable DNA markers; preferably cost-efficient to genotype. We tested the approach in breeding for Christmas tree traits in a Danish landrace of Abies procera. Initial analysis with simple sequence repeats (SSRs) revealed a substantial reduction in genetic diversity of Danish A. procera compared with direct imports from the USA, indicating a genetic bottleneck. Due to the reduced genetic variation, the SSRs were not variable enough for pedigree reconstruction, and 145 single nucleotide polymorphisms (SNPs) were therefore identified and genotyped via high-throughput targeted sequencing. In the field, we phenotypically selected 674 superior trees, based on criteria for good Christmas trees, among 21,000 trees in three Christmas tree plantations originating from an A. procera clonal seed orchard. By SNP-genotyping and parentage analysis, pedigree was reconstructed for the selected individuals, and genetic predictions for the Christmas tree traits were estimated. A final selection of 45 trees out of the 674 genotyped, based on an index of predicted values for different traits, foresees a gain in the Christmas tree score and branch angle score of 16% and 10%, respectively. The study clearly demonstrated the benefits of pedigree reconstruction, even though mass selection seemed to give similar gain. Primarily, the possibilities to control the level of inbreeding, and to check whether the production stands, from where we select trees, actually originate from the declared and desired parent pool.

International audience; The Viral Hemorrhagic Septicemia Virus (VHSV) is an OIE notifiable pathogen widespread in the Northern Hemisphere that encompasses four genotypes and nine subtypes. In Europe, subtype Ia impairs predominantly the rainbow trout industry causing severe rates of mortality, while other VHSV genotypes and subtypes affect a number of marine and freshwater species, both farmed and wild. VHSV has repeatedly proved to be able to jump to rainbow trout from the marine reservoir, causing mortality episodes. The molecular mechanisms regulating VHSV virulence and host tropism are not fully understood, mainly due to the scarce availability of complete genome sequences and information on the virulence phenotype. With the scope of identifying in silico molecular markers for VHSV virulence, we generated an extensive dataset of 55 viral genomes and related mortality data obtained from rainbow trout experimental challenges. Using statistical association analyses that combined genetic and mortality data, we found 38 single amino acid polymorphisms scattered throughout the complete coding regions of the viral genome that were putatively involved in virulence of VHSV in trout. Specific amino acid signatures were recognized as being associated with either low or high virulence phenotypes. The phylogenetic analysis of VHSV coding regions supported the evolution toward greater virulence in rainbow trout within subtype Ia, and identified several other subtypes which may be prone to be virulent for this species. This study sheds light on the molecular basis for VHSV virulence, and provides an extensive list of putative virulence markers for their subsequent validation.

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.

Piscine orthoreovirus (PRV) is a common and widely distributed virus of salmonids. Since its discovery in 2010, the virus has been detected in wild and farmed stocks from North America, South America, Europe and East Asia in both fresh and salt water environments. Phylogenetic analysis suggests three distinct genogroups of PRV with generally discrete host tropisms and/or regional patterns. PRV-1 is found mainly in Atlantic (Salmo salar), Chinook (Oncorhynchus tshawytscha) and Coho (Oncorhynchus kisutch) Salmon of Europe and the Americas; PRV-2 has only been detected in Coho Salmon of Japan; and PRV-3 has been reported primarily in Rainbow Trout (Oncorhynchus mykiss) in Europe. All three genotypes can establish high-load systemic infections by targeting red blood cells for principal replication. Each genotype has also demonstrated potential to cause circulatory disease. At the same time, high-load PRV infections occur in non-diseased salmon and trout, indicating a complexity for defining PRV's role in disease aetiology. Here, we summarize the current body of knowledge regarding PRV following 10 years of study.

