Development and application of a whole genome amplicon sequencing method for infectious salmon anemia virus (ISAV).

Infectious salmon anemia (ISA) is an infectious disease primarily affecting farmed Atlantic salmon, Salmo salar, which is caused by the ISA virus (ISAV). ISAV belongs to the Orthomyxoviridae family. The disease is a serious condition resulting in reduced fish welfare and high mortality. In this study, we designed an amplicon-based sequencing protocol for whole genome sequencing of ISAV. The method consists of 80 ISAV-specific primers that cover 92% of the virus genome and was designed to be used on an Illumina MiSeq platform. The sequencing accuracy was investigated by comparing sequences with previously published Sanger sequences. The sequences obtained were nearly identical to those obtained by Sanger sequencing, thus demonstrating that sequences produced by this amplicon sequencing protocol had an acceptable accuracy. The amplicon-based sequencing method was used to obtain the whole genome sequence of 12 different ISAV isolates from a small local epidemic in the northern part of Norway. Analysis of the whole genome sequences revealed that segment reassortment took place between some of the isolates and could identify which segments that had been reassorted.

Validation of a TaqMan one-step real-time RT-PCR assay targeting ISAV segment 7 spliced mRNA.

Infectious salmon anaemia virus (ISAV) can cause severe systemic infection in Atlantic salmon (Salmo salar L.), and a timely diagnosis is critical. Conventional real-time reverse transcription PCR (RT-qPCR) assays target unspliced RNA from either ISAV segment 7 or 8 and provide data on viral load. Here, we evaluate a TaqMan one-step RT-qPCR assay that detects explicitly a spliced messenger RNA (mRNA) of ISAV segment 7, thus providing evidence of active viral transcription. Assay performance was comparable with existing unspliced segment 7 and segment 8 assays. PCR efficiency as evaluated from dilutions of a synthetic DNA fragment was 98 % (R2 = 1.00). The assay also performed well on clinical heart samples with PCR efficiency of 108 % (R2 = 1.00). Finally, evaluation on kidney samples from experimental infection revealed higher levels of active transcription for high-virulent compared to low-virulent ISAV. At early, peak, and late infection, mean ratios of spliced to unspliced segment 7 RNA were 3.0 % (± 0.7), 1.7 % (± 0.3), and 1.5 % (± 0.1) for the low virulent and 9.4 % (± 2.2), 4.7 % (± 0.8), and 6.2 % (± 0.1) for the high virulent isolate, respectively. By detection and quantification of active ISAV transcription, this assay may provide a more detailed understanding of ISAV infection dynamics.

Factors influencing the immune response over 15 months after SARS-CoV-2 infection: A longitudinal population-wide study in the Faroe Islands.

BACKGROUND: The durability of SARS-CoV-2 antibody response and the resulting immunity to COVID-19 is unclear. OBJECTIVES: To investigate long-term humoral immunity to SARS-CoV-2. METHODS: In this nationwide, longitudinal study, we determined antibody response in 411 patients aged 0-93 years from two waves of infections (March to December 2020) contributing 1063 blood samples. Each individual had blood drawn on 4-5 occasions 1-15 months after disease onset. We measured total anti-SARS-CoV-2 receptor-binding domain (RBD) antibody using a qualitative RBD sandwich ELISA, IgM, IgG and IgA levels using an quantitative in-house ELISA-based assay  and neutralizing antibodies (NAbs) using an in-house ELISA-based pseudoneutralizing assay. IgG subclasses were analyzed in a subset of samples by ELISA-based assay. We used nonlinear models to study the durability of SARS-CoV-2 antibody responses and its influence over time. RESULTS: After 15 months, 94% still had detectable circulating antibodies, mainly the IgG isotype, and 92% had detectable NAbs. The distribution of IgG antibodies varied significantly over time, characterized by a biphasic pattern with an initial decline followed by a plateau after approximately 7 months. However, the NAbs remained relatively stable throughout the period. The strength of the antibody response was influenced by smoking and hospitalization, with lower IgG levels in smokers and higher levels in hospitalized individuals. Antibody stability over time was mainly associated with male sex and older age with higher initial levels but more marked decrease. CONCLUSIONS: The humoral immune response to SARS-CoV-2 infection varies depending on behavioral factors and disease severity, and antibody stability over 15 months was associated with sex and age.

