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1 AN ABSTRACT OF THE THESIS OF Hamdi Ogut for the degree of Master of Science in Fisheries Sicence presented on December Title: IN VITRO HOST RANGE OF INFECTIOUS PANCREATIC NECROSIS VIRUS (IPNV) AND ITS RELATIONSHIP TO VIRULENCE. Abstract approved: Redacted for Privacy Of 109 aquatic birnaviruses (AB) belonging to 9 serogroup A, all replicated and produced rapid and extensive cytopathology in CHSE-214 and RTG-2 cells, whereas only half produced significant levels of cytopathological effect (CPE) in two nonsalmonid cell lines tested (EPC and FHM). In many instances it was found that although CPE was not visible microscopically in EPC and FHM cells, virus replication occurred. Isolates belonging to Buhl, Canada 1, Canada 2, Ab and Tellina subtypes did not produce CPE on EPC and FHM cells. On the other hand, WB, VR-299, Jasper, Sp, and He subtypes replicated efficiently in both EPC and FHM cells. Serum sensitivity test results indicated that in 49 of 109 isolates in the presence of normal trout serum (NTS), 100 fold reduction in virus titers occured. Avirulent isolates belonging to various subtypes were more effectively inhibited in vitro by 1% normal trout serum (NTS), whereas, virulent isolates, especially Buhl subtype, were not affected by NTS at all and some replicated more efficiently in the presence of NTS. In an in vivo test of the relationship between virulence and serum sensitivity brook trout fry were exposed by immersion to four highly virulent Buhl subtype IPNV isolates which had been passed 11 times in the presence or absence of NTS. Significant mortalities occurred in the groups that were exposed to viruses that were passed fewer than three times after isolation from brook trout (45-70%). All four Buhl subtype virus isolates passed 11 times in RTG-2 cells in the absence of 1% NTS lost their virulence. In two cases (2/4), the isolates passed in the presence of 1% NTS retained their virulence.

2 In Vitro Host Range Of Aquatic Birnaviruses And Their Relationship To Virulence By Hamdi Ogiit A THESIS Submitted to Oregon State University In partial fulfillment of the requirements for the degree of Master of Science Completed: December 20, 1995 Commencement: June 1996

3 Master of Science thesis of Hamdi Ogut presented on. December, APPROVED: Redacted for Privacy Major Professor, represe 'n Fisheries Science. Redacted for Privacy Head of Department of Fisheries and Wildlife. Redacted for Privacy Dean of uate School I understand that my thesis will become part of the permenant Collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Redacted for Privacy Hamdi ()gilt, Author

4 ACKNOWLEDGMENTS: I would like to extend my deepest appreciation to Prof. Dr. Paul Reno for his advise, assistance, and encouragement during my research. Particularly, I would like to thank Dr. Paul Reno and Dr. Bob Olson for critically reading the manuscript as the preparation of the thesis would not have been possible without their comments and criticisms. I also thank Dr. Rich Holt for his support at the stage of preparation of this study. I would like also thank our laboratory assistants for their help, advice and especially for their patience with me. And, of course, Janet Webster, our librarian, was also very helpful. I also thank those not mentioned here but who shared their views in a constructive way. This study was in part funded through the Coastal Oregon Marine Experiment Station.

5 TABLE OF CONTENTS INTRODUCTION 1 LITERATURE REVIEW 3 History of IPN Virus 3 Geographic and Host Range 5 Epizootology 5 Virus Virulence 6 Page Host and Environmental Factors 9 In vitro models 10 Cell Susceptibility 10 Cell Culture Adapted Virus (CCA) 11 Persistent Infections 13 DI (Defective interfering) Particles 14 Interferon 15 Serum Inhibition and Virulence 15 MATERIALS AND METHODS 19 In Vitro Experiment 19 IPN Virus Isolates 19 Cell Lines 19 Determination of In Vitro Host Range 24 Effects of Normal Trout Serum 25 Single Passage 25 Multiple Passage 26 In Vivo Experiment 27 Fish 27 Experimental Design 28 RESULTS 29 Ability of aquatic birnaviruses to produce cytopathology (CPE) in teleost cell lines 29 Serotypes and in vitro host range 32 Effects of normal trout serum on aquatic birnaviruses replication 53

6 TABLE OF CONTENTS (Continued) Page Effect of in vitro passage of aquatic birnaviruses in the presence of NTS on virulence in brook trout. 58 DISCUSSION 65 Ability of aquatic birnaviruses to produce CPE in four teleost cell lines 65 Effects of Normal Trout Serum on Aquatic Birnaviruses replication 70 Effect of in vitro passage of aquatic birnaviruses in the presence of NTS on virulence in Brook trout 73 CONCLUSIONS 76 BIBLIOGRAPHY 78 APPENDICES 91 Appendix A 92 Appendix B 111

7 LIST OF FIGURES Figure Pages 1. a) Box and whisker plot of mean cytopathic effects (CPE) (MC) produced by 109 aquatic birnavirus isolates on four different cell lines 31 b) Geometric mean titer (GMT) (TCID50/ml.) yields of 109 aquatic birnaviruses in all four cell lines tested a) Box and whisker plot of mean cytopathic effects (CPE) (MC) produced by 109 aquatic birnavirus isolates in CHSE-214 cells 33 b) Geometric mean titer (GMT) (TCID50/ml.) yields of 109 aquatic birnaviruses in CHSE-214 cells a) Box and whisker plot of mean cytopathic effects (CPE) (MC) produced by 109 aquatic birnavirus isolates in RTG-2 cells 34 b) Geometric mean titer (GMT) (TCID50/ml.) yields of 109 aquatic birnaviruses in RTG-2 cells a) Box and whisker plot of mean cytopathic effects (CPE) (MC) produced by Buhl (n = 29) aquatic birnavirus isolates in all four cells 36 b) Geometric mean titer (GMT) (TCID50/ml.) yields of Buhl aquatic birnaviruses in all four cells a) Box and whisker plot of mean cytopathic effects (CPE) (MC) produced by VR-299 aquatic birnavirus isolates (n = 15 ) in all four cells 38 b) Geometric mean titer (GMT) (TCID50/m1.) yields of VR-299 aquatic birnaviruses in all four cells 38

