Evolution of the fish rhabdovirus viral haemorrhagic septicaemia virus

Katja Einer-Jensen1, Peter Ahrens2, Roald Forsberg3 and Niels Lorenzen1

1 Danish Institute for Food and Veterinary Research, Hangøvej 2, DK-8200 Århus, Denmark
2 Danish Institute for Food and Veterinary Research, Bülowsvej 27, DK-1790 Copenhagen V, Denmark
3 Bioinformatics Research Center, Department of Ecology and Genetics, University of Århus, Ny Munkegade, Building 540, DK-8200 Århus, Denmark

Correspondence
Katja Einer-Jensen
kej{at}dfvf.dk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Viral haemorrhagic septicaemia (VHS) caused by the rhabdovirus VHSV is economically the most important viral disease in European rainbow trout farming. Until 1989, this virus was mainly isolated from freshwater salmonids but in the last decade, it has also been isolated from an increasing number of free-living marine fish species. To study the genetic evolution of VHSV, the entire G gene from 74 isolates was analysed. VHSV from wild marine species caught in the Baltic Sea, Skagerrak, Kattegat, North Sea, and English Channel and European freshwater isolates, appeared to share a recent common ancestor. Based on the estimated nucleotide substitution rate, the ancestor of the European fresh water isolates was dated some 50 years ago. This finding fits with the initial reports in the 1950s on clinical observations of VHS in Danish freshwater rainbow trout farms. The study also indicates that European marine VHSV and the North American marine line separated approx. 500 years ago. The codon substitution rate among the freshwater VHSV isolates was found to be 2·5 times faster than among marine isolates. The data support the hypothesis of the marine environment being the original reservoir of VHSV and that the change in host range (to include rainbow trout) may have occurred several times. Virus from the marine environment will therefore continue to represent a threat to the trout aquaculture industry.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Viral haemorrhagic septicaemia (VHS) is best known as an infectious disease of rainbow trout (Oncorhynchus mykiss) in Western Europe, where it causes extensive losses among commercial farms (Wolf, 1988; Smail, 1999). The causative virus (VHSV) is an enveloped negative-strand RNA virus belonging to the Novirhabdovirus genus of the Rhabdoviridae. The VHSV genome consists of approximately 11 200 nucleotides and contains six genes in the order 3'-N-P-M-G-Nv-L-5', encoding a non-structural protein (Nv) with unknown function and five structural proteins: nucleocapsid- (N-), phospho- (P-), matrix- (M-), glyco- (G) and RNA polymerase (L) protein, respectively (Schutze et al., 1999). The G protein has been shown to be the target molecule for neutralizing and protective antibodies (Bearzotti et al., 1995; Lorenzen et al., 1990, 1999), which has also been reported for other rhabdoviruses (Huang et al., 1994; Kelley et al., 1972; Wiktor et al., 1973). The first isolation of VHSV and identification of this virus as the causal agent of severe disease outbreaks among cultured rainbow trout was carried out in Denmark in 1962 and, subsequently, a voluntary disease eradication campaign was started in Danish trout farms in 1965 (Jensen, 1965; Jørgensen, 1974). From 1980 to 1997, 20–40 farms carried out a controlled stamping out procedure each year and, as a consequence, the total numbers of infected farms in Denmark have decreased from approximately 400 to 40 over that period (Olesen, 1998). After the initial Danish isolation and identification, the virus was also reported in other European countries (e.g. Austria, Belgium, France, Germany, Italy and The Netherlands) (Olesen, 1998).

In the last decade, VHSV has been isolated from an increasing number of free-living marine fish species in the Northern hemisphere waters near Japan, Europe and North America (Hedrick et al., 2003; Mortensen et al., 1999; Takano et al., 2000), indicating a widespread occurrence of the virus in the marine environment. The isolation of VHSV at new geographical locations and in new hosts might reflect either the spread of the virus into new ecological niches or the existence of populations of virus that have not previously been discovered.

