1 Department of Morphology, Genetics and Aquatic Biology, Norwegian School of Veterinary Science, Oslo, Norway
2 National Veterinary Institute, PO Box 8156 Dep., 0033 Oslo, Norway
Correspondence
Agnar Kvellestad at National Veterinary Institute
Agnar.Kvellestad{at}vetinst.no
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ABSTRACT |
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INTRODUCTION |
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Members of the family Paramyxoviridae are responsible for a variety of diseases affecting humans and animals. Many of these diseases affect the respiratory organs. Over the past decades, several novel paramyxoviruses have been discovered in aquatic and terrestrial animals as a result of surveillance and disease investigations (Lamb et al., 2000; Lamb & Kolakofsky, 2001
; Wang & Eaton, 2001
). These include a paramyxovirus in diseased snakes (Clark et al., 1979
; Ahne et al., 1999
) and paramyxo-like viruses in carp (Body et al., 2000
) and in apparently healthy wild chinook salmon (Oncorhynchus tshawytscha Walbaum) (Winton et al., 1985
).
Paramyxoviruses are large (150300 nm), enveloped, pleomorphic viruses with a non-segmented, single-stranded, negative-sense RNA genome of 1516 kb. They replicate entirely in the cytoplasm. The virion has a nucleocapsid core containing the RNA genome and three nucleocapsid-associated proteins: an RNA-binding protein, a phosphoprotein and a large protein. The matrix protein resides between the core and the envelope. The envelope is covered with spikes consisting of one glycoprotein involved in cell attachment (haemagglutininneuraminidase, haemagglutinin or protein G) and another glycoprotein involved in the fusion of the viral envelope with the plasma membrane of cells (Lamb et al., 2000; Lamb & Kolakofsky, 2001
).
During attempts to culture the causative agents of epitheliocystis in Atlantic salmon, we isolated a hitherto-unknown paramyxovirus from a single fish. In this report we describe the isolation and partial characterization of this new virus, with special emphasis on morphology, replication and properties of the surface molecules.
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METHODS |
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Unless otherwise stated, RTgill-W1 cells were inoculated for virus propagation, incubated at 14 °C and inspected for cytopathic effects (CPE) by phase-contrast light microscopy. For primary virus isolation, cells in 24-well tissue culture plates were inoculated with tissue suspensions for 14 h, followed by washing and addition of fresh L15-10. The cultures were supplied with fresh L15-10 at intervals of 12 weeks. For higher passages of virus, supernatants from cultures with CPE were diluted in L15-5 and inoculated into new cultures. The characterization experiments were performed with virus from the third to fifth passage.
To test for susceptibility to this new virus, cultures of CHSE-214, BF-2, EPC and SHK-1 cells were inoculated with supernatants from parallel non-infected and infected RTgill-W1 cells with CPE and incubated at 15 °C.
Infectivity titre was determined by endpoint titration in 96-well culture plates with RTgill-W1 cells. CPE was read between 6 and 8 weeks post-inoculation (p.i.). The 50 % tissue culture infective dose (TCID50) was estimated by the method of Spearman and Kärber (Kärber, 1931).
Influenza A (A/PR/8/34) virus, Newcastle disease virus (F-strain, Weybridge) and influenza C virus (C/Johannesburg/1/66) were grown in the chorioallantoic membrane of 11-day-old embryonated eggs. Infectious salmon anaemia virus (Glesvaer/2/90) was grown in SHK-1 cells as described by Dannevig et al. (1995).
Clinical samples and sample processing.
Gills were sampled in September 1995 from 20 Atlantic salmon previously transferred to seawater netpens in May. From the beginning of August the fish displayed signs of respiratory distress. Cumulative mortality reached 40 % during the next 3 months. Mean water temperature decreased from 17 °C in August to 12 °C in September. Histological examination prior to sampling revealed extensive gill changes, with thrombosis of lamellar blood vessels, necrosis of epithelial cells, proliferation of apparently poorly differentiated epithelial cells, infiltration of epithelium with inflammatory cells and many inclusions indicating epitheliocystis. Areas of necrosis were also detected in the liver.