8 LIST OF FIGURES (Continued) 6. a) Box and whisker plot of mean cytopathic effects (CPE) (MC) produced by WB aquaticbirnavirus isolates (n = 14) in all four cells 40 b) Geometric mean titer (GMT) (TCID50/ml.) yields of WB aquatic birnaviruses in all four cells a) Box and whisker plot of mean cytopathic effects (CPE) (MC) produced by Jasper aquatic birnavirus isolates (n = 4) in all four cells 42 b) Geometric mean titer (GMT) (TCID50/ml.) yields of Jasper aquatic birnaviruses in all four cells a) Box and whisker plot of mean cytopathic effects (CPE) (MC) produced by Sp aquatic birnavirus isolates (n = 8) in all four cells 44 b) Geometric mean titer (GMT) (TCID50/ml.) yields of Sp aquatic birnaviruses in all four cells a) Box and whisker plot of mean cytopathic effects (CPE) (MC) produced by Ab aquatic birnavirus isolates (n = 5) in all four cells 46 b) Geometric mean titer (GMT) (TCID50/m1.) yields of Ab aquatic birnaviruses in all four cells a) Box and whisker plot of mean cytopathic effects (CPE) (MC) produced by CAN-1 aquatic birnavirus isolates (n = 5) in four cell lines 48 b) Geometric mean titer (GMT) (TCID50/ml.) yields of CAN-1 aquatic birnaviruses in four cell lines a) Box and whisker plot of mean cytopathic effects (CPE) (MC) produced by He aquatic birnavirus isolates (n = 2) in CHSE-214 cells 50

9 LIST OF FIGURES (Continued) b) Geometric mean titer (GMT) (TCID50/m1.) yields of He aquatic birnaviruses in four cells a) Box and whisker plot of mean cytopathic effects (CPE) (MC) produced by BC aquatic birnavirus isolates (n = 4) in four cell lines 52 b) Geometric mean titer (GMT) (TCID50/m1.) yields of BC aquatic birnaviruses in four cell lines a) Box and whisker plot of mean cytopathic effects (CPE) (MC) produced by 109 aquatic birnavirus isolates in EPC cells 54 b) Geometric mean titer (GMT) (TCID50/m1.) yields of 109 aquatic birnaviruses in EPC cells a) Box and whisker plot of mean cytopathic effects (CPE) (MC) produced by 109aquatic birnavirus isolates in FHM cells 55 b) Geometric mean titer (GMT) (TCID50/m1.) yields of 109 aquatic birnaviruses in FHM cells a) Box and whisker plot of the mean CPE differences of between virus replication in the presence and absence of NTS. 57 b) The mean login titer differences in the presence and absence of NTS a) Percent cumulative mortalities of 0.9 g brook trout fry exposed by immersion to 104 TOD50/ml. IPNV Buhl subtype isolate which three treatments applied to (90-11: virus passed only once in the absence of NTS, 90-11(-) virus passed 11 times in the absence of NTS, 90-11(+) virus passed 11 times in the presence of NTS.) 60 b) Virus titer (TCID50/m1.) of dead or moribund brook trout fry after exposure to IPNV propagated in the presence or absence of 1% NTS. 60

10 LIST OF FIGURES (Continued) 17. a) Percent cumulative mortalities of 0.9 g brook trout fry exposed by immersion to 104 TCID50/ml. IPNV H-VAT 9/86 isolate which three (H-VAT: virus passed only once in the absence of NTS, H-VAT(-) 11 times in the absence virus passed of NTS, H-VAT(+) virus passedll times in the presence of NTS.) 61 b) Virus titer (TCID50/m1.) of dead or moribund brook trout fry after exposure to IPNV H-VAT 9/86 propagated in the presence or absence of 1% NTS a) Percent cumulative mortalities of 0.9 g brook trout fry exposed by immersion to 104 TCID50/ml. IPNV csf (35-85 = virus passed only once in the absence of NTS, 35-85(-) = virus passed 11 times in the absence of NTS, 35-85(+) = virus passed 11 times in the presence of NTS.) 62 b) Virus titer (TCID50/m1.) of dead or moribund brook trout fry after exposure to IPNV csf propagated in the presence or absence of 1% NTS a) Percent cumulative mortalities of 0.9 g brook trout fry exposed by immersion to 104 TCID50/ml. IPNV csf Crayfish (Crayfish = virus passed only once in the absence of NTS, Crayfish(-) = virus passed 11 times in the absence of NTS, Crayfish(+) = virus passed 11 times in the presence of NTS) 63 b) Virus titer (TCID50/m1.) of dead or moribund brook trout fry after exposure to IPNV csf Crayfish propagated in the presence or absence of 1% NTS a) The relationship between CPE differences in RTG-2 cells infected with aquatic birnaviruses in the presence and absence of NTS and mortalityt levels in brook trout fry 72 b) The relationship between Logic) titer differences in RTG-2 cells infected with aquatic birnaviruses in the presence and absence of NTS and mortalityt levels in brook trout fry 72

11 LIST OF TABLES Table Pages 1. Aquatic Birnavirus isolates used in the experiments Isolates used in the multiple passage of IPNV in the presence and absence of NRTS and its effect on virulence Cytopathic effects (CPE) of aquatic birnaviruses on four teleost cell lines 30

12 LIST OF APPENDIX TABLES: Table A.1. Virus yields and destruction of cell monolayers by Buhl subtype Pages IPNV isolates 93 A. 2. Virus yields and destruction of cell monolayers by VR-299 subtype IPNV isolates 94 A. 3. Virus yields and destruction of cell monolayers by WB subtype IPNV isolates 95 A. 4. Virus yields and destruction of cell monolayers by Jasper subtype IPNV isolates 96 A. 5. Virus yields and destruction of cell monolayers by Sp subtype IPNV isolates 97 A. 6. Virus yields and destruction of cell monolayers by Ab and EVE subtype IPNV isolates 98 A. 7. Virus yields and destruction of cell monolayers by CAN -1, CAN-2 and CAN-3 subtype IPNV isolates 99 A. 8. Virus yields and destruction of cell monolayers by He and Te subtype IPNV isolates 100 A. 9. Shows CPE and virus yields of isolates whose serological identity have not yet investigated 101 A. 10. Probabilities of differences among 13 subtypes of virus in their ability to produce CPE in EPC cells 102 A. 11. Probabilities of differences among 13 subtypes of virus in their ability to produce CPE in FHM cells 103 A. 12. ANOVA table for geometric mean titers obtained in EPC cells infected with 13 different subtypes of IPNV subtypes of IPNV 104

