Detection of truncated virus particles in a persistent RNA virus ...

JOURNAL OF VIROLOGY, Apr. 1995, p. 2140–2147 0022-538X/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 69, No. 4

Detection of Truncated Virus Particles in a Persistent RNA Virus Infection In Vivo† BARBARA S. DROLET,1 PINWEN P. CHIOU,1 JERRY HEIDEL,2

AND

JO-ANN C. LEONG1*

Department of Microbiology, Center for Salmon Disease Research,1 and School of Veterinary Medicine,2 Oregon State University, Corvallis, Oregon 97331 Received 6 October 1994/Accepted 9 December 1994

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus which causes devastating epizootics of trout and salmon fry in hatcheries around the world. In laboratory and field studies, epizootic survivors are negative for infectious virus by plaque assay at about 50 days postexposure. Survivors are considered virus free with no sequelae and, thus, are subsequently released into the wild. When adults return to spawn, infectious virus can again be isolated. Two hypotheses have been proposed to account for the source of virus in these adults. One hypothesis contends that virus in the epizootic survivors is cleared and that the adults are reinfected with IHNV from a secondary source during their migration upstream. The second hypothesis contends that IHNV persists in a subclinical or latent form and the virus is reactivated during the stress of spawning. Numerous studies have been carried out to test these hypotheses and, after 20 years, questions still remain regarding the maintenance of IHNV in salmonid fish populations. In the study reported here, IHNV-specific lesions in the hematopoietic tissues of rainbow trout survivors, reared in specific-pathogen-free water, were detected 1 year after the epizootic. The fish did not produce infectious virus. The presence of viral protein detected by immunohistochemistry, in viral RNA by PCR amplification, and in IHNV-truncated particles by immunogold electron microscopy confirmed the presence of IHNV in the survivors and provided the first evidence for subclinical persistence of virus in the tissues of IHNV survivors. that virus persists at a level below this detection limit until sexual maturity. This explanation is compelling because it would help explain the resurgence of virus in adult populations in which reinfection seems unlikely as a sole source of virus. Defective interfering virus (DI) particles have been shown to contribute to the persistence of the rhabdoviruses vesicular stomatitis virus (VSV) and rabies virus (RV) (22, 24, 28, 41). Huang and Baltimore (26) were the first to propose that the action of DI particles was to modulate the yield of infectious virus in a cyclical manner. Over the years, several attempts have been made to understand how DI particles moderate virus infections in vivo (6, 21 27, 29, 40). VSV DI particles have been shown to play an important role in the relative virulence of different serotypes and in the establishment of persistent infections in cell culture. DI particles of many viruses have been shown to alter the pathogenesis and course of disease in animals given DI particles before or during an infection (5, 8, 36). Yet, defective virus has not been identified in natural infections in animals and often cannot be reisolated from animals inoculated with these particles to examine their effect on virus replication. There are two studies which have come close to detecting DI particles in natural infections. Pedley et al. (39) isolated rotaviruses with altered genomes from chronically infected immunodeficient children, but the particles were not confirmed as DI particles because of difficulties in culturing the rotavirus. Bean et al. (7) found ‘‘DI-type’’ RNAs in an avirulent strain of type A influenza virus after they had been passaged twice in embryonated eggs. Although the finding of DI RNAs explained the virulence difference, the possibility that the DItype RNA was made in the eggs was never ruled out. IHNV DI particles have been described in persistently infected fish (13, 34) and insect (46) cell cultures. These particles were postulated to cause the establishment of persistent cell culture infections and to inhibit subsequent viral infection in susceptible fish. It is possible that IHN disease is modulated by