The virus isolates from wild marine fish are serologically indistinguishable from the continental isolates (Benmansour et al., 1997; Einer-Jensen et al., 1995; Winton et al., 1989), but all marine isolates tested so far typically produce little or no mortality in rainbow trout fry following waterborne challenge (Dixon et al., 1997; Skall et al., 2004). However, some of the marine isolates have been shown to be highly pathogenic to turbot (Scophthalmus maximus) and Pacific herring (Clupea pallasii) (Kocan et al., 1997; King et al., 2001a). Genetic similarity between marine isolates from the North Sea and those responsible for outbreaks of disease in cultured turbot farms in Scotland has also been reported (Ross et al., 1995; Snow et al., 1999). Previous phylogenetic studies of VHSV isolates have identified the existence of discrete genetic groupings, related to geographical areas (Benmansour et al., 1997; Thiéry et al., 2002; Stone et al., 1997; Nishizawa et al., 2002). Although speculated (reviewed by Dixon, 1999), a close link between marine isolates and freshwater isolates has so far not been demonstrated. The present study focuses on a detailed phylogenetic characterization of the four known genotypes (Snow et al., 1999), with emphasis on determination of the origin of rainbow trout pathogenic isolates.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virus isolates and RNA extraction.
A panel of 74 virus isolates was selected in order to represent variations in geographical area, host species and year of isolation (Table 1 and Fig. 1). The analysed panel comprises 38 years of VHSV isolation and includes 40 freshwater isolates from rainbow trout, three isolates from sea-reared rainbow trout that had been infected before transfer to the sea, four rainbow trout isolates from sea-reared fish that were uninfected before transfer to the sea and 26 isolates recovered from a wide range of pelagic and bottom-dwelling wild marine fish species. The 74 isolates in this analysis represented 65 different collection sites covering wide geographic areas, although 34/43 rainbow trout isolates and 7/26 marine isolates came from Denmark or surrounding waters.


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Table 1. Data related to the 74 viral haemorrhagic septicaemia virus isolates used in this study

GenBank accession numbers representing available full-length G genes have been included. Only unique sequences (shown in bold) were included in the phylogenetic analysis. Isolates with identical sequences: DK-1p52=DK-1p55; DK-1p8=DK-1e62=DK-1p12; DK-5727=DK-5740=DK-5741; DK-9995361=DK-2000511=DK-2000791; DK-2000201=DK-2000273=DK-2000291=DK-2000704; SE-SVA29=SE-SVA31; SE-SVA30=SE-SVA32.

 


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Fig. 1. Map of the ICES statistical squares charted, from which the included European marine VHSV have been isolated. Relations between isolate code and ICES square coordinates are given in Table 1. Genotype distributions of the isolates are indicated by shaded or striped squares.

 
Propogated virus isolates with a low (usually 3–7) passage number were inoculated at low multiplicity on monolayers of BF-2 cells (Wolf et al., 1966; Lorenzen et al., 1988). When a total cytopathic effect was evident, the cell culture was centrifuged at 4000 g for 15 min. A 5 ml sample of each supernatant was ultracentrifuged (86 000 g, 1 h) and the pelleted virus particles were suspended in 95 µl RNase-free water. Viral RNA was extracted from the pellet by the use of RNA affinity spin columns (RNeasy Total RNA kit; Qiagen), according to the manufacturer's instructions. Purified RNA was eluted in 30 µl RNase-free water, aliquoted into six tubes and stored until used at –80 °C.

Primer design.
Primers for full-length RT-PCR amplification and sequencing of the G gene (Table 2) were designed on the basis of the published genomic sequences (GenBank accession nos: AF143863, AF143862, Z93414, Z93412, Y18263 and AJ233396), but the high level of genetic variation up- and downstream of the target gene necessitated the design of additional primer sets. Primers for sequencing were designed on the basis of the G gene sequence of the DK-3592B isolate (GenBank X66134) as well as on basis of the generated sequence data.


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Table 2. Primers used for RT-PCR amplification and sequence analysis

Primers for sequence analysis were designated GA–GD and GSeq. The + or – refer to orientation (sense or antisense, respectively).