13 LIST OF APPENDIX TABLES (Contunied) A. 13. ANOVA table for geometric mean titers obtained in FHM cells infected with 13 different subtypes of IPNV 105 A. 14. ANOVA table of CPE and virus yield responses of aquatic birnaviruses, and significance levels in different cell ines 106 B. 1. Replication of IPNV Buhl subtype isolates in the presence and absence of 1% NTS and their relationship to virulence 112 B. 2. Replication of IPNV VR 299 subtype isolates in the presence and absence of 1% NTS and their relationship to virulence 113 B. 3. Replication of IPNV WB subtype isolates in the presence and absence of 1% NTS and their relationship to virulence 114 B. 4. Replication of IPNV CAN subtype isolates in the presence and absence of 1% NTS and their relationship to virulence 115 B. 5. Replication of IPNV Sp subtype isolates in the presence and absence of 1% NTS and their relationship to virulence 116 B. 6. Replication of IPNV Jasper and BC subtype isolates in the presence and absence of 1% NTS and their relationship to virulence 117 B. 7. Replication of IPNV Ab and EVE subtype isolates in the presence and absence of 1% NTS and their relationship to virulence 118 B. 8. Replication of IPNV He and Te subtype isolates in the presence and absence of 1% NTS and their relationship to virulence 119 B. 9. Replication of IPNV (subtypes unknown) isolates in the presence and absence of 1% NTS and their relationship to virulence 120 B.9 Species from which IPNV isolated 121

14 In Vitro Host Range Of Aquatic Birnaviruses And Their Relationship To Virulence INTRODUCTION Infectious pancreatic necrosis virus (IPNV), a member of the family Birnaviridae, can cause mortality as high as % in 1-4 month-old rainbow trout fry (Frantsi and Sayan, 1971; McAllister, 1983). There have been hundreds of studies on IPNV characteristics since it was first isolated in 1957, yet there is still no effective vaccine or any other control method, except prevention of exposure. There are more than 3,000 IPNV isolates obtained from different aquatic hosts worldwide. Most were obtained from non-diseased fish. Knowledge on the virulence characteristics of IPNV is very limited. After isolating IPNV from a host, it is necessary to carry out a number of time consuming in vivo tests to determine whether it is virulent to trout fry or not. One of the main objectives of this study was to examine the relationship, if any, between in vivo and in vitro characteristics of the serotype of virus related to the virulence. The second main objective was to examine the effects of normal trout serum (NTS) on virus growth. Some studies suggest that serum obtained from normal rainbow trout (NRT) interacts with IPNV virus inactivating it and changing its virulence characteristics (Dorson et al., 1978; Hill, 1981 ). How the serum interacts with the virus and inactivates is unknown. Dorson et. al. (1978) suggested that although acquired sensitivity to NTS is not the only way that virus loses its virulence, it must be taken into account in the production of virus for infection trials. It has been suggested that passage of the virus in the presence of NTS prevents loss of virulence compared to virus passed in the absence of NTS (Hill, 1982).

15 2 In the first part of present study, the correlation between virulence of IPNV and its ability to replicate in certain fish cell lines was determined and described. Also, a study of the relationship between cytopathic effects (CPE) and virus titers in various cell lines was made. The second objective was studied by determining if virulence was correlated with the ability of the virus to replicate in the presence of NTS.

16 3 LITERATURE REVIEW History of IPN Virus: M'Gonigle (1941) first reported the signs of infectious pancreatic necrosis (IPN) disease in Canadian brook trout naming the disease " acute catarrhal enteritis". Later on, IPN disease was discovered and described in the US. by Wood et al., (1955) and named by Snieszko (Snieszko et al., 1957). Isolation of IPNV using finite teleost cell cultures occurred in 1957 (Wolf and Dunbar, 1957). Then, Wolf et al. (1959 and 1960) established its etiology as the first known viral disease of fish. Research on various aspects of IPN disease continued in 1960s. The development of the following continuous teleost cell lines during 1960s simplified studies of the virus: Rainbow trout gonad (RTG-2) (Wolf and Quimby, 1962), grunt fin (GF) (Beasly et al., 1965), fathead minnow (FHM) (Malsberger, 1965). The susceptibility of RTG-2 cells, as well as many other teleost cells to IPNV was also reported (Wolf and Mann, 1980). Early studies on the morphology of IPNV were controversial. Although originally described as 25 nm in diameter (Cerini and Malsberger, 1965), the correct size of the reovirus-like virion, 69 nm, was reported by Lightner and Post (1969), and confirmed by other researchers (Moss and Gravel, 1971). Another unresolved issue was whether the RNA genome of the virus is single or double stranded. Kelly and Loh (1972) reported that IPNV was a single stranded RNA virus. When the double stranded nature of the genome was described by Moss and Gravel (1971), other researchers agreed with and then verified the findings of Moss & Gravel (Argot and Malsberger, 1972; Cohen, 1973). Further biophysical studies proved that, IPNV has a double stranded RNA genome (Dobos, 1977; Dobos et al., 1977) consisting of two segments. These segments are RNA segment A and RNA segment B (Brown, 1986). RNA segment A encodes a major capsid protein (Vp2 = 54kd), a second structural protein (Vp3 = 31 kd) and a nonstructural protein (Vp4 = 29kd).

