Comparison of selected strains of infectious hematopoietic necrosis virus (IHNV) using neutralizing trout antisera

Abstract

Antisera against representative strains of infectious hematopoletlc necrosis virus (IHNV) from each of the 5 electropherotypes were prepared in rainbow trout Oncorhynchus mykiss following injections with live virus. Serum cross-neutralization comparisons by 50 % plaque neutralization tests (PNT) showed low antigenic heterogeneity between the vlruses, as calculated by l / r , a measure of the antigenic relationship between 2 virus types. The only vlrus strain clearly different from the others was the representative electropherotype 2 isolate (CST-82), with l / r values from 5.2 to 15.6 when compared to the other isolates. The l l r values obtained for the type 2 IHNV isolate were, however, not greater than 20, a value established as the cnterion for distinguishing serotypes. The other IHNV isolates tested formed a hon~ogeneous group sharing l / r values of 2.1 or less. These studies p rov~de the first evidence that rainbow trout respond distinctly with respect to the production of serum neutralizing antibodies between certain strains of IHNV. A further characterization of these responses will be important to the interpretation of ep~demiological studies and the development of vaccines for the control of IHNV. The salmonid rhabdovirus infectious hematopoietic necrosis virus (IHNV) causes commercially significant losses in susceptible populations of both captive and wild fish (Wolf 1988). This agent is endemic among populations of salmon on the west coast of the USA and Canada but is also known from Europe, Japan, Taiwan, Korea, and the People's Republic of China (Sano et al. 1977, Chen et al. 1985, Bovo et al. 1987, Hattenberger-Baudouy 1988, Luqi & Zhizhuang 1988, J . Park, Ulsan University, Korea, pers. comm.). Although fry are the most susceptible stage to virusinduced mortality (Amend & Nelson 1977), larger fish also suffer from both active infections and the sequelae of IHNV. 'Addressee for correspondence O Inter-Research 1993 Attempts to group or separate IHNV isolates have followed the virus since its initial identification. Three presumably different viral diseases were described among sockeye salmon Oncorhynch~is nerka in Oregon (OSV), Sacrament0 h v e r chmook salmon in Cahfornia (SRCV), and rainbow trout Oncorhynchus myhss and sockeye salmon in British Columbia (IHNV) (Wolf 1988). McCain et al. (1971) compared these 3 virus strains by neutralization using a polyclonal rabbit antiserum and found that OSV and IHNV were indistinguishable while SRCV was closely related to the other 2 viruses. Because of the inability to easily distinguish strains or serotypes of IHNV using polyclonal rabbit serum, alternative approaches were sought. Hsu et al. (1986) found that differences in the electrophoretic patterns of the N and G structural polypeptides using SDS PAGE could be used to separate IHNV strains into 5 groups or electropherotypes. The potential antigenic relationships of viruses in different electropherotypes were pursued by Engelking et al. (1991) who compared viruses from each of these 5 electropherotypes using 2 rabbit antisera prepared against types 1 and 4 whole virus and 2 antisera to the partially purified G protein of types 1 and 4 IHNV. Although their comparisons did not include full crossneutralizations for each of the 5 electropherotypes (5 sera X 5 viruses), they concluded that the antigenic differences observed between virus strains were sufficient to define variants but not multiple serotypes. Addtionally, they demonstrated that fish irnnlunized with partially purified G protein from type 1 virus were protected when challenged with type 2 or 5 virus. These comparisons and those of McCain et al. (1971) and others studying different phenotypic characters of the virus suggested that there are no great differences among strains of IHNV and that strains of the virus tend to be related more by geographic location than by host origin. Dis. aquat. Org. 15: 229-233. 1993 RB-7 6 Steelhead Deschutes River, OR 1976 1 CST-82 Rainbow trout Clear Springs, ID 1982 2 Trinity Coho salmon Trinity River, CA 1985 3 SRCV Chinook salmon Sacramento River, CA 1966 3 Coleman Chinook salmon Sacramento River, CA 1983 4 Cedar Sockeye salmon Cedar River, WA 1979 5 The use of monoclonal antibodies (mabs), both neusimilar to that described by Burke & Mulcahy (1980). tralizing and binding, has helped to overcome in part Modifications to that method included incubation of the poor resolving powers of low titered rabbit antisera virus dilutions at 4 "C for 16 h prior to a 1 h adsorption used in early comparisons (Winton 1991). Winton et on EPC cells at 15°C after which a methylcellulose al. (1988) and Ristow & Arnzen (1991) have shown that overlay was added. at least 4 groups of viruses can be distinguished by Preparation of fish antisera: