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.

Understanding host response to infectious salmon anaemia virus in an Atlantic salmon cell line using single-cell RNA sequencing.

BACKGROUND: Infectious Salmon Anaemia Virus (ISAV) is an Orthomixovirus that represents a large problem for salmonid aquaculture worldwide. Current prevention and treatment methods are only partially effective. Genetic selection and genome engineering have the potential to develop ISAV resistant salmon stocks. Both strategies can benefit from an improved understanding of the genomic regulation of ISAV pathogenesis. Here, we used single-cell RNA sequencing of an Atlantic salmon cell line to provide the first high dimensional insight into the transcriptional landscape that underpins host-virus interaction during early ISAV infection. RESULTS: Salmon head kidney (SHK-1) cells were single-cell RNA sequenced at 24, 48 and 96 h post-ISAV challenge. At 24 h post infection, cells showed expression signatures consistent with viral entry, with genes such as PI3K, FAK or JNK being upregulated relative to uninfected cells. At 48 and 96 h, infected cells showed a clear anti-viral response, characterised by the expression of IFNA2 or IRF2. Uninfected bystander cells at 48 and 96 h also showed clear transcriptional differences, potentially suggesting paracrine signalling from infected cells. These bystander cells expressed pathways such as mRNA sensing, RNA degradation, ubiquitination or proteasome; and up-regulation of mitochondrial ribosome genes also seemed to play a role in the host response to the infection. Correlation between viral and host genes revealed novel genes potentially key for this fish-virus interaction. CONCLUSIONS: This study has increased our understanding of the cellular response of Atlantic salmon during ISAV infection and revealed host-virus interactions at the cellular level. Our results highlight various potential key genes in this host-virus interaction, which can be manipulated in future functional studies to increase the resistance of Atlantic salmon to ISAV.

Transcriptome analysis revealed immune responses in the kidney of Atlantic salmon (Salmo salar) co-infected with sea lice (Lepeophtheirus salmonis) and infectious salmon anemia virus.

Sea lice (Lepeophtheirus salmonis) and infectious salmon anemia virus (ISAv) are two of the most important pathogens in Atlantic salmon (Salmo salar) farming and typically cause substantial economic losses to the industry. However, the immune interactions between hosts and these pathogens are still unclear, especially in the scenario of co-infection. In this study, we artificially infected Atlantic salmon with sea lice and ISAv, and investigated the gene expression patterns of Atlantic salmon head kidneys in response to both lice only and co-infection with lice and ISAv by transcriptomic analysis. The challenge experiment indicated that co-infection resulted in a cumulative mortality rate of 47.8 %, while no mortality was observed in the lice alone infection. We identified 240 differentially expressed genes (DEGs) under the lice alone infection, of which 185 were down-regulated and 55 were up-regulated, while a total of 994 DEGs were identified in the co-infection, of which 206 were down-regulated and 788 were significantly up-regulated. The pathway enrichment analysis revealed that single-infection significantly suppressed the innate immune system (e.g., the complement system), whereas co-infection induced a strong immune response, leading to the activation of immune-related signaling pathways such as Toll-like receptors and NOD-like receptors pathways, as well as significant upregulation of genes related to the activation of interferon and MH class I protein complex. Our results provide the first global transcriptomic study of gene expression in the Atlantic salmon head kidney in response to co-infection with sea lice and ISAv, and provided the baseline knowledge for understanding the immune responses during co-infection.

Description of spatiotemporal patterns of infectious salmon anemia virus (ISAV) detections in marine Atlantic Salmon farms in Newfoundland and Labrador.

OBJECTIVE: The objectives of this study were to describe spatiotemporal patterns of infectious salmon anemia virus (ISAV) detections in marine salmonid production sites in the province of Newfoundland and Labrador in Canada. METHODS: Infectious salmon anemia virus surveillance data between 2012 and 2020 from the province of Newfoundland and Labrador were used. Data comprised a total of 94 sampling events from 20 Atlantic Salmon Salmo salar production sites in which ISAV was detected. Using linear regression models, factors influencing time to detection (days from stocking to first ISAV detection) and time to depopulation (days from first detection to production site depopulation) were investigated. RESULT: Based on 28 unique cases, site-level annual incidence risk of ISAV detection ranged from 3% to 29%. The proportion of ISAV detection by PCR in fish samples ranged from 2% to 45% annually. Overall, ISAV variants from the European clade were more common than variants from the North American clade. The type of ISAV clade, detections of ISAV in nearest production sites based on seaway distances, and year of infectious salmon anemia cases were not associated with time to first ISAV detection. Time to depopulation for sites infected with the ISAV-HPRΔ variant was not associated with ISAV North American or European clades. CONCLUSION: Our results contribute to the further understanding of the changing dynamics of infectious salmon anemia detections in Newfoundland and Labrador since its first detection in 2012 and will likely assist in the design of improved disease surveillance and control programs in the province.