 
RT-PCR amplification and DNA sequencing.
RT-PCR-amplified products were sequenced directly without cloning in order to minimize the bias associated with minor heterogeneities in the virus population. The products were synthesized by single tube RT-PCR (Titan One Tube RT-PCR System; Roche) according to the manufacturer's instructions. Briefly, 5 µl extracted viral RNA (approx. 2 µg), 50 pmol of each of the desired primers (Table 2), 1 µl 10 mM dNTP, 10 µl 5xRT-PCR reaction buffer (enclosed buffer with 7·5 mM MgCl2 and DMSO), 2·5 µl 100 mM DTT, 1 µl enzyme mix and MilliQ water was used per 50 µl one-tube RT-PCR. The RT-PCRs were performed using a NMJ Research PCT-200 Peltier thermal cycler.

Quantity and quality of the RT-PCRs were evaluated by 1 % agarose gel electrophoresis of 5 µl PCR product (data not shown). In a few samples with multiple bands, the remaining 45 µl PCR product was run on a new gel. The band of expected size was then excised from the gel and the DNA was purified by affinity spin columns (QIAquick Gel Extraction; Qiagen) and eluted in 10 mM Tris/HCl pH 8·5. DNA in samples showing a single band of the expected size was purified immediately on affinity spin columns. The amplicons were additionally purified using QIAqiuck spin columns (Qiagen) and sequenced on ABI 373A or ABI 377 automatic sequencers using the PRISM BigDye Terminator cycle sequencing kit (Applied Biosystems).

Computer analysis.
Multiple sequence alignments of nucleotide sequences were performed with the CLUSTAL W program (Higgins & Sharp, 1989). Phylogenetic analysis was performed using the PAUP* software package, version 4.10 (Swofford, 2000). A starting tree was produced by the neighbour-joining algorithm with PAUP* default parameters. Using this tree, parameters of a general time reversible model of substitution (GTR) with invariable sites and a gamma distribution of rate heterogeneity were estimated from the data by maximum-likelihood. These parameters were: three base frequency parameters, six parameters of the substitution matrix, the proportion of invariable sites (PInvar), the transition/transversion ratio (Tratio) and one shape parameter from the gamma distribution describing rate heterogeneity among sites (Yang, 1993). This optimized substitution model was then used to re-estimate the tree and to assess the robustness of each node on the tree by the bootstrap resampling method with 1000 replicates (both by neighbour-joining). Bootstrap values exceeding 70 % were considered to indicate significant relatedness.

The US-Makah VHSV isolate was used as an outgroup root, as it has previously been established that the North American VHSV isolates are phylogenetically distinct from the European isolates (Benmansour et al., 1997; Nishizawa et al., 2002; Thiéry et al., 2002). Phylogenetic trees were displayed using TreeView (Page, 1996) and nucleotide identity within the VHSV population was determined using GeneDoc (Nicholas et al., 1997).

Nucleotide substitution rates and likelihood estimates for molecular clock tests were estimated using the TipDate model for evolution of a molecular clock (Rambaut, 2000) as implemented in the CODEML program (Yang, 1997; Goldman & Yang, 1994). This model incorporates the time-structure of serial samples, which means that the rate of substitution can be estimated directly from the data without the use of external calibration. The analyses were performed on fixed phylogenetic trees obtained by the neighbour-joining method (see above).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
RT-PCR amplification and sequence analyses
Nucleotide sequencing was performed on both strands for 66 G genes (1524 nt). Subsequent alignments, including available sequence from GenBank, revealed that 62/74 of the G gene sequences were unique (Table 1) and identical G gene sequences were only found in isolates originating from the same geographical region or from farms infected in the same epidemic. The identity level within the four observed genotypes varied from 94 to 99 % whereas it was 84 to 91 % between genotypes (Table 3). The North American isolate (genotype IV) had the lowest degree of similarity to the other isolates (Table 3).


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Table 3. Sequence identity between the VHSV isolates of the four genotypes as defined by Snow et al. (1999)

 
Differences between sequences published by different laboratories for the same virus isolate were observed in a number of cases, e.g. two GenBank sequences of DK-Hededam (U28798 and Z93412) differed in five positions, which resulted in four amino acid substitutions. Propagation on different cell lines, polymerase with or without proofreading and/or sequencing of cloned gene products rather than PCR amplicons might explain these differences. When available, sequence data from the present study were used for the phylogenetic analysis in order to minimize this bias (Table 1). The gene sequences obtained in this study were distinct, but mixed nucleotide positions occurred in nine isolates despite repeated amplification and subsequent sequence analysis (two or three times). This finding may indicate the presence of heterogeneous genetic populations within some of the virus samples. Sequencing of plaque-cloned virus populations remains to be performed to clarify such sequence differences.

