Structure and morphogenesis of Dugbe virus (Bunyaviridae, Nairovirus) studied by immunogold electron microscopy of ultrathin cryosections

Viru_sResew&, 21 (1991) 199-212 0 1991 Elsevier Science Publishers

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199 B.V. All rights resewed

0168-1702/91/$03.50

00715

Structure and morphogenesis of Dugbe virus (Bunyaviridae, Nairovirus) studied by immunogold electron microscopy of ultrathin cryosections T.F. Booth, E.A. Gould and P.A. Nuttall NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, U.K. (Accepted

5 August

1991)

Summary

We have studied the structure and morphogenesis of Dugbe (DUG) virus (Bunyaviridae, Nuirouirus) in cultured porcine kidney (PS) cells and a tick cell line (Ra 243) using immunogold electron microscopy. DUG virus is a tickborne arbovirus, considered to be a low health hazard, that is antigenically and genetically related to Crimean Congo haemorrhagic fever (CCHF) virus (Marriott et al., 1990). We have investigated the maturation and intracellular transport of DUG virus particles as a model for other more pathogenic nairoviruses using monoclonal antibodies for immunogold labelling of ultrathin cryosections and immunofluorescence techniques. The spherical DUG virus particle measures about 90 nm in diameter, with a 5 nm thick membrane covered by 5-7 nm long projections or “spikes”. These projections form hollow cylindrical morphological units, about 5 nm in diameter. DUG virus infection caused only a slight cytopathogenic effect in mammalian cells and none in tick cells. DUG virus particles assembled by budding from the Golgi complex, where the DUG virus glycoprotein Gl accumulated in vesicles originating from Golgi cisternae. The nucleocapsid protein N accumulated in scattered foci throughout the cytoplasm, and this appears to be related to the limited maturation of DUG virus particles that occurred. The reduced number of budding virus particles observed in tick cells was correlated with the reduced cytopathology observed. Correspondence to: T.F. Booth, NERC field Road, Oxford OX1 3SR, U.K.

Institute

of Virology

and Environmental

Microbiology,

Mans-

200

Dugbe virus; Bunyaviridae; Nairovirus; Immunogold labelling; Structure and morphogenesis; Golgi complex

Introduction

Dugbe (DUG) virus is a member of the ~~~~0~~~ genus in the Bunya~ridae, a diverse group of enveloped arthropod-borne viruses with several structural features in common (Bishop and Shope, 1979; Casals and Tignor, 1980). Bunyaviruses have a lipid bilayer envelope and a diameter of between 90 and 120 nm. They possess a tripartite single-stranded RNA genome, the three segments being designated large (L), medium (M) and small (S), (Bishop et al., 1987). The Nairovirus genus consists of seven serogroups, all transmitted by various species of ticks: Crimean-bongo haemo~rhagi~ fever (CCHF) group, Dera Ghazi Khan (DGK) group, Hughes group, Nairobi sheep disease (NSD) group, Qalyub group, Sakhalin group (Clerx et al., 1981) and Thiafora group (Zeller et al., 1989). CCHF is a major health risk to humans in parts of Africa, Asia and Europe (Casals and Tignor, 1974) and NSD virus causes economic losses of cattle and sheep in Africa (Davies et al., 1978). DUG virus, which is considered to present a low medical and veterinary health hazard, is both antigenically and genetically related to CCHF virus, thus providing a safe and relevant model for studying nairoviruses (Marriott et al., 1990; Ward et al., 1990). Using monoclonal antibodies and immunoblotting techniques, some nairoviruses have been shown to possess three structural proteins by radio-immune precipitation: a nucleoprotein (N: 48-54 kD) and two external glycoproteins (Gl: 72-84 kD and G2: 30-40 kD). The Gl protein contains epitopes that react with neutralizing monoclonal antibodies (Buckley et al., 1990). For some nairoviruses, however, a Gl protein has not been detected, and the G2 has only been detected in members of the Qalyub and CCHF serogroups (Clerx et al., 1981). Dugbe virus replicates and persists trans-stadially in orally infected ~~b~yo~~a LJu~eg~~~~ ticks and can subsequently be transmitted by tick feeding to a vertebrate host (Steele and Nuttall, 1989). Despite being highly efficient at transmitting DUG virus, no ultrastructural cytopathic effects were observed in DUG virus-infected ticks by electron microscopy, and mature virions were scarce (Booth et al., 1990). Therefore we have compared the cytopathology observed with DUG virus replication in mammalian tissue cultured cells with that in a tick cell line. DUG virus encodes at least six polypeptides (Cash, 1985). We have used monoclonal antibodies to investigate the structure and mo~hogenesis of DUG virus in cells using immunofluorescence and immunoele~tron microscopy. Materials