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus which causes devastating epizootics in trout and salmon hatcheries of the Pacific Northwest, Europe, and Asia. IHNV produces an acute infection in juvenile fish that can result in 100% mortality in affected populations. The main target of infection is the hematopoietic organ located in the kidney of the fish. During an epizootic, infectious virus can be detected in tissue homogenates of the infected fish by plaque assay until approximately 50 days postexposure (DPE), after which there is no demonstrable infectious virus. These animals remain apparently virus free until sexual maturity when virus can again be detected by plaque assay. It is not known whether infectious virus isolated from spawning adults is the result of reinfection from secondary reservoirs during their migration upstream or whether reactivation of a latent form of IHNV occurs in the survivor. With the exception of one study (1) the inability to detect reactivated latent virus in naturally and experimentally infected fish populations in carefully monitored studies has been the main support for the theory of reinfection. This theory, however, cannot explain several key observations. (i) Large amounts of virus are required to infect large fish (10), (ii) no secondary virus reservoirs have been found, and (iii) adult infection studies have shown that IHNV survivors are less susceptible than age-matched naive controls to reinfection by IHNV upon sexual maturation (10). The argument against persistence of IHNV has been based primarily on the finding that infectious virus cannot be detected in IHN survivors held in captivity. The inability to detect infectious virus may be the result of the methods used to detect infectious virus. The typical tissue homogenate plaque assay has a limit of detection of 200 PFU/g of tissue, and it is possible * Corresponding author. Phone: (503) 737-1859. Fax: (503) 7370496. † Oregon Agricultural Experiment Station Technical Paper no. 10,602. 2140

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DI particles in natural infections and may explain the apparent disappearance and reappearance of the virus during the life cycle of its host. Although IHN epizootic survivors have not been closely monitored in the wild, they have been assumed to be virus free and to suffer no long-term pathogenic effects that might alter their survivability in the wild. In this report we describe the pathologic changes found in the kidneys of rainbow trout 1 year after exposure to IHNV and the evidence for persistence of virus in these survivors by immunohistochemistry, PCR analysis, and immunogold electron microscopy. (This article reports a portion of the work encompassed by a thesis submitted to Oregon State University, Department of Microbiology, in partial fulfillment of the requirements for a Ph.D. degree.) MATERIALS AND METHODS Fish infection and sampling. Two thousand rainbow trout (Oncorhynchus mykiss) fry at 1 g of body weight were infected by static immersion with 3.7 3 103 PFU of IHNV per ml of water. After 30 days, 20% of the infected animals had died as a result of the virus infection. The survivors were reared in specific-pathogen-free water (10 to 128C) at the Center for Salmon Disease Research, Oregon State University, Corvallis, Ore. The fish were kept as a single group in tanks appropriate for their numbers and weight. Groups of 30 epizootic survivors were lethally sampled at 90 and 180 DPE and at 1 and 2 years postexposure (YPE). At each sampling time, pieces of kidney, spleen, and liver tissues were pooled for each fish and tested for infectious virus by plaque assay as recommended in the Fish Health Blue Book (2) on EPC cells (14). In addition, pieces of 11 different organs from each fish were fixed in 10% neutral buffered formalin (10% formalin, 33 mM sodium phosphate monohydrate, and 46 mM sodium phosphate dibasic), dehydrated in an alcohol-xylene series, and embedded in Paraplast paraffin (608C) (Oxford Labware) for immunohistochemistry. Mock-infected fish, which were exposed to noninfected cell culture medium, were used as negative controls. The IHNV stock was grown under conditions for low multiplicities of infection (.0.001 PFU per cell). The virus titer by 50% tissue culture infectious dose assay indicated that no biologically detectable concentration of DI particles was present in the stock. A newly initiated laboratory epizootic provided fresh positive and negative tissues for determining electron microscopic (EM) immunogold staining parameters. The same virus and infection procedures were used as described above. Fresh kidney tissues were cut into 1- to 2-mm3 blocks, fixed in paraformaldehyde-gluteraldehyde (2% paraformaldehyde–1% glutaraldehyde in 0.1 M sodium cacodylate buffer) (49), dehydrated with an ethanol series, and embedded in LR White resin (Ted Pella, Inc.). Kidney tissues from 1-YPE survivors, which had been fixed in formalin for 2 years but never embedded in paraffin, were treated as fresh tissues. Kidney tissues from 1-YPE survivors which had been previously embedded in paraffin were cut into blocks, deparaffinized, and rehydrated in a xylene-ethanol series and treated as fresh tissues. Ultrathin sections of these tissues were collected on nickel or gold grids (Ted Pella, Inc.). The average size of the different virus particles was determined by on-screen measurement in the Phillips CM12 electron microscope and by measurement of the particles on the photomicrograph negatives. More than 20 particles in two different sections from each fish were measured. Histology. Several histological stains were used to examine

