Genetic analyses reveal unusually high diversity of infectious haematopoietic necrosis virus in rainbow trout aquaculture

Ryan M. Troyer1,2, Scott E. LaPatra3 and Gael Kurath1,2

Department of Pathobiology, University of Washington, Seattle, WA 98195, USA1
Western Fisheries Research Center, United States Geological Survey, Biological Resources Division, 6505 NE 65th St, Seattle, WA 98115, USA2
Clear Springs Foods Inc., PO Box 712, Buhl, ID 83316, USA3

Author for correspondence: Ryan Troyer. Fax +1 206 526 6654. e-mail ryanmt{at}u.washington.edu


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Infectious haematopoietic necrosis virus (IHNV) is the most significant virus pathogen of salmon and trout in North America. Previous studies have shown relatively low genetic diversity of IHNV within large geographical regions. In this study, the genetic heterogeneity of 84 IHNV isolates sampled from rainbow trout (Oncorhynchus mykiss) over a 20 year period at four aquaculture facilities within a 12 mile stretch of the Snake River in Idaho, USA was investigated. The virus isolates were characterized using an RNase protection assay (RPA) and nucleotide sequence analyses. Among the 84 isolates analysed, 46 RPA haplotypes were found and analyses revealed a high level of genetic heterogeneity relative to that detected in other regions. Sequence analyses revealed up to 7·6% nucleotide divergence, which is the highest level of diversity reported for IHNV to date. Phylogenetic analyses identified four distinct monophyletic clades representing four virus lineages. These lineages were distributed across facilities, and individual facilities contained multiple lineages. These results suggest that co-circulating IHNV lineages of relatively high genetic diversity are present in the IHNV populations in this rainbow trout culture study site. Three of the four lineages exhibited temporal trends consistent with rapid evolution.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Infectious haematopoietic necrosis virus (IHNV) is a member of the family Rhabdoviridae that causes acute, systemic and often virulent disease in both wild and cultured salmon and trout (Wolf, 1988 ; Winton, 1991 ). IHNV is native to salmonids of the Pacific Northwest region of North America and its current geographical range extends from Alaska to northern California along the Pacific coast and inland to Idaho. Previous investigations of IHNV field isolates showed that numerous strains, which differ in many protein-based characteristics, exist (Hsu et al., 1986 ; Ristow & Arnzen de Avila, 1991 ; Winton et al., 1988 ; LaPatra et al., 1994 ). Oshima et al. (1995) differentiated 26 IHNV isolates by T1 ribonuclease fingerprint patterns and Nichol et al. (1995) produced a phylogenetic tree of 12 isolates from the north-west region of the USA based on sequence analysis of the glycoprotein and non-virion genes. These studies demonstrated that IHNV relatedness generally correlates with geographical origin.

To characterize the region-specific and overall genetic diversity of IHNV field isolates, our laboratory is studying genetic types of the virus throughout its range in North America. Surveys of Alaskan and British Columbian IHNV have documented relatively low genetic diversity (<2·0% nucleotide divergence) in these large geographical areas (Emmenegger et al., 2000 ; E. D. Anderson, G. S. Traxler & G. Kurath, unpublished). In apparent contrast, LaPatra et al. (1991 , 1994 ) found a high level of antigenic diversity among virus isolates at four aquaculture facilities along a 12 mile stretch of the Snake River in the Hagerman Valley, Idaho.

The 96 mile Hagerman Valley region in south-central Idaho is a major centre for aquaculture and currently produces 75% of the USA supply of food-size rainbow trout (USDA Economic Research Service, 1998 ). Historically, IHNV was only isolated occasionally in the Hagerman Valley and was associated with a chronic low virulence infection (Busch, 1983 ). In 1977, a new and highly virulent strain of IHNV appeared and caused the first documented IHNV disease outbreak in rainbow trout culture (Busch, 1983 ). From 1977 to 1980 the virus spread throughout the valley and became endemic by the end of 1980 (Busch, 1983 ). Since that time IHNV has continued to persist at multiple sites in the region causing epidemics which may approach 70–80% mortality depending on the life-stage of the fish and complications with other micro-organisms (LaPatra et al., 1991 ). IHNV disease typically occurs in rainbow trout fry maintained in the multiple outdoor rearing units of rainbow trout farm facilities. The first studies of diversity among IHNV isolates in this region were conducted by LaPatra et al. (1991 , 1994 ), who used two monoclonal and two polyclonal antibodies to examine the heterogeneity of serum neutralization profiles among 106 IHNV isolates taken from rainbow trout (Oncorhynchus mykiss) at four rainbow trout culture facilities between 1990 and 1992. Ten different serum neutralization groups were found, with three groups representing the majority (91%) of the isolates.

