Institutes of Infectology1, Diagnostic Virology2 and Molecular Biology3, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, Boddenblick 5 A, D-17498 Insel Riems, Germany
Institute of Immunology, Federal Research Centre for Virus Diseases of Animals, Paul-Ehrlich-Str. 28, D-72076 Tübingen, Germany4
Veterinary and Food Control Board of Saxonia-Anhalt, Haferbreiter Weg 132-135, D-39576 Stendal, Germany5
Author for correspondence: Harald Granzow. Fax +49 38351 7151. e-mail harald.granzow{at}rie.bfav.de
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Abstract |
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Introduction |
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Electron microscopy is being used regularly in diagnostic investigations of tissues from diseased fish or cell cultures infected with isolates from different fish species. Diagnostic studies of cyprinoid cell cultures incubated with homogenized fish tissues during a monitoring program for pathogens in wild fish populations in the Federal State of Saxonia-Anhalt (Germany) led to the isolation of a cytopathic virus from a white bream (Blicca bjoerkna L.; Teleostei, order Cypriniformes). This virus, detected in cell culture supernatants and infected cells by electron microscopy, showed an ultrastructure and morphogenesis that differed significantly from all known members or tentative members of the virus families established by the International Committee on Taxonomy of Viruses (ICTV) (van Regenmortel et al., 2000 ). Here, we present first results on characterization, primarily by electron microscopy, of the newly isolated virus.
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Methods |
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The cell lines (obtained from the Collection of Cell Lines in Veterinary Medicine of the Federal Research Centre for Virus Diseases of Animals, Germany) used in this study were the fish cell lines EPC, blue gill fry (BF-2), chinook salmon embryo (CHSE) and fathead minnow (FHM) (Fryer & Lannan, 1994 ), the foetal pig kidney cell line EFN-R, and the insect cell lines high five (Life Technologies) and EAA (generously provided by A. Gröner, Behringwerke, Marburg, Germany).
Fish cell cultures were grown to confluency in 75 cm2 cell culture flasks at 27 °C (EPC) or 24 °C (FHM, BF-2) in Eagles minimal essential medium (MEM) supplemented with 10% foetal bovine serum (FBS) buffered to pH 7·27·4 with NaHCO3. EFN-R cells were grown in medium 7 or MEM supplemented with 10% FBS at 37 °C. High five cells were grown in SF900 medium (Life Technologies) and EAA cells were grown in Grace medium with 10% FBS at 28 °C. The virus isolate DF 24/00 was propagated in EPC, FHM, CHSE and BF-2 cells at 20 °C. SVCV was grown in FHM cells at 20 °C. IHNV was cultured in EPC cells and VHSV was cultured in BF-1 cells, both at 15 °C.
Virus concentration and buoyant density determination.
Isolate DF 24/00 was propagated on EPC cells in 75 cm2 tissue culture flasks and harvested by two freezethaw cycles. The following procedures were performed at 12 °C. To remove cell debris, the collected culture medium was centrifuged at 4000 r.p.m. (SW28 rotor) (Beckman) for 10 min. The supernatant was layered onto a 15% sucrose cushion and centrifuged at 20000 r.p.m. (SW28 rotor) for 90 min. The virus pellet was then resuspended in STE (0·15 M NaCl, 0·01 M TrisHCl pH 8·3, 0·01 M EDTA pH 8·0) and centrifuged in a continuous gradient of 550% sucroseSTE at 20000 r.p.m. (SW28 rotor) for 30 min. The resulting opaque band containing the virus was removed with a syringe and dialysed against STE overnight at 4 °C.
The buoyant density of DF 24/00 was determined by equilibrium centrifugation in a continuous sucrose gradient. After one freezethaw cycle, the remaining cells of the infected monolayer were scraped into the medium and cellular debris was removed by centrifugation at 6200 g for 10 min. The supernatant was clarified by filtration through a 0·45 µm filter and virus was concentrated by sedimentation through a cushion of 10% sucrose in STE for 90 min at 20000 r.p.m. (SW28 rotor). The pellet was resuspended in STE, layered onto a continuous 3060% sucrose gradient in STE and centrifuged for 24 h at 40000 r.p.m. (SW40 rotor) (Beckman). Samples (1 ml) from the gradient were analysed by refractive index determination (ABBE Refraktometer, KRÜSS Optronic), titration on EPC cells and electron microscopy.
Antibodies.
For the generation of a polyclonal antiserum against isolate DF 24/00, a rabbit was immunized three times by intramuscular injection of 0·5 ml (approx. 5 µg) of purified virus suspended in 0·5 ml Freunds complete (for the first immunization) or incomplete (for booster immunizations) adjuvant. Serum obtained after the third immunization was used for labelling experiments.