17 4 The RNA segment B encodes a putative viral RNA polymerase (Vpl = 105kd), (McDonald & Dobos, 1981; Persson and MacDonald, 1982). Susceptibility of different species of trout to IPN disease has also been investigated (Silim et al., 1982). Seven different trout groups which have been chosen from three different species from different areas were tested. Brook trout had highest susceptibility to IPNV (30.0 to 46.25% mortalities). Lake trout were the most resistant (1.0 to 1.3% mortalities) and rainbow trout were less resistant than lake trout (6.0 to 10% mortalities). Almost all techniques for viral detection in terms of their reliability and rapidness have been compared and the superiority of one to another reviewed. The techniques for rapid detection and identification of IPNV used so far include fluorescent antibody (Swanson & Gillspie, 1981; Hattori, 1983), ELISA (Dixon & Hill, 1983; Caswell and Nicholson, 1986), coagglutination (Kimura et al., 1981), and immunodot blot (Caswell- Reno et al., 1989; Lipipun et al, 1989; Ya-li Hsu, 1989). During the 1980s and 1990s, serotyping of IPNV using monoclonal antibodies aided the classification of IPNV isolates. A polyclonal antibody classification system of IPNV strains based on virus neutralization was developed by Hill and Way (1988). According to this classification system, IPNV strains are divided into two serogroups; Serogroup A and Serogroup B. Serogroup A consists of 9 serotypes, Sp, Ab, West Buxton (WB), Jasper (Ja), Hecht (He), Tellina (Te), Canada 1 (C1), Canada 2 (C2) and Canada 3 (C3). It includes more than 2,000 isolates. Serogroup B comprises only one serotype and 10 isolates. Epitopes of these serotypes were defined using monoclonal antibodies (Caswell-Reno et al., (1986, 1989); Dobos (1976); Hetrick (1983)). The Ni strain previously was defined as a different serotype (Cristie et al., 1988) but later concluded to be a member of Sp serotype (Melby and Cristie, 1994).

18 5 Geographic and Host Range: IPNV has a wide geographic distribution. The first reports of IPNV disease came from Canada (M'Gonigle, 1941), and then the US (Wood et al., 1955). It was not reported in Europe until 1965 (Besse and DeKinkelin, 1965). It has since been reported from many other places: Japan (Sano, 1976), South Africa (Bragg and Combrink, 1989), Chile (MacAllister, 1983), Korea (Hah et al., 1984), Taiwan (Hedrick et al., 1983), China (Jaing et al., 1989), United Kingdom (Ball, et al., 1971) and Scandinavia and British Isles (Besse and de Kinkelin, 1965) In the countries given above not all waters are contaminated with IPNV. Many waters still are IPNV free. It is generally thought that IPNV has been transferred from country to country by shipping of IPNV infected trout eggs. Early on, it was thought that IPNV caused acute disease only in salmonid fishes. However, many studies have proven that IPNV could be isolated from non-salmonids. (Adair and Ferguson, 1981). IPNV was isolated from such molluscans as Asian clam (Corbicula fluminou), scallop (Patella vulgate), periwinkle (Littorina litorea), mussel (Mytilus edulis), American oyster (Crassostrea gigas), (Hill, 1982). IPNV was isolated from crustacea such as shore crab (Carcinus maenas) and Japanese shrimp (Peanaeus japonica) (Bovo et al., 1984). It was also isolated from lamprey (Lampetra fluviatilis) (Munro et al., 1976) and other teleosts (See Appendix B. Table 10, Paul W.Reno, personal communication). The vast mojority of aquatic birnavirus isolates were obtained from salmonid fishes. Epizootiology: IPN disease causes high mortality in young salmonids (Wolf et al., 1960; MacAllister 1983, 1995). Survivors are life time carriers of IPNV (Dorson, 1982). These asymptomatic, persistently infected fishes carry the virus in their visceral organs and shed

19 6 periodically (Hill, 1982), although the fish mounts a humoral immune response (Yamamoto, 1971; Reno 1976). IPNV can be transmitted both horizontally and vertically (Wolf et al., 1963). Billi and Wolf (1969) demonstrated that IPNV could be shed from feces and reproductive organs. Horizontal transmission has been then confirmed by many other researchers (Hill, 1982 ; Hedrick and Fryer, 1982; Boot land, 1986). Vertical transmission by carrier fish to progeny has also been shown by many researchers (Ahne & Negele, 1985; Dorson & Torchy, 1985; Boot land, 1991). Smail et al., (1992) reported that there has been an increased association of IPNV Sp strain with clinical disease in sea cage of Atlantic salmon post-smolts in Scotland. Peak mortalities occurred five weeks after transfer smolts to the sea sites. Mortality at the cage site rose from 0.3% to 2.4% and then again dropped to 0.2% over a period of 3 weeks. The worst mortalities (10-13%) were in the adjacent cages. Twelve out of 12 pools were positive for IPNV. Results of plaque neutralization tests from Atlantic salmon farms indicated that the strain was Sp. It was also suggested that IPNV Sp isolate is associated with clinical disease and there might have a synergistic effect with the infectious agent of exocrine pancreatic disease (PD). Virus Virulence: Virulence, a relative term describing the disease causing ability of a virus, is affected by different factors, including the physiological state of the animal, environmental factors and the nature of virus itself. To designate a virus as virulent, that virus must have ability to enter the host cell, replicate, overcome host cell defense mechanisms and destroy the cells which are important in the physiology of animal. In the case of an avirulent state, one or more stage described above may be blocked. The physical location responsible for virulence on the virion surface is variable among different kinds of viruses. Leavy et al., (1994) indicated that virulence of a virus is

20 7 often due to a single surface protein causing inhibition of synthesis of host cell macromolecules. Studies with rabies virus showed that antigenic alterations may influence viral pathogenesis (Detzschold et al., 1983). Highly pathogenic strains of rabies virus were selected by the presence of neutralizing monoclonal antibody. Avirulent strains of rabies virus were exposed to mice to confirm their avirulence. Sequencing of glycoprotein of the avirulent and virulent variants showed that substitution of a single amino acid altered virulence. In the case of arenaviruses, virulence is associated with the presence of the large [L] RNA segment. L segment encodes a large protein [L], presumably representing the viral polymerase (Riviere et al., 1985). In the case of bunyavirus, an RNA virus, surface glycoprotein is responsible for virulence (Bishop and Shop, 1980). Reassortment between virulent and avirulent strains were used in the determination of bunyavirus virulence (Janssen et al., 1986). Both the viral L genomic segment which encodes polymerase and S segment which encodes nucleocapsid protein (N) and small non-structural protein (NSs) can modulate the effect of the M segment on virulence. The 51 gene encoding the outer capsid protein is responsible for virulence of reoviruses (Fields and Greene, 1982). In most reported cases, attenuation of a reovirus occurred because the viral hemagglutinin lost its ability to react with host cell receptors (Fields and Greene, 1982). Two factors determine virulence of reovirus: The haemagglutinin is responsible for the host cell tropism and destruction. It is the major antigen for cellular and humoral immune responses. Second is the outer capsid protein (µ1c) providing a second site on the surface of virion that can be altered by mutation. g.1c protein alterations significantly reduces effectiveness of virus replication (Fields and Greene, 1982). Gene sequence analysis of Si gene taken from several virulent and avirulent variants indicated that the attenuated and wild types differ only in the single amino acid substitution at positions of sigma-1 protein (Bassel-Duby et al., 1985). The