First report of successful isolation of a HPR0-like variant of the infectious salmon anaemia virus (ISAV) using cell culture.

ISAV is the causative agent of the infectious salmon anaemia (ISA), a disease listed by the OIE that has caused important economic losses to the Atlantic salmon (Salmo salar) industry. ISAV variants are identified as pathogenic or non-pathogenic based on the presence or absence of a deletion in the highly polymorphic region (HPR) of segment 6 (S6). HPRΔ variants (pathogenic) are the only forms of the virus known to grow in cell culture. This is the first report of a HPR0 variant isolated in cell culture. The isolate is, however, atypical as it shows a marker of virulent variants on another segment (S5), which has never been reported for any other HPR0 variants. The significance of this finding remains unclear until more in-depth work is carried out but does challenge current knowledge.

Descriptive epidemiology of variants of infectious salmon anaemia virus in four Atlantic salmon farms in Newfoundland and Labrador, Canada.

An incursion of infectious salmon anaemia virus (ISAV) was detected in 2020 in southern Newfoundland, Canada. This resulted in an outbreak affecting four marine farms stocking Atlantic salmon (Salmo salar L.) vaccinated against ISAV. This study provides the first description of epidemiologic characteristics of an ISAV outbreak in 2020 and 2021, and detected ISAV variants at the population level. Fish kidneys were screened for ISAV by real-time RT-PCR and non-negative samples were submitted for genotyping and further diagnostic testing. Nine distinct ISAV variants were identified: five European and three North American (NA) HPRΔ ISAV, and one NA-HPR0 ISAV variant. A notable finding was the concurrent detection of both an HPR0 and an HPRΔ ISAV variant in one individual fish. In two farms, both European and NA variants were simultaneously detected, while in the other two farms either NA or European variants were identified, but not both together. Generally, mortality increases followed rises in ISAV prevalence and cycle threshold values on RT-PCR decreased with time. Epidemiologic descriptions of ISAV outbreaks in Atlantic Canada contributes to the understanding of local disease dynamics and identification of changes thereof. Such insights are essential for the strengthening of disease management plans.

Triploid Atlantic salmon (Salmo salar) may have increased risk of primary field outbreaks of infectious salmon anaemia.

The impact that escaped farmed fish may have on wild populations is of major concern for Atlantic salmon (Salmo salar) farming. Triploid fish, being infertile, were originally introduced to mitigate the genetic impact of escaped fish. In the recent years, an increase in the number of infectious salmon anaemia (ISA) outbreaks in Norway has been observed, mainly in the northern parts, which is also where farming of triploid fish has been licensed. The present study investigated the susceptibility of triploid Atlantic salmon to ISA both by field observations and experimental infections. Based on field observations, we found an increased susceptibility, with 9.4 increased odds to primary ISA outbreaks in triploid fish versus diploid fish at production-site level, and a tendency of increased odds (3.4) of ISA in triploid fish at individual cage level at sited with primary outbreaks. At some sites, ISA outbreaks were only diagnosed in cages with triploid fish and not in cages with diploid fish. Primary ISA outbreaks are the source for further spread of the disease, and it is noteworthy that in an experimental trial we found significantly more viral RNA in non-ISA-vaccinated triploid than in non-ISA-vaccinated diploid fish at the peak of the infection. Interestingly, the notable differences of susceptibility to ISA for non-ISA vaccinated diploid and triploid fish observed in field were not repeated experimentally. The possible increased risk of ISA should be considered when evaluating the costs and benefits of triploid salmon in farming. It is recommended to keep triploid and diploid fish in biosecure separated sites, or that triploid fish are not farmed at all.

Exploring genetic resistance to infectious salmon anaemia virus in Atlantic salmon by genome-wide association and RNA sequencing.