The commercial production of lumpfish

Simple Summary Replication studies are frequently based on viral production, which provides limited information to understand certain processes. Therefore, to discover which failures in the viral haemorrhagic septicaemia virus (VHSV) replication cycle might be involved in the differences in its virulence on fish, a different approach has been taken. Our results have demonstrated that adsorption and morphogenesis are the steps most involved in the modulation of virulence, although failures in the synthesis step were also observed. As a potential application of our results, we believe that this kind of knowledge relating in vivo virulence to in vitro markers could help reduce the need for experimental infections in animals, representing a step forward in ethical issues. Abstract The viral haemorrhagic septicaemia virus (VHSV), a single-stranded negative-sense RNA novirhabdovirus affecting a wide range of marine and freshwater fish species, is a main concern for European rainbow trout (Oncorhynchus mykiss) fish farmers. Its genome is constituted by six genes, codifying five structural and one nonstructural proteins. Many studies have been carried out to determine the participation of each gene in the VHSV virulence, most of them based on genome sequence analysis and/or reverse genetics to construct specific mutants and to evaluate their virulence phenotype. In the present study, we have used a different approach with a similar aim: hypothesizing that a failure in any step of the replication cycle can reduce the virulence in vivo, we studied in depth the in vitro replication of VHSV in different cell lines, using sets of strains from different origins, with high, low and moderate levels of virulence for fish. The results demonstrated that several steps in the viral replication cycle could affect VHSV virulence in fish, including adsorption, RNA synthesis and morphogenesis (including viral release). Notably, differences among strains in any step of the replication cycle were mostly strain-specific and reflected only in part the in vivo phenotype (high and low virulent). Our data, therefore, support the need for further studies aimed to construct completely avirulent VHSV recombinants targeting a combination of genes rather than a single one in order to study the mechanisms of genes interplay and their effect on viral phenotype in vitro and in vivo.

Viral haemorrhagic septicaemia (VHS) is one of the most serious viral diseases in salmonid and olive flounder farms. Various diagnostic methods for detecting VHS virus (VHSV) are described in the VHS chapter of the World Organization for Animal Health (OIE) Aquatic Diagnostic Manual. A conventional reverse transcription-PCR (cRT-PCR) targeting the viral nucleocapsid gene is recommended for the detection of VHSV and, to some extent, for genotypic classification. However, the recommended assay exhibits low sensitivity for the detection of VHSV genotype IVa isolates and often shows non-specific amplicons when the RNA template is extracted from non-infected fish cell lines. For these reasons, it is necessary to develop a new RT-PCR method for the foolproof detection of all VHSV genotypes and elimination of non-specific results. In this study, we selected five candidate primer sets that target the VHSV nucleoprotein (N) gene, and selected the most sensitive among them (3F/2R). We then established the optimal reaction conditions for these primers, and ensured that no non-specific amplification had occurred in the fish tissues, fish cell lines, or heterologous viruses. The analytical sensitivity of the novel cRT-PCR was compared to that of cell culture assays, real-time RT-PCR, and other cRT-PCR methods and was found to be as sensitive as or superior to the other methods for detecting all VHSV genotypes. Our newly developed cRT-PCR assay was tested with 80 isolates, representing a collection of all known VHSV genotypes worldwide. Clear and unique amplicons were amplified from all 80 VHSV isolates. The reproducibility, and partly the robustness, of the assay were confirmed by an inter-laboratory proficiency tests including nine laboratories. A high diagnostic sensitivity and specificity was confirmed on tissue material from affected fish. In conclusion a highly robust, sensitive and specific cRT-PCR for detection of VHSV was developed and validated.

Piscine orthoreovirus genotype 1 (PRV-1) is widespread in farmed Atlantic salmon (Salmo salar L.) populations in northern Europe, Canada and Chile. PRV-1 occurs in wild fish in Norway and Canada; however, little information of its geographical distribution in wild populations is currently available, and the effect of PRV-1 infection in wild populations is currently unknown. In this study, we present the findings of a survey conducted on 1,130 wild salmonids sampled in Denmark, Sweden, Ireland, Faroe Islands, France, Belgium and Greenland between 2008 and 2017. PRV-1 is reported for the first time in wild salmonids in Denmark, Sweden, Faroe Island and Ireland. The annual PRV-1 prevalence ranged from 0% in France, Belgium and Greenland to 43% in Faroe Islands. In total, 66 samples tested positive for PRV-1, including Atlantic salmon broodfish returning to spawn and Atlantic salmon collected at the feeding ground north of Faroe Islands. The phylogenetic analysis of S1 sequences of the PRV-1 isolates obtained in this survey did not show systematic geographical distribution. This study sheds light on the spread and genetic diversity of the virus identified in populations of free-living fish and provides rationale for screening wild broodfish used in restocking programmes.