and Methods

Virus, cells

The continuous porcine kidney cell line (PS cells) obtained from Dr. J.S. Porterfield (Sir William Dunn School of PathoIo~, Oxford) was grown in Lei-

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bowitz L15 medium supplemented with 10% tryptose broth and 3% foetal bovine serum (FBS). Virus titers were determined by plaque titration in PS cells (DavidWest and Porterfield, 1974). Maximum titers were obtained 48 h pi. (lo6 p.f.u./ml) after infection at an m.o.i. of 1. The DUG ArD 44313 strain was chosen as being the closest to the original tick isolate obtained from a pool of 20 male Amblyomma variegatum ticks collected in November 1985 at Bourbufaye, Senegal, suppliec

Fig. 1. DUG-virus infected porcine kidney cells stained by immunofluorescence; A, anti-G1 monoclonal antibody, 48 h pi.; B, control; C, anti-N monoclonal, 12 h p.i. (arrow: cell containing cytoplasmic antigen foci); D, anti-N monoclonal, 18 h p.i.; E, anti-G1 monoclonal, 12 h p.i.

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Dr. J.-P. Digoutte (Institut Pasteur, Dakar, Senegal). This virus was identified by using the complement fixation test and confirmed to be DUG virus by cross-immunofluorescence and cross-neutralization with the prototype strain, DUG Ib Ar 1792. A cell line from the tick Rhipicephalus uppendicufutus {Ra 234; (Leake et al., 1980)} obtained from Prof. M.G.R. Varma, London School of Tropical Medicine and Hygiene, was grown in Leibowitz L1.5 medium plus 10% FBS at 28°C. Cultures of Ra 234 cells infected with DUG virus produced a maximum titre of lo2 p.f.u./ml, and these cultures of Ra 234 cells became persistently infected with DUG virus as demonstrated by immunofluorescence microscopy, using DUGspecific antibody. Antisera

The DUG strain KT 281/75 (Buckley et al., 1990) was used to induce polyclonal rabbit hyperimmune antiserum and mouse monoclonal antibodies against DUG virus. This strain was antigenically indistinguishable from the ArD 44313 and the prototype strains. A hyperimmune polyclonal antiserum to DUG virus was raised in rabbits, and monoclonal antibodies to DUG virus were raised in Balb/c mice (Buckley et al., 1990). Spleen cells from the immunized mice were fused with NSO cells (Chanas et al., 1982). Two monoclonals were used for immunolabelling studies: H28.17, which reacts with the Gl protein of DUG virus, neutralizes the virus in vitro, and protects mice from DUG infection, and H 28.89, which reacts with the N protein of DUG virus (Buckley et al., 19901. Immunocytochemistry

PS cells inoculated with DUG virus at an m.o.i. of 1 were incubated for 12, 18, 36 or 48 h prior to fixation for immunolabelling. For immunofluorescence, cells were acetone fixed and stained by an indirect method using a biotinylated secondary antibody with a streptavidin-conjugated fluorescein isothiocyanate detection system (Amersham International, Amersham, U.K.). For electron microscopic (EM) immunolabelling, two methods of on-grid immunolabelling were used. After fixation in 1% paraformaldehyde, specimens were either embedded in LR White resin (London Resin Co., Basingstoke, U.K.) for sectioning (Newman et al., 1983), or infused with sucrose and frozen in liquid nitrogen for cryo-ultramicrotomy and immunolabelling according to the methods of Griffiths et al. (1984). Bound antibody was identified on sections by using a secondary incubation step with 10 nm immunogold conjugates (goat anti-mouse IgG, or goat anti-rabbit IgG) obtained from BioCell Labs., Cardiff, U.K. Controls included omitting the primary