Phylogenetic analysis
The genetic relationships among the 62 unique VHSV sequences are illustrated in the tree (Fig. 2), in which the four genotypes (I–IV; previously identified by Snow et al., 1999) were supported by significant bootstrap values. Trees with similar topology were consistently produced by both distance and parsimony methods (data not shown). The freshwater isolates all belonged to genotype I, whereas sea water isolates were represented in all of the four genotypes. Five sublineages were identified within the genotype I isolates (Fig. 2): sublineage I-a represented a continuous evolution of European freshwater VHSV isolates from Austria, Denmark, France and Switzerland, with the most recent isolates situated at the branch tips; I-b included isolates originating from the Baltic Sea, Skagerrak, Kattegat, North Sea and English Channel; I-c included a small group of older Danish freshwater isolates; I-d was represented by an old Norwegian isolate [NO-A163-68 EG46, supposedly imported with rainbow trout from Denmark; T. Håstein (personal communication)] and two recent isolates from rainbow trout farmed in brackish water in Finland; I-e included a Georgian isolate as the only representative. Among the rainbow trout isolates, the oldest isolates as well as the two recent isolates from brackish water (Finland) were positioned close to the common ancestor of marine (sublineage I-b) and freshwater isolates (indicated by asterisk in Fig. 2).



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Fig. 2. Phylogenetic trees showing the relationship of the 62 unique full-length VHSV G gene sequences (Table 1) illustrated by a radial tree (a) and a phylogram using an American isolate as outgroup (b). The best-fitting nucleotide substitution model (parameters: distance=GTR, rates=gamma, class=(a, b, c, d and e), Tratio=4·3, shape=1·235069 and PInvar=0·422062) was used during neighbour-joining distance analysis and the branch lengths in (a) and the horizontal branch lengths in (b) reflect genetic distances. Bootstrap values were obtained from 1000 resampled datasets and nodes with bootstrap values less than 70 % have been collapsed in (b). Genotype I sublineage identified by significant bootstrap values (>70 %) have been designated I-a to I-e. The asterisk denotes the shared ancestral node of European freshwater isolates and marine isolates. Scale bars refer to the expected numbers of substitutions per site.

 
Genotype II included only isolates from fish caught in a narrow region called the Eastern Gotland Basin (Baltic Sea) (Fig. 1). Due to very deep water in this area, the location holds a separate biotope with high salinity and is believed to host an endemic fish population. The presence of two relatively different genotypes (87 to 89 % identity, Table 3) within the Baltic Sea could therefore be a result of different genetic evolution within the biotopes or be a result of two separate introduction events in this area.

Genotype III included isolates from the North Sea/coastal waters of the UK and Ireland, as well as an isolate originating from an eel caught in northern France. As with genotype I, several distinct sublineages existed within genotype III. However, these lineages were not designated with letters, as only a few isolates covering a broad geographical zone were included. Virus genotypes I and III were both isolated from fish caught in the North Sea and Skagerrak, a finding that may reflect migration patterns of the host fish populations (Fig. 1).

Molecular clock analysis
A likelihood ratio test for the presence of a molecular clock, including all unique sequences, rejected the hypothesis, indicating that the rate of substitution is not constant throughout the tree (Table 4). Fig. 2 shows that a large group of freshwater isolates (I-a) protrudes further from the root than the marine isolates (I-b). This indicates that the rate of substitution varies as a function of the environment. On that assumption, the data were separated into two environmentally related phylogeny groups for further analysis: the large group of monophyletic freshwater isolates (denoted FW, corresponding to sublineage I-a) and all sea-water isolates (SW corresponding to I-b, II, III and IV). For these groups, the likelihood ratio test gave acceptance of the hypothesis of molecular clocks with substitution rates of 1·74x10–3 and 7·06x10–4 substitution per codon per year, respectively (Table 4). When the remaining genotype I isolates were included in either the fresh- or sea-water group, the molecular clock hypothesis was again rejected (Table 4). The estimated substitution rates of the fresh- and sea-water isolates were subsequently extrapolated onto the phylogenetic trees in order to identify the time of the most recent common ancestors (MRCA). This gave an estimated MRCA of the large group of freshwater isolates (I-a) approximately 50 (±4·8) years ago and an estimated MRCA of the marine isolates approximately 500 (±25·9) years ago.