Because of the development of high-resolution IHNV strain typing techniques and the previous indication of unusually high antigenic diversity in this region, we re-examined the genetic diversity of IHNV isolates from the same study site of four trout farms (LaPatra et al., 1994 ) over an expanded time period. A total of 84 virus isolates were characterized, representing three discrete time periods within a 20 year span (1978 to 1998) dating back to the emergence of IHNV-associated disease in this region. Slightly different selection criteria were used for virus isolates from each of the three time periods because of the nature of the virus isolates available. From the 1990 to 1992 isolates analysed serologically by LaPatra et al. (1994) , 42 were selected to include each of the ten identified antigenic groupings thus, presumably, representing the most diverse types of the 106 isolates examined. Isolates dating from 1997 to 1998 were examined in order to characterize the more recent virus types present at the same four facilities and to assess possible epidemiological trends. Since there were no neutralization group profiles on the numerous isolates available from these years, 32 isolates were selected by taking four isolates from each of the four facilities from both 1997 and 1998. Although this selection was basically random, information on isolation dates and locations within each facility was used to choose the most diverse set of isolates from this time period. During the years 1978 to 1988 few isolates of IHNV were saved and hence only 10 isolates were available. Six of these isolates were recovered from facilities within the Hagerman Valley separate from the four trout farms represented in the 1990 to 1992 and 1997 to 1998 sample sets.

The RNase protection assay (RPA) was used to provide rapid characterization of genetic differences over a large genomic region. A probe complementary to the entire glycoprotein (G) gene was chosen for the analysis because the glycoprotein forms the antigenic surface of IHNV and thus, presumably, reflects the serological variation observed by LaPatra et al. (1994) . In addition, sequencing of a 303 bp region in the middle of the G gene (referred to as mid-G) was undertaken for selected isolates in order to estimate nucleotide divergence and potentially infer virus lineage based on phylogenetic analyses. The mid-G region was previously shown to be variable in a study of 12 geographically dispersed IHNV strains (Nichol et al., 1995 ). However, in a subsequent study of 42 IHNV isolates from throughout Alaska, the diversity of the mid-G sequences was too low to generate an informative phylogenetic analysis (Emmenegger et al., 2000 ). Thus, both RPA and mid-G sequence analyses were carried out in order to provide a high level of confidence in the characterization of IHNV genetic heterogeneity in this study.


   Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Virus isolates and RNA extraction.
The majority of virus isolates were provided by Scott LaPatra (author) and three historical isolates were generously provided by D. Ramsey, Rangen Aquaculture, Hagerman, ID, USA. Isolates were generally recovered from single, acutely infected farm-reared rainbow trout fry. Viruses were isolated as described by Thoesen (1994) and stored as frozen aliquots of cell culture supernatant. Fish at four aquaculture facilities (referred to as facilities I–IV) along a 12 mile stretch of the Snake River in south-central Idaho were sampled to provide 78 out of 84 isolates. The remaining six isolates were recovered from fish at other aquaculture facilities within the 96 mile Hagerman Valley. Virus isolates were inoculated onto confluent monolayers of epithelioma papulosum cyprini (EPC) cells (Fijan et al., 1983 ) and total RNA was extracted from infected cells using a guanidinium thiocyanate procedure as described previously (Anderson et al., in press ). RNA aliquots of 1–3 µg/µl were used as viral target RNA in each RPA reaction.

{blacksquare} RNase protection assay (RPA).
IHNV isolates were characterized by the RPA using a [32P]UTP radiolabelled minus-sense RNA probe (prGF) complementary to the entire glycoprotein gene of IHNV strain RB1 (accession no. U50401). Probe synthesis and the RPA protocol were described previously by Anderson et al. (in press). Briefly, for each reaction, 105 c.p.m. of prGF was hybridized to viral target RNA overnight, followed by RNase digestion of mismatches, electrophoresis of cleavage products and autoradiography. Four control reactions were included with each set of assays: (1) viral RNA from strain RB1 prepared from infected cells as above; (2) plus-sense RNA synthesized in vitro, complementary to prGF; (3) prGF with no target RNA and no RNases in the digestion step; and (4) prGF with no target RNA. Each of the 84 isolates were analysed with prGF by RPA a minimum of two times. Banding patterns for all the isolates were compared visually and those isolates with identical banding patterns were considered to have the same haplotype. A haplotype was defined as a specific banding pattern of cleavage fragments. Isolates with at least one band difference were considered to have different haplotypes.

{blacksquare} Sequence analyses.
A 303 nt region of the glycoprotein gene from nucleotide 686 to 988 (accession no. U50401) on the IHNV G gene (mid-G region) was amplified and sequenced for 49 isolates. RT–PCR amplification and sequence analyses were carried out as described by Emmenegger et al. (2000) .