Antisera to the 6 IHNV panels of currently available mabs. strains were prepared in 2 yr old rainbow trout. At Although antibodies raised in rabbits and mice have least 3 trout were injected for production of antisera been and will continue to be useful in examining imagainst each virus isolate. These fish were obtained portant viral epitopes involved in virus neutralization, from the American River Hatchery, California Departfish antibodies to IHNV are the ultimate indicators of ment of Fish and Game, Rancho Cordova, CA, and the salmonid's response to viral infections. The use of were maintained at the Fish Disease Laboratory at this serological response for epidemiological studies Univ. of California, Davis, in 650 1 tanks receiving 15 "C and vaccine development therefore depends upon a well water and fed a commercial pelleted ration daily. better understanding of the fishes' capability to distinThe American River Hatchery has no history of IHNV guish between strains oi IHNV. Tne existence and and testing of serum prior to ir,'cctio:: shswed nc evisignificance of neutralizing antibodies in salmonids dence of anti-IHNV neutralizing activity. The fish were infected with IHNV were first demonstrated by Amend anesthetized with MS-222 (0.1 mg 1-') prior to & Smith (1974) and practical tests for the demonstraintraperitoneal injections with virus or coiiection of tion of antibodies were developed by Hattenbergerblood samples. Trout received 4 injections at 3 wk Baudouy et al. (1989). Using this latter technique we intervals with 0.1 m1 of cell-free supernatant containhave examined the relatedness of 6 IHNV isolates, ing approximately 107 PFU ml-' of IHNV. Blood samrepresenting each of the 5 electropherotypes, by their ples were drawn from the caudal vein at 10 d after the response in cross-neutralization tests to hyperimmune last injection. The antisera were heat inactivated for serum prepared in rainbow trout. 30 min at 45 "C prior to storage at -70 "C. Materials and methods. Cell culture: All virus iso50 % plaque neutralization test (50 % PNT): The lates were propagated in the EPC (epithelioma papuloPNT technique was performed as previously described sum cyprini) cell line, derived from common carp by Olesen & Jsrgensen (1986) for viral hemorrhaqc Cyprinus carpio (Fijan et al. 1983). The cells were septicemia virus (VHSV) but the neutralization time grown in minimal essential medium (MEM) containing was increased from 1 to 16 h (Hattenberger-Baudouy Eagle's salts supplemented with 7.5% fetal bovine et al. 1989). All virus-antibody mixtures in 96-well serum, 2 mM L-glutamine, 50 IU ml-' of penicillin and culture plates were performed in the presence of 50 ~g ml-' of streptomycin. normal trout serum diluted 1 : 40 as a source of compleVirus isolates: Six isolates were used in this study ment. Both negative and positive serum controls were (Table 1). They were selected to represent each of the included in each test. Initial positive control sera were 5 electropherotypes of IHNV as described by Hsu et al. kindly provided by Dr P. de Knkelin (INRA, Jouy-en(1986). A second type 3 virus isolate from coho salmon Josas, France) but later were replaced by anti-RB-76 0. kisutch adults at the Trinity River Hatchery (CA, IHNV trout serum. Negative control sera were from USA; LaPatra et al. 1989) was also included. The isovirus-free trout. Eight 3-fold dilutions of trout sera lates were not plaque purified before use. To estimate were mixed with 120 p1 of virus (2000 PFU ml-'). After concentrations needed for subsequent plaque neutral30 min at 15°C a complement source (trout sera) was ization tests (PNT), viruses were titrated by a method added and then the mixtures were incubated at 4OC for 16 h. The mixtures were then placed Table 1. Representative isolates of mfectious hematopoietic necrosis virus (W) onto EPC monolayers in 24-well culture from the 5 electropherotypes used for comparison by cross-neutrahzation plates, adsorbed for 1 h at 15°C prior to adding a methylcellulose overlay. The HSU et al. (1986); banadromous rainbow trout Isolate Host species Location (USA) Year Typea plaques were observed in the negative controls (6 to 7 d). The cells were then fixed and stained with 10 % formalin and 1 % crystal violet, the plaques counted, and the 50% PNT calculated as the reciprocal value of the highest serum dilution causing 50% reduction of the number of plaques found in the presence plates were incubated until clear virus Basurco et al.: Comparison of IHNV strains 23 1 Rainbow trout Isolateb antiserum' RB-76 CST-82 Trinity SRCV Coleman Cedar (1) (2) (3) (3) (4 (5) Anti-RB-76 21870 81

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@inproceedings{Basurco2006ComparisonOS, title={Comparison of selected strains of infectious hematopoietic necrosis virus (IHNV) using neutralizing trout antisera}, author={B. Basurco and Philip W Hedrick}, year={2006} }