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Table 4. Molecular clock analysis

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The present study focuses on the genetic evolution of the fish rhabdovirus VHSV in wild reservoir hosts and in cultured fish species. Previous findings of four main genotypes of VHSV (Snow et al., 1999), which correlated with the geographical regions of isolation, were confirmed (Fig. 2). However, this study is the most detailed to date with respect to the number of isolates included and the diversity of their historical and geographical background. The selected target gene region was (in contrast to previous studies, which focussed on gene fragments) the entire G gene (1524 nt) including several hypervariable regions. The data allowed a detailed description of the evolution of VHSV and also a comparison of codon substitution rates among freshwater and marine VHSV isolates.

Marine VHSV isolates were represented in all four genotypes, whereas all freshwater isolates belonged to genotype I (Fig. 2). The marine virus group therefore possesses the majority of diversity and has the freshwater isolates included in their phylogeny. This finding supports the assumption that freshwater VHSV originates from the marine environment.

Phylogenetic analyses indicated that the European genotype I lineages had a common ancestral source. The isolates closest to the ancestral source were either old freshwater isolates from rainbow trout or isolates from the marine environment. Recently, outbreaks of VHSV without physical linkage to freshwater VHSV have occurred in Scandinavian marine-reared rainbow trout. These VHSV isolates were located close to the ancestral source in the phylogenetic diagram (Fig. 2), and therefore most likely represent new introductions/adaptations to rainbow trout. This theory was further supported by the finding that virus isolated from outbreaks of VHS in Swedish net pens in Kattegat in the early spring of 1998 and again in 2000 (SE-SVA-14 and SE-SVA-1033, sea coordinate 45G1 in Fig. 1) were almost identical to isolates found in the surrounding marine environment (99 % identity within the G genes from DK-5e59, DK-4p37, SE-29SVA, SE-30SVA, SE-31SVA and SE-32SVA). The source of infection is unknown, but it has been demonstrated that VHSV can multiply to high titres in herring and other pelagic species (Hedrick et al., 2003; Hershberger et al., 1999). It may thus be speculated that virus excreted by shoals of migrating fish or the fish themselves have been the origin of the infection. Accordingly, raw marine fish was used for the feeding of rainbow trout in Finland, and could have been the source of the infection, as Dixon (1999) suggested for the first cases of VHS in Denmark. The isolation of a phylogenetically distinct VHSV isolate from a rainbow trout farm in Georgia, in the Caucasus, might represent an equivalent example of infection from wild fish, but it remains to be determined whether an uncharacterized marine reservoir of VHSV exists in the Black Sea.

Populations of RNA viruses are often complex mixtures of different, but closely related variants, and, as a consequence, they have a high potential for continuous adaptation (Quer et al., 1996). In light of this, it is interesting that Danish freshwater VHSV isolates grouped discontinuously into two separate sublineages (I-a and I-c) or as single representatives branching out from the same ancestral point as the Baltic marine lineage (I-b) (Fig. 2a). Taken together, the above findings indicate that host adaptation from marine environment/species to rainbow trout has occurred three or four times in freshwater farms in Denmark and more recently in fish reared at the coast of Finland and Sweden. Genetic relatedness between a virulent sea-water isolate from farmed turbots and VHSV isolated from marine fish species near the Shetland Islands was also reported by Stone et al. (1997) and Snow et al. (1999). VHSV in wild marine fish therefore represents a continuous potential threat to farming of VHSV-susceptible species in the coastal zones.