21 8 importance of this single amino acid substitution on the cell tropism and attenuated neurovirulence was also confirmed using reassortants (Kaye et al., 1986). In the case of IPNV, the portion of the virion responsible for virulence is unknown. Sano et al., (1992) and Dorson et al., (1978) reported that virulence is associated with RNA segment A which codes for VP2 and VP3, not RNA segment B which codes for VP1. But which part of the genome is responsible for virulence is not known. Darragh and McDonald (1982) studied IPNV-Jasper and IPNV-Oyster virus 3 noting the difference in the ability to infect FHM and CHSE-214 cells is related to RNA segment A. Dorson et al. (1978) reported that plaque size was not related to the virulence of IPN virus although this is the case in some other viruses such as poliovirus, coxsackievirus A9 and Venezuelan equine encaphalitis (VEE) virus (Holland, 1964). Intertypic reassortants of Buhl and EVE isolates were compared in terms of their plaque size (Sano and Okamoto, 1994). The genotype of P/E (P: RNA segment A from Buhl, and E: RNA segment B from EVE) produced large plaques ( mm) and IPNV-Buhl also produced large plaques (1.48±0.35 mm). However, E/P and EVE (E/E) formed only small plaques ( mm) indicating that plaque size is related to RNA segment A. In virulence experiments, there was no difference in virulence between parental and reassorted groups. Large plaque clones caused 44% average mortality (five large plaque clone groups) in rainbow trout and small plaque clone led to 48% mortality (average of five small plaque clones) showing no difference between two treatments (Sano and Okamoto, 1994). Virulent and avirulent isolates of IPNV are found within the same serotype (Sano et al., 1992). MacAllister and Owens, (1995) tested 15 different IPNV isolates for their virulence. The virulence was not associated with the species from which they were originally associated: salmonid, non-salmonid or molluscan hosts. Ab serotype isolates were found to be avirulent for brook trout, whereas Sp and VR-299 serotypes had high virulence (Vestegard-Jorgensen, 1971). Some of non-salmonid isolates were highly virulent for trout fry, while others were avirulent. For example, sand goby virus isolate

22 9 (Oxyeleotris mannoratus) was avirulent and striped bass isolate (Morone saxatilis ) showed high virulence. Out of three eel isolates tested, two were avirulent (0-3%) and one was virulent (87%). There was high variability in molluscan isolates in terms of their virulence on trout fry as well. Five molluscan isolates were tested, only one of them was virulent for brook trout (MacAllister and Owens, 1995). In short, virulence and plaque formation of IPNV are related to RNA segment A, but it is not know which part of genome is responsible for virulence. Host and Environmental Factors: Host factors. Host factors are important for the outcome of infectious disease and its severity (Fenner, 1968). Factors such as host cell background, immune status, genetic background, age of animal and nutritional status were studied intensively in vesicular stomatitis virus (Sabin, and Olitsky 1937). Hormones (Lodmell, 1983), nutritional status, which exerts a marked influence on the outcome of a disease (Chandra, 1979), the role of host immunological responses such as inflammation (Roberts, 1979), host cell enzymes (Scheid and Choppin, 1984) have also proven to be important in viral infection outcomes. The virulence of IPNV usually depends on the particular host species infected (Hill, 1982). Even in salmonids which are known to be susceptible to IPNV, there are marked differences between species in susceptibility to disease. Silim et al., (1982) reported that brook trout (Salvelinus fontinalis ) was the most susceptible species and lake trout (Salvelinus naymakush) the most resistant, and rainbow trout (Oncorhynchus mykiss ) of moderate susceptibility to IPNV. Fish age is also an important factor in IPNV disease outbreaks. There is a strong negative correlation between fish age and prevalence of IPNV (Dorson and Torchy, 1981; Bootland et al., 1990). IPNV is known to cause disease in 1-5 month-old fry (Wolf et al., 1960; MacAllister, 1983). Larger fish are often carriers of the virus (Dorson and Torchy, 1985; Ahne and Negele, 1985). Mortality caused by the PEM-PI-IPNV strain was 83% in

23 1-month-old brook trout, 75% in 2 month-old brook trout, in 4-month-old trout only 45% mortality (Frantsi and Savan, 1971). The mortality in 6 month-old trout was negligible. In another report, Lapierre et al., (1988) reported that IPNV caused mortality was highest in brook trout 6 to 11 weeks of age, and after 15 weeks of age, the fish did not seem to be sensitive to virus. MacAllister (1995) reported that the mortality caused by IPNV was highest (.70%) in day old brook trout fry with mortality peaking in 44 day-old fry. Temperature: Intense IPNV disease outbreaks occur at a temperature range optimum for efficient IPNV replication in the host cell. Dorson and Torchy (1981) and Hill (1982) reported that the temperature range of 8-12 C at fish hatcheries is an optimum range for IPNV replication as well as for fry growth. However, according to Frantsi and Savan (1971), the PEM-PI strain of IPNV caused 74% mortality of 2 month-old brook trout at 10 C and 46% at 15 C, while the VR- 299 strain caused only 31% mortality in 2 month-old brook trout at 15.5 C and caused no mortality at 10 C and 4 C. IPN disease mortality in rainbow trout fry was lower at 6 C and 10 C than at 14 C (Sano 1972). In a in vitro study reported by Dorson et al. (1978), the amount of virus released decreased with increasing temperature. It was highest at 14 C and 9 C and lowest at 20 C. These studies above also indicates that other factors beside temperature affect the disease outcome. Other factors include; age, immune status of host fish, nutrition, and characteristics of virus that affect the disease outcome. 10 In vitro Models: Cell susceptibility: IPN virus is routinely replicated in such cell lines as AS, BF-2, CHSE-214 (Nims et al., 1970; Wolf and Mann, 1980), EPC (Novao et al., 1993), and FHM and RTG-2