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the pathologic changes in 7-mm paraffin sections of kidney tissues from 1-YPE survivors. A hematoxylin and eosin stain (17, 31) was used to examine overall cell morphology. The Masson’s trichrome stain (33) was used to specifically examine fibrotic scar tissue. A periodic acid-Schiff (PAS) stain (35) was used to detect carbohydrate deposits. A PAS stain with a diastase predigestion and a PAS stain with phenylhydrazine HCl (30) were used to differentiate the carbohydrates as glycogen or glycoprotein, respectively. A mucicarmine stain (32) was used to detect epithelial mucins. A Perl’s iron stain (20) was used to detect released hemoglobin (hemosiderosis) which can result from erythrocyte destruction. Dunn-Thompson hemoglobin stain (12) was used to detect formalin-hemoglobin deposits which can occur during fixation of blood-rich tissues. Immunohistochemistry. Immunohistochemistry was done as described by Drolet et al. (11). Briefly, 7-mm paraffin-embedded sections were deparaffinized, hydrated, blocked with 5% nonfat milk, and incubated with a monoclonal antibody (MAb 1NDW14D) made to a conserved region of the highly conserved nucleocapsid (N) protein of IHNV (42). Sections were then incubated with a biotinylated goat anti-mouse antibody, phosphatase-conjugated avidin-biotin complexes, and a red phosphate substrate (Vector). Sections were then counterstained with hematoxylin for 1 min and with 0.2% ammonium hydroxide for 1 min. Mock-infected fish tissues were used as negative controls, and fish at 8 to 10 DPE were used as positive controls. Eleven different organ tissues from 15 survivor fish at 90 DPE and 10 survivor fish at 180 DPE were examined by immunohistochemistry. At 1 YPE, the kidney tissues of six survivors were also examined. Immunogold staining. All incubations were done in 0.5-ml microcentrifuge tubes with volumes of 25 ml, and all rinses were done in disposable glass dilution tubes. All reagents were filtered through a 0.2-mm Acrodisc membrane (Gelman Sciences). Grids were blocked with 10% normal goat serum in Tris buffer (20 mM Tris, 20 mM NaN3, 225 mM NaCl, 0.1% bovine serum albumin [pH 8.2]) for 10 min and were blotted and incubated in 1NDW14D MAb in Tris buffer overnight. Grids were rinsed in five changes of Tris buffer and then incubated with 5 nm of gold-conjugated goat anti-mouse immunoglobulin (heavy and light chains) (Ted Pella, Inc.) in Tris buffer for 1 h (16). Grids were rinsed in two changes of 103 salt-Tris buffer (20 mM Tris, 20 mM NaN3, 2.25 mM NaCl, 0.1% bovine serum albumin [pH 8.2]), five changes of Tris buffer, and five changes of nanopure water. Grids were air dried and stained on droplets of 1% uranyl acetate–0.2% lead citrate for 5 min each. Grids were examined by transmission EM (Phillips CM12). PCR sample preparation. Eight 7-mm paraffin sections were placed, four each, in 1.5-ml microcentrifuge tubes. Sections from mock-infected fish were used as negative controls, and sections from mock-infected fish with purified IHNV genomic RNA added were used as positive controls. Sections were deparaffinized with two changes of xylene and two changes of 100% ethidium hydroxide, centrifuged for 5 min between each step, and air dried (50). RNA was extracted by RNAzol (TelTest, Inc.) treatment, followed by chloroform extraction and isopropanol precipitation. RNA was washed twice in 75% ethanol and air dried. The RNAs in the two tubes were combined by dissolving the pellets in the same 10-ml volume of TrisEDTA (TE; 50 mM Tris, 1 mM EDTA, and 0.5% Tween 20 [pH 7.5]). PCR and Southern hybridization. Retrotherm PCR was done as described by Chiou et al. (9) with sense (nucleotides 319 to 338) and antisense (nucleotides 570 to 552) N gene primers to obtain a 252-bp N gene product (4). PCRs were

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FIG. 1. Kidney tissue, from a 1-YPE IHN epizootic survivor, stained with Masson’s trichrome stain. Fibrotic (f) connective tissue stained blue (arrowheads). Extensive fibrosis surrounds the renal tubule (t) and occludes the hematopoietic (h) tissue. Magnification, 3400.

