Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PEI, Canada C1A 4P3
Correspondence
Frederick Kibenge
kibenge{at}upei.ca
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ABSTRACT |
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Present address: Pacific Biological Station, Department of Fisheries and Oceans, Nanaimo, BC, Canada V9T 6N7.
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INTRODUCTION |
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Morphological, biochemical and replication properties of ISAV place the virus in the family Orthomyxoviridae (Falk et al., 1997; Kim & Leong, 1999
). Similar to influenza viruses, the ssRNA genome of ISAV comprises eight segments of negative polarity. Comparison of ISAV proteins with those of other orthomyxoviruses revealed low amino acid identity of between <13 and <25 % (Krossøy et al., 2001
; Kibenge et al., 2001b
; Ritchie et al., 2001
), supporting the proposal to assign ISAV to a new, fifth genus within the Orthomyxoviridae, genus Isavirus (Anon., 2001
).
The mechanisms of virus persistence and pathogenesis in ISAV infection at the cellular level are not well studied. The phenomenon of persistent ISAV infection in fish is unusual for orthomyxoviruses, as there is no evidence of persistence of influenza virus genetic material in any animal species. Influenza viruses are cleared from ducks in approximately 7 days (Webster, 1999), although they can persist in carrier cultures (Urabe et al., 1993
). In Atlantic salmon, ISAV seems to target leukocytic (Falk et al., 1995
), endothelial (Falk & Dannevig, 1995
) and endothelial-associated (Falk et al., 2001
) cells, while in vitro, it readily replicates with production of cytopathic effects (CPE) in salmon head kidney (SHK-1) (Dannevig et al., 1995
), TO (Wergeland & Jakobsen, 2001
) and Atlantic salmon kidney (ASK-2) (Rolland et al., 2002
) cells, which are macrophage-like cell lines (Dannevig et al., 1997
; Wergeland & Jakobsen, 2001
; Rolland et al., 2002
). Some strains of ISAV can also replicate and cause CPE in the Chinook salmon embryo (CHSE-214) cell line (Bouchard et al., 1999
; Kibenge et al., 2000
; Griffiths et al., 2001
). Virus replication may also occur in Atlantic salmon (AS) (Sanchez et al., 1993
; Sommer & Mennen, 1997
) and rainbow trout gills (RTgill-W1) (Bols et al., 1994
) cell lines, but, in these cases, the virus is non-cytopathic.
It is known that macrophages of mammals and birds contain Fc receptors, which allow them to internalize and digest virus particles coated with antibody (Mantovani et al., 1972). Antibody-enhanced infection occurs when monocytes and macrophages are more efficiently infected by virusantibody complexes via Fc receptor-mediated endocytosis than virus alone (Porterfield, 1986
). A number of human and animal viruses belonging to at least 11 different virus families have been shown to be capable of utilizing this mechanism of infection. These include Dengue virus and related flaviviruses (Halstead et al., 1984
; Peiris et al., 1981
), respiratory syncytial virus (Krilov et al., 1989
), rabies virus (King et al., 1984
), human immunodeficiency virus (Homsy et al., 1989
; Takeda et al., 1988
), influenza A virus (Ochiai et al., 1988
), feline infectious peritonitis virus (FIPV) (Stoddart, 1989
) and, most recently, coxsackievirus B3 (Girn et al., 2002
). Many of these viruses demonstrating antibody-dependent enhancement in vitro have been associated also with higher morbidity or mortality in cases of prior immunity (Sullivan, 2001
). However, in the case of influenza virus, antibody-mediated internalization and growth of influenza A virus NWS (H1N1) has been demonstrated only in the cultured murine macrophage-like cell line P388D1 in the presence of subneutralizing antiviral IgG (Ochiai et al., 1988
). Although Fc receptors for fish IgM have been demonstrated on fish leukocytes (O'Dowd et al., 1998
; Haynes et al., 1988
), there is no report to date of Fc receptor-mediated infection of macrophages by fish viruses. In this study, we investigated whether antiviral antibodies enable the fish orthomyxovirus ISAV to infect the macrophage-like fish cell lines SHK-1 and TO, thereby abrogating virus neutralization (VN) in vitro.
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METHODS |
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Polyclonal antibody preparations.
