Susceptibility and Permissivity of Zebrafish (Danio rerio) Larvae to Cypriniviruses.

The zebrafish (Danio rerio) represents an increasingly important model organism in virology. We evaluated its utility in the study of economically important viruses from the genus Cyprinivirus (anguillid herpesvirus 1, cyprinid herpesvirus 2 and cyprinid herpesvirus 3 (CyHV-3)). This revealed that zebrafish larvae were not susceptible to these viruses after immersion in contaminated water, but that infections could be established using artificial infection models in vitro (zebrafish cell lines) and in vivo (microinjection of larvae). However, infections were transient, with rapid viral clearance associated with apoptosis-like death of infected cells. Transcriptomic analysis of CyHV-3-infected larvae revealed upregulation of interferon-stimulated genes, in particular those encoding nucleic acid sensors, mediators of programmed cell death and related genes. It was notable that uncharacterized non-coding RNA genes and retrotransposons were also among those most upregulated. CRISPR/Cas9 knockout of the zebrafish gene encoding protein kinase R (PKR) and a related gene encoding a protein kinase containing Z-DNA binding domains (PKZ) had no impact on CyHV-3 clearance in larvae. Our study strongly supports the importance of innate immunity-virus interactions in the adaptation of cypriniviruses to their natural hosts. It also highlights the potential of the CyHV-3-zebrafish model, versus the CyHV-3-carp model, for study of these interactions.

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.

Production of infectious salmon anaemia virus (ISAV) ribonucleoprotein complexes using a mammalian cell based minigenome system.

Developments in recombinant virus techniques have been crucial to understand the mechanisms of virulence acquisition and study the replication of many different negatively stranded RNA viruses. However, such technology has been lacking for infectious salmon anaemia virus (ISAV) until recently. This was due in part to the lack of a Polymerase I promoter in Atlantic salmon to drive the production of recombinant vRNA. Therefore, the present study investigated a different alternative to produce ISAV recombinant vRNA, based on Mouse Pol I promoter/terminator sequences and expression in baby hamster kidney (BHK-21) cells. As a first step, a pathogenic ISAV was demonstrated to replicate and produce viable virions in BHK-21 cells. This indicated that the virus could use the mammalian cellular and nuclear machinery to produce vRNA segments and viral proteins, albeit in a limited capacity. Co-transfection of vRNA expressing plasmids with cytomegalovirus (CMV) promoter constructs coding for the three viral polymerase and nucleoprotein led to the generation of functional ribonucleoproteins (RNPs) which expressed either, green fluorescence protein (GFP) or firefly luciferase (FF). Further experiments demonstrated that a 21h incubation at 37°C was optimal for RNPs production. Inhibition by ribavirin confirmed that FF expression was linked to specific RNPs polymerase transcription. The present minigenome system provides a novel and alternative approach to investigate various aspects of ISAV replication and potentially those of other negatively stranded RNA viruses. Expression of RNPs in mammalian cells could also provide a method for the rapid screening of anti-viral compounds targeting ISAV replication.

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.

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.

Characterization and comparison of the full 3' and 5' untranslated genomic regions of diverse isolates of infectious salmon anaemia virus by using a rapid and universal method.

The 3' and 5' untranslated regions (UTRs) of the gene segments of orthomyxoviruses interact closely with the polymerase complex and are important for viral replication and transcription regulation. Despite this, the 3' and 5' RNA UTRs of the infectious salmon anaemia virus (ISAV) genome have only been partially characterized and little is known about the level of conservation between different virus subtypes. This report details for the first time, the adaptation of a rapid method for the simultaneous characterization of the 3' and 5' UTRs of each viral segment of ISAV. This was achieved through self circularization of segments using T4 RNA ligase, followed by PCR and sequencing. Dephosphorylation of 5' ends using tobacco acid pyrophosphatase (TAP) proved to be a specific requirement for ligation of ISAV ends which was not essential for characterization of influenza virus in a similar manner. The development of universal primers facilitated the characterization of 4 genetically distinct ISAV isolates from Canada, Norway and Scotland. Comparison of the UTR regions revealed a similarity in organization and presence of conserved terminal sequences as reported for other orthomyxoviruses. Interestingly, the 3' ends of ISAV segments including segments 1, 5 and 6, were shorter and 5' UTRs generally longer than in their influenza counterparts.

Atlantic salmon (Salmo salar L.) serum vitellogenin neutralises infectivity of infectious pancreatic necrosis virus (IPNV).

Vitellogenin is a phosphoglycoprotein which represents the main precursor of the egg yolk in teleost fish. This reproductive protein was also demonstrated to play an important role in innate immunity by acting as a pattern recognition molecule capable of binding to bacteria, fungi and enhancing macrophage phagocytosis. The presented results demonstrate that, egg homogenate, ovarian fluid and serum of mature female Atlantic salmon have high neutralising ability for infectious pancreatic necrosis virus (IPNV). Vitellogenin from mature female Atlantic salmon serum, purified by immuno-affinity on a column matrix coated with monoclonal anti-Atlantic salmon vitellogenin antibody, was able to neutralise between 9.1 x 10(4) and 3.09 x 10(5) TCID(50) IPNV mg(-1) of protein. To the author's knowledge, this is the first time that the neutralising activity of vitellogenin on a teleost virus has been demonstrated. The results may explain why IPNV is difficult to detect by culture methods in ovarian fluid and egg homogenates from carrier mature females and suggest a possible means of vertical transmission via the egg.