M. Snow

Identifying potential virulence determinants in viral haemorrhagic septicaemia virus (VHSV) for rainbow trout.

We identified viral haemorrhagic septicaemia virus (VHSV) isolates classified within Genotype Ib which are genetically similar (>99.4% glycoprotein amino acid identity) yet, based on their isolation history, were suspected to differ in virulence in juvenile rainbow trout. The virulence of an isolate recovered in 2000 from a viral haemorrhagic septicaemia disease episode in a marine rainbow trout farm in Sweden (SE-SVA-1033) was evaluated in juvenile rainbow trout via intraperitoneal injection and immersion challenge alongside 3 isolates recovered from wild-caught marine fish (DK-4p37, DK-5e59 and UKMLA98/6HE1) suspected of being of low pathogenicity to trout. Mortality data revealed that isolate SE-SVA-1033 caused VHSV-specific mortality in both intraperitoneal and immersion challenges (75.0 and 15.4%, respectively). The remaining Genotype Ib isolates caused significantly lower mortalities using the same experimental infection routes (<35.0 and <2.0%, respectively). Having identified VHSV isolates with clear differences in their pathogenicity, coding and inter-genic non-coding regions of 2 isolates (SE-SVA-1033 and DK-4p37) were determined and compared in order to identify potential markers responsible for the observed differences in virulence. Only 4 predicted amino acid substitutions were identified across the genome sequenced; these occurred in the N (R46G), G (S113G), NV (L12F) and L (S56A) proteins. These findings form the basis for further studies aimed at determining the biological significance of these mutations and suggest that small changes at the molecular level can cause significant changes in the virulence properties of VHSV isolates.

Interferon response following infection with genetically similar isolates of viral haemorrhagic septicaemia virus (VHSV) exhibiting contrasting virulence in rainbow trout.

Isolates of viral haemorrhagic septicaemia virus (VHSV) were identified which are genetically similar yet, based on their isolation history were considered likely to differ in virulence in juvenile rainbow trout. An experimental infection study was performed in order to verify this hypothesis and provide an experimental infectivity model with which to investigate the basis for susceptibility of rainbow trout to this commercially important virus. Significant differences in mortality were obtained following both intraperitoneal (IP) injection and immersion challenges with an early marine (DK-M.Rhabdo) and early rainbow trout VHSV isolate (DK-F1) respectively. Expression of Type I IFN, Mx1 (an IFN-inducible protein), and viral genes (encoding nucleo-, phospho-, matrix, glyco- and non-viron proteins) was studied in sequential tissue samples using real-time quantitative PCR (QPCR). Resulting data revealed a significant increase in IFN and Mx1 expression detected in fish challenged by IP injection with both isolates. Expression levels of these genes were directly related to the degree of viral replication as measured by the expression of VHSV RNAs. In immersion-challenged fish a significant increase in Mx1 was observed only when using the virulent isolate DK-F1; however no elevated host response was detectable in fish challenged with the marine isolate DK-M.Rhabdo. Quintessentially the inability to detect any virus in trout challenged with the marine isolate via immersion suggests the virus was incapable of establishing infection. The mechanisms for this appear to be more related to initial cellular entry and replication rather than due to the overcoming of initial infection via an elevated host innate immune response.

Development and validation of a range of endogenous controls to support the implementation of practical Taqman real-time PCR-based surveillance for fish diseases within aquaculture

The use of Taqman real-time PCR-based technology has recently become more frequent in the detection of pathogens in the aquaculture industry. This interest has necessitated the development of robust and reliable pathogen-detection assays. The development of a range of endogenous control assays to be run alongside these diagnostic assays works to further increase confidence in the latter. This study describes the design of a range of endogenous control assays based on the elongation factor 1-α (EF1-α) gene specific to a range of fish species including Atlantic salmon, Salmo salar; rainbow trout, Oncorhynchus mykiss; brown trout, Salmo trutta; cod, Gadus morhua; haddock, Melanogrammus aeglefinus; saithe, Pollachius virens; whiting, Merlangius merlangus; Norway pout, Trisopterus esmarkii; carp (family Cyprinidae), roach, Rutilus rutilus; European eel, Anguilla anguilla; and herring, Clupea harengus, as well as a number of fish cell lines. Evidence is provided of the validation of these assays for specific species, a range of tissue types and cell lines as well as an example of the potential uses of these assays.

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.

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.

Survey and management of mussel Mytilus species in Scotland

The important ecological role of Mytilus mussels in marine ecosystems, their high abundance in coastal waters and the demand for human consumption has made them a target species for aquaculture. Mussel cultivation is the most important and rapidly growing sector of the Scottish shellfish aquaculture industry and until recently production was considered to be based exclusively on the native species Mytilus edulis. However, the sympatric occurrence of M. edulis, M. trossulus, M. galloprovincialis and their hybrids in cultivation has recently been reported and significant production losses (over 50% at some sites) have been attributed to the presence of fragile-shelled M. trossulus. Given the ecological and economical importance of these species, an urgent need arose for a wider understanding of Mytilus species distribution on Scottish coasts and its implication for the sustainability of the Scottish shellfish industry. Here we present a summary of a 3-year project established within the “ECOsystem approach to SUstainable Management of the Marine Environment and its living Resources” (ECOSUMMER) Marie Curie network to address this need. We developed DNA-based molecular assays for the detection and surveillance of the different Mytilus species in Scotland. Several potential management strategies have been explored, aimed at favouring M. edulis production at mixed-species sites, but these have so far not been found to provide the reliable efficacy necessary for adoption by the industry. Complete eradication of M. trossulus from economically affected areas in Scotland may be unrealistic, especially considering that its introduction and distribution mechanisms in the environment remain uncertain. Area-specific solutions to managing the problem may thus be required, which may or may not involve eradication and fallowing (clearance of mussels from production sites for a given period of time). Nevertheless, the current distribution of M. trossulus is limited and its spread outside its existing range is clearly undesirable. Any management solutions must also be accompanied by an industry wide strategy and awareness, for example, through the development of an industry supported code of good practice.

Development of a real-time PCR assay for the identification of Gyrodactylus parasites infecting salmonids in northern Europe.

Gyrodactylus salaris is a monogenean freshwater parasite that causes high mortality in wild Atlantic salmon, and a number of countries employ monitoring programmes for its presence. A TaqMan-MGB (minor groove binding) probe real-time multiplex assay targeting the internal transcribed spacer ribosomal DNA (ITS rDNA) was developed to simultaneously identify G. salaris/G. thymalli and 2 other commonly occurring Gyrodactylus species infecting salmonids in northern Europe: G. derjavinoides and G. truttae. In addition, a Gyrodactylus genus-level assay was developed to assess parasite DNA quality. The species-specific real-time PCR method correctly identified target species from a wide geographical range and from a number of salmonid hosts. It did not amplify G. lucii or G. teuchis. These species were successfully amplified using the Gyrodactylus genus real-time assay. The species-specific real-time assay proved to be significantly faster than the currently employed molecular screening method of ITS rDNA PCR amplification followed by restriction fragment length polymorphism analyses (RFLP). However, as with ITS RFLP, the real-time method did not distinguish between G. salaris and the non-pathogenic G. thymalli, its principle advantage being a significant reduction in time to achieve an initial diagnostic screen before the employment of more in-depth analyses for those specimens giving a positive G. salaris/G. thymalli real-time result.