24 11 (Wolf and Quimby, 1962; Kelly, 1968; Novao et al., 1993). These cell lines have been compared in their susceptibility to IPNV and to virus yield. All IPNV isolates grow on CHSE-214 cell lines (Kelly, 1978). In terms of time required for destruction of cell monolayers, Kelly (1985) also demonstrated that assays using CHSE-214 cells were terminated after 3 days, whereas titrations in FHM cells enumerated after 5 days. The most susceptible cell lines to the turbot isolate were found to be CHSE-214, FHM and RTG-2 (Novao et al., 1993, Sherrer and Cohen, 1987). EPC and BB cell lines were less susceptible to this particular turbot IPNV isolate and the AS cell line was refractory. The titer of lysate from FHM at 15 C was 5x106 which was not as high as the value obtained from CHSE-214 (1.8x107) and RTG-2 (1.0x107). Further, the time required for monolayer destruction was longer in FHM cells than CHSE-214 cells (Novao et al., 1993; Lannan, 1984; Yamamoto, 1974). In a study comparing of RTG-2 to FHM, twelve out of twenty-four diagnostic samples (VR-299 and Buhl) inoculated onto RTG-2 cells yielded detectable IPNV, whereas only three out of twenty -four samples assayed in FHM were positive to IPNV after transfer (Kelly, 1978). In the same study it was concluded that the CHSE-214 cell line was more sensitive than FHM cell line and at least as sensitive as RTG-2 cell line. Rodriguez et al., (1993) reported that all IPNV isolates tested replicated (VR-299, Ab and Sp) on CHSE-214 and RTG-2 cell lines (n=194). 81.9% of them were positive on FHM cell line, and in EPC cells, 86.17% replicated. The IPN virus for this was isolated from carrier stocks of rainbow trout, however subtype of the virus was not determined. Cell Culture Adapted Virus (CCA): The cell line that is used for virus propagation is important for any studies with the virus. There are number of observations indicating that the virus loses its ability to destroy host cells after passage in different cell lines. It was reported that IPNV grown in RTG-2 cells adsorbs less efficiently to FHM cells than to RTG-2 cells, although there was no

25 12 difference in size, morphology and density of virions (Sheffer and Cohen, 1975). Adsorption of RTG-2-IPNV to RTG-2 cells was 29%, whereas the rate of adsorption to FHM cells was only 7%. It was stated that adsorption to FHM cells was significant, but virus did not replicate. Nicholson (1979) also reported that after passing VR-299 IPNV once in RTG-2 cell line, the plaque titer of RTG-2 IPNV in FHM cells was reduced by a factor of approximately 10-fold in comparison to the plaque titer in RTG-2 cell lines. In a further step, Nicholson et al., (1979) tried to isolate a FHM variant to compare wild type virus to a variant which passed only in FHM cells. Buhl, Idaho and Bonami, France isolates did not produce any plaques in FHM cells. Although undiluted virus was used, no FHM variant could be obtained. It was concluded that different isolates differ considerably in their ability to generate the FHM variant. Another important factor in virus virulence is the passage number of virus. Fewer than five passages of virus on cell monolayer did not alter the virulence of IPNV according to Hill and Dixon (1977). Kohlmeyer et al. (1986) studied Sp and Ab European strains in terms of their virulence, plaque formation and immunogenicity. Sp passed 11 times was characterized by small plaques and had low virulence (9%). The He and Ab isolates produced both small (0.52 mm) and large (>0.52 mm) plaques. In vivo tests indicated that Sp passed 5 times produced 50% mortality, whereas Ab caused 8%, and He was avirulent. Initial virulence of these isolates were not reported. Vestegard-Jorgensen (1971) reported that Sp caused 90% mortality in rainbow trout and 10% mortality was observed from Ab isolate. CCA (cell culture adapted) virus does not always mean that the virus has entirely lost its original structure. Wild type virus can be detected from cell culture adapted virus infected cells (Hill and Dixon, 1977) and from CCA virus infected trout fry (Dorson, 1978) (data were not provided).

26 13 Persistent Infection: Under certain circumstances, while virus infected cell cultures are multiplying normally and appear normal, they release significant amounts of virus. This phenomenon which occurs both in vivo and in vitro is called persistent infection (Joklik, 1985). Persistent infections are divided into two groups on the basis of the mechanism of persistance (Porter, 1985). One group is defined by the presence of the infectious virus which can be recovered in cell cultures. In the second group, the viral genome is present but infectious virus is not generally produced except during intermittent episodes of reactivation which are called latent infections. In order to be persistent, first, a virus under certain circumstances must not be cytolytic and must regulate its lytic potential (Fields and Knipe, 1990). Second, in vivo it must avoid detection and elimination by host immune system. There are many reports of viral persistence in cell monolayers. Parama virus in The Syrian hamster fibroblast cell line (BHK 21), (Staneck et al., 1972), vesicular stomatitis virus (VSV) in Aedes aegypti and A. albopictus cells (Artshop and Spence, 1974) and measles in Buffalo green monkey (BGM) cells (Menna et al., 1975 a, b) produced persistent infections. After a number of subcultures, the yield of infectious virus was decreased. It was suggested that passage of persistently infected cells might be the cause of decrease in the appearance of CPE (Rima 1976). There are at least three mechanisms that are involved with development of persistent infections in certain cell lines (Joklik, 1977). One is interferon production. Production of interferon controls cytopathic effects which accompany virus production (Sekellic & Marcus, 1978; Hedrick & Fryer, 1981). Second is selection of non-cytolytic mutant viruses (Preble and Younger, 1975) and the third one is integration of provirus into host cell by certain RNA viruses (Zhadnow, 1975; Simpson and linuma, 1975). In addition, defective interfering (DI) particles are another known factor on development of persistent infections (Hedrick and Fryer, 1981; Chu-Fang Lo et al., 1995)