Determination of the viral nucleic acid and lipid solvent sensitivity.
Cell cultures were infected with isolate DF 24/00, catfish iridovirus or infectious pancreatic necrosis virus (IPNV) at an m.o.i. of 0·1 and grown after the addition of 1000 or 100 µg/ml of 5-iodo-2'-deoxyuridine (IDU) (Serva). Virus infectivity was determined 4 days post-infection by titration. Lipid solvent sensitivity was determined by the addition of 10% chloroform (Roth) to pools of isolate DF 24/00, IPNV or VHSV for 4 h at 4 °C. After centrifugation at 2000 g, the supernatants were titrated.
Electron microscopy.
For negative staining, infected cell cultures were scraped off the plate and pelleted by low-speed centrifugation. The pellet was resuspended in PBS. Formvar-coated grids were placed for 7 min onto drops of cell culture supernatant or resuspended pellet. Negative staining was performed with 2% phosphotungstic acid (PTA, pH 7·4) for 7 min, PTA (pH 6·0) for 17 min, 2% ammonium molybdate (AMo, pH 6·5) for 1 min, 2% methylamine tungstate (MAT, pH 5·8) for 1 min and 1% aqueous uranyl acetate (UAc) for 545 s.
For immunoelectron microscopy studies on isolated DF 24/00, purified virions adsorbed to grids were incubated with the polyclonal serum in appropriate dilutions, followed by gold-tagged protein A (10 nm) (British Bio Cell). Finally, grids were stained with 2% PTA, pH 7·4.
Embedding of infected cell cultures in epoxy resin was performed as described previously (Granzow et al., 1997 ). Briefly, after cytopathic effects became obvious, infected cell cultures were fixed with 2·5% glutaraldehyde in 0·1 M cacodylate buffer (300 mosmol, pH 7·2) followed by 1% buffered osmium tetroxide (Polysciences). Cells were then dehydrated and embedded in Glycid ether 100 (Serva). Ultrathin sections were stained with UAc and lead citrate (Reynolds, 1963
) and examined with a Philips 400T (Eindhoven) or Zeiss 910 (Oberkochen) electron microscope.
Detection of viral glycoproteins.
Glycoproteins in purified virus preparations were detected after electrophoretic separation and blotting by specific insertion of Biotin-LC-Hydrazide (Pierce Chemicals) into the carbohydrate moieties followed by reaction with a streptavidinhorseradish peroxidase conjugate (Pharmacia Amersham). To this end, the protocol suggested by the supplier was modified as follows: an aliquot of purified virus containing 100 µg of protein was sedimented at 121000 g and resuspended in 50 µl of labelling buffer (sodium acetate buffer, 100 mM pH 5·5). Carbohydrates were oxidized by the addition of 5 µl of 30 mM sodium periodate. In a control sample, this oxidization step was omitted. Residual periodate was neutralized by the addition of 5 µl of 80 mM Na2S2O3. Then, 5 µl of 5 mM Biotin-LC-Hydrazide was added and the mixture was allowed to react for 1 h at room temperature. Virus was sedimented at 121000 g for 45 min and resuspended in 50 µl PBS. Proteins from 2 µl of this virus suspension were separated by SDSPAGE and blotted onto a nitrocellulose membrane (Towbin et al., 1979 ). After blocking with 1% BSA in TBS-T (10 mM Tris pH 7·4, 150 mM NaCl, 0·1% Tween-20), the blot was incubated for 20 min with streptavidinhorseradish peroxidase conjugate diluted 1:2500 in PBS-T and washed thoroughly with TBS-T. Glycoproteins were detected by chemiluminescence using the Super Signal West Pico kit (Pierce Chemicals).
Glycoproteins with affinity to concanavalin-A (con-A) were detected in an overlay blot assay. Samples were processed as described above. After blocking of the nitrocellulose sheet with BSA, it was incubated with a con-Ahorseradish peroxidase conjugate (Sigma) at a final concentration of 400 ng/ml in con-A buffer (20 mM HEPES pH 7·4, 500 mM NaCl, 0·1% Tween-20, 0·5 mM CaCl2, 0·5 mM MnCl2) for 1 h. After a thorough wash, glycoproteins with specificity for con-A were detected by chemiluminescence.
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Results |
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After staining with 1% UAc for 45 s, envelope spikes disappeared and the bacilliform shape changed to a more ovoid appearance. Staining with 1% UAc for shorter periods of time (515 s) reduced, but did not completely prevent, the structural alterations of the virus particles (data not shown). Table 1 summarizes the estimated sizes of the virions and their components using the different negative staining methods and after ultrathin sectioning.