Additional file 4. Distribution of discrepancies per sample. For each raw data, the number of inconsistencies found is expressed as stacked columns. Genome localization is marked with red, blue and green bars (CDS, intergenic regions and genome termini, respectively); SNPs and indels are marked by lighter and darker shades of the corresponding color, respectively. A horizontal double gray bar points the sequencing technology and the viral species for each raw data.

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. Electronic supplementary material The online version of this article (10.1186/s13567-019-0632-4) contains supplementary material, which is available to authorized users.

BackgroundNext generation sequencing (NGS) is becoming widely used among diagnostics and research laboratories, and nowadays it is applied to a variety of disciplines, including veterinary virology. The NGS workflow comprises several steps, namely sample processing, library preparation, sequencing and primary/secondary/tertiary bioinformatics (BI) analyses. The latter is constituted by a complex process extremely difficult to standardize, due to the variety of tools and metrics available. Thus, it is of the utmost importance to assess the comparability of results obtained through different methods and in different laboratories. To achieve this goal, we have organized a proficiency test focused on the bioinformatics components for the generation of complete genome sequences of salmonid rhabdoviruses.MethodsThree partners, that performed virus sequencing using different commercial library preparation kits and NGS platforms, gathered together and shared with each other 75 raw datasets which were analyzed separately by the participants to produce a consensus sequence according to their own bioinformatics pipeline. Results were then compared to highlight discrepancies, and a subset of inconsistencies were investigated more in detail.ResultsIn total, we observed 526 discrepancies, of which 39.5% were located at genome termini, 14.1% at intergenic regions and 46.4% at coding regions. Among these, 10 SNPs and 99 indels caused changes in the protein products. Overall reproducibility was 99.94%. Based on the analysis of a subset of inconsistencies investigated more in-depth, manual curation appeared the most critical step affecting sequence comparability, suggesting that the harmonization of this phase is crucial to obtain comparable results. The analysis of a calibrator sample allowed assessing BI accuracy, being 99.983%.ConclusionsWe demonstrated the applicability and the usefulness of BI proficiency testing to assure the quality of NGS data, and recommend a wider implementation of such exercises to guarantee sequence data uniformity among different virology laboratories.

Publisher's version (útgefin grein), 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., H2020 SFS-2014-2 ParaFishControl, Grant/ Award Number: 634429; European Union Reference Laboratory for Fish Diseases Grant Decision SI2.725290

Additional file 1. Proportion of fish showing histopathological findings not consistent with PRV-induced inflammation. The findings consisted of epicarditis and a focal inflammatory reaction involving the interface layer between stratum compactum and stratum spongiosum of the ventricle. These were randomly distributed throughout the experiment and observed in all groups.

Loss of introns in plant mitochondrial genes is commonly explained by retroprocessing. Under this model, an mRNA is reverse transcribed and integrated back into the genome, simultaneously affecting the contents of introns and edited sites. To evaluate the extent to which retroprocessing explains intron loss, we analyzed patterns of intron content and predicted RNA editing for whole mitochondrial genomes of 30 species in the monocot order Alismatales. In this group, we found an unusually high degree of variation in the intron content, even expanding the hitherto known variation among angiosperms. Some species have lost some two-third of the cis-spliced introns. We found a strong correlation between intron content and editing frequency, and detected 27 events in which intron loss is consistent with the presence of nucleotides in an edited state, supporting retroprocessing. However, we also detected seven cases of intron loss not readily being explained by retroprocession. Our analyses are also not consistent with the entire length of a fully processed cDNA copy being integrated into the genome, but instead indicate that retroprocessing usually occurs for only part of the gene. In some cases, several rounds of retroprocessing may explain intron loss in genes completely devoid of introns. A number of taxa retroprocessing seem to be very common and a possibly ongoing process. It affects the entire mitochondrial genome.