Destruction of the vascular viral receptor in infectious salmon anaemia provides in vivo evidence of homologous attachment interference.

Viral interference is a process where infection with one virus prevents a subsequent infection with the same or a different virus. This is believed to limit superinfection, promote viral genome stability, and protect the host from overwhelming infection. Mechanisms of viral interference have been extensively studied in plants, but remain poorly understood in vertebrates. We demonstrate that infection with infectious salmon anaemia virus (ISAV) strongly reduces homologous viral attachment to the Atlantic salmon, Salmo salar L. vascular surface. A generalised loss of ISAV binding was observed after infection with both high-virulent and low-virulent ISAV isolates, but with different kinetics. The loss of ISAV binding was accompanied by an increased susceptibility to sialidase, suggesting a loss of the vascular 4-O-sialyl-acetylation that mediates ISAV attachment and simultaneously protects the sialic acid from cleavage. Moreover, the ISAV binding capacity of cultured cells dramatically declined 3 days after ISAV infection, accompanied by reduced cellular permissiveness to infection with a second antigenically distinct isolate. In contrast, neither infection with infectious haematopoietic necrosis virus nor stimulation with the viral mimetic poly I:C restricted subsequent cellular ISAV attachment, revealing an ISAV-specific mechanism rather than a general cellular antiviral response. Our study demonstrates homologous ISAV attachment interference by de-acetylation of sialic acids on the vascular surface. This is the first time the kinetics of viral receptor destruction have been mapped throughout the full course of an infection, and the first report of homologous attachment interference by the loss of a vascular viral receptor. Little is known about the biological functions of vascular O-sialyl-acetylation. Our findings raise the question of whether this vascular surface modulation could be linked to the breakdown of central vascular functions that characterises infectious salmon anaemia.

Clinical characteristics of the Omicron variant - results from a Nationwide Symptoms Survey in the Faroe Islands.

OBJECTIVES: Omicron appears to lead to a milder illness for patients compared with previous COVID-19 variants. However, not all infected with Omicron would describe their illness as mild. In this study, we investigate the experienced severity and symptoms of the Omicron variant. METHODS: We conducted a nationwide cross-sectional study, including 5036 individuals of all ages, consisting of reverse transcription-polymerase chain reaction confirmed SARS-CoV-2 cases from 1 January to 31 January 2022 (n = 4506) and a control group without SARS-COV-2 infection in December 2021 or January 2022 (n = 530). Omicron was dominant during this period. Cases were asked about their acute symptoms and answered a web-based questionnaire 10-30 days after their positive test while controls were asked about symptoms during the past week. RESULTS: Among cases, 97% reported at least one symptom during the acute phase compared with 79% of controls. Just over half the cases assessed their illness as asymptomatic or mild, whereas 46% assessed their illness as moderate or severe. Children reported fewer symptoms and less severe illnesses than adults (P <0.001). The largest risk differences (RDs) between adult cases and controls due to symptoms were observed for fever (RD = 60.6%, confidence interval [CI] 57.4-63.6), fatigue (RD = 49.6%, CI 44.1-54.7), and chills (RD = 48.8%, CI 43.8-53.2). CONCLUSION: Most of those infected with Omicron experience symptoms, and the Omicron variant appears to lead to less severe disease. However, this does not mean that all the infected experience an Omicron infection as mild. The unprecedented rate of Omicron infections worldwide leads to urgent questions about the rate of long COVID after Omicron infections.

Salmon Erythrocytes Sequester Active Virus Particles in Infectious Salmon Anaemia.

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.