Fig. 2. Thin sections of A: uninfected PS cell and B: DUG virus infected PS cell, 48 h pi. Viral infection causes an increased number of vacuoles (v) compared to the normal Golgi complex (GC). Virus particles about 100 nm in diameter appear to be associated with these Golgi vacuoles (arrow). Inclusion bodies (ib) consisting of ribosome-like particles also accumulate cytoplasmically. Scales: A, 500 nm; B, 1 Grn.

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20.5

antiserum, or substituting an irrelevant one, and uninfected cells. Specimens were also fixed using conventional 2% ~utaraldehyde and 1% osmium tetroxide double fixation for embedding in epoxy resin (Emix, Emscope) for ultrathin sectioning and observation by transmission EM. DUG virus particles were negatively stained on carbon filmed grids using 2% ammonium molybdate. Specimens were observed in a JEOL 1OOCelectron microscope operated at 100 kV and micrographs were taken at magnifications of 10,000 to 30,000 times.

Results Localization

ofDUG

uiral antigens by immunofluorescence

Immunofluorescent antibody staining showed an initial signal in the Golgi region of infected PS cells 12-18 h post infection (p.i.), that could first be detected by the Gl monoclonal antibody (Fig. l.E). At 48 h pi., when the supernatant titres of DUG virus reached maximal levels, a strong Gl c~oplasmic reaction was present in 95% of infected cells (Fig. 1A) which was absent in control, mock-infected cells (Fig. 1B). Only very slight cytopathological changes were observed in infected cells by light microscopy, with slight rounding of infected cells. The anti-N protein monoclonal detected DUG virus antigen initially in scattered cytoplasmic foci (Fig. 1C) from which it spread throughout the cytoplasm (Fig. lC,D>. A similar pattern with both the Gl, N and polyclonal anti-DUG virus antibodies was observed in the tick cell line (data not shown) except that maximal infection of cells was not observed by immuno~uorescence until 6 days p.i., with about 20% of cells infected. Electron microscopy

In the specimens prepared for conventional electron microscopy, DUG virus infection was observed to cause an increased vacuolization of the Golgi apparatus, with an increased number of vesicles budding from the Golgi stack compared to that of control, uninfected cells (Fig. 2A,B). Virus particles approximately 90 nm in diameter appeared to be within distended vesicles, probably of Golgi origin (Fig. 23) and these particles were identified as virions by immunogold labeling (Fig. 3B). Infected cells cont~ned inclusion bodies consisting of arrays of ribosome-like particles (Fig. 2B, 6), and these areas were identified as containing the DUG virus N protein by immunogold labeling of ultrathin cryosections with the anti-N monoclonal antibody (Fig. 3A). The anti-G1 monoclonal did not bind to these

Fig. 3. Electron microscopic examination of immunogold-labelled ultrathin cryosections of DUG virus infected PS cells, 48 h pi. A: Monoclonal anti-DUG N protein labels the cytoplasm, inclusion bodies (IB) but not nucleus (n> or cell plasma membrane (arrow) of infected cells. B: Gold particles (arrows) bind to the sectioned surface of virus particles in large vacuoies in infected cells: (poiyclonal anti-DUG antibody). Scales: A and B, 200 nm.