incubated in an automatic thermal cycler (Coy Laboratory Products), electrophoresed on 1.5% GTG agarose gel (FMC Bioproducts), and blotted to Nytran Plus (Schleicher & Schuell, Inc.) by standard methods (43). PCR products were probed with a digoxigenin end-labeled (45) internal N gene primer (nucleotides 456 to 427) and detected by chemiluminescence with an alkaline phosphatase-linked anti-digoxigenin antibody (23). RESULTS Detection of infectious virus. Rainbow trout (O. mykiss) fry which had been infected with IHNV were positive throughout the epizootic for infectious virus by plaque assay until 46 DPE, with titers ranging from 103 to 106 PFU/g tissue. The cumulative mortality for the epizootic was 29%. After this initial period of virus production, there was no evidence of infectious virus in the 90- and 180-DPE and 1- and 2-YPE samples as tested by plaque assay. Histopathologic findings in survivors of IHNV infection. Kidneys of 1-YPE survivors stained with hematoxylin and eosin showed moderate to severe periglomerular and peritubular fibrosis as well as fibrotic foci within the hematopoietic tissue (Fig. 1). The kidneys of the survivors contained less than 50% of the hematopoietic cells found in the kidneys of agematched, mock-infected control fish. Glomeruli showed thickening and splitting of Bowman’s capsules and adherence of the glomerular tufts to the capsule. Renal tubules often contained large numbers of epithelial blebs, indicative of ongoing tissue injury. The PAS carbohydrate stain showed polysaccharidepositive deposits in the kidney tubules and in foci of the he-

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FIG. 2. Kidney tissue, from a 1-YPE IHN epizootic survivor, stained by alkaline phosphatase immunohistochemistry. Positive red IHNV deposits are shown in the renal tubules (t) and the hematopoietic tissue (h) (arrows). Also shown are individual IHNV-positive hematopoietic cells (c) and viral antigen deposition (arrowheads) surrounding the calcium inclusions of nephrocalcinosis (n). Magnification, 3400.

matopoietic tissue. When the tissues were predigested with diastase, which destroys the carbohydrate-staining properties of glycogen, the PAS-positive staining was unchanged in the tissues. This result indicated that the deposits were not glycogen. However, when the tissues were treated with phenylhydrazine HCl, which destroys the carbohydrate staining properties of glycoproteins, the PAS-positive staining was cleared. Iron deposition was seen with the Perl’s hemosiderin stain, and no formalin or hemoglobin artifacts were detected by the Dunn-Thompson stain. Nephrocalcinosis was prominent throughout the kidneys of the survivors. Kidneys from age-matched, mock-infected control fish showed no fibrosis or glomerular lesions. Hematopoietic tissue was dense, and few, if any, epithelial blebs were seen in the tubules. No carbohydrate or iron deposits were seen with the PAS and Perl’s hemosiderin stains. There was little or no nephrocalcinosis in the negative control fish. Immunohistochemistry of survivors of IHNV infection. Dense, positive deposits were seen within the renal tubules and hematopoietic tissue of survivor kidneys at 90 and 180 DPE and at 1 and 2 YPE when tested with the N-specific MAb (N-MAb) 1NDW14D for the viral nucleocapsid (Fig. 2). Positive results were also seen with a second N-MAb, 1NCO27G, and with two G-specific MAbs (G-MAbs 3GH135L and 3GH136J). Both N- and G-MAbs detected viral antigen at two different sites: (i) in deposits located within the renal tubules and the hematopoietic tissue and (ii) on the surfaces of individual hematopoietic cells. No evidence of virus was seen in any of the other tissues, with the exception of one positive liver

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FIG. 3. Kidney tissue from a 1-YPE IHN epizootic survivor. The tissue was negatively stained with uranyl acetate and lead citrate. Intracytoplasmic rhabdoviral inclusions in the renal epithelium are shown. The arrow indicates the 45-nm-diameter electron-lucent circular particles. Bar, 100 nm.