Sequence files were edited and analysed using MacVector 6.0 and AssemblyLIGN 1.0.9 applications (Oxford Molecular Group). In order to minimize the effect of random polymerase errors in the RT–PCR, the sequences that contained unique nucleotide substitutions within this set of sequences were confirmed by an independent repeat of RT–PCR sequence analysis from cell culture supernatant. Mid-G nucleotide sequences of selected IHNV isolates from each of the four virus lineages are available (accession nos AF237983–AF237992). All sequences obtained are also available from the authors upon request. Synonymous and nonsynonymous mutation analysis was performed using SNAP analysis (http://hiv-web.lanl.gov/SNAP/WEBSNAP/SNAP.html) based on the method of Nei & Gojobori (1986) . Phylogenetic analyses were performed with PAUP* ver. 4 (Swofford, 1998 ) and PHYLIP ver. 3.5c (Felsenstein, 1993 ). The Sacramento River Chinook virus (SRCV) isolate of IHNV, isolated in California in 1966 (Nichol et al., 1995 ), was used as an outgroup root. SRCV is the oldest isolate on the tree and it is well established that this isolate, as well as all California isolates analysed to date, are phylogenetically distinct from isolates from the rest of the IHNV range (Hsu et al., 1986 ; Oshima et al., 1995 ; Nichol et al., 1995 ). The nucleotide diversity within the IHNV population was calculated according to the method of Nei (1987) utilizing Kimura’s two-parameter model (Kimura, 1980 ) as applied in the Arlequin ver. 1.1 software package (Schneider et al., 1997 ).

{blacksquare} Virus virulence assays.
Virulence determinations were performed using the method of LaPatra et al. (1994) . Briefly, duplicate 16–24 fish groups of two sizes of rainbow trout (O. mykiss) juveniles (mean mass 0·8 g and 3·0 g) were challenged by immersion in 105 p.f.u./ml of representative IHNV isolates for 1 h with aeration in a volume of water that was 10 times the total mass (g) of the fish. Mock-infected control groups were exposed to cell culture media only. Experimental groups were held separately in 19 l aquaria at 15 °C and monitored for 21 days. A minimum of 20% of each day’s mortalities were examined for virus by plaque assay procedures (LaPatra et al., 1989 ). Proportions of mortalities (equivalent to cumulative per cent mortality) were compared using the chi-square test.


   Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
RPA confirms heterogeneity among study site IHNV isolates
RPAs were performed on 84 IHNV isolates using the probe prGF, which is complementary to the entire G gene. These analyses identified a total of 46 different haplotypes. Isolates from the three time periods were chosen based on slightly different selection criteria so the number of haplotypes found within each time period is not directly comparable. In the 1990 to 1992 sample set there were 25 haplotypes out of 42 isolates (Fig. 1a). Isolates from 1978 to 1988 included 10 haplotypes out of 10 isolates (Fig. 1b). Isolates from 1997 to 1998 included 14 haplotypes out of 32 isolates (Fig. 1c).



View larger version (107K):
[in this window]
[in a new window]
 
Fig. 1. RPA haplotypes generated for 84 IHNV isolates from (a) 1990 to 1992, (b) 1978 to 1988 and (c) 1997 to 1998 using the probe prGF, which is complementary to the full-length of the glycoprotein gene (1610 nt). Each lane represents a unique pattern of cleavage fragments, referred to as a haplotype, for a particular virus isolate. The number of isolates that share each haplotype is listed above the haplotype name. Rb, target RNA extracted from cells infected with the Round Butte 1 strain, which is complementary to the probe; probe, control with no RNase or target RNA; GF(+), plus-sense RNA transcript synthesized in vitro, which is complementary to the probe; no RNA, no target RNA. Autoradiograms were computer scanned using Adobe Photoshop version 3.0 for Macintosh.

 
Analyses of the banding pattern similarity between haplotypes revealed four haplotype groups based on a qualitative visual assessment of the number of shared bands between patterns (Fig. 1). Based on these analyses, haplotypes were designated by a capital letter denoting their group (haplogroups A–D) and a number specifying individual haplotypes within the group (e.g. haplotype A1). Several of the haplotypes from 1978 to 1988 isolates did not share enough bands with any of the other haplotypes to be placed into a group, so they were assigned the letter N to denote their lack of group designation.

Previous characterization of the isolates from 1990 to 1992 by LaPatra et al. (1994) classified 106 isolates into 10 antigenic profile types while the RPA classified 42 of these isolates into 25 haplotypes. A comparison of the RPA groups of the 1990 to 1992 isolates with antigenic types revealed a correlation between RPA haplotype group and antigenic reactivity (data not shown).