Omicron Outbreak at a Private Gathering in the Faroe Islands, Infecting 21 of 33 Triple-Vaccinated Healthcare Workers.

There are concerns that the severe acute respiratory syndrome coronavirus 2 Omicron variant evades immune responses due to an unusually high number of mutations on the spike protein. Here, we report a superspreading event of Omicron infections among 21 of 33 triple-vaccinated healthcare workers who attended a private gathering.

No Evidence of the Vertical Transmission of Non-Virulent Infectious Salmon Anaemia Virus (ISAV-HPR0) in Farmed Atlantic Salmon.

The nonvirulent infectious salmon anaemia virus (ISAV-HPR0) is the putative progenitor for virulent-ISAV, and a potential risk factor for the development of infectious salmon anaemia (ISA). Understanding the transmission dynamics of ISAV-HPR0 is fundamental to proper management and mitigation strategies. Here, we demonstrate that ISAV-HPR0 causes prevalent and transient infections in all three production stages of Atlantic salmon in the Faroe Islands. Phylogenetic analysis of the haemagglutinin-esterase gene from 247 salmon showed a clear geographical structuring into two significantly distinct HPR0-subgroups, which were designated G2 and G4. Whereas G2 and G4 co-circulated in marine farms, Faroese broodfish were predominantly infected by G2, and smolt were predominantly infected by G4. This infection pattern was confirmed by our G2- and G4-specific RT-qPCR assays. Moreover, the HPR0 variants detected in Icelandic and Norwegian broodfish were never detected in the Faroe Islands, despite the extensive import of ova from both countries. Accordingly, the vertical transmission of HPR0 from broodfish to progeny is uncommon. Phylogenetic and statistical analysis suggest that HPR0 persists in the smolt farms as "house-strains", and that new HPR0 variants are occasionally introduced from the marine environment, probably by HPR0-contaminated sea-spray. Thus, high biosecurity-including water and air intake-is required to avoid the introduction of pathogens to the smolt farms.

Multilocus sequence analysis reveals different lineages of Pseudomonas anguilliseptica associated with disease in farmed lumpfish (Cyclopterus lumpus L.).

The bacterium Pseudomonas anguilliseptica has in recent years emerged as a serious threat to production of lumpfish in Norway. Little is known about the population structure of this bacterium despite its association with disease in a wide range of different fish species throughout the world. The phylogenetic relationships between 53 isolates, primarily derived from diseased lumpfish, but including a number of reference strains from diverse geographical origins and fish species, were reconstructed by Multi-Locus Sequence Analysis (MLSA) using nine housekeeping genes (rpoB, atpD, gyrB, rpoD, ileS, aroE, carA, glnS and recA). MLSA revealed a high degree of relatedness between the studied isolates, altough the seven genotypes identified formed three main phylogenetic lineages. While four genotypes were identified amongst Norwegian lumpfish isolates, a single genotype dominated, irrespective of geographic origin. This suggests the existence of a dominant genotype associated with disease in production of lumpfish in Norwegian aquaculture. Elucidation of the population structure of the bacterium has provided valuable information for potential future vaccine development.

Seroprevalence of SARS-CoV-2-Specific Antibodies among Quarantined Close Contacts of COVID-19 Patients, Faroe Islands, 2020.

Close contacts of coronavirus disease (COVID-19) patients are at high risk for severe acute respiratory syndrome 2 (SARS-CoV-2) infection. We assessed the seroprevalence of SARS-CoV-2-specific antibodies among quarantined close contacts of COVID-19 patients in the Faroe Islands. We invited quarantined close contacts of COVID-19 index patients identified during March 3-April 22, 2020, to participate in this study; 584 (81%) contacts consented and underwent serologic testing. Among the 584 participants, 32 (5.5%) were seropositive for total antibody against SARS-CoV-2. Household and young or elderly contacts had higher risk for seropositivity than other contacts. We found a secondary attack rate of 19.2%. Seroprevalence among close contacts was almost 10-fold higher than among the general population of the Faroe Islands. Regularly testing household close contacts of COVID-19 patients might help track the transmission of SARS-CoV-2.