The gills of the fish, from which the described virus was isolated, were washed in buffers and disinfected with hydrogen peroxide and glutaraldehyde, essentially as described by Kvellestad et al. (2002), to prevent contamination of the cell cultures by normal surface microflora. The soft tissues were scraped from the gills and suspended in 10 ml L15-10 using a whirl mixer. Since infectious pancreatic necrosis virus (IPNV) is ubiquitous in Norwegian salmon farms (Melby et al., 1991
), rabbit antisera against Ab and Sp serotypes of IPNV were added to the tissue suspension in appropriate dilutions to inhibit replication of this virus in the cell culture.
Haemalum and eosin staining of cell cultures.
Infected and non-infected cells on glass coverslips were fixed in buffered formalin (10 %, v/v, in 135 mM sodium phosphate buffer, pH 7·0), permeabilized in 80 % aqueous acetone, stained with haemalum and eosin (Prentø, 1985; Prentø et al., 1985
), passed through a series of graded ethanols and xylene, and embedded in mounting medium.
Haemagglutination and haemadsorption assays.
Haemagglutination titrations were performed in microtitre plates by mixing 50 µl of virus supernatant diluted twofold in PBS (10 mM, pH 7·4) with 50 µl 0·5 % (v/v) washed erythrocytes. The endpoints were read after 1 h of incubation at room temperature except for influenza C virus, which was titrated at 4 °C. Erythrocytes from mammals, birds and fish were tested, including human (O), guinea pig, green monkey, rabbit, sheep, cow, horse, chicken, turkey, Atlantic salmon, brown trout (Salmo trutta L.), rainbow trout (Oncorhynchus mykiss Walbaum), Atlantic cod (Gadus morhua L.), crucian carp (Carassius carassius L.) and wolffish (Anarhichas lupus Olafsen). The haemagglutinating activity (HA units) was expressed as the reciprocal value of the highest dilution showing complete agglutination of the erythrocytes.
For haemadsorption, the cell cultures were first washed once with Hanks' balanced salt solution followed by incubation with 0·5 % (v/v) washed chicken erythrocytes in L15 for 30 min at room temperature. The cultures were then washed with L-15 to remove unattached erythrocytes and immediately examined by microscopy for adsorption.
Receptor-destroying enzyme activity was assessed by prolonged observation of the haemagglutination reaction at room temperature for up to 24 h. The elution of virus from the erythrocytes as shown by the conversion of positive haemagglutination patterns to a negative pattern indicated the presence of receptor-destroying enzyme activity. Following elution, fresh virus (4 HA units per well) was added to see whether the cells could be re-agglutinated.
Assays of neuraminidase and acetylesterase activity.
Cell culture supernatant or pelleted virus was used in both assays. Neuraminidase activity was assayed by incubating virus with either fetuin (Sigma) or N-acetylneuraminyl-lactose (Sigma) in phosphate buffer (0·5 M, pH 6·0) at 15, 25 and 34 °C. Following incubation for 18 h, samples were assayed for free N-acetylneuraminic acid using the thiobarbituric acid method as described by the Centers for Disease Control (1982). The acetylesterase activity was determined by incubating 10 µl of virus sample with 300 µl 1 mM p-nitrophenyl acetate (Sigma) in PBS. The release of acetate was monitored by determining the optical density at 405 nm (Vlasak et al., 1987
).
Metabolic inhibitors.
The nucleic acid type of the virus was presumptively determined by growing it in the presence of the thymidine analogues 5-bromo-2-deoxyuridine (Br-dU) or 5-iodo-2-deoxyuridine (IDU). RTgill-W1 cells in 25 cm2 flasks were infected with the virus and incubated with or without 100 µg Br-dU or IDU ml-1. After incubation for 9 and 22 days, the cells were examined by phase-contrast microscopy and the haemagglutination and infectivity titres were determined.
Inactivation studies.
