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.

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.

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.