27 14 Defective Interfering (DI) particles: Hedrick and Fryer (1981) mentioned that at least two mechanisms are involved with the absence of monolayer destruction in IPNV infected cells; interferon and DI virus production. They may function alone or together to spare PI (persistently infected) cell lines from cytocidal effects of infectious virus. Homologous viral interference is believed to affect the viral propagation of IPNV on cell monolayers ( Nicholson and Dunn, 1974; MacDonald and Yamamoto, 1977; Hedrick and Fryer, 1981, 1982). Lo et al., (1995) investigated the sequential changes of viral polypeptides of the virus preparation at high multiplicities of infection. The viral polypeptide VP2 was smaller in undiluted virus preparations than diluted preparation which caused CPE. The proportion of smaller VP2 and VP1 to normal molecules increased as the number of passage increased. Dorson et. al. (1978) reported an interesting observation on IPNV replication in RTG-2 cells. Cell culture adapted (CCA) virus, which was obtained by passing 13 times, prevented or reduced wild type virus replication on the cell monolayer. It was suggested that it might be because of having higher affinity receptors on the cell surfaces and or higher numbers of non-infecting interfering particles in CCA virus suspensions. Passage of IPNV in RTG-2 and CHSE-214 at high multiplicity of infection resulted in homologous viral interference (Nicholson and Dunn, 1974). The interference was described by reductions in CPE and infectious virus production. It was concluded that there is an evidence for production of defective interfering virus production (Hedrick and Fryer, 1982). There is, according to Nicholson and Dexter, (1975), evidence that the in vitro characteristics of DI particles are also true for in vivo conditions. Low dilutions of virus taken from carrier fish, did not cause any CPE which suggested that DI particles are responsible for absence of CPE (Nicholson and Dexter, 1975). Hedrick and Fryer, (1981) reported that they could not prove the presence of DI particles in CHSE-214 cells. However, STE-137 and RTG-2 cell lines which have similar

28 15 characteristics with CHSE-214 presented DI particles in autointerference. It was suggested that if DI mediates persistent infections in CHSE-214 cells, it must be unique mechanism for this cell line. Interferon: There are many reports on interferon or interferon-like substances. Some cell lines release interferon which exerts its activity intracellularly, some do not. It was found that the RTG-2 cell line releases interferon when infected with either IPNV or IHNV (DeSena and Rio, 1975; DeKinkelin and Dorson, 1973). An interferon-like substance has been found to be produced by FHM cells infected with IPNV (Gravel and Malsberger, 1965). MacDonald and Kennedy (1979) demonstrated that CHSE-214 were defective in interferon production in response to lytic infections with IPNV. It was also stated that persistent infection is mediated by DI (defective interfering) particles. This study also indicated that interferon may not be the only mechanism in prevention of destruction of cell monolayer. Both CHSE-214 and RTG-2 cell lines have approximately the same susceptibility, while one produce interferon, the other does not. Serum Inhibition and Virulence There are conflicting reports about the effects of normal trout serum (NTS) on in vitro virus propagation of IPNV. Vestegard-Jorgensen (1973) and Dorson and De Kinkelin (1974) reported that the serum taken from normal rainbow trout caused inactivation of cell culture adapted virus (CCA). Inhibition was due to an antibody-like non-virus-induced protein having 6S sedimentation coefficient. It was different from IgM which has 16S sedimentation coefficient. Sp strain IPNV was neutralized by serum taken from IPNV-free rainbow trout stock. Dorson (1975) also demonstrated that virulent Sp strain of IPNV when passaged in the absence of trout serum became avirulent. This

29 16 avirulent virus, which was originally resistant to inactivation by serum, became sensitive to inactivation by NTS. Hill and Dixon (1977) further analyzed the 6S factor. One virulent and three avirulent strains of IPNV were passed in RTG-2 cells in the presence (passed 10 times) or absence (passed 10 times) of NTS. The originally avirulent isolates passed in the absence of NTS remained avirulent, but passage of these avirulent strains in the presence of NTS increased virulence. However, specific data were not provided in this study. Hill et al., (1982) performed further work on 6S serum inhibition of IPNV. Three high passages of virus strains which were 6S sensitive and avirulent for fry, were passed in the presence of NTS. Two 6S sensitive viruses developed 6S resistance and virulence, the third avirulent strain remained avirulent, but developed 6S resistance indicating that 6S resistance does not always correlate with virulence. Cloning of four 6S resistant viruses in the absence of NTS resulted in 6S sensitive virus (No data were provided). Hill (1982) mentioned that normally virus in its 6S sensitive stage does not induce neutralizing antibody in rainbow trout, while 6S resistant virus does. However, Hill and Dixon, (1977) reported that a naturally avirulent strain of IPNV in its 6S (after serial passing in the presence of NTS) resistant form did not induce antibody production It was suggested that the virus may need to have virulence factor itself in order to induce immunity. CCA virus and wild type virus were compared for both plaque size and sensitivity to NTS (Dorson et al., 1978). It was found that CCA virus could be neutralized 50% by NTS at dilutions from 1/ 1,000 to 1/ 10,000 (Dorson, 1978). However, the wild type virus was not affected by 1/10 NTS. Cell culture adapted virus produced both small and large plaques sensitive to NTS indicating that plaque size is not related to the serum sensitivity. Kelly and Nielsen (1985) also demonstrated that adsorption of IPNV Sp subtype to FHM cells can be prevented by 1-2% rainbow trout serum. After incubation of FHM cells with virus in the presence of 1.3% NTS for 120 minutes, about 97% of virus