RT–PCR sequence and phylogenetic analyses
Sequence analyses of the 303 nt mid-G region were performed on representative isolates from all 46 individual haplotypes along with two additional isolates from haplotype A2 and one additional isolate from haplotype C9. A total of 49 isolates were analysed yielding 32 unique sequence types (data not shown). The sequences of the three isolates of haplotype A2 were all identical to each other, as were sequences of the two isolates from haplotype C9 (data not shown). The maximum nucleotide divergence among the isolates was 7·6%, which occurred within the isolates from 1997 to 1998. The maximum divergence within the 1990 to 1992 isolates was 5·9% and within the 1978 to 1988 isolates it was 3·6%. This trend of an increase in diversity over the three time periods examined was also observed in the intrapopulational nucleotide diversities of the Idaho study site isolates (Fig. 2a). Although these differences in intrapopulational diversity have overlapping error bars, the trend observed is the opposite of what might be explained by the differences in selection criteria for the isolates from the different time periods, suggesting that the increased heterogeneity with time may be real. When the same analysis was applied to compare the overall intrapopulational nucleotide diversity in the Idaho study site with that from the Alaska study (Emmenegger et al., 2000 ), a clearly significant difference was found in that the Idaho study site diversity was sixfold greater than that found throughout Alaska (Fig. 2b).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2. Intrapopulational nucleotide diversity. (a) Nucleotide diversities within time periods at the Idaho study site were calculated utilizing all 49 mid-G sequences generated in this study. (b) Overall nucleotide diversities within the Idaho study site and Alaska (Emmenegger et al., 2000 ) were calculated as populations that included one representative mid-G sequenced isolate from each G probe RPA haplotype (46 in Idaho and 21 in Alaska). It was necessary to randomly select one isolate to represent each Alaskan RPA haplotype so that the selection criteria for the Alaskan population matched that for the Idaho study site population. Intrapopulational nucleotide diversity is defined here as the probability that two randomly chosen homologous nucleotides are different within the population.

 
The predicted translation products of the 32 nucleotide sequences included 24 different amino acid sequences (data not shown). Several hotspots for amino acid substitutions were noted. There were four different amino acids present at glycoprotein amino acids 252 and 277 and five different amino acids were present at positions 256 and 270 (accession no. U50401). These codons, as well as codons 275, 276, 284 and 285, had an excess of nonsynonymous mutations relative to synonymous mutations (data not shown), suggesting that these sites may be under particularly strong positive selection or particularly low negative selection. These codons are located relatively near to previously identified linear neutralizing epitopes mapped to amino acids 230 and 231 and 272 to 276 (Huang et al., 1996 ).

Phylogenetic analyses included the 32 different mid-G sequences obtained in this Idaho IHNV study, along with the mid-G sequences of 12 isolates from the north-west USA (Nichol et al., 1995 ) and single representative sequences of isolates from Alaska (Emmenegger et al., 2000 ) and British Columbia (E. D. Anderson, G. S. Traxler & G. Kurath, unpublished). The isolates from Idaho formed a strongly supported monophyletic clade that was separate from virus isolates which represent the rest of the North American range of IHNV isolates examined to date (Fig. 3). Within the Idaho clade, four bootstrap-supported sub-clades were present, indicating four distinct virus lineages. The grouping of isolates into these four lineages matched the independently determined RPA haplogroups A–D isolate-for-isolate. Therefore, the four groups identified by both RPA and sequence phylogeny are hereafter referred to as lineages A–D. The four isolates from 1978 to 1988 that were not assigned to an RPA haplogroup (group N) did not fit into any of the four lineages. Branch lengths in Fig. 3 reflect genetic distance and thus it is directly observable that the genetic distance among the study site isolates is greater than that currently found within the rest of the North American range of IHNV. Within three of the four lineages (B, C and D), there was a temporal trend with a correlation between more recent time of isolation and greater genetic distance from the trunk of the tree (Fig. 3). The mid-G branching order of isolates sequenced by Nichol et al. (1995) , shown in Fig. 3, was equivalent to the phylogenetic branching order generated by Nichol et al. (1995) for the entire glycoprotein and non-virion genes. This provided validation for phylogenetic analyses based on sequence from the mid-G region.



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 3. Phylogenetic tree constructed from the mid-G sequences (303 nt) of Idaho and selected non-Idaho IHNV isolates. This neighbour-joining tree was generated with PAUP* (Swofford, 1998 ) using SRCV as an outgroup root. The significance of the branching order was assessed by bootstrap resampling of 1000 replicates. Branches with values of >70% that correspond to a confidence interval of >95% (Hillis & Bull, 1993 ) are indicated on each branch. Branches with values of <70% were collapsed. HO-7, LR-80, 193-110, CST-82, W, LWS, CRS89, RB-76, LR-73, SRCV, Col-80 and Col-85 were sequenced by Nichol et al. (1995) (accession nos L40871–L40882). BC represents a major IHNV type found in British Columbia (E. D. Anderson, G. S. Traxler & G. Kurath, unpublished data). AK represents a major IHNV type found in Alaska by Emmenegger et al. (2000) . All other numbered isolates were sequenced in this study. Isolates from the Hagerman Valley in Idaho are shown in colour to indicate the time period of isolation. Phylogenetic lineages A–D matched RPA haplogroups A–D exactly. Maximum parsimony and neighbour-joining analyses of 1000 bootstrap resampled trees generated from the same sequence data using PHYLIP (Felsenstein, 1993 ) produced a phylogeny with identical branching order at significant nodes and highly similar bootstrap values throughout (data not shown).