Lipid solvent sensitivity was tested by adding 1·5 ml chloroform to 3 ml cell culture supernatant, vortexing for 10 min at room temperature, centrifugation (2000 g, 10 min) and determination of the virus titre. The stability of the virus at different pH values was assayed by adding 0·5 ml cell culture supernatant to 4·5 ml aliquots of medium adjusted using HCl or NaOH. After incubation for 30 min at room temperature, an equal volume of medium containing NaOH or HCl was added to give a final pH of 7·4 and the virus titre determined. The temperature stability of the virus was tested by incubating aliquots of cell culture supernatant at 5, 14, 37 and 56 °C, followed by determination of virus titre. The effect of freezing and thawing on virus infectivity was determined by repeated freezing (-70 °C) and thawing in a water bath at 20 °C.
Isopycnic density.
Virus culture supernatant was clarified by centrifugation (6700 g, 20 min) and then precipitated by addition of polyethylene glycol (Mr 8000) and NaCl to final concentrations of 70 and 23·2 g l-1, respectively. The mixture was gently stirred at room temperature for 15 min, followed by further gentle stirring for 3 h at 4 °C. The precipitate was collected by centrifugation (6700 g, 1 h) and resuspended in TNE buffer (10 mM Tris/HCl, 100 mM NaCl, 1 mM EDTA, pH 7·2). One ml of virus concentrate was overlaid on either a preformed 565 % (w/v) sucrose gradient in TNE buffer or a discontinuous gradient composed of equal parts of 20, 30 and 40 % CsCl in TNE buffer. The gradients were centrifuged at 150 000 g for 1820 h (Beckman SW-41 rotor). Fractions of 0·25 or 0·5 ml were collected by upward displacement and tested for haemagglutinating activity. The buoyant densities of the collected fractions were calculated from the refractive index.
Gel electrophoresis of viral polypeptides and Western blotting.
Virus was purified by sucrose gradient centrifugation as described above. The virus band was collected by side puncture of the tube wall, diluted in TNE buffer, pelleted at 100 000 g for 1 h and resuspended in dissociation buffer (50 mM Tris/HCl, pH 6·8, 1 % SDS, 50 mM dithiothreitol, 8 mM EDTA, 0·01 % bromophenol blue). After heating for 5 min at 95 °C, the proteins were separated by SDS-PAGE using the discontinuous system devised by Laemmli (1970) with 0·5 mm thin pre-cast 12·5 % polyacrylamide gradient gels (ExcelGel SDS; Amersham) followed by staining with Coomassie blue. Gels were scanned in a desktop scanner (Image Scanner; Amersham) and subsequently analysed and printed out using Gel-Pro gel scanning software (Media Cybernetics). The molecular mass markers used were in the range 14·494·0 kDa (Amersham).
Electron microscopy of cell cultures.
Infected and non-infected cells on Thermanox plastic coverslips (Nunc) and in 25 cm2 flasks were fixed in glutaraldehyde (3 % in 0·1 M sodium cacodylate buffer, pH 7·4), post-fixed in osmium tetroxide (2 % in 0·1 M sodium cacodylate buffer, pH 7·4) for 2 h, dehydrated through a series of graded ethanols and embedded in Lx-112 embedding medium (Ladd Research Industries). Ultrathin sections were contrasted for 20 min in 5 % uranyl acetate and for 2 min in Coggeshall's 0·2 % lead citrate in 0·1 M sodium hydroxide and examined in a JEOL JEM-100S electron microscope operated at 60 keV.
Electron microscopy of purified virus preparations.
Virus was purified by CsCl gradient centrifugation as described above. To reveal the nucleocapsid of the virions, the virus was lysed by mixing equal parts of purified virus and 0·4 % Triton X-100 solution (pH 7·2). The mixture was kept at room temperature for 15 min before grid preparation. Negative staining was performed by applying droplets (5 µl) of lysed or non-lysed virus suspensions on carbon-filmed grids. After 1 min, the grids were washed in distilled water and stained with 0·5 % sodium phosphotungstic acid, pH 7·0, for 1 min. The grids had previously been glow discharged in air to facilitate spreading of both virus and stain. The specimens were examined in a JEOL 1010 electron microscope operated at 100 keV.
