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.

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.

Dual Mutation Events in the Haemagglutinin-Esterase and Fusion Protein from an Infectious Salmon Anaemia Virus HPR0 Genotype Promote Viral Fusion and Activation by an Ubiquitous Host Protease.

In Infectious salmon anaemia virus (ISAV), deletions in the highly polymorphic region (HPR) in the near membrane domain of the haemagglutinin-esterase (HE) stalk, influence viral fusion. It is suspected that selected mutations in the associated Fusion (F) protein may also be important in regulating fusion activity. To better understand the underlying mechanisms involved in ISAV fusion, several mutated F proteins were generated from the Scottish Nevis and Norwegian SK779/06 HPR0. Co-transfection with constructs encoding HE and F were performed, fusion activity assessed by content mixing assay and the degree of proteolytic cleavage by western blot. Substitutions in Nevis F demonstrated that K276 was the most likely cleavage site in the protein. Furthermore, amino acid substitutions at three sites and two insertions, all slightly upstream of K276, increased fusion activity. Co-expression with HE harbouring a full-length HPR produced high fusion activities when trypsin and low pH were applied. In comparison, under normal culture conditions, groups containing a mutated HE with an HPR deletion were able to generate moderate fusion levels, while those with a full length HPR HE could not induce fusion. This suggested that HPR length may influence how the HE primes the F protein and promotes fusion activation by an ubiquitous host protease and/or facilitate subsequent post-cleavage refolding steps. Variations in fusion activity through accumulated mutations on surface glycoproteins have also been reported in other orthomyxoviruses and paramyxoviruses. This may in part contribute to the different virulence and tissue tropism reported for HPR0 and HPR deleted ISAV genotypes.

Infectious salmon anaemia virus (ISAV) mucosal infection in Atlantic salmon.

All viruses infecting fish must cross the surface mucosal barrier to successfully enter a host. Infectious salmon anaemia virus (ISAV), the causative agent of the economically important infectious salmon anaemia (ISA) in Atlantic salmon, Salmo salar L., has been shown to use the gills as its entry point. However, other entry ports have not been investigated despite the expression of virus receptors on the surface of epithelial cells in the skin, the gastrointestinal (GI) tract and the conjunctiva. Here we investigate the ISAV mucosal infection in Atlantic salmon after experimental immersion (bath) challenge and in farmed fish collected from a confirmed outbreak of ISA in Norway. We show for the first time evidence of early replication in several mucosal surfaces in addition to the gills, including the pectoral fin, skin and GI tract suggesting several potential entry points for the virus. Initially, the infection is localized and primarily infecting epithelial cells, however at later stages it becomes systemic, infecting the endothelial cells lining the circulatory system. Viruses of low and high virulence used in the challenge revealed possible variation in virus progression during infection at the mucosal surfaces.

Low virulent infectious salmon anaemia virus (ISAV) replicates and initiates the immune response earlier than a highly virulent virus in Atlantic salmon gills.

Observations from the field and experimental evidence suggest that different strains of infectious salmon anaemia virus (ISAV) can induce disease of varying severity in Atlantic salmon. Variation in host mortality and dissemination of ISAV isolates with high and low virulence was investigated using immersion challenge; from which mortality, pathological, immunohistochemical and preliminary molecular results have been previously published. Here, real-time RT-PCR analysis and statistical modelling have been used to further investigate variation in virus load and the response of four select immune genes. Expression of type I and II interferon (IFN), Mx and γIFN induced protein (γIP) to high and low pathogenic virus infection were examined in gill, heart and anterior kidney. In addition, a novel RNA species-specific assay targeting individual RNA types was used to investigate the separate viral processes of transcription and replication. Unexpectedly, the low virulent ISAV (LVI) replicated and transcribed more rapidly in the gills compared to the highly virulent virus (HVI). Subsequently LVI was able to disseminate to the internal organs more quickly and induced a more rapid systemic immune response in the host that may have offered some protection. Contrary to this, HVI initially progressed more slowly in the gills resulting in a slower generalised infection. However HVI ultimately reached a higher viral load and induced a greater mortality.

Deletions in the highly polymorphic region (HPR) of infectious salmon anaemia virus HPR0 haemagglutinin-esterase enhance viral fusion and influence the interaction with the fusion protein.