Santalales is an order of plants consisting almost entirely of parasites. Some, such as Osyris, are facultative root parasites whereas others, such as Viscum, are obligate stem parasitic mistletoes. Here, we report the complete plastome sequences of one species of Osyris and three species of Viscum, and we investigate the evolutionary aspects of structural changes and changes in gene content in relation to parasitism. Compared with typical angiosperms plastomes, the four Santalales plastomes are all reduced in size (10-22% compared with Vitis), and they have experienced rearrangements, mostly but not exclusively in the border areas of the inverted repeats. Additionally, a number of protein-coding genes (matK, infA, ccsA, rpl33, and all 11 ndh genes) as well as two transfer RNA genes (trnG-UCC and trnV-UAC) have been pseudogenized or completely lost. Most of the remaining plastid genes have a significantly changed selection pattern compared with other dicots, and the relaxed selection of photosynthesis genes is noteworthy. Although gene loss obviously reduces plastome size, intergenic regions were also shortened. As plastome modifications are generally most prominent in Viscum, they are most likely correlated with the increased nutritional dependence on the host compared with Osyris.

A phylogenetic analysis of the early branching lineages of the monocotyledons is performed using data from two plastid genes (rbcL and matK), five mitochondrial genes (atp1, ccmB, cob, mttB and nad5) and morphology. The complete matrix includes 93 terminals representing Acorus, the 14 families currently recognized within Alismatales, and numerous lineages of monocotyledons and other angiosperms. Total evidence analysis results in an almost completely resolved strict consensus tree, but all data partitions, genomic as well as morphological, are incongruent. The effects of RNA editing and potentially processed paralogous sequences are explored and discussed. Despite a decrease in incongruence length differences after exclusion of edited sites, the major data partitions remain significantly incongruent. The 14 families of Alismatales are all found to be monophyletic, but Acorus is found to be included in Alismatales rather than being the sister group to all other monocotyledons. The placement is strongly supported by the mitochondrial data, atp1 in particular, but it cannot be explained as an artifact caused by patterns of editing or by sampling of processed paralogues.

The order Alismatales is a hotspot for evolution of plant mitochondrial genomes characterized by remarkable differences in genome size, substitution rates, RNA editing, retrotranscription, gene loss and intron loss. Here we have sequenced the complete mitogenomes of Zostera marina and Stratiotes aloides, which together with previously sequenced mitogenomes from Butomus and Spirodela, provide new evolutionary evidence of genome size reduction, gene loss and transfer to the nucleus. The Zostera mitogenome includes a large portion of DNA transferred from the plastome, yet it is the smallest known mitogenome from a non-parasitic plant. Using a broad sample of the Alismatales, the evolutionary history of ribosomal protein gene loss is analyzed. In Zostera almost all ribosomal protein genes are lost from the mitogenome, but only some can be found in the nucleus.

Piscine orthoreovirus (PRV-1) causes heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). Recently, a novel PRV (formerly PRV-Om, here called PRV-3), was found in rainbow trout (Oncorhynchus mykiss) with HSMI-like disease. PRV is considered to be an emerging pathogen in farmed salmonids. In this study, molecular and antigenic characterization of PRV-3 was performed. Erythrocytes are the main target cells for PRV, and blood samples that were collected from experimentally challenged fish were used as source of virus. Virus particles were purified by gradient ultracentrifugation and the complete coding sequences of PRV-3 were obtained by Illumina sequencing. When compared to PRV-1, the nucleotide identity of the coding regions was 80.1%, and the amino acid identities of the predicted PRV-3 proteins varied from 96.7% (λ1) to 79.1% (σ3). Phylogenetic analysis showed that PRV-3 belongs to a separate cluster. The region encoding σ3 were sequenced from PRV-3 isolates collected from rainbow trout in Europe. These sequences clustered together, but were distant from PRV-3 that was isolated from rainbow trout in Norway. Bioinformatic analyses of PRV-3 proteins revealed that predicted secondary structures and functional domains were conserved between PRV-3 and PRV-1. Rabbit antisera raised against purified virus or various recombinant virus proteins from PRV-1 all cross-reacted with PRV-3. Our findings indicate that despite different species preferences of the PRV subtypes, several genetic, antigenic, and structural properties are conserved between PRV-1 and-3. Molecular and antigenic characterization of piscine orthoreovirus (PRV) from rainbow trout (oncorhynchus orthoreovirumykiss)