BACKGROUND: Infectious Salmonid Anaemia Virus (ISAV) causes a notifiable disease that poses a large threat for Atlantic salmon (Salmo salar) aquaculture worldwide. There is no fully effective treatment or vaccine, and therefore selective breeding to increase resistance to ISAV is a promising avenue for disease prevention. Genomic selection and potentially genome editing can be applied to enhance host resistance, and these approaches benefit from improved knowledge of the genetic and functional basis of the target trait. The aim of this study was to characterise the genetic architecture of resistance to ISAV in a commercial Atlantic salmon population and study its underlying functional genomic basis using RNA Sequencing. RESULTS: A total of 2833 Atlantic salmon parr belonging to 194 families were exposed to ISAV in a cohabitation challenge in which cumulative mortality reached 63% over 55 days. A total of 1353 animals were genotyped using a 55 K SNP array, and the estimate of heritability for the trait of binary survival was 0.13-0.33 (pedigree-genomic). A genome-wide association analysis confirmed that resistance to ISAV was a polygenic trait, albeit a genomic region in chromosome Ssa13 was significantly associated with resistance and explained 3% of the genetic variance. RNA sequencing of the heart of 16 infected (7 and 14 days post infection) and 8 control fish highlighted 4927 and 2437 differentially expressed genes at 7 and 14 days post infection respectively. The complement and coagulation pathway was down-regulated in infected fish, while several metabolic pathways were up-regulated. The interferon pathway showed little evidence of up-regulation at 7 days post infection but was mildly activated at 14 days, suggesting a potential crosstalk between host and virus. Comparison of the transcriptomic response of fish with high and low breeding values for resistance highlighted TRIM25 as being up-regulated in resistant fish. CONCLUSIONS: ISAV resistance shows moderate heritability with a polygenic architecture, but a significant QTL was detected on chromosome 13. A mild up-regulation of the interferon pathway characterises the response to the virus in heart samples from this population of Atlantic salmon, and candidate genes showing differential expression between samples with high and low breeding values for resistance were identified.

Anguillid herpesvirus 1 (AngHV) ORF95 encodes a late, structural envelope protein.

Anguillid herpesvirus 1 (AngHV) is one of the vital pathogenic agents found in the wild and cultured eel populations, which has brought significant losses to eel culture industry in China. In this study, AngHV ORF95 was characterized. Bioinformatics analysis showed that ORF95 putatively encodes a structural protein that is homologous to hemagglutinin-esterase (HE) protein of infectious salmon anemia virus (ISAV). Temporal transcription and expression analysis indicated that ORF95 is a viral late gene. Subcellular localization analysis revealed that ORF95 was predominantly localized in the cytoplasm. Further, western blot analysis indicated that ORF95 is a structural protein of virion envelope. These results provide a novel basis to make further efforts to clarify the function of ORF95 in the process of AngHV infection and the possibility to use ORF95 as antigen to develop AngHV subunit vaccine.

Comparative transcriptome analysis of pilchard orthomyxovirus (POMV) and infectious salmon anaemia virus (ISAV).

The Tasmanian Atlantic salmon (Salmo salar) aquaculture industry had remained relatively free of major viral diseases until the recent emergence of pilchard orthomyxovirus (POMV). The virus originally isolated from wild pilchards in Southern Australia is of great concern to the industry as it can cause high mortality. Despite its classification in the Orthomyxoviridae family, POMV is genetically divergent from infectious salmon anaemia virus (ISAV) and potentially represents a new genus within the family. Previous research has produced a formal case definition for clinical POMV, but the molecular events that underpin viral infection have not been characterized. Here we have undertaken a comparative transcriptome analysis of the response of Atlantic salmon kidney cells (ASK) in vitro to both POMV and ISAV using RNA sequencing, by harvesting cells at 6 and 24 h post infection (hpi). Despite their genomic differences, both orthomyxoviruses induced significant, and in some cases similar, innate antiviral responses. Early up-regulation of pathogen recognition receptor genes, RIG-I and TLR3, was observed in response to both viruses and triggered downstream interferon (IFN) responses. Interferon transcripts (IFN-alpha1 and INF-alpha2) were only induced in POMV infected cells at 24 hpi, but IFN-alpha3 was up-regulated in all time points and with both viruses. In addition, a strong induction of antiviral response genes (Mx and ISG15) was observed during the early infection with both viruses. Analysis of transcription factor binding sites in the up-regulated gene sets indicated that the host response to both viruses was largely driven by interferon regulatory factors (IRF) 1 and 2. Only three genes (slc35f2, odf2, LOC106608698) were differentially expressed in opposite directions, up-regulated with POMV and strongly down-regulated with ISAV at 24 hpi. Differential expression of these transcripts is possibly a consequence of virus divergence, but could also be associated to higher viral loads observed in the infection with POMV. Results from this study improve our understanding of the innate immune responses and host-pathogen interactions between POMV and Atlantic salmon. Early host response genes could potentially be exploited as subclinical biomarkers specific to POMV, and improved the development of tools for disease surveillance.