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RESULTS |
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Electron microscopy of cell cultures
Structural changes were observed in the cytoplasm and plasma membrane of infected cells. The cytoplasmic inclusions in infected cells consisted of tightly packed coiled filaments approximately 17 nm in diameter (Fig. 2b, c). Small inclusions with loosely packed filaments of apparently greater diameter were seen in the cytoplasm of a very few cells (Fig. 2d
). The plasma membrane displayed many small electron-dense areas with outer surface projections extending approximately 10 nm from the outer surface, while on the inside more or less parallel aligned filaments of the type previously described with a diameter of approximately 25 nm were observed (Fig. 2e
). Highly pleomorphic virions were released by budding through these areas of altered plasma membrane (Fig. 2f
). Elongated virions in the budding phase measured at least 200600 nm in diameter and 4002100 nm in length, with prominent filaments beneath the envelope (Fig. 2g
). A few spherical particles with diameter 400800 nm were observed. In those that were fully released, many 17 nm thick filaments had apparently been released from the envelope and coiled up in the interior (Fig. 2h
). Rounded and shrunken cells contained a condensed nucleus and cytoplasm, inclusions and altered plasma membrane, but no budding was observed.
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Complete elution of virus from the agglutinated erythrocytes was observed after incubation at room temperature for 23 h indicating the presence of receptor-destroying enzyme activity. Erythrocytes previously agglutinated with the paramyxovirus could not be re-agglutinated with homologous virus or with Newcastle disease virus, indicating a relationship with this virus. In contrast, infectious salmon anaemia virus, influenza C virus and to a degree influenza A virus were able to re-agglutinate the paramyxovirus-treated erythrocytes.
Neuraminidase and acetylesterase activity
Significant neuraminidase activity was detected in the paramyxovirus preparation using both fetuin and N-acetylneuraminyl-lactose as substrates. In comparison with influenza A virus the magnitude of the reaction was similar at 34 °C but twice as strong at 15 and 25 °C, indicating that this new virus is adapted to cold-blooded animals. Acetylesterase activity was not detected in the paramyxovirus preparation.
Metabolic inhibition
No inhibition of virus replication was observed with either Br-dU or IdU.
Replication
To examine virus replication at different temperatures, RTgill-W1 cells in 162 cm2 flasks were inoculated with a virus dose of 105 TCID50 for 24 h, incubated at 6, 10, 16 and 21 °C and assayed for infectivity titre over a period of several weeks. The results are presented in Fig. 3 and show that the lag phase in this experiment lasted for approximately 2041 days depending on temperature. The highest titres were obtained at 10 °C, indicating a maximum rate of replication at around that temperature. Infected CHSE-214 cells showed CPE similar to that in RTgill-W1 cells, and the virus replicated at a slower rate, but the final titre was higher (data not shown). No CPE was observed in BF-2, EPC or SHK-1 cells inoculated with virus and incubated for 1 week.
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Isopycnic density
A weak opalescent band was observed after centrifugation in the CsCl gradient, corresponding to peak haemagglutinating activity at a density of 1·181·19 g ml-1 (Fig. 4). In later centrifugations, the band was frequently divided into two bands separated by approximately 1 mm. The sucrose gradient centrifugation gave essentially the same results (data not shown) although the virus band was less distinct.
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DISCUSSION |
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Structural changes were observed in the cytoplasm and plasma membrane, but not in the nucleus, indicating replication in the cytoplasm only. Nucleocapsid diameter of approximately 17 nm in virions studied by negative staining and in thin sections is within the size range (1318 nm) for members of the Paramyxoviridae (Lamb et al., 2000; Lamb & Kolakofsky, 2001
). Furthermore, the typical herringbone structure of the nucleocapsid of paramyxoviruses was also demonstrated by negative staining. The varying diameter of filaments in inclusions and beneath the plasma membrane may reflect different developmental states of the nucleocapsid. The inclusions formed within 7 days. Their purple colour was due to binding of both haemalum and eosin and demonstrated the presence of anionic and cationic groups, respectively, the former including phosphates (Prentø, 1985
) and the latter occurring in proteins (Prentø et al., 1985
). This is consistent with the presence of RNA and proteins, among them phosphoprotein, in the nucleocapsid (Lamb et al., 2000
; Lamb & Kolakofsky, 2001
). Inclusions may indicate an imbalance between assembly of the nucleocapsid and budding of the virions (Compans et al., 1966
). The nucleocapsid of inclusions with loose and tight packaging had similarities to the granular and smooth forms, described in cells persistently infected with Newcastle disease virus (McNulty et al., 1977
).