The preparation of rabbit polyclonal antisera to purified ISAV isolates RPC-980-049(1) and RPC-990-002(4) has been described previously (Kibenge et al., 2000). Atlantic salmon sera consisted of a pooled field sample collected from farmed fish in New Brunswick, Canada (Kibenge et al., 2002
), and pooled serum collected from Atlantic salmon experimentally infected with ISAV strain NBISA01. Rainbow trout serum was also collected from fish experimentally infected with ISAV strain NBISA01. For production of fish anti-ISAV sera, Atlantic salmon or rainbow trout were inoculated intraperitoneally with 105 TCID50 ISAV strain NBISA01 0·2 ml-1 per fish and kept in a fresh water tank at 10 °C for at least 84 days. Normal rabbit serum and sera from uninfected control rainbow trout and Atlantic salmon served as negative control sera. Polyclonal rabbit anti-IPNV serum to infectious pancreatic necrosis virus (IPNV) strain FVX-8 used for VN assays of IPNV was provided by Dr C. Yason (Atlantic Veterinary College Virology Diagnostic Laboratory). All serum samples were heat-inactivated at 56 °C for 30 min prior to use in order to destroy complement activity. This treatment also served to inactivate any ISAV (Falk et al., 1997
) that might have contaminated the fish sera used.
VN assays.
VN tests were carried out on 24-h-old cell monolayers in 48-well cell culture plates as described previously (Kibenge et al., 2001b). Cultures were examined microscopically for CPE to determine VN test results after 10 days of incubation (14 days in the case of the CHSE-214 cell line) at 16 °C.
Blocking of Fc receptors.
Staphylococcal Protein A (Sigma) was resuspended to a concentration of 1 mg ml-1 in sterile distilled deionized water. Parallel VN assays were conducted in TO cell monolayers using ISAV strain NBISA01 and rabbit or Atlantic salmon anti-ISAV sera in the presence or absence of Protein A, as described previously (Olsen et al., 1992), with slight modifications. Briefly, Protein A was added to a final concentration of 200 µg ml-1 to the pre-incubated virusantiserum mixtures, which were incubated for a further 1 h at room temperature. Virusantiserum mixtures were made in media without FBS in order to avoid competition for Protein A binding. TO cell monolayers in 48-well cell culture plates were inoculated with the virusantibodyProtein A mixtures and incubated for an additional 1 h at room temperature. Residual virusantibody complexes were then removed and fresh maintenance medium was added. Cultures were then incubated and monitored as described for the VN assays. Cells infected with virus in the presence or absence of Protein A served as controls.
Preparation of FITC-labelled ISAV.
ISAV strain RPC-980-280(2) was propagated in TO cells. Infected cell culture lysates were clarified at 3000 g for 30 min in a JA14 Beckman rotor. The cell pellet was saved and suspended in 1x TNE (10 mM Tris/HCl, 0·1 M NaCl and 1 mM EDTA, pH 7·9). Virus was purified on a Ficoll-400 (Amersham Pharmacia) step gradient and a sucrose cushion as described previously (Kibenge et al., 2000). Purified virus (1200 µl) with a concentration of 0·5 mg viral protein ml-1 was reacted with an equal volume of 0·1 mg FITC ml-1 dissolved in 0·5 M bicarbonate buffer (pH 9·5) for 1 h at room temperature, as described previously (Nichols et al., 1993
). Unconjugated dye was removed by passaging the virus preparation through a Bio-Gel P-6DG column (Bio-Rad). Labelled virus was eluted using an equal volume of 1x PBS and was then passed through a 0·45 µm syringe filter to eliminate virus aggregates. It was then stored at 4 °C. Labelled virus was divided into six equal volumes of 350 µl and each volume was used for each VN assay. A negative control for FITC-labelled virus was prepared by replacing purified virus with 1x PBS in the labelling reaction.
Demonstration of antibody-mediated virus uptake using FITC-labelled ISAV.
TO cells (1·4x105 ml-1) were grown in slide flasks (Fisher) (3 ml per flask) or 6-well tissue culture plates (Costar) (3 ml per well) overnight. Parallel VN assays were then set up using FITC-labelled ISAV and rabbit anti-ISAV serum in the presence and absence of staphylococcal Protein A. For this, 350 µl FITC-labelled ISAV was incubated with equal amounts of 1 : 640 dilutions of antiserum for 1 h at room temperature. Preliminary experiments established that Protein A, when used at a concentration of 200 µg ml-1, could completely block the enhancing property of the rabbit anti-ISAV serum at a dilution of 1 : 640 in TO cells. Protein A was, therefore, added to the virusantibody mixture to a final concentration of 200 µg ml-1 and incubated for 1 h at room temperature. Cell monolayers were then inoculated with the FITC-labelled ISAV antiserumprotein A mixture and incubated for 1 h at room temperature. TO cells inoculated with FITC-labelled ISAV alone were used as the positive control, whereas uninfected TO cells and TO cells inoculated with FITC were used as the negative controls. All flasks and plates were incubated at 16 °C for an additional 3 h and were then analysed using fluorescent microscopy or spectrofluorometry.