Infectious salmon anaemia virus-molecular biology and pathogenesis of the infection.

Aquaculture has a long history in many parts of the world, but it is still young at an industrial scale. Marine fish farming in open nets of a single fish species at high densities compared to their wild compatriots opens a plethora of possible infections. Infectious salmon anaemia (ISA) is an example of disease that surfaced after large-scale farming of Atlantic salmon (Salmo salar) appeared. Here, a review of the molecular biology of the ISA virus (ISAV) with emphasis on its pathogenicity is presented. The avirulent HPR0 variant of ISAV has resisted propagation in cell cultures, which has restricted the ability to perform in vivo experiments with this variant. The transition from avirulent HPR0 to virulent HPRΔ has not been methodically studied under controlled experimental conditions, and the triggers of the transition from avirulent to virulent forms have not been mapped. Genetic segment reassortment, recombination and mutations are important mechanisms in ISAV evolution, and for the development of virulence. In the 25 years since the ISAV was identified, large amounts of sequence data have been collected for epidemiologic and transmission studies, however, the lack of good experimental models for HPR0 make the risk evaluation of the presence of this avirulent, ubiquitous variant uncertain. This review summarizes the current knowledge related to molecular biology and pathogenicity of this important aquatic orthomyxovirus.

Chitosan-Based Nanoparticles for Intracellular Delivery of ISAV Fusion Protein cDNA into Melanoma Cells: A Path to Develop Oncolytic Anticancer Therapies.

Oncolytic virus therapy has been tested against cancer in preclinical models and clinical assays. Current evidence shows that viruses induce cytopathic effects associated with fusogenic protein-mediated syncytium formation and immunogenic cell death of eukaryotic cells. We have previously demonstrated that tumor cell bodies generated from cells expressing the fusogenic protein of the infectious salmon anemia virus (ISAV-F) enhance crosspriming and display prophylactic antitumor activity against melanoma tumors. In this work, we evaluated the effects of the expression of ISAV-F on the B16 melanoma model, both in vitro and in vivo, using chitosan nanoparticles as transfection vehicle. We confirmed that the transfection of B16 tumor cells with chitosan nanoparticles (NP-ISAV) allows the expression of a fusogenically active ISAV-F protein and decreases cell viability because of syncytium formation in vitro. However, the in vivo transfection induces a delay in tumor growth, without inducing changes on the lymphoid populations in the tumor and the spleen. Altogether, our observations show that expression of ISAV fusion protein using chitosan nanoparticles induces cell fusion in melanoma cells and slight antitumor response.

Development of an Isavirus minigenome system to study the function of the pocket RNA-binding domain of the viral nucleoprotein (NP) in salmon cells.

The Isavirus is an orthomyxovirus with a genome composed of eight segments of negative single-strand RNA (-ssRNA). It has been proposed that the eight genomic segments of the Isavirus are organized as a ribonucleoprotein (RNP) complex called a minigenome, which contains all the viral RNA segments, a viral heterotrimeric polymerase and multiple copies of the viral nucleoprotein (NP). Here, we develop an Isavirus minigenome system and show the importance of the formation of active RNPs and the role of viral NP R189, R194, R302 and K325 residues in the NP RNA-binding domain in the context of RNPs. The results indicate it is possible to generate a minigenome in salmon cells, a composite ISAV RNPs with EGFP-based chimeric vRNA with heterotrimeric polymerase (PB1, PB2, PA) and NP protein using CMV-based auxiliary plasmids. It was also shown that NP R189, R194, R302 and K325 residues are important to generate viral mRNA from the constituted RNPs and a detectable reporter protein. This work is the first salmon cell-based minigenome assay for the Isavirus, which was evaluated by a bioinformatic and functional study of the NP protein in viral RNPs, which showed that correct NP-vRNA interaction is key to the functioning of RNPs.

Rapid sequence modification in the highly polymorphic region (HPR) of the hemagglutinin gene of the infectious salmon anaemia virus (ISAV) suggests intra-segmental template switching recombination.