The virus acquires an envelope when it is assembled and released by budding through areas of the plasma membrane that have been modified by alignment of nucleocapsids on the cytosolic side and insertion of macromolecules visible as spikes on the outside. The spike length of approximately 10 nm was within the 812 nm size range of paramyxovirus outer projections and the attachment to erythrocytes was consistent with the presence of haemagglutinins among the envelope glycoproteins (Lamb et al., 2000; Lamb & Kolakofsky, 2001
). The detection of haemadsorption long before CPE showed that incorporation of haemagglutinin glycoproteins into the plasma membrane is an early event, which has also been observed after infection with parainfluenza virus (Fedova & Zelenkova, 1969
). The receptor-destroying enzyme activity, demonstrated by the elution of erythrocytes in the haemagglutination and the haemadsorption assays, was identified as a neuraminidase, which is consistent with the presence of this enzyme in most paramyxoviruses (Lamb et al., 2000
; Lamb & Kolakofsky, 2001
). Failure of Newcastle disease virus to re-agglutinate erythrocytes eluted for virus further substantiated the relationship with the paramyxoviruses. Finally, the detection of syncytia in infected cell cultures strongly indicated the presence of fusion activity in the virion. Hence, this new virus has all three surface activities typical of most paramyxoviruses and orthomyxoviruses including haemagglutinating, receptor-destroying (neuraminidase) and fusion activities.
The apparent loosening of the nucleocapsid from the inner envelope of released virions has also been observed in other viruses of this family and has been interpreted as part of the maturation process (Kim et al., 1979; Bächi, 1980
; Markwell & Fox, 1980
). Whilst the data on virion size in cell cultures are presumably not representative, due to loss of released particles during processing for electron microscopy, in negatively stained preparations the majority of virions were pleomorphic or spherical with a diameter of 150300 nm, which is in agreement with the size described for paramyxoviruses in general (Lamb et al., 2000
; Lamb & Kolakofsky, 2001
). The slow replication rate of this virus compared with several other fish viruses, for example infectious salmon anaemia virus in the same temperature range (Dannevig et al., 1995
), is consistent with the slow replication rate of at least some paramyxoviruses (Choppin, 1964
).
The paramyxo-like viruses isolated from chinook salmon (Winton et al., 1985) and carp (Body et al., 2000
) have optimal temperatures of approximately 18 and 21 °C, respectively, for in vitro replication, while the results presented here indicated a lower optimal temperature for the Atlantic salmon paramyxovirus. The optimal replication temperatures of all these viruses suggest that their host ranges are confined to cold-blooded animals. The majority of viruses accepted as members of the Paramyxoviridae infect warm-blooded animals or man (Lamb et al., 2000
; Lamb & Kolakofsky, 2001
), but a virus infecting snakes has also been assigned to the family (Clark et al., 1979
; Ahne et al., 1999
; Lamb et al., 2000
). The emergence of these new viruses from fish and reptiles provides opportunities to expand our knowledge of the evolution, epidemiology and pathogenesis of paramyxoviruses.
We do not know how long the Atlantic salmon paramyxovirus has been present in Norwegian aquaculture, as the extended length of time required to produce CPE on initial isolation may have allowed the virus to go undetected until now. However, gill diseases have for years been a problem in Norwegian aquaculture and the importance of this new virus as a possible aetiological agent should be further elucidated.
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ACKNOWLEDGEMENTS |
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Received 7 November 2002;
accepted 11 April 2003.
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