Slide flasks were processed for fluorescent microscopy by removing the inoculum from the flasks and washing the cell monolayers three times with 1x PBS. Slides were then detached from the flasks, fixed with 99 % ethanol for 10 min at 4 °C and air dried. Slides were analysed using a Fluoview 300 confocal laser scanning microscope (Olympus America) at a magnification of x400 and Fluoview software, version 3.0.
The 6-well tissue culture plates were processed for spectrofluorometry by removing the inoculum from the tissue culture plate and washing the cell monolayers with 1x PBS. Cells were detached using trypsin and resuspended in 1 ml growth medium containing 10 % FBS. Cells were pelleted at 500 g for 10 min and resuspended in 1x PBS. A 50 µl cell suspension from each sample was placed in a single well of a Nunclon F microwell plate and used to measure the intensity of fluorescence using a SpectraMAX Gemini XS spectrofluorometer (Molecular Devices) at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Each sample was tested in five replicates. Results were analysed by one-way ANOVA.
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RESULTS |
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VN antibody titres obtained against three different ISAV strains on the three cell lines (CHSE-214, SHK-1 and TO) with four different ISAV antisera are summarized in Table 1. Rabbit and Atlantic salmon antisera showed higher VN antibody titres in the CHSE-214 cell line than in the SHK-1 and TO cell lines for the three different strains of ISAV used. In the most extreme case, rabbit anti-ISAV serum to ISAV strain RPC-990-002(4) had a homologous VN antibody titre of 1 : 4800 in CHSE-214 cells but only 1 : 40 in SHK-1 cells and <1 : 10 in TO cells. With the Atlantic salmon and rainbow trout anti-ISAV sera, VN was observed only in CHSE-214 cells.
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ISAV VN in TO cells increases significantly in the presence of staphylococcal Protein A
To test the hypothesis that the poor neutralization of ISAV by anti-ISAV sera on macrophage-like fish cell lines was due to Fc receptor-mediated uptake of antibody-coated virus particles by the cells, staphylococcal Protein A was used to block Fc receptors on the antibody prior to use in VN assays. Appropriate VN controls in the absence of Protein A were also carried out. When Protein A was used in the VN assay with ISAV strain NBISA01 in TO cells, an antibody titre of 1 : 960 was obtained with rabbit anti-ISAV serum compared to a titre of 1 : 20 in TO cells without Protein A (Table 3). This was a 48-fold increase in ISAV VN titre in TO cells and a 1·5-fold increase compared to that obtained in CHSE-214 cells (Table 1
). Protein A by itself did not adversely affect ISAV infectivity. This finding indicated that ISAV VN in TO cells could be increased significantly by the pre-treatment of the virusantibody mixture with Protein A. However, Protein A did not affect the VN titre in CHSE-214 cells (Table 3
). Because Protein A is known to bind the Fc moiety of immunoglobulins (Olsen et al., 1992
; Stoddart, 1989
), these data support the conclusion that rabbit immunoglobulins could bind to receptors on TO cells (possibly Fc receptors) and that poor ISAV VN in TO and SHK-1 cells may be due to Fc receptor-mediated uptake and subsequent replication of ISAV in these macrophage-like fish cell lines. There was no VN when the rabbit anti-ISAV serum was used at dilutions of 1 : 40 to 1 : 320 in TO cells in the presence of Protein A, indicating that the binding effect of Protein A could be diluted out with higher antibody concentrations. In dilutions of 1 : 640 to 1 : 1280, Protein A could completely block antibody-mediated uptake of the virus. A similar biphasic response was also reported for FIPVantibodymacrophage interactions (Stoddart, 1989
). Protein A could not restore ISAV neutralization by fish antisera in TO cells (Table 3
), due either to the low affinity of Protein A to fish immunoglobulins or to insufficient amounts of Protein A used in the assay relative to the number of Fc receptors on fish immunoglobulins.