sample at 90 DPE. No positive staining was seen in mockinfected, age-matched negative control fish when tested with the four MAbs. Fifteen survivor fish at 90 DPE, 10 at 180 DPE, and 6 at 1 YPE were included in the study. The tissues from the six survivor fish at 1 YPE were found to contain viral protein by immunohistochemistry and were used for the following EM, immunogold, and PCR analyses. EM examination of 1-YPE kidney tissues. Examination of the kidney tissues from surviving fish was conducted by EM of negatively stained sections. Examination of the kidney tissues of 1-YPE survivors, previously embedded in paraffin and then processed for EM, showed intracytoplasmic rhabdoviral inclusions in renal epithelium (Fig. 3). These vesicles are similar to the RV cytoplasmic inclusions described by Ghadially (15). Inside the inclusion vesicles, uniform circular particles measuring 45 nm in diameter with electron-lucent cores were observed. There were no similar inclusion vesicles in the kidney tissues of negative control fish. Survivor kidney tissues that had been held in formalin for more than 2 years and then processed for EM contained viruslike particles in the tubules and hematopoietic tissue. Of the six fish sampled at 1 YPE and shown to contain IHNV protein by immunohistochemistry, four were examined by EM and all contained truncated viral particles. The average size of the complete IHNV virion in the positive control tissues was measured at 50 by 130 nm. The particles observed in the kidney tissues of survivors were 50 by 40 to 65 nm and appeared to be truncated rhabdovirus particles similar to those reported for RV and VSV (18, 28). They were distinguished from commonly known structures such as microvilli and cilia by size and morphology. Immunogold staining of virus particles. Despite EM evidence for rhabdoviruslike particles in the tubules and hematopoietic tissues of surviving fish, verification that the particles were IHNV virions required specific staining with an anti-

IHNV MAb. The parameters for specific labeling of virions with immunogold-conjugated MAb were established by using kidney tissues of fish sampled at 8 DPE, during the acute phase of the epizootic (Fig. 4). Positive staining was indicated by the binding of gold beads (5 nm) which appear as electron-dense dots. When the same staining procedures were used on the kidney tissues of fish that had survived an IHN epizootic at 1 YPE, the truncated particles were specifically labeled with the anti-IHNV N-MAb and the goat anti-mouse gold-conjugated antibody (Fig. 5). PCR amplification of viral N gene from fixed kidney tissues. Detection of specific IHNV RNA provided additional evidence that virus persists in survivors of an IHN epizootic. Nucleocapsid gene sequences were amplified by PCR of RNA extracted from 1-YPE survivor kidney tissues that had been fixed in formalin and embedded in paraffin. The expected 252-bp fragment amplified by primers to the nucleocapsid gene (Fig. 6a) was confirmed as an IHNV sequence by Southern blot hybridization. The digoxigenin-labeled probe used in the hybridization was to an internal region of the 252-bp sequence (Fig. 6b). DISCUSSION For more than 20 years, conflicting evidence has been reported to explain how IHNV is maintained in salmon and trout populations in the Pacific Northwest (1, 37). The available evidence for a virus-carrier state in fish came from recovery of infectious virus from rainbow trout surviving an IHN epizootic and held in the laboratory until maturity (1). Other reports have been made that no infectious virus can be recovered from survivors held in captivity (3). The uncertainty has led managers of the fisheries resources in the United States to release these fish in mitigation programs. Extensive searches for secondary carriers of the virus have also been made, but no con-

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FIG. 4. Immunogold labeling of IHNV particles in kidney tissues. (a) Immunogold labeling of IHNV particles with the anti-N-MAb 1NDW14D was followed by uranyl acetate-negative staining to define the virus particles. Arrows indicate the specific labeling (electron-dense dots) of the nucleocapsid core trailing from an IHNV particle and within a particle. (b) Immunogold labeling of IHNV particles with anti-G-MAb 3GH136J. Arrows indicate the specific labeling of the glycoprotein surface of the virion. These virus particles were not negatively staining. Bar, 50 nm.

vincing evidence for an alternative host for the virus has been found. The results of an extensive study on survivors of an IHN epizootic are reported here and show that IHNV persists and produces lesions in the kidneys of these survivors. Although 1-YPE survivors were negative for infectious virus by plaque assay, histological staining with hematoxylin and eosin showed prominent lesions in their kidneys. Glycoprotein was detected in deposits within the renal tubules and hematopoietic tissue. Epithelial blebs and hemosiderin deposits were prominent throughout the kidney, indicating ongoing renal

tissue injury and destruction of erythrocytes. These results suggest that renal and possibly hematopoietic function in IHN survivors is compromised. Intensely staining IHNV-positive deposits were seen in renal tubules and hematopoietic tissues of fish sampled at 90 and 180 DPE and at 1 and 2 YPE. Antigen was seen in fibrotic regions and renal tubules and in close association with hematopoietic cells. No infectious virus, IHNV or any other cytolytic fish virus, was ever recovered from these survivors after 46 DPE even though 11 different organs were sampled from 30 fish