Since the discovery of a non-virulent infectious salmon anaemia virus (ISAV) HPR0 variant, many studies have speculated on the functional role of deletions within the highly polymorphic region (HPR) of genomic segment 6, which codes for the haemagglutinin-esterase (HE) protein. To address this issue, mutant HE proteins with deletions in their HPR were generated from the Scottish HPR0 template (NWM10) and fusion-inducing activity was measured using lipid (octadecyl rhodamine B) and content mixing assays (firefly luciferase). Segment six HPR was found to have a strong influence on ISAV fusion, and deletions in this near-membrane region predominantly increased the fusion-inducing ability of the resulting HE proteins. The position and length of the HPR deletions were not significant factors, suggesting that they may affect fusion non-specifically. In comparison, the amino acid composition of the associated fusion (F) protein was a more crucial criterion. Antibody co-patching and confocal fluorescence demonstrated that the HE and F proteins were highly co-localized, forming defined clusters on the cell surface post-transfection. The binding of erythrocyte ghosts on the attachment protein caused a reduction in the percentage of co-localization, suggesting that ISAV fusion might be triggered through physical separation of the F and HE proteins. In this process, HPR deletion appeared to modulate and reduce the strength of interaction between the two glycoproteins, causing more F protein to be released and activated. This work provides a first insight into the mechanism of virulence acquisition through HPR deletion, with fusion enhancement acting as a major contributing factor.

A strand specific real-time RT-PCR method for the targeted detection of the three species (vRNA, cRNA and mRNA) of infectious salmon anaemia virus (ISAV) replicative RNA.

Three species of viral-derived RNA (vRNA, cRNA and mRNA) are produced during an infectious salmon anaemia virus (ISAV) infection. Conventional real-time RT-PCR (RT-qPCR) targeting ISAV segment 8 provides a very sensitive method for the detection of ISAV RNA, however it does not differentiate between these three individual RNA species. In this study, strand-specific tagged primers have been utilised in the RT reaction to specifically produce cDNA corresponding to each of the 3 viral RNA types produced from ISAV segment 8 for the subsequent detection by real-time PCR. The RNA species-specific assay was successfully used to specifically distinguish synthetic T7-produced RNA transcripts representing the 3 species of ISAV RNA at levels up to approximately 10(5)-fold higher than the other types. In addition, the method was applied to investigate the production of segment 8 RNA in time-course tissue culture experiments performed at optimal (15°C), sub-optimal (20°C) and inadequate (25°C) temperatures for replication or in the presence of a chemical inhibitor to vary the RNA populations and investigate its effectiveness. Variation in RNA production was observed between the optimal and sub-optimal temperatures and in the presence of the chemical inhibitor. Production of all RNA species was completely inhibited at 25°C indicating the potential usefulness of the assay as a tool in the understanding of ISAV replication and transcription dynamics.

Presence of a full-length highly polymorphic region (HPR) in the ISAV haemagglutinin-esterase does not affect the primary functions of receptor binding and esterase activity.

The putatively avirulent infectious salmon anaemia virus (ISAV) HPR0 variant has key phenotypic differences to isolates from disease outbreaks in Atlantic salmon farms. It appears to not cause disease, potentially displays a different tissue tropism and has yet to be isolated in conventional ISAV-permissive cell lines. This study focussed on identifying the biological basis for the observed differences by examining the properties of the haemagglutinin-esterase (HE) proteins derived from NWM10 (HPR0), Nevis 390/98 (HPR7 pathogenic strain) and mutant combinations of the two. Using a transfection-based system and haemadsorption analysis in salmon cell lines, this study demonstrated for the first time that an HPR0 HE was fully functional in terms of receptor-binding and -destroying activity and also suggested that the presence of a full-length HPR alone did not appear to affect these functions.

Surveillance for infectious salmon anaemia virus HPR0 in marine Atlantic salmon farms across Scotland.