Epidemiology and Clinical Course of First Wave Coronavirus Disease Cases, Faroe Islands.

The Faroe Islands was one of the first countries in the Western Hemisphere to eliminate coronavirus disease (COVID-19). During the first epidemic wave in the country, 187 cases were reported between March 3 and April 22, 2020. Large-scale testing and thorough contact tracing were implemented early on, along with lockdown measures. Transmission chains were mapped through patient history and knowledge of contact with prior cases. The most common reported COVID-19 symptoms were fever, headache, and cough, but 11.2% of cases were asymptomatic. Among 187 cases, 8 patients were admitted to hospitals but none were admitted to intensive care units and no deaths occurred. Superspreading was evident during the epidemic because most secondary cases were attributed to just 3 infectors. Even with the high incidence rate in early March, the Faroe Islands successfully eliminated the first wave of COVID-19 through the early use of contact tracing, quarantine, social distancing, and large-scale testing.

Seroprevalence of SARS-CoV-2-Specific Antibodies, Faroe Islands.

We conducted a nationwide study of the prevalence of severe acute respiratory syndrome coronavirus 2 infection in the Faroe Islands. Of 1,075 randomly selected participants, 6 (0.6%) tested seropositive for antibodies to the virus. Adjustment for test sensitivity and specificity yielded a 0.7% prevalence. Our findings will help us evaluate our public health response.

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

The commercial production of lumpfish Cyclopterus lumpus L. is expanding with the increased demand for their use as cleaner fish, to control sea-lice numbers, at marine Atlantic salmon Salmo salar 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 Gadus morhua L. and turbot Scophthalmus maximus 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.

Evolution of the Piscine orthoreovirus Genome Linked to Emergence of Heart and Skeletal Muscle Inflammation in Farmed Atlantic Salmon (Salmo salar).

Heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar) was first diagnosed in Norway in 1999. The disease is caused by Piscine orthoreovirus-1 (PRV-1). The virus is prevalent in farmed Atlantic salmon, but not always associated with disease. Phylogeny and sequence analyses of 31 PRV-1 genomes collected over a 30-year period from fish with or without HSMI, grouped the viral sequences into two main monophylogenetic clusters, one associated with HSMI and the other with low virulent PRV-1 isolates. A PRV-1 strain from Norway sampled in 1988, a decade before the emergence of HSMI, grouped with the low virulent HSMI cluster. The two distinct monophylogenetic clusters were particularly evident for segments S1 and M2. Only a limited number of amino acids were unique to the association with HSMI, and they all located to S1 and M2 encoded proteins. The observed co-evolution of the S1-M2 pair coincided in time with the emergence of HSMI in Norway, and may have evolved through accumulation of mutations and/or segment reassortment. Sequences of S1-M2 suggest selection of the HSMI associated pair, and that this segment pair has remained almost unchanged in Norwegian salmon aquaculture since 1997. PRV-1 strains from the North American Pacific Coast and Faroe Islands have not undergone this evolution, and are more closely related to the PRV-1 precursor strains not associated with clinical HSMI.

Presence and genetic variability of Piscine orthoreovirus genotype 1 (PRV-1) in wild salmonids in Northern Europe and North Atlantic Ocean.

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.

Carnitine levels and mutations in the SLC22A5 gene in Faroes patients with Parkinson's disease.