 
Distribution of RPA haplotypes
Analysis of the distribution of RPA haplotypes by date of isolation and aquaculture facility showed that multiple haplotypes were present at facilities during the same year (Table 1). Detailed analysis of the exact dates of virus isolation showed that in many cases multiple haplotypes were present at the same time or within several weeks at a single facility (data not shown). The four virus lineages identified by RPA grouping and sequence phylogeny were all present at multiple facilities. However, individual haplotypes within these lineages were generally confined to a single facility with the exception of haplotypes C1, A2 and D4, which were observed at multiple facilities. Of these exceptions, haplotype A2 was first observed in 1982, detected again in 1991, and by 1997 was observed at all four facilities, while haplotype D4 was also first observed at all four facilities during 1997. A number of interesting phenomena were observed within facilities. At facility I, only IHNV lineage B was observed during 1990 to 1992, three lineages (A, B and D) were present by 1997, and lineage C was not observed. At facility II, lineage C was highly prevalent from 1990 to 1992, but in 1997 to 1998, lineages A, B and D were all present and lineage C was undetected. At facility III, lineage C remained prevalent during 1990 to 1992 and 1997 to 1998. Lineages A and D were first detected at facility III in 1997, while the previously detected lineage B was not found during 1997 to 1998. Facility IV was found to have all four lineages from 1990 to 1992 and 1997 to 1998. The 1978 to 1988 time period isolates revealed the early presence of lineages A, B and D, as well as several haplotypes that were not placed in lineages.


View this table:
[in this window]
[in a new window]
 
Table 1. RPA haplotype distribution by year and facility for 84 IHNV isolates

 
Virulence
To determine if a change in the virulence of isolates from the four IHNV lineages had occurred between 1990 to 1992 and 1997 to 1998, groups of two sizes of naive rainbow trout fry were challenged with eight virus isolates from 1997 to 1998 as well as four virus isolates from 1990 to 1992. These isolates represented the four virus lineages found in this study during the two most recent time periods examined. Three of the four 1990 to 1992 isolates had been assayed previously for virulence (LaPatra et al., 1994 ) and these represented the least virulent isolate (no. 1, 039-82), the most virulent isolate (no. 3, HV-90) and an isolate of intermediate virulence (no. 2, 220-90). Virus challenge experiments with these isolates resulted in relative levels of cumulative per cent mortality (CPM) similar to that observed by LaPatra et al. (1994) . In both sizes of fish, the virulence of the 1997 to 1998 isolates, measured by the mean CPM, was not statistically different from the virulence of the 1990 to 1992 isolates (Table 2). It was noted that the two isolates of lineage A were less virulent than all the other lineages in both sizes of fish.


View this table:
[in this window]
[in a new window]
 
Table 2. Virulence of selected IHNV isolates

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Previous investigations of IHNV isolates from throughout North America have suggested that IHNV genetic diversity is less than 5% (Oshima et al., 1995 ; Nichol et al., 1995 ; Emmenegger et al., 2000 ). This level of diversity is much lower than that reported for field isolates of other rhabdoviruses such as vesicular stomatitis virus and rabies virus (Nichol et al., 1989 ; Bilsel & Nichol, 1990 ; Kissi et al., 1995 ; Bourhy et al., 1999 ). A related rhabdovirus of salmonids, viral haemorrhagic septicaemia virus (VHSV), has high genetic diversity between isolates from distant geographical regions, with up to 18% nucleotide diversity between European and North American virus isolates (Benmansour et al., 1997 ). Within regions, however, VHSV has similar diversity to IHNV, with 3·4% divergence within continental Europe and 0·9% divergence within North America (Benmansour et al., 1997 ). Thus, the observation of up to 7·6% divergence within the highly localized Idaho study site is novel among fish rhabdoviruses. This is especially significant for IHNV since it exceeds the previous estimate of divergence for the entire North American range of the virus. We considered the possibility that the high level of diversity observed within the Idaho study site might be due to the hypervariability of the G gene. However, RPAs of a limited number of Idaho isolates using probes for the NV gene and the 3' 910 nucleotides of the N gene showed variability of patterns similar to that obtained with the G gene probe (data not shown).

The picture that emerged of IHNV at this rainbow trout culture study site was the presence of four virus lineages of unusually high diversity that co-circulated among the four aquaculture facilities examined and which appeared to diverge over time. This pattern of evolution is quite different from the apparent genetic stasis of the virus in Alaska (Emmenegger et al., 2000 ). By phylogeny and RPA, lineage A exhibited relative genetic stasis while lineages B and C showed clear temporal trends consistent with directional evolution. Lineage D showed an intermediate pattern of evolution in that it exhibited a temporal trend in the phylogeny but was more conserved than lineages B and C by RPA (Fig. 1c). Additionally, lineage D exhibited a pattern of distribution similar to lineage A in that it was present at only one facility (IV) between 1990 and 1992 but had spread to all facilities by 1997. Phenomena at individual facilities demonstrated that virus traffic and micro-evolution are highly dynamic and complex at this site. At individual facilities we observed the apparent acquisition of lineages, spread of relatively stable lineages throughout the facilities (lineages A and D), the apparent loss of a lineage (lineage C at facility II), and the persistence of high diversity (facility IV).