Different nucleotide substitution rates were identified within the VHSV isolates from sea-water and the dominating freshwater sublineage I-a (Table 4). The rates of substitutions per codon per year were determined to be 7·06x10–4 and 1·74x10–3, respectively. This is within the range of evolutionary rates estimated for other RNA viruses (Jenkins et al., 2002).

The hypothesis of divergent substitution rates would be strengthened by performing maximum-likelihood estimation with a model allowing for local clocks in the tree and then by comparing this to estimates obtained with a model of no molecular clock, in analogy with the model of Yoder & Yang (2000). However, there is currently no such model that allows for time-structured data and the study was therefore restricted to the described exploratory analyses. The rejection of the molecular clock hypothesis for the full dataset and its subsequent recovery when the isolates from different environments are separated does, however, along with the more than twofold difference in the estimates of the rate of substitution, give a strong indication that the rate of substitution differs within the two environments.

The observed nucleotide substitution rate in the G gene of VHSV from farmed freshwater fish was approximately 2·5 times faster than in the G gene of VHSV from free living fish in sea-water. It could be speculated that rainbow trout is an unnatural host for VHSV and that host species difference in immune response and/or physiology have influenced the evolutionary rate of the virus (Moya et al., 2000). Alternatively, the higher substitution rate in freshwater may be related to the intense production procedures used in fish farms, where monocultures of rainbow trout are kept at high mean densities and stress levels, along with repeated introduction of naïve fish into the farms. Also, the generally higher temperatures within the freshwater farms than in the marine environment may have increased the speed of virus replication, as observed during in vitro replication. The elevated nucleotide substitution rates in freshwater isolates may therefore be a result of increased numbers of virus replications per year. A similar pattern has been observed for IHNV (Infectious Hematopoietic Necrosis Virus) in North America, where the evolution rate in a geographical zone with intense aquaculture was found to be three to four times higher compared with other regions (Troyer & Kurath, 2003).

The estimated mean substitution rates were related to the phylogenetic data and it was estimated that European freshwater VHSV had a common marine ancestor approximately 50 years ago, whereas European marine VHSV shared an ancestor with the North American type approximately 500 years ago. The first estimate coincides well with the initial reports of VHS-like symptoms in Denmark in the 1950s, followed by the first isolation of the causal virus in 1962 (Jensen, 1965). The second estimate is more difficult to confirm, but it could well be that the genotypes were separated a long time before fish farming was established on the two continents.

The present study demonstrates a close genetic linkage between VHSV in the marine environment and VHSV in farmed rainbow trout in Europe. Adaptation of the virus to rainbow trout is suggested to be a relatively recent event that has occurred more than once during the past 50 years in European rainbow trout farming.


   ACKNOWLEDGEMENTS
 
Warm thanks to Gael Kurath, Jim Winton and Marco Salemi for inspiration and fruitful discussions of the phylogenetic analysis and to Helle Hermansen, Mette Lorentsen, Lisbeth Troels, Sanne Madsen, Kirsten Vestergaard and Bill Batts for their excellent technical assistance. The authors would also like to thank the following for providing VHSV isolates; Anders Hellström (SE-SVA-14, SE-SVA-29, SE-SVA-30, SE-SVA-31, SE-SVA-32 and SE-SVA-1033), Oskar Schachner (AU-8/95), Hannele Tapiovaara (FI-ka-66/2000 and FI-ka-422/2000), Helle F. Skall (DK-5e59 and DK-6p403), Igor Shchelkunov (GE-1.2) and Thomas Wahli (CH-FC 262). Helle F. Skall is further thanked for providing the map illustrating the location of isolation of marine VHSV (Fig. 1). Thanks to Ellen Ariel for reviewing this manuscript. Part of this work was done while K. E.-J. visited the laboratory of Dr Jim Winton, Western Fisheries Research Center, WA, USA. This work was partly supported by the EC FAIR CT98-4064 project. R. F. was supported by grants 21-02-0206 and 51-00-0392 from the Danish Natural Science Research Council, by grant 1-R01-GM60729-01 from the National Institutes of Health, USA, by grant HAMJW from EPSRC and by grant HAMKA from MRC.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 18 November 2003; accepted 7 January 2004.