Within the palm family, molecular analyses have resulted in a need to re-evaluate characters previously considered useful to establish hypotheses of relationships among groups. Recent phylogenetic analyses of tribe Chamaedoreeae have shown that characters traditionally used as strong indicators of relationship, such as presence of solitary flowers and dioecy, are homoplasious within this tribe. In the largest genus, Chamaedorea, published molecular analyses recover well-supported groups not previously proposed based on morphological characters and at the same time resolve some of the current subgenera as polyphyletic. In this study we further explore the phylogenetic relationships in Chamaedoreeae and search for morphological synapomorphies for the monophyletic groups recovered. Phylogenetic analyses of morphological, nuclear (PRK, RPB2), and plastid (matK, ndhF, trnD-trnT, rpsl6 intron, trnL-trnF) data are performed and morphological characters are subsequently optimized on the resulting topologies. Although most of the morphological characters included are highly homoplasious, the inclusion of morphological characters in the phylogenetic analyses improves the resolution within Chamaedoreeae, particularly among species of Chamaedorea. With the exception of Synechanthus and Wendlandiella, all the genera and subgenera in Chamaedoreeae are defined by a combination of homoplasious characters, none of which is unique within the tribe. Two of the seven subgenera of Chamaedorea included in our analyses, C. subg. Eleutheropetalum and subg. Stephanostachys, are supported as monophyletic with a number of morphological synapomorphies also used in the original descriptions of these subgenera. Some morphological characters not used in the traditional subgeneric classification of Chamaedorea, such as leaf sheath structure and pistillode, gynoecium and filament connation, though homoplasious, are resolved as synapomorphies and, therefore, potentially useful when defining subgenera within this genus.

This study presents the first phylogenetic analysis of tribe Chamaedoreeae (Arecaceae), using parsimony and Bayesian analyses of plastid DNA sequences (matK, rps16 intron, 3′ region of ndhF, and trnD-trnT). The tribe includes more than 115 species, and has a disjunct distribution with four genera in Central and South America and one genus in the Mascarene Islands. While the placement of Chamaedoreeae within Arecaceae has been controversial, the monophyly of this tribe is well supported by plastid DNA sequence data. All genera in Chamaedoreeae are resolved as monophyletic with high support, but relationships among genera are not fully resolved. The placement of Hyophorbe and the monotypic Wendlandiella as sisters to the remaining genera indicates that solitary flowers and dioecy arose at least twice within this tribe, once in Wendlandiella and once in Chamaedorea. Although a low substitution rate of palm plastid DNA has been widely noted, the results of this study show high resolution at the speci...

Parasitism is a successful survival strategy across all kingdoms and has evolved repeatedly in angiosperms. Parasitic plants obtain nutrients from other plants and some are agricultural pests. Obligate parasites, which cannot complete their lifecycle without a host, may lack functional photosystems (holoparasites), or have retained photosynthesis (hemiparasites). Plastid genomes are often reduced in parasites, but complete mitochondrial genomes have not been sequenced and their mitochondrial respiratory capacities are largely unknown. The hemiparasitic European mistletoe (Viscum album), known from folklore and postulated therapeutic properties, is a pest in plantations and forestry. We compare the mitochondrial genomes of three Viscum species based on the complete mitochondrial genome of V. album, the first from a parasitic plant. We show that mitochondrial genes encoding proteins of all respiratory complexes are lacking or pseudogenized raising several questions relevant to all parasitic plants: Are any mitochondrial gene functions essential? Do any genes need to be located in the mitochondrial genome or can they all be transferred to the nucleus? Can parasitic plants survive without oxidative phosphorylation by using alternative respiratory pathways? More generally, our study is a step towards understanding how host- and self-perception, host integration and nucleic acid transfer has modified ancestral mitochondrial genomes.