The phylogenetic analyses presented here suggested four IHNV lineages radiating from a single source. This was in agreement with the known history, which suggests that isolates in the Hagerman Valley are likely to have a more recent common ancestor than the rest of the IHNV North American range (Busch, 1983 ). Our data indicated that it is unlikely that there were multiple introductions of IHNV from different sources. However, the possibility of multiple virus introductions from a single source cannot be ruled out. Also, this is a gene phylogeny and therefore cannot be assumed to completely reflect the true virus phylogeny.

The observation that multiple haplotypes and multiple lineages persist within the study site suggests to us that the haplotypes present are of relatively similar fitness and may indicate that there is a lack of purifying selection (genetic bottlenecks). There are several major biological features of rainbow trout farm aquaculture that may contribute to the different evolutionary pattern of IHNV seen in this study compared to IHNV from hatchery and wild salmonid populations. Within this trout farm study site, fish are held in captivity throughout their lives, while both hatchery-produced and wild salmonids migrate to the ocean for a major portion of their life-cycles. In the Idaho study site, photoperiod manipulation of fish is used to eliminate the annual spawning cycle and provide year-round fish production. This leads to a key difference in disease dynamics; there is no seasonal bottleneck in which susceptible fish are at low density. Instead, the frequent introduction of naive fish at a susceptible stage in their life-cycle into a high-density situation provides a year-round supply of susceptible host populations. Thus, there is no density restriction on virus transmission (Reno, 1998 ) and so there is little opportunity for purifying selection to occur. Additionally, the virus may potentially undergo a greater number of rounds of replication per time in this environment in which naive hosts are available all year round. The use of a geographically separate virus-free adult broodstock for egg production eliminates potential virus selection pressure on fish host evolution, so that fish in the farm environment do not develop virus resistance in successive generations. Additionally, the division of the virus population within the study site into separate facilities, and further into individual rearing units, may allow simultaneous amplification of virus variants with no competitive selection of one variant over the other. This partitioning of the virus population into multiple niches may also contribute to a high level of diversity. The restriction of most individual haplotypes to single facilities shows that there is some level of localized micro-evolution underlying the larger phylogeny. However, isolates within lineages do not clade together by facility. Geographical separation of facilities and physical separation of rearing units within facilities is likely to allow a limited amount of localized evolution although co-circulation of virus types clearly occurs. Other unique aspects of aquaculture at the study site should not be overlooked, including the presence of a single host species (rainbow trout) and a constant water temperature of 15 °C as opposed to river and ocean temperatures that are typically lower (ranging from 0–22 °C) and vary seasonally (Bjornn & Reiser, 1991 ).

Co-circulation of the virus lineages between facilities implies a vector. The water supplies for each of the four rainbow trout farm facilities are independently spring-fed and are considered virus-free. Susceptible eggs and young fry, which are kept in an enclosed and carefully controlled hatch-house and share the same water supply as the outdoor rearing units, very rarely experience IHNV-related mortality. Likewise, the eggs that produce the rainbow trout populations are routinely tested for virus and are considered virus-free. In addition, there is generally no transfer of fish, personnel or equipment between facilities. Thus, additional vectors may be involved in IHNV traffic. Examples may include resident fish, wind dispersion by aerosol, or birds. Facility IV maintained the highest lineage diversity observed throughout the different time periods examined. The presence of lineages A and D at facility IV in 1991 to 1992 and the subsequent presence of these lineages at the three remaining facilities in 1997 suggests that facility IV may potentially serve as a reservoir of virus that spreads to the other facilities.

It is important to note that the high and potentially increasing diversity of IHNV in trout farms did not appear to be associated with a change in virulence over the time periods examined. The introduction of naive fish into a high-density situation with apparently efficient waterborne transmission between fish may be likely to favour extensive replication in the host (virulence) with little fitness cost for this replication (Ewald, 1994 ). There has been some suggestion that virus strains present in the Hagerman Valley rainbow trout aquaculture have elevated virulence for rainbow trout compared to other strains of the virus (LaPatra et al., 1990a , b , 1993 ). However, we show here that the virulence of IHNV at four aquaculture facilities in the Hagerman Valley did not change significantly between 1990 to 1992 and 1997 to 1998. It is possible that IHNV virulence reached a threshold level for rainbow trout in the study site environment prior to 1990. While the two isolates of lineage A were less virulent than all other isolates in both sizes of fish, an association of lineage with virulence would require further study with a greater number of replicates.