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areas, (data not shown), but the polyclonal rabbit antiserum labeled these nucleocapsid regions in addition to the mature virus particles a~umulating in intracellular vacuoles (Fig. 3B). Neither the anti-N monoclonal (Fig. 3A), nor the anti-G1 monoclonal, labeled the surface of the plasma membrane or the nucleus of infected cells. Many of the increased numbers of budding Golgi vesicles seen in sections of DUG virus infected cells were labelled with the anti-G1 monoclonal antibody (Fig. 4) but these vesicles appeared smaller (about 50 nm in diameter) than mature virions, and did not label with the anti-N monoclonal. Background labeling on all control sections of non-infected cells was very low (Fig. 4A) and such cells showed normal organelle morphology. The changes in morphology of the Golgi complex associated with DUG virus infection were compared and found to be the same in both the cryosectioned (Fig. 4) and conventionally sectioned (Fig. 2) specimens, allowing for the slightly different appearance of the stained structures in the cryosections which show up partially in negative contrast. Structure of DUG viriom The structure of DUG virus particles was investigated in sections of both resin embedded and frozen PS cell cultures, and in negatively stained specimens of culture medium (Fig. 5). Sections of frozen specimens, (Fig. 5A) showed spherical virions possessing a bilayer membranous envelope; in this technique, stoning contrast was partially positive, partially negative, as compared with plastic sections (Fig. 5C) in which only a positive staining effect was apparent. Moreover, surface spikes or projections and pore-like passages in the envelope were evident (Fig. 5A). Ultrastructural detail deteriorated after immunolabelling (Fig. SC) although the spherical shape and bilayered nature of the envelope allowed easy identification of immunogold-labelled virus particles. In sections of plastic embedded specimens, the surface projections can be seen to be cylindrical in shape where they passed through the plane of the section at the correct angle (Fig. 5B) but pore-like passages were not seen by this technique. Surface envelope projections were also observed by negative staining (Fig. 5D).

Discussion Nairoviruses are the least well studied members of the Bunyaviridae. Dugbe virus is a good model for more pathogenic nairoviruses, such as those that cause CCHF and NSD, so it was important to determine the structural and morpho-

Fig. 4. DUG glycoprotein Gl accumufates in Goigi vesicles: ultrathin cryosections, immunogold labeled with anti-G1 monoclonal. A: Control uninfected PS cell culture: few gold particles; n: nucleus, G: Golgi complex; shows normal morphology, few gold particles bound to the surface. B: DUG-virus infected PS cell, 48 h pi. Gofd labeling on Golgi vesicles (arrows) which are increased in number. Little antigen is present in the er or elsewhere. N, nucleus; G, Golgi cisternae; er, endoplasmic reticulum. Scales A and B: 200 nm.

Fig. 5. A: Ultrathin cryosection of DUG virus infected PS cells. The DUG virion shows pore-like structures in the envelope (solid arrow) and an indication of spike-like projections (open arrow). B: Paraformaldehyde-fixed tissue, embedded in LR White resin, 60 nm section, stained in many1 acetate: 5 nm diameter circular profiles are visible (arrows). C: Immuno~old-labeled ultrathin cryosection of DUG virus infected PS cells ~paraformaldehyde Tied) labelled with rabbit polyclonal anti-DUG antibody and goat-anti-rabbit 10 nm immunogold. Some deterioration of ultrastructure due to immunolabelling is evident. D: Negatively stained virion in cell culture supernatant. Scale (A-D) = 100 nm.

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Fig. 6. DUG virus infected tick cell (Ra 243) culture, showing viral inclusion body (IB). Scale: 1 Frn.

genetic charcteristics of DUG virus in relation to other Bunyaviridae. The DUG virus particle had a spherical structure consisting of a lipid bilayer membrane envelope with integral glycoprotein spikes arranged in a surface mosaic. This is broadly similar to other Bunyaviruses, including CCHF virus (Ellis et al., 1980) and Uukuniemi virus (van Bonsdorff and Pettersson, 1975). The DUG virus particle however, was smaller in diameter (90 nm) compared to CCHF (about 120 nm), and the DUG virion had a much larger number of smaller spikes on the envelope surface (5-7 nm projection, 5 nm diameter), making a clear determination of their arrangement and symmetry from either negative stain or thin section electron micrographs impossible (Fig. 5). Some micrographs indicated that the spike projections of DUG virus are “hollow” and tube-like, in common with those of CCHF virus (Ellis et al., 1980), (Fig. 5B). In addition, “pores” similar to those of CCHF virus particles were observed (Fig. 5A). Further cryo-electron microscopy investigations should provide better resolution and allow models of the DUG virion to be reconstructed. A characteristic feature of bunyaviruses is their intracellular maturation by budding at smooth-surfaced membranes in the Golgi region (Murphy et al., 1973), which is associated with an expansion and increased vacuolization of the Golgi complex, and the accumulation of viral proteins in the Golgi region of infected