30 17 remained unadsorbed to the FHM cells, whereas in the absence of NTS 45% virus was adsorbed to FHM cells. Serum inhibition does not depend on the certain serotype or groups. Different subtypes of IPNV were tested for serum activity against IPNV on CHSE-214 and FHM cells (Kelly and Nielsen, 1985). Fish serum obtained from normal trout (Qu'Appelle) showed the greatest activity against Tellina virus (TV) (512). Anti-IPNV serum titers for VR-299 and Jasper were 128 and 256. Buhl isolate had the lowest titer (<8). Moreover, the 6S factor does not depend on geographic areas. Fish stocks from five different areas of the USA presented significant neutralizing activity against Sp and VR-299 isolates. Studies with radioisotope-labeled virus showed that inactivation of IPNV by serum is due to inhibiting of binding to host cell surface (Kelly and Nielsen, 1985) but it occurs prior to attachment. They also stated that this inhibition is not due to induction of interferon or binding to cellular receptors. Moreover, it does not depend on some cell variants, the viruses obtained by passing in different cell lines, like FHM-IPNV or RTG-2-IPNV. Overall serum titers in CHSE-214 and FHM cells were low but identical showing that serum activity was directed against FHM-IPNV or RTG-2 IPNV variants. There are some suggestions indicating that growth of an IPNV isolate in the presence of NTS is an important aspect of virus pathogenesis and must be considered in the infection trials (Dorson, 1978; Hill, 1981). On the other hand, MacAllister and Owens, (1986) passed IPNV VR times in CHSE-214 cells in the presence (5% NTS in MEM) and absence (5% FBS in MEM) of NTS. Replicate groups of 50 fish (brook trout) were exposed to viruses passed 1, 5, 10 and 15 times in the presence and absence of NTS. It was found that virulence of virus was not conserved by passing in the presence of NTS. Seventy percent mortality was observed after passing once in both treatments. Virulence was significantly decreased by passing 5 times in the present and absence of NTS. At passage number 5, mortalities from NTS treatment were 50% and with no NTS were 20%. At tenth passage, mortalities

31 18 respectively, were 20% and 5%. At 15th passage mortalities caused by two different treatments were 5% in NTS treatment and 2% in no NTS treatment indicating that the virulence, which was originally 70%, was not conserved by passing 15 times with or without NTS. However, the study also indicated that there was a significant difference in with and without NTS treatments at passage number 5 and even at passage number 10.

32 19 MATERIAL AND METHODS In vitro Experiment: IPNV isolates: IPNV isolates were available at Laboratory for Fish Disease Research at Hatfield Marine Science Center, Newport, OR. Isolates were stored at -80 C, or in the liquid nitrogen. Viruses were isolated from various species, geographic areas and belonged to several serotypes (Table 1). Their characteristics are listed in Table 1. Cell lines: The following cell lines were used in this study: - CHSE-214 (Chinook salmon embryo cells, Fryer, 1965.) EPC (Epithelioma papullosum cyprini, Fijan et al., 1979). - FHM (Fathead minnow, Gravel & Malsberger, et al., 1965). -RTG-2 (Rainbow trout gonad, Wolf and Quimby, 1962) Cells were grown in Eagle's Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (HyClone Laboratories, inc.). The procedures for the passage of these cell lines were carried out as described in Caswell-Reno (1989) and Lannan (1994). Medium: Cultures were grown in MEM supplemented with 10% fetal bovine serum (FBS). FBS was stored at -20 C, thawed at room temperature and kept at 4 C prior to use. PH of the medium which was buffered with HEPES (2.4%) was and was also kept at 4 C incubator. No antibiotics were used at the any stage of subculturing.

33 Table 1. Aquatic Birnavirus isolates used in the experiments. The data includes the name of isolate, serotype and subtype (by monoclonal antibody analysis), the fish species from which virus was originally isolated, country /state from which the virus was isolated and whether it was isolated from diseased or non-diseased fish. 20 Isolate Subtype Serotype Host species Coun./state d/nd(*) Unknown Unknown Unknown Idaho Unk. Domsea Unknown Al Oncorhynchus mykiss Idaho nd VR-299 VR-299 Al 0. mykiss W.Virginia d Crayfish Buhl Al Astarus astarus Idaho nd CSF Buhl Al 0. mykiss Idaho d H-VAT Buhl Al Salvelinus malma Idaho Unk. OLD-CSF Buhl Al 0. mykiss Idaho Unk Buhl Al 0. mykiss Idaho d Buhl AI 0. mykiss Idaho d Buhl Al 0. mykiss Idaho Unk Buhl Al 0. mykiss Idaho nd Buhl Al 0. mykiss Idaho nd Buhl Al 0. mykiss Idaho nd Buhl Al 0. mykiss Idaho nd Buhl Al 0. clarki Idaho Unk Buhl Al 0. mykiss Idaho nd Buhl Al 0. mykiss Idaho nd Buhl Al 0. mykiss Idaho nd 93-5 Buhl Al 0. nerka Idaho Unk Buhl Al 0. mykiss Idaho nd 93-7 Buhl Al 0. nerka Idaho nd Buhl Al 0. mykiss Idaho nd Buhl Al 0. mykiss Idaho d Buhl Al 0. clarki Idaho nd 93-3 Buhl Al 0. nerka Idaho nd Buhl Buhl Al 0. mykiss Idaho d CAL.Mojave Buhl Al 0. mykiss Ca d Cal.stoddard Buhl Al 0. mykiss Ca d nd:non-diseased; d:diseased; Unk.: Unknown

34 21 Table 1 (continued) Isolate Subtype Serotype Host species Coun./state d/nd CSF Buhl Al 0. mykiss Idaho d Buhl Al 0. mykiss Idaho nd ID-PASS-0 Buhl Al 0. mykiss Idaho Unk. Sawtooth Buhl Al 0. clarki Idaho Unk. 86-Q VR-299 Al Unknown Unknown Unk. Berlin VR-299 Al Salvelinus fontinalis N.Hampshire d Obanion VR-299 Al 0. mykiss Nevada Unk. CL-214 VR-299 Al 0. kisutch Oregon nd Coho VR-299 Al 0. kisutch Oregon nd lava lake VR-299 Al S. fontinalis Oregon nd LH(PA) VR-299 Al S. fontinalis Pa d Menhaden VR-299 Al Brevoortia tyrranus Maryland d Oswayo VR-299 Al S. fontinalis Pennsylvania d Pelton D. VR-299 Al 0. tsawytscha Oregon nd Reno VR-299 Al 0. clarki Nevada d SBV VR-299 Al Morone saxitilis Maryland d R.River VR-299 Al 0. mykiss Oregon nd WB Al S. fontinalis Idaho nd WB Al 0. clarki Idaho nd WB Al 0. mykiss Idaho nd WB Al 0. clarki Idaho d WB Al 0. mykiss Idaho nd CTI' WB Al 0. clarkii Oregon nd Tai WB Al 0. mykiss Taiwan d WB WB Al S. fontinalis Maine nd WB Al 0. mykiss Idaho nd WB Al S. fontinalis Idaho nd FR-21 Sp A2 0. mykiss France d OV-7 Sp A2 Ostrea edulis Uk nd