As a result of the sequential timing of IHNV emergence throughout the Hagerman Valley between 1977 and 1980, and throughout the Columbia River Basin between 1980 and 1982, it has been suggested that the Hagerman Valley was a source of virus for fish downstream throughout the rest of the Columbia River Basin (Groberg, 1983 ). Here we have characterized thoroughly the IHNV genetic types from a study site in the Hagerman Valley. However, comparisons with IHNV present downstream in the Columbia River Basin are premature at this time because very few isolates from the Columbia River Basin have been genetically characterized. To thoroughly address the question of relatedness between Hagerman Valley IHNV and IHNV downstream, representative isolates from numerous locations in the Columbia River Basin within the years 1990 to 1998 will be examined.


   Acknowledgments
 
The authors thank Doug Ramsey of Rangen Aquaculture, Hagerman, ID for generously providing several virus isolates. Jerry Jones and Bill Shewmaker provided excellent technical assistance. Bill Batts provided valuable DNA sequencing advice and assistance. The authors thank Jim Winton, Ronald DiGiacomo and Evi Emmenegger for discussion and careful reading of the manuscript. This work was supported by the Western Fisheries Research Center, Biological Resources Division, US Geological Survey, through the Washington Cooperative Fish and Wildlife Research Unit. The Washington Cooperative Fish and Wildlife Research Unit is supported by the US Geological Survey, University of Washington, and the Washington Department of Ecology, Fish and Wildlife, and Natural Resources.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Anderson, E. D., Engelking, H. M., Emmenegger, E. J. & Kurath, G. (2000). Molecular epidemiology reveals emergence of a virulent IHN virus strain in wild salmon and transmission to hatchery fish. Journal of Aquatic Animal Health (in press).

Benmansour, A., Basurco, B., Monnier, A. F., Vende, P., Winton, J. R. & de Kinkelin, P.(1997). Sequence variation of the glycoprotein gene identifies three distinct lineages within field isolates of viral haemorrhagic septicaemia virus, a fish rhabdovirus. Journal of General Virology78, 2837-2846.[Abstract]

Bilsel, P. A. & Nichol, S. T.(1990). Polymerase errors accumulating during natural evolution of the glycoprotein gene in vesicular stomatitis virus Indiana serotype isolates.Journal of Virology64, 4873-4883.[Medline]

Bjornn, T. C. & Reiser, D. W.(1991). Habitat requirements of salmonids in streams. In Influences of Forest and Rangeland Management on Salmonid Fishes and their Habitats, pp. 83-138. Edited by W. R. Meehan. Bethesda, MD:American Fisheries Society.

Bourhy, H., Kissi, B., Audry, L., Smreczak, M., Sadkowska-Todys, M., Kulonen, K., Tordo, N., Zmudzinski, J. F. & Holmes, E. C.(1999). Ecology and evolution of rabies virus in Europe.Journal of General Virology80, 2545-2557.[Abstract/Free Full Text]

Busch, R. A.(1983). Viral disease considerations in the commercial trout industry in Idaho. In Proceedings of a Workshop on Viral Diseases of Salmonid Fishes in the Columbia River Basin, special publication, pp. 84-100. Edited by J. C. Leong & T. Y. Barila. Portland, OR:Bonneville Power Administration.

Emmenegger, E. J., Meyers, T. R., Burton, T. O. & Kurath, G.(2000). Genetic diversity and epidemiology of the infectious hematopoietic necrosis virus in Alaska.Diseases of Aquatic Organisms40, 163-176.[Medline]

Ewald, P. W. (1994). Evolution of Infectious Disease. New York: Oxford University Press.

Felsenstein, J. (1993). PHYLIP: Phylogeny inference package, version 3.5c. University of Washington, Seattle, WA, USA.

Fijan, N., Sulimanovic, D., Bearzotti, M., Muzinic, D., Zwillenberg, L. O., Chilmonczyk, S., Vautherot, J. F. & de Kinkelin, P.(1983). Some properties of the Epithelioma papulosum cyprini (EPC) cell line from carp Cyprinus carpio.Annales de l’Institut Pasteur Virologie134E, 207-220.

Groberg, W. J.(1983). Priority research needs concerning fish viruses prevalent among Columbia River Basin salmonids. In Proceedings of a Workshop on Viral Diseases of Salmonid Fishes in the Columbia River Basin, special publication, pp. 159-167. Edited by J. C. Leong & T. Y. Barila. Portland, OR:Bonneville Power Administration.

Hillis, D. M. & Bull, J. J.(1993). An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis.Systematic Biology42, 182-192.