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FIG. 6. Detection of IHNV nucleic acid by PCR amplification of RNA from the kidney tissues of 1-YPE IHN epizootic survivors. (a) Retrotherm PCR amplification of a 252-bp fragment of the IHNV N gene electrophoresed on 1.5% agarose gel and stained with ethidium bromide. Lanes: 1, 123-bp ladder marker; 2, PCR amplification product of IHNV genomic RNA; 3, PCR amplification product of RNA extracted from a 1-YPE epizootic survivor. The bright band at the bottom of each lane is excess primers and nucleotides. (b) Southern blot hybridization showing that the 252-bp fragment is of IHNV N gene origin. Lanes: 1, 123-bp ladder marker; 2, PCR amplification product of a 1024 dilution of IHNV genomic RNA; 3, PCR amplification product of a 1023 dilution of IHNV genomic RNA; 4, linearized plasmid containing the IHNV N gene as a positive control; 5, PCR amplification product of the N plasmid; 6, PCR amplification product of RNA extracted from a kidney sample from an age-matched, mock-infected, negative control fish; 7, PCR amplification product of RNA extracted from a 1-YPE survivor kidney sample. The 500-bp band in lanes 2 and 3 is the result of self-priming (4).

FIG. 5. Immunogold-labeled IHNV DI particles in kidney tissues of a 1-YPE IHN epizootic survivor. Immunogold labeling of IHNV particles with the antiN-MAb 1NDW14D was followed by uranyl acetate-negative staining to define the viruslike particles (arrows). Bar, 50 nm.

every 3 to 6 months, throughout their captivity. Renibacterium salmoninarum, the causative agent of bacterial kidney disease, can cause renal lesions (44) similar to those seen in the survivor kidneys. However, bacterial contaminants were not detected in survivor populations. Lesions observed were therefore assumed to be caused by IHNV infection. All survivor kidney samples examined had moderate to severe deposits of calcium complexes in the renal tubules. This condition, nephrocalcinosis, has been associated with excessive carbon dioxide levels in the water (19, 48). Age-matched, mock-infected negative control fish had mild or no nephrocalcinosis. Routine sampling of rainbow trout populations at the Center for Salmon Disease Research fish rearing facility (Corvallis, Ore.) has shown a 30 to 60% incidence of nephrocalcinosis in IHN survivors versus a 0 to 10% incidence in age-matched, mock-infected survivors (10). The increased incidence of the condition in IHN survivors suggests that the general impairment of kidney function from IHNV infection predisposes fish to nephrocalcinosis. This, in turn, may result in the death of survivors before sexual maturation. Cytoplasmic rhabdoviral inclusions, like those reported in RV infections (15), were detected in a kidney sample which had been embedded in paraffin before processing for EM. We

were unable to confirm the inclusions as IHNV in origin, because the tissues were unsuitable for immunogold detection of viral antigen. Particles resembling rhabdovirus DI particles were observed in 1-YPE survivor kidney tissues that had been fixed in formalin. Rhabdovirus DI particles are typically spherical and one-third the size of standard virus particles (38). The particles observed in the EM of tissues from survivor fish at 1 YPE were round (50 nm diameter) to ovoid (50 by 40 to 65 nm) in shape, approximately one-third the size determined for standard IHNV particles (50 by 130 nm) in these studies. These truncated particles were confirmed as IHNV particles by immunogold labeling (Fig. 5). The finding of truncated IHNV particles resembling rhabdovirus DI particles suggests a model of how IHNV is maintained in salmon and trout populations. The model is on the basis of previous studies with VSV and RV grown in continuous culture in vitro. The production of particles is maintained so that the standard virus provides the necessary replication functions for DI particles which, in turn, interfere with the replication of standard virus and prevent them from completely destroying the cell culture (47). In animal infections, the result would be a subacute or latent form of the virus infection. This model in IHNV would account for the restriction of production of large quantities of infectious virus by the presence of DI particles. Then, when the balance is upset by the stressful conditions of spawning or the exposure of the fish to IHNV or other pathogens, infectious virus titers would increase to levels greater than 200 PFU/g of tissue, the detection limit for the plaque assay. Most, if not all, viruses have been shown to produce DI particles in tissue culture cells. In addition, animals given DI