Infectious salmon anaemia virus (ISAV) is a serious and commercially important pathogen of Atlantic salmon. Multiple viruses have been defined based on a highly polymorphic region (HPR) of the haemagglutinin-esterase (HE) protein encoded by genomic segment 6. The viruses causing disease outbreaks in farms to date all have deletions in this region with respect to a putative ancestral variant with a longer HPR (HPR0). The presence of HPR0 nucleic acid has been detected in many countries including Scotland, where it has mostly been associated with healthy wild and farmed fish. Pathogenic ISAVs appear to have been derived from HPR0 ancestors on multiple independent occasions, which suggests that the presence of HPR0 could represent a risk factor in the re-emergence of infectious salmon anaemia (ISA) disease. In order to better understand this potential risk factor, anonymous samples of gill and heart tissues from marine Atlantic salmon farms throughout Scotland were collected and screened for the presence of ISAV RNA. Since it has not been possible to isolate HPR0 in conventional ISA-permissive cell cultures, a sensitive real-time RT-PCR method was employed for the detection of viral RNA. DNA sequencing was carried out on the positive samples to determine their HPR sequence. ISAV RNA was detected in 6 samples originating from 4 different locations and sequence analysis indicated the viruses were of the HPR0 type. Full length segment 6 sequence analysis of 1 positive sample indicated that it was most similar to a European genotype sequence previously obtained from North America.

Development of a sensitive and controlled real-time RT-PCR assay for viral haemorrhagic septicaemia virus (VHSV) in marine salmonid aquaculture.

A survey was undertaken to determine the potential distribution of viral haemorrhagic septicaemia virus (VHSV) in marine cage-based salmonid farms in Scotland. A rapid, accurate and sensitive quantitative real-time RT-PCR (qRT-PCR) assay was developed, targeting a conserved region of the nucleoprotein (N) gene of the virus. The qRT-PCR assay was shown to be more sensitive than the conventional VHSV RT-PCR. A validation protocol included several different virus isolates as the target and confirmed that the assay could detect all European VHSV genotypes (I, II and III). Both endogenous and exogenous controls were designed to control for integrity of template and distinguish between true VHSV positives and contamination with the positive control material. In total, the universal European VHSV qRT-PCR assay with exogenous positive control was applied to screen 2040 individual Atlantic salmon Salmo salar and 150 individual rainbow trout Oncorhynchus mykiss. No evidence of the presence of VHSV in association with either salmonid species in Scottish marine farms was detected. However, both marine Atlantic salmon and rainbow trout are still considered possible carriers of VHSV, which remains a potential threat to freshwater farming. Therefore, a continued surveillance of these species in marine environment is recommended.

Identification of an interferon antagonist protein encoded by segment 7 of infectious salmon anaemia virus.

Infectious salmon anaemia virus (ISAV) is an orthomyxovirus and member of the genus Isavirus, which contains eight genomic segments coding for ten viral proteins. This study focussed on identifying the function of the largest protein encoded by ISAV genomic segment 7 (7i), which like influenza A segment 7 encodes two proteins, one of which is based on removal of an intron from the primary transcript. Using two independent methods, an Mx1 promoter-driven reporter system and real-time PCR of FACS-sorted transfected cells, we demonstrate that the non-structural ISAV 7i protein is an interferon-signalling antagonist. Other transfection studies indicated a predominantly cytoplasmic localisation of the expressed protein, which is consistent with this role. The demonstration that ISAV segment 7 encodes a putative non-structural IFN system antagonist reveals a difference with influenza A virus, where segment 7, which shares a similar coding strategy, encodes the structural matrix proteins.

Development and application of real-time PCR for specific detection of Lepeophtheirus salmonis and Caligus elongatus larvae in Scottish plankton samples.

Lepeophtheirus salmonis and Caligus elongatus are important parasites of wild and cultured salmonids in the Northern Hemisphere. These species, generically referred to as sea lice, are estimated to cost the Scottish aquaculture industry in excess of pound 25 million per annum. There is great interest in countries such as Ireland, Scotland, Norway and Canada to sample sea lice larvae in their natural environment in order to understand lice larvae distribution and improve parasite control. Microscopy is currently relied on for use in the routine identification of sea lice larvae in plankton samples. This method is, however, limited by its time-consuming nature and requirement for highly skilled personnel. The development of alternative methods for the detection of sea lice larvae which might be used to complement and support microscopic examinations of environmental samples is thus desirable. In this study, a genetic method utilising a real-time PCR Taqman-MGB probe-based assay targeting the mitochondrial cytochrome oxidase I (mtCOI) gene was developed, which allowed species-specific detection of L. salmonis and C. elongatus larvae from unsorted natural and spiked plankton samples. Real-time PCR is a rapid, sensitive, highly specific and potentially quantitative technique. This study demonstrated its suitability for the routine identification of L. salmonis and C. elongatus in mixed plankton samples. The real-time PCR assay developed has considerable potential for use in complementing, supporting and reducing reliance on time-consuming conventional microscopic examination for the specific identification of sea lice larvae in plankton samples.