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Genetic diversity patterns and evolution of an aquatic rhabdovirus

Gael Kurath, Ryan Troyer, Eric D. Anderson, and Eveline J. Emmenegger
Biological Resources Division, U.S. Geological Survey
Western Fisheries Research Center, 6505 NE 65th St., Seattle, WA 98115; gael_kurath@usgs.gov

Infectious hematopoietic necrosis virus (IHNV) is a rhabdoviral pathogen of salmonid fishes that is endemic throughout northwestern North America (reviewed in Wolf, 1988). Due to the significance of IHNV disease outbreaks in fish culture facilities, both wild and cultured fish populations are routinely surveyed for IHNV by fish health professionals. Therefore many hundreds of IHN virus isolates have been collected from throughout the viral range over the last 25 years. We have developed RNase protection and nucleotide sequencing methodologies to assess the genetic diversity of IHNV field isolates for epidemiological and evolutionary insights. To date we have characterized approximately 300 virus isolates, comprising broad scale regional studies of IHNV throughout Alaska and British Columbia, and an in depth study of IHNV within a localized study site of intensive trout farming in Idaho. IHNV incidence in each of these three areas has been high for the last 25 years, but our results show distinctly different levels of genetic diversity in different portions of the viral range.

The Alaska study involved 42 IHNV isolates from 23 different geographic sites, isolated over a 19 year period from 1977-1996. This collection included virus isolated from four different salmonid host species, from both hatchery and wild host stocks, and from asymptomatic spawning adults as well as juveniles undergoing disease epizootics. RNase protection fingerprint patterns representing the complete nucleocapsid (N), glycoprotein (G), and non-virion (NV) protein genes of each virus isolate distinguished 36 different composite haplotypes among the 42 isolates. These genetic haplotypes were used for epidemiological inferences, suggesting spatial patterns and both evolution and introduction events in the traffic patterns of IHNV. Nucleotide sequence analysis of a 303 nt region in the G gene (mid-G) and the complete 364 nt NV gene indicated surprisingly low genetic diversity within these Alaskan IHNV isolates. The maximum nucleotide divergence between isolates was 2% and 2.75% for the mid-G and NV sequences respectively, and phylogenetic analyses were uninformative due to low bootstrap values. A similar study of 48 IHNV isolates from throughout British Columbia also indicated very low genetic diversity, with a maximum of 1.6% nucleotide diversity in the mid-G sequencing region.

In contrast, a significantly higher level of genetic diversity was observed within a localized area of intensive trout farming in southern Idaho. This study site was selected due to a previous report indicating high antigenic diversity among the IHNV isolates in this area (LaPatra et al., 1994). In our study 84 IHNV isolates from four aquaculture facilities within a 12 mile stretch of the Snake River were analyzed for genetic diversity. These isolates were all from disease outbreaks in farmed rainbow trout fry over a 20 year period between 1978-1998. Both RNase protection fingerprint haplotypes and sequence analyses of the midG regions distinguished the Idaho isolates into four distinct phylogenetic lineages that co-circulated at each of the four sites and in all time periods examined. The maximum midG nucleotide sequence divergence was 7.6%, providing for informative phylogenetic estimations with significant bootstrap confidence values.

Thus, the genetic diversity of IHNV within this 12 mile area in Idaho was nearly 4-fold greater than the diversity observed throughout the entire regions of Alaska and British Columbia. This dramatic difference in genetic diversity levels is evident in the composite phylogenetic tree generated from the mid-G sequences of all these studies (Figure 1).

Figure 1. Phylogenetic analysis of IHNV isolates from throughout Alaska (42 isolates), throughout British Columbia (48 isolates), and within a localized study site in Idaho (84 isolates). Additional sequences of IHNV from California (3 isolates), Oregon (1 isolate) and Washington (4 isolates) were from Nichol et al., 1995. Numbers in parentheses indicate the number of different midG sequences (303 nt) identified within each set of virus isolates. Data were analyzed by the neighbor joining distance method (PHYLIP version 3.5, Joseph Felsenstein) and bootstrap values from a consensus tree generated with 100 bootstrapped data sets were overlaid on the non-bootstrapped tree to preserve branch length information. (click on figure to enlarge)

In this tree the 90 IHNV isolates analyzed from Alaska and British Columbia are represented by 20 distinct sequences that group together in a clade with small amplitude and no significant internal structure. In contrast, the majority of the tree topography represents the Idaho IHNV isolates, which are distinguished into four separate clades with several intermediate sequence types. California, Oregon, and Washington are regions that will be characterized in the future to complete the overall picture of IHNV genetic diversity throughout its North American range.

The conclusions from the composite tree at this time are that the Idaho IHNV lineages are phylogenetically distinct from the IHNV in Alaska and British Columbia, and that evolution within the Idaho site is proceeding with a different pattern than elsewhere in the viral range. The phylogenetic separation could be due to the reproductive isolation of the Idaho trout host populations, which are confined within the aquaculture facilities for their entire life cycles. In contrast, the IHNV from Alaska and British Columbia were from wild and hatchery fish stocks that are anadromous, meaning they all migrate out to sea for the majority of their life cycles before returning to fresh water to spawn. It is possible that the low genetic diversity of IHNV within these host populations is due to a larger effective virus population size as the hosts intermingle in the ocean environment. It is also possible that there is an unidentified marine reservoir of IHNV.

The clearly different pattern of evolution in Idaho in Figure 1 suggests that different selection pressures are acting on virus populations within the Idaho trout farms relative to their anadromous counterparts in Alaska and British Columbia. Among the potential variables that differ between these host environments, we hypothesize that factors related to the human intervention in the host biology may be involved in the generation and maintenance of high levels of genetic diversity on trout farms. Whereas salmonid fish naturally spawn annually in a specific seasonal cycle, in the trout farms photoperiod manipulation is used to produce broodstock that spawn in each month, to provide for constant production levels. This eliminates the annual bottleneck of virus transmission events, and results in the continual introduction of naive, susceptible populations of juvenile fish into facilities where the virus is endemic. High host population densities throughout the life of these fish also reduces transmission bottlenecks, and increases stress levels that may facilitate virus infection. Thus, we hypothesize that conditions within the trout farming environment may provide for more rounds of virus replication per time, and less genetic bottlenecks than in hatchery or wild environments. Controlled wet laboratory experiments to determine if these factors could result in the higher virus genetic diversity levels observed are planned for the future.

Acknowledgements: We would like to thank Dr. Ted Meyers of the Alaska Department of Fish and Game, Dr. Garth Traxler of the Canadian Department of Fisheries and Oceans, and Dr. Scott Lapatra of Clear Springs Foods, Inc., for providing the IHNV isolates and information that made this work possible.

References
LaPatra, S.E., Lauda, K.A., and Jones, G.R. 1994. Antigenic variants of infectious hematopoietic necrosis virus and implications for vaccine development. Dis. Aquat. Org. 20:119-126.

Nichol, S.T., Rowe, J.E., and Winton, J.R. 1995. Molecular epizootiology and evolution of the glycoprotein and nonvirion protein genes of infectious hematopoietic necrosis virus, a fish rhabdovirus. Virus Res. 38:159-173.

Wolf, K. 1988. Infectious hematopoietic necrosis. pp.83-114 In, Fish viruses and fish viral diseases. Cornell University Press, Ithaca, New York.

Abstract - Presented at the Virus Evolution Workshop
Ardmore, OK
October 21 - 24th, 1999

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