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particles before or during homolgous viral infections showed an altered infection course (25). Although both animal and human studies suggest a role for DI particles in viral pathogenesis, there is no definite report of DI particle isolation after a natural infection. In the study reported here, fish were infected by adding 103 standard virus particles per ml directly to the water, similar to the dose and route of exposure seen in natural infections. The observed daily and cumulative mortality was typical for an IHN epizootic, and clinical signs were similar to those seen in natural infections. Truncated viral particles resembling rhabdovirus DI particles were seen in epizootic survivor fish a year after infectious virus was no longer detectable. Although the truncated particles were not purified from the tissues and analyzed for interfering activity, the resemblance to the DI particles of VSV and RV is striking. Thus, under conditions which simulate natural infections in the wild, IHNV persists in these animals and this carrier state may be modulated by DI particles. ACKNOWLEDGMENTS Sandra Ristow (Washington State University, Pullman, Wash.) provided the MAbs used in this study. Linda Bootland and Harriet Lorz assisted in fish care, maintenance, and sampling. Pat Allison provided technical assistance with histological staining. Julie Duimstra provided technical assistance with immunogold labeling. Al Soeldner provided technical assistance in EM and photography. This study was supported by the School of Veterinary Medicine, Oregon State University; the Bonneville Power Administration, grant DE-FG79-88BP92431, project 88-152; the United States Department of Agriculture to the Western Regional Aquaculture Consortium under grant 92-38500-7195, project 92080441; and an Oregon Sea Grant with funds from the National Oceanic and Atmospheric Administration, Office of Sea Grant, Department of Commerce, under grant NA89AA-D-SG108, project R/FSD-16. Oregon Agricultural Experiment Station Technical Paper No. 10,602. REFERENCES 1. Amend, D. F. 1975. Detection and transmission of infectious hematopoietic necrosis virus in rainbow trout. J. Wildl. Dis. 11:471–478. 2. Amos, K. H. (ed.). 1985. Procedures for the detection and identification of certain fish pathogens, p. 114, 3rd ed. Fish Health Section, American Fisheries Society, Corvallis, Ore. 3. Amos, K. H., K. A. Hopper, and L. LeVander. 1989. Absence of infectious hematopoietic necrosis virus in adult sockeye salmon. J. Aquat. Anim. Health. 1:281–283. 4. Arakawa, C. K., R. E. Deering, K. H. Higman, K. H. Oshima, P. J. O’Hara, and J. R. Winton. 1990. Polymerase chain reaction (PCR) amplification of a nucleoprotein gene sequence of infectious hematopoietic necrosis virus. Dis. Aquat. Org. 8:165–170. 5. Bangham, C. R. M., and T. B. L. Kirkwood. 1990. Defective interfering particles: effects in modulating virus growth and persistence. Virology 179: 821–826. 6. Barrett, A. D. T., and N. J. Dimmock. 1986. Defective interfering viruses and infections of animals. Curr. Top. Microbiol. Immunol. 128:55–84. 7. Bean, W. J., Y. Kawaoka, J. M. Wood, J. E. Pearson, and R. G. Webster. 1985. Characterization of virulent and avirulent A/chicken/Pennsylvania/83 influenza A viruses: potential role of defective interfering RNAs in nature. J. Virol. 41:151–160. 8. Browning, M. J., B. S. Huneycutt, A. S. Huang, and C. S. Reiss. 1991. Replication-defective viruses modulate immune responses. J. Immunol. 147: 2685–2691. 9. Chiou, P. P., B. S. Drolet, and J. C. Leong. 1995. Polymerase chain amplification of infectious hematopoietic necrosis virus RNA extracted from fixed and embedded fish tissue. J. Aquat. Anim. Health 7:9–15. 10. Drolet, B. S., L. Bootland, H. V. Lorz, J. S. Rohovec, and J. C. Leong. Unpublished data. 11. Drolet, B. S., J. S. Rohovec, and J. C. Leong. 1994. The route of entry and progression of infectious hematopoietic necrosis virus in Oncorhynchus mykiss (Walbaum): a sequential immunohistochemical study. J. Fish Dis. 17:337–347. 12. Dunn, R. C., and E. C. Thompson. 1945. A new hemogloblin stain for histologic use. Arch. Pathol. 39:49–50. 13. Engelking, H. M., and J. C. Leong. 1981. IHNV persistently infects chinook salmon embryo cells. Virology 109:47–58.

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