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cells (Gahmberg et al., 1986; Kuismanen et al., 1982, 1984, Madoff and Lenard, 1982). It has also been shown that bunyavirus glycoproteins, expressed from cloned cDNA, accumulate intracellularly in the Golgi complex of cells rather than being transported to the surface (Matsuoka et al., 1988). In this study we investigated the structure and morphogenesis of DUG virus, using monoclonals specific for the Gl and N proteins, and a polyclonal hyperimmune rabbit antiserum. DUG virus was seen to mature by budding intra~eIluIarly at smooth membranes of the Golgi complex. The DUG virus Gl glycoprotein accumulated in vesicles of the Golgi complex which showed an increase in vacuolization. It seems probable that the va~uol~ation was caused by the accumulation of the glycoprotein in the Golgi region, as suggested by Gahmberg et al. (1986) for Uukuniemi virus (Bunyaviridae). The presence of Gl, and the absence of the N protein, on the surface of virus particles, and the presence of the N protein in a nucIeocapsid preparation, have been demonstrated previously (El-Ghorr et al., 1990). The N protein was identified in arrays of nucleocapsids and localized in inclusion bodies which formed initially in scattered foci throughout the cytoplasm of infected tick and mammalian cells. This is a similar situation to that observed in a non-cytopathi~ temperature-sensitive mutant of Uukuniemi virus, where the glycoproteins accumulated in the Golgi complex of cells at the restrictive temperature, and the nucleocapsids remained scattered in the cytoplasm. Virion maturation was thus defective, with few virions observed budding at Golgi membranes (Gahmberg et al., 1986). In infected ticks, almost no cytopathic effect of DUG virus infection was observed, yet viral antigen, replication, viral sense RNA, and viral infectivity, could all be detected in tick tissues (Booth et al., 1990). By contrast, in young mice, DUG virus infection caused mild histopathologic changes, including neuronal necrosis in the hippocampus, and mortaIity, (Coates and Sweet, 1990) although no haemorrhagic disease was caused. DUG virus produced only a slight cytopathic effect in PS cells (cell rounding, some cell detachment), and less than 10% of the cell section profiles contained virus particles at 48 h p.i. In the tick cell line, DUG virus grew to a low titre (lo2 p.f.u./ml). The relatively mild cytopathic effect of DUG virus observed in the tick cells (no cell detachment) as compared to the PS cells may be related to the ability of DUG virus infections to persist in tick hosts for months with no extensive cytolytic activity, or detrimental effect on the ticks’ subsequent ability to transmit the infection to a vertebrate during feeding (Steele and Nuttall, 19891. The Ra 243 tick cell line has been shown to support persistent infection with several other arboviruses (e.g. Louping ill virus, Semliki forest virus) in the absence of any cytopathic effects (Leake et al., 1980). Persistently infected DUG virus carrier cell cultures have also been obtained using mammalian (PS) cells by David-West and Porterfield (1974). This alternative mode of infection may be an important strategy for the maintenance of infections in the absence of severe ~opathologi~al effects, and is probably related to the observed reduction in virion maturation in tick cells. DUG virus is thus an excellent model for further studies of persistent subclinical nairovirus infections, particularly invoIving arthropod cells, and for further investigations of the, molecular interactions involved in control of viral maturation and pathogenicity.

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Acknowledgements This work was supported by the U.S. Army Medical Research and Development Command under contract DAMD 17-87-C-7176 and United Kingdom contract No. CB/SL/23c/1831. We also thank Dr. David Shotton, Zoology Dept., Oxford University for the use of equipment provided by the EPA cephalosporin fund.

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4 June

1991; revision

received

5 August

1991)