INTRODUCTION: Mitochondrial dysfunction, oxidative stress and energy production have been implicated in the etiology of Parkinson's disease (PD). Several agents are under investigation for potential neuroprotective effects including acetyl-l-carnitine (ALC). OBJECTIVE: To investigate whether low carnitine levels and mutations in the SLC22A5 gene encoding the carnitine transporter are associates with PD risk in the Faroe Islands where the prevalence of both PD and carnitine transporter deficiency (CTD) is high. METHODS: We conducted a case-control study with 121 cases and 235 randomly selected controls, matched by gender and age. Blood spots were analyzed for free and total carnitine levels by QUATTRO LC triple quadrupole mass spectrometry (MS/MS) and sequencing performed for five genetic mutations in the SLC22A5 gene with ABI PRISM 3130. RESULTS: PD cases had significantly lower levels of free and total carnitine levels compared with controls (P < .001). However, stratifying according to mutation status, the lower levels of carnitine was only evident among the non-mutation carriers. Specifically, no difference was found in c.95A > G mutation frequency in the SLC22A5 gene among cases and controls (p = .70). CONCLUSION: Low carnitine levels seem to be associated with PD, but only in individuals without the c.95A > G mutation rendering the carnitine transporter less efficient. Thus, the difference in carnitine levels is not caused by a higher frequency of c.95A > G mutation carriers in cases. The cause may be dietary or due to different gut microbiota among cases.

First field evidence of the evolution from a non-virulent HPR0 to a virulent HPR-deleted infectious salmon anaemia virus.

The putatively non-virulent subtype of infectious salmon anaemia virus (ISAV), ISAV-HPR0, is proposed to act as a progenitor and reservoir for all virulent ISAVs and thus represent a potential risk factor for the emergence of infectious salmon anaemia (ISA) disease. Here, we provide the first evidence of genetic and functional evolution from an ISAV-HPR0 variant (FO/07/12) to a low-virulent ISAV virus (FO/121/14) in a Faroese Atlantic salmon marine farm. The FO/121/14 virus infection was not associated with specific clinical signs of ISA and was confined to a single net-pen, while various ISAV-HPR0 subtypes were found circulating in most epidemiologically linked marine and freshwater farms. Sequence analysis of all eight segments revealed that the FO/121/14 virus was identical, apart from a substitution in the fusion (F) gene (Q266L) and a deletion in the haemagglutinin-esterase (HE) gene, to the FO/07/12 variant from a freshwater farm, which supplied smolts exclusively to the FO/121/14-positive net-pen. An immersion challenge with the FO/121/14 virus induced a systemic infection in Atlantic salmon associated with a low mortality and mild clinical signs confirming its low pathogenicity. Our results demonstrate that mutations in the F protein and deletions in the highly polymorphic region (HPR) of the HE protein represent a minimum requirement for ISAV to gain virulence and to switch cell tropism from a localized epithelial infection to a systemic endotheliotropic infection. This documents that ISAV-HPR0 represents a reservoir and risk factor for the emergence of ISA disease.

Localised Infection of Atlantic Salmon Epithelial Cells by HPR0 Infectious Salmon Anaemia Virus.

Infectious salmon anaemia (ISA) is an important, systemic viral disease of farmed Atlantic salmon, Salmo salar L. Endothelial cells are the main target cells for highly virulent HPR-deleted ISA virus (ISAV) types. Here we examine the pathogenesis of non-virulent ISAV HPR0 infections, presenting evidence of an epithelial tropism for this virus type, including actual infection and replication in the epithelial cells. Whereas all HPR0 RT-qPCR positive gills prepared for cryosection tested positive by immunohistochemistry (IHC) and immunofluorescent labelling, only 21% of HPR0 RT-qPCR positive formalin-fixed paraffin-embedded gills were IHC positive, suggesting different methodological sensitivities. Only specific epithelial cell staining was observed and no staining was observed in endothelial cells of positive gills. Furthermore, using an ISAV segment 7 RT-PCR assay, we demonstrated splicing of HPR0, suggesting initial activation of the replication machinery in the epithelial gill cells. Immunological responses were investigated by the expression of interferon-related genes (e.g. Mx and γIP) and by ELISA for presence of anti-ISAV antibodies on samples taken sequentially over several months during an episode of transient HPR0 infection. All fish revealed a variable, but increased expression of the immunological markers in comparison to normal healthy fish. Taken together, we conclude that HPR0 causes a localized epithelial infection of Atlantic salmon.