Hsu, Y. L., Engelking, H. M. & Leong, J. C.(1986). Occurrence of different types of infectious hematopoietic necrosis virus in fish. Applied and Environmental Microbiology 52, 1353-1361.[Medline]

Huang, C., Chien, M.-S., Landolt, M., Batts, W. & Winton, J.(1996). Mapping the neutralizing epitopes on the glycoprotein of infectious haematopoietic necrosis virus, a fish rhabdovirus.Journal of General Virology77, 3033-3040.[Abstract]

Kimura, M.(1980). A simple method for estimating evolutionary rate of base substitution through comparative studies of nucleotide sequences.Journal of Molecular Evolution16, 111-120.[Medline]

Kissi, B., Tordo, N. & Bourhy, H.(1995). Genetic polymorphism in the rabies virus nucleoprotein gene.Virology209, 526-537.[Medline]

LaPatra, S. E., Roberti, K. A., Rohovec, J. S. & Fryer, J. L.(1989). Fluorescent antibody test for the rapid diagnosis of infectious hematopoietic necrosis virus.Journal of Aquatic Animal Health1, 29-36.

LaPatra, S. E., Groberg, W. J., Rohovec, J. S. & Fryer, J. L.(1990a). Size-related susceptibility of salmonids to two strains of infectious hematopoietic necrosis virus.Transactions of the American Fisheries Society119, 25-30.

LaPatra, S. E., Groff, J. M., Fryer, J. L. & Hedrick, R. P.(1990b). Comparative pathogenesis of three strains of infectious hematopoietic necrosis virus in rainbow trout Oncorhynchus mykiss.Diseases of Aquatic Organisms8, 105-112.

LaPatra, S. E., Lauda, K. A. & Morton, A. W. (1991). Antigenic and virulence comparisons of eight isolates of infectious hematopoietic necrosis virus from the Hagerman Valley, Idaho, USA. In Proceedings of the Second International Symposium on Viruses of Lower Vertebrates, pp. 125–132. Corvallis, OR: Oregon State University.

LaPatra, S. E., Fryer, J. L. & Rohovec, J. S.(1993). Virulence comparison of different electropherotypes of infectious hematopoietic necrosis virus.Diseases of Aquatic Organisms16, 115-120.

LaPatra, S. E., Lauda, K. A. & Jones, G. R.(1994). Antigenic variants of infectious hematopoietic necrosis virus and implications for vaccine development.Diseases of Aquatic Organisms20, 119-126.

Nei, M. (1987). Molecular Evolutionary Genetics. New York: Columbia University Press.

Nei, M. & Gojobori, T.(1986). Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions.Molecular Biology and Evolution3, 418-426.[Abstract]

Nichol, S. T., Rowe, J. E. & Fitch, W. M.(1989). Glycoprotein evolution of vesicular stomatitis New Jersey virus.Virology168, 281-291.[Medline]

Nichol, S. T., Rowe, J. E. & Winton, J. R.(1995). Molecular epizootiology and evolution of the glycoprotein and non-virion protein genes of infectious hematopoietic necrosis virus, a fish rhabdovirus. Virus Research38, 159-173.[Medline]

Oshima, K. H., Arakawa, C. K., Higman, K. H., Landolt, M. L., Nichol, S. T. & Winton, J. R.(1995). The genetic diversity and epizootiology of infectious hematopoietic necrosis virus. Virus Research35, 123-141.[Medline]

Reno, P. W.(1998). Factors involved in the dissemination of disease in fish populations.Journal of Aquatic Animal Health10, 160-171.

Ristow, S. S. & Arnzen de Avila, J.(1991). Monoclonal antibodies to the glycoprotein and nucleoprotein of infectious hematopoietic necrosis virus (IHNV) reveal differences among isolates of the virus by fluorescence, neutralization, and electrophoresis.Diseases of Aquatic Organisms11, 105-115.

Schneider, S., Kueffer, J. M., Roessli, D. & Excoffier, L. (1997). Arlequin: a software for population genetic data analysis, version 1.1. University of Geneva, Geneva, Switzerland.

Swofford, D. L. (1998). PAUP*: Phylogenetic analysis using parsimony (* and other methods), version 4. Sinauer Associates, Sunderland, MA, USA.

Thoesen, J. C. (1994). Suggested procedures for the detection and identification of certain finfish and shellfish pathogens, 4th edn, vol. 1. Bethesda, MD: American Fisheries Society, Fish Health Section.

USDA Economic Research Service (1998). Aquaculture Outlook (October, 1998). http://usda.mannlib.cornell.edu/reports/erssor/livestock/ldp-aqs/.

Winton, J. R.(1991). Recent advances in detection and control of infectious hematopoietic necrosis virus in aquaculture.Annual Review of Fish Diseases1, 83-93.

Winton, J. R., Arawaka, C. K., Lannan, C. N. & Fryer, J. L.(1988). Neutralizing monoclonal antibodies recognize antigenic variants among isolates of infectious hematopoietic necrosis virus.Diseases of Aquatic Organisms4, 199-204.

Wolf, K. (1988). Infectious hematopoietic necrosis virus. In Fish Viruses and Fish Viral Diseases, pp. 83–114. Ithaca, NY: Cornell University Press.

Received 18 April 2000; accepted 8 August 2000.