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Immunolabelling of isolate DF 24/00
Labelling experiments with polyclonal rabbit antiserum showed the high reactivity of antibodies with the virion (Fig. 1E, F
). Only the surface of the virions, presumably the spikes, were labelled, whereas freely accessible nucleocapsids within partly disrupted virus particles and areas of the envelope without spikes were not labelled (Fig. 1F
). This indicated that the antiserum was directed primarily against viral envelope proteins.
Identification of viral glycoproteins
Preparations of purified virus were analysed for the presence of glycoproteins (Fig. 3) after SDSPAGE. Fig. 3(A)
shows a Coomassie-stained polyacrylamide gel with six distinguishable protein bands, designated p1, p2 and p4p7. Western blotting with the rabbit antiserum (Fig. 3B
, WB) demonstrated the presence of another protein designated p3. Carbohydrate-specific labelling with Biotin-LC-Hydrazide (Fig. 3B
, Bio) detected six viral proteins, p1p5 and p6 or p7. Here, the smallest protein p7 migrates with a different apparent molecular mass in lanes WB and Bio due to the integration of biotin into the protein or p6 was Biotin-LC-Hydrazide labelled. As a control, the critical oxidizing step has been omitted (Fig. 3B
, Co). This confirms that the major antigenic proteins (p1p4 and p7) are indeed glycoproteins. Three of them, namely p1, p2 and p7, show affinity to conA (Fig. 3C
), indicating that they contain
-mannose. These three proteins were also detectable using the lectins MAA, DSA, GNA and SNA from the DIG glycan differentiation kit (Roche) (data not shown).
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Discussion |
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The slight differences observed in the size of the isolated virions and their subunits, estimated by negative staining, seemed to be a result of the effect of different staining conditions. More strikingly, different staining conditions had a profound effect on the morphology of the virion. The acid pH of 6·0 of 2% PTA and a staining time exceeding 1 min resulted in the disruption of nearly all of the virion envelopes and also, although only partly, of the nucleocapsids of the virus particles. Staining with other acidic stains, such as 2% AMo and 2% MAT, for 1 min led to less dramatic alterations, whereas staining of virions with 1% UAc for about 45 s caused striking damage to the virions. This is obviously due to the sensitivity of the envelope to acid pH.
By comparison of our data on the size of DF 24/00 virions (1921x130160 nm) to baculo- (3060x250300 nm) and rhabdoviruses (45100x100430 nm) (van Regenmortel et al., 2000 ), it appears that DF 24/00 virions are distinctly smaller. In overall shape, DF 24/00 virions resemble most closely the bacilliform rhabdoviruses of plants (Jackson et al., 1987
). In contrast, the rigid rod-shaped nucleocapsid structure is similar to the BV nucleocapsids of baculoviruses, whereas rhabdovirus nucleocapsids appear as flexible structures of varying length. The long surface projections observed on DF 24/00 were never found on rhabdo- and baculoviruses but are characteristic for coronaviruses, which carry spikes of more than 15 nm in length. However, members of the Coronaviridae family are either spherical (genus Coronavirus) or toroid (genus Torovirus) in shape (van Regenmortel et al., 2000
) and do not contain a rod-shaped core as seen in DF 24/00.
Comparing the intracytoplasmic virus components, two typical inclusions that clearly differed from the intracellular nucleocapsid structures of DF 24/00 are present in rhabdovirus-infected cells. One of the rhabdovirus inclusion bodies evidently contains typical viral nucleocapsids (Dubois-Dalcq et al., 1984 ), whereas the second form, visible as a granular electron-dense matrix, possibly contains only the viral nucleoprotein (N), which is synthesized in excess early in infection and results in small cytoplasmic granula. Rhabdoviruses primarily bud at the plasma membrane where virus particles are immediately released from the cell. In contrast to rhabdo- and baculoviruses, DF 24/00 virions were only rarely observed during budding at the cell surface but acquired their envelope by budding through smooth intracytoplasmic membranes. This correlated with the consistent finding of enveloped virions inside cytoplasmic vesicles beneath the cell surface, probably on the way to release by exocytosis.
