Genotyping of marine viral haemorrhagic septicaemia virus isolated from the Flemish Cap by nucleotide sequence analysis and restriction fragment length polymorphism patterns

Abstract

A total of 14 viral haemorrhagic septicaemia virus (VHSV) isolates obtained from Greenland halibut Reinhardtius hippoglossoides caught at the Flemish Cap, a fishing ground in the North Atlantic Ocean near Newfoundland, were characterised using restriction fragment length polymorphism (RFLP) and nucleotide sequence analysis. RFLP analysis was performed on a 1259 bp fragment of the glycoprotein (G) gene, and a 305 nucleotide region within the nucleoprotein (N) gene was used for sequence analysis. Representative strains of the 4 established genotypes were employed for comparative purposes. Sequencing analysis indicated that the Flemish cap isolates grouped in Genotype 3, which also includes isolates from wild fish caught in the North Sea and coastal waters of the UK and Ireland, isolates derived from outbreaks of VHS in turbot farms in the British Isles, and an isolate from European eel Anguilla anguilla caught in northern France. Characterisation using RFLPs resulted in the development of a simple and reliable method of typing VHSV at the genotype level using a 2-step restriction analysis (2-SRA) assay. KEY WORDS: Viral haemorrhagic septicaemia virus · VHSV · Wild fish · Genotyping · Restriction fragment length polymorphism · RFLP · Sequencing analysis · Phylogenetics Resale or republication not permitted without written consent of the publisherDis Aquat Org 73: [23][24][25][26][27][28][29][30][31] 2006 lished by these authors, Genotype 1 groups isolates from continental Europe as well as several marine isolates from the Baltic Sea; Genotype 2 includes a number of marine isolates obtained from the Baltic Sea with no clear link to rainbow trout aquaculture; Genotype 3 comprises isolates obtained from fish cultured or caught around the British Isles, as well as a French isolate from European eel Anguilla anguilla caught on the Atlantic side of the Loire; Genotype 4 includes VHSV strains isolated from the Pacific coast of North America.Although there is no doubt about the usefulness and reliability of sequencing analysis, this technology is not practical or affordable for many laboratories. For this reason, other approaches for molecular typing of fish viruses have been developed. Ribonuclease protection assays (RPA) have been employed to characterise genetic differences in closely related RNA viruses, and this technique has been applied to the study of infectious haematopoietic necrosis virus (IHNV; Kurath et al. 1995, Troyer et al. 2000 and VHSV . Restriction fragment length polymorphism (RFLP) patterns have also been successfully used for genotyping aquabirnaviruses (Heppell et al. 1992, Lee et al. 1996, Biering et al. 1997, Cutrín et al. 2004, and have recently been applied to VHSV by Einer-Jensen et al. (2005), who proposed employing a set of 3 enzymes for typing VHSV isolates.Here we report the employment of both RFLP and sequence analysis to genotype 15 isolates of VHSV obtained from Greenland halibut caught at the Flemish Cap, a deep fishing ground in the North Atlantic Ocean in international wat...

Real-time RT-PCR for detection, identification and absolute quantification of viral haemorrhagic septicaemia virus using different types of standards

“…Upon infection of the host, the 6 genes are transcribed progressively from the 3' to 5' end of the template viral RNA, and in decreasing molar frequencies (Kurath & Leong 1985). Therefore, due to the initial position of the N-gene in the VHSV genome, N transcripts are the most abundant during viral infection and therefore have been the target of several molecular assays used to detect VHSV (López-Vázquez et al 2006, Matejusova et al 2008, Cutrín et al 2009.…”

Characterization of a VHS virus genotype III isolated from rainbow trout (Oncorhychus mykiss) at a marine site on the west coast of Norway

“…The third VHS virus genotype (III) represents isolates from marine fish species in Kattegat, Skagerrak and the North Sea [8] and a member of this genotype was in the autumn of 2007 associated with about 10% mortality in a rainbow trout farm in western Norway [9,10]. VHS virus genotype III has been found in eel ( Anguilla anguilla ), cod ( Gadus morhua ), herring ( Clupea harengus ), sprat ( Sprattus sprattus ), haddock ( Melanogrammus aeglefinus ), Norway pout ( Trisopterus esmarkii ), poor cod ( Trisopterus minutus ), blue whiting ( Micromesistius poutassou ), withing ( Merlangius merlangus ), turbot ( Scophthalmus maximus ), greenland halibut ( Reinhardtius hippoglossoides ) and lesser argentine ( Argentina sphyraena ) [cf [3,5,8,11]]. The outbreak of VHS in Norway is the first time an isolate belonging to genotype III is found in rainbow trout.…”

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

“…For Atlantic salmon, this finding was perhaps not surprising since, in Europe, the detection of VHSV in this species is very rare, including only a single isolation of virus in Spain (Jimenez de la Fuente et al 1988), which was further characterized by Lopez-Vazquez et al (2006b). In addition, results of experimental infection trials using bath immersion showed that all types of VHSV are non-or lowly pathogenic to Atlantic salmon (Skall et al 2005).…”