The ISAV has a genome composed of eight segments of (-)ssRNA, segment 6 codes for the hemagglutinin-esterase protein, and has the most variable region of the genome, the highly polymorphic region (HPR), which is unique among orthomyxoviruses. The HPR has been associated with virulence, infectivity and pathogenicity. The full length of the HPR is called HPR0 and the strain with this HPR is avirulent, in contrast to strains with deleted HPR that are virulent to varying degrees. The molecular mechanism that gives rise to the different HPRs remains unclear. Here, we studied in vitro the evolution of reassortant recombinant ISAV (rISAV) in Atlantic salmon head kidney (ASK) cells. To this end, we rescued and cultivated a set of rISAV with different segment 6-HPR genotypes using a reverse genetics system and then sequencing HPR regions of the viruses. Our results show rapid multiple recombination events in ISAV, with sequence insertions and deletions in the HPR, indicating a dynamic process. Inserted sequences can be found in four segments of the ISAV genome (segments 1, 5, 6, and 8). The results suggest intra-segmental heterologous recombination, probably by class I and class II template switching, similar to the proposed segment 5 recombination mechanism.

In vivo virulence and genomic comparison of infectious Salmon Anaemia Virus isolates from Atlantic Canada.

The infectious salmon anaemia virus (ISAV) is capable of causing a significant disease in Atlantic salmon, which has resulted in considerable financial losses for salmon farmers around the world. Since the first detection of ISAV in Canada in 1996, it has been a high priority for aquatic animal health management and surveillance programmes have led to the identification of many genetically distinct ISAV isolates of variable virulence. In this study, we evaluated the virulence of three ISAV isolates detected in Atlantic Canada in 2012 by doing in vivo-controlled disease challenges with two sources of Atlantic salmon. We measured viral loads in fish tissues during the course of infection. Sequences of the full viral RNA genomes of these three ISAV isolates were obtained and compared to a high-virulence and previously characterized isolate detected in the Bay of Fundy in 2004, as well as a newly identified ISAV NA-HPR0 isolate. All three ISAV isolates studied were shown to be of low to mid-virulence with fish from source A having a lower mortality rate than fish from source B. Viral load estimation using an RT-qPCR assay targeting viral segment 8 showed a high degree of similarity between tissues. Through genomic comparison, we identified various amino acid substitutions unique to some isolates, including a stop codon in the segment 8 ORF2 not previously reported in ISAV, present in the isolate with the lowest observed virulence.

Overview of infectious salmon anaemia virus (ISAV) in Atlantic Canada and first report of an ISAV North American-HPR0 subtype.

The infectious salmon anaemia virus (ISAV) is an important viral disease of farmed Atlantic salmon that has caused considerable financial losses for salmon farmers around the world, including Atlantic Canada. It is listed as a notifiable disease by the World Organization for Animal Health, and to this day, culling of infected cages or farms remains the current practice in many countries to mitigate the spread of the virus. In Atlantic Canada, ISAV was first detected in 1996 and continues to be detected. While some outbreaks seemed to have arisen from isolated infections of unknown source, others were local clusters resulting from horizontal spread of infection. This study provides a description of the detected ISAV isolates in Atlantic Canada between 2012 and 2016, and explores the phylogenetic relatedness between these ISAV isolates. A key finding is the detection for the first time of a North American-HPR0 ISAV subtype, which was predicted to exist for many years. Through phylogenetic analysis, a scenario emerges with at least three separate incursions of ISAV in Atlantic Canada. An initial ISAV introduction follows a genotypic separation between North America and Europe which resulted in the NA and EU genotypes known today; this separation predates the salmon aquaculture industry. The second incursion of ISAV from Europe to North America led to a sublineage in Atlantic Canada consisting of EU-HPR∆ isolates detected in Nova Scotia and New Brunswick, and the predominant form of ISAV-HPR0 (EU). Finally, we observed what could be the third and most recent incursion of ISAV in Newfoundland, in the form of an isolate highly similar to ISAV EU-HPR0 isolates found in the Faroe Islands and the one isolate from Norway.

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.

An updated proposal for classification of infectious salmon anemia virus strains.

Biological databases contain a wealth of valuable information that can contribute to the enrichment of virtually any area. However, the exponential growth of information together with its dissemination through virtual networks has become a double-edged sword, promoting synonymy that leads to confusion and chaos. Organization of data is a big effort that must be accompanied by clarity, both in the deposited data and in the publications arising from them. In this report, an effort is made to organize the information related to infectious salmon anemia virus and its classification based on the variability of genomic segment 6.