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DISCUSSION |
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Antibody-mediated growth of viruses is a well-described phenomenon in mammals and birds (Sullivan, 2001). However, this is the first report of antibody-mediated infection of fish cells by a fish virus. This phenomenon was demonstrated with rabbit anti-ISAV sera in TO cells, suggesting that this macrophage-like fish cell line may have Fc receptors for mammalian immunoglobulins. The possibility of this is supported by the fact that a mammalian homologue of Fc
RI
(the Fc receptor for IgE) chain exists in carp (Fujiki et al., 2000
). In mammals, the existence of specific Fc receptors for five classes of immunoglobulins (Fc
R, Fc
R, Fc
R, Fc
R and FcµR) has been recognized (Gessner et al., 1998
). Our study shows that other mammalian Fc receptor homologues may also exist in teleost macrophages. Fc receptors for shark IgM have been demonstrated on shark leukocytes and high levels of immune complex receptors, possibly Fc receptors, have also been demonstrated on Atlantic salmon leukocytes (O'Dowd et al., 1998
; Haynes et al., 1988
). Although mammalian IgM and complement can also mediate antibody-dependent enhanced virus infectivity via macrophage C3 receptors (Cardosa et al., 1983
), that mechanism was considered unlikely in the present study because we used heat-inactivated anti-ISAV sera.
Protein A could not restore the neutralization of ISAV by fish antisera in TO cells (Table 3), due either to the low affinity of Protein A for fish immunoglobulins or to insufficient amounts of Protein A used in the assay relative to the number of Fc molecules on fish immunoglobulins. An alternative hypothesis that could be derived from the data is that only a small proportion of ISAV is able to infect CHSE-214 cells and it is an epitope that permits virus entry into CHSE-214 cells that is primarily seen and neutralized by rabbit antiserum. However, since the pattern of neutralization of ISAV was the same for fish and rabbit antisera on the three cell lines (Table 1
), it is considered that poor neutralization of ISAV by fish antiserum in TO and SHK-1 cells is also Fc-mediated. It is also possible that macrophage-like fish cell lines have different types of receptors that allow binding of fish or mammalian immunoglobulins. Our preliminary observations on the ASK-2 cell line (another macrophage-like fish cell line) indicate that in this cell line, rabbit anti-ISAV serum, but not fish anti-ISAV serum, neutralizes the virus very well (unpublished data). A previous study in fish described antibody-enhanced infectivity of fish rhabdoviruses that did not appear to involve Fc or C3 receptors but an unknown mechanism (Clerx et al., 1978
).
Several different approaches have been used in the past to demonstrate conclusively antibody-mediated internalization of viruses in vitro via Fc receptors, including blocking with monoclonal anti-Fc receptor IgG and its Fab fragment (Peiris et al., 1981) with heat-aggregated IgG (Daughaday et al., 1981
; Lewis et al., 1988
) or by binding with staphylococcal Protein A before inoculation (Chanas et al., 1982
). The high affinity of Protein A for the Fc moiety of the antibodies was exploited elegantly by Stoddart (1989)
to demonstrate Fc receptor-mediated uptake of FIPV. In those studies, it was observed that Protein A reduced dramatically the level of FIPV infectivity in the presence of enhancing antibodies. This observation indicated that binding of Protein A to the Fc moiety could block the attachment of antibody-coated virus to Fc receptors and inhibit antibody-enhanced infection of the virus (Olsen et al., 1992
). Thus, Protein A can be used to demonstrate Fc-mediated uptake of viruses, as has been done in the present study.
Since ISAV in Atlantic salmon seems to target leukocytic, endothelial and endothelial-associated cells, it seems reasonable to speculate that Fc-mediated antibody-dependent enhancement of ISAV infection may occur in vivo. This would accelerate the disease process by efficiently and specifically delivering virus to the very target cells within which the virus replicates. Additionally, such an infection, if non-cytolytic, would facilitate virus persistence in fish infected with ISAV. Conceptually, such infection-enhancing antibodies may be more effective when neutralizing antibodies are either absent, such as following antigenic drift and shift of the virus, or consumed by virus excess.
In conclusion, our study has demonstrated for the first time antibody-mediated uptake and replication of a fish virus, ISAV, in macrophage-like fish cell lines SHK-1 and TO. Blockage of this mode of virus infection using Protein A suggests that this is an Fc-mediated infection of cells. Experiments with purified immunoglobulin Fc and Fab fragments need to be done to prove definitively that the antibody-mediated uptake of ISAV in fish macrophage-like cells demonstrated here is mediated by Fc receptors. Systematic analysis of ISAV proteins using both neutralizing and non-neutralizing mAbs is also necessary to identify the ISAV epitopes responsible for this phenomenon. Similarly, studies are necessary to test whether antibodies to ISAV could cause disease enhancement or not. Findings from these studies will bear directly on current efforts to develop ISAV vaccines that have substantial preventative value.
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ACKNOWLEDGEMENTS |
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Received 8 January 2003;
accepted 28 February 2003.