Isolation of viral hemorrhagic septicemia virus from Greenland halibut Reinhardtius hippoglossoides caught at the Flemish Cap

Abstract

Viral hemorrhagic septicemia virus (VHSV) was isolated from apparently healthy Greenland halibut Reinhardtius hippoglossoides caught in the Flemish Cap, a deep fishing ground in the North Atlantic Ocean in international waters near Newfoundland. The identity of the virus was confirmed by electron microscopy, immunodot, seroneutralization and reverse transcriptasepolymerase chain reaction. In the serology assays, all isolates reacted in the immunodot assay with a polyclonal antiserum against the European VHSV Type Strain F1, and were neutralized by the same antiserum, although most of the strains showed low or moderate neutralization titers. None of the isolates were detected by immunofluorescence using a specific monoclonal antibody against a nucleocapsid-related protein of VHSV F1. This is the first report of VHSV isolated from wild Greenland halibut, which represents a new host species for the virus, and it is also the first evidence of VHSV in a location close to the Atlantic coast of North America. This isolation indicates that VHSV is more widely distributed than has been thought, and appears to support a marine origin of this virus. KEY WORDS: Viral hemorrhagic septicemia virus · VHSV · Greenland halibut · Wild fish Resale or republication not permitted without written consent of the publisherDis Aquat Org 50: [171][172][173][174][175][176][177][178][179] 2002 time , and that in mainland Europe it may have originated from a marine source (Batts et al. 1993, Meyers & Winton 1995, Dixon 1999.In the present paper we report the first isolations of VHSV from Greenland halibut Reinhardtius hippoglossoides caught during a research campaign ('Flemish Cap'94') to evaluate fish stocks in a Newfoundland fishery in the North Atlantic Ocean, and discuss the possible origin of this virus. MATERIALS AND METHODSCell lines. Monolayers of chinook salmon embryo (CHSE-214), epithelioma papillosum cyprini (EPC) and rainbow trout gonad (RTG-2) cells were used for the primary detection of the virus. Other fish cell lines used to test the host range of the virus were brown bullhead (BB) and TV-1 (from turbot fin). All cell lines were grown in Eagle's minimum essential medium (EMEM, Gibco) supplemented with 10% foetal calf serum (FCS, Gibco), 100 I.U. ml -1 penicillin and 100 µg ml -1 streptomycin. The CHSE-214 cells were grown at 15°C, RTG-2 and TV-1 at 20°C and EPC and BB at 25°C. Confluent monolayers of all cell lines were maintained at 15°C, and the medium substituted by EMEM with 0 or 2% FCS.Virus isolation. Various species of fishes were caught in a research campaign ('Flemish Cap'94') during July 1994, led by the Instituto de Investigacións Mariñas (Vigo, Spain) as a part of an EU project to evaluate fish stocks in that fishery. Flemish Cap is a Newfoundland fishing ground located in international waters in the North Atlantic Ocean (Fig. 1A) where fishes are captured under NAFO (North Atlantic Fisheries Organization) surveillance.A total of 80 asymptomatic fishes (38 Atlantic cod, 30 Greenland halibut, 7 witch ...

Prevalence of viral haemorrhagic septicaemia virus in Danish marine fishes and its occurrence in new host species

“…Most of the fish were thus sampled in areas from which VHSV has either not been isolated from these species or has not been isolated at all. VHSV has previously been reported in Pleuronectidae, in Greenland halibut Reinhardtius hippoglossoides from the North American Atlantic area (Dopazo et al 2002) and English sole Parohrys vetulus from the North American Pacific area (Hershberger & Kocan unpubl. in Hershberger et al 1999).…”

“…Since the first isolation from marine fish of viral haemorrhagic septicaemia virus (VHSV) from Atlantic cod Gadus morhua in 1979 (Jensen et al 1979, Jørgensen & Olesen 1987, VHSV has been isolated from an increasing number of marine fish species all over the northern hemisphere (Meyers & Winton 1995, Traxler et al 1995, Dixon et al 1997, Kent et al 1998, Mortensen et al 1999, Smail 2000, Takano et al 2000, Cox & Hedrick 2001, King et al 2001b, Kocan et al 2001, Brudeseth & Evensen 2002, Dopazo et al 2002, Olivier 2002, Watanabe et al 2002, Kim et al 2003.…”

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

“…VHS outbreaks have also been reported in other cultured and wild fish species elsewhere (Schlotfeldt et al 1991, Meier et al 1994, Ross et al 1994, Meyers et al 1999, Isshiki et al 2001, and in recent years VHS viruses have been isolated from an increasing number of often asymptomatic wild marine fish (Dixon et al 1997, 2003, Mortensen 1999, Smail 2000, Brudeseth & Evensen 2002, Dopazo et al 2002, Watanabe et al 2002, Hedrick et al 2003.…”

“…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 waters near Newfoundland (Dopazo et al 2002). At least 1 reference strain belonging to each of the genotypes established by Snow et al (2004) was also included for comparative purposes and for the development of a simplified method of typing VHSV strains by RFLP analysis.…”

“…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 waters near Newfoundland (Dopazo et al 2002). At least 1 reference strain belonging to each of the genotypes established by Snow et al (2004) was also included for comparative purposes and for the development of a simplified method of typing VHSV strains by RFLP analysis.…”