Electrostatic Architecture of the Infectious Salmon Anemia Virus (ISAV) Core Fusion Protein Illustrates a Carboxyl-Carboxylate pH Sensor.

Segment 5, ORF 1 of the infectious salmon anemia virus (ISAV) genome, encodes for the ISAV F protein, which is responsible for viral-host endosomal membrane fusion during a productive ISAV infection. The entry machinery of ISAV is composed of a complex of the ISAV F and ISAV hemagglutinin esterase (HE) proteins in an unknown stoichiometry prior to receptor engagement by ISAV HE. Following binding of the receptor to ISAV HE, dissociation of the ISAV F protein from HE, and subsequent endocytosis, the ISAV F protein resolves into a fusion-competent oligomeric state. Here, we present a 2.1 Å crystal structure of the fusion core of the ISAV F protein determined at low pH. This structure has allowed us to unambiguously demonstrate that the ISAV entry machinery exhibits typical class I viral fusion protein architecture. Furthermore, we have determined stabilizing factors that accommodate the pH-dependent mode of ISAV transmission, and our structure has allowed the identification of a central coil that is conserved across numerous and varied post-fusion viral glycoprotein structures. We then discuss a mechanistic model of ISAV fusion that parallels the paramyxoviral class I fusion strategy wherein attachment and fusion are relegated to separate proteins in a similar fashion to ISAV fusion.

Discovery of variant infectious salmon anaemia virus (ISAV) of European genotype in British Columbia, Canada.

BACKGROUND: Infectious salmon anaemia (ISA) virus (ISAV) belongs to the genus Isavirus, family Orthomyxoviridae. ISAV occurs in two basic genotypes, North American and European. The European genotype is more widespread and shows greater genetic variation and greater virulence variation than the North American genotype. To date, all of the ISAV isolates from the clinical disease, ISA, have had deletions in the highly polymorphic region (HPR) on ISAV segment 6 (ISAV-HPRΔ) relative to ISAV-HPR0, named numerically from ISAV-HPR1 to over ISAV-HPR30. ISA outbreaks have only been reported in farmed Atlantic salmon, although ISAV has been detected by RT-PCR in wild fish. It is recognized that asymptomatically ISAV-infected fish exist. There is no universally accepted ISAV RT-qPCR TaqMan® assay. Most diagnostic laboratories use the primer-probe set targeting a 104 bp-fragment on ISAV segment 8. Some laboratories and researchers have found a primer-probe set targeting ISAV segment 7 to be more sensitive. Other researchers have published different ISAV segment 8 primer-probe sets that are highly sensitive. METHODS: In this study, we tested 1,106 fish tissue samples collected from (i) market-bought farmed salmonids and (ii) wild salmon from throughout British Columbia (BC), Canada, for ISAV using real time RT-qPCR targeting segment 8 and/or conventional RT-PCR with segment 8 primers and segment 6 HPR primers, and by virus isolation attempts using Salmon head kidney (SHK-1 and ASK-2) cell line monolayers. The sequences from the conventional PCR products were compared by multiple alignment and phylogenetic analyses. RESULTS: Seventy-nine samples were "non-negative" with at least one of these tests in one or more replicates. The ISAV segment 6 HPR sequences from the PCR products matched ISAV variants, HPR5 on 29 samples, one sample had both HPR5 and HPR7b and one matched HPR0. All sequences were of European genotype. In addition, alignment of sequences of the conventional PCR product segment 8 showed they had a single nucleotide mutation in the region of the probe sequence and a 9-nucleotide overlap with the reverse primer sequence of the real time RT-qPCR assay. None of the classical ISAV segment 8 sequences in the GenBank have this mutation in the probe-binding site of the assay, suggesting the presence of a novel ISAV variant in BC. A phylogenetic tree of these sequences showed that some ISAV sequences diverted early from the classical European genotype sequences, while others have evolved separately. All virus isolation attempts on the samples were negative, and thus the samples were considered "negative" in terms of the threshold trigger set for Canadian federal regulatory action; i.e., successful virus isolation in cell culture. CONCLUSIONS: This is the first published report of the detection of ISAV sequences in fish from British Columbia, Canada. The sequences detected, both of ISAV-HPRΔ and ISAV-HPR0 are of European genotype. These sequences are different from the classical ISAV segment 8 sequences, and this difference suggests the presence of a new ISAV variant of European genotype in BC. Our results further suggest that ISAV-HPRΔ strains can be present without clinical disease in farmed fish and without being detected by virus isolation using fish cell lines.