Although a similar rigid rod-shaped nucleocapsid was detected in DF 24/00 and BV of baculoviruses (Blissard & Rohrmann, 1990 ; Fraser, 1986
; McKinnon et al., 1974
; van Regenmortel et al., 2000
), pronounced differences exist regarding its size (3060 nm diameter for BV in comparison to 1925 nm for DF 24/00), morphology of the ends of the nucleocapsids, the intracellular location of the nucleocapsids and the site of capsid assembly. Morphogenesis of baculovirus nucleocapsids occurs in the nucleus and at their blunt end cap-like structures are present. In contrast, DF 24/00 nucleocapsids were only detected in the cytoplasm and lacked a cap-like structure. Similar to baculovirus nucleocapsids, DF 24/00 nucleocapsids were found arranged side by side at intracellular membranes, but these are located in the nucleus in baculoviruses and in the cytoplasm in DF 24/00. In contrast to baculoviruses, in the cytoplasm of DF 24/00-infected cells, smooth membranes seemed to proliferate and vesicles were often surrounded by more than one membrane. Envelopment of both viruses takes place by budding, but BV gain their final envelope by budding at the cell surface, whereas DF 24/00 primarily buds at intracytoplasmic membranes. Mature BV of baculoviruses are characterized by spikes only at the rounded ends of the enveloped particle, whereas DF 24/00 virions were completely surrounded by long and fuzzy spikes (van Regenmortel et al., 2000
; this report).
The composition of the viral envelope of rhabdoviruses, coronaviruses, baculoviruses and DF 24/00 virions also differs. Rhabdovirus envelopes contain one viral glycoprotein and coronavirions contain two or three viral glycosylated polypeptides (Dubois-Dalcq et al., 1984 ; van Regenmortel et al., 2000
). BV of baculoviruses contain one major envelope glycoprotein (gp64) (Rohrmann, 1992
; van Regenmortel et al., 2000
). Our preliminary biochemical analysis indicated the presence of at least six glycoproteins in purified virions. Five of them also reacted with an antiserum that had been produced against purified virions and which, in immunoelectron microscopy, only labels virion surface components but not nucleocapsids. Thus, in complexity of the viral envelope, DF 24/00 differs from baculo-, rhabdo- and coronaviruses.
In 1987, Ahne et al. (1987) isolated a syncytia-inducing virus from cultured grass carp (Ctenopharyngodon idella), which, in dimension and bacilliform shape, is similar to DF 24/00. However, contrary to our findings, in one electron micrograph after negative staining, the grass carp virus seemed to be without recognizable surface projections. Further detailed studies about the ultrastructure and morphogenesis in cells were not performed. Based on our experiences on the influence of different staining conditions on virion morphology, the absence of spikes might have been an artefact. Thus, both viruses could, in fact, have identical morphologies. More than two decades ago, Yudin & Clark (1978
, 1979
) and Chassard-Bouchaud et al. (1976)
each described a new virus (ecdysial gland virus and Y-organ virus) infecting the blue crab (Callinectes sapidus) and European shore crab (Carcinus maenas). Although not well characterized, these viruses also appear to be similar in virion morphology to DF 24/00. The highest similarity in ultrastructure and morphogenesis was found in newly discovered RNA-containing viruses isolated in Thailand from black tiger shrimp (Penaeus monodon), which suffered from yellow-head disease (YHD) (Chantanachookin et al., 1993
; Wongteerasupaya et al., 1995
). Similar viruses were also isolated from P. vannamei and P. stylirostris in Hawaii (Nadala et al., 1997
) and in Australia from shrimp with pathological signs that differed from YHD (Spann et al., 1995
, 1997
). Preliminary data indicate that DF 24/00 contains a nonsegmented ssRNA genome of more than 20 kb and the classification of these isolates has been a subject of controversial discussion. Based on the ultrastructure and the shape of the detected nucleocapsids as well as its animal host (crustacean), Boonyaratpalin et al. (1993)
and Chantanachookin et al. (1993)
reported it as a granulosis-like baculovirus (Anderson & Prior, 1992
; Edgerton et al., 1996
; Groff et al., 1993
; Hedrick et al., 1995
; Johnson & Lightner, 1988
; Mari et al., 1993
; van Regenmortel et al., 2000
). After Wongteerasupaya et al. (1995)
identified this virus as an RNA-containing pathogen, Nadala et al. (1997)
suggested that is was rhabdovirus-like. Until now, no exact classification of these virus isolates by the ICTV was performed. Therefore, these viruses are missing in the virus index of the seventh report of the ICTV (van Regenmortel et al., 2000
). In summary, we demonstrate that the novel virus isolate DF 24/00 from a white bream (B. bjoerkna L., order Cypriniformes) shares ultrastructural and morphogenetic features with three virus families, Coronaviridae, Rhabdoviridae and Baculoviridae, without meeting all of the criteria for inclusion into any of these families.
Therefore, additional comparative investigations are needed for the comprehensive characterization of the vertebrate and invertebrate viruses with similar morphology that may constitute members of a new virus family. Further studies on the biology and molecular biology of these viruses are underway. These studies will help to find the correct taxonomic status for these viruses.
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Acknowledgments |
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Received 19 June 2001;
accepted 13 August 2001.
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